Retinal Organoid–Based Evaluation of Icariin-Loaded Extracellular Vesicles for Hypoxia-Induced Retinal Injury via Mitochondrial Modulation

Methods

EV_ICA were isolated and characterized by morphology, EV marker expression, and zeta potential analysis. A CoCl₂-induced hypoxic injury model was established in R28 cells and hESC-derived retinal organoids to evaluate the therapeutic effects of EV_ICA. Gene and protein expression analyses, bulk RNA sequencing (RNA-seq), gene set enrichment analysis (GSEA), mitochondrial ROS measurements, and intracellular ATP assays were performed to assess neuroprotective efficacy and mitochondrial function.

Results: EV_ICA exhibited typical spherical morphology, expressed standard EV markers, and showed a shift toward a less negative zeta potential compared to Naïve EV. In hypoxia-injured retinal models, EV_ICA significantly restored the expression of key retinal and neuroprotective markers, including Syntaxin12, NeuN, Vegf, Rbpms, Brn-3a, and Bdnf, while suppressing Hif-1α accumulation. Transcriptomic and GSEA analyses revealed that EV_ICA reactivated biological pathways associated with phototransduction, visual perception, and retinal development that were impaired under hypoxic conditions.

Furthermore, EV_ICA improved mitochondrial homeostasis by up-regulating mitochondrial-related genes, reducing mitochondrial ROS accumulation, and recovering intracellular ATP production. Conclusion: These findings demonstrate that EV_ICA exerts neuroprotective effects by preserving mitochondrial quality control and restoring functional retinal gene programs under hypoxic stress. EV_ICA may therefore serve as a promising therapeutic candidate for hypoxia-related retinal degeneration and provide potential applications for cell-free regenerative medicine