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 × 106 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 αv 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 αv 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 × 109 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 αv. 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.

Retinal degenerative diseases, including age-related macular degeneration (AMD) and retinitis pigmentosa, lack effective therapies. Conventional monotherapeutic approaches fail to target the multiple affected pathways in retinal degeneration. However, the retinal pigment epithelium (RPE) secretes several neurotrophic factors addressing diverse cellular pathways, potentially preserving photoreceptors. This study explored human embryonic stem cell-derived, polarized RPE soluble factors (PRPE-SF) as a combination treatment for retinal degeneration. PRPE-SF promoted retinal progenitor cell survival, reduced oxidative stress in ARPE-19 cells, and demonstrated critical antioxidant and anti-inflammatory effects for preventing retinal degeneration in the Royal College of Surgeons (RCS) rat model. Importantly, PRPE-SF treatment preserved retinal structure and scotopic b-wave amplitudes, suggesting therapeutic potential for delaying retinal degeneration. PRPE-SF is uniquely produced using biomimetic membranes for RPE polarization and maturation, promoting a protective RPE secretome phenotype. Additionally, PRPE-SF is produced without animal serum to avoid immunogenicity in future clinical development. Lastly, PRPE-SF is a combination of neurotrophic factors, potentially ameliorating multiple dysfunctions in retinal degenerations. In conclusion, PRPE-SF offers a promising therapeutic candidate for retinal degenerative diseases, advancing the development of effective therapeutic strategies for these debilitating conditions.

Purpose: To characterize vitreous humor (VH) exosomes and to explore their role in the development of proliferative vitreoretinopathy (PVR) using mass spectrometry-based proteome profiling. Methods: Exosomes were isolated from undiluted VH from patients with retinal detachment (RD) with various stages of PVR (n = 9), macular hole (MH; n = 5), or epiretinal membrane (ERM; n = 5) using differential ultracentrifugation. The exosomal size, morphology, and exosome markers were analyzed using a nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and an exosome detection antibody array. The tryptic fragment sequencing of exosome-contained proteins was performed using liquid chromatography–tandem mass spectrometry (LC-MS/MS) and a Thermo Lumos Fusion Tribrid Orbitrap mass spectrometer. The pathway analysis of the MS data was performed. Results: The number of exosome particles were significantly increased only in the RD with severe PVR group compared with the control groups and the RD without PVR or with mild PVR groups. Of 724 exosome proteins identified, 382 were differentially expressed (DE) and 176 were uniquely present in PVR. Both DE proteins and exosome proteins that were only present in PVR were enriched in proteins associated with previously known key pathways related to PVR development, including reactive retinal gliosis, pathologic cellular proliferation, inflammation, growth of connective tissues, and epithelial mesenchymal transition (EMT). The SPP1, CLU, VCAN, COL2A1, and SEMA7A that are significantly upregulated in PVR were related to the tissue remodeling. Conclusions: Exosomes may play a key role in mediating tissue remodeling along with a complex set of pathways involved in PVR development.