(526a) Development of Phosphatidylserine Presenting Particles for Targeting Macrophages in Muscle Regeneration

Introduction. Efficient myoblast fusion to form multinucleated myotubes is essential for muscle regeneration^1,2. Macrophages facilitate myoblast fusion by mediating inflammation and subsequently secreting regenerative factors. Macrophage function and secretome can be modulated through receptor mediated binding of phosphatidylserine (PS) on the surface of apoptotic cells³. However, exogenously harnessing sufficient amounts of apoptotic cells to be utilized as a potential therapeutic are met with financial and technical challenges^4,5. Hence, this study aims to: 1) Synthesize PS-presenting microparticles that acts as synthetic apoptotic bodies and 2) Test these microparticles in an in vitromodel of muscle regeneration.

Methods. PS-presenting PLG microparticles (PS:PLG) were synthesized using the single oil-in-water emulsion/solvent extraction method⁶. An annexin V binding assay was used to investigate the presence of functional PS on microparticle surface. PS:PLG microparticles were loaded with coumarin 6 (C6), to track particle uptake by RAW 264.7 macrophages. To investigate the impact of PS particles on macrophage-induced myoblast fusion into myotubes (an in vitromodel of muscle regeneration), macrophages were treated with PS:PLG microparticles for 24 hours, after which macrophage conditioned media was collected and used to treat C2C12 myoblasts daily for 3 days. To quantitively assess number and size, myotube were probed with a fluorescent myosin heavy chain (myHC) antibody.

Results and Discussion. An annexin V binding assay demonstrated that PS:PLG particles bound fluorescently labeled Annexin V, while particles lacking PS did not (Figure 1). Flow cytometry indicated that particles made with increasing amounts of PS exhibited a greater fluorescent signal, indicating that more annexin V was bound (data not shown). The data indicates PS remains functional after incorporation into the particles and annexin V binding capacity can be modulated by varying the mass of PS added to the emulsion.

PS functions as an “eat me” signal to macrophages that promotes binding and uptake of apoptotic bodies.Fluorescent microscopy showed increased PS:PLG-C6 particle co-localization with macrophages compared to C6 loaded particles having no surface PS (Figure 2). The data indicates that macrophages more readily interact with PS:PLG particles, which could have implications for targeted drug delivery.

In the myotube formation assay, conditioned media from macrophages treated with PS:PLG particles significantly increased myotube width, an important index of muscle growth, compared to media from macrophage treated with or without particles lacking PS (Figure 3A & B). This result suggests that treatment of damaged or atrophied muscle, which contains macrophages, with PS:PLG particles may enhance muscle growth.

Conclusion and Future Work. Overall, these data demonstrates that PLG microparticles can be successfully surface functionalized with PS that remains able to bind Annexin V. The PS:PLG microparticles increased macrophage particle bindingand subsequently lead to the release of soluble factors that increase myotube width. Future studies aim to determine possible factors secreted by macrophages treated with the PS:PLG microparticles that led to increased myotube width. This information will inform development of future particle formulations that will deliver bioactive factors to enhance the macrophage regenerative secretome established by the PS:PLG particles.

References:

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