(526e) ATRA-Loaded PLG Microparticles to Direct Macrophage Regenerative Function

Statement of Purpose: Older adults experience a reduced capacity to regain muscle mass after atrophy that results from short bouts of disuse (illness, injury, surgery), which occurs often for this age group. Reduced responsiveness to therapy in aged muscle occurs due to low expression of growth factors, such as Insulin-like Growth Factor-1 (IGF1), which is produced by muscle-resident macrophages [1, 2]. Transcriptomics data indicates that activation of retinoic acid receptors (RAR) in macrophages enhances IGF1 expression [3], which can be achieved with all-trans-Retinoic acid (ATRA). This study aims to: (1) synthesize ATRA-loaded microparticles that remain bioactive after encapsulation and (2) test the myogenic ability of particle-treated macrophages in an in vitro model of muscle regeneration.

Methods: ATRA-loaded PLG particles were synthesized using a single emulsion/solvent-evaporation technique by adding 5 or 0.5 mg/mL of ATRA to the organic phase for high- and low-loaded ATRA-PLG particles. Drug loading was determined using UV-vis spectroscopy. To confirm the bioactivity of ATRA after encapsulation, RAW264.7 macrophages were incubated with high- or low-loaded ATRA-PLG particles for 24 hours, then images were analyzed to measure cell viability; IGF1 ELISA was conducted on the collected media. To investigate the impact of ATRA-PLG particles on macrophage-induced myoblast fusion into myotubes (an in vitro model of muscle regeneration), macrophages were treated with ATRA-PLG particles for 24 hours, after which macrophage conditioned media was collected and used to treat C2C12 myoblasts daily for 3 days. Control groups included conditioned media from macrophages without particle treatment and media conditioned with ATRA particles alone. After performing immunofluorescence microscopy for myosin heavy chain, image analysis was used to quantify myotube formation and growth.

Results and Discussion: ATRA was successfully incorporated into PLG microparticles, producing spherical and monodisperse particles with sizes ranging from 2.5 to 2.9 µm. Drug loadings for the high- and low-loaded ATRA-PLG particles were 78 and 6.6 µg of ATRA per milligram of particle, respectively. When incubated with macrophages, both high- and low-loaded ATRA particles increase IGF1 secretion into the media compared to blank PLG microparticles (Figure 1), indicating that the ATRA remained bioactive after encapsulation. Microscopy of cell morphology and cell density indicated that the amount of ATRA released did not induce cytotoxicity. In the myotube formation assay, media collected from macrophages treated with ATRA-PLG particles induced myoblasts to fuse more readily into myotubes (Figure 2A) compared to all other treatments, as quantified by the number of myotubes per image field (Figure 2B) and number of incorporated nuclei per myotube (Figure 2C). The data indicate that while untreated macrophages and ATRA released from the particles increase myotube formation, there is an enhanced effect when macrophages are pre-treated with the ATRA-PLG particles. Since macrophages are present in skeletal muscle, we expect that there are opportunities to modulate muscle growth with ATRA-PLG particles indirectly through macrophages and directly through ATRA acting on muscle.

Conclusion: ATRA can be encapsulated into PLG microparticles while maintaining bioactivity. Treatment of macrophages with ATRA-PLG particles changes the secretome in a way that enhances myotube formation compared to media from untreated macrophages or media containing ATRA, but no macrophage-derived factors. The data suggest that elevated levels of IGF1 play a role, but changes in other myogenic or myostatic factors cannot be ruled out. Future studies will further characterize the secretome of ATRA-PLG particle treated macrophages, confirm similar effects in primary macrophages, and assess the efficacy of ATRA-PLG particle delivery on muscle growth in a mouse model of disuse muscle atrophy.

References:

[1] Y. Zhang, X. Pan, Y. Sun, Y. J. Geng, X. Y. Yu, and Y. Li, "The molecular mechanisms and prevention principles of muscle atrophy in aging," Advances in Experimental Medicine and Biology, vol. 1088, pp. 347-368, 2018, doi: 10.1007/978-981-13-1435-3_16

[2] F. Haddad, G. R. Adams, F. sHaddad, and G. R. Adams, "Aging-sensitive cellular and molecular mechanisms associated with skeletal muscle hypertrophy," Journal of Applied Physiology, vol. 100, no. 4, pp. 1188-1203, 2006, doi: 10.1152/japplphysiol.01227.2005.

[3] L. Széles et al., "Research resource: Transcriptome profiling of genes regulated by RXR and its permissive and nonpermissive partners in differentiating monocyte-derived dendritic cells," Molecular Endocrinology, vol. 24, no. 11, pp. 2218-2231, 2010, doi: 10.1210/me.2010-0215.