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Agrin Nanoparticles In A Nanofiber Hydrogel-based Drug Delivery System To Reduce Neuromuscular Junction Degradation After Peripheral Nerve Injury
Erica Lee, MS, Chenhu Qiu, BS, Thomas Harris, MD, Nicholas von Guionneau, MD, Zohra Alsami, BS, Matthew Generoso, BS, Yicheng Zhang, BS, Zhicheng Yao, MS, Ahmet Hoke, MD PhD, Hai-Quan Mao, PhD, Sami Tuffaha, MD.
Johns Hopkins University School of Medicine, Baltimore, MD, USA.

PURPOSE: One of the most critical factors contributing to poor outcomes after peripheral nerve injury is the prolonged period of latency prior to reinnervation. Acetylcholine receptors (AChRs) within denervated muscle rapidly destabilize and consequently degrade neuromuscular junctions (NMJs); thereby limiting meaningful functional motor recovery. Agrin, a proteoglycan essential to NMJ formation and AChR aggregation, may have an essential role in preserving NMJ receptivity to reinnervation. This study aimed to (1) encapsulate agrin into biodegradable nanoparticles (NPs) that enable sustained release at target tissue sites and persist throughout the regenerative period; and (2) assess the efficacy of locally delivered agrin NPs in preserving neuromuscular junctions in denervated muscle to thereby improve functional recovery following nerve injury.
METHODS: (1) NP Fabrication: agrin was first complexed with dextran sulfate to create polyelectrolyte complex (PEC) cores, which were then encapsulated in biodegradable amphiphilic block co-polymers to form the NPs. Varying ratios of PEC:polymer were evaluated to maximize loading efficiency and release kinetics. In vitro NP release kinetics were evaluated and mitogenic activity of released agrin was compared to native agrin. (2) The effects of locally-delivered agrin-NPs on denervated muscle were assessed in a rat tibial nerve transection-without-repair model. Lewis-Norway rats were injected with low, medium, or high doses of agrin-NPs incorporated into a nanofiber-hyaluronic acid hydrogel composite (NHC) gel, empty-NPs within NHC, free agrin, or saline. After 6 weeks, animals were sacrificed and the soleus, lateral and medial gastrocnemius muscles were harvested for analysis.
RESULTS: (1) Fabrication of uniform NPs with an encapsulation efficiency of 85.6% was achieved. NPs composed of 1:5 PEC:polymer yielded optimal release of agrin. Near-zero-order release of agrin can be achieved for at least 70 days and released agrin exhibits comparable bioactivity to native agrin. (2) Agrin-NP treated animals retained significantly greater NMJ pretzel-like morphology after 6 weeks of denervation compared to free agrin and empty-NP groups with optimal benefit achieved by the medium dose (35.0% vs 23.1% free agrin, 35.0% vs 7.1% empty-NP; p<0.0001). Furthermore, both medium and high dose Agrin-NP treated animals demonstrated significantly lower NMJ plaque-like morphology than free agrin treated animals (p<0.0001). NMJ morphology of medium dose Agrin-NP treated animals were not significantly different than sham animals, suggesting optimal benefit was achieved at the medium dose. All Agrin-NP treated animals retained greater agrin levels in the soleus muscle as compared to free agrin-treated animals and endogenous agrin levels in sham animals at 6 weeks (p<0.001). Agrin levels were undetectable in serum at all Agrin-NP doses. No significant differences were seen in myofibril cross-sectional area between Agrin-NP, free agrin, and empty-NP groups. No foreign body response was detected in empty-NP or Agrin-NP treated animals.
CONCLUSION: Encapsulation of bioactive agrin with sustained release for over 70 days was achieved. Agrin-NP treatment in vivo limits neuromuscular junction degradation during denervation and thereby has potential as a therapeutic target to improve motor functional recovery.


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