Exosome Delivery to Retina and Active targeting of CNV by RGD-conjugated exosomes

Abstract

Purpose : This study assesses the transretinal penetration of intravitreally injected retinal tissue-derived naïve exosomes and the enhanced targeting of choroidal neovascularization (CNV) after decorating them with Arg-Gly-Asp (RGD), a well-known ligand for integrins which are essential in VEGF signaling in CNV.

Methods : Exosomes were recovered from either mouse Müller glial cell or whole mouse retina using differential ultracentrifugation. Their size, number, and morphology were characterized using nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Exosome markers were confirmed using an exosome detection antibody array. Intravitreal injection of fluorescent-labeled naïve or RGD-conjugated exosomes (5.3 x 10⁷ exosomes/μl) were given to wild type (n =9) or laser-induced CNV mouse model (n= 9). Retinal uptake of exosomes was assessed by in vivo retinal imaging microscopy and fluorescent staining with DAPI, GSA, and anti-integrin α_v antibody of retinal sections or choroid/RPE flat mounts. Active targeting of CNV was assessed by comparing retinal uptake between areas with and without CNV and by colocalization analysis of RGD-conjugated exosomes with integrin α_v within CNV.

Results : We recovered an average of 2.1 x 10⁹ particles/ml with a peak size of 140 nm from 10 whole mouse retinas, and 4.2 x 10⁸ particles/ml with a peak size of 147 nm from the supernatant of primary cultured mouse Müller glia. Rapid retinal penetration of intravitreally injected exosomes was confirmed by retinal imaging microscopy at 3 and 24 hours post-injection. Intravitreally injected naïve and RGD-conjugated exosomes penetrated into inner and outer retinal layers including IPL, INL, OPL, and ONL at 1 and 7 days after injection. Intravitreally injected RGD-conjugated exosomes were exclusively delivered to the area of CNV including ONL, RPE, and choroid in laser-induced CNV mouse model with 89.5% of colocalization with integrin α_v. Exosomal origination between mouse Müller glia cell and whole mouse retina did not show differences in retinal penetration.

Conclusions : Intravitreally injected exosomes demonstrated good penetration in both inner and outer retina. Further, intravitreally injected RGD-conjugated exosomes were selectively delivered to the area of CNV. This suggests that RGD-conjugated exosomes have a great potential to serve as an intraocular drug delivery vehicle, allowing an active targeting strategy.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.