The Antiangiogenic Effect of VEGF-A siRNA-FAM-Loaded Exosomes.

Abstract

Neovascular ocular diseases are caused by vascular endothelial growth factor A (VEGFA) overexpression. Thus, VEGFA inhibition is considered the main strategy for treating ocular neovascularization. However, existing anti-VEGF therapies have several limitations in stability and delivery efficiency. To overcome the limitations, exosome-based VEGF siRNA delivery technology has attracted attention since exosomes have the advantages of high in vivo stability and excellent intracellular delivery efficiency. Additionally, loading VEGFA siRNA into exosomes not only allows for targeting specific cells or tissues but can also improve therapeutic efficacy. Our research team purified and concentrated exosomes using chromatography techniques, added fluorescein amidite (FAM)-labeled VEGFA siRNA into exosomes, and observed the novel effect of drug delivery in vitro. This study successfully introduced hVEGFA siRNA-FAM into target cells, with high efficacy particularly at 48 h after treatment. Furthermore, the enhanced inhibition of VEGFA expression at 48 h post-treatment was confirmed. FACS analysis was performed using the apoptosis markers Annexin V-FITC (green) and PI-PE (red) to confirm the presence or absence of apoptosis. Both groups treated with hVEGFA siRNA-FAM-EXO (1) and hVEGFA siRNA-FAM-EXO (2) showed increased apoptosis as the exposure time passed compared to the untreated group (0 h). hVEGFA siRNA-FAM-EXO treatment effectively induced apoptosis. After 24 h, early apoptosis was 12.9% and 13.9% and late apoptosis was 1.5% and 3.7% in hVEGFA siRNA-FAM-EXO groups (1) and (2), respectively. After 48 h, early apoptosis was 23.9% and late apoptosis was 39.4% and 17.8% in hVEGFA siRNA-FAM-EXO groups (1) and (2), respectively, indicating a time-dependent pattern of apoptosis progression. Additionally, tube formation of human vascular endothelial cells (HUVECs) was induced to confirm the effect of VEGFA siRNA-loaded exosomes on the angiogenesis assay in vitro. Compared with controls, angiogenesis became significantly weakened in hVEGFA siRNA-FAM-EXO (1)- and hVEGFA siRNA-FAM-EXO (2)-treated groups at 48 h post-treatment and completely disappeared at 72 h, probably occurring due to decreased VEGFA, PIGF, and VEGFC in the intracellular cytosol and conditioned media secreted by VEGFA siRNA-FAM in HUVECs. In conclusions, FAM-tagged VEGFA siRNA was packed into exosomes and degraded over time after tube formation, leading to cell death due to a decrease in VEGFA, PIGF, and VEGFC levels. This study is expected to support the development of in vivo neovascularization models (keratitis, conjunctivitis, or diabetic retinopathy models) in the future.