Abstract
Purpose :
Exosomes are considered as versatile carriers for biomolecules; however, the effective delivery of therapeutic agents using exosomes presents several challenges. In this study, we explored the utility of exosomes as a delivery system for the mouse corneal endothelium (CE).
Methods :
Human corneal endothelial cell (CEC)-derived exosomes were isolated from culture media using differential ultracentrifugation. Their size, number, and morphology were characterized using nanoparticle tracking analysis and transmission electron microscopy (TEM). Exosome markers were confirmed using an exosome detection antibody array. PKH-67-labeled exosomes were injected into the anterior chamber of wild-type mice. Corneal endothelial uptake of exosomes was assessed by histological staining with DAPI on corneal whole mounts. Sonication method was used to load FITC-conjugated peptide into exosomes. Active targeting of CE was assessed by colocalization analysis of exosomes with FITC-conjugated peptide within CE.
Results :
An average of 3.7 x 109 particles/mL with a peak size of 120 nm exosomes were isolated. The morphology of exosomes was confirmed by TEM, and the presence of exosome markers, FLOT1, ICAM, ALIX, CD81, CD63, ANXA5, and TSG101, was examined by immunoblotting. Confocal microscopy of whole mounted corneas, performed two days after intracameral injection of PKH-67-labled exosomes containing FITC-conjugated peptide, revealed a scattered distribution of exosomes within the CE. Notably, no exosomes were observed in the corneal stroma. The FITC-labeled peptide was incorporated into exosomes through incubation at room temperature with saponin permeabilization and following sonication. Colocalization of PKH-67-labeled exosome and FITC-conjugated peptide signals in the CE showed successful loading of the peptide into exosomes and endothelial delivery of the exosomes.
Conclusions :
Intracamerally delivered CEC-derived exosomes exhibit effective penetration into the CE without reaching the stromal layer. Moreover, the delivery of synthetic peptide by these exosomes demonstrates effective targeting of the CE. We demonstrate proof of principle of using human CEC-derived exosomes for therapeutic agent delivery to the mouse CE via intracameral injection.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.