Osmotic Annealing Generates A Suite Of Mechanically-Activated Microcapsules For Tunable Drug Delivery PDF Download

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DISCLOSURES. No disclosures. INTRODUCTION. Drug delivery systems that employ physiological stimuli to trigger delivery (e.g. temperature, pH, etc.) allow for strict control and on-demand provision of biological factors [1]. To date, few systems have employed the mechanical environment as means for activation [2]. Many tissues in the body, such as musculoskeletal tissues, sustain loading during day-to-day activities. Upon injury or degeneration, normal loading patterns can shift towards aberrant, supra-physiological levels, which can intensify damage and promote degeneration [3]. Delivering therapeutic molecules in response to tissue loading could therefore provide strict spatiotemporal control over provision of biomolecules and lead to better outcomes. To that end, we developed mechanically-activated microcapsules (MAMCs) for the release of factors upon loading [4-5]. To further expand upon the therapeutic and tunable properties of this approach, this study aimed to expand the suite of MAMCs using osmotic annealing, to characterize their activation profiles in 2D and 3D environments in response to static and cyclic loading, and to investigate MAMC stability over time in u201cphysiological-likeu201d incubations. METHODS. MAMCs were fabricated using a glass capillary microfluidic device as in [6]. The inner phase contained bovine serum albumin (BSA) and Alexafluor488-BSA for visualization while the middle phase contained poly(lactic co-glycolic) acid (PLGA) 85:15 and Nile Red for visualization. Osmotic annealing was performed by collecting MAMCs in solutions containing different NaCl molarities, after which MAMC dimensions were characterized via confocal microscopy (Fig 1, A). To analyze MAMC resistance to compressive loads, MAMCs were compressed between parallel plates at 0.5% strain/sec. at various loads and imaged post overnight incubation to visualize inner solution retention (Fig 2, A). To characterize MAMC response in 3D environments, MAMCs were embedded in 500kPa PEGDA hydrogels (19%w/v). Using a custom confocal mounted compression device [7], MAMC-containing hydrogels were compressed from 0-30% strain at 5% strain steps and imaged at each step to measure MAMC deformation (Fig 2, B). To determine MAMC resistance to cyclic loading, MAMC-containing hydrogels were loaded between parallel plates from 2-20% strain at 5Hz for different durations and imaged to assess inner solution retention (Fig 2, C). Finally, to investigate MAMC stability in u201cphysiological-likeu201d solutions, MAMCs were incubated in PBS, basal media, or bovine synovial fluid at 37oC for up to 2 weeks and imaged at 1, 7 and 14 days of incubation to analyze microcapsule inner solution retention. Normally distributed data was analyzed via one-way ANOVA followed by Tukeyu2019s Multiple Comparison post-hoc test while non-normal data was analyzed via one-way or two-way ANOVA followed by Dunnu2019s Multiple Comparison test or Bonferroni post-hoc test respectively. RESULTS. Osmotic annealing led to decreased MAMC diameters and increased shell thicknesses as a function of NaCl molarity in the collecting solution (Fig 1). It is important to note that utilizing a NaCl molarity of 1.2M did not allow microcapsules to form, suggesting there is an upper limit to the NaCl molarity that can be used for effective osmotic annealing. Direct compression of MAMCs demonstrated that higher NaCl molarities increased MAMC resistance to static loads (Fig 2, B). In 3D environments, increased annealing also decreased MAMC deformation during static compression and increased resistance to cyclic loading (Fig 2, C-D). Finally, annealed MAMCs remained stable in bovine synovial fluid over a 2-week incubation period in comparison to BSA MAMCs (Fig 3), indicating the process increases stability of MAMCs in physiological conditions. DISCUSSION. Osmotic annealing expanded our suite of MAMCs and created microcapsules that possessed higher resistance to static and dynamic compressive loads in 2D and 3D environments and greater stability in synovial fluid. The manufacturing process did not require alteration of any microfluidic parameters, which provided ease of fabrication. The large suite of MAMCs presented in this study could allow for on-demand delivery of molecules to musculoskeletal tissues in response to the loading on the tissue and the surrounding matrix stiffness, ultimately allowing for strict mechano-regulation of biomolecule presentation.SIGNIFICANCE. This novel fabrication method allows for a wide suite of MAMCs to be manufactured in a single run, yielding microcapsules with varying resistance to loads and stability in synovial fluid. This drug delivery system can be easily tuned to loading patterns and injury degree to promote the healing of musculoskeletal tissues. REFERENCES. [1] Kost+ Adv Drug Deliv Rev 2001, [2] Korin+ Science 2012, [3] Sun+ Ann NY Acad Sci 2010, [4] Mohanraj+ 2016 ORS Annual Meeting #282, [5] Mohanraj+ 2017 ORS Annual Meeting #1405, [6] Tu+ Langmuir 2012, [7] Farrell+ Eur Cells Mat 2012 ACKNOWLEDGEMENTS. This work is supported by R01 AR071340 grant.