Cationic-motif-modified exosomes for mRNA delivery to retinal photoreceptors

Topical treatment of vitreoretinal diseases remains a challenge due to slow corneal uptake and systemic clearance. Exosomes are emerging nanocarriers for drug delivery due to biocompatibility and cellular targeting properties. To apply them for retinal targeting via the topical route, exosomes must traverse various ocular barriers including the cornea, lens, vitreous humor (VH), and the retina itself. Here we engineered high-purity milk-derived exosomes by anchoring arginine-rich cationic motifs via PEG₂₀₀₀ lipid insertion on their surface. Modification enabled exosomes to use weak-reversible electrostatic interactions with anionic glycosaminoglycan (GAG) and water content of the tissue to enhance their transport rate and retention. Addition of cationic motifs neutralized the anionic surface charge of exosomes (-24 to -2 mV) without impacting size or morphology. Cationic-motif-modified exosomes exhibited two-fold faster steady state diffusivity through bovine corneas compared to unmodified exosomes. Fluorescence recovery after photobleaching confirmed that cationic-motif-modified exosomes can diffuse through VH without steric hindrance. In healthy VH, cationic-motif-modified exosomes demonstrated stronger binding resulting in three-fold lower average diffusivity that enhanced by six-fold in 50% GAG-depleted VH recapitulating advanced liquefaction. Cationic-motif-modified exosomes penetrated through the full-thickness of porcine retinal explants resulting in ten-fold higher uptake in photoreceptors and three-fold greater transfection with encapsulated eGFP mRNA compared to unmodified exosomes. Cationic-motif-modified exosomes are safe to use as they did not adversely affect the mechanical swelling properties of the cornea or lens nor impact retinal cell viability. Cationic-motif-modified exosomes, therefore, offer themselves as a cell-free nanocarrier platform for gene delivery to retinal photoreceptors potentially via the topical route.