Sustained Agrin Nanoparticle Delivery Ameliorates Effects of Denervation and Preserves Neuromuscular Junction Morphology after Peripheral Nerve Injury

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) assess the efficacy of nanoparticle (NP) encapsulated agrin in preserving denervated NMJs over free agrin and (2) establish the optimal dosage schedule for sustained release. METHODS: (1) 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. (2) Using the same model, Lewis-Norway rats were injected with the medium dose agrin-NPs within NHC, free agrin, empty-NPs within NHC, or saline at the time of denervation. Animals were then sacrificed at 3, 6, 9, and 12 weeks. RESULTS: (1) 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. (2) Agrin-NP treated animals retained greater agrin levels in the soleus muscle as compared to free agrin-treated animals at all endpoints. Agrin levels in muscle remained above the EC50 in the 6-week endpoint Agrin-NP treated animals whereas 9-week endpoint Agrin-NP treated animals fell below the EC50, suggesting an optimal dosage schedule of 6 weeks. Furthermore, NMJ morphology of Agrin-NP treated animals were not significantly different than sham animals at 6 weeks while a decline in pretzel-like NMJ morphology was seen at the 9-week endpoint. 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.