New nanoparticles for topical ocular delivery of erythropoietin

Erythropoietin (EPO) is known for its neuroprotective and neuroregenerative properties. EPO topical ocular administration has not been tested yet and its bioavailability could be improved by mucoadhesive hydrogels. Thus, this study aimed to develop and evaluate a chitosan (CS) and hyaluronic acid (HA) nanoparticulate system for topical ocular delivery of EPO. Nanoparticles were prepared by ionotropic gelation using six different HAs (HAI -HA6), and characterized by size, zeta potential (ZP), polydispersity index (Pdi), cytotoxicity and mucoadhesion. Encapsulation efficiency and drug loading capacity were also determined. Ex vivo permeation was tested using fresh porcine corneas, scleras and conjunctivas. The permeated EPO was quantified by ELISA, and its presence in the membranes was confirmed by immunohistochemistry. Nanoparticles (NPs) presented size <= 300 nm, ZP around + 30 mV and low Pdi (0.167-0.539) at a 1:1 CS:HA mass ratio. The most suitable HA was HA6 (300 kDa - Eye), which had the best mucoadhesive properties. CS/HA6-EPO nanoformulation permeated more rapidly through porcine conjunctiva, followed by sclera and thirdly by cornea, as assessed by immunohistochemistry. All formulations were noncytotoxic on ARPE-19 and HaCaT cell lines, as evaluated by metabolic and membrane integrity tests. In conclusion, CS/HA6-EPO NPs could be a promising formulation for increasing EPO ocular bioavailability by enhancing its retention time and permeation through the different ocular membranes.