Skeletal muscle biomechanics drives intramuscular transport of locally delivered drugs

Abstract

Introduction: Effective local drug delivery to contractile tissues such as skeletal muscle requires a thorough understanding of the impact of mechanical loads on intramuscular pharmacokinetics. Current preparations for studying skeletal muscle biomechanics typically use: mounting techniques that lead to mechanical disruption of the tissue, which can create drug transport artifacts. In order to accurately study mechanical influences on drug transport, experimental techniques and setups need to meet the particular design requirements of both biomechanical testing setups and local drug delivery preparations. Studies of intramuscular pharmacokinetics require anatomically physiologic and functionally viable conditions for accurate drug transport. In this study, we invent a method for the surgical isolation and mounting of whole skeletal muscles of small rodents that maintains the physiologic configuration of the tissue. We also invent a mounting assembly and dynamic loading system designed appropriately for in vitro drug transport studies. We present an effective protocol for tissue processing and visually quantifying intramuscular distribution of drug. With the primary objective of investigating muscle pharmacokinetics, we use these techniques in a study to elucidate the influence of mechanical loading on the intramuscular transport and distribution of locally delivered drug. Methods and Results: The dynamic loading system was characterized and used to investigate intramuscular transport of aqueous macromolecular drug. The loading system was designed to achieve a maximal force, velocity, and acceleration of up to 72N, 0.45m/s, and 8.5m/s2, respectively, for imposing cyclic strain on soleus muscle samples. Total compliance of the series assembly from the motor to muscle mounting blocks was less then 0.0057 ± 0.002mm/N.

(cont.) Under proportional-integral-derivative (PID) control with a positional resolution of 20gpm, the loading system achieved a positional precision of +60gm or better for sinusoidal reference curves required in our studies. Tissue architectural and functional integrity as well as a technique for quantifying intramuscular fluorescent dextran were validated using the loading system. Histologic studies of rat soleus showed that interstitial porosity was consistent in tissues subjected to mechanical loading for 70 minutes, and changes in porosity were independent of the nature of imposed static (0-15% fixed strain) and cyclic (3Hz sinusoidal strain with amplitude of 2.5% oscillating about mean strains of 5-15%) loads. Permanent changes in architectural integrity depended only on the duration of time spent in vitro after isolation, in which porosity increased at the tissue edge from 11.1 + 3.3% to 21.0 + 6.1% over the course of a 70-minute incubation. The source solution used for local delivery of drug (dextran) preserved tissue functional viability, allowing muscle samples to maintain isometric twitch contractile activity at a rate of 3Hz for at least 1 hour. The active twitch force- length characteristic of soleus samples showed 0.24 + 0.06N at 0% strain, a maximum of 0.35 + 0.06N at 10% strain, and a decrease to 0.19 + 0.06N at 20% strain. Isometric twitch contractile force was at least 0.19N when measured every 15 minutes over a 2 hour period. Fractional volume of distribution for dextran was 84% of the bulk source concentration over the range of 0.1 M-lmM bulk concentrations, and demonstrated the non-binding properties of dextran. Fluorescence intensity of FITC-dextran equilibrated in soleus tissue exhibited a linear dependence on dextran concentration.

(cont.) Dextran penetration and distribution in soleus muscles under either cyclic (3Hz, 0-20% peak-to- peak) or static (fixed at 0%) tensile strain for 80 minutes was quantified by fluorescent imaging. Penetration depth of 1mM 20kDa FITC-dextran at the planar surfaces of the soleus increased significantly from 0.52 + 0.09mm under static strain to 0.81 + 0.09mm under cyclic strain. Penetration at the curved margins of the soleus was significantly greater than at planar surfaces by a factor of 1.57 and 2.52 under static and cyclic strain, respectively. Penetration at curved surfaces increased to a greater extent, by a factor of 1.6, than at planar surfaces under cyclic strain. Discussion: This investigation demonstrated that dynamic, or cyclic, tensile strain impacts the penetration and distribution of aqueous drug in skeletal muscle. In the course of this study, we established an effective and robust experimental system and protocol for studying mechanical influences on intramuscular pharmacokinetics. The innovation of our surgical isolation and mounting technique allowed for the investigation of an isolated soleus muscle without disrupting the muscle, tendons, or physiologic bone attachments. The mounting device enabled muscles to be secured in a physiologic in situ configuration, to undergo more physiologically distributed tensile stresses and strains, and to be mechanically loaded while incubated in vitro in drug. Thus, the method and device eliminated the artificial tissue stresses typically introduced by current tissue handling techniques that could result in drug transport artifacts.

(cont.) While effective as a standalone biomechanical testing preparation, characterization and validation of the dynamic loading system with a protocol for tissue processing and quantitative assessment of intramuscular fluorescent drug distribution demonstrated that it is a novel and robust preparation for investigating both tissue biomechanics and pharmacokinetics. With the finding from the present study that dynamic loading influences intramuscular drug transport in an architecturally dependent manner, we intend to investigate the isolated effects of different mechanical loading regimens on drug transport to establish a broader understanding of muscle pharmacokinetics. It is hoped that the insights from this work will guide the design and application of future local drug delivery strategies to mechanically active tissues.