Systematic engineering of controlled, localized oligonucleotide delivery systems for wound angiogenesis

Abstract

The standard of care for diabetic wounds has remained relatively unchanged for decades, resulting in patients with wounds that do not heal on meaningful time scales, referred to as ulcers, and high rates of recurrence for patients whose wounds do heal. This common complication of diabetes decreases quality of life, increases mortality, and raises health care costs. New paradigms to treat these wounds remains a formidable but critical challenge.

Addressing diabetic ulcers at the molecular level may decrease healing time and prevent recurrence. Impaired blood vessel formation, or angiogenesis, in diabetic ulcers is an important target pathway. Angiogenesis is needed to bring oxygen, nutrients, signaling cues, and cells to newly formed tissue while removing waste. Nucleic acid oligonucleotide therapies, such as small interfering RNAs (siRNAs) or microRNA inhibitors (anti-miRs), that regulate gene expression at the post-transcriptional level, hold particular promise for promoting angiogenesis and wound healing; however, the large size and negative charge of these therapies require drug carriers to mediate their biological effect.

In this thesis, we leverage sequential electrostatic adsorption of oligonucleotide therapy and polyelectrolytes into thin film coatings on commercial wound dressings through the layer-by-layer (LbL) process. These dressings package oligonucleotide, enhance its transfection efficacy, and control its temporal release locally to the wound bed. After initial validation experiments, we sought to systematically understand our drug carrier system and use this insight to engineer better wound dressings. First, we developed a proof-of-concept anti-miR-coated dressing and showed its efficacy in promoting both wound closure and sex-dependent angiogenesis. We found that therapy released from coated dressings had a preferential association with different wound cell types, particularly endothelial cells. We then sought to uncover how changes in the oligonucleotide structure itself may alter interactions with transfection polymers in thin film coatings. We found that binding with certain polyelectrolytes differed based on whether the therapy was a flexible single stranded anti-miR or a more rigid double stranded helix siRNA. We also showed how chemically modified nucleotides, such as locked nucleic acid and 2’-O-methyl RNA, can modulate affinity to polyelectrolytes and ultimately impact transfection efficacy. We also elucidated how physicochemical properties of the hydrolysable transfection-enhancing poly(β-aminoester) polymer mediate its efficiency in transfecting oligonucleotide therapy. We demonstrated that a more hydrophobic polymer enhanced transfection efficacy through its ability to facilitate permeation of biological barriers. Finally, we identified how modulation of the anionic excipients contained in these thin film coatings can be leveraged to vary the release kinetics from coated wound dressings. We engineered formulations that released on a fast or slow time scale. We observed that while both release time scales promoted efficacy in wound closure, they did so through potentially different mechanisms despite the same putative pro-angiogenic anti-miR therapy.

In sum, this thesis elucidates how physicochemical properties and formulation of coated wound dressings alter their interfacial effects with biological systems. We use this knowledge to rationally design better drug carriers that can deliver pro-angiogenic oligonucleotide therapeutics to the wound bed. The findings have broad applications in the delivery of nucleic acid therapies for a wide host of diseases where local delivery to the injured tissue could prove beneficial. Ultimately, we also advance our pro-angiogenic coated wound dressing strategy towards clinical translation. Our strategy has the potential to provide a new, targeted therapeutic paradigm to help those suffering from diabetic ulcers.