Metabolic reprogramming of macrophages via engineered nanoparticles after volumetric muscle loss

Volumetric muscle loss (VML) injuries are characterized by an exacerbated and chronic inflammatory state that inhibits muscle stem-cell mediated repair, leading to fibrosis and attenuation of neuromuscular strength. The excessive infiltration of myeloid cells in VML injuries leads to the depletion of nutrients needed by myogenic progenitors for tissue repair. Mitigating the inflammatory response and fibrosis are potential approaches to enhance therapies for VML. The adenosine monophosphate-activated protein kinase (AMPK) pathway plays a critical role in myeloid responses to injury by regulating cellular metabolism, suppressing glycolysis, and attenuating fibrosis-inducing pathways such as transforming growth factor-beta (TGFβ). AMPK Activation reduces fibrosis and promotes muscle stem cell differentiation, however, a limiting factor of AMPK agonists such as AICAR (5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside) is that these therapies display low bioavailability. As such, there remains an unmet need for strategies to target AMPK in skeletal muscle to enhance therapeutic efficacy and prevent muscle degeneration. To address this need, we used synthetic protein nanoparticles (SPNPs) to encapsulate AICAR. SPNPs can have high drug loading (>10wt%), allow for a sustained release of the packaged drug (72 hours), and have a demonstrated targeting capacity. Increasing the bioavailability of AICAR through packaging into SPNPs would be enabling for improving metabolism after trauma and extensible to other AMPK agonists and small molecules, which in turn can further augment existing regenerative therapies to promote healing Quadriceps muscles from mice were given a VML injury and treated with either AICAR and Vehicle loaded SPNPs (x1011 particles) at 1,4,7 days post-injury (dpi). Muscles were collected at 8 dpi, digested into single cell suspensions, FACS sorted to obtain live cells and processed for single cell sequencing.

体积性肌肉丢失（Volumetric Muscle Loss, VML）损伤以加剧且慢性的炎症状态为特征，该状态会抑制肌肉干细胞介导的修复过程，进而引发纤维化，并导致神经肌肉强度减弱。VML损伤部位髓系细胞的过度浸润，会耗尽肌源性祖细胞进行组织修复所需的营养物质。缓解炎症反应与纤维化，是提升VML损伤治疗效果的潜在策略。腺苷酸活化蛋白激酶（adenosine monophosphate-activated protein kinase, AMPK）通路在损伤后的髓系细胞应答中发挥关键作用，其可通过调控细胞代谢、抑制糖酵解，以及削弱诸如转化生长因子-β（transforming growth factor-beta, TGFβ）这类促纤维化通路来实现相关功能。AMPK激活可减少纤维化并促进肌肉干细胞分化，然而，诸如AICAR（5-氨基咪唑-4-甲酰胺-1-β-D-呋喃核糖苷，5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside）这类AMPK激动剂的一个局限性在于，其生物利用度较低。因此，目前仍亟需能够在骨骼肌中靶向AMPK的策略，以提升治疗效果并预防肌肉退行性变化。为满足这一需求，本研究采用合成蛋白质纳米颗粒（Synthetic Protein Nanoparticles, SPNPs）包裹AICAR。该类纳米颗粒可实现高载药量（>10wt%），对包裹的药物具备72小时的持续释放能力，且已被证实具备靶向能力。通过将AICAR包裹于SPNPs中来提升其生物利用度，将有助于改善创伤后的代谢状态，且该策略可拓展至其他AMPK激动剂与小分子化合物，进而进一步优化现有的再生治疗方案以促进组织愈合。本研究对小鼠股四头肌制造VML损伤，并分别在损伤后第1、4、7天（dpi）施加负载AICAR与赋形剂的SPNPs（剂量为1×10¹¹颗粒）。于损伤后第8天（8 dpi）采集肌肉样本，将其消化为单细胞悬液，通过荧光激活细胞分选（Fluorescence-Activated Cell Sorting, FACS）分离活细胞，随后进行单细胞测序。