Intraocular RGD-Engineered Exosomes and Active Targeting of Choroidal Neovascularization (CNV)

Purpose: To assess the transretinal penetration of intravitreally injected retinal multicell-derived exosomes and to develop exosome-based active targeting of choroidal neovascularization (CNV) by bioengineering with ASL, which is composed of a membrane Anchor (BODIPY), Spacer (PEG), and targeting Ligands (cyclic RGD peptide). Methods: Retinal multicell-derived exosomes were recovered from a 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 (PKH-26)-labeled or engineered ASL exosomes (1 × 10(6) exosomes/μL) were given to the wild-type mouse or laser-induced CNV mouse model. Retinal uptake of exosomes was assessed by in vivo retinal imaging microscopy and histological staining with DAPI, GSA, and anti-integrin α(v) for 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 ASL exosomes with integrin α(v) within CNV. Staining with anti-F4/80, anti-ICAM-1, and anti-GFAP antibodies on retinal sections were performed to identify intracellular uptake of exosomes and immediate reactive retinal gliosis after exosome treatment. Results: An average of 2.1 × 10(9) particles/mL with a peak size of 140 nm exosomes were recovered. Rapid retinal penetration of intravitreally injected exosomes was confirmed by retinal imaging microscopy at 3 and 24 h post-injection. Intravitreally delivered PKH-26-labeled exosomes reached inner and outer retinal layers including IPL, INL, OPL, and ONL at 1 and 7 days post-injection. Intravitreally injected ASL exosomes were predominantly delivered to the area of CNV including ONL, RPE, and choroid in laser-induced CNV mouse models with 89.5% of colocalization with integrin α(v). Part of exosomes was also taken intracellularly to vascular endothelial cells and macrophages. After intravitreal injection, neither naive exosomes nor ASL exosomes induced immediate reactive gliosis. Conclusions: Intravitreally delivered retinal multicell-derived exosomes have good retinal penetration, and ASL modification of exosomes actively targets CNV with no immediate reactive gliosis. ASL exosomes have a great potential to serve as an intraocular drug delivery vehicle, allowing an active targeting strategy.