Review
Cite This: Chem. Rev. XXXX, XXX, XXX−XXX pubs.acs.org/CR
Intracellular Delivery by Membrane Disruption: Mechanisms,
Strategies, and Concepts
Martin P. Stewart,*,†,‡,@ Robert Langer,*,†,‡ and Klavs F. Jensen*,†
†Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States
‡The Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139,
United States
@School of Life Sciences, Faculty of Science, University of Technology Sydney, Ultimo, NSW, Australia
ABSTRACT: Intracellular delivery is a key step in biological research and has enabled
decades of biomedical discoveries. It is also becoming increasingly important in indus-
trial and medical applications ranging from biomanufacture to cell-based therapies.
Here, we review techniques for membrane disruption-based intracellular delivery from
1911 until the present. These methods achieve rapid, direct, and universal delivery of
almost any cargo molecule or material that can be dispersed in solution. We start by
covering the motivations for intracellular delivery and the challenges associated with the
different cargo typessmall molecules, proteins/peptides, nucleic acids, synthetic nano-
materials, and large cargo. The review then presents a broad comparison of delivery
strategies followed by an analysis of membrane disruption mechanisms and the biology of
the cell response. We cover mechanical, electrical, thermal, optical, and chemical strategies
of membrane disruption with a particular emphasis on their applications and challenges to
implementation. Throughout, we highlight specific mechanisms of membrane disruption
and suggest areas in need of further experimentation. We hope the concepts discussed in
our review inspire scientists and engineers with further ideas to improve intracellular delivery.
CONTENTS 4.1.4. Structure and Properties of the Cell
Surface Q
1. Introduction C 4.2. Defect Formation in Lipid Membranes Q
2. Intracellular Delivery Cargo and Applications C 4.2.1. Mechanical and Electrical Q
2.1. Overview of Key Applications C 4.2.2. Chemical R
2.1.1. Intracellular Delivery is Moving Beyond 4.3. Cell Response to Membrane Disruption R
Traditional Transfection C 4.3.1. Plasma Membrane Repair S
2.1.2. Intracellular Delivery for Cell-Based 4.3.2. Cell Swelling T
Therapies D 4.3.3. State of the Resealed Cell T
2.1.3. Intracellular Delivery in Stem Cell 4.3.4. Stress Response After Membrane Dis-
Reprogramming D ruption U
2.2. Cargo Categories for Intracellular Delivery E 4.3.5. Manipulating Cell Response to Opti-
2.2.1. Small Molecules E mize Outcomes U
2.2.2. Proteins and Peptides F 4.3.6. Semi-intact Cells W
2.2.3. Nucleic Acid Transfection I 5. Intracellular Delivery by Direct Penetration W
2.2.4. Synthetic Nanomaterials and Devices K 5.1. Microinjection W
2.2.5. Large Cargo L 5.1.1. Advances in Technical Precision of
3. Approaches for Intracellular Delivery M Microinjection X
3.1. Carrier-Mediated M 5.1.2. Attempts toward Higher Throughput
3.2. Membrane Disruption-Mediated N Microinjection X
3.2.1. Direct Penetration N 5.1.3. Microinjection Summary Y
3.2.2. Permeabilization O 5.2. Penetrating Projectiles (Biolistics) Y
4. Membrane Disruption-Mediated Delivery: Back- 5.2.1. Cell Type Applicability Y
ground Concepts O 5.2.2. Cargo Applicability Z
4.1. Cell Structure and Properties O 5.2.3. Biolistic Systems and Variations Z
4.1.1. Plasma Membrane Function O
4.1.2. Plasma Membrane Composition and
Properties P Received: November 9, 2017
4.1.3. Intrinsic Membrane Permeability Q
© XXXX American Chemical Society A DOI: 10.1021/acs.chemrev.7b00678
Chem. Rev. XXXX, XXX, XXX−XXX
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5.2.4. Penetrating Projectiles (Biolistics) Sum- 6.2.2. Mechanisms of Electroporation in Cell
mary Z Suspensions AR
5.3. Nanowires and Nanostraws Z 6.2.3. Targeting Subcellular Structures Across
5.3.1. Expanding the Repertoire of Deliverable the Pulse Strength-Duration Space AR
Cargo AA 6.2.4. Cargo-Dependent Influx Mechanisms AS
5.3.2. Nanowire Penetration Mechanisms AA 6.2.5. Cargo-Dependent Influx Mechanisms:
5.3.3. Nanowire Effects on Cells AB Small Molecules AS
5.3.4. Nanostraw Arrays for Injection and 6.2.6. Cargo-Dependent Influx Mechanisms:
Extraction AB Proteins & Other Macromolecules AT
5.3.5. Mechanisms of Cargo Delivery by 6.2.7. Cargo-Dependent Influx Mechanisms:
Penetrating Elements AB Plasmid DNA AU
5.3.6. Nanowire and Nanostraw Summary AC 6.2.8. Cargo-Dependent Influx Mechanisms:
6. Intracellular Delivery by Permeabilization AC siRNA & Other Oligonucleotides AV
6.1. Mechanical Membrane Disruption AC 6.2.9. Summary of Cargo-Dependent Influx
6.1.1. Mechanical: Solid Contact AC Mechanisms AW
6.1.2. Solid Contact: Sudden Cell Shape 6.2.10. Tailoring Pulse Parameters for Optimal
Changes & Protease Treatments AD Delivery AW
6.1.3. Solid Contact: Projectile Permeabiliza- 6.2.11. Dual Pulse Strategies AW
tion AD 6.2.12. Nucleofection Mechanisms AX
6.1.4. Solid Contact: Filtroporation AE 6.2.13. Electroporation Challenges & Techni-
6.1.5. Solid Contact: Microfluidic Cell Squeez- cal Advancements AX
ing AE 6.2.14. Technical Innovations: Bulk, Micro- &
6.1.6. Solid Contact: Nanowires for Transient Nano- Electroporation BA
Permeabilization AG 6.2.15. Capillary Electroporation BA
6.1.7. Solid Contact: Summary AH 6.2.16. Microfluidic Electroporation BA
6.1.8. Fluid Shear-Mediated Permeabilization AH 6.2.17. Nanochannel Electroporation BB
6.1.9. Fluid Shear: Syringe Loading AH 6.2.18. Nanostraw Electroporation BB
6.1.10. Fluid Shear: Microfluidic Control of 6.2.19. Nanofountain Probe Electroporation BC
Shear Forces AI 6.2.20. Summary of Micro- and Nano-electro-
6.1.11. Fluid Shear: Other Examples of Cell poration BC
Permeabilization Through Shear 6.2.21. In Vitro & Ex Vivo Applications of
Forces AI Electroporation BC
6.1.12. Fluid Shear: Sonoporation AI 6.2.22. Intracellular Delivery of Impermeable
6.1.13. Fluid Shear: Shock Wave-Mediated Drugs BC
Permeabilization AK 6.2.23. Biomanufacture Through Transfection BC
6.1.14. Fluid Shear: Laser-Induced Cavitation 6.2.24. Large Volume Flow Electroporation BC
Bubbles AK 6.2.25. Delivery of Genome-Editing Proteins
6.1.15. Fluid Shear: Laser-Induced Cavitation and RNPs BD
via Absorbent Particles AL 6.2.26. Hard-to-Transfect Cells BE
6.1.16. Fluid Shear: Laser-Induced Cavitation 6.2.27. T Cells & Other Immune Cells BE
at an Interface AL 6.2.28. Ex Vivo Intracellular Delivery for Cell-
6.1.17. Fluid Shear: Summary AM Based Therapies BE
6.1.18. Pressure Change-Mediated Permeabi- 6.2.29. Electroporation Summary BG
lization AM 6.3. Thermal Membrane Disruption BG
6.1.19. Osmotic Shock and Plasma Membrane 6.3.1. Thermal Shock of Competent Bacteria BH
Disruption AM 6.3.2. Freeze−Thaw and Other Temperature
6.1.20. Hypotonic Loading of Red Blood Cell Cycling Strategies BH
Ghosts AN 6.3.3. Supraphysiological Heating BI
6.1.21. Hypotonic Shock for Intracellular De- 6.3.4. Thermal Inkjet Printers BI
livery AN 6.3.5. Laser−Particle Interactions BI
6.1.22. Osmotic Gradients Acting on Part of 6.3.6. Laser−Membrane Interactions BI
the Plasma Membrane AO 6.3.7. Thermal Membrane Disruption Sum-
6.1.23. Hydrostatic Pressure and Hydrody- mary BJ
namic Delivery AO 6.4. Optical Membrane Disruption (Optopora-
6.1.24. Disruption of Endosomes by Osmotic tion) BJ
Forces AO 6.4.1. Optoporation: Pioneering Studies BJ
6.1.25. Induced Transduction by Osmocytosis AP 6.4.2. Mechanisms of Optoporation BJ
6.1.26. Pressure Changes: Summary AP 6.4.3. Femtosecond Optoporation BK
6.2. Electrical Membrane Disruption (Electro- 6.4.4. Toward High Throughput and More
poration) AP User-Friendly Optoporation BK
6.2.1. Mechanisms of Membrane Disruption 6.4.5. Optoporation Summary BK
and Cargo Entry AQ 6.5. Biochemical Membrane Disruption BK
B DOI: 10.1021/acs.chemrev.7b00678
Chem. Rev. XXXX, XXX, XXX−XXX
Chemical Reviews Review
6.5.1. Organic Solvents and Penetration En- delivery have come about. It also clarifies why membrane-
hancers BL disruption-based delivery has become a key approach.
6.5.2. Detergents BM In this review we cover literature from 1911 to the present.
6.5.3. Membrane-Active Peptides BQ However, the field of membrane disruption-mediated intra-
6.5.4. Pore-Forming Proteins and Toxins BS cellular delivery was small until the mid 1980s, which coin-
6.5.5. Chemical Destabilization BU cided with the rise of electroporation along with other means
7. Gated Channels and Valves BV of cell permeabilization. We have narrowed the discussion of
7.1. Endogenous Channels (ATP-Activated) BV membrane disruption-mediated delivery primarily to cells in
7.2. Endogenous Channels (Swelling-Activated) BV vitro, as opposed to in vivo scenarios. The review will focus
7.3. Engineered Channels/Valves BW mostly on cells of animal and human origin, although we will
7.4. Optogenetic Control of Cell Permeability BW sometimes venture beyond this scope to highlight particular exam-
7.5. Stimuli-Sensitive Channels for Larger Cargo ples in bacteria, microorganisms, and plants.
Delivery BW To begin the review, we will first cover the types of cargo
7.6. Nanodevice Gating BW that researchers seek to deliver and their applications. This
8. Summary and Outlook BW includes an overview of cargo chemical and dimensional prop-
8.1. Summary BW erties, as these characteristics are inextricably linked to the chal-
8.2. Outlook CC lenges involved in their delivery. We then survey all methods of
Author Information CD cargo delivery, defining what is membrane disruption-mediated
Corresponding Authors CD and what is not. Specifically, we break this into two areas:
ORCID CD (1) carrier-mediated delivery, which comprises endocytic and
Notes CD fusion entry pathways, and (2) membrane disruption-based intra-
Biographies CD cellular delivery, which includes direct penetration and plasma
Acknowledgments CD membrane permeabilization mechanisms. Next, we provide rel-
References CE evant background on cell membranes, their function, and
mechanisms of disruption and cell recovery. We then explore
1. INTRODUCTION each membrane disruption technique in depth, highlighting its
history, the mechanisms by which it operates, pros and cons,
Cells transmit information through molecules. Just as com- and, where appropriate, a perspective on opportunities and poten-
puter chips process information using electronic signals, the tial feasibility.
currency of information exchange in cells is molecules. DNA
encodes RNA and proteins. Proteins perform work, transmit 2. INTRACELLULAR DELIVERY CARGO AND
signals, and act as building blocks of cellular structure. Lipids APPLICATIONS
form membranes and store energy. The cell is infinitely more
complex than an electronic device; we are still learning how it 2.1. Overview of Key Applications
works. In addition to the natural molecules that comprise cells,
new technologies are enabling synthetic materials to be deployed For decades researchers have been developing, synthesizing,
within cells. Introducing molecules and materials into cells is and adapting molecules and materials for deployment to the
an important step in decoding cell function, guiding cell fate, intracellular environment. Most of these “cargo” are membrane
and reprogramming cell behavior. Thus, intracellular delivery is impermeable and thus require intracellular delivery. In this sec-
central to our ability to understand biology and treat disease. tion, we provide an overview of the key applications of
This review is intended for anyone interested in intracellular intracellular delivery and the categories of cargo that research-
delivery: the biologist looking for the most appropriate method ers seek to deliver along with related challenges.
for their project, the chemist investigating a novel molecule 2.1.1. Intracellular Delivery is Moving Beyond Tradi-
that requires verification in live cells, the engineer searching to tional Transfection. Transfection refers to the intracellular
develop innovative new intracellular delivery technology, the delivery of nucleic acids: DNA and RNA. Nucleic acids have
cell physiologist seeking a deeper understanding of the mech- been the most popular category of cargo material delivered
anisms underlying membrane disruption-based delivery, or the into cells over the last decades. Genetic modulation with DNA
biomanufacturing expert examining ways to improve produc- or RNA is viewed as a robust route for controlling cell
tion efficiency. This review seeks to deconstruct the literature function. Increasingly, however, researchers are discovering
into a clear and understandable framework. More than 1500 new ways to manipulate cells with other forms of cargo, for
15−18
papers are referenced, but we have examined almost 4000 in example, genome-editing nucleases, synthetic intra-
the process of compiling this paper. cellular probes,
19,20 and combinations of proteins and/or inhib-
itors that guide cell fate.21,22The scope of this review is focused on membrane disruption- This reflects a transition from the
based intracellular delivery, as opposed to carrier-mediated meth- narrowly-focused delivery of nucleic acids to a wider concept
ods. There are many more reviews on carriers (also known as of “intracellular delivery”. To illustrate this, Figure 1 depicts
vectors, vehicles, nanocarriers, and delivery nanoparticles), par- the diversity of cargo that can be delivered into cells and the
ticularly for nucleic acid delivery,1−9 including in this jour- potential outcomes. The schematic highlights the progression
nal.10−15 Comparatively fewer reviews exist on membrane from input cargo to cellular output states and end-point appli-
disruption-based delivery, possibly due to the diverse array of cations. In all these cases, the prime challenge is that imper-
approaches for creating holes in membranes. Part of the scope meable cargo must be introduced to the cell interior without
of this review also covers the different cargo types that research- untoward damage or perturbation to the cell. The five horizon-
ers seek to deliver to the intracellular space. This analysis tal tiers in Figure 1 are not mutually exclusive, having signif-
illustrates why and how such diverse methods of intracellular icant overlap between inputs and outputs. This “menu” of options
C DOI: 10.1021/acs.chemrev.7b00678
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Figure 1. Example motivations for intracellular delivery. Combinations of cells and cargo molecules/materials are shown on the left. Through
intracellular delivery these cargoes are able to confer the outcome or application depicted on the right. The horizontal tiers are not mutually
exclusive and substantial overlap exists between them. Abbreviations: TCR = T cell receptor. CAR = chimeric antigen receptor. CNT = carbon
nanotube. HSCs = hematopoietic stem cells.
reflects the combinatorial potential of intracellular delivery to in vitro manipulation step. Ex vivo cell-based therapies have
analyze cell properties and engineer cell function. demonstrated efficacy in treating several human diseases in clin-
2.1.2. Intracellular Delivery for Cell-Based Therapies. ical trials.28,29 Examples include hematopoietic stem cell (HSC)
In cell-based therapies, cells can be viewed as a living drug to transplantation30 and engineering of immune cells for cancer
be administered to the patient. Cells that have been modified, immunotherapy,23,25,31,32 as mentioned above. Disease-causing
repaired, or reprogrammed are introduced into a patient to confer mutant HSCs can be genetically corrected with ex vivo gene
a therapeutic effect or restore lost function. For example, when therapy, whereby stable genomic modifications are used to
endogenous immune cells lose their ability to eliminate cancer confer a durable therapeutic effect.28 Recent successes include
cells, modified T cells can be introduced to compensate.23 viral vector-mediated gene therapy for correction of monogenic
In the case of CAR-T cells, novel function is conferred through diseases such as severe combined immunodeficiency (SCID-X1),
induced expression of specific T cell receptors (TCRs) or chi- Wiskott-Aldrich syndrome (WAS), and β-thalassemia.29 The
meric antigen receptors (CARs) that guide the T cells to bind future delivery of genome editing components for precise gene
to, and attack, speci c cancer cells.24,25fi Recent clinical trials correction is anticipated to improve the safety and efficiency of
against B cell malignancies validate the power of this approach,26 HSC gene therapy above what is currently attained with viral
which was approved in 2017 by the United States Food and vectors.16,33,34
Drug Administration (FDA).27 2.1.3. Intracellular Delivery in Stem Cell Reprogram-
Currently, most cell-based therapies are carried out through ming. In 2006 it was shown that expressing a combination
ex vivo manipulation, where cells extracted from the patient are of transcription factors can induce a state of pluripotency in
manipulated in vitro and then reintroduced to the body to pro- somatic cells, now known as induced pluripotent stem cells
duce a therapeutic e 28ffect. Intracellular delivery is critical to the or iPSCs.35 Early results were achieved with expression from
D DOI: 10.1021/acs.chemrev.7b00678
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Chemical Reviews Review
Figure 2. Size scale of cargoes of interest for intracellular delivery. The top left quadrant represents 5 nm in scale. The top right quadrant represents
50 nm in scale, including a pink box showing the size of the 5 nm quadrant. The bottom right quadrant represents 500 nm in scale, including a
green box showing the size of the 50 nm quadrant. The bottom left quadrant represents 5 μm in scale, including a blue box showing the size of the
500 nm quadrant. The properties of each of the cargoes and their applications are discussed throughout section 2. PBFI is a potassium indicating
dye. ASO means antisense oligonucleotide. siRNA is small interfering RNA. miRNA is micro RNA. GFP stands for green fluorescent protein. RNP
stands for ribonucleoprotein. TALEN means Transcription activator-like effector nuclease. ZFN means zinc finger nuclease. The pressure sensor is
actually 6 μm long but is scaled to half size for presentation purposes. Several images are reprinted with permission from ref 46, Copyright 2012
Frontiers; ref 47, Copyright 2012 RSC; ref 48, Copyright 2015 Springer Nature; and ref 49, Copyright 2013 Springer Nature.
potentially mutagenic viral vectors, an approach that is con- encompasses a diversity of origins, from typical biomolecules
sidered problematic for medical applications. To address this like proteins, DNA, and RNA to synthetic materials such as
concern, iPSCs have since been produced via direct intracellular carbon nanotubes (CNTs), quantum dots, nanoparticles, and
delivery of proteins,36 mRNA,37,38 and microRNA39 together with microdevices. In the following, we categorize these cargoes for
small molecules.40 Medical applications of iPSCs include in vitro discussion of their properties, delivery challenges, and intracel-
expansion for drug screening of patient cells and gene therapy lular applications. This analysis sheds light on how such diverse
before reimplantation.41 Reprogrammed iPSCs also offer poten- methods of intracellular delivery came about and the factors
tial for cell-based regenerative medicine,42 for example to gen- underpinning the emergence of membrane disruption-based
erate immune-compatible organs for patient transplants,43 off- delivery as a key approach.
the-shelf T cells for cancer immunotherapy,44 or gene-edited 2.2.1. Small Molecules. 2.2.1.1. Small Molecule Drugs.
endothelial cells to correct hemophilia.45 Small molecule drugs are organic compounds of 900 Da or
less, a molecular weight which corresponds to a physical size of
2.2. Cargo Categories for Intracellular Delivery 1 nm or less (Table 1). The first small molecule drugs were
Cargoes of interest for intracellular delivery are highly variable natural products isolated from plants, microbes, marine inver-
in size, shape, architecture, and chemical properties (Figure 2). tebrates, or other lifeforms. An early example is morphine, a
They range from small hydrophilic molecules around 1 nm, metabolite purified from opium extract in 1815 and dispensed
such as the cryoprotectant trehalose, to large micron-sized organ- by Merck as a pain relieving medicine from 1827.50 Today
elles and microorganisms approaching the size of the cell itself. thousands of small molecule drugs are used as medicines.
This scale represents more than 3 orders of magnitude. It also Advances in chemistry have enabled the purification of countless
E DOI: 10.1021/acs.chemrev.7b00678
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Chemical Reviews Review
Table 1. Characteristics of Common Cargo Molecules of Interest for Intracellular Deliverya
cargo typical size (units) approx. mass (Da) dimensions in solution (nm) charge at neutral pH
small molecules N/A <900 Da <1 nm variable; often neutral to promote permeability
peptides <40 amino acids ∼110 Da per amino acid ∼0.2−3 nm varies according to amino acid composition
proteins 20 to 1000s of amino acids ∼110 Da per amino acid ∼2−25 nm varies according to amino acid composition
Cas9 RNP ∼1400 amino acids, ∼188 kDa (∼158 kDa ∼12−15 nm ∼−80 (+22 protein, −100 RNA)
∼100 base RNA protein, ∼30 kDa RNA)
nucleic acids
ASO 13−25 bases (single stranded) 4−8 kDa length of 4−8 nm if linear −1 per base
siRNA/miRNA 21−23 basepair duplex 13−15 kDa 2 nm wide × 7.5 nm long −1 per base
mRNA 0.5−10 kilo-bases RNA (single ∼320 Da per base tens to hundreds of nm −1 per base
stranded)
plasmid DNA 2−10 kilo-basepairs DNA ∼650 Da per base pair hundreds of nm; depends on −1 per base
(double stranded) supercoiling
aThe cargoes are ordered down the table in approximate size order. RNP = ribonucleoprotein. ASO = antisense oligonucleotide.
natural products, production of derivatives and mimics of them, methods such as osmotic lysis of pinosomes, microinjection, or
or production of completely synthetic compounds.50 electroporation. Alternatively, it can be acetoxymethyl-esterified
If a drug target is intracellular, one of three scenarios makes (AM-esterified) to neutralize the carboxyl groups, as described by
it feasible: (1) passive diffusion across the membrane, (2) active Tsien in the early 1980s.68 This process shields the charge of the
transport via membrane proteins, or (3) intracellular delivery. dye molecule, making it cell-permeable. Once the molecules
Small molecules that exhibit passive membrane permeability are inside cells, the acetoxymethyl ester is hydrolyzed by
usually align with Lipinski’s classic “rule of 5”.51 Such molecules esterases (intracellular enzymes) and the dye molecule returns
should ideally be less than 500 Da, of intermediate lipo- to the natural, impermeable state.63 This approach has become
philicity, of limited hydrogen bonding capacity, and uncharged. a standard practice for loading cells with PBFI to monitor
These requirements have been used to narrow drug discovery intracellular potassium concentrations. Other probe molecules,
efforts to candidates that are likely to be bioavailable. This is such as the calcium-sensitive dyes fura-2, fluo-4, and indo-1,
especially important for synthetic molecules, which lack endog- can also be acetoxymethyl-esterified for intracellular delivery
enous transport processes. On the other hand, a number of and accumulation. Furthermore, the strategy of acetoxymethyl-
natural products undergo active transport and therefore do not esterification has been used for modification and delivery of
need to be permeable or obey Lipinski’s rule of 5.52 Oxidized small signaling molecules, such as inositol trisphosphate.69
ascorbate, for example, is membrane impermeable due to its Another example of small molecule probes requiring intracellular
hydrophilic nature but readily undergoes transport into cells delivery are terbium cryptates (∼1 nm).70 Researchers have
through GLUT1, a glucose transporter that is overexpressed in delivered these to the cytosol by osmotic lysis of pinosomes or
many cancer cells.53 transient permeabilization with pore-forming toxins.71,72 Upon
In instances where small molecules are neither permeable loading, the terbium-based probe TMP-Lumi4 enables lumi-
nor actively transported, intracellular delivery is required. One nescence resonance energy transfer (LRET) for imaging of
of the simplest strategies is to administer the molecule along- specific protein−protein interactions in live cells.72
side a solvent such as ethanol or DMSO. Not only do these 2.2.1.3. Cryoprotectants. Cryoprotectants are chemicals
solvents improve the solubility of the small molecule, but they used to protect biological cells and tissues from freezing damage
also increase the incidence of nanoscale membrane defects that incurred by ice crystal formation. Membrane permeable cryo-
assist the passage of small molecules across membranes.54 Alter- protective agents include DMSO, glycerol, and ethylene glycol
natively, several small molecule anticancer drugs have been encap- and are typically nontoxic, low molecular weight molecules that
sulated in nanocarriers such as liposomes to improve their can penetrate the cell membrane. Unfortunately, these agents are
intracellular delivery.1 Intracellular delivery enables the deploy- limited in their cryoprotective capability. Impermeable sugars
ment of drugs that are larger than 500 Da. An example is may be better cytoprotectants but are highly hydrophilic and
bleomycin (Mw = 1.4 kDa, ∼2 nm diameter), an anticancer drug do not readily diffuse across cell membranes. For example,
with poor permeability due to its positive charge and hydro- trehalose (Mw = 342 Da) is a natural disaccharide synthesized
philicity. By performing intracellular delivery with electro- by a range of organisms to withstand desiccation or freezing.
poration, bleomycin potency can be increased more than a Studies have shown that intracellular loading of trehalose into
hundred fold.55,56 This strategy has been demonstrated both in animal cells at concentrations up to 0.2 M can provide superior
vitro and in vivo.56 cryoprotection to animal cells when compared to alternative
2.2.1.2. Small Molecule Probes. In addition to drugs, another methods.73,74 Techniques for intracellular delivery of trehalose
category where small molecules are useful is as intracellular include influx during thermal shock,75 stimuli-responsive
probes.57 Probe molecules are capable of optically reporting nanocarriers,76 engineered pores,77 and electroporation.78,79
membrane potential,58,59 pH,60,61 and intracellular concen- 2.2.2. Proteins and Peptides. Proteins are polymers of
trations of K+,62−65 Na+,64 or Ca2+66,67 by changing their fluo- amino acids that self-organize into three-dimensional, tertiary
rescent properties according to concentration or other stimuli. structures with specific biological functions. Proteins catalyze
Most of these probes require intracellular delivery. One example biochemical reactions, transmit signals, form receptors and trans-
is PBFI (∼1 kDa), a fluorescent dye that can be employed for porters in membranes, and provide intracellular and extracellular
the measurement of intracellular potassium concentration, structural support. Peptides are smaller than proteins, with
however, it is naturally cell impermeable.63,64 The native form generally less than 40 amino acids. Depending on the peptide,
of PBFI can be loaded into cells via intracellular delivery they may or may not form defined three-dimensional structures.
F DOI: 10.1021/acs.chemrev.7b00678
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Chemical Reviews Review
2.2.2.1. Brief History of Intracellular Delivery of Proteins. comparative study of available techniques for antibody delivery
Intracellular delivery of purified proteins began in the 1960s, indicated that electroporation is the leading option.125
even before the advent of nucleic acid transfection. In proof-of- 2.2.2.2. Motivations for Intracellular Delivery of Proteins.
concept demonstrations, amoebae were microinjected with ferritin Straightforward intracellular delivery of proteins and peptides
(450 kDa)80 and mouse eggs with bovine albumin (67 kDa).81 holds significant, yet currently unrealized, potential for many
In the 1970s, more advanced studies used intracellular delivery areas of science and medicine.21,22 Delivery of proteins into
of proteins conjugated with fluorescent dyes to investigate living cells, such as genome-editing nucleases,128 active
intracellular processes and structures.82−86 During this time, inhibitory antibodies,125 or stimulatory transcription factors,36
further advancements in the intracellular delivery of proteins represents a powerful toolset for manipulating and analyzing
were reported using methods that encapsulate proteins within cell function.21,22 For example, the localization and visual-
red cell ghosts87−89 and liposomes.90,91 This was followed by ization of engineered antibodies within living cells and pertur-
methods that induce transient permeabilization of cell mem- bation of their associated cellular processes may allow a more
branes including hypotonic shock,92,93 osmotic lysis of direct study and functional analysis at a level not possible with
pinosomes,94,95 Paul McNeil’s scrape,96 bead97 and syringe98 genetic methods.125 As well as classical antibodies (∼150 kDa),
loading methods, detergent exposure,99 electroporation,100,101 a number of recombinant small antibody-based molecules such
and treatment with the pore-forming toxin Streptolysin O as immunoglobulin (Ig) derived Fab (∼50 kDa) and scFv
(SLO).102,103 Since 2000, a new generation of membrane (∼25 kDa), non-Ig derived monobodies (∼10 kDa), nanobodies
disruption delivery techniques has been developed using micro- (∼14 kDa), and a bodies (∼6.5 kDa) have been developed.129ffi
fluidics and nanotechnology,19,104−107 such as cell squeezing108 When combined with fluorescent labels these antibodies are able
and nanowires.109,110 to serve as precise functional probes for intracellular imaging
Reagents for the intracellular delivery of proteins were applications.130 Further, there are applications where direct pro-
adapted from reagents that were initially used for the delivery tein delivery is favorable over indirect expression from nucleic
of nucleic acids, including lipid and polymer compounds acids, for example to avoid the risk of insertional mutagenesis
developed in the 1990s (see the review in ref 111). Protein associated with DNA transfection. However, one significant
delivery mediated by chemical carriers is also referred to as challenge is that the amount of protein delivered has to be
protein transduction or less often by the misnomers pro- sufficient to generate the desired effect, whereas plasmid DNA
tein transfection or profection.111 The following categories can be amplified by replication. Unlike nucleic acids, with their
have been reported: (1) lipid and polymer compounds analo- uniform properties, one-size-fits-all protein delivery has been
gous to transfection reagents,112−114 (2) cell penetrating pep- elusive due to the inherent variance in size, structure, and
tides (CPPs), also known as protein transduction domains charge among proteins.21,22,131
(PTDs),115,116 (3) bacterial toxins and viral components,117−121 2.2.2.3. Expanding Protein Therapeutics Through Intra-
and (4) engineered nanocarriers.122−124 Lipid and polymer cellular Delivery. Since the advent of human recombinant insulin
reagents, while successful for some proteins, are not appro- in 1982, the number of protein therapeutics has been growing
priate for most situations. Unlike DNA and RNA, proteins are rapidly.132 There are now more than 200 FDA-approved pro-
vastly different in size, charge, and structure. Thus, lipid and tein therapeutics, of which around half are monoclonal anti-
polymeric reagents designed for nucleic acids exhibit a limited effi- bodies. According to market reports, annual worldwide revenue
cacy for use across a range of different proteins.111 On the other from protein therapeutics is anticipated to reach USD 200 billion
hand, PTDs and CPPs can be attached to most proteins, but by 2020. Protein therapeutics can be grouped into molecular
they are prone to endocytic entrapment, cell toxicity, and poor types that include antibody-based drugs, anticoagulants, blood
e ciency of cytosolic delivery.125ffi Despite promise, the history factors, bone morphogenetic proteins, engineered protein
of PTD and CPP research is troubled by disagreement scaffolds, enzymes, Fc fusion proteins, growth factors, hormones,
regarding delivery mechanisms.116,126,127 Intracellular delivery interferons, interleukins, and thrombolytics.132,133 Rather than an
of proteins using bacterial toxins and viral components is sim- intracellular site, these therapeutics exert their action outside
ilar in many ways to PTDs and CPPs, but with more precise, the cell, by modulating molecular interactions in the blood, in
well-defined mechanisms.118,121 Use of bacterial toxins and interstitial fluids, or at the cell membrane. Part of the success of
viral components aims to mimic pathogenic entry processes by protein therapeutics is due to their precision as inhibitors or
targeting a protein of interest to a particular endocytic pathway binding partners. In particular, proteins and peptides can gen-
and then triggering natural mechanisms of endosome escape. erate surfaces capable of recognizing targets that their small
Unfortunately, this strategy has to be tailored to particular cell molecule counterparts fail to.21
types, however, and can be highly labor-intensive or inacces- Around two-thirds of the human proteome lies inside the
sible for most researchers seeking to achieve protein delivery. cell, inaccessible to binding by impermeable molecules.134 Because
The final category of engineered nanocarriers has seen a huge of this, intracellular proteins have a limited potential for therapeutic
rise in interest over the last 15 years. They can be designed as modulation. While an extensive discussion of intracellular protein
higher ordered structures with multifunctional and stimuli- delivery in vivo is beyond the scope of this review, protein
responsive properties. Such nanocarriers are constructed from, delivery has been critical to medical developments and scien-
and functionalized with, combinations of biomolecules, lipids, tific understanding when used in ex vivo cell-based therapies.
polymers, and inorganic materials. They have yet to be trans- One example is the preparation of antitumor vaccines for can-
lated into commercial products. cer immunotherapy. Loading mutant tumor proteins into den-
Overall, The intracellular delivery of proteins lacks straight- dritic cells can program an immune response that primes cyto-
forward universal techniques and typically requires significant toxic T cells to attack and kill tumor cells that exhibit those
amounts of optimization for robust effects to be observed. The same mutant proteins. This strategy has been verified in animal
limited success of the above-mentioned approaches to intra- models135,136 and is beginning to be tested for safety and fea-
cellular delivery of proteins is re ected by the fact that a sibility in clinical trials.137,138fl
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2.2.2.4. Gene Editing through Intracellular Delivery of particularly important when comparing proteins to nucleic acid,
Nucleases and RNPs. Gene editing allows precise, targeted as proteins denature much more easily than nucleic acid (e.g.,
changes in the genomic DNA of a cell.17 Recent advances rely due to heat, salt concentrations, or pH changes) restricting the
on enzymes known as nucleases, protein machinery that can treatments that can be used in their formulations.
cut or alter DNA. Key examples include zinc fingers (ZFNs), 2.2.2.6. Effect of Charge on Intracellular Delivery of Pro-
transcription activator-like effector nucleases (TALENs), mega- teins. The overall charge of proteins and peptides is dependent
nucleases, and the clustered regularly interspaced short on their amino acid composition. Generally, in a solution of
palindromic repeats (CRISPR)/Cas system of RNA-guided neutral pH, arginine and lysine will confer a positive charge,
nucleases. CRISPR-based gene editing is usually performed whereas glutamate and aspartate will confer a negative charge.
with the bacterial nuclease Cas9, which forms a complex, or The majority of proteins, such as antibodies, carry a mild neg-
ribonucleoprotein (RNP), with a single guide RNA (sgRNA) ative charge in physiological solutions. However, the charge of
to become targetable and active.139 Genome editing requires that peptides can be highly variable. Molecular charge is an impor-
nucleases enter the nucleus to exert their action on genomic tant consideration as molecules with a positive charge tend to
DNA.17,140 In the case of CRISPR, initial studies in live cells be more efficient at penetrating negatively charged cell mem-
introduced Cas9 indirectly via expression from plasmids or branes to gain entry to cells. Examples include so-called super-
mRNA.140,141 Subsequent experimentation with delivery of the charged proteins,161 cationic cell-penetrating peptides (CPPs)
preformed Cas9 RNP indicates this to be a more efficient and such as the arginine-rich TAT peptide from HIV,162 and
straightforward approach,142,143 particularly when used with cationic lipids and polymers commonly used as transfection
therapeutically relevant cells types, such as iPSCs, primary T agents.10 Cationic molecules are thought to associate robustly
cells and HSCs.144−146 to the cell surface (for example via attachment to anionic
Since the initial reports in 2014, Cas9 RNPs have been proteoglycans) where they induce endocytosis and/or generate
delivered by methods as diverse as electroporation,143,144,146,147 membrane defects.116 For membrane disruption-mediated deliv-
microinjection,148,149 lipid nanoparticle formulations,150 osmoti- ery, however, highly charged molecules are often less amenable to
cally induced endocytosis followed by endosome disruption,151 delivery as they may need to overcome unfavorable energetic
micro uidic deformation,152 and CPPs.153fl Typically, sgRNA is barriers to diffuse through holes in the plasma membrane.163 One
about 100 base pairs of single-stranded RNA (∼30 kDa, −100 way to overcome this is to provide an electrophoretic driving force,
charges) while native Cas9 is ∼158 kDa (∼10 nm diameter) such as via voltage pulses supplied during electroporation.163
with theoretical net charges of +22.150,154,155 Thus, the resultant 2.2.2.7. Permeability of Peptides. Unlike nucleic acids and
RNP complex has about −80 negative charges, ∼188 kDa mass, proteins, some peptides possess an intrinsic ability to permeate
and is up to 15 nm in size (Table 1). These properties make through cell membranes and into the cell. However, such mem-
electroporation methods particularly tractable for RNP delivery, brane permeable peptides exhibit permeability coefficients sub-
as the negative charge facilitates electrophoretic delivery.128 stantially below typical small molecule drugs. One example is the
Furthermore, the negative charge on Cas9 RNPs makes them 11 amino acid cyclic peptide cyclosporin A (Mw ≈ 1.2 kDa),
electrostatically amenable to complexation with cationic lipid and which is a useful inhibitor of cyclophilin in T cells. Cyclosporin
polymer reagents for carrier-mediated delivery.15,150,156 Indeed, A is a feasible drug for oral delivery due to its relatively high
other types of RNPs have previously been delivered with cationic permeability coefficient that is similar to that of small mol-
polymer reagents.157 RNP delivery strategies are currently a topic ecules (2.5 × 10−7 cm s−1), its low concentration required for
of intense research for the purpose of therapeutic genome intracellular activity (7−10 nM),164 and relative chemical sta-
editing, especially for ex vivo cell-based therapies.15,16,18,34 bility conferred by its cyclic conformation. Despite the success
Recently, CRISPR-based gene therapy for correction of disease- of cyclosporine A, most inhibitory peptides are limited in their
causing genes was achieved in human embryos.158 The correc- usefulness due to inconsistent or low cell permeability or sen-
tion of a common four base pair deletion in the MYBPC3 gene sitivity to degradation by proteases. To this end, researchers in
known to cause hypertophic cardiomyopathy was achieved the field have sought to understand the rules governing peptide
through microinjection of Cas9-sgRNA RNPs and a 200-mer permeability in the hope of applying this knowledge to design
ssODN correction template into zygotes.158 better peptides for intracellular delivery.116,165−167 Understanding
2.2.2.5. Delivery-Relevant Properties of Proteins and Pep- peptide permeability is complicated by observations that suggest
tides. The molecular weight of most proteins is in the range of many different entry mechanisms are possible. The simplest
5 kDa up to several hundred kDa. This corresponds to physical mechanism is passive diffusion as a result of the foreign mol-
dimensions of 2−20 nm, ∼10× smaller than the encoding ecule partitioning the hydrophobic cores of membranes, such
mRNA. Peptides are smaller than proteins with a typical molec- as is believed to be the case for cyclosporin A.168 Alternatively,
ular weight below 5 kDa and physical dimensions less than 3 nm transmembrane transporters have been proposed to shuttle short
in size. The molecular weights and dimensions of some common peptides across the membrane.168 Other peptides are believed to
proteins include green fluorescent protein (GFP, 28 kDa, a 2 × induce endocytosis and subsequent endosomal escape. Most
4 nm barrel), bovine serum albumin (BSA, 67 kDa, a 12 × 4 × cell-penetrating peptides (CPPs) are thought to enter cells via
4 nm rod), Cas9 (158 kDa, a globular endonuclease of >10 nm endocytosis,116 although other routes such as direct translocation
diameter), and immunoglobulin antibody (∼150 kDa, 14 × across the membrane, inverted micelle formation, transient pore
8 × 4 nm).159 Because the structure of proteins is critical to formation, adaptive translocation, and local electroporation-like
function, any modification for intracellular delivery including effects have been suggested.127
chemical modifications or packaging in carrier particles should Some general characteristics have been found to promote
not compromise protein structure and function. Accordingly, peptide permeability. For example, the most potent CPPs are
the varied charge and dimensional and structural properties of usually between 8 and 20 amino acids long and possess
each protein may be considered unique, thus requiring a cus- somewhere between 5 and 8 positively charged residues (usually
tom solution for efficacious intracellular delivery.160 This is arginines) in various configurations that confer a favorable charge
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Figure 3. Concept map displaying the main application areas of transfection. In terms of market share and research, medical, or industrial activity,
transfection is the largest subcomponent of intracellular delivery. HSCs = hematopoietic stem cells.
for membrane interactions and cell entry.116 Other strategies product lipofectamine in 1993. This was shortly followed
involve the use of “stapled peptides”, where a synthetic brace by dendrimers such as PAMAM180 from 1993 (“superfect”
(typically a covalent cross-link between two residues) is added to reagent launched in late 1990s) and cationic polymers such as
lock small peptides into a stable active conformation (most often PEI in 1995179 (marketed as “polyjet” soon after). Cationic
an α helix).169,170 Using stapled peptides, Verdine and colleagues polymers such as Polybrene181 and poly- -lysine182,183L also
produced a synthetic, cell-permeable, stabilized α-helical peptide formed the basis of several transfection technologies. The tech-
of 16 amino acids that targets a critical protein−protein interface nique of electroporation was first used for DNA transfection in
in the difficult-to-drug NOTCH transactivation complex.171 the early 1980s.184 Electroporation remains particularly useful
Ongoing research efforts are expected to decode the size, for hard-to-transfect cell types and was commercialized from
conformation, charge, polarity, and amphiphilicity that optimize the mid-1980s by Biorad and others. Today, most transfection
the intracellular delivery of peptides and their cargo. is performed with lipid reagents, while polymer reagents and
2.2.3. Nucleic Acid Transfection. The word “transfection” electroporation are the next most popular options. The popularity
is derived from the terms transformation and infection. It has of these techniques over more efficacious virally-mediated trans-
paradoxically come to refer to nonviral (i.e., noninfectious) meth- duction methods is due to the relative simplicity of transfection
ods of nucleic acid delivery. Transfection has mainly referred to procedures, lower cost, and smaller time investment.
the intracellular delivery of plasmid DNA, mRNA, and oligonu- By 2020 the transfection market is predicted to be worth one
cleotides of DNA or RNA. Nucleic acid−based constructs/ billion USD, with applications across three core areas: (1)
devices, however, represent an emerging scenario. The analogous basic research, (2) biomanufacture, and (3) cell-based
term “transduction” refers to the introduction of nucleic acids to therapies (Figure 3). Because genetic material underlies almost
the intracellular space by viruses or viral vectors. Viral mediated all biological function, transfection is central to biological
delivery of nucleic acids is the gold standard for their intracellular research, in both academic and industrial settings. Transfection
delivery. The use of viruses for transduction leverages the impacts fields from cell biology and genetics to immunology
naturally occurring mechanisms that viruses use to enter cells. and drug discovery. In the context of biomanufacture,
2.2.3.1. Brief History and Motivations of Transfection. Start- transfection is used for bioproduction of proteins, antibodies,
ing from the 1960s, researchers observed that mixing nucleic viral vectors, and virus-like particles for vaccines. In cell-based
acids, which are negatively charged, with cationic molecules therapies, transfection is critical for ex vivo gene therapy (correct-
leads to the formation of macromolecular complexes that can ing aberrant genes),29 hematopoietic stem cell engineering,30,185
enter cells and degrade, thereby releasing nucleic acids inside a production of induced pluripotent stem cells,37 and prepara-
cell. Two early examples of transfection complexes were the tion of cells for immunotherapy.186−188 As shown in Figure 3,
polymer diethylaminoethyl-dextran/nucleic acid combination nucleic acid transfection is currently the dominant category of
(1968)172−174 and the insoluble ionic salt calcium phosphate/ intracellular delivery. In future, however, demand for delivery
nuclei acid precipitant (1973).175 Today, the most widespread of non-nucleic acid materials (for example, antibodies, genome
reagents employed for transfection take advantage of the editing nucleases, and synthetic materials) is expected to com-
attraction between cationic lipids and nucleic acids. The first pete with transfection in several applications.15,18,19,21,22,107
reports of lipid-based transfections were as early as the 1980s, 2.2.3.2. DNA Vectors. A vector is a DNA molecule that acts
rst with liposomes (1980)176,177fi and then via “lipofection” as a vehicle for the expression or replication of DNA. Some
with cationic lipids (1987).178 The most effective methods examples of different types of vectors include plasmids, cos-
were commercialized, with the launch of the cationic lipid-based mids, viral vectors, and artificial chromosomes. Plasmids are
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circular double-stranded DNA molecules that were originally “hairpin miRNA”, named as such due to their shape, are pro-
discovered in bacteria.189 Cosmids are similar to plasmids but cessed by enzymes within the cell into smaller pieces similar to
exhibit the ability to be packaged into phage capsids.190 Viral siRNAs, which then silence genes through antisense or RNAi
vectors pack a limited amount of DNA within a viral envelope, mechanisms.
an efficient configuration that confers self-delivery through Overall, oligonucleotides may modify cell behavior through
viral-mediated cell entry.191 Artificial chromosomes have a a number of mechanisms.218 These include (1) activating toll-
larger DNA capacity than other vectors, containing up to a like receptors in the endosome (nonspecific), (2) siRNAs,
million base pairs with dimensions in the micron size range. (3) miRNA mimics, (4) antagomirs, sterically blocking endog-
Artificial chromosomes are used in specialized situations where enous miRNA, (5) ASOs such as gapmers that induce RNase
their larger capacity and natural chromosome-like behavior are H degradation or sterically block target RNA, (6) oligonucleo-
advantageous.192 tides directed against nuclear regulatory RNA species such
The most commonly used vectors are plasmids, which are as long noncoding RNAs (lncRNAs), (7) splice switching
usually around 5−10 kilo-base pairs. DNA engineering tech- oligonucleotides that perturb mRNA maturation, (8) antigene
niques enable the manipulation of all vectors through recom- oligonucleotides that bind to genomic DNA, perturbing trans-
bination, thus allowing specific sequences to be cut and pasted cription or binding of other proteins, and (9) aptamers, which
into them. Pioneering studies in the 1970s inserted foreign bind to and alter the function of proteins.218 Aptamers are
DNA into viral vectors193 and plasmids194 for subsequent intra- distinct from the rest of these oligonucleotides in that they
cellular delivery and gene expression. By decoding the genetic form higher order structures with conformations exhibiting
elements of vectors, such as expression promoters and origins affinities to specific target molecules. With the exception of the
of replication, it became possible to introduce and express first mechanism (activation in endosomes), oligonucleotides
genes from one organism into another and vice versa.195 For must enter the cytoplasm or nucleus to exert their effects.
example, plasmids were exploited to first express eukaryotic The chemical and dimensional properties of oligonucleo-
genes in bacteria196,197 and then foreign genes in animal cells, tides affect their function and capacity for intracellular delivery.
via calcium phosphate transfection198,199 or microinjec- Because oligonucleotides are negatively charged polar mole-
tion.200−202 That plasmids must enter the nucleus to undergo cules in the size range of small proteins (Figure 2), their pro-
expression was established by microinjection experiments that pensity for interaction with negatively charged cell membranes
compared cytoplasmic with nuclear injection.202 and consequential cellular permeability is low. siRNA duplexes
A 5−10 kilo-basepair plasmid is >100 nm in diameter when have approximate dimensions of 7.5 nm length by 2 nm
uncondensed203,204 (Table 1). Each nucleotide carries a single diameter219 (Table 1). miRNA is only slightly larger than
negative charge due to repeating phosphate groups along the siRNA because it is a single stranded hairpin shape with an
DNA polymer backbone. Cationic compounds, such as lipids extraneous loop. An ASO of 16 bases is about 5 nm long by
and polymer reagents, condense plasmids into solid nanopar- 1 nm wide. In addition to their large size and relative negative
ticles with dimensions of tens of nanometers.10,205,206 Conden- charge, challenges associated with oligonucleotide delivery
sation of DNA promotes cellular uptake by reducing the include susceptibility to enzymatic degradation and binding to
plasmid size and somewhat shielding its negative charge. The undesirable targets.220 One approach to prevent unwanted oli-
level of supercoiling also influences the durability and compac- gonucleotide degradation and chemical cleavage is chemical
tion. In general plasmids bearing a smaller footprint are capa- modi 9,221fication. Another method to improve intracellular
ble of more efficient transfection and expression.207,208 delivery of oligonucleotides is to neutralize the negatively charged
2.2.3.3. Oligonucleotides. Oligonucleotides are single- or portions of the polymer backbone. This strategy prevents repul-
double-stranded sequences of DNA or RNA, generally less than sion between oligonucleotides and cell membranes, thus increas-
30 nucleotides in length. Antisense oligonucleotides (ASOs) ing permeability. Oligonucleotides can be neutralized by replace-
were first discovered in 1978, when it was shown that a single- ment of natural bases with morpholinos,222 peptide nucleic acids
stranded 13-mer of DNA hybridized with complementary (PNAs)223 or by the addition of specific functional groups.224
mRNA to inhibit its translation.209 Antisense inhibition occurs Of the different approaches for intracellular delivery of oligo-
because mRNA is either sterically blocked and thereby nucleotides, lipid reagents have been the most prevalent.229−231
unavailable for translation or designated for enzymatic degra- However, delivery strategies can also include combinations of
dation. In the 1980s ASOs were established as tools for per- the approaches listed above, including chemical modification
forming genetic loss of function studies.210−212 In these cases, of the oligonucleotide itself, use of lipid or polymeric nano-
ASOs were either expressed from plasmids or microinjected carriers, and linking oligonucleotides to cell targeting agents
after in vitro transcription. Thereafter, several companies began such as carbohydrates, peptides, or aptamers.220,225 In these
developing antisense therapeutics, with the first approved approaches, it is thought that the biological effects of oligonu-
medication in 1998 being fomivirsen, a 21-mer oligonucleotide cleotides are due to the small amount of oligonucleotides that
that blocks the translation of cytomegalovirus mRNA.213,214 escape from endosomes and reach target cytosolic or nuclear
The discovery of RNA interference (RNAi) by Fire and compartments.226−228 In cells that are recalcitrant to such
Mello in 1998215 led to the revelation of double-stranded RNA reagents, success has been obtained with electroporation232−237
for silencing gene expression. Subsequently, it was shown that and pore-forming agents.238,239
RNAi in mammalian cells could be mediated by intracellular 2.2.3.4. Intracellular Delivery of Messenger RNA. One alter-
delivery of short 21−22 base pair duplexes, termed small native to the delivery of DNA vectors is the use of mRNA.
interfering RNAs (siRNAs).216 Once in the cytoplasm, siRNAs This alternative is particularly attractive on therapeutic
bind to protein machinery known as the RNAi-induced silencing applications (discussed below). Pioneering studies for the intra-
complex (RISC), which binds with matching RNA to enzymati- cellular delivery of mRNA expression were conducted in the
cally degrade it. Micro RNAs (miRNAs), discovered in 1993,217 1970s via microinjection methods.240−242 Following that, mRNA
represent the endogenous mechanism of gene silencing. Small was transfected into mammalian cells using the cationic polymer
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DEAE-dextran243,244 and with cationic lipid complexes,245,246 the barrier to the study of immune cells, where cells types such as
latter of which became the standard approach.247 Transfection T cells, B cells, natural killer cells, dendritic cells, and macro-
of mRNA via electroporation has also been demonstrated as an phages are known to be di 234,274−286fficult to transfect. Primary
effective option in a number of cell types.248,249 stem cells, cells of the hematopoietic lineage, and neurons are
Protein expression from the intracellular delivery of other cell types that have historically proven difficult to trans-
mRNA has a number of advantages when compared to that fect.287−290 The ability to conduct biological studies in these
of DNA vectors.250,251 First, there is no risk of adverse genomic important cell types is often restricted by limitations on
integration that can occur after the intracellular delivery of transfection efficiency and tolerance to treatment. Thus, while
DNA. Second, mRNA expression is based upon interaction there has been a huge amount of work on refining transfection
with ribosomes located in the cytoplasm and thus does not approaches over the last decades, there is still significant room
require delivery across the nuclear envelope as DNA does. for improvement in key cell types.
Third, protein expression resulting from mRNA delivery is 2.2.4. Synthetic Nanomaterials and Devices. Synthetic
dose-dependent and rapid, commencing within minutes. Fourth, nanomaterials and devices represent another frontier where
additional control over the location of protein expression can be demand for suitable intracellular delivery solutions exceeds
conferred by using mRNA, as its specific subcellular delivery supply.19,20 Probes engineered from functional nanomaterials,
can localize protein expression.252 Fifth, mRNA can be less including carbon nanotubes (CNTs),291−293 quantum dots,294,295
toxic and less immunogenic than DNA vectors, making it a magnetic nanoparticles, and various fluorescent reporter sys-
preferred option for sensitive cells or applications that require tems19,296−299 have potential as sensors for intracellular pro-
cell high viability. These beneficial aspects of mRNA delivery cesses. Yet limitations to their successful intracellular delivery,
make it attractive for therapeutic applications.253−255 One such a poor understanding of their interaction with biological
therapeutic application used intracellular delivery of mRNA for environments, and the toxicity issues related to these novel
expression of tumor antigens in dendritic cells and T cells materials have retarded their deployment inside the cell. Many
ex vivo, a promising immunotherapy strategy for eradication of of these materials and devices still await systematic intracellular
cancer.188,256−259 Electroporation of mRNA has become a testing due to ineffective delivery, and as a consequence, their
preferred option for delivery of mRNA in therapeutic cell types true effectiveness remains unknown.19,20,300 The delivery
that are difficult to transfect with cationic lipids, such as challenges of these molecules and unconventional materials
dendritic cells.260−262 must first be addressed before their potential in research or
Similar to DNA, mRNA is a large negatively charged poly- therapeutic and diagnostic applications can be fully realized.
mer that can be condensed into cationic nanoparticles to Below we highlight several examples of progress in the field.
reduce size and promote uptake.247,255 mRNA is a single CNTs have been proposed as sensors, labels, electrical field
stranded nucleic acid that usually forms secondary structures enhancers, and next-generation devices in biological applica-
featuring various loops and hairpins (Figure 2). The dimen- tions.292,301 The smallest single-walled configurations exhibit
sions of mRNA are normally ∼10 times larger than the protein diameters from 1.2 nm and lengths spanning from tens of
it encodes, putting it in the range of 20−200 nm.263 The dis- nanometers up to microns.302 Chemical functionalization can
advantages of using mRNA are that it may invoke an immune be employed to increase the solubility and biocompatibility of
response or be unstable; however, these disadvantages can be CNTs;302 however, their toxicity profiles and suitability for
circumvented with appropriate chemical modi cations.9,264 intracellular applications are still a matter of controversy.303fi
2.2.3.5. Nucleic Acid−Based Constructs and Devices. One One example where they have been useful in probing the intra-
emerging area of nucleic acid research involves DNA constructs cellular environment was published by Fakhri et al. in which
and devices engineered to form higher-order two- or three- functionalized CNTs were loaded into cells by electropor-
dimensional shapes. These precision DNA nanostructures have ation.293 By tracking the near-infrared luminescence of kinesin-
become known as DNA origami, a concept that rose to prom- targeted single-walled CNTs, they observed a regime of nonequi-
inence in 2006.265 Using DNA origami, precise nanostructures librium stirring dynamics driven by active cellular motors.293
of calculable size and shape can be assembled into template Another recent study used microinjection to load high con-
structures via specific folding interactions. Tian et al. recently centrations of single-walled CNTs of length ∼150 nm into frog
developed DNA octahedrons of ∼60 nm with encoded sites for embryos.304 The localization of CNTs and potential toxicity
molecular positioning, allowing multiple nanoparticles with were tracked throughout the growth of the animal. They found
different functions to be integrated into a single structure.48 CNTs tended to localize to the perinuclear region within most
In another example, DNA icosahedra found use as vehicles for cells; however, there were no obvious structural defects, devel-
the delivery of quantum dots.266 DNA origami, with a de ned opmental abnormalities, or toxicity to report.304fi These studies
number of binding sites, has recently been used to calibrate suggest CNTs might be safe for intracellular applications.
fluorescence for determination of protein copy number inside Quantum dots are semiconductor crystal configurations in
cells.267 Oligonucleotides may also be deployed inside cells as the size range <10 nm. Due to their advantageous optical pro-
probes. For example, oligonucleotide-based molecular beacons perties, intracellular labeling and analysis applications have been
are short (∼25 base) hairpins featuring internally quenched proposed.295,305 Quantum dots are usually negatively charged,
fluorophores that alter their fluorescence upon hybridizing with and surface passivation with a polyethylene glycol (PEG) shell
a target sequence.268,269 Aptamers, described above as inhib- is a standard strategy to increase the biocompatibility of the
itors, can also be used as conjugates, receptor-targeting moieties, structure, with a final diameter of 20 nm being typical for this
intracellular biosensors, and imaging probes.270−273 configuration.300 An early study compared microinjection,
2.2.3.6. Hard-to-Transfect Cells. Effective transfection electroporation, and lipid transfection reagents for quantum
remains a significant hurdle for many primary cells. Moreover, dot delivery into cultured cells.294 The investigators found that
even when high transfection efficiencies are achieved, toxic and lipid reagents and electroporation failed to disperse the dots
off-target effects may confound results. This is a well-known homogeneously into cells, instead leading to aggregation or
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endosomal entrapment. On the other hand, low throughput were capable of generating a new animal.327,328 Based on this
microinjection was able to deliver quantum dots homoge- work, Gurdon later shared the Nobel prize for “the discovery
neously to the cytoplasm. Since then a number of approaches that mature cells can be reprogrammed to become plurip-
have been tested for quantum dots delivery. They include osmotic otent”. Microinjection was also required for the nuclear trans-
loading of pinosomes,306 CPPs,307 microfluidic cell squeezing,308 plant that led to the first mammalian cloning, as exemplified by
controlled laser-induced cavitation,309,310 detergent permeabiliza- the birth of Dolly the sheep in 1997.329 Furthermore, in an
tion,311 and successful examples of electroporation.312,313 We unconventional form of gene therapy, transplant of pronuclei
point the reader to dedicated reviews on intracellular delivery from human eggs with pathological mitochondria to donor
of quantum dots for further information.300,314,315 eggs with functional mitochondria has been shown to correct
Magnetic nanoparticles in biomedical science are useful for diseases of mitochondrial inheritance.330
magnetic resonance imaging (MRI), generating local heat Other examples emphasizing the importance of microinjection
effects, and providing a handle for attractive forces.316 In some in biotechnology include in vitro fertilization (IVF) and chrom-
of these applications it is necessary for magnetic nanoparticles osome or mitochondrial transplantation. IVF occurs through
to be delivered to the cell interior. One group developed a cell the artificial delivery of sperm into eggs cells. The IVF concept
labeling approach using short cell penetrating peptides (HIV- was first demonstrated through microinjection of sperm into
Tat) to derivatize ∼45 nm superparamagnetic nanoparticles.317 sea urchin eggs.331 Subsequently, IVF generated the first human
The particles were internalized into hematopoietic stem cells pregnancies in the early 1990s.332 Chromosome transplantation
before being reintroduced for in vivo homing to the bone mar- techniques have also been described with microinjection appa-
row where they were subsequently detected and retrieved.317 ratus.333 Indeed, artificial chromosomes have been engineered
In another example, Nitin et al. developed ∼10 nm and transferred into cells by microinjection for transgenic
superparamagnetic iron oxide nanoparticles with a PEG-modified, studies or proof-of-concept gene therapy.334,335 In another
phospholipid micelle coating, functionalized with cell-penetrating example of large cargo delivery, transplant of mitochondria
peptides.318 The particles were demonstrated to enter cells and (∼1−2 μm) via microinjection has been demonstrated in
act as MRI contrast agents for intracellular molecular imaging several different cell types and model systems.336−338
in deep tissue.318 Furthermore, polymer nanoparticles with While microinjection has traditionally dominated large cargo
iron oxide cores have been deployed for DNA transfection pur- delivery, it is not the only option. Indeed, several rival methods
poses.319 The magnetic core enables their uptake and local- have arisen mainly out of the need for greater throughput. For
ization to be tracked by MRI.319 In another case, the mech- example, Chiou and colleagues pioneered an approach using
anical and physiological properties of fibroblasts were measured laser-triggered cavitation bubbles to deliver ∼2 μm bacteria
as a function of intracellular uptake of biocompatible magnetic into cultured cells at both single cell339 and high throughput
nanoparticles and the applied magnetic field.320 scales.340 The same approach was extended to delivery of
Various nanoparticle systems have also been deployed as functional mitochondria for studies of mitochondrial dysfunc-
intracellular temperature probes.321 In one report, temperature- tion in metabolic diseases.341 Another method of mitochon-
responsive nanodiamonds of approximately 100 nm were intro- drial transfer is cell fusion, where the mitochondria are sup-
duced into cells via nanowires.322 The nanodiamonds were then plied from donor cells.342,343 In studies involving gene therapy
used as local temperature gauges to perform nanometer-scale with human artificial chromosome, they can also be transferred
thermometry in living cells at microkelvin resolution.322 Another by cell fusion, in a process termed microcell-mediated
study used smaller, but less accurate, particles for intracellular chromosome transfer (MMCT).192,344−347 Engineered CHO
temperature measurements.323 Okabe et al. prepared a fluo- donor cells carry the human chromosome and are triggered
rescent polymeric thermometer of ∼9 nm diameter, function- to fuse with the acceptor cell, thus transferring the genetic
alized it with hydrophilic residues, and microinjected it into the material.344
cytoplasm of living cells. With a temperature measurement Apart from delivery of organelles and subcellular components,
resolution of 0.18−0.5 K, they claimed to measure temperature insertion of large synthetic materials and devices is another area
differences between various organelles.323 of interest. As a case in point, micron-scale particles, spheres, and
2.2.5. Large Cargo. Relative to most cells, large cargo is beads have been loaded into cells for intracellular microrheology
anything from hundreds of nanometers up the range of the cell studies that analyze the internal mechanics and dynamics of
itself (usually tens of microns). Examples of large cargo that cells. So far they have been delivered by microinjection348,349
have been delivered into cells are shown in the bottom left or ballistic propulsion.350−353 A recent study microinjected
quadrant of Figure 2 and include bacteria, mitochondria, whole PEGylated tracer beads of up to 0.5 μm into cells to show that
chromosomes, microbeads, sperm, nuclei, and microelectro- motor-driven cytoplasmic mixing substantially enhances intra-
mechanical systems (MEMS) devices. The first demonstration cellular movement of both small and large components.354
of large cargo delivery occurred alongside the invention of In another study of cell mechanics, ∼1 μm melamine particles
microinjection itself in 1911.324 Marshall Barber demonstrated coated with PEG were fired into HeLa cells by a gene gun to
that a single bacterium, once inside the cytoplasm of a plant study glassy dynamics in the cytosol.355 Magnetic cargoes have
cell, was sufficient to kill it.324,325 also been used to probe the intracellular environment. Moch
For a century microinjection has been the dominant method et al. loaded 2.8 μm magnetic beads into the cytoplasm of
for introducing large cargo into cells. Microinjection was used mammalian cells for probing cell mechanical properties by mag-
for the first nuclear transplant experiments that surgically netic tweezers.356 Garzon-Coral et al. measured mitotic spindle
dissected the nucleus from blastula cells and inserted them into forces in one- and two-cell Caenorhabditis elegans embryos in vivo
living frog eggs.326 To the amazement of the researchers, these by injecting 1.0 μm diameter superparamagnetic beads and using
eggs then had the potential to grow and produce a new animal. magnetic tweezers to exert calibrated forces of up to 200 pN
Building on this breakthrough, Gurdon and colleagues showed on mitotic spindles.357 In other instances, MEMS can mea-
that nuclei transplanted from fully differentiated somatic cells sure intracellular properties, such as cytoplasmic pressure.49
L DOI: 10.1021/acs.chemrev.7b00678
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Chemical Reviews Review
Figure 4. Map of intracellular delivery methods and their mechanisms. Current intracellular delivery methods are shown sorted within the four
indicated mechanisms: (1) permeabilization, (2) penetration, (3) endocytosis, and (4) fusion. Methods that overlap on more than one mechanism
may promote intracellular delivery via multiple pathways depending on the context. For example, most viral vectors are believed to go through
endocytosis, but a limited number fuse directly with the plasma membrane.
One group deployed a MEMS-based intracellular hydrostatic plasmids and other nucleic acids into compact nanoparticles
pressure sensor, about 6 μm in size, that was claimed to be deliv- with dimensions down to tens of nanometers.10,205,206 This
ered into HeLa cells via lipofection.49 The same researchers makes the task of delivering these molecules significantly more
also microinjected silicon MEMS barcodes up to 10 μm in manageable. Moreover, the positive charge of these particles
length into mouse embryos for tracking and labeling pur- facilitates their interactions with the cell surface, which is nega-
poses.358 tively charged due to the typical −35 to −80 mV membrane
potential of cells. The positive charge is also believed to promote
3. APPROACHES FOR INTRACELLULAR DELIVERY binding to certain receptors.10 Upon binding, subsequent internali-
As outlined in the previous section, a wide range of cargoes have zation via endocytosis is thought to be most efficient for particles
in the size range below 100 nm.363been introduced to the intracellular space through a diverse Complexation into nano-
range of delivery approaches. Here, we categorize these approaches particles also confers protection for nucleic acids against
according to the mechanism at the plasma membrane (Figure 4), degradation in the cytoplasm.
364 Potential disadvantages of com-
rather than traditional classi cations of biological, physical, and plexation, however, may include delayed unpacking (making itfi
chemical techniques.105,359−362 As the cell is agnostic to our inaccessible for gene expression
365) or excessive toxicity.366
distinction between scienti c disciplines, we believe this In the last two decades researchers have expanded the scope offi
categorization is more mechanistically relevant.107 Broadly, transfection strategies to include carriers designed from lipids,
methods may involve either (1) disruption of the cell membrane liposomes, polymers, inorganic nanomaterials, carbon nano-
to facilitate entry of cargo or (2) packaging with carriers, which tubes, protein-based nanoassemblies, and functionalization with
then undergo uptake into endosomal tra cking routes or fuse various peptides, ligands, and chemical modi cations.
6,7,9,10,364
fi
ffi
with the target cell membrane. Although chemical or structural The other major type of carriers for nucleic acid delivery are
modifications can be used to increase the passive permeability viral vectors, which exploit the viral infection pathway to enter
of some small molecules or short peptides, most cargo mole- cells but avoid the subsequent expression of viral genes that
cules and materials require an active intracellular delivery method. leads to replication and pathogenicity.
191 This is done by
deleting coding regions of the viral genome and replacing them
3.1. Carrier-Mediated with the DNA to be delivered, which either integrates into host
Most of the early developments in carrier-mediated delivery chromosomal DNA or exists as an episomal vector. At present,
were directed toward nucleic acid transfection, particularly viral vectors are the most clinically advanced nucleic acid deli-
for DNA plasmids. As mentioned in the transfection section very agents owing to their high efficiency and specificity. They
(see section 2.2.3), cationic lipids and polymers can condense were first employed from the 1970s, constructed from SV40367
M DOI: 10.1021/acs.chemrev.7b00678
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Chemical Reviews Review
Figure 5. Cargo delivery trajectories for the main intracellular delivery categories. (A) Viral vectors only deliver nucleic acids but do so very
efficiently (endocytosis example). (B) Most nonviral carriers are optimized for nucleic acid delivery although some adaptations can carry other
materials. Nonviral carriers are endocytosed into the cell with small amounts of nucleic acid breaking out into the cytoplasm while the majority are
degraded in lysosomes or recycled back out to the extracellular space. (C) Membrane disruption is able to deliver almost any cargo that can be
dispersed in solution provided it is small enough to fit through transient openings in the plasma membrane. Nucleus is depicted in purple.
or retroviruses.368,369 Newer generations of viral vector plat- fusion intrinsic properties. Examples include (1) cell ghosts, dead
forms have been produced based on components from lenti- cells that have had their cytoplasm replaced with cargo,89,384
virus, retrovirus, adenovirus or adeno-associated virus, and (2) virosomes, cargo-loaded vesicles reconstituted to display
other viruses.370−372 While highly e cient for DNA delivery, functional viral proteins,385 and (3) fusogenic liposomes.386,387ffi
notable weaknesses of viral vectors are (1) labor-intensive and In the latter case, a new generation of liposomes have been
expensive protocols, (2) safety issues, (3) risk of causing immune/ reported to become fusogenic by modulating the lipid
inflammatory responses, (4) integration into the genome with composition without any need for fusogenic protein or peptide
recombinant vectors, (5) risk of insertional genotoxicity, and (6) conjugation.387,388
limited packaging capacity (Adeno and AAV typically restricted Recently, a subset of natural cell-derived vesicles known as
to carry 5−7.5 kb).290,373 The problems with viral vectors exosomes have been discovered to fuse with target cell mem-
continue to motivate the development of nonviral carriers.9,10,374 branes for the exchange of RNA and proteins between immune
Beyond nucleic acid transfection, researchers initially explored cells.389 Although the exact fusion mechanisms are yet to be
protein delivery through the use of red cell ghosts87−89 and described, it is anticipated that exosome-inspired systems may
liposomes.90,91 Newer generations of nanocarriers are now represent a new generation of vehicles for efficient and biocom-
being designed to address intracellular delivery of proteins on a patible intracellular delivery.390,391
broader scale,6,124,131,375 although these developments are 3.2. Membrane Disruption-Mediated
more at a nascent stage. Intracellular delivery of genome edit-
ing complexes is a particularly important application that is Unlike carriers that may be restricted in the feasibility of cargo-
driving the evolution of next-generation nanocarriers.15,18,156 carrier combinations, membrane disruption-mediated strategies
Mechanistic investigations indicate that most carriers enter are near-universal, being able to rapidly deliver almost any cargo
cells via endocytosis before escaping into the cyto- that can be dispersed in solution (Figures 4 and 5). In this review
plasm226,363,376,377 (Figure 5). Mechanisms of endocytosis the term “membrane disruption” refers to the generation of any
available to nanocarriers include phagocytosis and pinocytosis hole that would increase the permeability of the plasma mem-
through clathrin-dependent and clathrin-independent path- brane to cargo. This includes pores, defects, inhomogeneities,
ways.363,377 The pathways employed by target cells are tears, lesions, holes, and perforations of all sizes and shapes.
dependent upon the particle size, shape, material composition, The challenge for membrane disruption-mediated appro-
surface chemistry, and/or charge.226,363,376−378 Cargo not able aches is (1) to open up the right kind of holes in the plasma
to escape endosomes are trafficked through lysosomes for membrane to achieve substantial delivery of the cargo and
degradation or recycled back out to the cell surface.379−381 (2) to avoid undesirable cell perturbation or death associated
Maximal efficiencies of around 1% endosomal escape have with membrane damage. The main two ways membrane dis-
been reported for the most advanced nonviral carrier strategies, ruption is accomplished are through direction penetration or
including lipid nanoparticles380,382 and cell-penetrating pep- permeabilization.
tides.116 Moreover, the exact mechanisms of endosome escape 3.2.1. Direct Penetration. Strategies involving direct
remain unclear and are a matter of ongoing research.379−381,383 penetration use a conduit or vehicle to break through the mem-
Alternatively, some carriers are able to fuse directly with the brane, thereby creating a passage for the cargo. Prevalent
plasma membrane. These systems were first inspired by viruses examples are microinjection, ballistic particles, and nanoneedles,
that deploy specialized surface proteins to induce fusion with as shown in Figure 4. Microinjection was the first intracellular
target membranes.89,384 Fusogenic carriers are bound by a delivery method to be invented and represents a classic case
phospholipid bilayer that hosts the fusion machinery or confers of a direct penetration strategy.325,392 The cell membrane is
N DOI: 10.1021/acs.chemrev.7b00678
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Chemical Reviews Review
disrupted with a miniaturized pipette-like element, which is then important to note that the imposed membrane disruptions are
used to pump fluid containing the molecule of interest inside not stable and thus only allow passage of cargo for a limited time.
the cell. Nanoneedles operate on a similar principle except that Upon membrane insult, the target cell responds with active
they are scalable in parallelized arrays and typically consist of membrane repair processes. Healing of the plasma membrane
109,110,393
finer, more intricately fabricated structures. Ballistic can take from a few seconds up to several minutes to complete.
particles are coated with the material to be delivered and fired Once membrane integrity is restored, the cell may engage meta-
at high velocity into the cell.394 They are categorized as mem- bolic and transport processes to restore cytoplasmic composition
brane disruption in this review (rather than carriers) due to the and bring itself back to full health.398,399 Most permeabilization
critical role of active force in puncturing the cell membrane to strategies apply specific conditions, such as temperature and
achieve access. In all direct penetration strategies, the target cell buffer composition, to first promote permeabilization and delivery
must respond to repair damage sustained to the plasma mem- and subsequently facilitate cell recovery. The membrane disrup-
brane or other cellular structures. tion effect must not be too severe or prolonged, otherwise the
3.2.2. Permeabilization. In contrast to direct penetration, cells will be unable to repair and recover. Effective permea-
permeabilization strategies make the cell transiently permeable bilization strategies must therefore find a balance, optimizing
to cargo present in the extracellular solution. The plasma mem- both the membrane damage and cell treatment conditions.
brane is considered permeable when membrane disruptions are The remainder of this review will focus on membrane
of sufficient size and lifetime to permit passage of the cargo mole- disruption-based delivery. This exploration will mostly be
cules or materials. Thus, the threshold level of permeabilization centered around animal and mammalian cells in vitro and
needed depends on the properties of the cargo. ex vivo. In the next section we will discuss background con-
As seen in Figure 4, many different permeabilization strat- cepts helpful in understanding how and why membrane disruption
egies have been attempted. They range from mechanical and can be a successful approach. Following that, we will offer a
laser-based to electrical and chemical.105,395−397 The key events detailed appraisal of the various delivery methods. Each section
associated with permeabilization-based intracellular delivery are will cover content areas that include history, mechanisms, fea-
shown in Figure 6. First, the cargo of interest is dispersed into sibility, performance, toxicity, applications, technical advances,
and envisaged future opportunities.
4. MEMBRANE DISRUPTION-MEDIATED DELIVERY:
BACKGROUND CONCEPTS
In this section we will cover the basics of (1) cell and mem-
brane properties, (2) mechanisms of membrane disruption,
and (3) cell response to membrane disruption. These back-
ground concepts lay a foundation to explore the common
issues that arise in membrane disruption-based intracellular
delivery. The following sections then examine direct pene-
tration (section 5) and permeabilization (section 6) methods.
4.1. Cell Structure and Properties
4.1.1. Plasma Membrane Function. The primary barrier
to intracellular delivery is the plasma membrane, which defines
the essential boundary between the inside and outside of a cell.
Figure 6. Key events associated with permeabilization-based intra- The plasma membrane enables cells to control their compo-
cellular delivery. Acute membrane disruption triggers an increase in sition and properties. Its central component is a ∼5 nm thick
permeability to the cargo of interest (green). For a limited window of phospholipid bilayer with polar heads facing the aqueous
time (seconds), cargo diffuses into the cell according to its concentra- environment and fatty acyl chains pointing inward to form a
tion gradient while some cytoplasmic materials may be lost (orange). hydrophobic core. This hydrophobic core is the main limiting
Upon membrane disruption the cell responds with active membrane barrier to the passage of macromolecules and polar molecules.
repair processes that can take from a few seconds up to several The permeability of a given molecule or cargo material across
minutes to complete. Once membrane integrity is restored, the cell the plasma membrane depends on the properties of the
engages metabolic and transport processes to restore membrane and
cytoplasmic homeostasis. It may take hours for the cell to return to membrane (e.g., composition, heterogeneity, and thickness),
the preperturbation state. active cellular regulation of the plasma membrane (e.g., protein
activity and cytoskeletal interaction), the properties of the
molecule itself (e.g., charge, size, and polarity), and environ-
solution at a concentration conducive to influx. Second, the cells mental factors (e.g., temperature).168,400
are exposed to the membrane disruption event. Physical methods The plasma membrane allows compartmentalization of elec-
of permeabilization generally have better control of the intensity, trolyte concentrations between the cell interior and external
duration, and placement of the membrane disruption effect.105,396 solutions (Figure 7A). For example, relatively high intracellular
Biochemical methods, such as exposure to pore-forming toxins or potassium (140 mM) and low sodium (5−15 mM) are gene-
detergents, are more scalable but can be harder to control since rated by the action of the Na+/K+ ATPase, a plasma
they are not associated with a discrete event.395 Upon membrane membrane-embedded transport protein.401 Intracellular chlor-
disruption, cargo begins to diffuse into the cell according to its ide, calcium, and magnesium are all lower than their corre-
concentration gradient while some cytoplasmic contents are lost. sponding extracellular concentrations. The maintenance of these
In certain cases, additional effects, such as electrophoretic force, electrolyte gradients is key for the typical negative membrane
can also be harnessed to augment influx of cargo. Third, it is potential (−35 to −80 mV) of most animal cells and a host of
O DOI: 10.1021/acs.chemrev.7b00678
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Chemical Reviews Review
4.1.2. Plasma Membrane Composition and Proper-
ties. The plasma membrane has characteristic properties dis-
tinct from other types of lipid membranes (Figure 7B). It is
much more complex and dynamic than pure lipid bilayers,
containing hundreds of different lipid species and up to 50%
membrane proteins by weight. Proteins associated with the
plasma membrane include various transporters, receptors, and
enzymes. They may span the membrane via transmembrane
domains or be anchored to one side via lipophilic appendages.
The spatial organization of plasma membranes feature both
lateral heterogeneity (lipid domains) and uneven distribution
between inner and outer leaflets (lipid asymmetry).402 Cells
use up to 5% of their genes for synthesis of a diverse array of
lipids, reflecting the importance of the functions arising from
this diversity.403
The different types of lipids are distributed in a highly
regulated and distinct manner across the various membranes of
the cell.402 This gives the different membranes unique pro-
perties (Figure 7B). In eukaryotes there are three main cate-
gories of membrane lipids: glycerophospholipids, sphingolipids,
and sterols. Glycerophospholipids are the major structural lipids
of membranes, of which common species are phosphatidylcho-
line (PtdCho), phosphatidylethanolamine (PtdEtn), phospha-
tidylserine (PtdSer), and phosphatidylinositol (PtdIns). Their
hydrophobic tail is a diacylglycerol (DAG), which contains
saturated or cis-unsaturated fatty acyl chains of varying lengths.
Unsaturated tails do not pack as tightly, increasing the lateral
space between lipids and promoting lateral fluidity in the mem-
brane. PtdCho is the most common lipid, accounting for >50%
of the phospholipids in most eukaryotic membranes.402 PtdSer
and PtdIns exhibit negatively charged head groups and localize
to the inner (cytoplasmic) leaflet. The major sphingolipids in
mammalian cells are sphingomyelin (SM) and sugar-decorated
glycosphingolipids (GSLs). The sphingolipids feature a ceramide
Figure 7. Relevant structure and properties of the cell interior and as their hydrophobic backbone, having saturated (or trans-
surface. (A) Overview of typical animal cell structure with basic unsaturated) tails so they tend to form a taller, narrower cylin-
organelles, intra- and extracellular ion concentrations, and negative der shape than their glycerophospholipid counterparts.
membrane potential (ΔV). ER: endoplasmic reticulum. (B) Features Sterols are highly abundant in the plasma membrane, contrib-
of the plasma membrane including lipid asymmetry across bilayer uting greatly to barrier function and lateral organization.404,405
leaflets and lateral segregation into domains, such as raft phases. In mammals, the predominant species of sterol is cholesterol,
Abbreviations are phosphatidylcholine (PtdCho), phosphatidyletha- which represents up to 40% of the lipid molecules in the plasma
nolamine (PtdEtn), phosphatidylserine (PtdSer), phosphatidylinositol
(PtdIns), sphingomyelin (SM), and glycosphingolipids (GSL). membrane.
405 This is in contrast to other internal membranes,
Carbohydrate residues are depicted in black and cholesterol in such as the endoplasmic reticulum (ER), where the correspond-
purple. Phosphatidylcholine (PtdCho) lipid heads are light blue. Note ing number is only ∼5%. Cholesterol tends to straighten out
the highly regulated heterogeneous distribution of molecules between hydrophobic chains and fill in structural defects in membranes.
different types of membranes, lateral domains, and leaflets. As a result, Thus, it serves to stiffen and thicken the plasma membrane,
the ER membrane is thinner and sparser than the plasma membrane, improving its durability. Cholesterol is also essential to the for-
with more unsaturated lipid tails. (C) Plasma membrane reservoirs mation of lipid rafts, which are characterized by the assemblage
and their relationship with the underlying actin cortex, which is potential of sterol-sphingolipid interactions and particular pro-
connected by membrane-cortex linker proteins (green). Actin rods teins that have affinity for the raft phase (i.e., raft proteins).405
support filopodia and microvilli. Blebs are typically devoid of actin
until they are pulled back in. Remodeling of the actin cytoskeleton These lateral raft domains are thought to serve as platforms for
facilitates formation and stabilization of endocytic pits. key structural, signaling, and membrane trafficking phenomena,
such as the nucleation of caveolae pits in the plasma mem-
brane.406 In contrast to the plasma membrane, internal mem-
other essential functions. The cell also has elevated intracellular branes, such as the ER, feature less cholesterol, more unsat-
concentrations of metabolites such as ATP (typically 2−5 mM), urated lipids, and less diversity of lipid species.402 Internal
amino acids, and other biomolecules. The difference between membranes are thinner, sparser, and less durable, being more
intracellular and extracellular composition is an important con- adapted for biogenesis rather than the comparatively robust
sideration in membrane disruption-based intracellular delivery, and stable barrier function of the plasma membrane.402
as strategies that take this into account can lead to more The unique characteristics of the plasma membrane are a
efficient treatments and better cell health. Minimizing the key factor in certain membrane disruption strategies. For exam-
depletion of intracellular contents, for example, can improve ple, certain pore-forming toxins, such as cholesterol-dependent
cell treatment outcomes (see section 4.3). cytolysins (CDCs),407 and detergents, such as saponins,408 are
P DOI: 10.1021/acs.chemrev.7b00678
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Chemical Reviews Review
specific for high cholesterol-containing membranes. This shape and volume changes without tearing the membrane,415,416
makes it possible to disrupt plasma membranes in a relatively a key property to ensure durability of the cell in mechanically
speci c manner without damaging internal membranes.397fi challenging environments.
4.1.3. Intrinsic Membrane Permeability. Although the In cases where the plasma membrane is significantly rein-
plasma membrane comprises a highly regulated barrier to con- forced by other components, it may become more difficult to
trol the intracellular composition, it is naturally permeable to mechanically disrupt. This is an important factor to consider
certain substances. Phospholipid bilayers are permeable to gas particularly for mechanical membrane disruption techniques.
molecules such as O2, CO2, and N2 (permeability coefficients For example, the cell surface has been reported to exhibit an
101−10−2 cm s−1); solvents such as H2O, ethanol, and dimethyl impressive ability to conform to nanoneedles and other pene-
sulfoxide (DMSO; permeability coefficients 10−3−10−4 cm s−1); trating objects, making intracellular delivery less efficient than
and to some extent other small uncharged polar molecules anticipated.417,418
like urea and glycerol (permeability coefficients 10−6− As living cell membranes are much more complicated, dynamic,
10−7 cm s−1).168,409 Most cell-penetrant small molecule drugs and heterogeneous than artificial lipid bilayers, insights from
and peptides have permeability coefficients approaching a simplified model systems and simulations must be interpreted
maximum of about 10−6 cm s−1.168 Despite their small size, the with caution.410 The full complexity of the properties and behav-
cations Na+ and K+ are relatively impermeable with coefficients ior of the cell surface must be accounted for when thinking about
of 10−14−10−15 cm s−1. intracellular delivery approaches and the cell response. Further-
In live cells it is often a challenge to decipher whether more, plasma membrane variability across cell types is a fron-
permeability arises due to the passive properties of the plasma tier that must be addressed in order to better understand how
membrane, the presence of membrane transporters and solute to target specific cell types.
carriers, or fluctuations in transient bilayer defects (such as can
be promoted by ethanol or DMSO).168,410
4.2. Defect Formation in Lipid Membranes
In many instances
the apparent permeability of a molecule is actually regulated by Membrane disruption-based delivery approaches rely on var-
the cell. For example, membrane proteins called aquaporins ious methods to nucleate and expand defects in the plasma
increase the transmembrane flux of water and glycerol,411 the membrane. Mechanistically, the most well-studied examples
expression of which can vary significantly across a cell popu- are electroporation and mechanical tension, partly due to their
lation or between cell types. The cell actively opens and closes relative simplicity and ease of modeling and simulating. There
sodium channels to dynamically alter the Na+ permeability are also a host of molecules that can bind to and disrupt mem-
during action potentials. Furthermore, many small molecule branes by chemical means. Here we provide a theoretical over-
drugs have been postulated to enter cells via metabolite trans- view of the various mechanisms underlying membrane dis-
porters whose structures they often mimic.412 In other cases, ruption. Further details on the individual disruption methods
peptide transporters, such as PepT1 and OATP, have been are discussed later in their respective sections.
reported to pump small peptides and peptide-based drugs into 4.2.1. Mechanical and Electrical. Theories seeking to
or out of cells.168 Regardless of the mechanisms, few candidate explain the energetics and formation of membrane disruptions
drug molecules exhibit passive permeability or are amenable to by mechanical tension and electrical potential have arrived
419−421
active uptake by the cell. Chemical modifications or conju- at similar models. At near-physiological temperatures,
gations can be conferred to increase the permeability in some there is a finite probability of thermally-driven defect formation
cases, but this is not feasible for most macromolecular cargo, in lipid membranes. This defect formation is associated with
particularly for those larger than one nanometer in size. random lateral diffusion of lipids, which is made possible by
4.1.4. Structure and Properties of the Cell Surface. fluctuations in the void volume between lipid molecules.
The durability of the plasma membrane may be reinforced by Thermally-driven defects take the form of a so-called hydro-
intra- or extra-cellular scaffolds. Some lipids (e.g., glycosphin- phobic pore, where a small gap opens up between hydrophobic
golipids) and proteins (glycoproteins) have extracellular carbo- tails (Figure 8A). Hydrophobic pores are thought to be at a
hydrate domains. When sufficiently dense, these carbohydrate local free energy maximum when the radius is around 0.5 nm,
moieties can form a thick outward coating known as the which is slightly larger than the width of one lipid headgroup.
glycocalyx, which is prominent in animal epithelial/endothelial From there, further lateral growth permits the rearrangement
cells and some types of bacteria.413 On the interior side, the of hydrophobic tails into a hemispherical conformation at the edge
plasma membrane may be reinforced by the underlying actin of the pore. Once polar head groups face the aqueous solution, the
cytoskeleton and the proteins that link it to the membrane, pore becomes hydrophilic, thereby permitting the passage of
to form a cortical structure hundreds of nanometers thick414 water and becoming conductive to electrical charge. Hydro-
(Figure 7C). Other cytoskeletal elements such as microtubules, philic pores are thought to occupy a local energy minimum and
intermediate filaments, septins, spectrins, and clusters of cell thus exhibit notable stability at a minimum radius of around
adhesion molecules (e.g., integrins) can also assemble into sup- 0.8 nm, which is the width of around 2 to 2.5 lipid head groups.422
porting structures that affect membrane properties. Because the Over time the most likely outcome is that thermal fluctu-
actin cortex is often more mechanically robust than the plasma ations lead to closure of a hydrophilic pore. This happens
membrane, in many cases it is thought to control cell shape through a reversal over the energy barrier represented by the
and apparent surface area.414 Indeed, the plasma membrane hydrophobic pore, thus returning the membrane to a defect-
features a plethora of small folds, wrinkles, and reservoirs in the free state. Conversely, there is the low probability of crossing
form of outward-protruding actin-filled filopodia/microvilli the much larger energy barrier toward destruction of the whole
and actin-void blebs or inward-bending endocytic pits, such as membrane bilayer via infinite expansion of the pore. Increased
caveolae. The excess of plasma membrane surface area is thought input of mechanical tension or electrical potential into the
to be in the range of 2−10 fold the apparent cell surface area.414 system tilts the energy landscape toward the possibility of
These excess reservoirs allow the cell to accommodate rapid total destruction. Opposing pore expansion is line tension, an
Q DOI: 10.1021/acs.chemrev.7b00678
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Chemical Reviews Review
population of hydrophilic defects of various sizes that can be
modeled by a probability density function.420 In real world
numbers, biomembranes can generally withstand up to 3% mech-
anical area strain425 or 200 mV electrical potential419 before
persistent loss of membrane integrity occurs.
For mechanical membrane disruption in live cells that pos-
sess membrane reservoirs, further considerations are the defor-
mation force and velocity. In the case of a nanowire impaling a
small local area (Figure 9), it is evident that the deformation
velocity, rather than the final deformation magnitude, deter-
mines whether a membrane disruption event occurs. Slow impale-
ment of a nanowire allows sufficient time for lipids to flow in
from the surrounding reservoirs (such as blebs and folds) to
compensate for the local increase in membrane tension. On the
other hand, rapid impalement does not give the system suf-
ficient time to pull lipids from adjacent areas. Instead, the local
membrane tension ramps up to a point where formation of a
hole/pore becomes energetically favorable.426 This helps to
explain why larger contact forces are known to increase the
probability of membrane penetration when deforming membranes
with sharp AFM tips or nanowires,427−432 because the size of
the impaling object and the force upon contact determine the
local deformation rate, which is in turn related to the pro-
bability of membrane disruption. Further mechanistic studies
are required to quantify these parameters in different types of
cells.
4.2.2. Chemical. Apart from physical insults, a host of
chemical agents and effects can lead to membrane perforation
(Figure 10). Chemical disruption of lipid barriers can occur
through modification of constituent lipids, for example by oxi-
dation, insertion of pore-forming proteins and peptides, and
exposure to agents acting as detergents and surfactants. Because
Figure 8. Theory of mechanical and electrical disruption of lipid the modeling of these phenomena is more complicated, energy
bilayers according to energy landscape of defect formation.
(A) Energy landscape according to hydrophilic pore theory. Energy landscapes have not been described for most of these sce-433
is required to open up hydrophobic defects with radius ∼0.5 nm. narios. Instead, simulations are increasingly being exploited410,434
Further growth to a hydrophilic, toroidal pore with lipid head groups to capture, model, and visualize critical molecular events.
facing inward is associated with a local energy minimum at pore radius Membrane disruption can proceed via localized chemical
∼0.8 nm. W1 represents the energy landscape at rest with no external reactions, especially oxidation/peroxidation
435 (Figure 10A).
mechanical or electrical input, W2 (yellow) represents an intermediate Simulations and experiments suggest that oxidized lipids
mechanical of electrical stress, while W3 (orange) indicates the effect exhibit distorted hydrophobic tails that decrease the lateral
of a large mechanical or electrical potential. Low temperature is ordering of lipids and cause an increased area per lipid head.
synonymous with increased barrier heights, while high temperature This, in turn, triggers bilayer thinning and variations in the
favors membrane destabilization. (B) Illustration of pore formation
due to mechanical stress where the membrane is rst stretched before lateral diffusion coefficients, which is associated with a declinefi
pore formation. The applied in-plane tension (T ) and the line in bending rigidity and increase in membrane deformation andM 436−438
tension (T ) within a lipid pore are diametrically opposed. permeability. If these effects are sufficiently localized, itL
(C) Illustration of pore formation due to application of electrical can lead to formation of membrane pores, as suggested by sim-
potential normal to membrane where E is the electric field strength ulations.410,438
and TL = line tension within a hydrophilic pore. Hydrophilic pores are Another biochemical trigger for membrane disruption involves
conducting, thus leading to relaxation of charge buildup and a the exposure of bilayers to pore-forming agents, predominantly
reduction of entropy in the system. in the form of amphiphilic peptides or proteins (Figure 10B).
Subunits associate with the membrane before assembling into a
inward-directed force produced around the rim of a hydrophilic pore complex with variable size ranges, some being as large as
pore (Figure 8B,C). Under certain conditions, line tension has several tens of nanometers.439,440 Membrane disruption can
been observed to drive closure of micron-scale holes in giant also occur via detergents or surfactants (Figure 10C). These
vesicles and is directly related to the composition of the amphiphilic molecules integrate into the membrane and distort
membrane, being boosted by the incorporation of cholesterol, or buckle the bilayer, inducing conformational stresses that
for example.423 The line tension may also be influenced by relax via pore formation and loss of bilayer integrity.441,442
supporting structures, such as the actin cortex, which can be Detergents and surfactants thereby solubilize membranes in a
regulated by the cell to modulate membrane resealing.424 concentration-dependent manner.441,442
Thus, electroporation and mechanical disruption can be
described according to the following. For a given cell, the com- 4.3. Cell Response to Membrane Disruption
bined effects of temperature, expansive electrical or mechanical The previous sections 4.1 and 4.2 covered some basic proper-
forces, and line tension within the pores conspire to yield a ties of cells and their membranes as well as mechanisms of how
R DOI: 10.1021/acs.chemrev.7b00678
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Chemical Reviews Review
Figure 10. Chemical mechanisms for generating disruptions in lipid
bilayers. (A) Localized chemical effects within a specific region (red
circle) can lead to a change in the structure of lipid molecules,
triggering their dissociation from the bilayer as free molecules or
Figure 9. Dependence of deformation rate on mechanical membrane micelle-like formations. The dissociated molecules then leave behind a
disruption. In the schematic, impalement with a solid nanowire only hole in the membrane. As an example, an intense laser pulse can break
leads to membrane disruption in the case of rapid impalement. Slow bonds within lipid tails or cause them to become distorted through
impalement allows the system sufficient time for lipids to flow in from unsaturation. (B) Pore-forming agents can interact with a membrane
adjacent reservoirs. Rapid deformation ramps up the local membrane to assemble an oligomeric pore that allows the passage of cargo
tension (TM) to a point where membrane disruption (hole formation) molecules and materials. (C) Surfactants and detergents can embed
becomes energetically favorable. into the bilayer and induce curvatures that distort the membrane,
leading to pore formation and loss of bilayer integrity.
membranes can be disrupted. Here, we will examine how cells
respond to membrane disruption (summarized in Figure 11). Since Steinhardt’s discovery, a number of different mechanisms
The first response is an urgent call to action to repair the and pathways have been implicated in membrane repair. The
breached membrane. If this is not accomplished rapidly, the topic has been discussed in detail in recent reviews.398,445−453
cell will die. The second major response from the cell is after Overall, up to six repair variations have been proposed.447
membrane repair, where it seeks to rebalance the homeostasis As illustrated in Figure 12, the mechanisms include contraction,
of its membrane composition and intracellular contents. This patching, plugging, exocytosis, internalization, and external-
response takes place over minutes to hours and will determine ization.447 Multiple membrane repair processes may cooperate
whether the cell returns to its previous state, lives with perma- together to achieve resealing at time scales of anywhere from
nent alterations, or dies through a form of programmed cell death. a few seconds to several minutes.447 The type of membrane
This section provides an overview of these events and the repair is thought to depend on factors such as environmental
strategies and concepts associated with their manipulation in conditions (e.g., temperature and extracellular ions), size of the
order to optimize membrane disruption-based intracellular hole, and cell type.
delivery. Studies have shown that, while large holes (>0.2 μm) cause
4.3.1. Plasma Membrane Repair. Plasma membrane more immediate trauma in cells, they tend to be detected and
resealing was thought to be a passive process until the mid- repaired more quickly.399,444,454 Rapid exocytosis, plugging,
1990s when Steinhardt and colleagues discovered that rapid and patching are typical mechanisms that cells deploy to repair
exocytosis drives plasma membrane repair.443 In a mechanism large holes.444 For smaller disruptions, internalization through
analogous to neurotransmitter release, exocytosis was found to endocytosis or externalization through shedding serves to
be triggered by calcium influx.443 The calcium concentration extract lesions into a disposable vesicle.454−456 Very small holes,
difference between inside (∼100 nM) and outside (∼1 mM) is particularly from electroporation or lingering pore-forming
∼4 orders of magnitude and serves as an acute alarm signal to toxins, can persist for longer durations and drain the cell of
detect and repair plasma membrane breaches.444 resources.399,454,457,458 Thus, strategies to reseal small disruptions
S DOI: 10.1021/acs.chemrev.7b00678
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Chemical Reviews Review
Figure 11. Cell response to membrane disruption. First, plasma membrane repair (PMR) engages within seconds to rescue the cell. If PMR fails the
cell depolarizes, swells, and dies. Shown are the altered cytoplasmic contents that eventuate if membrane disruption is conducted in a physiological
buffer. If PMR is successful, the cell is left in a perturbed state with loss of cytosol. Stress response guides the cell to return to the preperturbation
homeostatic state or into apoptosis. In some cases, trauma or off-target damage associated with the disruption-recovery cycle may cause mutations,
fate changes, or loss of cell potency.
post-treatment should be of benefit to membrane permeabiliza-
tion-based methods. In this regard, additives such as PEG,
poloxamer 188, and other poloxamers (which may also exhibit
antioxidant activity459) have shown potential as cell recovery
agents.98,460−465 Vitamin C, Vitamin E, and lipid antioxidants
represent further options for promoting restoration of membrane
integrity after delivery.466−468
4.3.2. Cell Swelling. Although rarely mentioned in the
membrane disruption literature, cells tend to swell when their
membranes are disrupted in physiological buffers. From Figure 7A
one can see that Na+ and Cl− will flow into a compromised cell
while only K+ ions will exit. The net influx of osmolytes and
osmotically-obliged water causes cell swelling through a colloid
osmotic effect, a process that goes hand-in-hand with depolar-
ization of the cell membrane potential. Cell swelling has been
observed with electroporation,469−478 microinjection,479 laser
optoporation,480−486 and exposure to cavitation487 or fluid
shear.488 In these reports swelling usually reaches a maximum Figure 12. Proposed mechanisms of membrane resealing. In each
within 1−2 mins of membrane disruption before plasma mem- case, the black line with gap represents the plasma membrane with awound-induced hole and healing progresses from top to bottom.
brane repair and regulatory volume mechanisms synergize to Black circles represent vesicles in the cell. Red lines in “Contraction”
bring cells back to normal volume. represent cortical cytoskeleton; orange dots in “Internalization”
Interestingly, cells can survive up to 50% volume increase represent machinery powering endocytic invagination and pinching;
and still recover.469,470,475,477,485,489 Above that, the risk of blue dots in “Externalization” represent ESCRT machinery powering
instant death from bursting becomes imminent.490 It is known scission; green dots in “Plugging” represent proteins cross-linking
that swelling activates speci c stress signaling events491 and is a membranous compartments. Adapted from ref 447. Copyright 2015,fi
classic hallmark associated with necrotic cell death.492,493 Inhib- with permission from Elsevier.
ition of cell swelling has been explored as a strategy to improve cells. Despite this, exactly how volume changes influence delivery
cell function during and after membrane disruption-based intra-
490 performance and cell survival is yet to be extensively explored.cellular delivery. Related to this notion, cell shrinkage has 4.3.3. State of the Resealed Cell. When the plasma mem-
been observed in electroporation conditions where the induced brane is compromised to allow cargo influx, there is uncontrolled
membrane disruptions are small and the buffer is composed of exchange of molecules between the inside and outside of the cell.
osmolytes that are too big to flow into the cells (for example, In standard physiological buffer (see Figure 7A), disrupted
an isotonic large molecular weight PEG buffer).475,494 Unlike cells will suffer elevated Na+, Cl−, and Ca2+, and reduced levels of
physiological media, such a buffer is devoid of electrolytes that K+, ATP, metabolites, amino acids, proteins, and other intra-
can flow into the cell, and thus K+ and Cl− ions exit the cyto- cellular contents (Figure 11). Even after plasma membrane integ-
plasm along with water.475 Such results demonstrate how rity is fully restored, cells may still undergo necrosis, a type of
buffer composition can affect the volume response of disrupted cell death caused by irreversible disturbance of cellular
T DOI: 10.1021/acs.chemrev.7b00678
Chem. Rev. XXXX, XXX, XXX−XXX
Chemical Reviews Review
Table 2. Disruption Buffers from Papers Analyzed in the Process of Compiling This Reviewa
mode of membrane disruption cell media Na-rich “physiological” buffer K-rich “intracellular” buffer buffered sugar solution not specified/other
all 37% (n = 110) 34% (n = 101) 9% (n = 27) 17% (n = 52) 12% (n = 35)
electroporation 28% (n = 47) 31% (n = 52) 7% (n = 12) 25% (n = 42) 17% (n = 28)
physical (nonelectroporation) 58% (n = 49) 32% (n = 27) 7% (n = 6) 5% (n = 4) 7% (n = 6)
biochemical 28% (n = 13) 43% (n = 20) 22% (n = 10) 15% (n = 7) 0% (n = 0)
aNote that some papers use multiple buffers so percentages may not add to 100%. Not specified is likely to be cell media or Na-rich buffer by
default.
homeostasis.399 In particular, dramatically reduced levels of disruption.518,519 Whether or not preactivation of MAP kinase
ATP and potassium can trigger necrotic cell death due to pathways can improve survival upon membrane disruption-
deregulation of mitochondrial activity.399 Necrotic cell death is mediated delivery remains unexplored.
almost indistinguishable from an initial failure to reseal, also being Many of the characteristic responses elicited from pore-forming
characterized by swelling and loss of membrane integrity.492 toxins are also shared with electroporation and mechanical
Once the cell reseals its plasma membrane, homeostatic pro- wounding, further reinforcing that membrane disruption is the
cesses will kick in to restore intracellular contents. In this regard, key event.399 In the early days of the field, McNeil and col-
the most critical molecules are thought to be ATP, potassium, leagues witnessed that expression of c-fos and NF-κB, two trans-
and calcium.399 ATP is a particularly crucial metabolite as it is criptional activators, are strongly and selectively increased in cells
the primary energy source for the cell. Studies have shown it that suffered and resealed a mechanically-induced membrane
can take from 2495,496 to 5497 hours to recuperate ATP levels after disruption.520 Detectable NF-κB and innate inflammatory
electroporation496 or treatment with pore-forming toxins.495,497 responses were also measured in endothelial cells subject to
Potassium has been observed to drop from ∼140 to ∼20 mM membrane attacks with pore-forming toxins.521 Furthermore,
when cells are exposed to transient membrane damage498 and mechanical micropuncture was found to activate MAP kinases,
recovery can take from minutes to hours.399 Influx of calcium CREB1, and protein kinase C (PKC) to promote cell sur-
can be viewed as a double-edged sword, although it assists the vival.522−524 Interestingly, engagement of PKC is thought to prime
cell in detecting and repairing damage, excessive amounts can cells to cope with future membrane wounding events522 and has
be toxic and lead to cell death.456,499−501 High intracellular similarly been observed upon SLO exposure512 and electro-
calcium serves as an activator of certain proteases, such as poration.525 Recently, electroporation was also demonstrated
calpains, enzymes that promote apoptosis and degradation of to activate MAP kinase pathways526 and trigger transcriptional
cytoplasmic components.399 changes to support MAP kinase activity, membrane repair, and
Membrane disruption and recovery is often paralleled by cyto- recovery from oxidative stress.527 Complementing this picture,
skeletal disruption and recovery. In particular, microtubule depoly- reports have emerged that electroporation triggers autophagy
merization has been observed upon electroporation,502−505 in response to nanosecond pulsed electric fields.528
mechanical wounding,506,507 and exposure to pore-forming A key implication in all of these findings is that activation
toxins.508 Microtubule depolymerization manifests locally of stress response pathways prioritizes cell survival and threat
around the wound sites due to calcium influx.506,507 This is surveillance at the expense of proliferation and anabolism.
in congruence with the observation that electroporation does If stress levels reach a critical threshold, cells trigger a shutdown
not alter microtubule structure in media devoid of calcium.503 response via apoptosis or other forms of regulated cell death.492
In standard calcium conditions recovery of microtubule integ- In certain cell types delayed cell death has been a significant
rity has been reported to take from minutes up to 1 hour.503,504,507 problem after electroporation, even when the initial membrane
In some cases, membrane disruption also appears to cause repair is successful.490,529 In other cases, cell outcomes may be
depolymerization of F-actin and intermediate laments.504,509fi improved by adding inhibitors of apoptosis.530 As more inhib-
4.3.4. Stress Response After Membrane Disruption. itors of specific cell death processes become available, they may
A number of secondary consequences occur as a result of the find use in such situations.
perturbations associated with membrane disruption.399,495 For 4.3.5. Manipulating Cell Response to Optimize
example, a decrease in cytosolic potassium can lead cells into a Outcomes. The concept of optimizing intracellular delivery by
quiescent state characterized by autophagy (recycling of cel- manipulating cell response has received sporadic attention over
lular building blocks), formation of lipid droplets to conserve the past decades. As mentioned above, some positive results
energy, and arrest in global mRNA translation.495 Time taken to have been reported from supplementation with membrane
restore intracellular potassium homeostasis correlates with the healing polymers98,460−465 and/or antioxidants.461,466−468
duration of these effects.495 Furthermore, a drop in potassium Most of the work to date, however, has focused on engineering
is thought to be responsible for activation of MAP kinase stress the permeabilization buffer. The electroporation literature, in
response and proteolytic signaling cascades such as the inflam- particular, has extensively explored this aspect in an effort to
masome, which in turn trigger downstream effectors including optimize cargo delivery and cell health outcomes.
caspase proteins and the unfolded protein response.399,510−513 An analysis of 300 membrane disruption-based delivery
In all systems tested so far, pore-forming toxins activate the papers analyzed in the process of compiling this review reveals
three main MAP kinase stress response pathways: p38, JNK, four main types of buffers: (1) Na-rich “physiological” buffers
and ERK.399,514−517 Cell permeabilization in media containing such as PBS, (2) cell media, which is essentially physiological
high potassium prevents MAP kinase activation, indicating that buffer plus nutrients, (3) K-rich “intracellular” buffers, and
potassium depletion is the key trigger.518,519 MAP kinase (4) buffered sugar solutions. In our analysis, cell media (37%) and
and its downstream effectors promote cell survival and their Na-rich buffers (34%) are the most popular, ahead of buffered
inhibition appears to worsen cell death after membrane sugar solutions (17%) and K-rich buffers (9%; Table 2).
U DOI: 10.1021/acs.chemrev.7b00678
Chem. Rev. XXXX, XXX, XXX−XXX
Chemical Reviews Review
Deconstructing these trends by modes of membrane disruption slightly antagonistic to calcium, possibly helping to blunt some of
reveals further insights. For example, buffered sugar solutions the damaging aspects of calcium influx.443 It is also a cofactor
have historically been used by the electroporation community to hundreds of intracellular enzymes, including those involved
to avoid electrolytic effects associated with higher conductivity in energy metabolism and stabilization of mitochondrial
salt-based bu ers.531,532 Their origins can be traced back to the membranes.561,562ff ATP supplementation might be beneficial
beginnings of the field in the late 1970s and onward.502,531,533−538 not only in preventing its loss from the cytoplasm,397 but also in
In contrast, physical nonelectroporation-based methods, such as engaging extracellular receptors to activate “purinergic” signaling,
mechanical wounding and optoporation, have mostly opted for which is thought to prime cells against the danger of membrane
cell media (58%) or Na-rich buffers (32%). Biochemical disruption.456,563 As an example of its potential benefits, elec-
methods, of which detergents and pore-forming toxins are the troporation buffers supplemented with ATP help to achieve
main options, have been the most likely to experiment with faster gene expression after plasmid delivery.564 Glucose is added
K-rich “intracellular” buffers (22%) but most often used their to some buffer formulations146,551 and would tend to prevent
Na-rich counterparts (43%). Biochemical permeabilization cell energy depletion due to cytoplasmic leakage. Antioxidants
methods, which have less control over the timing of membrane have been reported to promote membrane repair and overall
disruption, seem more concerned with maintaining intracellular cell health by neutralizing reactive oxygen species (ROS).466−468
homeostasis through implementation of K-rich bu ers.539,540ff ROS may damage proteins, lipids, and nucleic acids, the latter
K-rich buffers have been in use since the pioneering days of of which can lead to mutations in DNA. Most of the optimized
membrane permeabilization, with detergents,541 electropora- buffers also tend to contain little or no Ca2+. Although it is the
tion,542 and mechanical scraping543 being early examples. The prime trigger for membrane repair, precise studies have shown
argument in favor of these bu ers is simple: by mimicking the that only ∼5−20 μM is required.565−567ff Normal extracellular
intracellular composition as closely as possible, homeostasis and Ca2+ levels (∼1 mM) are probably only helpful when cells are
cell health should theoretically be maintained.397,542,544 One returned back to standard media for final recovery.
study compared K-rich buffers to Na-rich ones, concluding that Other potential supplements for augmenting cell health could
K-rich are superior for gene expression and cell recovery after be zinc568 and recombinant proteins that participate in mem-
delivery by mechanical membrane disruption.545 A different brane repair, such as MG53,568−571 annexins,572 and ASMase.573
investigation found that electroporation in buffers designed to Conducting cell membrane disruption and/or recovery in the
match intracellular contents (with appropriate levels of ATP, presence of certain inhibitors may also be beneficial in guiding
GTP, amino acids, K+, Mg2+, and Ca2+) accelerated recovery of cell fate, however, has received little attention to date.
protein synthesis to within 5 min compared to >1 hour for Recombinant proteins and specific inhibitors might be worth
standard PBS.546 Another group observed electroporation in using in clinical scenarios, such as for ex vivo cell-based
intracellular mimicking buffer featuring high K+, Mg2+, ATP, therapies.
and glutathione promoted cell survival compared to cell media Temperature is a core consideration for any in vitro cell treat-
or PBS.547,548 Furthermore, a cold-storage solution for organ trans- ment procedure, and deliberate membrane disruption is no
plants, containing high K+, Mg2+, and antioxidants, was reported to exception. Despite this, there is no consensus in the literature
markedly improve survival of electroporated cells.549 Although on which temperatures are best for membrane disruption-based
most of the commercial electroporation buffers today are based intracellular delivery. An analysis of more than 300 membrane
on high sodium,550 nucleofection offers a K-rich variant with disruption-based delivery papers analyzed in compiling this
high magnesium, ATP, and glucose, which appears to be useful review reveals three categories of temperature that have been
in treating primary human cells.146 Whether K-rich intracel- used: (1) ≤ 4 °C, (2) room temperature (usually in the range
lular mimicking buffers are under-utilized in membrane disruption- 18−25 °C), and (3) ∼ 37 °C (Table 3). The rationale for
mediated delivery remains to be established.
Commercial electroporation systems such as nucleofection Table 3. Disruption Temperatures Used in Papers Analyzed
appear to have placed significant e affort into optimizing pro- in the Process of Compiling This Review
prietary buffers, mostly arriving at formulations featuring high
+ mode of membrane RT notNa , 10−20 mM Mg2+, strong pH buffering, and extra organic disruption 37 °C (∼18−25 °C) <4 °C specified
osmolytes.550 A number of academic groups have lifted the lid on all 22% 46% 16% 16%
these formulations and screened their effectiveness in an attempt (n = 65) (n = 139) (n = 47) (n = 49)
to lower costs.551−553 Indeed, several studies testing nucleofec- electroporation 9% 67% 11% 13%
tion buffers found only marginal bene ts over PBS554fi or cell (n = 15) (n = 112) (n = 19) (n = 22)
media,555 suggesting that the high cost of these proprietary buff- physical (non- 34% 25% (n = 22) 12% 27%
electroporation) (n = 30) (n = 11) (n = 24)
ers may not be justified. On the other hand, Biorad electropo-
biochemical 43% 13% (n = 6) 38% 6%
ration guides recommend more basic options such as cell media, (n = 20) (n = 18) (n = 3)
strongly buffered Na-rich saline, or buffered sugar solutions.556,557 aNote that some papers use multiple temperatures, so percentages
Neon electroporation buffers seem to be based on PBS bolstered
558,559 may not add to 100%. RT denotes room temperature and variesby extra pH buffering, sugar, and magnesium. Interestingly, considerably between publications from 18 to 25 °C. Not specified is
many of the electroporation-based preclinical or clinical studies most likely to be room temperature by default.
simply use OPTIMEM (a popular cell media) in place of
commercial electroporation bu ers.187,560ff treating cells at ≤4 °C is that it can facilitate a preservative effect.
Taken together, consistent benefits seem to be obtained by Most stress responses and programmed cell death pathways are
supplementing buffers with Mg2+, ATP, glucose, antioxidants, inhibited at 4 °C, so unless the cell is killed by the treatment
and by lowering or avoiding Ca2+. Additionally, in the case of itself, the long-term cell survival may be improved. One detergent-
electroporation, strong pH buffering probably helps to negate based protocol credited low temperature and intracellular buffer
the detrimental effects of electrolytic reactions. Magnesium is as the two main factors increasing cell survival.539 Biochemical
V DOI: 10.1021/acs.chemrev.7b00678
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Chemical Reviews Review
protocols employed ≤4 °C 38% of the time compared with early 1900s. Particle bombardment and nanowires/nanostraws
11% for electroporation and 12% for physical nonelectropora- were invented in the late 1980s and early 2000s, respectively.
tion. Low temperatures probably slow down membrane repair, In this section we discuss the key details of each of these methods.
but also make cells more resistant to disruption, particularly
574,575 5.1. Microinjectionelectroporation. Furthermore, many pore-forming toxins
do not assemble at 4 °C, so a switch to warmer conditions can In 1911 Marshall Barber reported the invention of micro-
be used as a trigger to control the timing of permeabilization.576 injection.
324 By pulling glass capillaries over a flame, Barber
The rationale for treating cells at room temperature is simply generated pipettes with sharp micron-sized ends suitable for
convenience, as it does not require any additional temperature injection into living cells. Combined with micromanipulators
control equipment. Membrane repair in mammalian cells seems and pressure control systems, dual pipettes were demonstrated
to proceed quite normally at 25 °C, as evidenced by studies of with holding, dissecting, extraction, and injection capabilities.
annexin-mediated resealing.500,566,577,578 Electroporation pro- The apparatus was used to extract nuclei from living amoebae,
tocols, in particular, favor room temperature (67% of papers inject various fluids into cells, and deliver single bacteria into
325
analyzed). Because Joule heating associated with electropor- plant cells. Barber rightly predicted that “The introduction
ation can spike the temperature of a solution by up to 20 °C,579 of foods, poisons, stains, and fixatives is made possible and cells
using a baseline of 37 °C may be harmful to cells undergoing may be probed or dissected under high powers, methods which
electroporation. On the other hand, the rationale for treating may be of use in the study of the structure, chemistry, and
cells at 37 °C is maintenance of physiological function. Most physiology of cells. Finally, materials may be withdrawn from
nonelectroporation protocols choose to employ such physio- one cell and injected into another, and it is possible that
logical conditions, with biochemical procedures using 37 °C investigations on fertilization and heredity may be extended by
43% of the time and physical nonelectroporation 34% (Table 3). this technic”. After inventing microinjection, Barber trained
Membrane repair and stress response are expected to be at their others in its use before leaving the field.325 In 1915, Kite used
most efficient at 37 °C. it to inject dyes into the cytoplasm of living animal and plant
4.3.6. Semi-intact Cells. Although most applications of cells to investigate their permeability.595 Chambers then intro-
intracellular delivery by membrane permeabilization aim for a duced an improved version of the instrument in the early
transient permeabilization from which the cell recovers, there 1920s, featuring more precise micromanipulators and pressure
are situations where a persistent ongoing permeabilization is control.596
chosen. Such systems have been referred as semi-intact cells,580 As microinjection spread to other researchers, it was initially
semipermeable cells,581 or perforated cells.582 They involve adopted by plant, developmental, and microbiologists, for exam-
irreparable disruption of cell membranes by mechanical580−582 ple to determine cytoplasmic pH, introduce viruses into cells,
or biochemical means.583−590 Strategies such as low temperature or perform nuclear transplants.597−600 Moreover, it became the
and low calcium concentrations may be employed to deliberately basis for patch clamp and a host of similar pipette-based cell
prevent membrane resealing.580 Efflux of cytoplasmic constitu- manipulation and analysis techniques.325,601 As covered in
ents follows, but the extracellular media is manipulated to “recon- section 2.2.5, microinjection has long been the dominant method
stitute” the cytoplasmic composition replete with desired inhib- for intracellular delivery of large cargo. It was used for the first
itors, activators, antibodies, metabolites, ATP-regenerating nuclear transplants in 1952,326 cloning frogs in 1958,327 cloning
systems, and other macromolecules of interest.586,587,591 Semi- mammals in 1997,329 mitochondrial transplants in 1974,336
intact systems have therefore been useful for functionally recon- chromosome transplant protocols in 1973,333 intracellular deliv-
stituting intracellular processes while being able to manipulate ery of sperm into egg cells in 1962,331 and the first human preg-
the buffer. Apart from high potassium, such buffers usually con- nancies achieved by IVF in 1992.332 More recent examples of
tain high magnesium, low calcium, ATP at mM concentrations, large cargo delivery include micron-sized beads for intracellular
strong buffering, and reducing agents or antioxidants. The major microrheology analysis,348,349,354 magnetic beads for applica-
concern in using these methods is that it has been difficult to tion of forces,356,357 and silicon MEMS barcodes up to 10 μm
assess to what extent the semi-intact cells are a valid model for in size.358
intact cells.395 The concept of semi-intact cells illustrates the Although microinjection was employed for large cargo deliv-
lengths biologists have pursued to address intracellular delivery ery from the beginning, it took more than half a century for it
and manipulation challenges. Despite their limitations, these recon- find routine use for intracellular delivery of proteins, DNA, and
stituted systems have been valuable in discovering fundamental other such biomolecules into animal cells. Purified proteins began
mechanisms of secretory pathways and principles underlying traf- to be injected into animal cells in the 1960s. The protein fer-
ficking of proteins, lipids, and nucleic acids between intracellular ritin was introduced into amoebae to monitor its intracellular
organelles, for example to decode the rules that govern nuclear distribution.80 Then mouse oocytes injected with bovine gamma
import.592,593 Semi-intact cells remain popular for certain types globulin were shown to be capable of developing into defect-free
of studies, such as probing mitochondrial function in muscle animals.81 In 1972, the calcium-sensitive protein aequorin was
cells.594 injected into the squid giant synapse to track intracellular cal-
cium concentrations.602 Other studies in the 1970s used fluo-
5. INTRACELLULAR DELIVERY BY DIRECT rescently labeled proteins and dextrans to examine nuclear per-
PENETRATION meability82,83 and autophagy.84 Microinjection of peptides also
Direct penetration mechanisms are utilized in the techniques of emerged around that time.603 Fluorescently labeled actin85 and
microinjection, particle bombardment, and nanowires/nanostraws. alpha-actinin86 were injected into cells to visualize and eluci-
In each of these cases, penetrating elements provide direct date their role in the cytoskeleton. A classic example where intra-
access to the intracellular space. Microinjection is the classic cellular delivery of a protein led to discovery of its function was
embodiment of the direct penetration mechanism and was the the case of vinculin.604 Microinjection of the uncharacterized
first intracellular delivery technique to be introduced in the protein labeled with fluorescent dyes was used to identify its role
W DOI: 10.1021/acs.chemrev.7b00678
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Chemical Reviews Review
Figure 13. Intracellular delivery via microinjection. (A) Depiction of an adherent cell being microinjected with a glass micropipette.
(B) Microinjection of a suspended cell that is held in place by a secondary holding pipette. (C) Nanopipette injection (nanoinjection) where the
penetrating aperture consists of a nanotube. In this illustration an intracellular organelle is being injected. (D) Use of a hollow AFM cantilever to
inject cells (FluidFM). (E) Microfluidic microinjection where a cell is pushed onto a sharp micropipette via flow. Pressure is then generated in the
micropipette to deliver fluid into the cell. Reversing the flow of the main microfluidic channel can be used to eject the cell.
as a mediator of cytoskeletal adhesion assemblies by observing impact on other cellular structures (Figure 13C). Exploiting a
localization dynamics in living fibroblasts.604 different mechanism of volume control, an electrochemical atto-
Along with protein delivery, researchers began experiment- syringe with tip diameter of 100−400 nm achieved picoliter to
ing with microinjection of DNA and RNA. The first mRNA attoliter volume control.611 Such fine electrochemical control
expression studies were carried out by microinjection from 1973 of fluid motion allowed the accurate dispensation of precise
onward.240−242,605 Viral DNA was injected into cells to inves- volumes from the fabricated “nanopipette”.611 Another group
tigate its ability to transform cells.606 Recombinantly engineered employed carbon nanotubes as the pipette. The device, termed
plasmids were expressed in cells postinjection in 1977.200 Several a nanotube endoscope, demonstrated delivery of fluorescent
years later, Capecchi demonstrated that nuclear injection of molecules to subcellular localizations at a resolution down to
plasmid DNA led to successful expression in 50−100% of cells. 100 nm.612 Recently a system that takes advantage of elec-
Yet the same plasmid injected into the cytoplasm led to 0% trophoretic cargo propulsion was claimed to provide higher cell
expression in hundreds of cases.202 Thus, microinjection studies viability postinjection.613 This was based on a 100 nm diameter
were used to prove that plasmids must be delivered to the nanoinjector that drives materials into cells via electrophoretic
nucleus to undergo expression. In 1980, transgenic mice were force rather than bulk pumping of uid.613fl
successfully produced by microinjection of recombinant Some interesting adaptations to the microinjection concept
plasmid DNA into the nucleus of fertilized ooctyes.607 have been produced by modifying atomic force microscope
Following the elucidation of antisense oligonucleotides in the (AFM) systems to allow injection or extraction.614 One tech-
1980s, antisense RNA was injected into cells to inhibit protein nology, called FluidFM, was first demonstrated by the use of
expression in studies of developmental biology.211,608 The Nobel hollow cantilevers with fluid control capabilities for force-con-
Prize winning experiments that elucidated RNAi were trolled injection of soluble materials into cells (Figure 13D).615
performed by microinjection of double stranded RNA into AFM force feedback was reported to enable unprecedented
C. Elegans cells in 1998.215 control of contact force thereby facilitating the determination
As illustrated in the above examples, microinjection is a ver- of required penetration forces.614 Recently, the FluidFM sys-
satile delivery platform, being able to deliver almost any cargo tem has been used for nondestructive sampling from cells for
to most cell types. In its current form, microinjection is com- time-resolved analysis of molecular composition616 and metab-
monly performed with commercial systems fitted with glass micro- olite profiles.617 It also features the precision to deliver or extract
pipettes of tip diameters from 0.3 to 1.0 μm (Figure 13A). from the nucleus.616,618 In a similar approach to FluidFM,
It is important to note that microinjection does suffer some another group used a scanning probe system to detect cell
degree of cell type-dependence. Small cells, such as blood cells surfaces and provide voltage pulses to deliver fluorescent dyes
with diameters less than 10 μm, can be challenging to micro- into individual cells.619
inject due to their small volume and poor tolerance for needle 5.1.2. Attempts toward Higher Throughput Micro-
penetration.609 For nonadherent or suspension cells an addi- injection. The primary limitation of standard microinjection
tional holding pipette is employed to keep cells in place is the serial, low throughput, and tedious nature of the process.
(Figure 13B), but this adds to the complexity and time- Even an experienced operator is limited to approximately one
consuming nature of the procedure. Researchers and clinicians successful injection per minute. An early attempt at automated
most often use microinjection for experiments or procedures microinjection was published in 1988, with a reported through-
involving single cells or small batches of cells where high fidel- put of 1500 cells per hour when performed on adherent
ity of intracellular delivery is ensured. For example, due to its cells.620,621 For unknown reasons, this innovation was not widely
accuracy and control, microinjection has been a routine tech- adopted. Other attempts at high throughput microinjection
nique to achieve human pregnancies by in vitro fertilization. include a vacuum-enabled embryo holding array, which allows
5.1.1. Advances in Technical Precision of Micro- injections based on robotic motion control and image recogni-
injection. Significant advantages of microinjection include pre- tion by computer vision processing.622 The reported through-
cise control of dosage volume and injection location. In one put of 15 cells per minute was demonstrated to yield a high
innovation, organelle targeting was demonstrated with an ultra- survival rate (98%) for large nonadherent cells such as embryos
fine tip and femtoliter to attoliter volume control provided by a and oocytes. In a semiautomatic approach, a microrobotic sys-
galinstan expansion syringe.610 Using a tip diameter of ∼100 nm, tem achieved up to 25 injections per minute on adherent endo-
researchers were able to inject single chloroplasts in plant cells thelial cells.623 The human operator selects injection destinations
without dissipation of intracellular turgor pressure or untoward through mouse clicking on a computer screen and the system
X DOI: 10.1021/acs.chemrev.7b00678
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Chemical Reviews Review
executes with a reported survival rate of >95% and a success biolistic process, ballistic particle delivery, microprojectile
rate of >80%.623 bombardment, and, in certain embodiments, the “gene gun”.
Apart from automation, microfluidic systems have been Biolistic delivery came onto the scene in 1987, where it was
explored to address microinjection throughput challenges. Adamo first invented for the purpose of DNA transfection in plants.394
and colleagues reported a microfluidic version of microinjection In the late 1980s and early 1990s it was adapted for trans-
that works by suction of cells onto a 0.5 μm diameter hollow-tip fection of diverse microorganisms (yeast, fungi, algae, and bac-
glass needle embedded in a PDMS device (Figure 13E).624 teria), many of which are difficult to transfect with other
Several picoliters of liquid could be injected into the cell in methods.626,627 It was also attempted for transfection of an
∼0.5 s followed by flow reversal of the main channel to dislodge assortment of animal cells and tissues. Given the limited
the cell.624 The cell could be then routed through an exit channel penetration distance of particle bombardment into tissue, it
for collection after delivery.624 Problems with cell clogging and was initially tested with cell cultures in vitro and skin or
fouling from biological debris, however, prevented the device exposed tissue sections in vivo.627,629−631 For cell cultures in
from achieving consistent operation. A follow-up concept sought vitro, particles are sprayed down onto monolayer of adherent
to address this problem with high-pressure fluid jet injection, but cells or a thin dispersion of suspension cells. As a rule of
synchronization of jet firing with cell passage at the injection thumb, particle sizes should be no larger than one tenth the
nozzle presented a signi cant unsolved challenge.625fi size of the target cells.626 Heavy metal particles are durable and
5.1.3. Microinjection Summary. Microinjection was the dense and do an excellent job of maintaining the momentum
first intracellular delivery method to be invented and has been needed for breaching the plasma membrane.626 Particles used
in use for over a hundred years. It is a method of choice to deli- in biolistic systems tend to be tungsten (occasionally toxic),
ver almost any cargo, whether large or small, to single cells or gold, or silver (less toxic) and in the size range 0.5 to 2 μm.632
small groups of cells (<100). Despite technical advances, how- 5.2.1. Cell Type Applicability. Several early studies on
ever, the intrinsic low throughput of microinjection remains biolistic intracellular delivery sought to test applicability to
a serious limitation for the great majority of applications. hard-to-transfect mammalian cells, particularly immune cells,
An effective platform for high throughput microinjection would blood cells, and neurons. It was shown that both adherent and
be ground-breaking but remains elusive. suspension cell cultures can be transfected with plasmid-coated
5.2. Penetrating Projectiles (Biolistics) metal particles. Transfection efficiencies in T cells were reported to
Biolistic intracellular delivery employs high-velocity micropro- be a maximum of 2%,
633 6%,634 and 3%,635 respectively. In ex vivo
jectiles to deliver nucleic acids and other substances into intact HSCs, efficiencies were either not directly reported
636 or
cells and tissues. The microprojectile particles are accelerated achieved a maximum of 6% alongside 75% viability.
637
to su cient velocity by use of a gas shock wave, which can be Both adherent and suspension tumor cells could be trans-ffi
generated by various means. For example, it may be derived by fected with the plasmid-coated ∼1−2 μm gold particles shot
a chemical explosion (gunpowder), high-voltage electronic from a helium driven gene gun.
638 However, this study reported
discharge, release of pressurized inert gas, or helium shock only the yield of expressed protein and not percentage cells638
generated via a rupture-membrane mechanism.626−628 The gas transfected. A comparison across many cell types observed
shock wave is used to either (1) accelerate a macroprojectile from 2% to 40% transfection efficiency depending on cell639
into a stopping plate to dislodge adhered microprojectiles or line. Upper limits of 30−40% were obtained for common640
(2) blast the microprojectiles o the surface of a screen or the adherent cell lines such as prostate cancer cell lines or HEKff
626−628 cells.628inside of a barrel. Particles then collide with target cells, Due to the random spray of particles over a cell
bursting through the plasma membrane and releasing cargo sample, it is unlikely that particles will penetrate the nucleus
molecules from their surface into the cytosol (Figure 14A). of every cell to deliver DNA cargo for subsequent expression.
Biolistic intracellular delivery has been referred to as the For large cells that “catch” many particles, such as myotubes,
20−70% transfection can be obtained.641
Some reports claim biolistic delivery is a highly efficient
DNA transfection method in mammalian cells.642 However, it
is only efficient in its use of DNA, not necessarily in the per-
centage of cells treated. It has been estimated that about 200
plasmids are delivered per gold particle.643 Hence, the amount
of DNA required to produce a given yield of protein can
be very e 644fficient. In comparison, electroporation and lipid
reagents are highly wasteful of DNA (most is lost in solution)
but produce a large proportion of cells that are successfully
transfected. Empirical optimizations aimed at improving the per-
formance of biolistic delivery in animal cells identified parameters
such as size of the particles, the target distance, extent of vacuum,
and the size of the cell culture plate.626,642 Tuning of such
parameters, however, has yielded limited success. Thus, after
Figure 14. Intracellular delivery via penetrating projectiles. (A) Biolistic an initial period of excitement surrounding biolistic transfection,
projectiles consisting of metal beads are propelled toward a cell with electroporation and viral vectors have risen to prominence as the
enough force to burst through the plasma membrane. The metal
beads are coated with cargo, which then releases inside the cell. Inset preferred methods for hard-to-transfect cells such as HSCs and
shows an example of a single cargo-covered bead disrupting the immune cells.
plasma membrane. (B) A magnetic field is used to attract magnetically One area where the biolistic process gained notable traction
functionalized particles (such as modified CNTs) through the plasma is delivery to neurons and organotypic brain slices.628,645−650
membrane into the target cell for delivery of attached cargo. Neurons are regarded as very difficult to transfect with
Y DOI: 10.1021/acs.chemrev.7b00678
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Chemical Reviews Review
conventional methods. Early studies of plasmid delivery into ∼80 nm silver nanoparticles were also evaluated and found to
neural cell cultures have achieved transfection efficiencies of transmit less damage to cells.673 In both cases delivery effi-
<2%,651 2−8%, depending on the type of neurons,648 and up to ciency of cargo was not reported to be adversely compromised
10%.647 Although most of the protocols hover below by using nanoparticles instead of microsized beads, and the
10%,647−651 maximums of 20−30% were reported with a higher surface area to volume ratio of nanoparticles could be a
highly optimized protocol.628 As the alternatives are generally potential advantage. Overall, implementation of biolostic
poor, such performance has proven sufficient to carry out particle bombardment approaches to animal cells requires a
several interesting studies in neuronal cultures.628 Particle number of empirically determined parameters to be optimized.
bombardment has been particularly useful in organotypic brain These include size of particles, distribution, density, impact
slices, where alternative methods such as electroporation lack speed, and loading technique.396
access to cells.650 In a nanoinspired adaptation of the projectile delivery approach,
After three decades of experimentation, the main cells and tis- Cai et al. used DNA-carrying nickel-embedded nanotubes
sues that have proven amenable to biolistic delivery are (1) plants, propelled by magnetic fields to “spear” cells.674 Nanotubes in
especially for generating transgenic crops,394 (2) neurons and solution were attracted to a magnet placed underneath the
organotypic brain slices,628,645−650 (3) microorganisms that are substrate, thus creating the driving force for penetration of cells
difficult to transfect with other methods,626 and (4) inocula- placed on the substrate (Figure 14B). With this method they
tion of skin or muscle for applications such as vaccina- demonstrated efficient GFP expression in primary mouse
tion.629,652−654 E cient DNA immunizations against in uenza B cells and neurons with minimal cell death.674ffi fl More recently,
have been achieved by using a gene gun to deliver DNA-coated magnetic nanospears composed of Au/Ni/Si with dimensions
gold beads to the epidermis in mice and chicken.655 Projectile of ∼5 μm long and <50 nm diameter have been used to
bombardment is suitable for these applications because the transfect adherent cell lines with absorbed plasmids at >80%
immunization is thought to be effective even when only a small efficiency.675 Thus, for in vitro and ex vivo applications, smaller
fraction of cells are transfected. For intracellular delivery to projectiles that minimize damage to cellular structures may
skin cells, there is a notable trade-off between power, size and present an opportunity to improve projectile-mediated intra-
number of bombarding particles, and cell viability.656 cellular delivery.
5.2.2. Cargo Applicability. The biolistic process has been 5.2.4. Penetrating Projectiles (Biolistics) Summary.
used mainly for plasmid transfection. Additionally, it has Since its introduction in 1987, a range of different types of
proven particularly advantageous for delivery of larger DNA cargo-laden projectiles have been fired into cells for the
vectors such as cosmids and arti cial chromosomes.643,651,657fi purpose of transfection and intracellular delivery. The field is
In the early 2000s researchers successfully experimented with witnessing a trend toward smaller, less damaging projectiles
attaching dyes and indicators to the projectiles,658−661 mostly and attempts to improve the consistency of cell treatment.
for delivery to neural cell types and brain slices. Following that, If limitations around 1) cargo and cell type applicability, and 2)
mRNA and siRNA were shown to be feasible for transfection consistency of cell treatment can be overcome, biolistic
into a variety of cells and organisms.662−666 Biolistic methods intracellular delivery has the potential to deliver beneficial
have also been deployed for delivery of large beads to the cyto- outcomes not yet realized.
plasm for analysis of intracellular mechanical properties. In these 5.3. Nanowires and Nanostraws
cases cytoplasmic microrheology was assessed by monitoring
fluctuations in polymer beads within the cytoplasm.350−353 In Nanowires, also referred to as nanoneedles, nanosyringes, nano-
one example, ∼1 μm melamine particles coated with PEG were fibers, and high aspect ratio nanostructures, are thin elongated
shot into HeLa cells to study glassy dynamics in the cytosol.355 structures typically with diameters of hundreds of nanometers
More recently, protein delivery has been demonstrated with or less and lengths on the micrometer scale. For intra-
particle bombardment, first in plants667,668 and then in mam- cellular delivery at high throughput, nanowires are fabricated
malian cells.669 Protein delivery protocols have been further into vertically aligned arrays that can interface with thousands
adapted for biolistic Cas9 RNP delivery.670 RNPs were dried of cells. Nanostraws are hollow versions of nanowires, which can
onto gold particles and fired into immature wheat embryos to deliver fluid from an external reservoir directly to the intracellular
produce gene-edited crops.670 space.
5.2.3. Biolistic Systems and Variations. Biorad is the Intracellular delivery by penetrating nanowires was first dem-
main supplier of commercial biolistic delivery platforms. The onstrated by McKnight and colleagues in the early 2000s.109,676
gene gun is a hand-held device with a “point and fire” mode of They produced conical spikes of 6−10 μm in length, tip
operation. The more advanced biolistic systems employ a vac- diameters of 20−50 nm, and base diameters of ∼1 μm. These
uum chamber for higher momentum and evenness of micro- carbon/nitrogen-based structures were grown via plasma-enhanced
particle dispersion. The vacuum systems are typically used for chemical vapor deposition off nickel-spotted silicon wafers.109
in vitro applications where the sample is more amenable to The first cargo to be delivered with them was DNA plasmids,
manipulation. A major weakness of biolistic delivery is the which were physically absorbed or covalently tethered to the
damage that high velocity particles can cause to cells. This is tips of the conical nanowires. CHO cells were then forced against
one of the reasons why it is popular for plants, which have stiff the array by centrifugation at 600 g followed by sandwiching
cell walls that can tolerate harsh mechanical impacts.626 Damage against an opposing substrate. This provided an active force for
from gene guns has been identified as a key limiting factor in penetration, which proved to be necessary for efficient trans-
treatment of cell cultures in vitro, as well as skin and muscle fection in this system (Figure 15A). The nanowires were able
tissues.671 In general, damage is intensified as the projectile to achieve nuclear penetration as evidenced by rapid GFP expres-
diameter increases relative to the cell size. Nanoparticles of sion. Interestingly, GFP plasmids that were physically absorbed
∼40 nm have been tested with the biolistic method and found to the nanowires were passed on to cell progeny, while cova-
to provide better cell survival, especially with small cells.672 lently tethered plasmids were not, suggesting that the former
Z DOI: 10.1021/acs.chemrev.7b00678
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Chemical Reviews Review
Figure 15. Intracellular delivery via penetrating nanowires/nanoneedles and nanostraws. (A) Cell pushed onto an array of nanowires with active
force (F), such as centrifugation. The number of penetrating nanowires increases given the same needles as in B. (B) Passive settling and adhesion
of a cell onto an array of nanowires coated with cargo molecules at the tip (green). In this case some nanowires may penetrate through the plasma
membrane into the cytosol to release their contents inside the cell (green cloud). (C) Hollow nanowires (nanostraws) used for intracellular
delivery by pumping cargo from a fluid reservoir connected to the nanostraws.
dissociate in the cell interior while the latter are able to mediate recombinase682 and antibodies against cytoskeletal proteins.427
gene expression even though they remained attached to the Apart from large cargo, nanowire arrays have been proven to
nanostructures. In follow-up studies the same researchers deliver most categories of macromolecules into the cytosol of
upgraded their nanowire array method to feature spatially various cell types.
indexed substrates for long-term cell tracking676 and simul- 5.3.2. Nanowire Penetration Mechanisms. Despite the
taneous delivery of multiple different plasmids.677 reports of successful delivery of multiple cargo types, it is not
5.3.1. Expanding the Repertoire of Deliverable Cargo. fully understood how nanowires breach the plasma membrane.
As mentioned above, nanowire arrays were first used for Indeed, the mechanisms and efficiency of nanowire penetration
DNA transfection.109,676−678 Since then the delivery of have been a matter of debate for almost a decade. For example,
siRNA,110,679−681 proteins,110,427,680,682 molecular beacons,683 several groups claim that active force is not required if the
quantum dots,684 DNA nanocages,685 and impermeable drugs110 density, length, and diameter of nanowire arrays is optimized
has been further demonstrated. One of the first such examples for a particular cell type.110,678,679,687 On the other hand,
was achieved by Park et al., who produced nanosyringes of conflicting reports indicate that a majority of nanowires fail to
50 nm outer diameter and 120 nm height.684 The cup-like hollow penetrate cells that passively settle on top.688−690 For example,
nanostructures were prefilled with DNA or ∼3 nm quantum nanowires ranging from 2 to 11 μm in length and 100 nm
dots, which were then released into cells upon penetration.684 diameter were found to be excluded from the cytoplasm as
This was one of the first reports where passive settling of cells observed by confocal imaging.690 TEM images also revealed
onto penetrating nanostructures appeared sufficient for effi- that both the plasma membrane and nuclear envelope resist
cient delivery (Figure 15B). nanowire penetration, and overall DNA transfection efficiency
In 2010 Shalek et al. showcased the multifaceted potential was low in the absence of active forces.688 Using ∼100 nm
of nanowires by demonstrating successful intracellular delivery diameter hollow nanostraws to conduct a time-resolved GFP
of a wide range of materials to various cell types. Functional quenching assay, researchers from the Melosh lab determined
siRNA, plasmid DNA, peptides, proteins, and membrane imper- that only 7 ± 3% of features were penetrant, even in adherent
meable drugs were noncovalently and nonspecifically bound to cells.418 Studies of the mechanism suggest that puncture does
the surface of silicon nanowire arrays, and cells were allowed to not occur upon initial cell contact but requires active cell
settle on top, thus taking advantage of passive penetration. spreading and coincident accumulation of traction forces from
These materials were successfully introduced into a range of focal adhesions.418,678,691 Once penetrant, however, a given nano-
immortalized cell lines and primary cell types, including hard- wire continues to provide sustained intracellular access as long as
to-transfect mammalian neurons.110 Patterning of target the cell remains adherent.418
molecules on the nanostructure arrays was a further advantage The majority of the literature indicates that provision of
of this approach, as it can enabled spatially encoded delivery of active forces is necessary or at least helpful for penetration and
cargo materials.110 Shalek et al.’s nanowire platform was then subsequent cargo delivery. In several studies with hard-to-
adapted for hard-to-transfect primary immune cells.679,686 transfect immune cells, it was found that intracellular delivery
By screening nanowire density and height against different of plasmid DNA, siRNA, and proteins was only possible with
cell types and sizes, optimal parameters were supposedly estab- the addition of g-forces to push cells against vertically aligned
lished for each cell type. Efficient delivery of molecules to pri- nanowires.680,684 This was the case even when the same nano-
mary B cells, dendritic cells, macrophages, natural killer cells, wire architecture was previously successful with common cell
and T cells was reported without the adverse immune responses lines.680,684 This raises the possibility that some cell types, par-
that confound common transfection reagents.679 ticularly those that naturally exist in a nonadherent state, may
Kim et al. also used a nanowire strategy to deliver molecular require active forces to achieve nanowire-mediated intracellular
beacons for the quantitative detection of mRNA.683 In their delivery. Larger penetration forces would also increase the
strategy, ZnO nanowires were incorporated into a PDMS device velocity of impalement upon contact with the cell membrane,
whereby pneumatic pumping provided the force to push cells thereby elevating the chance of membrane disruption events
down onto nanowires. Another group reported the delivery of (Figure 9).
peptide-functionalized DNA nanocages by passive incubation Several strategies have been used to provide active forces for
of cells on 1 μm long 150 nm diameter cargo-coated nanowire nanowire penetration. As mentioned above, one technique is
arrays.685 Other modes of nanowire delivery have been shown to generate g-forces from centrifuging cells onto nanowire
to be capable of intracellular loading of proteins such as Cre arrays.109,676,680 Another method is to sandwich the cells between
AA DOI: 10.1021/acs.chemrev.7b00678
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Chemical Reviews Review
nanowires and an opposing surface. For example, DNA delivery et al. obtained results suggesting that the growth rate of HEK
in hard-to-transfect algae was augmented by using an engineered cells may be stimulated by arrays of nanoneedles.699 Although
PDMS microvalve to press cells against an array of ZnO nano- nanowire induced-perturbations appear minor in most reports,
wires.692 Other strategies have been inspired by cell printing, details of their effects on cell physiology should remain open
whereby jetting velocity upon ejection from the printing nozzle for further investigation.
is directly proportional to penetration force and can be tuned 5.3.4. Nanostraw Arrays for Injection and Extraction.
to balance efficiency of cell impalement versus cell bursting.693 Nanostraws, which are essentially hollow nanowires, can be
Movement of nanoneedles by a piezoelectrically actuated stage used for injection of cargo-laden fluid from an external res-
has also been tested.682 In this case an inverted array of ervoir (Figure 15C). In one of the first examples of nanostraw
nanoneedles was oscillated with an amplitude of 10 μm against delivery, researchers from the Melosh lab fabricated beds of
an immobile monolayer of cells to improve plasmid trans- aluminum nanostraws on polycarbonate track-etched substrates
fection.682 followed by seeding of HeLa cells and CHO cells. By controlling
How large are the forces required for nanowire penetration? the composition and pressure of the fluidic reservoir underneath
Researchers have attempted to address this question with a the nanostraws, temporal control over delivery of dyes and
number of different methods and calculations. Using a model quenching agents was achieved, thus providing direct fluidic
that estimates traction forces associated with long-term cell access to the cell interior.700 In a different study, hollow nano-
adhesion, calculations of 1.5 to 6 nN were obtained for cells straws were fabricated from silicon oxide. Only nanostraws that
cultured on ∼100 nm diameter nanowires.428 In another case, pumped a mixture of membrane-perturbing saponin and cargo
active centrifugation of a grid of diamond nanowires was used were able to introduce fluorescently labeled dextran, indicating
to poke holes in cells for diffusive delivery of cargo from the that nanostraws acted to localize the membrane permeabilizing
extracellular solution.694 They estimated a force of ∼2 nN was effects of saponin and to function as conduits for delivery into
needed to breach the membrane with ∼400 nm diameter nano- cells.701 In an analogous fashion, nanostraws have been reported
wires. Other groups have used AFM to directly quantify the to localize the membrane-perturbing effects of electric 702fields.
forces of penetration for different diameter objects. For exam- Low voltage pulses acted as a gating mechanism to enable access
ple, it was observed that 30−40 nm wide multiwalled CNTs to the cytosol for delivery of membrane impermeable dyes and
had a penetration force of 100−200 pN and required an inden- plasmid DNA.702 A key benefit of hollow nanostraws, as opposed
tation depth of only 100−200 nm.429 Obataya et al. found that to solid nanowires, is the temporal control over delivery, volume,
silicon AFM tips sculpted into thin nanowires of 200−800 nm and dosage concentration.
diameter exhibited penetration forces in the range of 0.65 to In further studies of nanostraw technology, intracellular admin-
1.9 nN when tested on cultured human epidermal cells.430,431 istration of calcium with complex signal patterns, such as
Nanowires of 200 nm were found to breach the plasma mem- oscillations over time,703 and delivery of cell impermeable
brane after ∼1−2 μm indentation and be much more efficient small molecule probes,704 has been achieved. Nanostraws were
at both plasma membrane and nuclear envelope penetration also adapted for cytoplasmic extraction, being capable of con-
compared to pyramidal tips.430,431 As evidence of penetration, tinuous time-resolved sampling from the intracellular space for
a 200 nm diameter nanowire inserted into the nuclei of HEK up to 5 days.705 In another example, ∼6 μm long conical nano-
cells successfully induced expression of attached plasmid DNA.695 straws were employed for delivery of ∼10 nm quantum dots
Another study with larger AFM probe tips estimated that the into single cell microalgal organisms.706 Moreover, Golshadi
forces required to penetrate supported lipid membranes range et al. showed that an array of short, dense nanotubes of 200 nm
from 5 nN for a sharp (<300 nm diameter) nanoneedle probe outer diameter, 140 nm inner diameter, and 180 nm protrusion
to 20 nN for a standard pyramidal tip.432 However, the sup- height were capable of intracellular dye delivery and efficient
ported lipid membranes may be more difficult to break through plasmid transfection in HEK cells.707 Because of the dense
than a cell plasma membrane, depending on approach speed, clustering of these structures, fully adherent cells could cover
temperature, and other factors. One group used antibodies almost 1000 nanotubes.707
attached to nanowires to detect membrane penetration and 5.3.5. Mechanisms of Cargo Delivery by Penetrating
found that lowering temperature to 4 °C appeared to improve Elements. The mechanisms by which nano- and microscale
nanowire penetration by reducing membrane adaptability.427 penetrating elements can deliver molecules into cells are 3-fold:
Together, mechanistic studies indicate that biological mem- injection, dissociation, and permeabilization (Table 4). Micro-
branes under physiological conditions are able to passively injection, mostly featuring tip diameters of ∼0.3−1 μm, is the
adjust to nanowire conformations, and therefore factors such classic example of delivery by injection (see section 5.1).
as small tip area, low temperature, high forces, and/or Advanced versions of microinjection have also been introduced
approach velocities may facilitate effective penetration of the with ∼100 nm diameter tips (nanoinjection610−613) and AFM
plasma membrane. control (FluidFM615). Nanostraws can be considered a highly
5.3.3. Nanowire Effects on Cells. Long-term culture of parallelized adaptation of the microinjection mechanism with
cells on nanowires is not thought to be damaging; however, capability for much higher throughput.418,700−702 However,
there are concerns over unexpected changes in the behavior of some degree of control over the penetration and injection pro-
cells cultured on nanowires.696 Early studies indicated that cess is sacrificed.
nanowires altered the growth rate and cell cycle progression of To date, most of the nanowire systems deliver cargo by dis-
cells.676 Nanowire arrays have also been reported to interfere sociation. These include the original nanowire arrays intro-
with cell division in broblasts and lead to a higher frequency duced by McKnight et al.109,676fi and Shalek et al.110,679 for
of multinuclear cells, an effect that was more pronounced with simultaneous treatment of thousands of cells as discussed
longer nanowires.697 Moreover, when nanowire density increases, above. Single cell versions of nanowire delivery have also been
it may inhibit stable cell adhesion and trigger cells into a more explored. One system attached multiwalled CNTs of 10−20 nm
motile and less proliferative state.698 On the other hand, Bonde diameter and up to 1.5 μm length to AFM tips to deliver
AB DOI: 10.1021/acs.chemrev.7b00678
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Chemical Reviews Review
Table 4. Cargo Delivery Mechanism versus Scale of Throughput for Nano- and Micro-mechanical Membrane Disruption
Techniques−a
aFor injection mechanisms, the nano- or micro-mechanical element is hollow, thus allowing injection of cargo. Dissociation-based delivery works by
enabling cargo to detach from the penetrating element once inside the cell. For permeabilization, the cargo is in the extracellular solution and flows
into the cell by diffusion upon withdrawal of the penetrating element. References for each example are included.
quantum dots to selected single cells.708 Dissociation was information for the future implementation of nanowires and
achieved by the action of intracellular enzymes that cleave the nanostraws.
linker holding the cargo to the penetrating CNT.708 In another
approach, AFM-controlled nanoneedles sculpted by focused 6. INTRACELLULAR DELIVERY BY
ion beams have been shown to provide nuclear penetration PERMEABILIZATION
and mediate gene expression.430,431,695,709 In a non-AFM-based As specified in section 3, permeabilization methods work by
strategy, one group used a ∼500 nm diameter gold nanowire to transiently permeabilizing the cell to cargo in the extracellular
penetrate mouse embryos and release plasmids inside. The solution. Here we will discuss methods for intracellular delivery
plasmids are released through dissociation triggered by an that rely on mechanical, electrical, optical, thermal, and chem-
electric pulse. Because the technique is thought to be less ical means of permeabilizing the plasma membrane. A major
violent, embryo survival was reported to be significantly higher advantage of permeabilization-based delivery is that it is
than traditional microinjection.713 near-universal, being able to deliver almost any material that
Finally, nanowire delivery can also be mediated by perme- can be dispersed in solution. Because most cells can recover
abilization whereby the mechanism involves diffusive influx of from micron-sized membrane disruptions,444 delivery of large
cargo from the extracellular solution. In this case the pene- cargo is also feasible.
trating element is withdrawn from the cell and the influx occurs 6.1. Mechanical Membrane Disruption
before completion of plasma membrane repair (see Figure 6).
Both single cell714 and parallelized694 versions of this Mechanical methods of membrane permeabilization have been
approach have been published. They will be further discussed performed by (1) solid contact of foreign objects with cells
in section 6.1.1 below, which deals with delivery by per- (such as is the case for direct penetration mediated delivery
meabilization. discussed in the previous section), (2) fluid shear forces, and
5.3.6. Nanowire and Nanostraw Summary. In the (3) hydrostatic or osmotic pressure changes. These three
reported nanowire and nanostraw delivery modalities demon- mechanisms of membrane permeabilization are categorized
strated thus far, the cargo material is delivered by (1) disso- and discussed separately below.
ciation from the penetrating structure upon cytosolic entry, 6.1.1. Mechanical: Solid Contact. 6.1.1.1. Scrape andBead Loading. Among the earliest reported mechanical cell
(2) direct injection through hollow nanostraws, or (3) per- permeabilization methods were those published by McNeil
meabilization of the plasma membrane (Table 4). In most and colleagues in the 1980s, which include scrape loading96
cases active forces and/or rapid approach velocities improve and glass bead loading.97 In scrape loading a rubber spatula is
penetration and resultant delivery efficiency. So far, high aspect passed over a cell-laden substrate to dislodge adherent cells
ratio nanowires for intracellular delivery have been successfully and bring them into solution; hence, the technique is only
fabricated out of carbon, diamond, silicon, silicon oxide, zinc applicable to adherent cells (Figure 16A). Moreover, the
oxide, gold, and various other inorganic semiconductors, metals,
696,720−722 amount of damage to each cell is stochastic, with some cellsand metal oxides. Polymer coatings have been sug- being instantly killed while others remain almost unaffected.
gested to improve delivery performance and cell health, for In cells that receive optimal amounts of membrane damage,
example, in the case of siRNA delivery681 and DNA cargo molecules dispersed in solution diffuse through transient
transfection.723 The physiological effect of exposing nanowire membrane disruptions to achieve delivery.
materials to the intracellular space will be essential knowledge Glass bead loading involves shaking the adherent cells with
if nanowires are to proceed toward biomedical applications. medium containing glass beads and the cargo to be delivered
Furthermore, open questions remain regarding the membrane (Figure 16B). The impact of collisions between beads and cells
conformation adopted around nanowires and the subsequent imparts sufficient strain to generate disruptions in the plasma
degree of penetration. Understanding the effect of nanowire membrane. Again, the magnitude of plasma membrane damage
dimensions and density, the requirement of active forces, sur- that each cell sustains is highly variable and may lead to
face functionalization and chemistry, as well as the influence of inconsistent delivery and cell survival. The generation of
culture conditions, cell properties, and cell type will be key cellular and biological debris may be another problematic
AC DOI: 10.1021/acs.chemrev.7b00678
Chem. Rev. XXXX, XXX, XXX−XXX
Chemical Reviews Review
Figure 16. Mechanical membrane permeabilization by direct contact. (A) Scrape loading, where a rubber spatula or similar scraping object can be used
to simultaneously dislodge cells and permeabilize them. (B) Bead loading, wherein micron-scale beads can be rolled across a cell monolayer for
controlled cell injury via collisions. (C) Filtroporation, where a solution of cells is passed through holes in filter membranes, such as a track-etched
polycarbonate filter. (D) Microfluidic cell squeezing, where cell membranes are disrupted by rapid deformations in cell shape that occur with passage
through microfabricated constrictions. (E) Permeabilization with nanoneedle/nanowire arrays. (i) The array is first centrifuged or otherwise pressed
against cells adhered to a rigid substrate. (ii) The array is then removed to enable cargo influx through membrane disruptions in the target cells.
aspect of cell scraping and bead loading. Moreover, delivery of collectively generated by the fibroblasts then trigger compac-
expensive reagents that need to be concentrated into small tion of the collagen matrix into a dense body one tenth of its
volumes can be di cult to achieve with these protocols. On original size.767ffi This process induces plasma membrane
the other hand, potential benefits include the low-cost and disruptions in the contracting fibroblasts. Membrane perme-
accessible nature, as these methods can be performed with abilization is thought to be due to the tearing of focal adhesion
common lab equipment. In applications where high cell sites associated with rapid cell shape change and compression
viability is not a priority, scraping and bead loading may of the collagen matrix.576,766 The lesions are resealed in a Ca2+-
represent convenient options. A later adaption of bead loading dependent fashion, with the fibroblasts reported to be imper-
termed “immunoporation” used beads functionalized with meable to uptake several seconds after return to standard physi-
antibodies to bind to cells and permeabilize them by ripping ological media.576,766 Fibroblasts that detach from their
off bits of their membranes.724−730 substrates to round up in mitosis also exhibit permeability to
Bead and scrape loading techniques have been used to dextrans up to 150 kDa, peptides, proteins, or oligonucleo-
deliver a variety of cargoes into cells. Bead loading has been tides.768 This observation is in congruence with other studies
used to deliver dye-conjugated dUTP for fluorescent visual- that have observed plasma membrane damage and dye uptake
ization of chromosome formation,731 antibody loading into macro- during mitotic cell rounding.769,770
phages732,733 and fibroblasts,734 intracellular delivery of pro- In what could be a related phenomenom, permeabilization has
teins,735−737 peptides,738 fab fragments,739,740 peptide nucleic been observed when attached fibroblasts are treated with strong
acid probes,741 SNAP-reactive dyes,742 CNTs,743 and quantum doses of the proteases trypsin, Pronase, or collagenase.771,772
dots up to 15 nm in several cell lines.744 Scrape loading has Cytoplasmic delivery of the proteins insulin (6 kDa), lysozyme
achieved intracellular delivery of proteins,96,543,745−751 anti- (14 kDa), BSA (76 kDa), and thyroglobulin (660 kDa) was
bodies,752−754 peptides,755,756 morpholinos,757 high molecular achieved with this simple treatment. Although the mechanisms
weight dextrans,96,758 lipopolysaccharides,759 dyes,760,761 were not investigated, cells presumably become permeable as
pH-sensitive probes,762 and transfection of plasmids.545 they detach from the substrate.766 Indeed, membrane ripping
A variant of the scrape loading technique is scratch loading.763 has previously been observed when certain cell types move
Also introduced by McNeil, it involves dragging a needle or other across or detach from surfaces.766 However, intracellular delivery
kind of sharp object across a layer of adherent cultured cells. of proteins by protease permeabilization has been reported for
The cells that brush the edge of the needle undergo membrane both adherent773 and nonadherent cell types.774 If protease-
damage but remain adherent to the substrate. Intracellular mediated permeabilization is not due to membrane ripping
delivery of dextrans,763 dyes,764 fluorescently labeled nucleo- during detachment, it could be that cells are permeabilized
tides,765 and quantum dots744 has been achieved in cells adjacent through the action of the proteases themselves. Trypsin can
to the scratch zone. Although the method has lower throughput trigger signaling events that culminate in vigorous contractile
than scrape loading, one advantage of scratch loading is that cells activity at the cell surface and loss of coherence between the
remain adherent for immediate analysis by imaging. cortex and plasma membrane.775 Such events could potentially
6.1.2. Solid Contact: Sudden Cell Shape Changes & induce transient plasma membrane disruptions. Thus, further
Protease Treatments. Sudden contraction of cells studies may be needed to identify the mechanisms of membrane
from an adherent, elongated shape to a rounded shape has the disruption by rapid cell shape changes and the action of proteases
potential to generate membrane disruptions. Grinnell and as well as whether these phenomena can be made more widely
colleagues found that the sudden contraction involved in the useful for intracellular delivery.
fibroblast-driven collapse of collagen matrices is able to induce 6.1.3. Solid Contact: Projectile Permeabilization.
permeabilization and uptake of dextrans up to 150 kDa in Sautter et al. pioneered a variation of the biolistic approach
size.766,767 In this approach, broblast-colonized collagen that retains free DNA in solution.776fi It is distinct from the
matrices that are stabilized by substrate attachment are peeled projectile bombardment methods covered in section 5.2 in that
away from their support. The isometric contractile forces the particles are used to permeabilize the cells rather than carry
AD DOI: 10.1021/acs.chemrev.7b00678
Chem. Rev. XXXX, XXX, XXX−XXX
Chemical Reviews Review
Figure 17. Different variations of cell squeezing for intracellular delivery by mechanical membrane permeabilization. (A) The original microfluidic
platform for cell squeezing.108 The rapid deformation a cell experiences upon passage through a constriction transiently permeabilizes the plasma
membrane, allowing influx of macromolecules into the cytosol. Reprinted with permission from ref 108. Copyright 2013 PNAS. (B) Similar to cell
squeezing in panel A but with addition of a downstream electric field. The electric field enhances delivery of large nucleic acids, such as plasmid
DNA, into the cell by electrophoretic forces. In this case the device was optimized for delivery of plasmids into the cell nucleus at high
throughput.787 Panel (i) shows the delivery concept. Panel (ii) shows the close-up architecture of the constriction and electrode zones on the chip.
Panel (iii) shows a view of the whole chip including holes (white) for inlet (left) and outlet (right). Scale bar: 1 mm. Reprinted with permission
from ref 787. Copyright 2017 Springer Nature. (C) Cell squeezing with different constriction geometries in a PDMS device. (i) Comparison of 45°
pyramidal pattern, 90° sawtooth pattern, and 135° reverse wishbone pattern of repeated constrictions. (ii) COMSOL modeling indicates the stress
(N m−2) that the cell membrane would undergo upon passage through the different shapes of constrictions. Experiments and modeling show the
reverse wishbone pattern as the most effective for localized membrane disruption in this device.788 Figure reprinted from ref 788. Copyright 2017,
with permission from John Wiley and Sons.
cargo. Projectiles are accelerated toward target cells in a Bernoulli optimal conditions of 8 μm pore size and driving pressure of
air stream as a fine mist of droplets. The projectile particles create 35 kPa were identified in ∼13 μm CHO cells. Thus, the cells
membrane disruptions that allow influx of plasmid DNA dispersed experienced a ∼40% constriction of their diameter as they passed
within the droplets. This stream of droplets can be targeted toward through the polycarbonate filter. These conditions permitted
150 μm areas of cells or tissue for localized targeting with dynamic uptake of a luciferase reporter plasmid, which resulted in
adjustment of particle density and velocity. transfection of the cells with a stated transfection efficiency
6.1.4. Solid Contact: Filtroporation. In 1999 a above 50% after 2 days in culture. Despite these results, further
constriction-based method for generating disruptions in the work on filtroporation is absent from the literature as the
plasma membrane was reported.777 The technique, termed technique does not appear to have gained traction.
“filtroporation”, works by forcing cell suspensions through 6.1.5. Solid Contact: Microfluidic Cell Squeezing.
uniformly sized micropores in commercially available track- Microfluidic and lab-on-chip methods of plasma membrane
etched polycarbonate filters (Figure 16C). In the reported perturbation offer the opportunity for precise control of the
study, a polycarbonate lter of approximately 12 μm thick with membrane disruption process.19,104,106,107fi In 2013, Sharei and
pore sizes ranging from 5−18 μm was used. Plasmid DNA colleagues reported on the development of a microfluidic
and dextran-conjugates up to 500 kDa were successfully delivered platform for intracellular delivery by rapid cell deformation (or
into CHO cells of nominal diameter ∼13 ± 2 μm. The cell squeezing) through channel constrictions (Figure 16D). This
suspensions were driven through the polycarbonate filter by a innovative method has demonstrated delivery of diverse macro-
pressure regulator supplying constant pressures of 0−175 kPa. molecular materials into a wide range of cell types.108,308,778 The
Delivery efficiency and cell damage were both increased as a delivery mechanism is via diffusion of macromolecular cargo
function of driving pressure. Severity of the treatment also through membrane disruptions generated by rapid deforma-
increased as the micropore diameter was decreased when all tions of cell shape (Figure 17A). The device is comprised of
other parameters were held constant. By tuning parameters, parallel constrictions generated by deep reactive ion etching in
AE DOI: 10.1021/acs.chemrev.7b00678
Chem. Rev. XXXX, XXX, XXX−XXX
Chemical Reviews Review
silicon wafers, followed by bonding to pyrex glass and drilling generally quite low in many cell types. Ding and co-workers
holes for inlet and outlets. Gas pressures of 10−100 kPa are explored the idea of adding a downstream electric field to
then used to drive cell suspensions through constrictions of investigate whether it could improve DNA transfection results
4−8 μm width, 10−50 μm length, and 20 μm channel depth. (Figure 17B).787 The strategy, termed “disruption and field
The ability to engineer angle of entry and repeated con- enhancement” (DFE), was compared with standard cell squeezing,
strictions is also possible. In the first published study, the bona microfluidic flow-based electroporation, commercial electro-
fide cytoplasmic delivery of unaggregated quantum dots was poration (Neon−Thermo Fisher), microinjection directly to
demonstrated in HeLa cells.308 Then a wider range of cell the nucleus, and lipofection.787 In HeLa cells, DFE was able to
types was screened to showcase efficacy with primary blood achieve similar transfection efficiencies as lipofection and com-
derived immune cells (T cells, B cells, and macrophages), pri- mercial electroporation. Surprisingly, plasmid expression
mary dendritic cells, embryonic stem cells, and primary fibro- approached its maximum within 1−2 hours of treatment,
blasts, as well as a panel of immortalized cell lines.108 Efficient which was also the case with microinjection. This contrasts with
cytosolic delivery of siRNA, carbon nanotubes, quantum dots, the delayed onset of expression after lipofection and standard
antibodies, transcription factors, and dextran-conjugated dyes electroporation, which can take 24 hours or longer due to the
was observed in many of these cell types. requirement of endocytosis and other intracellular trafficking
A major strength of cell squeezing is the simplicity of the processes to deliver DNA to the nucleus.787 Fixation and
approach: no moving parts or external power supply are imaging of cells directly after treatment indicated that DFE,
required, simply a pressure source and controller to modulate flow like microinjection, could deliver plasmids directly into the
rate. Weaknesses include cell type and size dependence for a par- nucleus for immediate expression. To determine whether DFE
ticular device geometry, and the potentially narrow range of was permeabilizing the nuclear envelope to permit DNA uptake,
flow rates required to achieve optimal balance between delivery a HeLa cell line expressing the protein CHMP4B−GFP was
and viability. However, a variety of constriction geometries imaged with confocal microscopy. CHMP4B is a component
have been developed to address a broad range of cell types. of the ESCRT-III complex, recently discovered to be involved
Furthermore, experiments with different buffer compositions in the repair of both plasma membrane and nuclear envelope
(e.g., Ca2+ concentration) indicate that it can successfully be disruptions.454,789−791 While squeezing and standard electro-
tuned to optimize membrane recovery kinetics and cell poration only permeabilized the plasma membrane, DFE was
survival.779 In line with what is known from the cell biology found to also generate disruptions in the nuclear envelope.
of membrane repair (see section 4.3), it was observed that After treatment, nuclear envelope repair appeared to be com-
buffers with calcium promoted rapid (∼30 s) closure of pleted within ∼15 min, in agreement with previous stud-
membrane wounds while no calcium conditions allowed the ies.789,790 It was speculated that, by first disrupting the plasma
membrane to remain open for several minutes.779 By modu- membrane, subsequent exposure to the electric field was able
lating treatment parameters as well as temperature, a further to electroporate the nucleus. Indeed, specific types of electro-
demonstration of immune cell engineering with siRNA, poration have previously been found to selectively permeabi-
antibodies, and proteins was shown in T cells, B cells, lize intracellular compartments (reviewed in ref 579). DFE
dendritic cells, and monocytes/macrophages at throughputs of thus represents a useful strategy for high throughput nuclear
millions of cells per second.780,781 These results suggest cell delivery and rapid expression of DNA.787 Further work should
squeezing is a promising path toward engineering cell function clarify the exact mechanisms of cargo influx upon two-step
for immune cell therapy at high throughput. mechanical/electrical hybrid treatments such as DFE.
The cell squeezing platform has been used for protein 6.1.5.2. Variations on Microfluidic Cell Squeezing and Cell
delivery to primary mammalian plasmacytoid dendritic cells Deformation Strategies. In 2015 the Qin lab introduced micro-
with a device consisting of 10 μm long and 4 μm wide con- fluidic intracellular delivery devices featuring various types of
strictions repeated 5 times in series.782 Zoldan and colleagues PDMS-based microconstrictions.792 Until this point, most
employed microfluidic cell squeezing to perform high through- results had been obtained in microfabricated silicon devices.108
put delivery of fluorescently labeled tRNAs into multiple By using repeated arrays of constrictions fabricated from
myeloma cells with a transfection e ciency of ∼45%.783ffi PDMS, Qin and co-workers reported delivery of single-stranded
Delivery of fluorescently labeled tRNAs enabled the monitoring DNA, siRNA, and plasmids into HEK cells and several other
of protein synthesis inside cells in real time.783 Delivery of cell lines.792 Moreover, they demonstrated genome editing in
otherwise impermeable JAK inhibitors into human primary MCF7 and HeLa cells via delivery of plasmids that express
cells was achieved by squeezing cells through constrictions of Cas9 and gRNA, although transfection efficiencies were not
10 μm long and 4 μm wide.784 Intracellular delivery of small directly reported.792 In a subsequent study, the group modified
fluorescent tags for protein labeling and subsequent live cell their device architecture to perform siRNA delivery to cancer
imaging has also been demonstrated by cell squeezing.785 cells with a repeated pattern of 5 μm constrictions in a reverse
Because of the sensitivity of cells to constriction size, it was wishbone configuration788 (Figure 17C). Experiments and sim-
tested whether the squeeze platform could exploit size dif- ulations both indicate that the sharper constrictions conferred
ferences of cells to facilitate selective intracellular delivery.786 by the reverse wishbone intensified the local stress on the
As a proof of concept, Saung et al. showed that the system is plasma membrane to increase the magnitude of membrane dis-
able to selectively deliver molecules to pancreatic cancer cells ruption.788 Another of this group’s publications featured sharp
within a heterogeneous mixture containing T cells.786 One future star-shaped constrictions to facilitate delivery of dextrans,
application of this concept would be the selective tagging of siRNA, and Cas9 RNPs to the intracellular space of hard-to-
CTCs or other abnormally large cells in the blood.786 transfect suspension cell lines and T cells.152 By delivering
6.1.5.1. Electric Field-Enhanced Microfluidic Cell Squeez- RNPs targeting GFP, they were able to achieve CRISPR-medi-
ing. Like most other mechanical membrane disruption tech- ated GFP knockout in several standard cell lines. Also demon-
niques, DNA transfection efficiencies upon cell squeezing are strated was low-efficiency CRISPR-mediated knock-in editing
AF DOI: 10.1021/acs.chemrev.7b00678
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Chemical Reviews Review
of the PD-1 gene in primary T cells, an application that could constrictions (unpublished observations). However, further
be relevant for cell-based therapies.152 investigations with different types of cells (particularly those of
So far, the results on cell squeezing indicate that the rapid clinical relevance) may be required to fully address the ques-
deformation of cells in suspension is able to create holes in the tion of off-target DNA damage.
plasma membrane in a relatively well-controlled and repro- 6.1.6. Solid Contact: Nanowires for Transient Per-
ducible manner. In an extension of this concept, it is possible meabilization. Arrays of sharp nanowires have been used to
to asymmetrically deform cells by flowing them past an abrasive permeabilize cells by transiently piercing their plasma
object positioned on one side of a microfluidic channel. Such a membranes. In these cases, nanowires are thrusted into the
strategy would presumably disrupt the plasma membrane in a cells followed by withdrawal to promote diffusive influx from the
more localized manner, preferentially permeabilizing one side surrounding media (Figure 16E). This mode of plasma
of the cell. To explore this idea, the Qin lab introduced a membrane penetration is similar to the nanowires/nanostraws
device with sharp silicon nanoblades protruding from one side described in section 5.3, except that the delivery mechanism is
of PDMS microfluidic channels.793 The protruding edge of the via diffusion through a permeabilized plasma membrane rather
silicon nanoblade essentially formed a spike of ∼200 nm than dissociation from the nanowires themselves. In one
radius, creating a gap of ∼2 μm for cell passage. By optimizing notable example, a grid of diamond nanowires was centrifuged
the flow rate and number of nanoblade constrictions, they onto cultured cells at controlled forces using standard lab
achieved ∼70% delivery efficiency of 70 kDa dextan with centrifuges.694 Thin diamond nanowires were fabricated by
∼80% cell viability in hard-to-transfect HSCs.793 Compared to first depositing a nanodiamond film on silicon wafers followed
electroporation, the delivery efficiency was the same; however, by microwave plasma chemical vapor deposition to grow a
survival and ability of HSCs to remain pluripotent were uniform field of needles. In the versions used for experiments,
claimed to be superior with the nanoblade device. Cas9 RNPs dimensions were optimized to ∼300 nm diameter and ∼4.5 μm
were successfully delivered into HSCs, but the actual gene height with straight sidewalls at a density of ∼6 nanowires per
editing efficiencies as a percentage of total cells treated were 10 × 10 μm2. It was found that nanowires of diameter >800 nm
not reported.793 caused excessive damage to cells, but those <400 nm produced
Another method of microfluidically controlled cell deforma- a suitable balance between delivery efficiency and cell damage.
tion is to flow cells through a T-junction and cause them For this geometry, it was calculated that centrifugation at
to collide with the channel wall.794 A recent report from 300 r.p.m. yields ∼2 nN penetration force per nanowire, which
Deng et al. showed that cells can be propelled into a spike-like was claimed to be an ideal penetration force for monolayers of
structure protruding from the channel wall to permeabilize cells grown in culture. Upon withdrawal of nanowires from
them at a throughput of a million cells per minute.794 As with cells, influx of IgG antibodies, ∼20 nm quantum dots, and
cell squeezing, the extent of permeabilization is proportional to ∼200 nm polystyrene nanoparticles into the cytoplasm of
the flow speed of the cells.794 Using this strategy, intracellular primary neurons was demonstrated. Furthermore, by pack-
delivery of dextrans, Cas9 RNPs, siRNA, plasmid DNA, and aging DNA with lipid-based lipofectamine complexes, plasmid
DNA nanostructures was demonstrated at efficiencies around transfection in neurons was boosted from around 1−5%
50% in several cells lines.794 (lipofectamine alone) to almost 50% with additional nanowire
6.1.5.3. Potential Off-Target Effects of Cell Squeezing. Cell permeabilization. If nanowire permeabilization were used with
squeezing strategies often rely on significant cell deformations, naked DNA alone, transfection efficiency was <1%, suggesting
sometimes up to 70% of the cell diameter. An unresolved issue that (1) centrifuged nanowires did not consistently permea-
is whether off-target damage may be inflicted upon intracellular bilize the nucleus and (2) lipid complexes may facilitate nuclear
structures, such as the cytoskeleton, nucleus, and even genomic targeting and protect the DNA from premature degradation.
DNA. For example, it has been observed that cells migrating Thus, direct cytosolic delivery of DNA−lipid complexes may
through tight constrictions undergo transient nuclear ruptures boost transfection efficiency in otherwise difficult-to-transfect
and DNA damage.789 As the stiffest large object in the cell, the cells such as neurons.
nucleus is widely regarded as the determining factor governing Several other groups have also used arrays of nanowires to
passage of cells through micron-sized constrictions.795,796 It has permeabilize cells for intracellular delivery. In one case arrays
also been observed that apoptotic and cell stress response can of silicon lances were pressed against cell monolayers with a
significantly impact cell survival after passage of cells through compliant suspension system in lieu of centrifugation.719 The
constrictions.797 Lamins, which mechanically reinforce the silicon lances were larger than typical nanowires, with lengths
nuclear envelope, play a protective role in physically buffering of 8 μm, diameters around 0.5−1.0 μm, and sharpened tips.
the nucleus from mechanical stress and their depletion is Although this setup yielded diffusion-based intracellular deliv-
known to make cells more vulnerable to death after passage ery of propidium iodide, delivery of larger molecules of bio-
through constrictions.797 Moreover, DNA damage has pre- logical interest was not tested.719 Matsumoto produced nano-
viously been observed with imposed cyclic mechanical stresses wire arrays of 25 μm length and 200 nm diameter.800 They
in certain cell types.798 Experiments from Ding and colleagues were attached to a piezoelectric actuator stage and lowered
that visualized nuclear disruptions with CHMP4B-GFP onto cell monolayers before being vertically oscillated at a
indicated that squeezing HeLa cells (nuclear diameter ∼8− frequency of 5 kHz and an amplitude of ∼0.5 μm for up to
12 μm) through 7 μm constrictions did not disrupt the nuclear 2 min.800 Continuous delivery of molecules from solution
envelope.787 Because disruption of the nuclear envelope can be appeared to be augmented by the agitation associated with
associated with DNA damage, it indicates genomic DNA may nanowire oscillation. Up to 50% of cells retained detectable
be safe even when cells are squeezed by more than 50% of their levels of 70 kDa dextran after treatment. Efficiency of plasmid
initial diameter. Moreover, measurements of DNA damage transfection, however, was only ∼7%, which was less than the
with a high throughput COMET assay799 showed no signifi- 18% achieved when plasmids are directly attached to nano-
cant DNA damage in HeLa cells forced through 6 μm wires.682 Interestingly, the above-mentioned examples of
AG DOI: 10.1021/acs.chemrev.7b00678
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Chemical Reviews Review
Figure 18. Mechanical membrane permeabilization by fluid shear forces. (A) Syringe loading, where a cell solution is repeatedly aspirated and
ejected through the terminal aperture of a syringe needle. Shear forces at the nozzle promote cell membrane disruption. The inset illustrates cell
deformation associated with shear forces. (B) Microfluidic shear-based permeabilization. Similar to syringe loading but exploiting the increase of
shear forces associated with flow through narrowing microfluidic channels. The inset illustrates cell deformation upon flow through a single
constriction. (C) Cone−plate viscometer. Generation of permeabilizing shear forces via rotation of a viscometer plate above a monolayer of cells.
(D) Generation of local shear forces via collapse of a cavitation bubble. (E) Generation of local shear forces via oscillation of a cavitation bubble.
(F) Induction of cavitation bubbles on the basal side of a cell through a seed structure that absorbs laser energy. The cavitation bubble can produce
a large hole in the plasma membrane that allows influx from a separate fluid reservoir underneath the cell.
nanowire permeabilization are essentially scaled-up versions of a sufficiently rapid velocity, it can tilt the lipid heads in the
single cell permeabilization previously performed with sharp- direction of the shear and lead to buckling instabilities that
ened AFM tips. In 2006, a method introduced by Hara et al. eventuate in bilayer rupture.801 Alternatively, a jet of water
demonstrated stab and withdraw permeabilization by using molecules propelled perpendicularly into a membrane can
AFM tips that had been sharpened by focused ion beam pierce it in an analogous way to a mechanical object.802 Unlike
technology.714 Expression of plasmid DNA that flowed into membrane disruption via solid contact (discussed above), fluid
cells from the culture media was achieved with serial pen- shear forces are less invasive. On the flipside, fluid shear forces
etrations of sharpened tips into HeLa cell nuclei using a in aqueous environments tend to be more difficult to control.
computer controlled device called the “CellBee”.714 In this section we discuss the strategies and methods that have
6.1.7. Solid Contact: Summary. Classic methods of been used to perform membrane disruption-based intracellular
mechanical contact-mediated permeabilization such as scrape delivery by harnessing fluid shear forces. First, we will explore
and bead loading provide low-cost, accessible, and crude shear forces generated by flow of fluid past microscale channels
solutions for delivery of certain cargoes, especially proteins, and objects. Second, acoustic sonoporation, which is thought
small molecules, and oligonucleotides. However, delivery to depend mainly on the forces associated with cavitation
efficiency and cell survival may not be sufficient for certain bubbles, will be discussed. Third, we will cover laser-induced
applications, particularly in sensitive cell types. Recent progress cavitation as a strategy for generating highly localized and
in solid contact-mediated mechanical membrane disruption intense zones of fluid shear.
takes advantage of the increased precision afforded by MEMS, 6.1.9. Fluid Shear: Syringe Loading. One of the simplest
microfluidics, lab-on-chip, and nanotechnology capabilities to approaches for generating zones of high fluid shear force is to
finely control the level of cell injury.19,104−107 Prominent drive a liquid through tight constrictions. In 1992 McNeil and
examples include microfluidic constrictions for squeezing of colleagues introduced an intracellular delivery method called
cells in suspension108,787 and nanowires to transiently per- syringe loading, where cell suspensions mixed with high
meabilize adherent cell monolayers for high throughput intra- concentrations of a cargo to be loaded are repeatedly aspirated
cellular delivery.694 and expelled through fine-gauge syringe needles to transiently
6.1.8. Fluid Shear-Mediated Permeabilization. Lipid permeabilize cells (Figure 18A).98 The flow rate is a critical
bilayers can be disrupted by fluid shear forces in a number of parameter as it determines the velocity of the cells traversing
ways. If water molecules flow parallel to a membrane surface at the constriction zone and thus the shear forces they experience.
AH DOI: 10.1021/acs.chemrev.7b00678
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Chemical Reviews Review
A typical syringe loading protocol consists of eight passes of cell simplex virus replication, herpes virus and nucleoporin anti-
suspension through a 1 mL syringe affixed with a 30 G needle, bodies were introduced into Vero cells by 50 passages through
which has an inner diameter of 160 μm.98 In the initial publi- a 27 gauge needle.819
cation, delivery of cargo sizes up to 150 kDa were obtained in 6.1.10. Fluid Shear: Microfluidic Control of Shear
several mammalian cell lines.98 Furthermore, the addition of Forces. Syringe loading presumably works by creating regions
pluronic F-68 (also known as poloxamer 188) was found to of significant shear force around the entrance and exit of the
increase the tolerance of cells to membrane permeabilizing syringe needle (Figure 18A). Because the fluid flow is con-
shear forces, thereby enabling the cells to undergo harsher trolled manually, however, it may require extensive empirical
treatments and improve cell survival. In the cell types tested, testing and skill to reproducibly obtain optimal cell treat-
syringe loading in the presence of pluronic F-68 appeared ments.820 Improved precision and reproducibility could
more e cient than both bead and scrape loading.98ffi Low- potentially be achieved by using microfluidic devices to
volume versions of the protocol were also developed, using a generate consistent zones of fluid shear. Along these lines,
25 μL Hamilton syringe with 25 G fixed needle (inner Prausnitz and colleagues fabricated a simple flow-through
diameter 260 μm) for 80 passes. A 5 μL version of the protocol microfluidic device with parallel constrictions821 (Figure 18B).
was described with a 10 μL micropipette tip (inner diameter Lasers were used to bore out 50−300 μm conical micro-
not reported) involving 60 passes. channels from 100 to 250 μm thick mylar sheets, and syringe
In subsequent reports, syringe loading has demonstrated pumps were employed to flow cell suspensions through the
utility in a variety of delivery applications, mostly to conduct channels at controlled flow rates, thereby subjecting cells to well-
studies in basic biology. In one example, it has been used to defined shear forces. The resultant loading of fluorescently
perform DNA transfection.803 Using a selection strategy, stable labeled dextrans and proteins into DU145 prostate cancer cells,
integration of plasmid DNA into the genome of host CHO and as well as the viability, however, turned out to be less favorable
mouse Ltk(−) cells was estimated in approximately one of every than syringe loading. Further attempts toward plasma
50 000 cells treated, which was considered a success given the membrane permeabilization through microfluidic control of
low cost of the technique.803 Ghosh and colleagues found that shear forces have not been reported and therefore present an
syringe loading could deliver neutrally charged antisense phos- opportunity for future investigations.
phorodiamidate morpholinos into cells for the purpose of gene 6.1.11. Fluid Shear: Other Examples of Cell Perme-
silencing.804 Moreover, the same delivery strategy has been abilization Through Shear Forces. Driving fluid through
used for loading of small molecular weight nucleotides, GTP narrow constrictions is not the only way to generate fluid shear
and GDP (∼0.5 kDa), and their analogues to explore G-protein forces for cell permeabilization. Indeed, researchers have used
biology in immune cells and endothelial cells.805,806 In another cone−plate viscometers to produce hydrodynamic shear forces
application, fluorescent labeling of the neuronal cytosol was above cell monolayers, obtaining uptake of fluorescent mole-
achieved when trypsinized ganglia were syringe-loaded with cules in neuronal and endothelial cultures (Figure 18C).822,823
dye-conjugated 10 kDa dextrans.807 In 1997, LaPlaca and colleagues confirmed permeabilization of
The most common application of syringe loading has been neurons by observing an increase in intracellular Ca2+, release
delivery of proteins and antibodies to the intracellular space. of intracellular enzymes to the extracellular solution, and cell
GST-FAK fusion proteins were loaded into fibroblasts by swelling.822 Later, Blackman and colleagues used a modified
passing them through a 30 gauge syringe needle 30 times.808 cone−plate setup to expose endothelial cell monolayers to
HEp-2 cells were loaded with monoclonal antibodies by consistent fluid shear forces.823 When forces were too high,
20 cycles through a 27 gauge needle.809 A modified version of cells peeled away from the substrate. Upon empirical optimi-
the protocol was employed by Sydor et al. to deliver fluo- zation, however, conditions were identified where all cells
rescently labeled antibodies into trypsinized neurons by using remained attached to the substrate yet 16% of cells retained
∼100 cycles of aspiration-expulsion though pipette tips.810 For 4kDa Dextrans.823 The Blackman cone−plate viscometer was
delivery of monoclonal antibodies to fibroblasts, a mixture of then used to permeabilize cultured neurons, investigate their
cells and antibodies was cycled 20 times through a 30 gauge physiological response, and test strategies to improve post-
needle.811 Kasier et al. syringe loaded a fluorescently labeled injury neuron survival.824 Relative permeabilization efficiency
version of the protein profilin into amoebas and human cells to was analyzed by influx of small molecular weight fluorescent
study its binding to intracellular actin.812 In other studies of dyes.824
the actin cytoskeleton, FITC-conjugated antifascin immuno- Intense pulses of fluid shear can be directed at cells by firing
globulins were delivered into ∼95% of fibroblasts or myoblasts jets of pressurized inert gas toward them.825,826 Similar to the
by four passages through a 1 mL syringe fitted with a 25 gauge case of cone−plate viscometers, it was found that excessive
needle.813 Researchers from the Schwartz lab loaded endo- shear forces can rip cells away from the underlying substrate,
thelial cells with alexa-labeled versions of the p21 binding but if modulated just below this range, they were capable of
domain of PAK1 to investigate mechanobiology of the Rac1 permeabilizing cell membranes while leaving adherent cells in
pathway.814,815 Several studies have also employed syringe place. With the appropriate optimizations, intracellular delivery
loading to study the effect of bacterial and viral proteins inside of dextrans, plasmids, and other cargo has been demonstrated
cells. For example, fibroblasts were syringe-loaded with HIV in common adherent cell lines permeabilized by inert gas
proteins to examine their impact on intracellular and nuclear jets.825,826
architecture.816 In another case, CHO cells were drawn up and 6.1.12. Fluid Shear: Sonoporation. Sonoporation is the
expelled slowly (∼0.2 mL s−1) through a 30 gauge syringe disruption of cell membranes by acoustic pressure waves,
needle six times for intracellular delivery of the bacterial toxin mostly in the ultrasound frequency range (20 kHz to GHz). Its
ExoU.817 Moreover, Xu et al. delivered the Legionella deployment for intracellular delivery purposes first arose in the
pneumophila protein SidK into macrophages by 100 cycles of mid 1980s through the use of ultrasound to permeabilize
pipetting through a 200 μL pipette tip.818 In studies of herpes cultured cells.545,827−829 Permeabilization was achieved by
AI DOI: 10.1021/acs.chemrev.7b00678
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placing cell suspensions in a plastic tube and applying three contrast agents. A bubble that expands and contracts in
half-second pulses of the ultrasonic transducer directly to the synchrony with the acoustic field (stable cavitation) generates
tube. With this rudimentary approach, Fechheimer et al. local oscillatory shear forces due to microstreaming.848,849 The
demonstrated intracellular loading of dextrans and proteins microstreaming forces are sufficiently potent to permeabilize
into Amoebae.545,827,829 Ultrasound-mediated permeabilization nearby cells. On the other hand, a bubble that implodes
was compared head-to-head with scrape loading.545 However, (inertial cavitation) can trigger extreme phenomena including
scrape loading was found to yield superior delivery of dextran- electromagnetic radiation (sonoluminescence), severe temper-
conjugated dyes and DNA plasmids into HeLa cells, hepatic ature spikes up to thousands of degrees, sonochemical reac-
tissue cultures, and mammalian fibroblasts.545 tions such as the production of free radicals, and intense micro-
About a decade later, sonoporation began to be taken jetting. Although any of those phenomena can perturb lipid
seriously as a method for DNA transfection.830−832 Several bilayers, the permeabilizing effects of bubble collapse have
factors converged to motivate this trend.833 First, high intensity primarily been ascribed to the potent fluid shear forces
focused ultrasound was gaining prominence as a noninvasive generated by microjetting.840,842,846 As a cavitation bubble
method for therapeutic treatment of targeted cells and tissues implodes, surrounding water molecules rush in to fill the void.
in vivo.834,835 Examples include local tissue ablation, local drug If there is a surface nearby (such as a lipid membrane) less
delivery stimulated by ultrasound, and gene therapy by tar- water molecules are available to flow from that region. This
geted nucleic acid transfection.836 Second, the mechanisms of biases the flow toward that surface and results in the microjet
ultrasonic effects were being increasingly clarified, with cavi- being oriented in that direction. Thus, imploding cavitation
tation bubbles implicated as the prime instigators of membrane bubbles can result in the selective puncture of an adjacent cell
disruption.837 These mechanistic insights enabled a more (Figure 17D). High pressure ultrasound is more likely to
rational approach toward sonoporation that greatly boosted its trigger inertial cavitation while low pressure protocols bias the
efficiency. Particularly key was the deployment of gas body system toward stable cavitation.842
ultrasound contrast agents to act as cavitation nuclei. This 6.1.12.2. Cargo Delivered by Sonoporation. Because of the
modification was found to drastically improve transfection variation in magnitude and mode of fluid shear phenomena
efficiency compared to ultrasound alone.831,838 For example, related to sonoporation, it is perhaps not surprising that the
commercially available microbubbles were mixed with cultured resultant holes have been reported to range from nanometers
immortalized human chondrocytes and exposed to 1.0 MHz up to several microns.840,842,846,850−852 Under conditions
ultrasound transmitted through the bottom of a six well culture where large holes are generated, sonoporation can be expected
plate. The addition of microbubble cavitation nuclei, along to enable delivery of small and large cargoes alike. Because of
with other empirical optimizations, enhanced DNA trans- the motivation for gene therapy, significant efforts have gone
fection nearly 20-fold over previous reports and indicated that into optimizing sonoporation for DNA transfection over the
ultrasound could be a feasible DNA transfection technique.838 last two decades.830,831,833,838,853−859 Transfection of other
6.1.12.1. Mechanisms of Sonoporation. As the field nucleic acids, such as antisense oligonucleotides,860
currently stands, hundreds of studies have been published on siRNA,861,862 and mRNA,863 have received less attention but
the subject of understanding and improving sonoporation. have also been demonstrated. To study mechanisms, much
Although noninvasive in vivo applications may be the final work in the field has exploited delivery of fluorescently labeled
goal, many of these efforts have exploited in vitro experiments dextrans of varying molecular weight (∼1−30 nm hydro-
for in-depth mechanistic investigations and proof-of-principle dynamic radius)545,827,828,849−851,864−872 and low molecular
studies. Recent reviews have covered the sonoporation field in weight dyes (<1 nm).802,845,850,851,857,865,871,873−881 Also dem-
detail.839−844 The mechanisms underlying sonoporation are onstrated has been intracellular delivery of small molecule
diverse and may involve (1) microstreaming caused by stable drugs,871,882−886 polymer nanoparticles of 25−75 nm,867 viral
cavitation, whereby a cavitation bubble oscillates in synchrony particles,887 proteins,865 antibodies,888 and peptides.889 In some
with the acoustic field (Figure 18E), (2) jetting forces from cases delivery has been ascribed to endocytosis and not influx
inertial cavitation, which is triggered by the collapse of a cavi- after permeabilization.868 This explanation could be applicable
tation bubble (Figure 18D), (3) a shrinking cavitation bubble to larger cargo such as plasmid DNA, where delayed expression
pulling against the plasma membrane,845 (4) an expanding kinetics akin to electroporation have been observed.846 As with
cavitation bubble pushing against the plasma membrane,845 other mechanical delivery methods,787 the plasmid transfection
(5) bubble translation, whereby acoustic radiation forces push efficiency of acoustic shear-based methods may be improved
a bubble through the plasma membrane, (6) nucleation of a by addition of downstream electrophoretic forces to augment
cavitation bubble between bilayer leaflets, rupturing the mem- delivery of charged cargoes.890
brane upon expansion, and (7) nonbubble acoustic effects, The majority of reports on sonoporation-mediated delivery
such as acoustic streaming due to pressure differences of the have focused on technology development and not its use to
acoustic 840,842,846field. The literature consensus indicates that carry out basic research. In the early days of sonoporation in
the first two mechanisms are the most prevalent. Below we the late 1980s and early 1990s, however, there were several exam-
discuss how such cavitation phenomena generate membrane ples of biologists using it to carry out basic research.827,829,873,887,888
disruptions. Although at least one commercial sonoporation system has been
Cavitation bubbles form and/or expand when the low- available for more than a decade (Sonidel SP100), its use for
pressure part of the acoustic wave passes through a liquid intracellular delivery appears confined within the ultrasound
medium. Conversely, the high pressure peak of the wave community.891,892 The most significant challenge for sonopo-
corresponds with compression and/or implosion of cavitation ration in vitro remains the random and uncontrolled nature of
bubbles.840,842,846,847 The bubbles may be created by the pres- cavitation events leading to excessive cell damage and death.844
sure waves themselves or provided by the supplementation of A 2012 review of 26 published studies conducted over more
stabilized microbubbles in the form of commercially available than a decade concluded that conventional in vitro sonoporation
AJ DOI: 10.1021/acs.chemrev.7b00678
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with nucleation agents almost never yielded above 50% for
both delivery efficiency and cell viability.844 Poor viabilities are
perhaps due to cavitation-related side effects such as high local
temperatures and generation of reactive oxygen species.893
Thus, bulk sonoporation may be inherently limited as a
delivery approach in vitro. In vivo applications have been more
promising,836 especially in skin where optimal parameters have
been identified and barriers to delivery of therapeutic cargo are Figure 19. Mechanisms of laser-induced membrane disruption.
more on the tissue, rather than cellular, level.839 (A) Laser optoporation occurs when incident energy is absorbed by
6.1.13. Fluid Shear: Shock Wave-Mediated Perme- the plasma membrane, directly disrupting it. This is known as
abilization. Shock waves di er from acoustical waves in that “Optoporation” and is covered in section 6.4. (B) Membraneff
disruption eventuates as the result of laser absorption by an absorbing
they are higher pressure and propagate at supersonic speed.894 agent in contact with the cell (such as a particle or interface), which
They are best known as the byproducts of explosions. Various then generates secondary effects (heat, fluid shear, near-field plasma
devices and strategies have been employed for producing shock etc.) to disrupt the plasma membrane. (C) Membrane disruption
waves to permeabilize cell membranes. They include shock eventuates through laser absorption by an absorbing agent distant
wave lithotripters,895−898 shock tubes,899−901 underwater spark from the plasma membrane. In these cases, fluid shear from cavitation
discharge,902 and laser-induced shock waves.900,901,903−909 and/or shock waves is the most likely cause of membrane disruption.
These systems mostly administer pulses one at a time instead
of the continuous waves characteristic of acoustic ultrasound. membrane absorbs laser energy and is disrupted (Figure 19A),
Lithotripters generate potent high pressure pulses that are used this is known as optoporation and is covered in section 6.4.
to break down tissue obstructions such as kidney stones If the absorbing agent is in direct contact with the plasma
(requiring up to 4000 individual pulses). In 1994, Gambihler membrane, the membrane will likely be perforated by a
and colleagues placed polypropylene vials containing a mixture complex combination of secondary effects including extreme
of suspended mouse L1210 lymphocytic leukemia cells and heat, near-field plasmas, and phenomena related to growth and
fluorescent dextrans under the focal point of lithotripter shock collapse of cavitation bubbles (Figure 19B). If the absorbing
waves.897 After treatment, the uptake and retention of dextran agent is distant from the plasma membrane, membrane
molecules was detected by flow cytometry. Although the disruption is much simpler and cleaner: it most likely occurs
authors admitted electroporation was more consistent and by fluid shear (Figure 19C) as thermal effects and near-field
efficient, lithotripter treatment showed a significant uptake of plasmas do not propagate very far in an aqueous environment.
2000 kDa dextran (∼50 nm diameter) with reasonable cell In any of the above three scenarios (Figure 19A−C), the
survival. membrane may be disrupted by laser-induced cavitation. Upon
Kodama et al. employed shock tubes to generate intense absorption, the energy supplied by the laser is transduced into
shock waves in cell suspensions and obtained intracellular heat and/or chemical effects that lead to vaporization of
delivery of labeled dextrans.899−901 Shock tubes generate a surrounding liquid to create cavitation bubbles.911,912 The
mechanical pulse when a thin diaphragm between a high bubbles disrupt cell membranes in the same way as sonopor-
pressure and low pressure chamber ruptures. The pulse then ation, either by microjetting after collapse (Figure 18D),
propagates through a second diaphragm and is focused into the through microstreaming from bubble oscillation (Figure 18E),
cell solution via a reduction nozzle, thereby achieving mem- or through secondary effects. Most reports of laser-induced
brane permeabilization.900 cavitation suggest bubble collapse, but there are a few cases
A number of studies have employed laser-induced shock where laser pulsing regimes can be tuned to sustain bubble
waves for cell membrane permeabilization.900,905−909 Laser- oscillations.913
induced stress waves can be generated by optical breakdown, In a series of studies by Ohl and colleagues, microfluidic
ablation, or rapid heating of an absorbing medium.907 In one confinement was used to investigate the relationship between
configuration, laser irradiation of an absorbing polymer film membrane damage and proximity to laser-induced cavitation
produces shock waves that emanate into a solution containing bubbles.914 The photoabsorbent molecule phenol red was
cells and cargo.906,908,909 Depending on experimental con- added to the solution to allow generation of cavitation bubbles
ditions, the mechanism of cell membrane disruption may or from the laser focal region. Their results showed that the prob-
may not rely on cavitation. In one set of examples, the rise time ability of cell permeabilization by cavitation bubble collapse
of the stress wave and its duration was associated with could be modeled as a function of the distance of cells from the
membrane permeabilization, probably due to shear forces bubble and maximum cavitation bubble radius.914 In a follow
involved with the wavefront itself.900,905−907,909 Conversely, in up study, they took advantage of arrayed microfluidic cell traps
other studies cavitation was implicated as the critical determ- to immobilize myeloma cells and systematically analyze the
inant of shock wave-induced membrane damage.895,903,910 conditions for controlled permeabilization at single cell
6.1.14. Fluid Shear: Laser-Induced Cavitation Bub- level.487 Again, phenol red was used as an absorbing agent to
bles. So far, we have covered membrane disruption arising facilitate the production of laser-induced cavitation bubbles
from acoustic pressure waves and shock waves, as well as that expand to ∼100 μm diameter and collapse within tens of
cavitation phenomena triggered by these stimuli. Cavitation microseconds.487 High frame rate imaging clearly visualized the
can also be triggered and/or controlled in a more direct expansion and shrinkage of cavitation bubbles in a non-
manner by the action of lasers incident upon an absorbent symmetric manner due to the presence of a nearby structure.
agent in an aqueous environment.911,912 The absorbent agent During bubble collapse, a fast microjet was directed toward the
may be the membrane itself, a photoabsorbent molecule added cell to generate a single large pore with diameters ranging from
to solution, a particle suspended in solution, or a material 0.2 to several μm. The diffusive uptake of trypan blue dye into
interfacing with the solution (Figure 19). When the plasma the cell then took place over several seconds. If the standoff
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distance between cell and bubble were greater than 30 μm, no with less cytotoxicity.923 Building on this approach, the same
membrane disruption occurred. One concern highlighted by the group further improved their methods to deliver quantum dots
authors was whether cavitation bubbles perturb cells through into cells at high throughput with efficiencies and viabilities
temperature spikes. To address this issue, Ohl and colleagues above 80%.310 Interestingly, in primary human T cells the vapor
performed another study with fluorescence-based thermometry nanobubble approach was reported to yield greater siRNA
to measure local temperature gradients around laser-induced transfection efficiency and cell survival when compared with
bubbles.915 Under similar conditions as their previous experi- nucleofection.924 In congruence with these results, other groups
ments, it was found that the temperature rises are moderate have presented experimental and theoretical work that demon-
(<12.8 °C), localized (<15 μm), and short-lived (<1.3 ms). strates nanobubble formation from the generation of a nanoscale
Thus, by developing a cavitation regime that damages cell plasma around an irradiated particle due to enhanced near-field
membranes purely through mechanical forces, laser-induced effects rather than from the heating of the particle.925,926
cavitation may be amenable to implementation on a wider 6.1.16. Fluid Shear: Laser-Induced Cavitation at an
scale. It was suggested that arraying cells in microfluidic traps Interface. Absorbing materials can be placed along a solid−
would allow for potential scale-up with predetermined laser liquid interface to convert laser energy into membrane-per-
protocols to control the size and position of membrane- turbing cavitation bubbles or shock waves. In recent studies,
permeabilizing cavitation bubbles. Ohta and colleagues fabricated a channel of defined height,
6.1.15. Fluid Shear: Laser-Induced Cavitation via with cells cultured on one side apposing an optically absorbing
Absorbent Particles. To transduce laser energy into composite layer of 1 μm amorphous silicon and 200 nm
cavitation, some approaches employ a deliberate seed particle indium tin oxide.927 Instead of generating an exploding bubble,
to absorb the laser energy. One of the first papers to do this they oscillated a bubble using a 980 nm laser with 90 μs pulses
was published by Pitsillides et al.916 They labeled lymphocytes over a duration of 10−15 s. Up to three oscillations of 8−10 μm
with antibody-functionalized metal microspheres and irradi- without collapse were able to induce microstreaming shear forces
ated them with a 565 nm laser at a fluence of 0.35 J cm−2 and to trigger plasma membrane permeabilization in adjacent cells.
pulse duration of 20 ns.916 Rapid eminence of microbubbles In this configuration, the bubble had to be pressed tightly
was observed around the seed particles and cell membranes against the cell to induce membrane disruption. For 70 kDa
were subsequently permeabilized. By adjusting particle fluorescently labeled dextran, up to 80% delivery at >95%
numbers, size, and laser energy delivered to the metal micro- viability was achieved. The pore-size was estimated to be about
spheres it was possible to tune the treatment either toward 30 nm based on exclusion of 500 kDa dextran, and the closure
killing cancer cells for potential therapeutic purposes or trans- dynamics indicated plasma membrane healing within ∼20 s.
iently increasing the permeability of the plasma membrane for In a follow-up study, the same authors lowered the channel
intracellular delivery.916 Another group used femtosecond laser height to 10 μm and generated stronger shear forces over 0.4 s
irradiation of gold nanoparticles to produce plasmonic nano- with 60−100 μs pulses applied at a frequency of 50 Hz.913
bubbles and permeabilize primary human cells for ex vivo By generating larger pores with more powerful shear forces,
intracellular delivery.917,918 Selective delivery of plasmids and delivery efficiency of 500 kDa dextran improved to 70% and
dextrans was demonstrated in primary human cancer cells, expression of 5.7 kb DNA plasmid was recorded at 86%.
T cells, and hematopoietic stem cells with reportedly good cell Permeabilization of adherent cells can be achieved by cul-
viability.917,918 turing them on patterned thermoplasmonic substrates followed
In 2010, Prausnitz and colleagues introduced an intracellular by laser irradiation.509,928,929 In a strategy introduced by Mazur
delivery strategy involving laser irradiation of dispersed carbon and colleagues, a substrate patterned with microscale gold-
black nanoparticles919 Adherent cells were exposed to the cargo coated pyramids was fabricated by photolithography and
molecule to be delivered and sprinkled with ∼200 nm aggre- template-stripping. A nanosecond pulsed laser was then
gates of carbon black followed by irradiation with femtosecond scanned across the substrate to produce intense heating at
lasers.919 Rather than thermal effects, they proposed that the the apex of each pyramid, thereby generating bubbles through
mechanism of membrane disruption was primarily due to a plasmonic effects.930 A large beam spot can be scanned across
carbon-steam reaction at the particle surface, which sub- the substrate to permeabilize millions of cells over the course
sequently propagates cavitation-related acoustic forces.919,920 of minutes.929 Growth and collapse of the bubbles presumably
Delivery of dyes, proteins, siRNA, and plasmid DNA were disrupts cell membranes by mechanical shear forces, although
achieved with acceptable cell viabilities in several cancer cell plasmonic chemical effects or heat cannot be ruled out.
lines.919,921 Control experiments verified that neither the Delivery of dextrans up to 2000 kDa have been obtained with
carbon black particles nor laser exposure alone were su cient high cell viabilities929ffi through membrane holes estimated to be
to enable molecular uptake.919 This intracellular delivery con- in the range of 20 nm.509
cept was then extended beyond adherent cells to homogeneous In a different approach, the Chiou lab developed a “pho-
suspensions of carbon black nanoparticles and cells, which may tothermal nanoblade” capable of addressing single cells.339
be more amenable to treatment at higher throughputs.922 A metallic nanostructure was placed at the tip of a micropipette
In a different strategy from Braeckmans and co-workers, gold as a seed structure to harvest short laser pulse energy and
nanoparticles were employed as absorbing agents and laser convert it into highly localized explosive vapor bubbles. Upon
excitation parameters were screened to test for and manipulate placement of the device next to cells, laser irradiation triggered
the balance between pure heating and bubble nucleation.923 cavitation events that yielded controlled pore sizes of up to
By tuning the laser energy, they identified conditions where it several microns on the apical surface of adherent cells. Delivery
was possible to produce vapor nanobubbles around ∼70 nm of large cargo such as ∼2 μm bacteria, mRNA, plasmid DNA,
gold nanoparticles without transfer of heat to the surrounding polystyrene beads, and quantum dots was achieved.309,339
environment. This analysis revealed that vapor nanobubbles Furthermore, in an intriguing biological application, the photo-
without heat enabled superior delivery and siRNA transfection thermal nanoblade was used for mitochondrial transplants
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Figure 20. Mechanical membrane disruption via osmotic pressure changes. (A) Cells in suspension subject to hypotonic shock will first swell,
which unravels membrane reservoirs. If the membrane strain is sufficient, permeabilization will occur. The inset shows microscale conformation of
the plasma membrane. (B) Cells in an adherent monolayer cultured on a porous substrate can be subject to a perturbing osmotic gradient via
hypotonic shock at their apical surface. Swelling and subsequent permeabilization occur similarly as in panel A, but the permeabilization is localized
to the apical side of the cell. (C) In a scenario where endosomes are preloaded with osmolytes and cargo to be delivered, a hypotonic shock can be
used to cause lysis of endosomes and release of cargo to the cytoplasm.
between cells.341 By delivering functional mitochondria to cells the use of lab-on-chip, microfluidic, and nanotechnological
with normally dysfunctional mitochondria, it was pos- systems.
sible to identify mechanisms involved in restoration of metab- 6.1.18. Pressure Change-Mediated Permeabilization.
olism.341 Consistent with what is known about membrane Osmotic and hydrostatic pressure gradients can be imposed
repair in healthy cells, electrical impedance measurements across cell membranes leading to their rupture. The geometry
showed that it takes 1−2 min to recover membrane integrity of these gradients can vary, for example between a suspended
after treatment with the photothermal nanoblade.931 cell and the extracellular solution, across a select part of the
A high throughput version of the photothermal nanoblade plasma membrane (such as the apical membranes of an
concept was unveiled in 2015.340 Substrates arrayed with pores adherent cell monolayer), or between an intracellular vesicle
lined by metallic absorbers were irradiated to generate exploding (e.g., endosome) and the surrounding cytosol. Although they
cavitation bubbles underneath the basal side of adherent cells may be difficult to control in time and space, transient pressure
(Figure 18F). Membrane permeabilization was synchronized gradients achieved by osmotic or hydrostatic means represent a
with active pumping of cargo through the pores to successfully low-cost and simple avenue for membrane disruption-mediated
introduce living bacteria into the cytoplasm of several cell intracellular delivery. These methods have not been heavily
types. Showcasing the potential of the approach, it was dis- pursued to date, however, perhaps due to a poor understanding
covered that the iglC gene from the bacterial species F. novicida of their effects and hesitance of researchers to excessively
is required for intracellular proliferation after cytosolic delivery. perturb cells.
932
Such a high throughput strategy to deliver micron-sized cargo 6.1.19. Osmotic Shock and Plasma MembraneDisruption. One of the simplest perturbations that a cell
clearly has broad utility with adherent cells, showcasing the can experience is an osmotic shock, whereby a hydrostatic pre-
power of well-controlled fluid shear forces to permeabilize vast ssure is generated across the cell membrane due to differences
populations of cells. in osmotic potential. Most mammalian cells normally exist in
6.1.17. Fluid Shear: Summary.Methods that exploit fluid an aqueous environment of ∼300 mOsm and significant devia-
shear forces to permeabilize cell membranes range from tions from this condition will induce the flow of water mole-
constriction-mediated fluid shear zones in microfluidic devices cules into (hypotonic swelling) or out of (hypertonic shrinkage)
and syringe nozzles to cone−plate viscometers and cavitation- the cell. When a cell is placed into a low osmolarity solution,
related phenomena. The use of cavitation bubbles for plasma water rushes into the cell through the plasma membrane and
membrane disruption has received the most attention to date, aquaporin channels to solvate impermeable intracellular electro-
probably because of the ability to control local shear forces lytes and osmolytes. The subsequent swelling of cell volume leads
remotely via acoustic fields and lasers. Because of the to the unfolding of loose membrane, followed by well-
inherently unstable and powerful nature of cavitation, however, described lipid bilayer rupture if area strain exceeds 2−3%
challenges remain in how to harness them for reproducible and (Figure 20A). Cells have been reported to possess membrane
consistent cell treatments. Nevertheless, cavitation bubbles reservoirs of 2−10× their apparent surface area depending on
have demonstrated the ability to delivery almost any cargo to a the cell type and state.415 Caveolae, endocytic pits, membrane
wide range of cell types. In the future we anticipate intriguing folds, filopodia, and microvilli are all examples of membrane
and innovative devices able to deploy highly localized fluid reservoirs that can unfold to buffer membrane strain and
shear forces for precise membrane disruption, perhaps through accommodate cell surface area increase.414,416 It is thought that
AM DOI: 10.1021/acs.chemrev.7b00678
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these reservoirs should be exhausted globally or locally before They found that treatment times of 10−30 s were ineffective in
membrane stretch can result in the opening of holes. loading aequorin (21 kDa) or GFP (28 kDa). Upon 2 min
6.1.20. Hypotonic Loading of Red Blood Cell Ghosts. exposure, long-term cell viability up to 18 hours was more than
If the magnitude and duration of osmotic shock is optimal, 50% with sufficient delivery to determine intracellular calcium
partially burst cells can recover membrane integrity in the form concentrations, which turned out to be ∼0.9 μM in follicular
of hollowed out “ghosts”. Although dead, ghosts can reseal and lymphoma cells. Ten minutes hypotonic exposure led to robust
regain a limited set of functions. The concept was first estab- delivery but a gradual loss of viability in almost all cells after
lished in red blood cells (RBCs) throughout the 1960s.933−935 10 hours, probably due to delayed cell death responses (see
Although RBCs possess little surface reservoirs compared to section 4.3). Major advantages of Klabusay’s protocol are its
most nucleated cells, their capacity to reseal after a brief applicability to treat difficult-to-transfect suspension cells and that
hypotonic shock is well-proven.936,937 Indeed, RBC ghosts it appears agnostic to cell size and type of cargo material to be
were able to enclose molecular cargo and even retain some delivered.
basic biological functions despite being hollowed out of In 1999 Koberna and co-workers unveiled a method they
cytoplasmic components.937,938 In one early study, by adding termed “hypotonic shift” to achieve intracellular delivery of
ferritin at various times after the onset of hemolysis, it was modified nucleotides, nucleosides, dyes, and peptides into a wide
determined that most cells were permeable for 15−25 s after range of cell types.951 Cells were exposed to the hypotonic buffer
hypotonic shock.939 Furthermore, the size and shape of for 5 min before a return to isotonic media for recovery. The
membrane disruptions, as seen in fixed cells by SEM imaging, hypotonic buffer consisted of 30 mM KCl saline and 10 mM
resembled long, narrow tears up to 1 μm long.940 Later more HEPES for pH buffering (∼70 mOsm combined). After
accurate studies, however, indicated smaller holes around tens treatment, metabolic production of DNA, RNA, and protein
of nanometers or less.941 After such initial studies, hypotonic was inhibited and took ∼4 hours to return to normal levels. No
lysis procedures were optimized to result in high efficiency loss of viability or apoptosis was observed. The hypotonic shift
loading of proteins and enzymes into RBC ghosts.938,942,943 method was reported to be highly effective for delivery of smaller
RBC ghosts have been proposed as drug carriers for decades, around molecules ∼1 kDa but efficiency decreased for cargo of
in part due to their ease of hypotonic loading and potential for greater molecular weight. For example, the procedure was unable
autologous biocompatibility.944−947 Interestingly, fusion of pre- to deliver large proteins such as labeled antibodies. Nonetheless,
loaded RBC ghosts into recipient cells was a popular method the hypotonic shift approach has been particularly popular for
of intracellular delivery in the 1970s and 1980s87,938,948,949 intracellular delivery of labeled nucleotides.952−960 It has also
before falling out of favor with the rise of electroporation and been adapted for the successful intracellular loading of the
other alternatives.544 peptide actinomycin D,961 dye-conjugated dextrans,962 and 5 nm
6.1.21. Hypotonic Shock for Intracellular Delivery. gold particles.963
Unlike RBCs that can passively reseal, most cell types mobilize Apart from severe hypotonic shock, intracellular delivery has
active repair processes to recover from membrane disrup- also been accomplished with milder hypotonic shocks in the
tion.424 It was not until the early 1980s that osmotic delivery range of ∼150 mOsm. Mills et al. used hypotonic swelling for
methods would be translated beyond RBCs into other cell types. intracellular loading of antibodies into rat submandibular acini
In 1982, Borle and Snowdowne devised a simple procedure to cells.964 This application is notable in that cells are not indi-
deliver the calcium-sensitive protein aequorin (21 kDa) into vidually isolated in suspension; acini are small clusters of cells
nucleated cells.92,93 Washed pellets of monkey kidney cells organized in a quasi-circular arrangement to form a hollow
were suspended and immersed in a ∼10 mOsm hypotonic duct in the center. In the procedure, acini were exposed to a
solution consisting of 3 mM MgATP, 3 mM HEPES buffer, mild hypotonic solution (∼150 mOsm) containing 5 mM ATP
and a given concentration of aequorin for 2 min at 4 °C. This and the antibody of interest for 1 min following a switch back
was followed by sufficient addition of buffered KCl to restore to isotonic conditions. The loaded antibody was found capable
isotonicity. Cells were then incubated in standard cell media of inhibiting its target CTFR protein in the cytoplasm,
for 1 hour at 37 °C to promote restoration of homeostasis verifying that delivery had indeed occurred. The procedure has
before experiments. Optical readouts of aequorin activity indi- also been used to deliver the calcium chelator BAPTA965 and
cated that it had been loaded successfully into fully functional enzymes966 into acini cells.
cells, and it was used to measure accurate intracellular calcium In studies that require intracellular delivery of lanthanum-
concentrations of ∼50 nM. based contrast agents, milder hypotonic shocks (∼90−
Citing Borle and Snowden’s method as an inspiration, the 160 mOm) have been used to load normally impermeable
hypo-osmotic approach for cytoplasmic delivery of aequorin tracers, such as Gadolinium ions, into adherent or suspension
was re-examined in greater detail by Klabusay et al.950 They cells.967 In this case, a 30 min ∼160 mOsm hypotonic exposure
were motivated by the need to accurately measure intracellular at 37 °C was used for cytoplasmic delivery of lanthanide
calcium dynamics in follicular lymphoma B cells, an application complexes and dyes in various macrophage and cancer cell
where the aequorin protein offers superior signal-to-noise ratio, lines.967 A comparison with electroporation and osmotic lysis
better dynamic range, and more reliable calcium readouts than of pinosomes concluded that hypotonic shock was the most
commonly used small fura dyes. In their method, cell suspen- advantageous method for delivery of these small (<1 nm)
sions of 30 μL were added to 200 μL of pH buffered hypo- molecules.967 Other reports appear to verify this strategy, as
osmotic solution (∼2 mOsm) and 0.1 mg mL−1 aequorin before Gadolinium complexes have been delivered into HeLa cells
gentle mixing. After a predetermined duration of hypotonic with the same protocol.968 In further cases, a more severe
exposure, addition of 230 μL hyperosmotic solution was used shock of ∼90−110 mOsm for 60 min at 37 °C produced load-
to bring the suspension back to isotonic conditions and enable ing of Lanthanide complexes into HeLa cells.969,970 Interest-
membrane recovery. To test the response of cells to hypo-osmotic ingly, iron oxide nanoparticles of up to 60 nm were loaded into
conditions, the exposure time was varied from 10 s to 10 min. RBCs with hypotonic shocks of 90−110 mOsm.969 Other
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reports in RBCs employed a 30 min ∼160 mOsm hypotonic DNA-containing saline solution into a 20 g animal. The
shock at 4 °C to load gadolinium-based complexes into the introduced solution is close to 10% of the body weight, thus
cytoplasm without loss of cell functionality.971−973 representing a rapid expansion of blood volume that cannot be
In a strategy that synergizes hypotonic shock with the immediately pumped through the vena cava of the heart. This
membrane-perturbing effects of detergents, Medepalli and causes sudden distension and hydrostatic pressure build-up in
co-workers demonstrated quantum dot loading into adherent the surrounding tissues. A weak point is typically retrograde
H9C2 cells by exposure to a mild hypotonic buffer (150− flow into the liver, where it has been observed that fenes-
200 mOsm) combined with low concentrations of the detergent trations in hepatic tissue expand to generate disruptions in
saponin.311 Presumably saponin reduces the threshold for cell membranes, thereby allowing influx of cargo molecules
induction of plasma membrane defects under hypotonic stretch, from the blood directly into the cytosol of hepatocytes.980
thereby synergizing the permeabilization effects of both Considerable delivery efficiencies have been achieved using
approaches. After delivery, quantum dots of hydrodynamic hydrodynamic tail vein injection, with up to 40% transfection
diameter 20−25 nm were observed to be evenly dispersed in of liver hepatocytes from a single injection.979 Macromolecules
the cytoplasm of treated cells. such as labeled dyes, proteins, oligonucleotides, siRNA,
6.1.22. Osmotic Gradients Acting on Part of the bacterial artificial chromosomes, and linear or circular DNA
Plasma Membrane. When cells form a tight monolayer fragments as large as 175 kb have also been delivered to rodent
across a porous substrate, they form an impermeable barrier hepatocytes by this method, lending credence to a membrane
between two bodies of liquid media. An osmotic shock in one permeabilization mechanism without reliance on endocyto-
of those solutions creates an osmotic gradient across the cells. sis.980−987 More recently, hydrodynamic tail vein injections
Taking advantage of this principle, Widdicombe et al. cultured have found use in CRISPR-based genome editing in mouse liver,
epithelial or endothelial cells into confluent polarized mono- albeit at lower efficiencies.988−991 A major limitation of
layers on substrates with 0.45 μm pore size.974 The apical hydrodynamic injection is that it is only available in rodents.
media was then exchanged with water containing macromole- Apart from injection into veins, intracellular delivery of
cules to be loaded while retaining the basal media as physi- nucleic acid cargo has been observed by direct injection of
ological saline (Figure 20B). This resulted in a ∼300 mOsm solutions into skeletal muscle, heart, thyroid, skin, and liver.992
osmotic gradient across the cell monolayer. Disruption of the Mechanistic studies indicate that this also occurs by membrane
apical cell membrane was evidenced by uptake of 67 kDa permeabilization, but a role for endocytosis has not been
uorescent albumin and 2000 kDa dextrans but was reversible completely ruled out.992−995fl The degree to which membrane
within ∼5 min when apical water was replaced with normal cell permeabilization or active uptake processes underlie delivery is
culture media. By adding fluorescently labeled molecules at probably dependent upon the properties of the solution and
di erent times after hypotonic shock, it was found that the manner in which the injection is performed.992,994ff Further
majority of uptake occurred within the first 4 min. This tech- investigations are required to clarify these matters.
nique was reported to be temperature insensitive, working In 1999 Mann et al. introduced a method for hydrostatic
equally well at 4 or 37 °C, thereby indicating that endocytic pressure-mediated transfection in human vein segments and rat
activity had a minimal role and suggesting plasma membrane myocardium ex vivo.996 Segments of veins (∼1−2 cm) were
disruption as the prime mechanism. After the procedure, cell cannulated, encased in a plastic sleeve to prevent distension,
layers were able to recover full trans-epithelial resistance within and infused with pressurized solutions of up to ∼100 kPa
several hours. above baseline pressure.996 Ten minutes of this treatment was
In a complementary study by Widdicombe’s co-workers, reported to yield intracellular delivery of fluorescently labeled
Tawa et al. demonstrated successful transfection of airway antisense oligonucleotides into ∼90% of endothelial cells lining
barrier cells in rat lungs by exposure to apical water containing the vein segment.996 Moreover, ex vivo treatment of rat hearts
DNA.975 A follow-up report argued that the hypotonic trans- pressurized inside and out at up to ∼200 kPa showed ∼50%
fection of DNA to airway barrier cells could be due to active transfection in myocardial cells.996 Although the exact delivery
uptake by membrane trafficking, which is known to stimulate mechanisms were not revealed, imaging of cells after treatment
exocytosis and endocytosis associated with regulatory volume suggested it was nonendocytic.996 Variants of this technique
mechanisms.976 However, this model would not t with have been used to achieve intracellular delivery of siRNA,997fi
the original observation of rapid delivery by Widdicombe et al. antisense oligonucleotides,996,998−1002 plasmid DNA,998,1003,1004
In an analogous situation, hypotonic aerosols have been and ∼100 nm polystyrene microspheres.1005
observed to facilitate intracellular delivery of PEI-complexed 6.1.24. Disruption of Endosomes by Osmotic Forces.
DNA by a membrane permeabilization mechanism in mouse In 1982 Okada and Rechsteiner described an intracellular delivery
airway epithelium.977 Thus, a hypo-osmotic delivery principle technique termed osmotic lysis of pinosomes. It works by
may prove feasible when applied to exposed cell monolayers harnessing osmotic forces to rupture endosomes preloaded
in vivo, particularly in the lungs. with cargo of interest, thereby obtaining cytosolic delivery
6.1.23. Hydrostatic Pressure and Hydrodynamic (Figure 20C).94 In the first step, endocytic uptake is promoted
Delivery. Membrane disruption due to a sudden increase in by a ∼10 min incubation of cells in a ∼800 mOsm hypertonic
hydrostatic pressure is believed to be the mechanism of buffer containing 0.5 M sucrose, 10% polyethylene glycol
so-called “hydrodynamic delivery”, where a rapid injection of (PEG)-1000, and molecules to be delivered. Exchange to a
fluid into the cardiovascular system causes transient disruption hypotonic solution (∼180 mOsm) consisting of diluted media
in the plasma membrane of cells in certain tissues. A prime for ∼2 min then generates a rush of water into the cell. During
example is tail vein injection, where robust transfection of this hypotonic shock phase endosomes laden with cargo and
hepatocytes and sometimes other cardiovascular tissues has osmolytes expand and rupture, thus releasing their contents.
been observed in rodents.978,979 In a mouse model, trans- The pendulum swing from hypertonic to hypotonic conditions
fection is achieved by fast injection (∼5 s) of almost 2 mL of may also disrupt the plasma membrane; however, cells are
AO DOI: 10.1021/acs.chemrev.7b00678
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generally able to release osmolytes to counteract swelling.491 endosomes or an unmet need for destabilizing agents to assist
Endosomes, on the other hand, have no volume regulation and with endosome rupture, a role that PEG was later suggested to
therefore swell uncontrollably until bursting.1006,1007 This means play.1044
the imposed hypotonic shock may impact endosomes signifi- Interestingly, an in vivo application of the osmotic lysis of
cantly more than the plasma membrane. Okada and Rechsteiner pinosomes concept was accomplished in rat arteries. Without
reported that the osmotic lysis of pinosomes method was capable surgical removal, isolated, pressurized mesenteric arteries of
of introducing antibodies, various proteins, and 70 kDa labeled rats were cycled through hypertonic and hypotonic solu-
dextrans into the cytosol of L292, 3T3 fibroblasts, and HeLa tions. Endothelial cells were found to take up dyes, dextrans,
cells.94 peptides, and labeled antibodies into the cytoplasm without
Following the example of the original paper, osmotic lysis compromising the structure and function of the surrounding
of pinosomes has been particularly useful for intracellular tissues.1026 This strategy was used to identify a critical role for
delivery of proteins,94,95,1007−1021 antibodies,1013,1022−1026 connexin 40 protein in endothelium-derived hyperpolarizing
dextrans,94,1026−1028 and peptides.1029−1031 In a landmark factor(EDHF)-mediated dilation of endothelial cells in rat
paper in 1988, osmotic lysis of pinosomes was used to prove mesenteric arteries.
that cytosolic loading of proteins could mediate their presen- 6.1.25. Induced Transduction by Osmocytosis. Moti-
tation as antigens through the major histone compatibility I vated by limitations of the osmotic lysis of pinosomes method
pathway to invoke a specific immune response.95 In other reports, in primary cell types, D’Astolfo et al. introduced an adaptation,
osmotic lysis of pinosomes has found success in intracellular termed iTOP, which stands for induced transduction by osmo-
delivery of cell lysates,1032 hyaluronan,1033,1034 trehalose,1035 cytosis and propanebetaine.151 Instead of relying on hypotonic
Lanthanide imaging probes,71,72 various small molecule solutions for endosome disruption, propanebetaine appears
dyes,1026,1036 uridine triphosphate-glucuronic acid,1037 antisense sufficient to trigger cargo leakage specifically from macro-
oligonucleotides,1038 antisense morpholinos,804 virus particles,1039 pinosomes. The method relies on NaCl-related hypertonicity
and nanomaterials such as quantum dot-labeled motor proteins of the extracellular medium to induce macropinocytosis fol-
for biophysical studies.306,1040,1041 lowed by spontaneous endosomal leakage. A high extracellular
With the advent of RNAi-mediated gene silencing in the concentration of Na+ ions was shown to be necessary to
early 2000s, researchers tested the ability to perform siRNA stimulate NHE1-mediated macropinocytosis. Unlike osmotic
transfection via osmotic lysis of pinosomes. By using up to lysis of pinosomes, however, no discrete trigger was required
1.6 μM siRNA in solution, gene silencing of >50% was for endosomal rupture. Instead, intracellular macropinosome
reproducibly achieved in common cell lines such as HEK and leakage was a stochastic event promoted by the presence of
HeLa.1042 In a subsequent study by a different group, improved propanebetaine or other compounds with similar physico-
RNAi transfection was demonstrated in hard-to-transfect chemical properties. The osmotic pressure created by hyper-
immune cell lines.1043 Their modified procedure was more tonic endosomes may also contribute to destabilize endo-
extreme, involving hypertonic sucrose solutions of up to 2 M somes. Using iTOP, RNPs of Cas9-sgRNA were delivered into
and siRNA concentrations of 10 μM.1043 Difficult-to-transfect KBM7 cells and H1 human embryonic stem cells to produce
immune cell lines, including mouse macrophages RAW264.7 CRISPR-mediated gene deletions. Various other proteins were
and J774.1 as well as the T lymphocyte cell line DO11.10, were also delivered, demonstrating efficient delivery of a number of
shown to be transfectable with this approach. Other benefits cargo materials into a variety of primary cell types.
were minimal cytotoxicity and immunomodulatory responses 6.1.26. Pressure Changes: Summary. Rapid changes in
compared to the synthetic cationic lipid reagents lipofectamine hydrostatic and osmotic pressure are an obvious, straightfor-
and oligofectamine, or the polymer reagent jetPEI. In a micro- ward, and low-cost strategy for placing cell membranes under
fluidic adaption of the approach, a device was deployed for mechanical force. This insult has been harnessed against both
rapidly cycling suspended cells through the various solutions to the plasma membrane (external) and endosomal membrane
induce osmotic lysis of pinosomes, thus avoiding the need for (internal) systems for the purposes of intracellular delivery.
centrifugation to exchange solutions.1028 Results were reported Because current procedures rely on buffer changes, however,
to be superior to the conventional protocol for loading fluores- there is a significant stress placed on the entire cell. Concepts for
cent dextrans into Jurkat cells.1028 better controlling the spatiotemporal application of mechanical
It is important to note that the osmotic lysis of pinosomes pressure changes at the scale of the cell may provide more
method has several caveats: (1) cell stress, (2) delivery capac- precise membrane permeabilization in future.
ity is limited by the extent of endocytosis, and (3) absence of
reports on larger cargo such as plasmid DNA and mRNA. First, 6.2. Electrical Membrane Disruption (Electroporation)
the hypertonic media imposes significant stress on cells and has In the 1980s, electroporation, which involves the transient
been observed to actually inhibit endocytosis in some cell permeabilization of cell membranes with electric pulses, rose to
types.1044 Second, the extent of endocytosis during the hyper- prominence as a powerful approach for intracellular delivery,
tonic exposure window is a limiting factor that affects the final applicable to a wide range of cell types, from animal cells to
concentration of cargo delivered.1044 Multiple rounds of the plants and lower organisms. Prior to its introduction, the stage
procedure may be conducted to boost delivery efficiency but had been set by more than a decade of research exploring the
are time-consuming and must be balanced with considerations effect of voltage pulses on arti cial lipid bilayers, vesicles1045,1046fi
of cell stress.1027 Several publications indicate that cell function and red blood cells.533,534,1047 In nucleated mammalian cells,
and health may be compromised as a function of duration and Neumann and colleagues published a groundbreaking report in
intensity of the osmotic challenges.94,932 The third consid- 1982 which demonstrated that electroporation led to the effi-
eration is that certain combinations of cell types and cargo cient transfection of plasmid DNA in mouse lyoma cells.184
molecules appear to be unfeasible with the procedure. This can The study also stimulated electroporation theory by introduc-
be due to degradation of cargo in the acidic environment of ing a generalized van’t Hoff relationship to model the extent of
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Figure 21. Energy landscapes and theory of pore formation in membranes by electric fields. (A) Schematic of pore formation showing the
transition from a hydrophobic pore to a hydrophilic (conducting) pore. (B) Graphs of relationship between free energy of pores ΔW and pore
radius r for ΔΦm = 0 (upper curve) and at ΔΦm > 0 (lower curve). r* is the critical radius corresponding to the transition from hydrophobic to
hydrophilic pore. ΔWf corresponds to the height of the energy barrier for pore formation while ΔWres relates to the energy barrier height for pore
resealing. rIRE is the pore radius corresponding to the state of irreversible electroporation. ΔΦm is the electrical potential difference across the
membrane. The higher the electrical potential, the more probability of pore formation. Figure reprinted by permission from Springer Nature from
ref 163. Copyright 2015. (C) Calculations of the effect of applied voltage on the energy landscape of pore formation with transmembrane potentials
ranging from 0 to 0.5 V. Reprinted figure with permission from ref 420 and the authors. Copyright 1999 by the American Physical Society.
permeabilization in an electroporated cell, whereby poration better conductors, however, the electrical expanding pressure
phenomena are viewed as structural rearrangements of lipids decreases, resulting in a decay in the rate of their growth.
and water.184 Electroporation, while initially emphasized for This explains two phenomena characteristic of electroporation:
DNA transfection, has subsequently shown utility for delivery (1) longer pulses (usually tens of ms) are required to grow
of a huge variety of cargo: from small molecule drugs, dyes, larger pores, and (2) electroporation is not very good at
and tracers to larger proteins/antibodies and multiple forms of producing large (e.g., >50 nm) pores.579,1052
DNA and RNA.163,1048−1050 In this section we first cover the Apart from the energy landscape of electroporation, theo-
mechanisms of electroporation before exploring the challenges, retical models and simulations have been used to decipher the
technical advances, and applications. chemical thermodynamic and kinetic aspects of pore for-
6.2.1. Mechanisms of Membrane Disruption and mation.184,1053−1058 Upon application of an electric field across
Cargo Entry.Mechanistically, electroporation is the formation a lipid bilayer, an early event is tilting of the electrical dipoles
of pores in a membrane by the application of a potential associated with the lipid headgroups to align with the direction
difference across that membrane. When the potential differ- of the applied electric eld.184,1053,1058fi This causes rotation of
ence reaches a critical magnitude of voltage, the probability of lipid molecules, thus thinning the bilayer, perturbing its
electroporation taking place drastically increases. According to organization, and facilitating the entry of water molecules into
theory, the increase in electric field energy within the membrane the hydrophobic core.184,1053,1058 Pore formation follows, and
and ever-present thermal fluctuations combine to create and the rate of transition between pore states is subject to a
expand a heterogeneous population of pores.163,419,1051 Although hysteresis where their opening (microseconds range) is
there is no fixed voltage threshold that triggers electroporation, thought to be much faster than the time scales of their closure
the critical parameter of electroporation is the trans-membrane (seconds to minutes).1057 Such models of electroporation have
potential. This is because the maintenance of a trans-membrane been developed to a degree where analytical expressions are
electrical potential incurs a probability of generating a membrane available to optimize electroporation in several biotechno-
defect for a given field strength, time, and temperature. logical and medical applications.1054−1056 For further explan-
Membrane defects originate as so-called hydrophobic pores of ation of the formulas and analytical frameworks, see the fol-
radius <0.5 nm, which form due to random thermal lowing referenced publications.184,1053−1058
fluctuations of the individual lipid molecules that make up Electroporation is thought to be primarily related to changes
the membrane (Figure 21A). Fueled by the external electrical in electrical conductance, but chemical, thermal, and electro-
energy provided, these defects may then traverse their energy mechanical membrane deformation effects may also contrib-
landscape to become hydrophilic pores, which are typically ute.458,1059 The application of mechanical tension has been
lined by at least 8−10 phospholipid head groups and defined observed to lower the electric voltage threshold required for
by their ability to permit free passage of water molecules membrane disruption.1060,1061 This is because mechanical
(Figure 21B). Hydrophilic pores (r > 0.5 nm) can be stable forces contribute to bias the energy landscape toward defect
because the energy barrier also exists in the reverse direction. formation (see Figure 8). In keeping with this notion, lower
Current theory posits that small pores are not very good temperatures have been observed to increase the field strength
conductors; hence the continued application of an electric field required for electroporation575,1062 and slow the kinetics of
is not only critical for their formation, but also their enlarge- resealing.1054,1055 Furthermore, mathematical descriptions and
ment.419,1051 Pore formation and expansion are energetically models have been developed to assess, for example, the effect
favorable because they relax the charge buildup that would other- of applied voltage on the distribution of pore radii420,1063
wise become entropically unfavorable. As the pores become (Figure 21C). More recently, simulations have also assisted in
AQ DOI: 10.1021/acs.chemrev.7b00678
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illuminating the molecular events associated with electro- less numerous population of defects.1067 In general, it is
poration, although (due to limitations in computational power) thought that coverage area of permeabilization is controlled by
they currently only cover very short time scales on the order of pulse strength while the pore growth size is more strongly
nanoseconds or less.1053,1064,1065 It is imperative to note, correlated with the pulse duration.1048 Once pores are formed
however, that many of the mathematical models and and begin conducting, the local electroporation effect dimin-
simulations are challenging to verify experimentally. ishes somewhat as charge is free to flow through these defects.
6.2.2. Mechanisms of Electroporation in Cell Suspen- Therefore, the amount of energy channeled into the growth of
sions. In suspensions of isolated cells electroporation is pores declines through the lifetime of a particular pulse.458
observed with applied trans-membrane potentials in the range Upon electroporation, the response within cell populations
of 0.2−1.5 V. Pulse times are typically on the order of and between cell types is somewhat heterogeneous, reflecting
microseconds to almost a second. The membrane charges like differences in cell size, orientation, surface area, and physiolog-
a capacitor with a characteristic charging time proportional to ical state, as well as variances in membrane composition and
the surface area of the enclosed membranous body.579 For the presence of local inhomogeneities in the electric field itself.
conventional cuvette-style parallel plate setups, a cell suspen- The microenvironment of the cell surface is characterized by
sion in conducting buffer is placed between two electrodes the distribution of nearby or adhered macromolecules, mem-
connected to a generator of high electric voltage (Figure 22). brane proteins, lipid phases and lateral domains, extracellular
protrusions, membrane reservoirs, and underlying cytoskeletal
linkages (see Figure 7B,C). It is currently not well understood
how these complexities influence the generation of defect
nucleation and growth under an electric eld.1049fi A recent
study to visualize the behavior of membrane defects in artificial
planar bilayers found that electropores form preferentially in
the liquid disordered phase.1068 This preference is also likely to
be true in live cells, but lack of experimental methods to
measure such phenomena has made it challenging to
validate.1068 Another mystery is the lifetimes of electropores
in live cells. Once hydrophilic pores of >1 nm open up in the
plasma membrane, they are thought to either spontaneously
close or require active cellular processes for the bilayer to heal.
For active repair processes, many researchers observe time
scales of seconds to minutes.1048,1069 The electroporation lit-
erature, however, suggests rapid shrinkage of pores after ces-
sation of the electric 1070field. A memory effect, where changes
in the membrane porosity remain on a longer time scale of
hours has also been suggested.1069 For further reading on the
Figure 22. Conventional parallel plate cuvette con guration for theory and mechanisms of electroporation as pertaining to livefi
electroporation of suspended cells (left). Zoom-in (right) shows the cells, we recommend other more comprehensive reviews on
the topic.163,419,544,579,1048,1049,1051,1054,1055,1069−1072approximate distribution of pores over the cell surface as a function of
orientation and polarization under applied electric field. The surface 6.2.3. Targeting Subcellular Structures Across the
area of poration and number of pores is greater on the hyperpolarized Pulse Strength-Duration Space. The parameter space for
side compared to the depolarized side. Further zoom-in (bottom) electroporation is vast. As discussed, there is no fixed threshold
illustrates the capacitor-like function of the lipid bilayer before electroporation voltage because formation of electropores
poration and the flow of positive charges once a conducting pore is depends on a combination of voltage strength, pulse duration,
formed (opposite movement of negative charges not shown). Electric number of pulses, pulse waveform, temperature, buffer con-
field lines are displayed in gray. ductivity, and cell properties.1048,1049 This large variable space
presents a challenge in optimizing electroporation. All other
This type of setup produces a near-homogeneous electric field variables being held constant, most approaches focus on tuning
across the cell suspension. Upon application of voltage, the the “pulse strength-duration space”.579 Manipulating this
various regions of the plasma membrane take different times to parameter space can exert a measure of spatiotemporal control
reach their characteristic trans-membrane threshold potentials. over which cellular membranes are permeabilized (Figure 23).
This results in growth of a heterogeneous distribution of pores In general, high voltage ultrashort pulses have been reported to
over the cell surface, both in terms of number and size. perturb internal and organelle membranes while longer and
Moreover, because of the negative resting potential of cells milder pulses emphasize permeabilization of the plasma mem-
(−35 to 80 mV for most cell types; see Figure 7A), brane and bias the effect toward larger cell types.579
permeabilization occurs first at the hyperpolarized side of the The charging time for the plasma membrane is on the order
cell facing the positive electrode.1048 This creates an inherent of 1 μs. It is even shorter in highly conductive buffers such as
anisotropy in the area and degree of permeabilization between PBS. Pulses of duration less than the plasma membrane
the two poles.1066 The hyperpolarized side of the cell is charging time are thought not to efficiently porate the plasma
supposed to carry smaller but more numerous pores. The membrane of a cell.419 For example, high voltage ultrashort
depolarized half, which faces the negative electrode, has fewer pulses in the nanosecond range may rupture subcellular
pores due to fewer nucleation events. The pores on the structures and organelles while leaving the plasma membrane
depolarized side may, however, be larger in diameter as the essentially untouched.458,579 A pioneering study by Schoen-
prolonged electrical field exposure is focused on expanding a bach et al. in 2001 demonstrated short nanosecond pulses at
AR DOI: 10.1021/acs.chemrev.7b00678
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>10 kV cm−1 field strength selectively target intracellular
organelles.1073 Specifically, human eosinophils were exposed to
a field strength of 53 kV cm−1 applied in a train of 5 pulses of
60 ns each. In response, the cells formed intracellular granules
even without extensive plasma membrane permeabilization.
Follow up studies by the same group indicated these nano-
second pulses induced apoptosis, as signified by exposure of
annexin-V at the cell surface and the absence of ethidium
homodimer fluorescence.1074 Further hallmarks of apoptosis
were observed with fluorescent probes that report on caspase
activation and the release of mitochondria-associated protein
cytochrome c into the cytoplasm. It is thought that apoptosis
occurs due to a release of cytotoxic factors from permeabilized
mitochondria and breakdown of intracellular calcium stores.
It was therefore concluded that apoptosis triggered by nano-
second pulsed electroporation can occur in the absence of
disruption to the plasma membrane. This is of widespread
interest for two reasons. (1) The targeted induction of apopto-
sis by ultrashort electrical pulses could avoid the immune
response associated with lysing or necrotic cells.1075 (2) For
intracellular delivery applications it is an effect that should be
avoided to maintain cell survival. Unwanted disruption of
intracellular organelles could explain observations of delayed
cell death that sometimes occur after high field strength elec- Figure 23. Relationship between the pulse strength-duration
troporation. parameter space and subcellular targeting. High intensity short pulses
As nanosecond pulses increase in duration, the chance of are biased toward perturbing small membrane bound bodies like
permeabilizing the plasma membrane also increases.1076 Pulses organelles, while milder, longer pulses are more specific for the plasma
− membrane and larger cells. At large field strengths and longerin the 1 10 ns range have less chance of permeabilizing the durations thermal damage due to heating becomes an issue, being also
plasma membrane, while pulses in the 10−1000 ns range tend
to generate very small pores (≤1 nm).477,1076,1077
dependent on buffer conductivity.
Conventional electroporation systems almost exclusively
target the plasma membrane. Short pulses in the microsecond 6.2.5. Cargo-Dependent Influx Mechanisms: Small
to millisecond range result in numerous but smaller sized pores Molecules. Small neutral molecules enter cells via diffusion163
distributed evenly over the poles of the plasma membrane and throughout the duration of a pore’s lifetime (Figure 24B).
sometimes nucleus.579 The longer pulse space >0.1 ms is If the molecules are charged, such as propidium iodide (PI,
limited to lower voltages; otherwise Joule heating becomes a ∼660 Da), which carries two positive charges, there is an
problem for treated cells, a factor also dependent on con- added electrophoretic component that can augment delivery
ductivity of the medium. Because voltages must be lower in during the pulse (Figure 24C). In this case, delivery will beaugmented at the side of the cell facing the positive electrode,
this regime, the dependence on size of the membrane-bound as PI will be attracted toward the negative electrode and into
body biases poration toward larger objects at their poles, the cell.1067,1116 Due to its small size and high diffusion
therefore favoring plasma membrane disruption of larger cells
579 coefficient, PI will also diffuse into the opposite side of the cell(>tens of micron diameter). At these longer durations the but to a lesser extent. Because the lifetime of the electropores is
membranes of larger cells such as skeletal muscle and nerve much longer than the pulse duration, diffusion has been
cells are much more responsive to electroporation. Taken observed as the dominant mechanism of entry with only a
together, data compiled from multiple reports suggest that minor contribution from electrophoresis.1117,1118 Electropores
manipulation of the pulse strength-duration parameter space is have been reported to remain open to small molecule diffusion
able to mediate a significant measure of control over the sub- for up to several minutes after pulsing.1084,1117
cellular localization and distribution of membrane disruptions For very small pore sizes (∼1 nm) diffusion alone may be
generated in cells (Figure 23). insufficient for influx of charged molecules. This is because of
6.2.4. Cargo-Dependent Influx Mechanisms. Electro- Born’s energy barrier, which describes the energetic cost of
poration has been used to deliver a diverse range of cargo mol- moving an ion or small charged molecule through a hole in a
ecules and materials to the intracellular space. This includes dielectric membrane.163,1119 The charged entity interacts with
dyes,100,742,1056,1078−1080 radiotracers,1081,1082 sugars,79,470,534,1083 the pore wall, increasing the energy required for translocation.
metabolites,1081,1084 poorly permeable drugs,55,56,1085,1086 For pore sizes close to the molecule size, the energy barrier for
ions,1087,1088 molecular beacons,1089,1090 proteins,100,546,1091−1097 crossing the membrane strongly correlates with the charge
antibodies,101,125,537,1098−1102 Cas9 protein or RNP com- number on the molecule. For example, Venslauskas et al. com-
plexes,143,144,146,147,1103 antisense oligonucleotides,1104 pared delivery of bleomycin (radius = ∼1.2 nm and charge =
siRNA,235,1105−1109 mRNA,257,260,261,1110,1111 plasmid +1) to tetra-sulfonato-porphyrin (TSPP, radius = ∼1.0 nm and
DNA,184,1112,1113 quantum dots,294,312,313,1114 and gold nano- charge = −4) under pulsing conditions designed to generate
particles.1115 The mechanisms of uptake of these cargoes vary only small pores.1120 Their experiments revealed that the
as a function of their size, charge, and conformational flexibility electric field strength required to deliver the more highly
(Figure 24). charged molecule, TSPP, was several times greater than for
AS DOI: 10.1021/acs.chemrev.7b00678
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Figure 24. Relationship between size and charge of cargo molecules and mechanisms of entry through a given pore size for electroporation.
(A) Depiction of approximate size and charge properties of molecules illustrated in scenarios from panels B to E. The depictions are based on
knowledge from the literature and explained in the text.
bleomycin. Other groups claim to have identified ultrashort proteins, antibodies and dextrans.1048 Most proteins and dextrans
pulse electroporation conditions (∼60 ns) where plasma mem- tend to be weakly charged or neutral; thus, the electrophoretic
brane pores are so small that they do not allow transmission of contribution is thought to be minimal. Early experiments with
PI, although they are conductive for smaller ions.1121 In such a proteins claimed efficient loading (>80% of cells), sometimes
scenario, an electric field pulse can help overcome Born’s up to micromolar cytoplasmic concentrations, in a variety of
energy barrier and promote influx. mammalian cell lines at high survival rates (>80%).101,1092,1122
6.2.6. Cargo-Dependent Influx Mechanisms: Proteins Dye-conjugated dextrans of known molecular weights (from 3
& Other Macromolecules. Diffusion is the most likely to 2000 kDa) have also been electroporated into cells to
mechanism underlying electroporation-mediated intracellular analyze delivery efficiency and decipher the rules governing
delivery of larger macromolecules (∼10−1000 kDa), such as uptake.532,1080,1091,1123−1125 In comparison to small molecules,
AT DOI: 10.1021/acs.chemrev.7b00678
Chem. Rev. XXXX, XXX, XXX−XXX
Chemical Reviews Review
Figure 25. Model for endocytosis of electroporation-induced DNA aggregates at the cell surface. During the electric field pulse, negatively charged
plasmid DNA is propelled into the side of the cell facing the negative electrode. Due to conformational flexibility some parts of the DNA may be
threaded through pores in the cell membrane. Aggregates are then endocytosed, from which they either escape and find their way to the nucleus for
the purpose of expression or are degraded by lysosomes.
which can diffuse into cells for minutes, proteins and larger proteins and dextrans to become aggregated or trapped at the
molecules (>10 kDa) exhibit a narrow window of opportunity plasma membrane.1092,1122 Such membrane-bound proteins
to enter cells, constituting just a few seconds.1095 It is known can be removed with the protease trypsin while dextrans could
that electroporation produces mostly small pores with a subset not, demonstrating that proteins were stuck to the cell surface
of larger pores that grow as a function of the pulse duration.419 and not inside the cell.1122 If electroporation causes cargo to
When the electric field is turned off the large pores shrink aggregate at the cell surface, this would make it amenable for
almost instantly, while the small pores may linger in the plasma uptake by endocytosis.1130 The degree to which this occurs for
membrane for minutes.163 Thus, the entry of larger cargo different cargo molecules, however, has not been thoroughly
coincides with the pulse timing and is more efficient for longer investigated.
pulse durations.532 The smaller pores that prevail for minutes 6.2.7. Cargo-Dependent Influx Mechanisms: Plasmid
are unable to facilitate di usive in ux of proteins.1126ff fl DNA. In contrast to small molecules, proteins, and dextrans,
Although less well-accepted, some researchers have proposed the mechanisms of nucleic acid delivery via electroporation are
alternative delivery mechanisms. For example, the imposed regarded to be almost entirely dependent upon electrophoretic
electric field might augment macromolecule delivery through forces provided during the pulse.163,1048,1131 In particular, the
electrophoretic or electro-osmotic e ects.1123,1127,1128ff The case of DNA plasmids has been extensively studied due to a
models based on electrophoresis, however, have not addressed broad interest in exogenous gene expression over the past
how they would be relevant to uncharged molecules. The decades.1132,1133 After pioneering efforts demonstrating DNA
electro-osmotic explanation, on the other hand, proposes that transfection in mouse cells in the early 1980s,184,1134 it was not
the application of an electric field causes a convective flow of until a decade later that researchers realized that plasmids were
electrolytes and osmotically obliged solution that sweeps the not immediately crossing the cell membrane but rather
cargo molecules along with it. Although discussed in some aggregating at the cell surface as a result of electrophoretic
papers, the few studies that have sought to investigate electro- forces (Figure 24E).531,1113,1135 A correlation between longer
osmotic contributions to molecular delivery in live cells are pulse durations, more prominent aggregates, and higher
inconclusive.1123,1127 Most of the electroporation literature transfection efficiency also lent support to this view.469,1135
favors explanations that emphasize cargo influx by diffusion or Moreover, it was observed that preadsorption of DNA to the
electrophoresis.163,1048,1049,1056,1129 cell surface dramatically increased transfection efficiency and
Another idea is that electroporation-stimulated endocytosis contributed to pore formation and stabilization, most likely by
via macropinocytosis may contribute to protein uptake in the spearing of plasmid molecules into the membrane.1135,1136
minutes following electric field exposure.1130 Strong electro- In 2002 Golzio et al. advanced our understanding of
poration treatments have sometimes been reported to cause electroporation-mediated plasmid transfection with single-cell
AU DOI: 10.1021/acs.chemrev.7b00678
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Chemical Reviews Review
Figure 26. Schematic of the mechanisms of influx in relation to disruption size, molecule size, molecule charge, and conformational flexibility. For
charged objects approaching the disruption size or larger, electrophoretic forces are crucial for delivery. (A) Shown is the case for a molecule much
smaller than the size of the membrane disruption. Regardless of charge, delivery is mostly via diffusion. (B) Shown is the case for a negatively
charged molecule of similar size to the transient membrane disruption. Delivery requires an electrophoretic driving force. (C) Shown is the case for
a flexible molecule (here a DNA plasmid) that is much larger than the membrane disruption. Electrophoretic force can thread part of the molecule
into the cell.
imaging experiments that visualized the interaction of DNA at Overall, there are a protracted series of steps required for
the cell surface during electroporation.1137 It was found that electroporation-mediated transfection and many of them require
DNA aggregated exclusively on the side of the cell facing the membrane trafficking and other active cellular processes. Only a
negative electrode (cathode) and formed localized clumps of small fraction of electroporated DNA vectors will arrive in the
0.1−0.5 μm in size. At the cell surface, it is believed that the nucleus for successful expression.1132 Despite this, electro-
highly negatively charged DNA plasmids are threaded through poration is one of the few membrane disruption-based meth-
small pores where they become stuck in the negative electrode- ods that can achieve high rates of DNA expression in millions
facing region of the plasma membrane.1067,1135,1136 These of cells at acceptable throughputs. Several other methods are
aggregates are then internalized via endocytosis over tens of able to introduce DNA to the cytosol, but it is often unable to
minutes. Some of the plasmids eventually arrive at the nucleus migrate through the tight cytoplasmic meshwork and is there-
over a timecourse of ∼2 hours or longer.1132 Collectively, these fore degraded before reaching the nucleus, as has been shown
results led to the emergence of an endocytic model of plasmid for plasmids after microinjection.
202 In rare cases electropor-
electrotransfer that has mostly gained acceptance (Figure 25). ation appears to mediate rapid expression of plasmids within1143
As membrane remodeling via endocytosis is a core pathway an hour. Most often, however, it takes anywhere from 4 to1132,1140
used by cells to repair their membranes,447,455 endocytic 24 hours for peak expression. Electroporation’s para-
uptake could be an active cellular response to the perturbation digm of plasmid aggregation and endocytosis may thus serve to
caused by DNA entanglement in the membrane, as earlier concentrate and protect DNA for the prolonged journey to the
predicted by Tsong and colleagues.1070 Subsequent studies cell nucleus.
have shown that, in CHO cells for example, ∼50% of DNA is 6.2.8. Cargo-Dependent Influx Mechanisms: siRNA &
internalized by caveolin/raft-mediated endocytosis, ∼25% by Other Oligonucleotides. Electroporation-mediated delivery
clathrin-mediated endocytosis, and ∼25% by macropinocyto- of oligonucleotides and siRNA is similar to the case of DNA in163
sis.1138 Within 2 hours, more than half of the DNA ends up in that it also relies on electrophoretic forces. A key distinction,
however, is that siRNA undergoes direct delivery into the
lysosomes, as revealed by colocalization with the lysosomal
1138 cytoplasm without relying on endocytosis (Figure 24D). Thismarker LAMP1. Furthermore, single-particle tracking is by virtue of its smaller dimensions (2 × 7.5 nm)219
experiments of fluorescently labeled plasmids indicate that compared to DNA plasmids (∼100−200 nm;206 see Table 1).
cytoskeletal processes, involving both actin and microtubule Imaging of fluorescently labeled siRNA has shown that it
networks, are involved in trafficking of DNA-associated
1138−1140 enters during application of the electric field at the side of theendosomes toward the cell nucleus. It is important cell facing the cathode and disperses throughout the cytosol
to recognize, however, that many of the basic mechanisms of within tens of seconds.1144 siRNA influx was reported not to
the plasmid trafficking from the membrane to the nucleus
1131,1132 occur after cessation of applied voltage, indicating that elec-remain underexplored and poorly defined. trophoretic forces are probably required for delivery.1144 For
How plasmids enter the nucleus is poorly understood, short (10 ns) pulses applied to GUVs, influx of siRNA also
as DNA plasmids are invariably many times larger than the relied on electrophoretic drive and some siRNA remains trapped
∼40 kDa cutoff for passive influx through nuclear pores. DNA in the bilayer at the end of the electric pulse.1145 However,
transfection is known to be greater in proliferating cells that electrophoretic driving forces might only be necessary for
undergo transient nuclear envelope breakdown through mitosis, delivery through small pores of <10 nm where there is a Born
which allows plasmids to be entrapped inside the freshly energy barrier.163 Such a scenario is analogous to that
reformed postmitotic nucleus.1141 The revelation that nuclear discussed for small charged molecules (Figure 24C). It is
membrane disruptions are not an uncommon event in the life likely that transient large pores (>10 nm) can facilitate entry of
of a cell, and thus generate a stochastic pathway of exchange siRNA via free diffusion, since siRNA knockdown has been
between cytosol and nucleus, could also provide clues.789,790 observed with membrane disruption-based methods that lack
Alternatively, internalization motifs, such as nuclear targeting electrophoretic forces, including with pore-forming toxins,238
sequences, have been reported to promote import of plasmids micro 108,780fluidic cell squeezing, and laser-nucleated cavita-
into cell nuclei with varying success rates.1142 tion bubbles.923 Therefore, one can speculate that siRNA
AV DOI: 10.1021/acs.chemrev.7b00678
Chem. Rev. XXXX, XXX, XXX−XXX
Chemical Reviews Review
Figure 27. Examples of dual-pulse electroporation protocols from the literature. (A) The −1first pulse has a field strength of 1 kV cm and duration
of 1 ms. The second pulse is 0.3 kV cm−1 in strength and 10 ms in duration. Figure reprinted from ref 1124. Copyright 2014, with permission from
Elsevier. (B) Schematic of a pulse sequence consisting of AC first followed by a preprogrammed delay then a second DC pulse of lower voltage.
In this case, the first pulse is 1 ms and the second one is 30 ms. Figure reprinted from ref 1125. Copyright 2015, with permission from Elsevier.
delivery is mediated by a combination of electrophoretic and/ Electroporation can therefore be viewed as a balancing act
or diffusive mechanisms depending on the size and lifetime of between a large number of parameters and conditions. There is
the pores. significant debate surrounding the optimal electroporation pro-
6.2.9. Summary of Cargo-Dependent Influx Mecha- tocols for intracellular delivery, and this is further complicated
nisms. Taken together, the literature indicates electro- by variation between cell types.1147,1148 Another issue is the
poration-mediated intracellular delivery is influenced by the lack of understanding associated with postelectroporation cell
pore diameter (ddisruption) and the cargo dimensions (dmolecule), death, where loss of viability sometimes manifests after hours
as well as the charge and conformational flexibility of the cargo or even days.490,529 This is an especially striking problem
molecule (Figure 26). For d 1149molecule ≪ ddisruption both neutral and regarding electroporation of primary or sensitive cell types.
charged molecules should diffuse across their concentration When wanting to optimize the delivery of a particular cargo
gradient whilever the pore is large enough. Although the molecule into a specific cell type, the starting point is usually
majority of delivery is via diffusion, electrophoretic or electro- to screen three core parameters: (1) field strength (voltage),
osmotic phenomena may assist translocation during the pulse. (2) pulse duration, and (3) number of pulses.
For dmolecule ≈ ddisruption charge will play a critical role. Neutral Based on a large number of electroporation studies, several
molecules may diffuse through pores while their charged types of pulsation strategies have been devised. In a review by
counterparts will face the Born’s energy barrier, only being able Gehl,1048 three categories of approaches for DNA transfection
to translocate while driven by sufficient electrophoretic forces. were described, all of which have achieved some measure of
For the case of dmolecule ≫ ddisruption only molecules that are success: (1) exclusively short, high-amplitude pulses,184,1113,1150
both conformationally flexible and significantly charged will for example, a series of six pulses of 100 μs at field strengths of
have a chance of penetrating. As exempli ed by the case of 1.4 kV cm−1;1151fi (2) exclusively long, low-amplitude
DNA plasmids, parts of the molecule may be threaded into pulses,469,532,1112,1135 for example, eight pulses of 20 ms at
pores and therefore become embedded in the membrane. This eld strengths of 0.2 kV cm−1;1152fi and (3) a short, high-
makes the molecule available to be taken up via endocytosis, a amplitude pulse followed by a long, low-amplitude pulse;1153
result that may or may not be desirable for a given application. for example, a first pulse of 10 μs at 6 kV cm−1 followed up
6.2.10. Tailoring Pulse Parameters for Optimal with a second pulse of 10 ms at 0.2 kV cm−1 as pioneered by
Delivery. In the most elementary electroporation scenario, Sukharev et al.531 The rationale behind this dual pulse strategy
one wants to open up pores of sufficient size and duration to is that the first pulse is thought to nucleate many pores over a
allow the desired influx of cargo molecules via diffusion. In large segment of the cell surface, while the second pulse should
more complicated cases, however, involving charged molecules simultaneously grow the pores and electrophoretically propel
close to or larger than the pore size (Figure 26), the efficiency of charged molecules into the cell. Indeed, several studies have
delivery depends critically on magnitude and duration of elec- confirmed that the duration of the second low-voltage pulse
trophoretic forces.469,531,532,1113,1135 Regarding plasmid deliv- correlates with DNA transfection efficiency.531,532,1135
ery, for example, longer pulse durations are often found to 6.2.11. Dual Pulse Strategies. The dual pulse strategy
heavily improve transfection efficiency. Yet longer pulses can has captivated considerable attention from the field and
bring the problem of Joule heating and excessive cell inspired a number of further investigations.1124,1125,1150,1154,1155
damage.1146 One strategy to mitigate Joule heating is the use Figure 27 shows examples of pulse parameter sequences that
of low conductivity buffers that have lower electrolyte con- constitute typical dual pulse strategies. The first example
centrations than standard physiological buffers or media. The consists of two consecutive DC square wave pulses (Figure
osmolarity of the buffer will have an effect as well, because it 27A),1124 while the second uses an AC signal for the first pulse
can alter the size of the cell, tension on the plasma membrane, followed by a delay then a second low-voltage DC pulse
conformation of membrane reservoirs, and the interaction (Figure 27B).1125 The AC pulse is designed to increase the con-
between cargo molecules and the cell surface.1049 Temperature sistency of permeabilization at each pole of the cell and reduce
will also affect the properties of the membrane and energy side effects at the electrodes. These reports are a few among
barriers for electroporation, as well as the active cell response many to suggest that dual pulse strategies optimize delivery
and membrane repair dynamics.1049 while preserving cell viability not only for DNA transfection
AW DOI: 10.1021/acs.chemrev.7b00678
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but also for delivery of other molecules like proteins and high stem cells,1156,1163,1164 human monocyte-derived dendritic
molecular weight dextrans.1124,1125 cells,1165,1166 monocytic cell lines,1159 primary leukemia cells
6.2.12. Nucleofection Mechanisms. Nucleofection, one and cell lines,1167,1168 primary natural killer cells and cell
of the most popular electroporation systems of all time, was lines,1169,1170 primary lymphocytes,1171,1172 embryonic and
introduced in the early 2000s and rapidly gained traction as an adult stem cells,1173,1174 and mammalian neurons.1157,1175 These
effective intracellular delivery method. It is based upon a papers and others contributed to the emergence of nucleofection
classical cuvette configuration with parallel plate electrodes, as a leading method for transfection of recalcitrant cell types.
but the novelty comes from the systematic selection of optimal Overall, there are many examples of pulsing strategies that
pulsing parameters and cell-type speci c bu ers.550,1156,1157fi ff have been successfully employed to electroporate molecules
Although the exact pulsing parameters are proprietary, patents into cells.163,1048,1049,1132 Nucleofection is but one example of a
indicate that it is based around a dual pulse approach.1158 The dual pulse strategy that has been systematically honed for
first pulse is administered at field strengths of 2−10 kV cm−1 application with a wide range of cell types, including difficult-
for durations ranging from 10 to 100 μs. The second pulse lasts to-transfect cells. A deeper understanding of the mechanisms
1−100 ms at a lower, unspecified, field strength. Dozens of of electroporation phenomena on both cells and cargo
different pulsing protocols are programmed into the nucleo- molecules could yield even further advancements in delivery
fector control unit, presumably based on variations on this performance and cell health.
theme. The user then finds optimal electroporation con- 6.2.13. Electroporation Challenges & Technical
ditions by screening the programs against delivery and viability Advancements. As with most membrane disruption-based
outcomes for different cell types. To facilitate best results, cell intracellular delivery strategies, a major challenge with elec-
type-specific buffers are also recommended. Patents on nucle- troporation is cell mortality post-treatment. Cell death may
ofection buffers report near-physiological concentrations of occur immediately due to irreversible electroporation, lysis, or
extracellular (high) Na+ and (low) K+ augmented by >10 mM excessive thermal damage490 (see Figure 11). Or it may take
Mg2+ and robust pH bu 550ffering. This is in contrast with the the form of a delayed necrosis, possibly due to failure of mem-
trend of literature promoting the benefits of low conductivity brane repair or prolonged apoptotic responses, taking place
buffers featuring organic osmolytes1148 or high K+ cytoplasm- hours or days after treatment.529 As an example of this
mimicking bu ers.548ff The high conductivity of nucleofection problem, early reports on nucleofection of human monocyte-
buffers (ionic strength >200 mM) is thought not to cause Joule derived dendritic cells yielded unprecedented plasmid trans-
heating problems due to the emphasis on small volumes and fection results, with up to 60% gene expression. However,
shorter pulse durations.550,1157 Users have reported adapting long-term functional assays indicated that cells were hampered
nucleofection for use with phosphate buffered saline without a by gradual loss of proliferative potential and poor viability.1165
decline in performance.552,554 A number of publications share In this section, we discuss the problems with electroporation
protocols for homemade nucleofection buffer formulations to and the efforts that have gone into reducing its toxic burden on
increase transparency of the protocols and lower costs.551,552 cells.
A notable appeal of the nucleofector system has been the 6.2.13.1. The Problem of Joule Heating. When electric
assertion that it delivers plasmid DNA rapidly and directly to current passes through an aqueous solution, it triggers tem-
the nucleus.1156,1159 This speculation, however, is controversial perature increase (Joule heating) concurrent with various chem-
and difficult to find experimental support for in the literature. ical reactions at the solution−electrode interface (electrolysis).
An alternative explanation is that endocytic trafficking directs Electrolysis itself produces changes in the temperature, pH,
DNA to the nucleus, as has been observed for other types of and the chemical composition of the adjacent solution. The
electroporation.1132 A number of factors lend credence to the degree of Joule heating is influenced by the conductivity of the
endocytic explanation. First, the cytoplasm is a highly crowded buffer, electrode architecture, electric field parameters, and
and viscous environment laced with cytoskeletal filaments and capacity of the system for dissipation. For cuvette style setups,
organelles. The mobility of microinjected plasmid DNA is temperature spikes of more than 30 K above ambient
extremely small or even negligible in the cytoplasm or cell conditions have been measured in physiological saline at
nucleus.1160−1162 To be electrophoretically propelled through millisecond pulse durations.1146 Such observations have led
the cytoplasm into the nucleus, a combination of significant some researchers in the field to assert that Joule heating is a
plasmid compaction and large electrophoretic forces would signi cant problem.579fi For example, an 8 kV cm−1 pulse of 100 μs
potentially be required, although this has not been directly has been calculated to lead to a temperature increase from 23
proven.1132 Second, the reported timing of gene expression is to 42 °C in PBS solution.579 Lipid membranes and proteins are
in the range of 6 h after treatment,1156,1159 which is actually destabilized by temperatures above 42 °C.1176 Therefore, Joule
longer than achieved with standard electroporation that relies heating is not just an issue for the plasma membrane but also
on endocytosis.1132 In contrast, microinjection of DNA directly for intracellular membranes and proteins throughout the cell.
into the nucleus can mediate gene expression within 30 min. To mitigate the negative effects of Joule heating, electro-
Some authors have speculated whether nucleofection per- poration procedures can be performed at room temperature
meabilizes the nucleus with its first high-voltage pulse, thus (20−25 °C) or on ice (0−4 °C). Lower temperatures,
assisting in nuclear delivery.105,579,1157 This hypothesis has not however, make cells more resistant to pore formation,574,575
been rigorously tested in experiments to date. thereby reducing delivery efficiency. Another approach to
Regardless of the actual mechanisms, nucleofection has combat Joule heating is to use low-conductivity buffers, which
shown significant success rates for DNA transfection and feature lower concentrations of electrolytes and instead maintain
expression in traditionally di cult-to-transfect cell types.1108ffi osmolarity by inclusion of organic osmolytes or sugars like
This has been demonstrated in various types of stem cells, sucrose and mannitol.532 Low-conductivity buffers reduce
primary cells, and postmitotic cells, for example: primary human Joule heating while enabling the long pulses that are preferred
melanocytes, smooth muscle cells, chondrocytes, mesenchymal for some protocols, such as for DNA transfection.
AX DOI: 10.1021/acs.chemrev.7b00678
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6.2.13.2. The Problem of Metal Contamination. A number fraction they behave as single cells, while for volume fractions
of publications have assessed the detrimental effects of metal greater than 10% (or for clusters of cells), the suspension density
ions released into solution by electrolysis.574,1177−1180 For large will distort the conferred transmembrane potential.1087,1189
surface area electrodes, such as cuvette style electroporation 6.2.13.5. Counteracting Electrolysis. The above-mentioned
chambers, the most commonly used materials are aluminum, studies show that electrolytic effects and electrode corrosion
copper, and stainless steel. Analysis of stainless steel and are a critical consideration for electroporation. This is espe-
aluminum electrodes found that, after a train of pulses similar cially important for cells and biological material bound for
to a standard electroporation protocol, metal ions were found medical applications, such as for cell-based therapies. Tactics
in solution at up to milliMolar concentrations.574,1178,1180,1181 that may be used to mitigate the negative effects of electrolysis
Aluminum ions and aluminum hydroxides can wreak havoc on include lowering solution conductivity, changing the pulsing
cellular processes, such as inositol phosphate activity.574,1177 schemes, buffering more strongly against pH changes, and
Moreover, Stapulionis et al. found that released copper, iron, reducing the surface area of electrodes adjacent to cells.
and aluminum ions can interact with nucleic acids and cause Another strategy is to switch the polarity of electrodes between
their precipitation out of solution.1178 Other studies have found successive pulses, which has been shown to minimize
Fe2+/Fe3+ to be toxic to in vitro cell cultures at milliMolar cumulative electrolysis and decrease the contamination of
concentrations.1180 Fe2+/Fe3+ ions released from the anode metal ions in solution by an order of magnitude.1180 The idea
behave as Lewis acids and hydrolyze the water molecules in the of using more inert gold or platinum or replacing metal
solution. This effect can reduce pH and potentially alter the electrodes with plastic, graphite, or liquid systems has also
medium conductivity.1181 Metal ions released from the elec- been explored.
trodes can also contribute to local distortion of the electric 6.2.13.6. Cell Damage from the Electric Field. Aside from
field, further compounding the problems associated with metal cell damage due to electrolytic effects (e.g., Joule heating, con-
ion contamination.1182 tamination via corrosion of electrodes, and pH changes), the
6.2.13.3. The Problem of pH Changes. As touched upon electric field itself may harm cell components more directly.
previously, pH changes that take place at the electrodes can For example, the application of strong electric fields to cells has
have a substantial impact on cell health. The changes in pH been suggested to trigger lipid peroxidation,1190−1192 gen-
values in solution have been measured to exceed 1−2 pH units eration of reactive oxygen species,1193,1194 protein denatura-
under conditions similar to those used in standard electro- tion, and DNA damage1195,1196 among other responses. Under
poration.1183 As with Joule heating, any shift in pH (ΔpH) electroporation conditions compatible with cell survival, it was
depends on the medium conductivity. ΔpH of a solution shown that electroporation can trigger an “oxidative jump” where
in which sucrose was substituted for NaCl was reported to the level of reactive oxygen species (ROS) rises sharply.1193 The
be about 5 times less than phosphate buffered saline. The measured generation of ROS was to some extent dependent on
electrode material also contributes, with aluminum cathodes extracellular calcium and magnesium but could be prevented
yielding a 2-fold greater ΔpH in comparison with platinum, by addition of antioxidants. In subsequent studies, lipid perox-
copper or stainless steel cathodes. Such results led to a recom- idation, as evidenced by the presence of lipid hydroperoxides,
mendation against using aluminum electrodes.1183 Several was observed in the membranes of both plant and animal cells
studies have successfully visualized the changes in pH at elec- following electroporation.1191,1192 Further investigations using
trodes by using pH sensitive dyes.574,1184,1185 Acidic fronts the chemiluminescent probe lucigenin found that CHO cells
form at the anode while the cathode becomes basic. A study by subject to millisecond pulses undergo a threshold level of
Li et al. used microchip-based electroporation to determine oxidation to their plasma membrane lipids but that this effect
that hydroxyl ions at the cathode are more toxic than protons at only partially correlates with cell survival.1194 Interestingly,
the anode.1185 They observed that strong pH buffering can, to lipid peroxidation of unsaturated phosphatidylcholine species
some extent, neutralize the problem, thereby bringing cell has also been observed during electroformation of giant
viability up above 90% in comparison to 60% for inadequately unilamellar vesicles.1197 Membranes characterized by a high
buffered and 40% for unbuffered solutions.1185 The idea of degree of peroxidized lipids tend to be weaker and more
switching the polarity of electrodes between pulses has also been susceptible to disruption, including by electroporation.1198
suggested to prevent cumulative pH biases at the electrodes.1183 Indeed, lipid peroxidation is well-known to influence mem-
6.2.13.4. The Problem of Non-Uniformity in the Electric brane behavior, including domain formation and mechanical
Field. Nonuniformity of the electric field can cause some cells properties, which could have implications for cell recovery
to be treated too harshly while others are insufficiently postelectroporation.
permeabilzied. Indeed, significant heterogeneity in electro- The reactive oxygen species produced by electroporation
poration arises due to a lack of consistency within the electric will not only target lipids but can also degrade proteins and
eld.1049,1146fi One effect of excessive electrolysis is deterio- nucleic acids. DNA damage in proportion to the applied
ration of the electrode performance. For example, a study with voltage and duration has been reported in HL60 cells, although
stainless steel electrodes in parallel plate geometry showed no specific mechanisms were pinpointed.1196 It could be that
significant pitting of the anode.1186 The increase in the DNA damage was due to influx of oxidative agents from the
roughness of the electrode was proposed to contribute to extracellular environment. Regarding proteins, Chen and col-
heterogeneity and loss of consistency of the field applied across leagues have suggested nonthermal electroconformational
the cell suspension. Subsequent studies also showed that the damage to ion channels following exposure to strong electric
pulsing frequency and the presence of chloride ions a ected elds.1199−1202ff fi More general models describing electrocon-
the corrosion of iron electrodes.1187 Furthermore, in a dense formational damage of membrane proteins and other cellular
suspension of electroporated cells, neighboring cells will affect components have subsequently been described.1203,1204 In par-
the geometry of the electric field due to mutual electrical ticular, it was proposed that charged amino acids in membrane
shading.1147,1188 When cells represent 1% of the volume proteins or voltage-sensing segments in voltage-dependent
AY DOI: 10.1021/acs.chemrev.7b00678
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Figure 28. Electroporation (EP) configurations. (A) Bulk (conventional) electroporation in parallel plate cuvette (i) and capillary (ii) geometries.
(B) Microscale electroporation examples showing (i) electroporation in droplets, (ii) the use of channel architecture to manipulate voltage pulses,
(iii) hydrodynamic focusing to generate liquid electrodes, and (iv) hydrodynamic vortices to rotate cells through electric fields for more
homogeneous permeabilization. (C) Nanoscale electroporation with examples of (i) nanochannel electroporation, where cells are pressed against
nanoscale apertures; (ii) nanostraw electroporation, in which the electric field is concentrated onto the end of a nanostraw; and (iii) nanofountain
electroporation, which exploits a hollow AFM tip for addressing individual cells.
transporters could be vulnerable to sharp changes in electrical species generated by nanosecond pulsed electric elds.1209fi
potential. These effects were thought to be more pronounced for Although undesirable for intracellular delivery, nonthermal elec-
shorter pulses of higher amplitude.1205 Indeed, several studies trical destruction of proteins, cells, and tissue has been proposed
showed that high voltage nanosecond pulses are likely to perturb for a host of other medical and industrial applications.1210
the function of voltage-gated channel proteins1206 and possibly Molecular dynamics simulations have shown that the
other proteins in general.1207 presence of hydrophilic pores can augment the process of
Although not typically used for intracellular delivery, nano- lipid flip-flop, whereby lipids translocate from one leaflet of a
second pulsed electric fields are of interest for understanding bilayer to the other.410 Partial abolition of the naturally uneven
how electric fields can affect cells on different time scales bilayer distribution of lipids has been observed in RBCs as a
and in various compartments. One study examined generation consequence of electric fields.1211 Vernier and others found
of ROS in response to nanosecond pulsed electric fields that nanosecond electric pulses can facilitate phosphatidylser-
(30 kV cm−1 at 100 ns).1208 They found that ROS was ine (PS) exposure to the outer leaflet within seconds,1212−1214
inhibited by both calcium chelators, and the antioxidant trolox, indicating a biophysical mode of action rather than cell sig-
in agreement with earlier observations that the presence of naling. Rols et al. performed a follow-up study with millisecond
divalent ions appears to participate in ROS generation.1193 permeabilizing pulses to examine membrane disorganization
Other reports have shown that H2O2 is among the damaging and phospholipid scrambling.
1215 Under the chosen conditions,
AZ DOI: 10.1021/acs.chemrev.7b00678
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Chemical Reviews Review
PS exposure could not be detected. The threshold conditions also means that gold plating becomes economical. Chemical
that trigger PS exposure thus remain to be precisely determined; stability of the electrodes is superior to those made from less
however, it appears that PS scrambling may only be relevant inert metals like iron, aluminum, or copper. The inventors
under regimes of very high field strength. Scrambling of the compared pH deviations in the capillary system to those of
membrane asymmetry has implications for the long-term conventional cuvette style chambers, and it appeared to confer
survival of cells, particularly in vivo where immune recognition substantial advantages in protecting cells from the toxic
mechanisms tend to destroy cells exhibiting wayward external- electrolytic processes that can occur at electrodes. Together
ization of lipids. these features are thought to increase the viability of cells
6.2.13.7. Cargo Incompatibility with the Electric Field. treated in capillary electroporation systems.558
Apart from damage to the cell, administration of the field On the other hand, one disadvantage of the Neon system is
strengths commonly used for electroporation may also cause the reduced flexibility in determining pulse parameters. The
problematic issues with the cargo molecules. For example, the pulse duration is limited in the range 1−100 ms and voltage
Bhatia group reported aggregation of quantum dots upon from 500 to 2000 V. Given the distance of the conductive path
electroporation, indicating it is not a suitable technique for between the electrodes, this means the field strength does not
intracellular delivery of quantum dots.294 Electric pulse- exceed 1 kV cm−1. The user may increase the number of pulses
induced precipitation of nucleic acids and other biological but there is no option to program pulses of different voltage or
macromolecules has also been observed under certain con- frequency. Thus, the dual pulse strategies that have become so
ditions,1178,1216 although it is unclear why other groups have popular with the Nucleofector system are not possible with the
not seen such problems. These studies noted that nucleic acids Neon platform. High cost of capillary tips, electrodes, and
aggregated into a nonfunctional state under the conditions of buffers is another factor that users dislike.559 In response to
their experiment. If they can be identified, it seems likely that this, some researchers have published protocols advising users
the conditions leading to precipitation must simply be avoided. on how to recycle the components and employ homemade buf-
Nevertheless, it is worth noting that not all molecular cargo can fers, such as one consisting of PBS supplemented with 250 mM
be assumed to be compatible with strong electric fields. sucrose and 1 mM MgCl .5592
6.2.14. Technical Innovations: Bulk, Micro- & Nano- 6.2.16. Microfluidic Electroporation. Motivated by the
Electroporation. The early generation of electroporation sys- shortcomings of conventional electroporation equipment, a
tems were con 1134figured with a cuvette style geometry. Sub- number of researchers and engineers have explored alternative
sequently, the first commercial electroporator, the BioRad solutions. Electroporation combined with microfabricated, micro-
Gene Pulser, was launched with this configuration in the mid- fluidic, and nanotechnology concepts has received a great deal of
1980s. Since then, the cuvette geometry has become the attention in the last two decades as evidenced by a spate of
standard platform for electroporation, being simple, robust, reviews on the topic.1217−1222 Compared to bulk electroporation
and reasonably well understood (Figure 28A(i)). The nucle- systems, it has been argued that micro- and nano- electroporation
ofector is no exception, and as discussed previously, its novelty can provide the following advantages:1220,1221 (1) lower voltages
arises not through a deviation from this geometry but rather due to smaller scale, thus obviating the need for high powered
from the systematic use of optimized pulsing protocols and cell pulse generators, (2) ability to concentrate, trap, and position
type-specific buffers. Despite its widespread adoption, however, cells and cargoes for higher efficiency delivery, (3) real time
the cuvette style geometry is not without problems. As discussed monitoring of device performance at single cell level, and
above, the large surface area of the metal electrodes presents (4) scalable solutions from single cells up to large populations.
issues concerning electrolysis, such as Joule heating, corrosion, One of the first microfluidic electroporation systems was
pH deviations, and inconsistent field profile. Second, cuvette constructed by Huang and Rubinsky in the late 1990s.1223
style electroporation is difficult to perform with low volumes It was essentially a small hole of 2−10 μm diameter that a
(<20 μL). As the intracellular delivery of a molecule via per- single cell could be sucked onto. The application of an electric
meabilization is directly related to extracellular concentration, pulse from below was used to permeabilize the basal side of the
it is often advantageous to concentrate the cells into a minimal trapped cell and study the mechanisms of electroporation
volume in the range of 10 μL or less. This maximizes the con- at single cell level. Although only demonstrated as a proof of
centration, which is especially useful for expensive or precious concept, such developments spurred the field on toward fur-
reagents. Below we discuss the innovations that have devel- ther efforts. Several years later the first microfluidic flow elec-
oped in the electroporation field, including different setups for troporation devices appeared on the scene. Huang and Rubinsky
bulk, micro-, and nanoelectroporation. were again pioneers in this department, demonstrating loading
6.2.15. Capillary Electroporation. One of the first of small molecule dyes and transfection with GFP-encoding
commercial setups to challenge the dominance of the cuvette plasmids, albeit at low throughput.1224 In the following, we will
style geometry came in the form of capillary electroporation highlight several examples of flow-based microfluidic electro-
(Figure 28A(ii)). This design was introduced by a company poration.
called NanoEntek in Korea and subsequently commercialized Droplet-based microfluidics enables the use of microscale
by Invitrogen/Thermo Fisher as the “Neon” electroporation compartments to expose cells to a particular chemical envi-
system.558 In the Neon system, cells and bu er solution are ronment within picoliter reaction volumes.1225ff Zhang et al.
pipetted into a narrow capillary (0.56 mm wide and 30 mm encapsulated cells in aqueous droplets before flowing them over a
long) featuring a wire gold electrode with minimal surface area pair of electrodes subjected to a constant DC voltage.1226 Due to
at the top. The other electrode, also made of gold, is located the nonconductivity of the oil phase, cells only experience a
within a conductive electrolyte bath outside the capillary. Because transient electric pulse when the conductive droplets pass the
of the small surface area and distance from the cells, bubbles, electrodes (Figure 28B(i)). The cell is then permeabilized to
Joule heating, and pH waves are more effectively separated the molecular cargo loaded within the droplet. In this case a
from the electroporated cells. The small size of the electrodes DNA plasmid encoding for GFP was successfully delivered
BA DOI: 10.1021/acs.chemrev.7b00678
Chem. Rev. XXXX, XXX, XXX−XXX
Chemical Reviews Review
into CHO cells.1226 The pulse parameters were governed by appears to introduce agents faster and deeper into the cyto-
the flow speed, size of the droplet, distance between the elec- plasm, a result attributed to enhanced and concentrated electro-
trode pair, and the positioning of the cell inside the droplet. phoretic forces. In support of this, finite element simulations
Owing to the rise in droplet-based microfluidics for high- found that fringe fields extend into the cell and could possibly
throughput single-cell manipulation, techniques that can per- be used to propel molecular cargo through the permeabilized
form intracellular delivery on cells within droplets are expected section of the cell periphery and deep into the cytoplasm.
to be important. Compared to conventional electroporation and other forms of
In a second example of flow-based microfluidic electro- microfluidic electroporation, it was proposed that the nano-
poration, electric pulse parameters are again determined by the channel delivery mechanism was based on electrophoretic forces
device geometry and flow speed under constant DC voltage. rather than diffusion and/or endocytosis. Nanochannel elec-
However, in this case electroporation occurs at narrow con- troporation was able to deliver dyes, oligonucleotides, siRNA,
strictions within the main flow channel.489 The geometry of plasmids and quantum dots into recipient cells. Moreover, only
the device channel controls the field amplification so that cells nanochannel electroporation could deliver quantum dots into
experience an electric pulse as they passage through a con- Jurkat cells, while conventional or microfluidic electroporation
striction (Figure 28B(ii)). Pulse duration imposed on the cell could not. One drawback of the method, however, was the low
is therefore determined by flow speed, while amplitude is given throughput nature of the technique. Prior to electroporation
by width ratio of the constriction to main channel. The number each single cell required placement against the nanochannel
of constrictions in series will effectively determine the number with optical tweezers.
of pulses. In subsequent efforts, Geng et al. scaled up this In 2016 the Lee group published a scaled-up version of
concept to process 20 mL min−1 of cells in continuous flow nanochannel electroporation able to process up to 40 000 cells
mode with a minimalist setup featuring low-cost components, a on a single chip over a 1 cm2 area.1234 In this version, termed
syringe pump, and a benchtop DC power supply without the “3D nanochannel electroporation”, the aperture dimensions
need for a pulse generator.1227 For plasmid transfection in were expanded to 300−650 nm. Positive dielectrophoresis was
CHO cells a transfection efficiency of up to 75% was achieved. employed to simultaneously position thousands of cells across
In a different microfluidic electroporation strategy, hydro- the array and press them against the nanochannel openings.
dynamic flow focusing was exploited to create parallel laminar This was necessary because a tight seal between the cell
flow streams of different conductance (Figure 28B(iii)). Using membrane and nanochannel is critical to ensure consistent
a three-inlet approach, the top and bottom sheath flows were electroporation performance across the device. Molecules to be
composed of highly conductive 3 M KCl solutions, which loaded are filled into a reservoir below the substrate and
acted as liquid electrodes, while cells in standard aqueous solu- delivered into cells concurrently with application of the electric
tion were flowed through the center of the configuration.1228 field. The system was used for transfecting plasmid DNA into
By applying a DC voltage of only 1.5 V, electric field intensities batches of natural killer cells, which are otherwise difficult to
of more than 1 kV cm−1 could be generated across the central transfect. A predecessor to this idea was published in 2006 by
zone to electroporate the passing cells. The device showed up to Kurosawa et al. using an insulating substrate with an array of
70% delivery efficiency of 1228 1235fluorescein dyes into yeast cells. 2 μm holes in it. Just like in 3D nanochannel electro-
Moreover, distancing the metal electrodes from cells using poration, the field was concentrated at the holes and molecules
hydrodynamic focusing had the advantage of separating cells to be delivered were supplied from underneath. This design is
from electrolysis issues such as heating, bubble generation, essentially a scaled-up version of the original microfluidic
pH changes, and production of toxic ions.1228 Thus, the use of electroporation system published by Huang in 1999.1223
nonmetal liquid electrodes in hydrodynamic flow mode may The Lee lab also published a series of papers where cells
mitigate some of the problems associated with cuvette-style were sucked into microchannels made of PDMS. In effect, this
electroporation. design was not too dissimilar from a parallel array of micro-
In a fourth example of microfluidic ingenuity, a spiral-shaped pipettes.1236−1238 An electric field was introduced to focus the
microfluidic channel was implemented to generate flow vor- electroporation effects to the region of the cell sucked into the
tices.1229 As cells traverse through the curved channels, vortices microchannels, thereby locally permeabilizing them.1236−1238
caused by Dean flows facilitate their rotation in reference to The concept was later combined with electrophoresis for
the electric field (Figure 28B(iv)). This results in permeabiliz- increasing the efficiency of delivery, where the delivery of
ing the entire cell surface, rather than just the cell poles. By molecules could be optically monitored in real time.1239 Again,
increasing the cell surface area that can be electropermeabilized a similar concept to take advantage of using channels as
high delivery efficiency was achieved with both dyes and DNA trapping arrays was used to transfect plasmid DNA into stem
plasmids.1229 Other vortex-based micro uidic systems have cells.1240fl Collectively, these innovations show the power of
been implemented to achieve a similar effect1230,1231 and have localizing electric fields to the subcellular scale. If the problem
demonstrated intracellular deliver of dyes, miRNA, siRNA, of scale-up to high throughput can be solved at an acceptable
proteins, and plasmids.1232 cost, this strategy can be expected to benefit the intracellular
6.2.17. Nanochannel Electroporation. Inspired by early delivery toolkit.
work on electroporation through micron-sized apertures,1223 6.2.18. Nanostraw Electroporation. Another form of
Lee and colleagues introduced the concept of nanochannel elec- nanoscale electroporation takes the form of so-called nano-
troporation.1233 By scaling the aperture size down to ∼90 nm, the straws (Figure 28C(ii)). The key difference is that the nano-
membrane disruption effect of electroporation could be concen- scale aperture protrudes into the target cell as a hollow nano-
trated onto a very small spot on the cell surface (Figure 28C(i)). needle. Although cell membranes appear to be resistant to
A significant claim of this strategy is dose control, i.e., the finding penetration by such nanoneedles under passive conditions, the
that the amount of delivered material directly correlates with addition of an electric field permeabilizes the cell membrane at
the voltage pulse duration. Nanochannel electroporation also the tip of the nanostraw.702 One benefit of this approach is that
BB DOI: 10.1021/acs.chemrev.7b00678
Chem. Rev. XXXX, XXX, XXX−XXX
Chemical Reviews Review
active forces, such as optical tweezers or positive dielectropho- our discussion on the in vitro and ex vivo applications relevant
resis, are probably not required to establish optimal contact to intracellular delivery in human and animal cells. Within this
between cells and the nanostraw. Rather, a consistent period of context, electroporation has been employed mainly for nucleic
settling may be sufficient to facilitate uniform contact between acid transfection, of which there are three main market areas:
cells and the substrate.428 Furthermore, if cells properly adhere (1) biomedical research, (2) biomanufacture of biologics (pro-
to the nanostraw array, substantial pumping forces can pre- teins, antibodies, and viral vectors/particles), and (3) ther-
sumably be used to flow molecules into the cytoplasm without apeutics (cell-based therapies, gene therapy, and cell manipu-
cell detachment. In light of poor results with aluminum elec- lation for regenerative medicine; see Figure 3). Furthermore,
trodes in bulk conditions, however, the choice of aluminum intracellular delivery of non-nucleic acid cargo is beginning to
nanostraws as the fabrication material may need to be revised enjoy increased attention, especially with the rise of genome
in future versions of this device. editing and new forms of cell-based therapies. Below we
6.2.19. Nanofountain Probe Electroporation. A highlight a selection of key applications where electroporation
scanning probe-based approach for localized electroporation, had made an impact.
termed nanofountain probe electroporation, has been intro- 6.2.22. Intracellular Delivery of Impermeable Drugs.
duced by Espinosa and colleagues.1241,1242 It is essentially an Permeabilization via electroporation has been proposed for
atomic force microscope cantilever engineering with a hollow pharmacological applications to identify the cytoplasmic
channel for fluid flow. Target cells are cultured on a grounded activity of otherwise impermeable drugs and small mole-
coverslip and positive or negative voltages are applied to the cules1249 (Figure 29A). In the 1980s a study by Melvik et al.
conductive cantilever, thereby focusing the electric field at the showed that electroporation of cell lines significantly enhanced
site of contact between the cantilever and cell (Figure 28C(iii)). the efficacy of cis-dichlorodiammineplatinum(II) (cisplatin) up
By coordinating the movement of the tip and the flow of fluid, to 3-fold greater than controls.1085 Using radiolabeled tracers,
introduction of dextrans and proteins into cells can be they found electroporation rendered cells permeable to small
achieved.1241 In follow-up applications of this system, it has molecules for up to 10 min. Subsequently, electroporation has
successfully been employed to deliver molecular beacons to the been used to screen for cytotoxicity of drugs that are otherwise
cytoplasm for detection of mRNA transcription.1242 susceptible to be pumped out of cells by the activity of cellular
6.2.20. Summary of Micro- and Nano-electropora- efflux pumps.55,1086 Bleomycin (∼1.4 kDa) represents a par-
tion. Innovations in micro- and nanoelectroporation have ticularly striking example of a drug where activity is drastically
showcased a number of interesting proof-of-concept proto- increased with electroporation-mediated intracellular deliv-
types. Diverse architectures have been developed, including the ery.55,1086 Thus, electroporation can be leveraged to test for the
use of micro- or nanochannels smaller than the cell, channels cytoplasmic activity of otherwise impermeable small molecules,
larger than the cell, chambers, compartments, and droplets, peptides, and biochemical agents.
and hydrodynamic effects such as sheath focusing and vor- 6.2.23. Biomanufacture Through Transfection. Bio-
tices.1218,1220 Some of these reports claim improved delivery manufacture refers to the production of biomaterials or
efficiency and viability over conventional bulk electroporation. biomolecules by the harnessing of biological systems. Trans-
They have also provided elegant solutions to problems that fection of common cell lines can be used for production of
have long troubled traditional electroporation setups, such as proteins, antibodies, viral vectors, or viral particles1250−1255
electrolytic reactions at the electrodes, gas bubble formation, (Figure 29B). These are often produced in mammalian cell
pH deviations, Joule heating, inconsistent cell treatment, lines such as CHO, HEK-293T, HeLa, and A549 cells or insect
inability to scale down reagent volumes, excessive power con- cell lines, depending, for example, on the need for species-
sumption, and requirements for cumbersome equipment. Yet specific post-translational modifications. Significant efforts have
the technical advancements of miniaturized approaches have gone into engineering these systems for maximum yield and
not translated to widespread adoption, most likely due to high economies of scale. Both stable genetically modified cell lines
cost, impractical throughput, lack of focus on clinical or indus- and transient transfection are key strategies for biomanufac-
trially relevant problems, or lack of user-friendly designs.1221 ture. Although lipid and polymer reagents are most commonly
Thus, it remains to be seen what the next generation elec- used for transfection in biomanufacture, electroporation is
troporation systems will look like and whether they will currently the leading option among membrane disruption-
challenge the dominance of existing methods. Apart from mediated methods.
technical upgrades, recent literature emphasizes that further 6.2.24. Large Volume Flow Electroporation. The
theoretical studies on the mechanisms of cell membrane concept of large volume flow-based electroporation for cell
permeabilization and cargo uptake are needed to obtain further processing emerged in the late 1980s. A flow-based electro-
progress in the eld.163,1118,1131,1132,1138,1144,1219fi fusion system for processing several milliliters of suspended
6.2.21. In Vitro & Ex Vivo Applications of Electro- cells per minute was introduced by Teissie ́ and col-
poration. Of the membrane disruption-based approaches, leagues.1256,1257 Following that, the same group reported on
electroporation is currently the most mature in regard to a flow-based intracellular delivery system with similar
industrial applications and clinical translation. Electroporation- throughput capable of transfecting plasmid DNA into different
based technologies have been deployed in vivo as well as cell types at efficiencies of 25−35%.1258 In 2002, a commercial
in vitro. The in vivo applications include electrochemotherapy, large volume flow electroporation system for clinical and
nonthermal tissue ablation, DNA vaccines, and transdermal industrial bioprocessing was reported in the scientific literature
drug delivery. These have already been discussed in other by the company Maxcyte.1259 Initial reports claimed that
reviews.1048,1050,1243−1247 In biotechnology, electroporation has common suspension and adherent cells lines could be loaded
also been used for extraction of biomolecules, sterilization/ with 500 kDa dextran at >90% efficiency and >90% viability
pasteurization of solutions, and transformation of microorgan- while gene transfection rates could reach up to 75%. The latest
isms.1248 In keeping with the focus of this review, we will center versions of this technology are capable of tunable scale, from
BC DOI: 10.1021/acs.chemrev.7b00678
Chem. Rev. XXXX, XXX, XXX−XXX
Chemical Reviews Review
Figure 29. In vitro and ex vivo applications of intracellular delivery achieved with electroporation. (A) Delivery of impermeable drugs to the
intracellular space for drug testing and/or cell manipulation. (B) Transfection with plasmid DNA encoding proteins, antibodies, and viral
components for biomanufacturing purposes. (C) Loading of protein antigens or mRNA encoding such into dendritic cells. Presentation of antigen
fragments through MHC pathways is able to prime T cells against cells carrying the antigens and may be useful for cancer immunotherapy.
(D) Transfection of cytotoxic immune cells with mRNA encoding TCRs and/or CARs can be used to direct immune cells against specific cell
targets, such as cancer cells. TCR = T cell receptor. CAR = chimeric antigen receptor. (E) Genome-editing molecules can be delivered into stem
cells for the purposes of adding, deleting, or correcting genes. Modified stem cells can then be expanded for potential deployment in cell- and
tissue-based gene therapy. Red signifies areas of the genome that have been edited. ZFN = zinc finger nuclease.
tens of thousands of cells up to 200 billion cells packed into uniform spacing.1261 The microfluidic design of the needle
liters of solution. The run time for a batch of 200 billion cells is electrode array brings the benefit of lowering the required
approximately 30 min in a single run. Moreover, the system is voltage. The system enables processing rates of 20 million cells
sterile and compliant with current good manufacturing pro- per minute and was suggested to be suitable for in vitro and
cesses (cGMPs) for biological clean room facilities. In further ex vivo batch mode applications. Another group published a
demonstrations of its utility in manufacturing scenarios, the similar concept constructed from custom-made microfluidics
flow electroporation platform was used to batch transfect components as a solution for batch flow electroporation of
HEK293T cells for large-scale bioproduction of lentiviral mRNA into tens of millions of dendritic cells.1262
vectors.1260 6.2.25. Delivery of Genome-Editing Proteins and
Recently, Zhao et al. published a different strategy for large RNPs. Recent advances in genome editing via programmable
volume flow electroporation with a device that integrates a nucleases have spurred an interest in intracellular delivery of
flow tube and a miniaturized needle electrode array with these proteins, particularly Cas9 RNPs. In the past few years
BD DOI: 10.1021/acs.chemrev.7b00678
Chem. Rev. XXXX, XXX, XXX−XXX
Chemical Reviews Review
RNP delivery has been successfully accomplished with cell lines,1159 primary leukemia cells and cell lines,233,1167,1168
electroporation,143,144,146,147 microinjection,148,149 lipid nano- primary natural killer cells and their derivative cell lines,1169,1170
particle formulations,150 osmotically induced endocytosis primary lymphocytes,1171,1172,1266 embryonic and adult stem
followed by endosome disruption,151 microfluidic cell cells,1173,1174 and mammalian neurons.1157,1175
deformation,152 and CPPs.153 Electroporation, however, is Other electroporation platforms have also achieved a mea-
reported to be more efficient with a number of primary, blood, sure of success in hard-to-transfect cells. Minimalist setups fea-
and immune cell types in vitro. RNP delivery via electro- turing standard 2 or 4 mm cuvettes, commercial pulse gener-
poration has been demonstrated in a range of cell types, from ators (such as the BioRad Gene Pulser or BTX units), and
common cell lines to blood and immune cells of clinical an electroporation buffer consisting of OPTIMEM media
relevance, with both conventional cuvette style (Nucleofec- (or equivalent) have attained favorable results with macro-
tion)30 ,143 ,146 ,147 ,287 and capillary electroporation phages,234,275 T lymphocytes,187,257,551,1267−1271 dendritic
(Neon)45,144,145,1263 platforms. cells,186,260−262,1110,1272,1273 and B cells.1274,1275 Some of
The mechanisms of RNP entry via electroporation have not these groups have even used such setups to perform small
been heavily studied yet. Given what we already know about the scale clinical trials.560 In other cases, the Maxcyte system for
influx behavior of nucleic acids and proteins (Figures 24−26), large-scale clinical-grade flow electroporation has demonstra-
it is worth considering the possibilities. As discussed in ted e 1276ffectiveness with leukemia cells, natural killer
section 2.2.2, an sgRNA-Cas9 RNP complex should have about cells,1277,1278 dendritic cells,136−138 T cells,187 and CD34(+)
−80 negative charges, be ∼188 kDa, and be up to 15 nm in hematopoietic cells.185 The Neon capillary electroporation
size (Table 1). The mechanisms of electroporation-mediated system has successfully delivered molecules into iPSCs,45,145
delivery could thus be similar to siRNA, namely direct trans- T cells,144 and HSCs.1263 Together these studies suggest that
location of a highly negatively charged molecule into the no one electroporation system has a monopoly on effectiveness
cytoplasm at the side of the cell facing the negative electrode with sensitive or difficult to treat cell types.
during the pulse (Figure 24D). Once in the cytoplasm a 6.2.27. T Cells & Other Immune Cells. Immune cells are
nuclear localization sequence (NLS) on the Cas9 would then a key category of cells for biomedical investigations and
promote its shuttling to the nucleus. Another possibility is that therapeutic applications. In T cells it has been asserted that
RNPs are endocytosed after being entangled in the destabilized RNA delivery to the cytoplasm is not difficult, but DNA plasmid
plasma membrane, such as is the case for plasmid DNA transfection, which requires nuclear penetration, remains a
(Figures 24E and Figure 25). Indeed, postelectroporation signi cant hurdle.279,1279fi This is an example where primary
aggregation or trapping of proteins at the plasma membrane cells may exhibit an innate toxic reaction against delivered
has been observed in several cases.1092,1122 The ground-breaking material. T cells, in particular, appear to display little tolerance
potential of genome editing will no doubt stimulate the eld to plasmid transfection regardless of delivery technique.146,279fi
toward studying mechanisms of protein and RNP delivery to Electroporation is counted among the techniques that perform
the nucleus. For example, the optimal nuclear concentrations well in delivering siRNA and mRNA into T cells; however, the
of Cas9 RNP needed for efficient genome editing are still margin of error leading to loss of viability can be narrow,279
unknown. In the future, it will also be interesting to see how and changes in the activation state, signaling pathways, and trans-
other membrane disruption-based delivery approaches (which criptional responses of cells must be taken into account.1280,1281
do not supply electrophoretic forces) compare in their effi- Many of the published electroporation protocols underscore
ciencies of RNP delivery. the narrow window of appropriate parameters, emphasizing
6.2.26. Hard-to-Transfect Cells. A number of sensitive that there exists a fine line between effectiveness and cell
primary cell types do not easily tolerate foreign nucleic acids or death.275,1168 The challenge for electroporation appears to be
the toxic side-effects of common transfection reagents. For exam- the long-term survival, potency, and functionality of treated
ple, dendritic cells, T lymphocytes (T cells), B lymphocytes cells, not so much the initial delivery. Indeed, post-treatment
(B cells), natural killer (NK) cells, leukemia cells, hema- loss of viability, proliferative potential, or potency has been
topoietic stem cells (HSCs), macrophages, and neurons have reported for immune cells and other primary cell types.279,1165,1264
all been reported to be recalcitrant to polymer- or lipid-based Moreover, electroporated immune cells have sometimes been
transfection.233,260,261,282,286,290,1172,1264,1265 Lentiviral trans- observed to exhibit an unfavorable response or poor engraftment
duction and electroporation have emerged as the two leading when infused back to the in vivo setting.279 On the other hand,
alternatives. However, procedures with viral vectors are some- several studies have shown electroporated cells to recover well
times unfavorable because they can (1) be labor-intensive, and exhibit decent potency in clinical contexts.187,560,1282,1283
inconsistent, and expensive; (2) present safety hazards; (3) cause 6.2.28. Ex Vivo Intracellular Delivery for Cell-Based
untoward immune or inflammatory responses in vivo; and Therapies. Scientists have long envisaged the power of
(4) carry a risk of insertional genotoxicity via genomic inte- ex vivo cell manipulation for cell-based therapies, especially in
gration. Electroporation, on the other hand, is rapid and sim- regard to gene therapy, immunothereapy, and regenerative
ple, but its core weakness is poor viability or loss of cell func- medicine.28,29,31,41 The concept is to remove cells or tissues
tionality, as has been reported for nucleofection of dendritic from the patient, engineer their function, and reimplant them
cells or T cells.279,1165,1264 to confer a therapeutic effect. Many of the relevant cell types,
Nucleofection, in particular, has sought to build a reputation however, fall into the category of “hard to transfect” cells as
on e ectiveness with hard-to-transfect cells.289,1108ff Nucleofec- outlined above. In the following we will highlight several areas
tion has demonstrated significant success with DNA and RNA where electroporation has been attempted for ex vivo cell-
transfection in various types of stem cells, primary cells, and based therapies.
postmitotic cells. Published examples include primary human 6.2.28.1. Protein Loading for Antigen Display in Cancer
melanocytes, smooth muscle cells, chondrocytes and mesen- Immunotherapy. Loading of exogenous proteins into the
chymal stem cells,1156,1163,1164 dendritic cells,1165,1166 monocytic cytoplasm of antigen-presenting cells leads to their processing
BE DOI: 10.1021/acs.chemrev.7b00678
Chem. Rev. XXXX, XXX, XXX−XXX
Chemical Reviews Review
and display through the MHC-I pathway95,1284 (Figure 29C). novel specificity to kill any cell that carries molecules to which
This primes cytotoxic T cells against any cells carrying the the CAR binds. The goal is to target the killing action of TCR-
antigens, such as cancerous cells that produce mutant proteins or CAR-modified immune cells against cancer cells carrying
(Figure 29C). Thus, intracellular delivery of tumor proteins complementary surface markers. Electroporation has been used
into antigen presenting cells, especially dendritic cells, has been to deliver mRNA for expression of TCRs or CARs, chemokine
proposed as a strategy for cancer immunotherapy.1285 Kim receptors, or cytokines in T cells.187,257,1293,1294 Similar to the
et al. were among the first to use electroporation to load dendritic case of dendritic cells, switching from plasmid DNA to mRNA
cells with exogenous antigens ex vivo before implanting them was reported to allow >90% gene expression with >80%
back into the body to elicit a robust antitumor response in viability in T cells postelectroporation, even while using a basic
mouse models.135 The Maxcyte clinical electroporation system cuvette-style electroporation protocol in OPTIMEM buffer.257
was also used to achieve similar results by loading tumor cell Using such methods, it was shown that multiple infusions of
lysate into dendritic cells.136 In recent years this concept has mRNA-electroporated CAR-T cells mediated shrinkage of
been put to the test in human clinical trials. In 2013, a Japanese large vascularized flank mesothelioma tumors of human origin
group confirmed the safety and feasibility of administering in a genetic mouse model.187 CAR expression and antitumor
dendritic cell vaccines generated by cytosolic loading of auto- activity of mRNA-electroporated T cells was detected up to a
logous tumor lysates via the Maxcyte system.137 This strategy week after electroporation. This is important because mRNA
was reported to produce a significant antitumor effect com- electroporation for transient expression of CARs in T cells is
pared to passive incubation (pulsing) of dendritic cells with seen as a far safer alternative to permanent integration of CAR
tumor lysate.138 genes into the genome.1291,1295 T cells electroporated with
6.2.28.2. mRNA Transfection for Antigen Display in mRNA encoding for a CAR against CD19 showed cancer
Cancer Immunotherapy. For induction of the MHC-1 antigen killing capacity in immunodeficient mice bearing xenografted
presentation pathway, mRNA transfection may be preferred to leukemia.259 Even a single injection of CD19 mRNA CAR-T
protein loading1286 (Figure 29C). Van Tendeloo et al. cells yielded a significant prolongation in survival in this model.
published a paper in 2001 showcasing the efficacy of such an Because mRNA electroporation is a cost-effective and efficient
mRNA-based strategy in dendritic cells.260 Using a basic path to engineer T cells for pilot studies, this approach has
cuvette style electroporation setup with OPTIMEM buffer, been pursued for high throughput and iterative testing of
they were able to achieve >80% expression with >80% viability novel constructs and targets in small scale clinical trials in
compared with much poorer results from plasmid DNA in humans.32,1283,1291
earlier studies.1287 Their comparison of methods for mRNA 6.2.28.5. Electroporation to Produce Cytotoxic NK Cells
transfection to dendritic cells suggested that electroporation for Cancer Immunotherapy. Although most work with CARs
was far superior to lipofection and other methods.1288 Based has been carried out with T cells, NK cells represent an
on these studies, the idea of electroporation-mediated mRNA alternative option.1292 Among the first attempts to investigate
transfection for ex vivo immunotherapy and gene therapy this possibility were a series of experiments in 2005 by Imai
gained significant momentum.1289 Using similar electropora- et al. that used retroviral transduction to guide the activity of
tion methods as those described by Van Tendeloo et al.,1273 NK cells expressing CD19 CARs against patient leukemia cells
several groups have pressed ahead with small-scale clinical in in vitro assays.1296 Next, electroporation of CAR mRNA
trials to treat human patients suffering from melanoma and into NK cells was attempted in 2010. Members of the Maxcyte
other cancers.560,1290 Results gathered to date indicate positive team used their clinical-scale large-volume electroporation
long-term survival rates and safety of the treatments. platform to transfect mRNA encoding a CD19 CAR into NK
6.2.28.3. Electroporated B Cells for Antigen Display in cells.1277 The engineered cells demonstrated cytotoxic killing
Cancer Immunotherapy. Apart from dendritic cells, several of acute lymphoblastic leukemia and B-lineage chronic lympho-
other types of professional antigen-presenting cells have been cytic leukemia cells for up to 3 days after electroporation.1277
tested for their ability to prime T cells against a tumor antigen. Shimasaki et al. then employed the Maxcyte system to scale up
Coughlin et al. employed nucleofection to demonstrate that mRNA transfection to large batches of expanded NK cells with
B cells from pediatric patients can be efficient antigen presenting numbers reaching up to 250 million cells per run.1278 Under
cells upon loading with tumor mRNA.1266 As a proof of concept, these conditions CD19 CAR expression reached >80% after
mRNA-transfected B cells were used to successfully prime a 24 hours and mediated significant antitumor cytotoxicity in a
T cell response against cultured neuroblastoma cells in vitro.1266 mouse xenograft model of B cell leukemia.
According to another study, electroporation of multiple RNAs 6.2.28.6. Electroporation for Ex Vivo Gene Therapy of
into activated B cells with a standard cuvette style system Blood & Immune Cells. Ex vivo cell-based therapies have long
elicited in vitro antigen-specific cytotoxic T cell responses with been pursued as an avenue for treatment of blood cells to
similar e ciencies as those of mature dendritic cells.1275 Thus, address hematological diseases.29ffi However, only recently have
the use of intracellular delivery for ex vivo activation of B cells gene therapy clinical trials in T cells and HSCs shown sig-
may represent an alternative source of antigen presenting cells nificant progress. These trials mostly used lentiviral trans-
for cancer immunotherapy, especially in pediatric cases where duction, which can carry a risk of genotoxicity due to random
dendritic cells are not as readily available. genomic integration.26,1297−1299 To address this problem, new
6.2.28.4. Electroporation to Produce CAR-T Cells for approaches that deliver genome editing molecules directly into
Cancer Immunotherapy. A more direct way of inducing an cells have attracted interest for ongoing studies.1300 As dis-
immune response against cancer is to express a T cell receptor cussed in previous sections of this review, electroporation is
(TCR) or chimeric antigen receptor (CAR) directly into among the techniques that can deliver genome-editing mole-
cytotoxic immune cells, such as T cells or natural killer (NK) cules in the form of mRNA, sgRNA, proteins, and RNPs into
cells32,1283,1291,1292 (Figure 29D). A CAR is a genetically clinically relevant cell types at reasonable efficiencies and
engineered immunoreceptor that endows modified cells with a viabilities.
BF DOI: 10.1021/acs.chemrev.7b00678
Chem. Rev. XXXX, XXX, XXX−XXX
Chemical Reviews Review
Here are two examples where electroporation of one autologously sourced iPSCs for therapeutic gene editing before
component is combined with nonintegrating viral transduction reimplantation.41
of another. First, integrase-defective lentiviral expression of 6.2.29. Electroporation Summary. Electroporation can
donor DNA combined with nucleofection of zinc finger mRNA deliver a vast range of molecular cargo into a wide variety of
was used for HDR-mediated correction of monogenic muta- cell types with precise temporal control. With conventional
tions in the IL2RG gene of patient HSCs.30 This strategy has electroporation the pulse parameters (field strength, pulse
the potential to provide a one-time cure for the immune dis- duration, pulse number, and frequency) are flexible; therefore,
order X-linked severe combined immunodeficiency (SCID-X1) the same piece of hardware can be programmed to address a
as gene-edited HSCs give rise to functional lymphoid progen- large number of scenarios. Parameters can be manipulated to
itors that exhibit a selective growth advantage over disease focus the membrane-perturbing effects on different regions of
mutants. Second, a recent study by DeRavin et al. used the cell, such as certain parts of the plasma membrane or
targeted integration of a corrected gene into CD34(+) HSCs membranes of intracellular organelles (Figure 23). Addition-
as a treatment strategy for X-linked chronic granulomatous dis- ally, the dual mechanisms of pore formation and electro-
ease.185 Similar to the previous example, they used electro- phoretic propulsion of cargo may be beneficial for delivery of
poration (in this case, the MaxCyte platform) to transfect zinc charged cargoes, such as plasmid DNA or mRNA (Figure 24).
finger mRNA into cells while donor DNA for gene correction Fundamentally, it is not well understood how cell structure,
was supplied by adeno-associated viral (AAV) vectors. cytoskeleton, membrane proteins, domain phases, and mem-
By targeted integration of a corrected gene into the AAVS1 brane reservoirs influence electroporation in live cells, making
safe harbor locus of the genome, it was argued that genotoxicity it difficult to decipher critical molecular events. Additionally,
associated with random integration can be avoided. In mice the intrinsic pore-formation mechanisms bias electroporation
transplanted with corrected HSC progenitors, 4−11% of toward the formation of numerous small pores, somewhat
human cells in the bone marrow expressed the therapeutically limiting the delivery of large cargoes.
corrected gp91phox protein. Electroporation has a number of challenges, especially post-
6.2.28.7. Electroporation for Gene-Editing of Blood & treatment cell death. Indeed, the window for effective
Immune Cells. Other proof of concept studies for therapeutic treatment can be quite narrow, especially in primary cells. Detri-
genome editing in HSCs and T cells have been carried out mental effects of electroporation can be attributed to electro-
with Nucleofection,146 Neon electroporation,144,1263 or stand- chemical phenomena at the electrodes including Joule heating,
ard BTX cuvette-based electroporation.1271 In these cases, pH waves, bubble formation, corrosion, and contamination of
delivery of Cas9 RNPs144,146 or mRNA encoding Cas9, ZFNs, the solution. Other potential issues include electric field-based
TALENS, or megaTAL nucleases was demonstrated.146,1263,1271 perturbation of native proteins, scrambling of lipid membranes,
In comparison, plasmid DNA encoding for these components generation of ROS, and incompatibility with certain cargo
usually led to comparatively lower efficiencies or poorer tol- molecules. Technical innovations featuring different electrode
erance in these cell types.146 Also of note, electroporation- designs or microfluidic and nanochannel designs have been
based codelivery of RNPs and a single-stranded oligonucleo- developed to overcome some of these issues (Figure 28), but
tide DNA template (HDR template) with 90 nucleotide they have not yet superseded the basic cuvette-style electro-
homology arms mediated up to 20% knock-in in primary poration, which remains the most widely used platform for
human T cells,144 obviating the need to express DNA template common use.
from plasmids or viral vectors. The challenges of current electroporation techniques
6.2.28.8. Electroporation for Genome Editing of Stem notwithstanding, for many applications the benefits outweigh
Cells. iPSCs, HSCs, and embryonic stem cells hold potential the weaknesses. Consequently, it has become the most widely
for regenerative medicine as a source of autologous cells and used membrane disruption-mediated intracellular delivery
tissues for patients. By introducing genome-editing molecules approach. Electroporation has shown promise for treatment
through intracellular delivery, stem cells can be prepared for of a wide variety of patient derived cells and stem cells, with
gene therapy (Figure 29E). Using nucleofection, Kim et al. even the most basic electroporation platforms finding use
were among the first to determine the advantages of RNP among in vitro and ex vivo medical and industrial applications,
delivery versus plasmid transfection by observing higher site- from biomanufacture and clinical trials of cancer immunother-
specific editing rates with reduced off-target mutations in stem apy to ex vivo cell-based gene therapy and regenerative medicine.
cells.143 They reported that RNP delivery was less stressful to
human embryonic stem cells, producing at least 2-fold more 6.3. Thermal Membrane Disruption
colonies than plasmid transfection strategies.143 In keeping Membrane formation, dynamics, and properties are temper-
with this notion, recent CRISPR protocols for implementation ature-dependent. At sufficiently high temperatures, lipid
in human stem cells and primary cells indicate a preference for bilayers will dissociate due to kinetic energy of the constituent
Nucleofection of Cas9-sgRNA RNPs over plasmids.287 Fur- molecules being greater than the forces that maintain the
thermore, Neon capillary-based electroporation was used to membrane formation, namely the hydrophobic forces that
introduce CRISPR-Cas9 nucleases via plasmids and/or RNPs repel water from the lipid tails. The thermodynamic consid-
to correct disease-causing mutations in patient-derived iPSCs.45 erations of lipid bilayer behavior dictate that temperature is key
This strategy mediated functional correction of large factor in determining the energy required for a given membrane dis-
VIII gene chromosomal inversions in patient cells, a mutation ruption event. The key role of temperature has been empha-
that underlies hemophilia A. Endothelial cells derived from sized in the electroporation literature, for example, where theory
these iPSCs were competent in rescuing factor VIII deficiency posits that electric potential differences across membranes can
in an otherwise lethal mouse phenotype of hemophilia. Thus, tilt the energy landscape of stochastic thermally-driven defect
direct intracellular delivery of genome editing molecules takes formation.419 The implications of temperature must be fully
us closer to the long-standing goal of exploiting patient-derived considered in any membrane disruption event. This applies
BG DOI: 10.1021/acs.chemrev.7b00678
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Chemical Reviews Review
Figure 30. Thermal membrane disruption. (A) Membrane disruption by freeze−thaw cycles. Formation of ice crystals leads to volume expansion
due to the changes in hydrogen bonding arrangement. Volume expansions are thought to be related to cracking of membranes during ice crystal
formation. (B) Heating of cells above 42 °C increases the chances of spontaneous defect formation in membranes. (C) Microfluidic geometries
may be used to confine the heating locally to a part of the cell, such as is possibly the case for thermal inkjet printing. (D) Absorbent nanoparticles
may be used to locally convert laser power into local heating for membrane perturbation. (E) A focused laser can generate local heating at the
membrane with selection of appropriate parameters.
both to the physical properties of lipid membranes and the bacteria undergo transient incubation at 0 °C, a brief pulse up
active response of the cell (see section 4.3). to 37−42 °C, and subsequent return to normal growth con-
Membrane permeability is known to increase during thermal ditions where the genes of interest are expressed.1305,1306
phase transitions.73,1047,1301 Both magnitude and rate of tem- Multiple cycles are sometimes conducted to boost efficiency.
perature changes influence the molecular rearrangements in Mechanistic studies suggest that phase transitions of mem-
membrane domains that are linked to the stochastic for- brane lipids cause damage to the outer membrane, and are
mation of defects.1302 Close to phase transitions, ion channel- necessary for DNA entry.1307,1308 Some data indicates that cold
like events are known to occur, even in the complete absence shock may not need to go down as low as 0 °C, as the rate and
of proteins.1301,1303,1304 The occurrence of purely lipid ion magnitude of temperature changes are more critical than
channels depends on temperature, hydrostatic pressure, lateral specific temperature extremes.1307 However, more recent reports
pressure, voltage, pH, and ion concentrations. Such pore claim that a brief freeze in liquid nitrogen for 20 s increases the
formation is expected to be especially probable adjacent to efficiency of freeze−thaw transfection, even obviating the need
domain interfaces and protein clusters. for standard pretreatment steps normally employed to make
Strategies for permeabilizing cells by thermal means include bacteria competent.1309 Interestingly, microwave irradiation of
(1) cycling cells through a cooling−heating cycle, which may or frozen bacteria/DNA samples was also found to improve DNA
may not involve freezing, (2) heating cells to supraphysiological transfection.1309 Finally, microfluidic reactors have been
temperatures, and (3) transient intense heating of a small part employed for temperature shock transfection of bacteria.1310
of the cell. The literature includes examples of each of these The advantages include fewer materials, smaller sample volume,
approaches, which will be discussed here in this section. and increased precision compared to conventional bulk pro-
Overall though, thermal methods of membrane perturbation cedures.1310
have not been widely employed with animal cells, despite being 6.3.2. Freeze−Thaw and Other Temperature Cycling
universal and obvious. This can probably be attributed to Strategies. Apart from bacteria, rapid freeze−thaw procedures
challenges in spatiotemporal control of temperature exposure have also been demonstrated to facilitate exchange between
and concerns related to off-target damage. In future there exists intracellular and extracellular solutions when conducted with
an opportunity to address these challenges with emerging animal cell membranes (Figure 30A). In 1989 this was shown
lab-on-chip, microfluidic, optical, and nanotechnological sys- with synaptosomes, which are vesicular sacs reconstituted from
tems.19,104−107 synaptic terminal membranes by mild homogenization of
6.3.1. Thermal Shock of Competent Bacteria. nervous tissue.1311 In the reported procedure rat brain synapto-
In bacteria, thermal shock has been used for decades to trans- somes were frozen and thawed in the presence of 5% DMSO.1311
fect “competent” bacteria with DNA plasmids. The method Impermeant proteins, inhibitors, and metabolites were success-
was described in early papers from the 1980s where agents fully introduced to study neural signaling processes.1311
such as divalent cations (typically in the form of CaCl2) and An updated “cryoloading” procedure was reported by Nath
dimethyl sulfoxide (DMSO) were added to make E. coli et al. where molecules of at least 150 kDa were successfully
amenable or “competent” to DNA transfection. Next, the delivered into chick synaptosomes.1312 After recovery ∼80% of
BH DOI: 10.1021/acs.chemrev.7b00678
Chem. Rev. XXXX, XXX, XXX−XXX
Chemical Reviews Review
the synaptosomes were properly functional and capable of membranes more susceptible to fluid shear from laser-induced
recycling synaptic vesicles.1312 stress waves.1317
Intracellular delivery by extreme cooling−heating cycles has 6.3.4. Thermal Inkjet Printers. Thermal inkjet printers
rarely been attempted in animal cells, probably due to the delicate that disperse small volumes of fluid have been successfully
and complex nature of cell recovery and growth from the frozen deployed for mammalian cell gene transfection and intra-
state. In one of the few cases where it was tested, trehalose cellular delivery.1318,1319 By replacing standard ink with media
(∼0.34 kDa) was loaded into suspensions of adult islet cells by and cells, these printers not only perform intracellular delivery
cooling them through their membrane phase transition.73 Under but can additionally pattern cells over a substrate. In thermal
conditions where cells were cooled at a rate of 1 °C per inkjet printers, a metal plate is heated at one side of the nozzle,
minute, permeability to trehalose was greatest around the which creates small air bubbles that collapse to provide
region 0−5 °C.73 Loaded trehalose exhibited cryoprotectant pressure pulses to eject tiny drops of fluid. Over several micro-
properties and was able to significantly increase cell survival seconds the plate temperature may transiently rise to 300 °C.
and insulin production of islet cells. Building on this approach, It is not known whether membrane permeabilization is
Puhlev et al. compared intracellular delivery via cooling in obtained by fluid shear forces or transient thermal disruption
suspension versus adherent fibroblasts. In their procedure cells at the nozzle. In the studies performed so far Xu et al. achieved
were exposed to 50 mM trehalose for 5 min on ice, followed by transfection efficiencies of 10% with GFP plasmids in porcine
10 min at 37 °C.1035 As with the previous paper, maximal aortic endothelial cells at 90% cell viability,1318 while Cue and
delivery was estimated to occur below 5 °C and was more Boland obtained above 30% transfection efficiency in CHO
efficient in suspended cells versus their adherent counterparts. cells with similar viabilities.1319 Further mechanistic insights
A similar strategy was also tested by the Toner lab.75 Temper- may improve the efficiency of this approach. A potential bonus
ature cycling from 0 to 39 °C was able to load trehalose into a of thermal inkjet printing is the ability to array cells into
target cell population of suspended rat hepatocytes without com- specific geometries and perform intracellular delivery in a
promising cell viability.75 Using an extracellular concentration single step, thereby facilitating the possibility of in vitro tissue
of 0.4 M in diluted culture medium, 1 h of temperature oscil- engineering.1320 The results with thermal inkjet printers point
lations conducted every 10 min produced an average cytoplasmic to an opportunity for future studies with microfabricated
concentration of 0.13 M (∼3% of extracellular concentra- devices, where it should be possible to gain spatiotemporal control
tion) as detected by high-performance liquid chromatogra- over temperature exposure through microfluidics (Figure 30C).
phy.75 Extended periods of incubation at 39 °C increased 6.3.5. Laser−Particle Interactions. As discussed in the
loading efficiency but came with the caveat of reducing cell sections on fluid shear, laser irradiance of an absorbent object
survival. in an aqueous environment can produce a variety of effects
6.3.3. Supraphysiological Heating. As temperature including cavitation, plasma production, chemical reactions,
moves above 37 °C, the probability of membrane defects and heat.1321−1323 Although it is sometimes difficult to be sure
arising increases. In experiments on mammalian cells, Bischof of the mechanisms, we report here on studies that claim to
et al. exposed fibroblasts and muscle cells to temperatures disrupt membranes by laser-mediating temperature changes.
ranging from 37 to 70 °C and monitored membrane integrity In most cases nanoparticles are used as nucleation sites for
in real time. Permeability was assessed by tracking the leakage intense local heating (Figure 30D). Umebayashi et al. showed
of calcein (0.62 kDa) with timelapse fluorescence microscopy. that laser irradiation of unbound latex particles dispersed in
Slow leakage, which starts above 40 °C, was found to be a solution leads to the uptake of impermeant dye molecules.1324
function of both temperature and time. Cells held at 45 °C The mechanism was proposed to be through thermal pertur-
were completely depleted of calcein within 25 min. This bation at the particle−membrane interface, pore formation,
corroborates well with other data indicating cells must work and subsequent diffusive influx of extracellular molecules.1324
harder to maintain their relatively high potassium concen- A similar thermal delivery concept was shown by Yao et al.
trations during treatments at 43 °C.1313 In Bishof et al.’s with selectively bound antibody-conjugated gold nanoparticles,
experiment, leakage takes slightly less than 10 min at 50 °C. featuring a strong correlation between nanoparticle size and
Above 55 °C, almost 50% of calcein leaks out of the cell within heating intensity.1325 Follow up studies investigated the effects
1 min and efflux is fully complete by 2 min. To explain the of laser pulsing parameters (pulse duration, exposure intensity,
increase in permeability, contributions from both protein and irradiation mode) and found conditions where more than
denaturation and increased kinetic diffusion of lipid molecules 50% of the treated suspension cells could take up a labeled
were suggested. Other studies in red blood cells indicate that 150-kDa IgG antibody.1326 In other studies, cancer cells were
thermally induced membrane disruption occurs at about 60 °C targeted by folate-conjugated gold nanorods. Under femto-
and protein denaturation temperature depends on the specific second laser irradiation the nanorods were shown to thermally
protein.1314,1315 Interestingly, addition of poloxamers, which disrupt the membranes as evidenced by flux of dye molecules
are both membrane-healing and antioxidant,459 are able to across the plasma membrane.1327 Gu et al. reported using low
rescue viability of thermally challenged cells.1316 This indicates power continuous wave near-infrared (NIR) lasers to thermally
that loss of membrane integrity is a key aspect of immediate excite inert crystalline magnetic carbon nanoparticles for
cell toxicity upon heating.1316 For intracellular delivery pur- delivery of impermeable dyes and plasmids.1328 Gold nano-
poses, supraphysiological temperatures have rarely been employed particles have also been packed into a dense surface layer
(Figure 30B), probably due to concerns of nonspecific cell where >10 s of infrared laser irradiation heats the underside of
damage and toxicity as exemplified by the trehalose experi- cells to trigger permeabilization and delivery of dyes, dextrans
ments discussed above.75 Baseline temperature is a critical and plasmids.1329
parameter for any delivery protocol, however, and there have 6.3.6. Laser−Membrane Interactions. In the absence of
been a few rare reports of supraphysiological regimes. For absorbing structures, lasers alone can be harnessed for local
example, 43 °C was employed in one study to make cell heating of cell membranes within the focal region (Figure 30E).
BI DOI: 10.1021/acs.chemrev.7b00678
Chem. Rev. XXXX, XXX, XXX−XXX
Chemical Reviews Review
The mechanisms of laser interaction with lipid membranes are 5−8 μm was focused onto the surface of NIH 3T3 fibroblasts
complex, usually being underpinned by a mixture of thermal, with an exposure time of 0.25 s to puncture the plasma
chemical, and mechanical components.1321−1323 Hence, only membrane.1330 After conducting the procedure in the presence
under a narrow range of conditions are lasers thought to of 10 μg mL−1 plasmid DNA, repair of a single large hole in the
produce purely thermal membrane disruption. One example membrane was found to take 1−2 min, followed by detectable
was published by Palumbo et al. where 0.25 s exposure to a gene expression after 2 h.1330 Plasma membrane disruption
488 nm continuous wave argon laser of spot size 5−8 μm was mechanisms were reported to be thermal, and laser exposures
focused onto the cell surface.1330 Their report indicated that of greater than 0.5 s were observed to permanently damage
the poration mechanism was via heating; however, other effects cells.1330
cannot be ruled out. More information on laser optoporation is The next major breakthrough in optoporation occurred in
presented the next section of this review. 2002, with the implementation of femtosecond-pulsed lasers.1337
6.3.7. Thermal Membrane Disruption Summary. Tirlapur and König used a high-intensity, near-infrared (wave-
Baseline temperature is a basic consideration in any intracel- length 800 nm), femtosecond-pulsed laser beam from an
lular delivery technique. Moreover, fast temperature fluctua- 80 MHz titanium−sapphire laser, with a mean power of
tions within, and deviations outside, the physiological temper- 50−100 mW. The laser was tightly focused to a subfemtolitre
ature range can result in thermally-driven membrane defects. focal volume just above the cell membrane. Under 16 ms
Transfection assisted by thermal membrane disruption has exposure time, CHO and Ptk2 cells were transfected with GFP
been harnessed in bacteria for decades. In metazoan cells, using only 0.4 μg mL−1 of DNA plasmid in solution. Unprec-
strategies to control the spatiotemporal control of temperature edented high transfection efficiency and viability were
exposures may yield fruit, as evidenced by encouraging reports reported, with both coming in at close to 100%. A prime lim-
with microfluidics, thermal inkjet printing, and laser-particle itation of the procedure, however, was the need to manually
interactions. refocus on each cell, yielding a throughput of only a few cells
6.4. Optical Membrane Disruption (Optoporation) per minute. Since this landmark report (1) femtosecond lasers
gained prominence as the most effective pulsing strategy for
A wide variety of laser procedures have been implemented to optoporation and (2) the number of optoporation publications
selectively perform nanosurgery on cells and their compo- has increased dramatically. In terms of cargo delivery, the field
nents.1331 Targets include individual chromosomes, organelles, has placed particular focus on delivery of small molecule dyes
mitochondria, cytoskeletal structures, and lipid membranes. for mechanistic studies and DNA transfection to demonstrate
Optoporation is the permeabilization of lipid membranes by applications. Indeed, laser optoporation has achieved success-
high intensity light. In some studies it has also been referred to ful delivery of plasmid DNA,481,483,484,1330,1332−1358 mRNA,252,1353
by terms such as photoporation, optoinjection, laserfection, siRNA,1333,1343,1352 antisense morpholinos,1353 peptides,486,1359
and optical transfection.486,1332,1333 The aim of optoporation is proteins,1333,1343 dextrans,1333,1343,1349,1353,1360,1361 dyes [see
to permeabilize the plasma membrane to cargo while leaving refs 252, 480−484, 486, 1333, 1340, 1341, 1343, 1345, 1346,
other cellular structures intact, thus preserving the health of the 1348, 1354, 1357, and 1361−1366], sucrose,485 molecular
cell to the maximum extent possible. In this review, we define beacons,1367 ions,1333,1343,1368 semiconductor nanocrystals,1333,1343
optoporation as membrane disruption arising from direct inter- gold nanoparticles,1369 quantum dots,1370 and ∼1 μm polystyrene
action of a laser focal region with the plasma membrane and beads.1371 Moreover, many of these studies have sought to
not absorption of laser energy by an intermediate structure compare the mechanisms of various laser treatment regimes in
such as a nanoparticle or metal surface. Those strategies per- order to optimize delivery efficiency and minimize off-target
meabilize membranes by secondary effects such as fluid damage.
shear (section 6.1.2) and chemical effects (section 6.5.5) and 6.4.2. Mechanisms of Optoporation. The mechanisms
are covered in the respective sections dealing with those of laser-mediated membrane disruption are complex, involving
phenomena. combinations of mechanical, thermal, and chemical effects.
6.4.1. Optoporation: Pioneering Studies. DNA trans- Possibilities include burning/evaporation, thermoelastic mechan-
fection by laser optoration was first reported in 1984.1334 ical stress, generation of low-density free-electron plasma and
Nanosecond pulses of an Nd:YAG UV laser (wavelength 355 nm) reactive oxygen specifies (ROS), and effects beyond the focal
at an energy of 1 mJ with a spot size of ∼0.5 μm were focused region, such as shock wave emission and growth/collapse of
on the surface of adherent NRK cells. A single pulse of 5−10 ns cavitation bubbles, which themselves produce fluid shear stress,
was sufficient to open up a hole several microns wide and extreme heat, and sonochemical phenomena1321−1323,1331
promote the influx of DNA plasmids from an extracellular (Figure 31). The relative dominance of these phenomena
concentration of 10 μg mL−1 before closure of the wound. depends on factors such as wavelength, frequency, whether the
When manually targeting the laser pulse above the nucleus, source is continuous wave or pulsed, laser power, exposure
10% transfection efficiency was achieved while random scan- time, spot size, and absorbance properties of the focal region. For
ning of the laser over the substrate resulted in only 0.6% example, membrane wounding from continuous wave irradi-
chance of success.1335 Laser transfection with a similar laser but ation is thought to arise primarily from local heating, which
different cell types was repeated several years later, this time intensifies in proportion to exposure time. Nanosecond pulsed
establishing that a small percentage of target cells stably lasers have been suggested to produce a combination of
integrated the plasmid into their genome.1336 Addition of dyes heating, bubble formation, and thermoelastic or dielectric
to change absorption properties of the media was another var- mechanical stresses to damage the membrane. Femtosecond
iable that was examined, with the presence of standard cell laser mechanisms appear tunable based on irradiance strength,
culture media additive phenol red shown to decrease the laser pulse duration, and frequency. Mechanisms range from almost
power needed for optoporation.1330 A 488 nm continuous purely chemical degradation to combinations of thermal and
wave argon laser with nominal power of 2 W and spot size of mechanical. In cases where laser energy is transduced into fluid
BJ DOI: 10.1021/acs.chemrev.7b00678
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Chemical Reviews Review
distinct regions of adherent primary rat neurons to assess
localization-dependent biological functions.252 mRNA-medi-
ated expression of the transcription factor Elk-1 was found to
produce different responses whether delivered to the soma or
axon of the neurons.252 This optoporation protocol involved
an 840 nm titanium-sapphire laser delivering 100 fs pulses at a
repetition rate of 80 MHz for 1−5 ms at a power of 30 mW.252
Other studies have quantitatively measured the loading
efficiency of femtosecond optoporation and found that
targeted cells can incorporate up to 40% of the concentration
of extracellular molecules before resealing.483 Furthermore,
sub-20 fs pulses at MHz frequencies with submillisecond
exposure times have been demonstrated for the effective
transfection of human primary pancreatic and salivary gland
stem cells.1348
6.4.4. Toward High Throughput and More User-
Friendly Optoporation. A major rate-limiting step for
Figure 31. Optoporation strategies for membrane disruption. Focused optoporation is the reliance on precise positioning of the
laser can inflict (A) thermal, (B) cavitation, (C) chemical, or (D) laser focal spot and alignment with target membranes.1322,1337
mechanical effects (such as dielectric strain) against lipid bilayers. A misfocus of as little as 3 μm results in greater than 50%
reduction in membrane disruption efficiency.1344 One strategy
shear that travels far beyond the focal region, such as cavitation to mitigate this limitation is the implementation of a “bessel
or shock waves, the mechanisms of membrane damage are not beam”, where the focal region is stretched into a rod of light
strictly optoporation and these scenarios are covered elsewhere over 100 μm in length and a few microns wide.1344 Bessel beam
in the section on fluid shear (section 6.1.2). Alternatively, if setups have been combined with microfluidics for hydrodynamic
transmission of thermal energy from an absorbing object in flow focusing to reach throughputs of tens of cells per second.1365
immediate contact is the mechanism of membrane disruption, However, cell viability and delivery efficiency were substantially
these accounts are covered in the thermal section (section 6.3). less than standard femtosecond optoporation.1365 Whether or
6.4.3. Femtosecond Optoporation. Most recent work not bessel beams cause off-target damage to nonmembranous
favors the use of a laser regime characterized by wavelengths cellular structures appears to be unknown.1321
>700 nm administered at high frequencies (∼MHz range) and Other attempts to increase the throughput of optoporation
femtosecond pulse timings with a cumulative exposure of include a user-friendly “point and click” touchscreen software-
milliseconds or less.1321 For example, a typical protocol might integrated approach.1355 With this system throughputs of up to
involve 5 ms of exposure to a cycle of 100 fs pulses with gaps of 100 cells per minute were obtained on adherent neurons.1355
10 ns (∼100 MHz frequency) for cooldown. When operating An extension of this strategy relies on automated image ana-
at wavelengths >700 nm the mechanisms are related to multi- lysis of cell morphology, centering of the microscope stage to
photon effects inherently concentrated within the focal region, the laser focus, and execution of a femtosecond laser illu-
thus offering increased precision and high spatial resolu- mination protocol.1357 With this system, software-controlled
tion.1321 NIR and IR wavelengths also have the advantage of meandering of the sample stage allows adherent cells in a
being less toxic to cells, as UV and blue light in particular are typical cell culture dish to be automatically targeted at a rate
notorious for causing damage to DNA and other cellular struc- around 10 000 cells per hour.1357 If optoporation is to be
tures. By using extremely short femtosecond pulses, absorbing adopted by users outside of specialized laboratories, further
material in the focal region does not have sufficient time to efforts will need to address the challenge of how to precisely
transmit heat to adjacent regions. This enables extremely high- focus the laser spot onto thousands of cells for rapid treatment.
powered lasers to be deployed while avoiding excessive heating Other issues that need to be addressed are portability, instru-
of cells. In such a scenario the resultant membrane disruption ment complexity, and high cost.
mechanisms have been reported to be due to chemical effects, 6.4.5. Optoporation Summary. Optoporation has
such as the breakdown of bonds in lipid tails by low-energy captured a significant level of attention over the last several
plasma1321−1323,1331 (Figure 31C). In other cases, femtosecond decades, particularly for transfection. One of the main pro-
pulsing generates a well-controlled cavitation bubble originat- blems is the high cost of lasers and the optical systems required
ing within the focal region, the presence of which can destroy to operate them, as well as poor adoption outside of expert com-
the membrane (Figure 31B). In many of these reports, dis- munities. A second main problem is how to increase the through-
tinctions between exact mechanisms are difficult to determine put of treatments, which is an area where microfluidics and
and could be multifactorial. computer automation have made positive contributions. Future
A number of elegant studies have been performed with progress in optoporation is likely to require creative ideas to
femtosecond pulsed lasers. For example, in optical setups that move beyond traditional limitations.
combine laser tweezing and optoporation, optical tweezers may
be used to guide a microbead (∼1 μm) or nanoparticle through a 6.5. Biochemical Membrane Disruption
hole formed by the laser, thus delivering large cargo.1369,1371 A number of of chemical effects and biochemical agents can be
In studies with frog embryos, quantum dots were delivered by used to disrupt cell membranes. These range from synthetic deter-
NIR femtosecond lasers. Neither the quantum dots nor gents, surface-active agents (surfactants), organic solvents, and
optoporation retarded the ability of these embryos to grow oxidizing agents to naturally secreted proteins and metabolites
into tadpoles. In another case, cargo was introduced into from a diversity of organisms. For example, organic solvents
BK DOI: 10.1021/acs.chemrev.7b00678
Chem. Rev. XXXX, XXX, XXX−XXX
Chemical Reviews Review
Figure 32. Simulations of membrane bilayer perturbation with DMSO and ethanol. (A) Presented are side views of the final structures for the
bilayer systems containing 0, 5, 10, and 40 mol % of DMSO. Lipids are shown in cyan, water in red, and DMSO in yellow. Reproduced from ref
1378. Copyright 2007 American Chemical Society. (B) Formation of nonbilayer structures within the membrane interior with 15 mol % of ethanol:
(1) 3100, (2) 13 180, (3) 19 920, and (4) 30 000 ps. Shown are water molecules (red and white) and phosphorus (green) and nitrogen (blue)
atoms of lipid head groups. The rest of the lipid atoms as well as ethanol molecules are not shown. Reproduced from ref 1382. Copyright 2009
American Chemical Society.
have been used for decades as penetration enhancers for trans- between polar lipid headgroups and reduces membrane thick-
dermal delivery by fluidizing, destabilizing, or extracting com- ness, thereby weakening the membrane.1374 This has been experi-
ponents from lipid bilayers.1372 Since the dawn of life, living mentally verified by the use of X-ray diffraction techniques.1376
organisms have evolved a range of potent molecules to attack Moreover, the association of DMSO with solute molecules
and disrupt the membrane integrity of competing lifeforms. lowers the activation energy needed for them to enter into,
Pore-forming proteins (PFPs), which are produced by humans, and ultimately cross, the bilayer.1374 This interpretation is
animals, plants, fungi, protists, and bacteria for self-defense, are supported by vibrational spectroscopic studies.1377
one such example.399 Many plants synthesize and secrete In addition to physical measurements, simulations have been
metabolites like saponins to serve as an innate immune barrier used to visualize the molecular events associated with mem-
to disrupt the membranes of invading microbes or other threat- bane disruption by DMSO.410 In simulations by Gurtovenko
ening organisms.1373 These natural compounds tend to be et al., it was observed that at low concentrations, DMSO causes
relatively specific, relying on unique characteristics of the target membrane thinning and increases fluidity of the membrane’s
membrane for their action, such as composition of membrane hydrophobic core.1378 In agreement with experimental data,
lipids and presence of external receptors. Several artificially DMSO molecules are seen to penetrate into the bilayer, both
produced detergents and solvents also exhibit a useful ability to expanding the distance between the lipids and reducing the
disrupt plasma membranes in a relatively controlled manner. thickness of the bilayer (Figure 32A). Consequently, the lipid−
Furthermore, emerging concepts from nanotechnology, such as water interface becomes more prone to structural defects,
near-field ionizing plasmas, present opportunities to confine chem- especially due to thermal fluctuations. At higher DMSO
ical destabilization phenomena to small membrane patches for concentrations, water molecules enter the membrane interior
short durations. This section will cover artificial and natural via DMSO-mediated structural defects. As the number of
biochemical permeabilization strategies that hold demonstrated penetrating water molecules increases, a significant reorientation
or theoretical potential for intracellular delivery applications. of lipid headgroups toward the membrane interior is required
6.5.1. Organic Solvents and Penetration Enhancers. to minimize the free-energy of the system, resulting in the
6.5.1.1. DMSO. Organic solvents are low-molecular weight formation of hydrophilic channels spanning the membrane
compounds that can perturb bilayer structures by burying their bilayer.410 The emergence of hydrophilic channels occurs
hydrophobic residues into the membrane. A classic example of spontaneously between 10 and 20% molar concentration of
a membrane-active organic solvent is dimethyl sulfoxide (DMSO), DMSO.1378 The addition of sterols (i.e., cholesterol) can
often used as a penetration enhancer to increase the permeability provide stabilization to the membrane and thus increase the
of drugs and other small molecules.54,1374 DMSO is amphiphilic, DMSO concentration required for pore formation.1379
containing one hydrophilic sulfoxide group and two hydrophobic 6.5.1.2. Ethanol and Other Alcohols. In contrast to DMSO,
methyl groups. It is known to promote permeation of both ethanol’s hydrophobicity is rather limited as a short-chain
hydrophilic54 and hydrophobic1374 species across lipid bilayers. alcohol. Rather than embed deeply, ethanol molecules tend to
DMSO’s penetration enhancing effect can be attributed to two remain at the water−lipid interface forming hydrogen bonds
mechanisms: first, its ability to increase the solubility of small with hydrophilic lipid headgroups.1380,1381 Ethanol has a
molecules and, second, because of increased incidence of mem- disordering effect on lipid hydrocarbon tails, increasing fluidity
brane defects that allow passage of normally impermeant mol- of the membrane and reducing rigidity. Simulations confirm
ecules. Experiments with phospholipid vesicles have found that compromising the water−lipid interface induces ingres-
leakage of carboxyfluorescein (∼376 Da) at concentrations of sion of water pockets into the membrane as inverse micelles,
DMSO >10%.1375 For a given DMSO concentration, leakage rather than pores that span the whole membrane1382 (Figure 32B).
also increases as a function of temperature.1375 The bilayer structure is partly destroyed due to lipid desorp-
Experiments have been conducted to understand the mech- tion.1382 Both experimental and simulation studies have
anisms by which DMSO increases lipid bilayer permeability. shown that the bilayer structure cannot be maintained beyond
DMSO incorporation into the bilayer increases the distance an ethanol concentration around 12% molar or 30% v/v.
BL DOI: 10.1021/acs.chemrev.7b00678
Chem. Rev. XXXX, XXX, XXX−XXX
Chemical Reviews Review
Correspondingly stronger results can be expected with longer
chain alcohols, such as propanol, butanol, and pentanol, as the
concentration required for defect formation is inversely
proportional to hydrocarbon chain length.1383 As an example,
significant membrane defects have been reported in mem-
branes exposed to only 1% butanol.1384
One case where ethanol was used for intracellular delivery
purposes was reported recently by O’Dea et al.1385 Reversible
cell permeabilization was achieved by temporally and volu-
metrically controlling the contact of the target cells with a
hypotonic solution of 75% H2O, 25% ethanol, 32 mM sucrose,
12 mM potassium chloride, 12 mM ammonium acetate, and
5 mM HEPES. An atomizer was employed to spray a small
volume of this solution to form a thin film over a monolayer
of cells. After 2 min, permeabilization was terminated with a
neutralizing solution. Using this protocol intracellular delivery
of proteins, mRNA, and plasmids was reported.1385 The observa-
tion that ethanol permeabilization is optimal at 25% v/v fits well
with the results from simulations that predict the effects of
ethanol should be reversible at concentrations <30% v/v.1382
6.5.1.3. Organic Solvents and Penetration Enhancers
Summary. Although widely used for permeabilizing fixed
cells1386 and increasing the permeability of small molecules,54
organic solvents and other low molecular weight penetration
enhancers have generally not been used as the sole membrane Figure 33. Proposed mechanisms of membrane permeabilization by
disruption agents to deliver cargo molecules. This is probably detergents that flip flop. Integration of detergent monomers perturbs
due to their nonspecific nature and lack of spatiotemporal membrane integrity while stochastic local enrichment of detergents
control over the membrane disruption process. They may, leads to formation of pores.
however, be useful as nonspecific and relatively inert adjuvants
to modify other membrane permeabilization strategies such as curvature strain. The major structural consequence of this
electroporation.1387−1389 curvature strain is a disordering of the hydrophobic chains.
6.5.2. Detergents. Detergents are water-soluble surfac- In turn, the membrane becomes thinner and more flexible.
tants capable of solubilizing phospholipids found in biological Monolayer curvature strain can be partially relaxed by the
membranes. Solubilization refers to the dissolution of the bilayer sequestering of surfactants into highly curved rims covering the
structure by sequestration into detergent-lipid micelles.441,442 For hydrophobic edges of toroidal pores.441 Over time, thermal
the purposes of intracellular delivery, complete solubilization of fluctuations will give rise to such events. Moreover, reduction
membranes is lethal and undesirable, thus detergents must be of the pore’s line tension by detergents may massively increase
used at intermediate concentrations for limited durations to the lifetimes of induced pores or even stabilize them indefinitely.
yield optimal levels of cell permeabilization. Although the Above a critical surfactant concentration, pores appear sponta-
mechanisms of detergent solubilization of biological mem- neously so that permeabilization becomes effectively persistent.441
branes have been discussed for decades,441,1390−1392 the milder 6.5.2.2. Membrane Disruption by Detergents that Do Not
intermediate regime of nonlethal permeabilization is less well Flip Flop. Detergents that embed into the outer leaflet but can-
understood. As well as intracellular delivery applications, not flip flop expand the bilayer asymmetrically (Figure 34).441,442
motivations to investigate this regime include understanding If the bilayer is unable to bend to assume its spontaneous bilayer
the action of membrane-perturbing secondary metabolites and curvature, it develops a bilayer curvature strain by compressing
characterizing new candidates for antimicrobials. the molecules in the overpopulated (outer) leaflet and/or
6.5.2.1. Membrane Disruption by Detergents that Flip expanding those in the underpopulated (inner) leaflet. Bilayer
Flop. Owing to their amphiphilic properties, detergent mole- curvature eventually leads to mechanical failure of the
cules integrate into lipid membranes. Most detergents are cone- membrane because the outer monolayer forms mixed micellar
shaped, in that the headgroup of the detergent is disproportion- structures that bud off from the membrane. Shedding of these
ately larger than the hydrophobic chains. They generally work micelles into the aqueous solution results in emergence of
by inserting into lipid bilayers and distorting their structure. defects and subsequent permeabilization.442 These disruptions
Several mechanisms have been suggested for detergent- can have several effects. First, relaxation of the curvature strain
mediated permeabilization of lipid bilayers depending on the allows the membrane leaflets to anneal, and second, they
type of detergent.441 Those capable of flip-flopping to the inner permit the passage of detergent molecules inside the cell to
leaflet will distribute throughout both leaflets of the bilayer access the inner leaflet, thereby promoting further infiltration
(Figure 33). Because of the cone-shaped nature of detergents, of the membrane by mechanisms akin to detergents that flip
the structure of the monolayer wants to assume a degree of flop (Figure 33).
convex intrinsic curvature. However, this is impossible if the 6.5.2.3. Membrane Disruption by Detergents that Do Not
monolayer is part of a bilayer, because it competes with the Embed. A third possibility is that collision of detergent micelles
opposite spontaneous curvature of the other leaflet since both with the cell membrane recruits lipid units into the micelles,
are coupled with each other. Instead, the monolayers are “bent thereby generating defects in the membrane (Figure 35).461,1393
straight” by an elastic deformation giving rise to a monolayer There is little theory to support this third scenario; however, it
BM DOI: 10.1021/acs.chemrev.7b00678
Chem. Rev. XXXX, XXX, XXX−XXX
Chemical Reviews Review
Figure 35. Proposed mechanisms of membrane permeabilization by
detergent micelle collisions. Micelles colliding with the membrane
may create defects by sequestering lipid molecules from the bilayer.
sulfate (SDS), and lauryl maltoside, which destroy the mem-
brane through homogeneous disordering when a critical cur-
vature stress is reached. In contrast, the heterogenous category
included the fungicidal lipopeptides surfactin, fengycin, and
iturin, as well as digitonin, CHAPS, and lysophosphatidylcho-
Figure 34. Proposed mechanisms of membrane permeabilization by line, which perturb membranes without substantial overall
detergents that do not flip flop. Once detergent monomers gain access disordering. Rather, they disrupt membranes locally in surfactant-
to the interior side of the membrane, they can distribute to both rich defect structures. Nazari et al. proposed that such hetero-
leaflets and perturb the membrane by mechanisms similar to geneous perturbation mechanisms may account for the superior
detergents that flip flop (see Figure 33). activity, selectivity, and mutual synergism of antimicrobial
biosurfactants, such as lipopeptides and saponins, to efficiently
should be mentioned as a possibility. In order to first achieve permeabilize target cell membranes in discrete loci at minimal
micelles, the detergent will need to be at a concentration above concentrations.1394
the critical micelle concentration (CMC). This will only be a 6.5.2.5. Detergent Permeabilization of Live Cells. A fur-
realistic scenario in the case of detergents that do not readily ther consideration influencing detergent-mediated membrane
embed into cell membranes. Thus, integration of individual permeabilization is the composition of the target membrane of
detergent molecules into the target membrane may not be living cells. The permeabilizing activity of certain antimicrobial
strictly necessary to cause defect generation and subsequent peptides and surfactants is strongly modulated by cholesterol,
permeabilization. proteins, and other raft domain components.441 Owing to
6.5.2.4. Relationship between Strain and Emergence of the heterogeneous and dynamic nature of living cell mem-
Defects.Most of the detergents used to permeabilize biological branes, it has been a challenge to predict how detergents will
membranes integrate into the bilayer.441 Curvature-driven dis- permeabilize cells. One study by Vaidyanathan and colleagues
tortion and disordering of membranes leads to perturbation of used patch clamp to analyze permeabilization behavior of deter-
the bilayer structure and subsequent permeabilization. As dis- gents as a function of concentration.1395 They observed that
cussed, the key property of a micelle-forming amphiphile anionic SDS, cationic cetyltrimethylammonium bromide (CTAB),
inserting into a lipid bilayer is its preference for a locally curved and cationic fluorescent octadecyl rhodamine B (ORB) increased
interface that is in conflict with the (on average) planar topology the membrane permeability of cells substantially within a second of
of a bilayer. Indeed, strongly curvature-inducing detergents are exposure. It was reported that SDS ≤ 0.2 mM (below SDS’s CMC
known to be far more effective in membrane permeabiliza- of ∼1 mM) and CTAB and ORB ≤ 1 mM (above CTAB’s CMC
tion.441 When local concentrations of detergents are high of ∼50 μM) induced transient cell membrane permeability
enough (perhaps due to random fluctuations), local mechan- without causing acute or permanent toxicity.1395 Thus, careful
ical distortions can cause defects in the form of spontaneous titration of detergent concentrations enabled the identification
pores or shedding of micelles. of conditions from which cells can recover after permeabilization.
A comprehensive study from Nazari et al. compared the In another study of detergent permeabilization in live cells,
membrane-perturbing effects of a number of different deter- Koley and Bard used electrochemical microscopy to monitor
gents and surfactants on lipid vesicles, categorizing them into the permeability of HeLa cells to the hydrophilic anion ferrocy-
homogeneously and heterogeneously perturbing surfac- anide (∼0.2 kDa) in the presence of increasing concentrations
tants.1394 In the homogeneous category were typical synthetic of the nonionic detergent triton X-100.1396 No effect on per-
detergents, such as C12EO8, octyl glucoside, sodium dodecyl meability was seen at triton X-100 concentrations of 0.15 mM
BN DOI: 10.1021/acs.chemrev.7b00678
Chem. Rev. XXXX, XXX, XXX−XXX
Chemical Reviews Review
for up to 1 hour. At 0.17 mM, initial permeabilization was
observed followed by a recovery of cell viability. From 0.19 mM,
which approaches the CMC, rapid irreversible permeabilization
and cell death resulted. Thus, the effective concentration
window of triton X-100 treatment with live cells is narrow
under the tested set of experimental conditions. The above
results underscore the importance of conducting systematic
permeabilization studies in live cells.
6.5.2.6. Saponins. Saponins are steroid and triterpinoid
glycosides produced by plants and certain marine organisms
as secondary metabolites in response to environmental sti-
muli.1373,1397 By perturbing the membranes of competing life
forms, saponins constitute a form of innate immune system to
poison threatening microbes, parasites, insects, and herbi-
vores.1373,1398 The detergent phenomena of saponins originates
from their amphiphilic properties, featuring a lipophilic
sapogenin part (usually a triterpene or steroid group) and a
hydrophilic glycoside moiety. A wide range of applications for Figure 36. Interactions of 50 μM digitonin (c < CMC) with SOPC
saponins relating to their membrane-perturbing activity have (1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine) phospholipid bilayer
been proposed. They include augmenting the penetration of membranes containing varying amounts of cholesterol. Reproduced
drugs and cytotoxic agents to cancer cells, vaccine adjuvants, or from ref 1406. Copyright 2014 American Chemical Society.
deployment as microbials and pest control agents.1399−1401
For applications with mammalian cells, studies usually employ saponins.408 Hence, saponins can be been exploited to target
generic saponins or pure digitonin. Generic saponins are com- the plasma membrane while leaving those of cholesterol-poor
mercially available cocktails typified by a sapogenin content organelles, such as the ER and mitochondria, largely
>10% while digitonin is a prototype member of the saponin una ected.397,585,1405ff Calcium stores within intracellular
family isolated from the foxglove plant Digitalis purpurea. organelles are generally not eroded by the saponin concen-
Other less-studied saponins that have been reported to disrupt trations that permeabilize plasma membranes.583
membranes include α-tomatine, glycyrrhizin, α-chaconine, and How do saponins interact with cholesterol to disrupt mem-
α-hederin.1393 Saponins in general, and digitonin specifically, branes? Frenkel et al. conducted investigations into the mech-
have been used with live cells for two main applications: anism using quantitative physical techniques in model mem-
(1) persistent permeabilization to produce “semi-intact cells” branes. Their measurements indicate that digitonin extracts
for real-time manipulation of cytoplasmic constituents (see cholesterol out of the bilayer core to form a surface complex,
section 4.3.6) and (2) to transiently disrupt the plasma which then induces curvature and disordering of the mem-
membrane for intracellular delivery. Early work emphasized the brane.1406 The magnitude of these effects was directly propor-
first of these two applications. tional to the amount of cholesterol in the bilayer (Figure 36).
6.5.2.7. Characteristics and Mechanisms of Saponin- At 0% cholesterol, digitonin could not bind to the membrane
Induced Membrane Disruption. Saponins were initially and thus had no effect. At 5% cholesterol, exposure to
characterized as membrane-perturbing agents in the scientific digitonin triggered the formation of sterol-aglycone complexes
literature of the 1960s and 1970s.940,1402 Electron micrographs without significant membrane distortion. At 20% cholesterol,
captured their membrane disrupting capabilities in reconsti- digitonin extracts cholesterol into aggregates, thus removing it
tuted membranes, indicating arrays of holes around 8 nm.1402 from the hydrophobic core region. The steric hindrance between
Serial section electron microscopy of fixed hemolysing saccharide residues in these aggregates may induce changes in the
erythrocytes revealed lesions of 4−5 nm after saponin treat- curvature of the membrane outer leaflet leading to compro-
ment.940 Most cell permeabilization studies have employed misation of the membrane integrity and concomitant increase
saponins in the concentration range 10−1000 μg mL−1, which in membrane permeability.1406 In essence, digitonin binds to
represents ∼8−800 μM. In this range, disruption sizes from a cholesterol and transforms it into a detergent.
few nanometers up to one micron have been reported. Differ- Beyond digitonin, studies have explored a wider range of
ences are probably related to variations in cell type, concentra- individual saponins for membrane permeabilization. Recently a
tion, duration of exposure, and other experimental condi- set of oleanane saponins (glycyrrhizic acid, Gypsophila,
tions.397,1403,1404 The inconsistency of these reports may also Saponaria, and Quillaja saponins) and digitonin were tested
stem from the variety of analysis techniques. For example, in live cells. These saponins showed variable permeabilizing
misleading artifacts can occur during fixation of membranes for effects on cellular membranes from 6 μM, as measured by an
AFM and SEM imaging. Thus, our knowledge on saponin- impedance-based plate reader with ECV-304 human urinary
based permeabilization and the characteristics of holes formed bladder carcinoma cells.1407 The results indicated that the
may require revision with more current methods and stricter molecular charge may be a relevant consideration in explaining
control of environmental conditions. the action of oleanane saponins. Further studies with α-hederin
Most saponins preferentially interact with cholesterol- and indicate that the critical micelle concentration (CMC) plays a
hydroxysterol-rich membranes, a property that makes them key role in its mechanism. At concentrations lower than the
relatively specific for the plasma membranes of animal cells. CMC, α-hederin monomers bind to cholesterol and induce
The efficiency of their membrane-perturbing effects are directly vesiculation and lateral phase separation.1408,1409 These effects
correlated with sterol content. Indeed, cholesterol-rich bilayers are analogous to the action of detergents that do not flip flop,
are thought to be about 20- to 100-fold more sensitive to as depicted in Figure 34. At concentrations higher than the
BO DOI: 10.1021/acs.chemrev.7b00678
Chem. Rev. XXXX, XXX, XXX−XXX
Chemical Reviews Review
CMC, α-hederin aggregates promote pore formation and the More recently, saponins have been exploited for the delivery
loss of membrane material by analogy to the scenario illus- of quantum dots and nanoparticles. Lukyanenko published a
trated in Figure 35. Thus, the self-aggregating properties and protocol for the delivery of nanoparticles up to 20 nm with a
co-operative action of saponins may also be important for their transient 30−60 s exposure to 0.01% saponin (10 μg mL−1 or
effects. Most studies agree that the permeabilizing activity of ∼8 μM) in high potassium low calcium permeabilization
saponins relies on the presence of sterols, from which they bu er.1416ff Depolymerization of cytoplasmic actin with cyto-
form complexes to distort the membrane into nonbilayer chalasin D was reported to boost the efficiency of nanoparticle
structures (Figure 36). As an exception to this rule, some penetration into the cytoplasm, as the actin meshwork that
bidesmosidic saponins, such as avicin D,1410 appear capable underlies the plasma membrane may be considered another
of porating cell membranes through detergent properties inde- barrier to delivery.1416 Medepalli and co-workers demonstrated
pendent of cholesterol binding.1393 quantum dot loading into adherent H9C2 cells with a com-
6.5.2.8. Saponin-Mediated Permeabilization for Studies in bination of 50 μg mL−1 (∼40 μM) saponin and 180 mOsm
Semi-intact Cells. Detergent-permeabilized semi-intact cells hypotonic media for 5 min at 4 °C.311 Whether or not
have led to advances in several areas of biology, including hypoosmotic shock produces a membrane tension to synergize
decoding the rules governing nuclear import of proteins and with the membrane perturbing effect of saponin or generates
DNA,592,593 studying mammalian protein synthesis and inward fluid flux to promote delivery remains to be determined.311
secretion machinery,590,591 and the analysis of functional For intracellular analysis with fluorescently-labeled antibod-
mitochondria in muscle fibers, tissues, and cells in situ.594 ies, Jacob et al. developed a saponin-based permeabilization
The emergence of saponins for the production of semi-intact protocol to load immune cells with monoclonal antibodies for
cells began around the early 1980s. In 1982 Wakasugi et al. the detection of cytoplasmic antigens by 1417flow cytometry.
used saponin or digitonin in the range 20−100 μg mL−1 They incubated primary lymphocytes and lymphoma cell lines
(∼16−80 μM) to permeabilize acini from rat pancreases and at 4 °C in HBSS buffer with antibodies in a buffer containing
probe the e ect of ATP on intracellular calcium dynamics.1411ff 2% FBS and 0.1−0.3% saponin (10−30 μg mL−1 or ∼8−24 μM)
A year later, the plasma membranes of isolated guinea pig for 30 min. As judged by flow cytometry analysis, monoclonal
hepatocytes were made permeable with 75 μg mL−1 (∼60 μM) antibody delivery was achieved while cell integrity and mor-
saponin to study the ATP-dependent uptake of calcium into phology remained intact.1417 Interestingly, this protocol did
the endoplasmic reticulum.583 Upon saponin treatment, cells not rely on fixation with paraformaldehyde, a step that was
were suspended in a medium resembling cytosol with an ATP- only incorporated in later adaptations, presumably to prevent
regenerating system consisting of ATP, creatine phosphate, leakage of cytokines from the cell or to avoid dealing with
and creatine phosphokinase. Dunn and Holz used 20 μM apoptotic cells.1418−1420 An earlier method featuring lysophos-
digitonin to permeabilize chromaffin cells, and this protocol phatidylcholine as permeabilization agent was similarly
became a popular system to study intracellular processes in independent of xation.99fi
this cell type.584,1412 Human platelets were also treated with 6.5.2.10. Detergent-Like Lipids and Other Surfactants for
saponins for the loading of the secondary messenger inositol Intracellular Delivery. Surfactants include synthetic deter-
1,4,5-trisphosphate into the cytoplasm and studying of the gents, physiological compounds such as bile salts, lysolipids
metabolic signaling response.1413 Several groups reported that, and certain amphiphilic peptides and amphiphiles. A widely
with optimal conditions, 50% or more of the cytoplasmic enzyme used example is the naturally occurring lipid lysophosphati-
lactate dehydrogenase (∼140 kDa) is able to remain inside cells dylcholine (also known as lysolecithin). Miller et al. employed
for extended periods, indicating the possibility of maintaining a lysophosphatidylcholine exposures to permeabilize CHO cells
feasible balance between plasma membrane permeabilization and maintain them as semi-intact cells capable of DNA syn-
and cell function in these experiments.584,585,1414 In most thesis for several hours.541 The protocol was used to explore
of these papers the plasma membrane resealing dynamics for soluble factors that inhibit or stimulate DNA synthesis.
were not discussed. Thus, it is difficult to ascertain whether or A follow-up paper outlined generalized protocols for delivery of
not the cells were persistently permeabilized or whether they cargo molecules to a wide range of monolayer and suspension
recovered upon plasma membrane repair. cells.1421 In it, lysophosphatidylcholine concentrations from
6.5.2.9. Saponin-Mediated Permeabilization for Intra- 30−250 μg mL−1 (60−500 μM) were chosen depending on
cellular Delivery. An optimized protocol for peptide delivery the balance between delivery, viability, and leakage of the rep-
into cardiac myocytes employed a 10 min incubation at 4 °C resentative endogenous protein lactase dehydrogenase. Balinska
with 50 μg mL−1 (∼40 μM) saponin.539 Along with saponin, employed lysophosphatidylcholine to introduce the exogenous
the permeabilization buffer was designed to mimic aspects of nucleoside dTTP into the DNA of hepatoma cells via
the intracellular environment by including high potassium, permeabilization-mediated intracellular delivery.1422 Because
extracellular ATP to maintain energy stocks, and ascorbic acid there was only a slight loss (20−25%) of lactate dehydrogen-
as an antioxidant.539 The authors reported loading of peptides ase, they concluded permeabilization of cells does not per-
at up to ∼10% of the extracellular concentration without loss sistently disrupt membrane integrity and resealing could be
of long-term viability. In another method, Miyamoto et al. used achieved by exchanging back to standard media.1422 Nomura and
7.5 μg mL−1 (∼6 μM) digitonin to induce reversible per- colleagues used lysophosphatidylcholine permeabilization for the
meabilization of the plasma membrane in bovine, mouse, and delivery of larger proteins: diphtheria toxin (A fragment), horse-
porcine somatic cells.1415 By optimizing the procedure, high radish peroxidase, and antibodies against SV40 T-antigens.1423
efficiency (∼80%) loading of 70 kDa dextrans was achieved in These macromolecules were successfully introduced into living
bovine cumulus cells. It was also used to introduce cytoplasmic mouse erythroleukemia cells, baby hamster kidney, and mouse
extracts from Xenopus laevas eggs into several mammalian cell fibroblast cells.1423 Furthermore, lysophosphatidylcholine has
types for successful induction of nuclear reprogramming and been used to permeabilize primary human lymphocytes and
activation of pluripotent genes.1415 monocytes for detection of intracellular antigens by flow
BP DOI: 10.1021/acs.chemrev.7b00678
Chem. Rev. XXXX, XXX, XXX−XXX
Chemical Reviews Review
cytometry.99 50 μg mL−1 (100 μM) lysophosphatidylcholine can be harnessed to produce nanoscale cargo with more potent
was incubated at 4 °C for 5 min before recovery with anti- cell penetration properties. If membrane-perturbing nano-
bodies inside, thus avoiding the need for fixation. particles can be made switchable by light or other environ-
Along with lysophosphatidylcholine, similar compounds have mental stimuli, they may confer the level of control required
been investigated for their detergent-like mechanisms. For exam- for reversible permeabilization at discrete locations on the cell
ple, simulations have been performed on plant-derived resor- surface.
cinols1424 and dioctanoyl-phosphatidylcholine, a cone-shaped 6.5.2.12. Summary of Permeabilization by Detergents.
counterpart of the native lipid DPPC.1425 Studies with The above-mentioned studies suggest saponins, detergents,
dioctanoyl-phosphatidylcholine reveal a curvature stress that and other membrane permeabilizing surfactants can be used to
can be relieved upon pore formation.1425 Such mechanisms introduce a wide range of cargo molecules into various cell
may also be applicable to lysophosphatidylcholine, which is types. The emergence of membrane defects depends on var-
also a cone-shaped lipid. In the case of resorcinols, micelles are iables such as exposure time, temperature, diffusion, random
observed to bind to the membrane. If micelles remain compact, fluctuations, mixing effects, and spontaneous interactions.
they displace phospholipid head groups into the bilayer center, This is in contrast to physical methods where a well-defined
thereby disrupting the structure of the leaflet and causing the stimulus triggers a clean disruption event. Electroporation, in
lipids to surround the micelle.1424 However, if resorcinols are particular, has often been reported to achieve superior results
already embedded within the bilayer their presence leads to in the hands of researchers when compared with deter-
stabilization instead, just like cholesterol. Thus, simulations are gents.1098 The use of physically controllable or light-switchable
a useful tool to gain insight into the mechanisms and molecular surfactant systems may aid in developing more precise mem-
events that underlie detergent-mediated membrane disruption. brane perturbation strategies. Furthermore, it is worth con-
In future, insights arising from such studies could be leveraged sidering that a wide range of organisms produce secondary metab-
to inform intracellular delivery. olites with membrane-disrupting properties. As an increasing
6.5.2.11. Microfluidic and Nanotechnological Control of abundance of these natural detergents and lipopeptides are
Detergent Exposure. For detergents and surfactants applied in characterized, new possibilities for ideal membrane permeabi-
bulk solution, a key weakness is that the nature of the mem- lization agents may become available. For example, anabaeno-
brane injury lacks precise spatiotemporal control. Molecules lysin lipopeptide toxins have recently been proposed as a
are added indiscriminately to solution, and it is difficult to get rid potent alternative to digitonin for the selective disruption of
of them once the job is done. Thus, it is challenging to balance cholesterol-containing biological membranes.1428 Finally, the use
the required level of membrane permeabilization against of microfluidics and nanotechnology for local and transient
excessive toxicity (Figure 37A). Recently, Kilinc et al. used exposure of cells to surfactants is another frontier where spatio-
temporal control of membrane disruption may increase the
effectiveness of intracellular delivery.
6.5.3. Membrane-Active Peptides. Various membrane-
active peptides are known to disrupt lipid bilayer mem-
branes.433,440 Antimicrobial peptides (AMPs), which are usually
both amphiphilic and cationic, can induce pore-formation at
critical concentrations.1429,1430 Under certain circumstances,
cell-penetrating peptides (CPPs) and pathogenic amyloid pep-
tides can also permeabilize lipid bilayers, although the mech-
anisms are less well-defined.433 Most membrane-active pep-
Figure 37. Schematic of exposure to membrane-perturbing detergents tides are thought to be intrinsically disordered in solution but
and/or surfactants by (A) bulk mixing, (B) microfluidic hydro- adopt more defined structures upon contact with biological
dynamic focusing, and (C) localization to a nanoscale particle. membranes, giving rise to their membrane-disrupting proper-
ties.433 Membrane-active peptides are often conjugated to
microfluidics to demonstrate controlled flux of localized sapo- cargo to facilitate intracellular delivery.1431,1432 However, there
nins to perform precise axotomy (cut off an axon) on neurons are also reports of membrane-active peptides permeabilizing
cultured on microchips.1426 In a variation on this theme, the cells to enable cytosolic delivery of dyes,1433,1434 small pro-
detergent sodium dodecyl sulfate (SDS) was employed in teins,1435 low-molecular weight dextrans,1436 and short oligo-
laminar flow mode in a microfluidic device to damage speci 1437fic nucleotides.
sections of neurites and investigate the recovery process.1427 6.5.3.1. AMPs. The best-characterized membrane-active pep-
Saponin has also been combined with nanostraws to localize tides are the AMPs. To date, more than 5000 of them have been
membrane disruption to the nanostraw openings.701 These catalogued,1438,1439 with frog skin alone representing a source
examples showcase the potential of nano and micro uidic of more than 300 variants.1440fl Only a small selection of AMPs
systems to localize and control damage conferred by detergents have been studied for their molecular mechanisms of action.
to subcellular regions (Figure 37B). It remains to be seen A common feature is their ability to adopt a conformation with
whether such strategies can be feasible for intracellular delivery hydrophobic segments distinct from hydrophilic/cationic seg-
at high throughput, although we anticipate that inventors will ments.1429 For a given AMP, the ability to disrupt membranes
test this possibility in the near future. also depends on the lipid composition of the target membrane.
Membrane-perturbing nanoparticles are another concept In contrast to the plasma membrane of animal cells, most micro-
worth considering (Figure 37C). Multifunctional nanocarriers bial membranes, including those of Gram-positive (mono-
that switch to a membrane disrupting state are already being derm) and Gram-negative (diderm) bacteria, fungi, and protozoa,
developed for endosomal escape purposes.6 Similarly, conjuga- feature many negatively charged lipid headgroups on their
tion with membrane-active peptides116 or pore-forming toxins119 outer lea ets.1441fl This allows a combination of electrostatic
BQ DOI: 10.1021/acs.chemrev.7b00678
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and hydrophobic interactions to drive adsorption of cationic
AMPs to the surface of microbes with high affinity.1429 Once at
the interface, hydrophobic segments integrate into the mem-
brane to disrupt it, with several different models proposed to
explain pore formation.440,1430,1442 Due to their higher affinity
for microbial membranes, AMPs can lyse microbes at μM con-
centrations while having less effect on animal cell membranes.
This enables them to kill microorganisms without being sig-
nificantly toxic to mammalian cells. Moreover, in an opposite
manner to saponins, cholesterol in the plasma membrane of
animal cells serves to suppress the activity of AMPs due to its
stabilizing effect. At high enough concentrations, however,
AMPs will disrupt plasma membranes of mammalian cells, and
this is the regime of interest for potential intracellular delivery
applications.
6.5.3.2. Mechanisms of Membrane Disruption by AMPs.
The main models used to describe AMP-mediated pore
formation mechanisms share a common aspect, namely two Figure 38. Schematic overview of the possible interaction pathways of
distinct peptide−lipid states: an inactive surface-bound state an antimicrobial peptide with a lipid bilayer. Possible thermodynamic
and a pore-like insertion state.1429,1443 One of the best studied states (either stable or metastable) are indicated by black labels and
AMPs is melittin, a peptide extracted from bee venom.1444 It is the major kinetic pathways connecting them by gray arrows and red
a 26 amino acid chain containing +6 positive charges in total. labels. Short black arrows represent additional interconversion
Amino-terminal residues 1−20 are mostly hydrophobic while pathways. Outside the target membrane, peptide monomers and
carboxyl-terminal residues 21−26 are hydrophilic due to a small aggregates exist in equilibrium. At the target membrane, the
peptides bind to the interface (Adsorption). At the interface an
string of positive charges. Pores produced by melittin exposure equilibrium may exist between monomeric and polymeric aggregation
have been estimated at 2.5−3 nm in palmitoyloleoylphospha-
1445 states. For a symmetric bilayer, the asymmetric membrane boundtidylcholine (POPC) vesicles. Experiments with GUVs state is not thermodynamically stable. Eventually the peptides will
held by micropipettes revealed that melittin first increases the distribute equally between the two monolayer leaflets. This can occur
membrane surface area due to adsorption/integration before via two alternative translocation pathways. In the nonleaky variant the
rearranging to induce stable pores without vesicle rupture.1446 peptides are able to cross the bilayer without the formation of a pore.
Subsequent studies suggested that melittin partitions to both In some cases, the intermediate transmembrane state is thermody-
sides of the bilayer, probably via transient defects, before finally namically stable (e.g., hydrophobic peptides which adopt a trans-
reaching a concentration where stable pore formation occurs. membrane orientation). The key feature of many antimicrobial
The critical concentration lies in the μM range and corre- peptides is that they permeabilize the membrane following a leaky
1447 translocation pathway. Above a certain peptide−lipid ratio, thesponds to a peptide-to-lipid ratio of 1:100 or greater. peptides insert into the bilayer to form a porated lamellar phase
Another heavily studied AMP is magainin 2. Tamba et al. (poration). A variety of different pore structures may be formed,
showed that pore formation is triggered when magainin 2 including the barrel-stave, the toroidal, and the disordered toroidal
reaches a critical concentration at the membrane interface.1448 state. These separate states should be interpreted as extreme cases
Their studies predicted that the initial disruption size could be with mixed varieties of these models, and conversion between
as large as tens of nanometers before shrinking to a more stable alternative states is likely to occur. The porated states can be stable
pore of several nanometers.1449 The pores are thought to be themselves, but they can also be transient structures in the translo-
“chaotic”, lined by a mixture of peptides and lipids acting in cation pathway. In that case, once enough peptides are adsorbed at
cooperation, rather than a well-de ned peptide lined channel.1450 the opposing monolayer leaflet, the pores seal. On the other hand,fi
In keeping with the notion of a two-state model, the human increased accumulation of certain peptides may lead to a detergent-like disintegration of the membrane resulting in formation of
LL37 peptide has been observed to first absorb parallel to the nonlamellar, e.g., micellar, systems (solubilization pathway). Note
surface as an α helix before inserting and rotating normal to that the secondary structure of the peptides could vary along the
the membrane to form pores with an estimated diameter of various pathways. The helical or random configurations drawn here
2.3−3.3 nm.1451 AMPs can, to some extent, exhibit detergent- are merely illustrative of these processes and should not be taken
like effects including membrane thinning, bilayer stresses, literally. Figure and legend reproduced from ref 1454. Copyright
toroidal pore formation, and micellization.1452 Unlike deter- 2008, with permission from Elsevier.
gents, however, they tend not to dissolve the membrane
structure but rather induce smaller pores for the passage of of several peptides together cooperatively results in defect
low-molecular weight molecules.1447 One report suggested, formation.410 AMP aggregation leads to a high local density of
however, that AMPs can form larger holes in certain types of positive charges. This dense concentration of positive charges
membranes.1453 Atomic force microscopy imaging of the at the membrane interface can result in a highly localized elec-
supported lipid bilayers was used to visualize a population of tric field, which could destabilize the bilayer by an electro-
pores that could grow as a function of AMP concentration.1453 poration-like effect.1455−1457 Interestingly, simulations indicate
In many cases the exact structure of AMP-mediated pores is that the emerging defects appear to exhibit a significantly dis-
unknown. Multiple models have been proposed such as ordered shape, rather than a classic toroidal pore.1454 Studies
toroidal, disordered toroidal, and barrel stave. The depictions of magainin MG-H2 peptide reveal that its binding creates a
of these pore models are shown in Figure 38.1454 Molecular local tension in the exposed leaflet, which then generates a
dynamics simulations have played a part in elucidating possible compressive stress that is relieved upon pore formation.1458
molecular events.410 They indicate that synergistic aggregation Simulations of melittin1454 and cateslytin1459 support a similar
BR DOI: 10.1021/acs.chemrev.7b00678
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Chemical Reviews Review
interpretation. Overall, the prerequisites for AMP-mediated The structure and lifetime of such pores in live cells remain to
pore formation appear to be (1) a high concentration of be determined.
peptides in solution and (2) aggregation. The simulations used 6.5.3.4. Summary of Permeabilization by Membrane-
to visualize pore formation favor a model whereby membrane Active Peptides. Reviews of the literature increasingly look to
defects occur as disordered nonuniform pores.1454 examine common principles underlying the action of AMPs,
6.5.3.3. Cell-Penetrating Peptides and Amyloid Peptides. CPPs, and amyloid peptides.433,440,1452,1464 Further studies will
In contrast to the case of AMPs, cell-penetrating peptides be required to uncover their mechanisms of action in live cell
(CPPs) and amyloid peptides do not adhere to the principle of membranes and to what degree they can be harnessed for intra-
well-defined hydrophilic/cationic and hydrophobic segments. cellular delivery.1432 So far, there are examples of membrane-active
Though most CPPs tend to be cationic, they may also be peptides permeabilizing cells to enable cytosolic delivery of
uncharged and hydrophilic. Well-studied CPPs include pen- dyes,1433,1434 small proteins,1435 low-molecular weight dex-
etratin, HIV-1 TAT peptide, and poly arginines of 8 or 9 units. trans,1436 and short oligonucleotides.1437 It remains to be seen
For these peptides molecular dynamics simulations have observed whether membrane-active peptides can create pores large enough
only very transient pores.1460 Other simulations reveal defor- for siRNA, mRNA, RNPs, or larger protein influx without
mations and bending phenomena without actual pore forma- excessive cell toxicity. Provided that treatment with membrane-
tion, although this is controversial and it has been argued that active peptides can be made sufficiently reversible and tolerable,
some simulations of CPP behavior could be artifactual.410 their specificity for different types of membranes suggests they
When attached to bulky cargo molecules, CPPs are believed to could be an intriguing strategy for intracellular delivery.1465
enter cells via endocytosis rather than direct translocation 6.5.4. Pore-Forming Proteins and Toxins. Organisms
through the membrane, arguing that pore-formation in the from all kingdoms have evolved pore-forming proteins (PFPs)
plasma membrane might play very little role in the actual deliv- that can permeabilize the membranes of competing lifeforms.399
ery.116 Thus, the mechanisms could be different when CPPs PFPs are produced by prokaryotes, eukaryotic parasites, fungi,
are present as lone molecules versus when they are conjugated marine organisms, and plants either as a defense mechanism or to
to a cargo molecule. access nutrients, especially under conditions of high competition
To explain the observations gathered from various studies, or stress. Vertebrates also produce PFPs, such as the complement
Miranker and colleagues proposed a common mechanistic land- membrane attack complex (MAC) produced to kill bacteria
scape for membrane-active peptides.433 The initial formation of and the perforins expressed by immune killer cells to destroy
a pore is catalyzed by peptide-induced membrane tension that malignant or infected cells. The best-characterized and largest
lowers the activation energy of spontaneous poration to a class of PFPs, however, is that of the bacterial pore-forming
regime more accessible to thermal fluctuations (Figure 39).433,1463 toxins (PFTs).
PFTs are generally secreted as soluble monomers that can
assemble into oligomers, undergo conformational changes, and
insert into the membrane as an assembled pore complex
(Figure 40).399,439 Depending on the PFT, pore assembly may
either take place (1) before reaching the target cell surface or
(2) via lateral diffusion and binding of monomers once
embedded within the target cell plasma membrane. For many
PFTs, the stoichiometry of the assembled pore is around seven
subunits, such as is the case for S. Aureus α-hemolysin or the
aerolysin family. These PFTs form 1−3 nm pores to permit the
passage of ions, ATP, and nucleotides.399,439,1466
Alternatively, cholesterol-dependent cytolysins (CDCs) form
multimeric assemblies of >30 units and generate large pores in
the range of 20−50 nm (Figure 41A).439 Atomic force
microscopy images of the prototype CDC perfringolysin O
Figure 39. Schematic of the e ect of peptide binding on lipid bilayer (PFO) embedded into cholesterol-containing supported lipidff
integrity. (i) The reference state for energy change is an intact bilayers reveals the formation of ring-like pores with diameters
of ∼25 nm (Figure 41B).1467,1468phospholipid bilayer. (ii) Spontaneous fluctuations result in the Many PFTs rely on the
sampling of membrane defects. These are energetically unfavorable presence of specific surface receptors to bind and insert. CDCs,
and therefore sampled infrequently. (iii) Widening of the defect to for example, exploit the presence of cholesterol or other lipid
permit leakage results in a further energetic penalty. (iv) In the raft components, making them quite specific for the plasma
presence of surface-bound peptides (magenta), membrane tension is membrane of animal cells.407 This cholesterol-specific action
induced. (v) Peptide binding increases the frequency of defect makes CDCs reminiscent of saponins in their selectivity.
formation. (vi) Surface tension is released by pore formation1461 and Owing to this specificity and their large pore size, CDCs are
stabilized by peptide binding resulting in equilibrium poration (vii).
1462 the PFTs that have primarily been used for intracellularNote, many forms of defect, such as chaotic pores, can be
accommodated by this model, and defect characteristics may di er delivery of larger cargo (>1 nm) and will be the focus offf
between alternate peptides or the same peptide under alternate subsequent discussion in this section.
conditions. Figure and legend reproduced from ref 1463. Copyright 6.5.4.1. Cholesterol-Dependent Cytolysins for Intracellular
2013, with permission from John Wiley and Sons. Delivery. The most widely used PFTs for permeabilization-
mediated intracellular delivery are the cholesterol-dependent
In other words, membrane-active peptides distort the structure cytolysins (CDCs), of which Streptolysin O (SLO), Lister-
of lipid bilayers to a point where pore formation becomes the iolysin O (LLO), and PFO are the best-known examples. SLO
most energetically favorable option at a given temperature. is secreted by the bacteria Streptococcus pyogenes and has been
BS DOI: 10.1021/acs.chemrev.7b00678
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Chemical Reviews Review
Figure 40. Schematic representation of the pore formation pathway of pore-forming toxins (PFTs). Soluble PFTs are recruited to the host
membrane by protein receptors and/or specific interactions with lipids (for example, sphingomyelin for actinoporins or sterols for cholesterol-
dependent cytolysins (CDCs)). Upon membrane binding, the toxins concentrate and start the oligomerization process, which usually follows one
of two pathways. In the pathway followed by most β-PFTs, oligomerization occurs at the membrane surface, producing an intermediate structure
known as a prepore (mechanism 1), which eventually undergoes conformational rearrangements that lead to concerted membrane insertion. In the
pathway followed by most α-PFTs, PFT insertion into the membrane occurs concomitantly with a sequential oligomerization mechanism, which
can lead to the formation of either a partially formed, but active, pore (mechanism 2), or the formation of complete pores. Although classified as
β-PFTs, CDCs also share some of the features of this second pathway, as they can also form intermediate structures (known as “arcs”, named after
their shape) during pore formation. In both α-PFT and β-PFT pathways, the final result is the formation of a transmembrane pore with different
architecture, stoichiometry, size, and conduction features, which promote the influx or efflux of ions, small molecules, and proteins through the host
membrane and trigger various secondary responses involved in the repair of the host membrane. Note that, although the host membrane shown
here is the eukaryotic plasma membrane, some PFTs are antibacterial and form pores in the inner membranes of Gram-negative bacteria or the cell
membranes of Gram-positive bacteria. Figure and legend reproduced by permission from Springer Nature from ref 439. Copyright 2015.
population, possibly because electroporation is to some extent
cell size-dependent. In another comparative study, SLO
treatment, electroporation, and lipid-carriers were tested for
delivery of antisense ODNs that neutralize BCR-ABL mRNA
to reduce protein expression.1104 Contrasting the earlier report,
greater variation in ODN uptake was seen for SLO
permeabilized cells when compared with electroporated cells
in the chronic myeloid leukemia model cell line KYO-1. The
authors suggested that SLO exposure led to relatively under-
permeabilized and overpermeabilized populations. Compared
Figure 41. Structure of pores created by Perfringolysin O (PFO), a to SLO and electroporation, lipid delivery vehicles were found
CDC pore-forming toxin. (A) PFO oligomerizes into large prepores, to be ineffective for KYO-1 cells. A separate study in primary
which, after an extended conformational change, form a membrane- rat ventricular myocytes used SLO to successfully deliver
inserted β-barrel pore. Top view of the pore is on the right. Figure FITC-dextrans up to 148 kDa and bovine albumin serum
adapted by permission from Springer Nature from ref 439. Copyright (67 kDa), followed by cell recovery.1473
2015. (B) AFM images of the PFO pore complexes in supported lipid In 2001, Bhakdi and co-workers published a report that
bilayers that contain cholesterol. Scale bar 25 nm. Figure reprinted from significantly advanced our understanding of SLO-mediated intra-
ref 1467. Copyright 2004, with permission from John Wiley and Sons. cellular delivery.497 First, pretitrated concentrations of high-quality
SLO were administered to cells to determine precise concen-
used since the 1970s to selectively permeabilize the plasma trations for permeabilization in a variety of mammalian cell
membrane for the study of intracellular processes in semi- lines. Second, they deliberately employed calcium to trigger
intact cell models.1469,1470 In the 1990s SLO began to be used plasma membrane repair. With this approach, effective delivery
widely for intracellular delivery purposes.1471 Barry et al. demon- of proteins and dextrans was achieved in 60−80% of cells with
strated that antisense phosphodiester oligodeoxynucleotides >50% long-term viability. Third, they explored the size limits of
(ODN) could be introduced into cells during a brief perme- cargo influx to estimate pore size. SLO-mediated permeabiliza-
abilization step with SLO.1471 Cells were able to recover full tion was able to deliver 150 kDa dextrans but failed to permit
function and showed maximum ODN-induced down regu- the passage of 250 kDa dextrans (approximate diameter
lation of gene expression at 18 hours before recovery to normal ∼23 nm).866 These results suggest that SLO pores exhibit a
expression at 48 hours.1471 A subsequent study compared cutoff size in the range of 20 nm. This is in reasonably good
SLO-mediated permeabilization to electroporation for delivery agreement with AFM images of another CDC family member
of a restriction enzyme. It concluded that electroporation was PFO, which features pore diameters of ∼25 nm.1467 A fourth
more cytotoxic and that SLO was a better option for both observation was that calmodulin activity, microtubule integrity,
CHO cells and human fibroblasts.1472 In their hands, SLO and cytoplasmic ATP only returned to normal levels after
provided a more uniform permeabilization across the cell ∼4 hours. Under various conditions screened, the method
BT DOI: 10.1021/acs.chemrev.7b00678
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Chemical Reviews Review
permitted proteins to be delivered to approximately 50% of the subsequently trigger rupture to release endosomal contents
total cell population under near-full retention of viability, a and cytotoxic granzymes, which then induces the death of
performance level that has since been confirmed by others.1474 target cells.1528 In an analogous scenario, adenovirus employs
In subsequent studies it has been shown that delivery per- the viral membrane-lytic protein-VI to first generate small
formance can be better for siRNA-mediated gene knockdown, pores that trigger plasma membrane repair processes.1529 This
where the molecule to be introduced is significantly smaller results in the endocytosis of protein-VI into leaky compart-
(∼13 kDa). Transfection with an optimized SLO permeabi- ments from which it, and potentially other viral components,
lization method showed >80% RNAi-mediated knockdown can subsequently escape.1529
in difficult to transfect myeloma cell lines (JIM-3, H929, Recently, the natural AB-toxin mechanism has been repur-
RPMI8226, and U266 cell lines) with minimal effect on cell posed for intracellular delivery through protein engineering
viability (<10% death) and cell cycle.238 However, as noted efforts. Yang et al. showed that a neutralized version of
by Bhakdi and colleagues,497 several caveats exist for the use perfringolysin (PFO) can be targeted to the EGF receptor of
of SLO. Primary among them is that the quality of SLO cancer cells and preferentially activated in endosomes to
preparations is important, because contaminations with proteases deliver toxic gelonin into the cytoplasm.119 To do this, they
or DNases may create deleterious artifacts. Due to variations designed a bispecific antibody, where one terminal binds PFO
in batch quality, the appropriate SLO concentration window while the other targets the EGF receptor for endocytosis. Once
usually needs to be precalibrated by titration experiments prior in endosomes, the acidic environment triggers PFO to disrupt
to cell treatment. Moreover, an oxygen-stable C530A substitution the endosomal membrane. In another example of this strategy,
mutant obviates the need for a reducing agent to maintain SLO Pentelute and colleagues showed that the protective antigen
activity in the permeabilization bu er.497ff Thus, protein engineer- component of anthrax toxin generates a pore that can mediate
ing efforts have contributed toward improved versions of pore- egress of polypeptides, impermeable small molecule drugs, and
forming proteins for cell permeabilization. Despite these cave- antibody mimics from endosomes to the cytosol.1530 The
ats, SLO permeabilization represents a relatively cheap, simple, power of these bioinspired approaches lies in their specificity
and effective method to introduce molecular cargo up to ∼20 nm against different types of membranes and endosomal compart-
into living cells. SLO has been used to perform cytoplasmic ments.118 Such studies indicate the utility of pore-forming
delivery of siRNA,238,239,1475 antisense oligonucleoti- toxins and their components not just for plasma membrane
des,1104,1471,1476−1485 proteins,102,121,497,1472,1473,1486−1488 pep- permeabilization but also for controlled disruption of cargo-
tides,1474,1489,1490 cytoplasmic extracts,508,540,1491−1506 dex- laden endosomes.
trans,1473 PNA probes,1507−1509 molecular beacons,1089,1510−1516 6.5.4.3. Summary of Permeabilization by Pore-Forming
photosensitizers,1517 phosphatidic acid,1518 Rb+ ions,102 ATP,102 Proteins and Toxins. Pore-forming proteins are produced by a
various RNA probes,1519−1521 lanthanum probes,72,1522 and wide range of organisms, many of which still remain to be char-
gold nanoparticles.1523 acterized. While most of them produce small pores that are
Beyond SLO, permeabilization-based delivery with other limited in their usefulness for intracellular delivery, some fam-
CDC family members, such as LLO and PFO, has also been ilies, such as the cholesterol-dependent cytolysins, are com-
reported in the literature.1524−1526 Recently LLO, which monly used for permeabilizing cell membranes to introduce
is produced by the bacteria Listeria monocytogenes, was found molecular cargo. Limitations are similar to detergents, sur-
to be useful for delivery of small to midsized molecular cargo factants, and membrane-active peptides, namely the delayed
of <10 kDa.1526 By screening for passage of dextrans of size 3, kinetics of pore formation/membrane disruption and the lack
10, 40, 70, and 150 kDa, Murakami and coauthors found a size of spatiotemporal control. Significant improvements in cell
cutoff between 10 and 40 kDa. Thus, LLO-mediated perme- treatment and intracellular delivery may be attainable if these
abilization could be used to efficiently deliver the nucleotide problems are addressed. Moreover, we anticipate that pore-
analogue 8-OH-cAMP (∼0.4 kDa, a PKA activator) and the forming proteins will continue to find utility in molecular con-
small peptide Akt-in (∼2 kDa, an Akt inhibitor) inside cells jugates and multifunctional nanoparticles designed to over-
without loss of proteins, such as GFP (∼28 kDa), from the come the various types of membrane barriers.
cytosol.1526 6.5.5. Chemical Destabilization. Chemical destabiliza-
6.5.4.2. Pore-Forming Proteins as Endosome Disruptors. tion of lipid molecules can occur due to oxidative damage from
There are a number of naturally occurring scenarios where a variety of sources. In fact, membrane-perturbing lipid perox-
organisms use pore-forming proteins to deliver cargo into idation events are thought to be a normal part of cell phys-
target cells. So-called AB toxins can mediate this e ect.399ff The iology. In a recent study, for example, endogenous production
B component permeabilizes membranes, often triggered by the of reactive oxygen species (ROS) by the NOX2 enzyme medi-
acidic environment of endosomes, while the A subunit exerts ates disruption of endosomal membranes to trigger leakage of
separate enzymatic activity when unleashed into the cyto- antigens into the cytosol of dendritic cells for subsequent
plasm.399 In other words, A is the cargo and B is the membrane immune activation.1531 ROS and other free radicals cause
disruptor. Under this principle, the vertebrate immune system peroxidation of lipid tails, which can lead to similar effects as
has evolved perforins for the purpose of permeabilization to those seen for surfactants, including distortion, buckling, curvature
deliver toxic granzymes.1527 strain, and peeling o of micelles from lipid bilayers.461,1532,1533ff
One model for how AB toxins operate was presented in an Common species of peroxidized lipids have been proposed to
elegant study from Lieberman and colleagues. They observed exist in two main classes: (1) phosphatidylcholines with a
that sublytic perforin permeabilization of the plasma mem- hydroperoxide side chain and (2) phosphatidylcholines with
brane (featuring small 1−2 nm pores) triggers endocytosis in oxidized and truncated chains terminated by an aldehyde or
response to calcium influx. This results in endocytic uptake of carboxylic group (Figure 42).1532 Lipid tails become more
perforins plus cytotoxic granzymes.1528 Perforins then lodge in polar due to the presence of hydroperoxides, aldehyde groups,
the membrane of endosomes, inhibit their maturation, and or carboxyl groups. Consequently, these groups bend toward
BU DOI: 10.1021/acs.chemrev.7b00678
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Chemical Reviews Review
Figure 42. Chemical structures of oxidized phosphatidylcholines and their effects on bilayer packing. (A) Hydroxy- (HOSAPC and HOPLPC) and
hydroperoxy-(HPSAPC, HPPLPC, and 9-tc) phosphatidylcholines. Different cis/trans isomers are possible. 13-tc refers to trans-11, cis-9 isomer of
HPPLPC. (B) Truncated (cleaved chain) phosphatidylcholines with aldehyde (12-al, PONPC, POVPC, ox1-DOPC, and ox2-DOPC) and
carboxylic (PAzPC and PGPC) functional groups. Figure reprinted from ref 1532. Copyright 2012, with permission from Elsevier. (C) Example of
conformation changes that lipid molecules undergo due to peroxidation. In this case singlet oxygen adds the more hydrophilic (hydroperoxy-)
group −OOH at either the 9 or 10 position, which migrates to the bilayer surface. This imposes a kink in the acyl chain, with an accompanying
increase in area δA per lipid. Figure reprinted from ref 436. Copyright 2009, with permission from Elsevier.
the water phase to allow hydrogen bonding with water and the damage310,923,925,926,929,930 and have been used to load cells
lipid headgroups. The result of these distortions is an increase with cargoes such as dyes, dextrans, siRNA, and quantum dots.
in area per lipid headgroup, which leads to membrane The diffusive range of singlet oxygen species in aqueous
thinning, a decrease in lateral ordering, and membrane area environments has been estimated at about 100 nm.436 Thus,
expansion.436 local confinement of lipid oxidation may be a feasible strategy
Using GUVS as a model system, Riske et al. artificially for transient and precise membrane perforation without
converted the native lipid phosphatidylcholine to an oxidized damage to the bulk of the cell.
version with hydroperoxides groups at the 9 or 10 chain pos-
ition (Figure 42C). This was accomplished by using a membrane- 7. GATED CHANNELS AND VALVES
localized amphiphile photosensitizer that generates singlet So far, we have discussed membrane disruption approaches
oxixdation under irradiation with visible light. They found a sub- whereby cells recover through active plasma membrane repair
stantial increase in GUV membrane surface area without mem- (see section 4.3). In some cases, however, it may be possible
brane disruption or evidence of poration.436 It was hypothesized to deliver cargo into cells by actuating opening and closing
that more intense treatment would eventually lead to of “windows” in the cell membrane. Such a strategy can be
compromisation of membrane integrity, just like with detergents. executed by external manipulation of native transmembrane
Compared to the hydroperoxy-phospatidylcholines investi- proteins (e.g., channels and transporters), insertion of engineered
gated by Riske et al., oxidized lipids with truncated chains molecular valves, or deployment of synthetic nanodevices.
featuring aldehydes or carboxyl termini (see Figure 42B) are 7.1. Endogenous Channels (ATP-Activated)
much more potent perturbants of membrane organiza-
tion.1532,1533 In the latter scenarios, simulations and experi- Since the 1980s several reports have demonstrated the influx of
ments both observe pore formation and micellization as a func- small molecules through the manipulation of particular endo-
tion of concentration, concomitant with an increased susceptibility genous membrane transporters and channels. Impermeable
to bilayer rupture.437,438 dyes have been observed to enter a number of cell types in the
6.5.5.1. Con nement of Oxidative Damage through presence of high concentrations (up to 5 mM) of extracellularfi
ATP.1535Ionizing Plasmas. How is it possible to con ne lipid oxidation This is because ATP-gated channels permitting deliv-fi
to subcellular regions? Under certain regimes, lasers exert a ery are present in certain cell lines and primary immune cells.
1536
chemical oxidation e ect on membranes through generation of Steinberg et al. showed that only cargoes with a molecularff
near-field ionizing plasmas, as opposed to thermal or mech- weight less than 900 Da were able to enter cells in the presence1537
anical affects. For example, femtosecond lasers can produce of ATP. Specifically, it was found that ATP permeabilizes
near-field ionizing plasmas under speci c intensities, pulse dura- the plasma membrane of mouse macrophages to 6-carboxy-fi
tions, and frequencies1321,1331 (see optoporation section 6.4 and fluorescein (376 Da), lucifer yellow (457 Da), and fura-2
Figure 31D). Furthermore, near-field ionizing plasmas emanating (831 Da) but not to trypan blue (961 Da), evans blue (961 Da),
from laser-irradiated gold nanoparticles have been proposed as or larger dye conjugates. These studies led to the idea that
the primary mechanism of membrane permeabilization in a purinergic (i.e., ATP-mediated) activation of membrane chan-
recent study.1534 Theoretical simulations and experiments both nels can enable passage of cations and other small molecules.
indicate that generation of a low-density plasma is able to Toner and colleagues later used ATP-activated channels to1538
perforate the cell membrane via oxidative effects. This strategy load cells with trehalose, a 342 Da disaccharide with
was reported to transfect siRNA into cells with >90% e ciency widespread applications in cryopreservation.ffi
and viability.1534 Other delivery strategies that rely on fast 7.2. Endogenous Channels (Swelling-Activated)
pulse laser irradiation of metal nanoparticles or microscale Osmotic swelling is one form of stimulus that can trigger the
features may work through a similar mechanism of plasma-induced opening of mechanosensitive channels for influx of certain
BV DOI: 10.1021/acs.chemrev.7b00678
Chem. Rev. XXXX, XXX, XXX−XXX
Chemical Reviews Review
molecules. For example, osmotic swelling of Jurkat cells at 100
mOsm but not 200 mOsm was found to trigger opening of
channels for the delivery of monomeric sugars and sugar
alcohols but not larger molecules.1539 It was observed that
extensive hypotonic swelling rendered the cell membrane
permeable to PEG300−400, but not to PEG600−1500. By
reference to the hydrodynamic radii of these PEG molecules,
the size-selectivity of membrane permeation yielded an
estimate of ∼0.74 nm for the cutoff radius of the swelling-
activated channel.1540 Further work identified SLC5A3 as an
osmotically sensitive myo-inositol transporter that opens at
imposed extracellular osmolarities of less than 200 mOsm.1541
Figure 43. Synthetic nanodevices for use as membrane-embedded
valves or channels. (A) DNA origami nanostructures assembled to
Thus, this set of endogenous channels may be manipulated by form a membrane channel. Reprinted with permission from AAAS
osmotic stimuli to transport small molecules into cells. from ref 1548. (B) Carbon nanotubes embedded within lipid bilayers
7.3. Engineered Channels/Valves for molecular transport. Figure reprinted by permission from Springer
Nature from ref 1549. Copyright 2014.
One of the first efforts toward engineering a switchable channel
for intracellular delivery was reported by Toner, Bayley, and switch the channel between open and closed conformations.
colleagues. Using a strategy that exploits of site-directed As a demonstration of utility, this system was exploited to load
mutagenesis of S. Aureus α-toxin, they developed a self- the bicyclic peptide phalloidin (789 Da) into CHO cells to
assembling, proteinaceous, 2 nm pore equipped with a Zn2+-
1542 label actin filaments.actuated switch. Toxin monomers added to solution
integrate into the plasma membranes of target cells and assem- 7.6. Nanodevice Gating
ble to form an oligomeric pore complex. By adjusting the More radical concepts for engineering switchable permeability
concentration of extracellular Zn2+, reversible permeabilization into cell membranes have been demonstrated with synthetic
of the plasma membrane to small molecules (1 kDa or less) nanodevices. Langecker et al. created an artificial membrane
was achieved.1542 In a follow-up study, the switchable pore was channel based on DNA origami nanostructures that anchor to
used to load trehalose into fibroblasts at up to 0.5 M the lipid membrane by cholesterol side chains (Figure 43A).1548
concentration.77 Thus, protein engineering can be leveraged to The shape of the DNA-based channel was inspired by the
generate membranes with inbuilt permeability switches trig- bacterial channel protein α-hemolysin with some differences in
gered by chemical, enzymatic, or physical stimuli.1543,1544 physical properties such as charge, hydrophobicity, and size.
7.4. Optogenetic Control of Cell Permeability Although not implemented in cells, future applications in cell
membranes could include their deployment as antimicrobial
The emergence of optogenetics heralded the concept of agents or as intracellular delivery conduits.1548
engineered light-activated transporters for manipulating cell Carbon nanotubes (CNTs) represent another form of nano-
permeability.1545,1546 Kocer and colleagues modified the technology with engineering potential at the scale of the cell
mechanosensitive channel of large conductance (MscL) from membrane. CNTs feature narrow hydrophobic inner passage-
E. coli into a light-addressable nanovalve sensitive to 366 nm ways that mimic structural motifs typical of biological channels.1549
UV irradiation.1545 They verified the system by controlling the In a recent study, CNTs were inserted into lipid bilayers and
flux of calcein across proteoliposome membranes for both live cell membranes to form conducting channels capable of
one-way and two-way exchange. In a parallel approach, Boyden transporting water, protons, small ions, and DNA under phys-
et al. exploited the naturally occurring algal protein iological conditions (Figure 43B). It was found that the local
channelrhodopsin-2 as a rapidly gated light-sensitive cation channel and membrane charges control the conductance and
channel in mammalian cells.1546 Lentiviral transduction was ion selectivity of the CNT pores, thus suggesting potential
used to express these channels in neurons, whereby photo- starting points for engineering gating function.
stimulatation with blue light enabled cation influx and sub- Recently one group devised molecular motors that can bur-
sequent spatiotemporal actuation of neuron action potential row through lipid membranes upon excitation with light.1550
firing, which was a long-sought goal in the field. Although limited After the molecular motors bind to lipid membranes they are
to cations, this optogenetic proof of concept can conceivably available to be excited by ultraviolet light. They then convert
be extended to a wider range of synthetic and bioinspired this input into mechanical energy to drill through the cell
nanovalves. membrane. The study demonstrated intracellular delivery of
7.5. Stimuli-Sensitive Channels for Larger Cargo Delivery the motors themselves, small molecule dyes such as PI, and
accelerated cell death as a result of apoptosis or necrosis.1550
The mechanosensitive bacterial MscL channel can be func- Experimental results suggest permeabilization occurs through
tionally expressed in mammalian cells to afford controlled
1547 the transduction of light energy into nanomechanical actionuptake of membrane-impermeable molecules. The pore rather than chemical or thermal e 1550ffects.
diameter of >2.5 nm allows passage of large organic ions and
small proteins up to 6.5 kDa. Furthermore, gating of the 8. SUMMARY AND OUTLOOK
channel was found to be responsive to changes in membrane
tension, both in native bacteria and mammalian cell 8.1. Summary
membranes. To devise more convenient gating, charges were Motivations for better intracellular delivery range from basic
engineered within the pore of MscL to induce spontaneous research to the potential of therapeutic applications including
channel closure. Fortunately, exposure to charged methane- cell-based therapies, gene therapy, and regenerative medicine
thiosulfonate agents (such as MTSET) at 1 mM was found to (Figure 1 and Figure 3). Cargoes of interest vary from small
BW DOI: 10.1021/acs.chemrev.7b00678
Chem. Rev. XXXX, XXX, XXX−XXX
Chemical Reviews Review
BX DOI: 10.1021/acs.chemrev.7b00678
Chem. Rev. XXXX, XXX, XXX−XXX
Table 5. Summary of Membrane Disruption Approaches Covered in This Reviewa
Throughput/
Modality Methods Membrane Disruption Mechanisms Spatial Distribution across Cell Disruption Size Scalability Suspension/Adherent
Direct Penetration
Mechanical Microinjection Mechanical forces at contact zone. Membranes only tolerate At contact zone Depends on size of injection tip, usually Low, could be Mostly adherent.
2−3% lateral strain.425 Can be strain rate dependent (see 0.3−1 μm improved via Suspension cells
Figure 9) and refs 421 and 1551 automation require secondary
holding pipette
Penetrating Projectiles Depends on size of projectile - usually Potentially high Primarily adherent.
(Biolistics) micron-size Some reports on
suspension cells
Nanowires, Nanoneedles and Depends on size of tip: reported range Potentially high Mostly adherent.
Nanostraws 50−1000 nm Suspension cells
must be forced onto
the array
Permeabilization
Mechanical (Solid Cell Scraping Mechanical forces transmitted by direct contact or cell Presumably at contact zone Probably depends on force, strain rate, High Adherent
Contact) Bead Loading deformation. Membranes only tolerate 2−3% lateral strain.
425 otherwise at weak points/defects size of contact zone, direction of strain High Adherent
Can be strain rate dependent (see Figure 9) and refs 421 and due to global membrane strain
Scratch Loading 1551 Low/Medium Adherent
Microfluidic Cell Squeezing/ High Suspension
Constriction-Mediated Cell
Deformation
Nanowire Permeabilization Potentially high Adherent
Sudden Cell Shape Changes/ Possibly tearing forces at adhesion sites Possibly at adhesion sites Unknown Potentially high Adherent
Protease Treatments
Mechanical (Fluid Syringe Loading/ Fluid shear forces causing membrane strain Unknown Unknown Potentially high Suspension
Shear) Microfluidic/Bulk Fluid
Shear
Sonoporation/Shockwaves Stable Cavitation (Microstreaming), Inertial Cavitation Presumably a single hole per From nanometers to several microns High Both
Laser-Induced Cavitation (Jetting), or other Acoustic Effects cavitation bubble depending on cavitation intensity and Medium to High Both
stand-off distance
Mechanical Hypo-osmotic Shock Mechanical forces transmitted by osmotic/hydrostatic pressure. Presumably at weak points or Probably depending on membrane High Both
(Pressure) Membranes only tolerate 2−3% lateral strain.425Hydrostatic Pressure Can be strain nucleating at membrane defects reservoirs, attachment/reinforcement High Both
rate dependent (see Figure 9) and refs 421 and 1551 of membrane, and magnitude/rate of
pressure
Osmotic Rupture of Limited by endosome High Both
Endosomes
Electroporation Conventional Electroporation Probability of defect formation for given pulse-strength duration At cell poles. More Nucleate as small defects then grow as a High Primarily Suspension,
at a given temperature. See section 6.2.1 for details. permeabilization expected on function of voltage and duration but Adherent also
hyperpolarized side possible
Micro-electroporation Depends on geometry Potentially high Primarily Suspension
Nano-electroporation Usually single hole at nanoaperture Currently Low/ Both, depending on
Medium system
Thermal Freeze−Thaw Expansive mechanical strain due to ice crystal formation Location of ice crystals Presumably variable High Both
Rapid Temperature Defect formation due to phase transitions Probably near lipid domain Presumably small defects High Both
Transitions/Cycles boundaries and protein clusters
Supraphysiological Heating Dissociation of bilayer structure leading to defect formation Site of maximal heat Presumably small defects High Both
Laser Absorption at Membrane Absorption causes high local temperature to trigger membrane Laser focal point or location of Presumably variable depending on local High Both
or Absorbent Particle/ disruption absorbent structure temperature effects
Structure
Optoporation Laser Optoporation Can be a mix of (1) Chemical (low energy ionizing plasma); Maximal in focal region. Usually Depending on parameters and Low to high - Primarily Adherent,
(2) Mechanical (cavitation, shock waves, thermoelastic one hole mechanisms. Nanometers to several limited by laser but suspension also
stress); (3) Thermal (Heat in focal region) microns possible
Chemical Reviews Review
BY DOI: 10.1021/acs.chemrev.7b00678
Chem. Rev. XXXX, XXX, XXX−XXX
Table 5. continued
Throughput/
Modality Methods Membrane Disruption Mechanisms Spatial Distribution across Cell Disruption Size Scalability Suspension/Adherent
Permeabilization
focusing
approach
Biochemical Organic Solvents and Perturb bilayer structure by burying their hydrophobic residues Indiscriminate in bulk, otherwise Presumably small defects then High Both
Penetration Enhancers into the membrane depends on local concentration disintegration of the whole bilayer at
high concentration
Detergents/Surfactants: Insert into bilayer and distort the structure, leading to defects, Indiscriminate in bulk, otherwise Presumably small defects then High Both
Generic pore formation, and micellization depends on local concentration disintegration of the whole bilayer at
high concentration
Detergents: Saponin Family Extracts cholesterol out of the bilayer core to form a surface Cholesterol rich sites. From nanometers to micron High Both
complex, induces curvature and defect/pore formation Indiscriminate in bulk, otherwise
depends on local concentration
Pore-Forming Toxins: CDC Insertion and oligomerization into pore structure in Cholesterol rich sites. <30 nm High Both
Family cholesterol-rich membranes Indiscriminate in bulk, otherwise
depends on local concentration
Membrane-Active Peptides Adopt active conformation upon membrane binding. Depend on membrane Presumably small defects High Both
Concentration dependent aggregation/insertion composition. Indiscriminate in
bulk, otherwise depends on local
concentration
Lipid Peroxidation Lipid peroxidation leads to structural interference/distortion of Depends on source of oxidation. Presumably small defects, but large holes High Both
membranes to form pores and defects If local, can be confined are conceivable
Gated Channel Endogenous or Engineered Appropriate stimuli (e.g., mechanical, chemical, optical) to Depends on location of the Limited by size of the channel. Usually High Both
and Valves Membrane Transporters/ “gate” opening and closing activity membrane transporters/channels only amenable for transport of small
Channels molecules <1 kDa
Synthetic Nanodevices Insertion of constructs into host membrane. Gating may be Depends on location of the Limited by size of the engineered central Potentially High Both
engineered nanodevices within host channel
membrane
aSeveral are widely used for intracellular delivery while others have barely been attempted. Techniques marked in bold text represent methods that are either commercially available or accessible with
common lab equipment.
Chemical Reviews Review
BZ DOI: 10.1021/acs.chemrev.7b00678
Chem. Rev. XXXX, XXX, XXX−XXX
Table 6. Cargo Loaded versus Membrane Disruption Approacha
Cargo
Nucleic Acids and Their Analogues
Small Molecule Peptides/Proteins/ Generic Synthetic Large Cargo:
Drugs/Probes/ Antibodies/ Macro-molecules Nanomaterials/ Bacteria, Organelles,
Modality Method Dyes/Sugars/Ions etc. RNPs etc. (e.g., Dextrans) Oligos mRNA Vector DNA qDots/CNTs etc. Beads etc.
Direct Penetration
Mechanical Microinjection Dyes,595 Mercury,324 Proteins,82−86,604,1553 Dextrans1556 Antisense mRNA240,1557 pDNA,200−202,607 qDots,294,1559,1560 Bacteria,324 Nuclear
Trehalose1552 Antibodies,1554 Oligonucleotides,211,608 viral DNA200, MW-CNTs,315 transplant,326−328
Peptides,603,1555 Cas9 siRNA215 606,1558 SW-CNTs304 Chromosome
protein/RNP149 transplant,333
Sperm/IVF,331,332
Mito-chondria,336
Artificial
vesicles1561
Beads,1562,1563
Superparamagnetic
beads,357 Silicon
barcodes358
Penetrating Dyes,660,1564 Proteins,667−669 Cas9 siRNA665,666 Cas9 mRNA,1565 pDNA394,642,674,675 PEBBLE Beads,351,353 Latex
Projectiles Indicators659 protein/RNP670 mRNA662−664 nanosensors1566 particles352
(Biolistics)
Nanowires and Drugs,110 Proteins,110,680 siRNA110,680,681 pDNA109,110,676, qDots,684 DNA
Nanoneedles Molecular Beacons683 Peptides,110 Cre 684,713,1567 Nanocages685
Recombinase,682
Antibodies427
Nanostraws Dyes,700,702 Co2+ Proteins707 Dextrans701,707 pDNA700,702,707 qDots706
ions,418 Ca2+
ions,703 Small
molecule probes704
Permeabilization
Mechanical Cell Scraping Dyes760−762 Proteins,96,543,745−751 Dextrans,96,758 Antisense pDNA545
(Solid Antibodies,752−754 Lipopolysaccharide759 Morpholinos757,1568−1570
Contact) Peptides755,756
Bead Loading Nucleotides,731,956 Proteins,735−737 Fab Dextrans97,735,1572 pDNA729 qDots,744 SW-
RNA probes,1571 Fragments,739,740 CNTs743
PNA probes,741 Antibodies,732−734
SNAP-reactive dyes742 Peptides738
Scratch Loading Dyes764 Dextrans763 qDots744
Microfluidic Cell Dyes,108,777,793 Proteins,108,780,781,785,787 Dextrans108,152, siRNA,108,152,780,788,792,794 mRNA108,787 pDNA152,777,787,792−794 qDots,308 CNTs,108
Squeezing/ Tags,785 Drugs784 Cas9 RNPs152,793,794 777,780,788,794 tRNA783 DNA
Constriction- nanostructures794
Mediated Cell
Deform-ation
Nanowire Dyes714,719 Antibodies694 Dextrans800 Lipid-pDNA qDots694 ∼200 nm polystyrene
Permeab-ilization complexes,694 beads694
pDNA800
Sudden Cell Shape Dyes769 Proteins,768,771,773,774 Dextrans766,768,1573 Oligonucleotides768
Changes/ Peptides768
Protease
Treatment
Chemical Reviews Review
CA DOI: 10.1021/acs.chemrev.7b00678
Chem. Rev. XXXX, XXX, XXX−XXX
Table 6. continued
Cargo
Nucleic Acids and Their Analogues
Small Molecule Peptides/Proteins/ Generic Synthetic Large Cargo:
Drugs/Probes/ Antibodies/ Macro-molecules Nanomaterials/ Bacteria, Organelles,
Modality Method Dyes/Sugars/Ions etc. RNPs etc. (e.g., Dextrans) Oligos mRNA Vector DNA qDots/CNTs etc. Beads etc.
Permeabilization
Mechanical Syringe Loading/ Dyes,824 Nucleotides805,806 Proteins,808,812, Dextrans98,807,821,823,825 Antinsense morpholinos804 pDNA803,825
(Fluid Microfluidic/Bulk 814−818,821
Shear) Fluid Shear Antibodies809−811,
813,819
Sono-poration Dyes,802,845,850,851,857,865, Proteins,828,865 Dextrans545,827,828,849 siRNA,861,862 Antisense mRNA863 pDNA545,830,831,833, 25−75 nm Viral particles887
871,873−881,906 Antibodies,888 −851,864,865,867−872,890 oligonucleotides,860 838,853−859,890,1574 nanoparticles867
Drugs871,882−886,1574 Peptides889 ssDNA1574
Shock Wave- Dyes,899,903,905,906,909 Proteins,901 Dextrans897,899,900 Antisense pDNA1317,1577−1579
Mediated Drugs1575 Peptides904 oligonucleotides1576
Permeab-ilization
Laser-Induced Dyes487,879,914,918,919,922, Proteins,340,919 Dextrans913,918,922, siRNA921,923,924 mRNA339 pDNA339,913,917, qDots,309,310,1584 Bacteria,339,340
Cavitation 926,928,929,1580,1581 Antibodies1325 924,927,929,1581−1584 919,1585 Gold Mitochondria,341
nanoparticles1586 ∼200 nm
polystyrene
beads339
Mechanical Hypo-Osmotic Dyes,951,1587 Proteins,92,93,950,964, Dextrans962,974,1588 pDNA975 qDots,311 ∼5 nm
(Pressure) Shock Lanthanide ions/ 966,974 gold
complexes,967−972 Peptides951,961 nanoparticles963
Nucleotides,951−960
Nucleosides,951 BAPTA965
Hydrostatic Dyes980,984 Proteins,980,1589 ssODN,988,989 gRNA,991 mRNA984 pDNA,978,979,981, ∼100 nm Polystyrene
Pressure Antibodies1589 siRNA,981,982,997,1590 984,989,990,998, microspheres1005
Antisense 1003,1004,1590,
morpholinos,1591 1592,1593 Bacterial
Antisense artificial chromo-
oligonucleotides996, somes
998−1002 (BACs)983
Osmotic Rupture of Trehalose,1035 Proteins,94,95,1007−1021 Dextran,94,1026−1028 siRNA,1042,1043 Antisense qDots,1594 Protein- Virus particles1039
Endosomes Lanthanide imaging Antibodies,1013, Hyaluronan1033,1034 oligonucleotides,1038 conjugated
probes,71,72 Dyes,1026,1036 1022−1026 Antisense morpholinos804 qDots306,1040,1041
UDP-glucuronic acid1037 Peptides,1029−1031
Cell lysate1032
Electro- Conventional Dyes,100,1056,1078−1080 Proteins,100,135,546, Dextrans546,1078,1126,1259 Antisense mRNA30,146,185,187, pDNA184,1112,1113 qDots,294,312,313,1114
poration Electro-poration Radio-tracers,1082 1091−1097 oligonucleotides,1104 257,259−261,560, 20 nm gold
SNAP-reactive dyes,742 Antibodies,101,125, siRNA235,1105−1109 1110,1111,1263, nanoparticles1115
Sugars,79,470,534,1083 537,1098−1102 1266,1271,1275,
Metabolites,1081,1084 Cas9 protein/ 1277,1278,1290,
Drugs,55,56,1085,1086 RNP143,144,146,147, 1293,1294
Ions,1087,1088 287,1103
Molecular Beacons1089,1090
Microelectroporation Dyes489,1224,1228−1232 Proteins1232 Dextrans1231 siRNA,1232,1595 miRNA1232 pDNA1224,1226,1227,
1229,1230,1232,1595
Nanoelectroporation Dyes,702,1233 Proteins1241 Dextrans1241,1242 siRNA,1233 pDNA702,1233,1234 qDots1233
Molecular Beacons1233,1242 Oligonucleotides1233
Chemical Reviews Review
CB DOI: 10.1021/acs.chemrev.7b00678
Chem. Rev. XXXX, XXX, XXX−XXX
Table 6. continued
Cargo
Nucleic Acids and Their Analogues
Small Molecule Peptides/Proteins/ Generic Synthetic Large Cargo:
Drugs/Probes/ Antibodies/ Macro-molecules Nanomaterials/ Bacteria, Organelles,
Modality Method Dyes/Sugars/Ions etc. RNPs etc. (e.g., Dextrans) Oligos mRNA Vector DNA qDots/CNTs etc. Beads etc.
Permeabilization
Thermal Rapid temperature Trehalose73,75,1035
transitions/cycles
Supra-physiological Dyes1176 Proteins1596 pDNA1318,1319
Heating
Laser Absorption Dyes1324,1325,1327−1329,1597 Antibodies1326 Dextrans1325,1329 siRNA1598 pDNA1328,1329
Converted to Heat
Opto- Laser Opto-poration Dyes,252, Peptides,486,1359 Dextrans1333,1343,1349, siRNA,1333,1343,1352 mRNA252,1353 pDNA481,483,484, Semiconductor ∼1 μm Polystyrene
poration 480−484,486,1333,1340,1341,1343, Proteins1333,1343 1353,1360,1361 Antisense 1330,1332−1358 nanocryst- beads1371
1345,1346,1348,1354,1357, morpholinos1353 als,1333,1343 Gold
1361−1366 nanoparticles,1369
Sucrose,485 Molecular qDots1370
beacons,1367
Ions1333,1343,1368
Biochemical Organic Solvents Proteins1385 mRNA1385 pDNA1385
and Penetration
Enhancers
Detergents/ Nucleosides,1422 Dyes,1422 Proteins,1423
Surfactants: Nutrients and Antibodies99,1423
Generic Metabolites,1422
Ferro-cyanide1396
Detergents: Inositol1413 Proteins,539 Peptides,539 Dextrans1415 ∼20 nm
Saponin Family Antibodies,1417 nanoparticles,1416
Cyto-plasmic qDots311
extracts1415
Pore-Forming PNA probes,1507−1509 Proteins,102,121,497, Dextrans1473,1526 siRNA,238,239,1475 Antisense Gold
Toxins: CDC Molecular Beacons,1089,1510 1472,1473,1486−1488 oligonucleoti- nanoparticles1523
Family −1516 Photosensitizers,1517 Peptides,1474,1489, des1104,1471,1476−1485
Phosphatidic acid,1518 1490,1526 Cyto-plasmic
Rb+ ions,102 ATP,102 extracts508,540,1491−1506
Various RNA probes,1519
−1521
Lanthanum probes,72,1522
Nucleotide analogues1526
Membrane-Active Dyes1433,1434 Proteins1435 Dextrans1436 Oligonucleotides1437
Peptides
Lipid Per-oxidation siRNA1534
Gated Membrane Trans- Dyes,1535,1537,1545,1550,1599,1600 Proteins,1547
Channels porters and Ions,1546 Trehalose77 Peptides1547
and Channels
Valves
aTechniques marked in bold text represent methods that are either commercially available or accessible with common lab equipment. Cargoes are ordered across the table in approximate size order.
Chemical Reviews Review
molecules that can naturally permeate the lipid bilayer to Throughput and applicability to suspension or adherent cells
highly charged molecules and large complexes, genetic con- are further considerations. In microinjection, for example,
structs, synthetic materials, or organelles approaching the size almost any cargo can be delivered to any cell type but only one
of the cell itself (Figure 2). For the majority of these cargoes, cell at a time (Figure 13). The challenges involved in scale-up
the plasma membrane is the primary barrier to intracellular to high throughput are yet to be surmounted. Other methods,
delivery. Cells exhibit a distinct set of properties that can be such as scrape loading (Figure 16A), are low cost and high
exploited to overcome this barrier. For example, delivery throughput but may lack consistency and precision across
methods can take advantage of the negative membrane cell populations. In a further example, large cargo delivery can
potential, cholesterol-rich nature of the plasma membrane, or be accomplished with laser-controlled cavitation bubbles
presence of specific extracellular receptors (Figure 7). (Figure 18F), but these systems require complex equipment
A broad assortment of approaches have been designed to and may only be applicable to adherent cells. Such a scenario
deliver cargo into cells. They can be categorized as either would rule out delivery to most immune and blood cells that
carrier-mediated or membrane disruption-mediated strategies naturally exist in suspension. Electroporation is currently the
(Figure 4). Cells generally respond to the presence of carriers dominant high throughput method in the field. As covered in
by processing them through endocytosis and other membrane section 6.2.3, it has been demonstrated in applications ranging
trafficking pathways. On the other hand, they react to from testing of impermeable drugs and biomanufacture to
membrane disruption by deploying membrane repair processes engineering cells for cancer immunotherapy and stem cell-
to heal the plasma membrane and restore cell homeostasis based gene therapy (Figure 29). However, electroporation is
(Figure 11). Due to their perturbing nature, most delivery not without its drawbacks. Post-treatment cell death and
strategies involve a trade-off between effective delivery and inability to deliver large uncharged cargo are two such examples.
tolerable cell damage. Membrane disruption-mediated delivery Overall, no single method has a monopoly on all applications,
strategies have the advantage of rapid and near-universal and further work is required to identify the optimal delivery
delivery of almost any cargo that can be dispersed in solution strategies for a given application.
(Figure 5). The latest understanding of membrane repair Table 6 compares membrane disruption approaches versus
pathways indicates that membrane disruption is a common the cargoes they have been reported to deliver. It is important
event in the life of cells, and they are well equipped to deal to note that many combinations have simply not been attempted.
with it. More challenging is the selection of appropriate Moreover, many reports use a certain technique to deliver a
membrane disruption modalities and their precise implemen- particular cargo only because they adapt the protocol from an
tation to large batches of cells at high throughput. This is an earlier publication. Thus, certain techniques seem to have an
engineering challenge that involves elements of both arbitrary emphasis on a particular cargo. For example, opto-
technological innovation and mechanistic understanding of poration publications have tended to focus heavily on plasmid
the cell itself. Theories have been developed to explain defect transfection while neglecting other cargoes. Filling out the table
formation in lipid bilayers and the phenomena that can be by screening all possible combinations would be extremely
leveraged to achieve controlled disruption of cell membranes informative for the field. Comparisons of cost and cell type
(Figure 8, 9, 10). In parallel, empirical studies have identi ed applicability would also add value to such an analysis and assistfi
key modalities, such as electroporation and mechanical defor- in guiding experimentalists toward optimal solutions. In the
mation, which can be deployed to obtain a relatively scalable future, we expect to see more publications move beyond trivial
and reproducible control of plasma membrane disruption. delivery of small molecules dyes (<1 kDa) to showcase delivery
Tables 5 and 6 summarize the membrane disruption of a smorgasbord of diverse cargo, especially proteins,
approaches that have been discussed in this review. Table 5 nanomaterials, and larger cargo.
lists each method alongside what is known about disruption 8.2. Outlook
mechanisms, size and distribution of resultant holes, treatment Several membrane disruption-based methods are in widespread
throughput, and whether it is applicable to adherent or use in academic, industrial, and medical laboratories across
suspension cells. If there is one theme that emerges from this the world, such as electroporation and microinjection. Yet the
analysis, it is that we still lack a clear mechanistic under- majority of modalities are either in nascent development or are
standing of how many membrane disruption-mediated intra- yet to be pursued to their full potential. By identifying where
cellular delivery methods actually work. Indeed, many of the the field can reduce costs and complexity, the potential exists
methods suffer from a lack of mechanistic insights needed to to lower the barrier of entry to interdisciplinary scientists and
properly hone and optimize the salient parameters. Sonopora- researchers in resource-poor settings. This would no doubt
tion is an example of a delivery strategy that has been strengthen global discovery. Overall, we believe that better and
challenging to optimize because of such complexity. In other more streamlined intracellular delivery is more likely to arise
cases, a membrane disruption method may work well but a lack out of a deeper understanding of current approaches and their
of knowledge of appropriate environmental conditions leads to capabilities.
underperformance. For example, how living cell membranes The field has a number of frontiers where opportunities are
behave and recover at different temperatures and buffer ripe. One is the huge repository of unexplored membrane-
osmolarities still remains poorly understood (Tables 2 and 3). perturbing compounds in the form of natural and synthetic
Other methods feature clearly defined mechanisms but face detergents, surfactants, pore-forming toxins, membrane-active
intrinsic limitations because of the nature of the membrane peptides, and other secondary metabolites. Another is the rise
disruption effect. For example, conventional electroporation of new microfluidic and nanotechnological tools that provide
and pore-forming toxins tend to generate membrane disrup- an unprecedented level of control to the membrane disruption
tions of less than 50 nm and are therefore limited in their process. This may be via high throughput systems for
ability to deliver large cargo. mechanical deformation, such as microfluidic cell squeezing,
CC DOI: 10.1021/acs.chemrev.7b00678
Chem. Rev. XXXX, XXX, XXX−XXX
Chemical Reviews Review
or advanced fabrication of nanostructures, including nanowires Biographies
and nanostraws. Combining the strengths of multiple modal-
Robert Langer is one of 13 Institute Professors at MIT (an Institute
ities may be a prudent approach toward better technologies.
Professor is the highest honor awarded to a faculty member). His
For example, electroporation is biased toward producing small h-index of 242 is the highest of any engineer in history. He has over
pores but provides a convenient electrophoretic force for the 1300 issued and pending patents which have been licensed or
delivery of charged molecules. Methods that combine large sublicensed to over 350 companies. He served as Chairman of the
disruption sizes with electrophoretic driving forces could FDA’s Science Board (highest advisory board) from 1999 to 2002.
potentially harness the benefits of both techniques. Future Langer is one of very few individuals elected to the National Academy
strategies could also be based on synthetic valves and nano- of Medicine, the National Academy of Engineering, the National
devices that embed within the membrane and enable remote Academy of Sciences, and the National Academy of Inventors. He is
control of permeability via external triggers. Light-gated methods one of four living individuals to receive both the U.S. National Medal
that confer switchable control of membrane disruption are only of Science and the U.S. National Medal of Technology and
beginning to be explored. In the coming years cost and Innovation. In 2015, Dr. Langer received the Queen Elizabeth Prize
convenience will be another important factor, as many of the for Engineering. He has also received the Draper Prize (considered
current methods are either too expensive or overly reliant on the engineering Nobel Prize), Albany Medical Center Prize, Wolf
cumbersome equipment. Prize for Chemistry, Millennium Technology Prize, Priestley Medal
As our insights into membrane repair processes and cell (highest award of the American Chemical Society), Gairdner Prize,
recovery deepen, it may become possible to provide stimuli Kyoto Prize, Breakthrough Prize, and the Lemelson-MIT prize, for
that switch membrane repair on and off or to modulate stress being “one of history’s most prolific inventors in medicine.” He holds 31
responses that would otherwise lead to untoward cell fate honorary doctorates including honorary degrees from Harvard and Yale.
changes or death. How can we understand the energy landscape Klavs F. Jensen studied chemical engineering at the Technical
of defect formation to generate ideal membrane disruptions? University of Denmark and completed his Ph.D. in chemical
What kinds of disruptions are optimal for delivery of specific engineering at the University of WisconsinMadison. He started
cell-cargo combinations? How does the composition of the his independent career at the University of Minnesota in 1980 and
external buffer determine which pathways are activated in moved to MIT in 1989 as professor in chemical engineering and
response to permeabilization? The answers to these, and materials science and engineering. In 2007 he became Warren K.
similar, questions will be more attainable with the establish- Lewis Professor and head of MIT’s department of chemical
ment of better approaches to investigate plasma membrane engineering until 2015. His research interests revolve around
homeostasis and the cellular response (Figure 11). Thus, along miniaturized systems for chemistry and biological discovery and
with technical advances in membrane disruption, our toolbox manipulation. Professor Jensen is a member of the U.S. National
for studying cells must also improve. Academies of Engineering and Sciences, as well as the American
For ex vivo cell-based therapies in particular, quality control Academy of Arts and Science.
procedures may be essential to ensure the safety and efficacy of Martin P. Stewart was born in Sydney, Australia in 1983. He received
engineered cells. Methods for assessing DNA damage, fate a B.Sc. (Hons) from the University of Technology Sydney in 2007.
changes, and cell functionality will possibly be required to Martin then obtained his Ph.D. from TU Dresden, Germany in 2012
avoid reintroduction of malignant or undesirable cells in cGMP working under the supervision of Professors Daniel Müller and Tony
settings. More accurate assays to evaluate cell function are Hyman. His Ph.D. research focused on the mechanisms of cell shape
expected to inform the appropriate and safe use of membrane in mitosis. After a postdoctoral stint at ETH Zürich, Switzerland with
disruption-based delivery methods going forward. Combined Professor Daniel Müller, he joined the laboratories of Professors Klavs
with further technological innovations in the way we disrupt Jensen and Robert Langer at MIT in 2014. Martin’s current research
membranes, we expect future progress in the field to catalyze interests are in cell manipulation and analysis, specifically in the areas
breakthroughs in delivery applications ranging from funda- of intracellular delivery and cell biophysics. He has been a recipient of
mental research to ex vivo cell-based therapies. postdoctoral fellowships from the Swiss National Science Foundation
and the Life Sciences Research Foundation. He has also been awarded
AUTHOR INFORMATION grants from the American Australian Association and the Broad
Institute of MIT and Harvard. In 2018 Martin commenced as a
Corresponding Authors research group leader and lecturer in medical technologies at the
*E-mail: martin.stewart@uts.edu.au.. University of Technology Sydney.
*E-mail: rlanger@mit.edu.
*E-mail: kfjensen@mit.edu. ACKNOWLEDGMENTS
ORCID This work was supported by the U.S. National Institute of
Martin P. Stewart: 0000-0003-4112-6622 Health (R01GM101420-01A1). M.P.S. was supported by the
Robert Langer: 0000-0003-4255-0492 Swiss NSF through advanced postdoc mobility fellowship
Klavs F. Jensen: 0000-0001-7192-580X P300P3_151179. M.P.S. acknowledges support from a Keith
Murdoch Fellowship via the American Australian Association,
Notes
a Life Sciences Research Foundation Fellowship sponsored by
The authors declare the following competing financial Good Ventures, and a Broadnext10 Catalytic Steps funding
interest(s): RL and KFJ are on the board of SQZ Biotech- gift from the Broad Institute of MIT and Harvard. We are
nologies, a company that uses cell squeezing for membrane grateful for discussion and feedback from Xiaoyun Y. Ding,
disruption. James C. Weaver, Eric Van Leen, and Ronan W. O’Connell.
CD DOI: 10.1021/acs.chemrev.7b00678
Chem. Rev. XXXX, XXX, XXX−XXX
Chemical Reviews Review
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