Hur Woojune, Bhujel Basanta, Oh Seheon, Lee Seorin, Chung Ho Seok, Park Jin Hyoung, Kim Jae Yong
Department of Ophthalmology, Asan Medical Center, University of Ulsan College of Medicine, Seoul 05505, Republic of Korea.
Department of Medical Science, University of Ulsan Graduate School, Seoul 05505, Republic of Korea.
Bioengineering (Basel). 2025 Aug 26;12(9):919. doi: 10.3390/bioengineering12090919.
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.
新生血管性眼病是由血管内皮生长因子A（VEGFA）过度表达引起的。因此，抑制VEGFA被认为是治疗眼部新生血管形成的主要策略。然而，现有的抗VEGF疗法在稳定性和递送效率方面存在若干局限性。为了克服这些局限性，基于外泌体的VEGF siRNA递送技术引起了关注，因为外泌体具有体内稳定性高和细胞内递送效率优异的优点。此外，将VEGFA siRNA加载到外泌体中不仅可以靶向特定细胞或组织，还可以提高治疗效果。我们的研究团队使用色谱技术纯化和浓缩外泌体，将荧光素酰胺（FAM）标记的VEGFA siRNA添加到外泌体中，并在体外观察了药物递送的新效果。本研究成功地将hVEGFA siRNA-FAM导入靶细胞，尤其是在治疗后48小时具有高效性。此外，证实了治疗后48小时VEGFA表达的抑制增强。使用凋亡标记物膜联蛋白V-FITC（绿色）和PI-PE（红色）进行流式细胞术分析以确认凋亡的存在与否。与未处理组（0小时）相比，用hVEGFA siRNA-FAM-EXO（1）和hVEGFA siRNA-FAM-EXO（L）处理的两组随着暴露时间的延长凋亡增加。hVEGFA siRNA-FAM-EXO处理有效地诱导了凋亡。24小时后，hVEGFA siRNA-FAM-EXO组（1）和（2）的早期凋亡分别为12.9%和13.9%，晚期凋亡分别为1.5%和3.7%。48小时后，hVEGFA siRNA-FAM-EXO组（1）和（2）的早期凋亡分别为23.9%，晚期凋亡分别为39.4%和17.8%表明凋亡进展呈时间依赖性模式。此外，诱导人血管内皮细胞（HUVECs）的管形成以确认加载VEGFA siRNA的外泌体对体外血管生成测定的影响。与对照组相比，在治疗后48小时，hVEGFA siRNA-FAM-EXO（1）和hVEGFA siRNA-FAM-EXO（2）处理组的血管生成明显减弱，在72小时完全消失，这可能是由于HUVECs中细胞溶质和VEGFA siRNA-FAM分泌的条件培养基中VEGFA、PIGF和VEGFC减少所致。总之，FAM标记的VEGFA siRNA被包装到外泌体中，并在管形成后随时间降解，由于VEGFA.PIGF和VEGFC水平降低导致细胞死亡。本研究有望支持未来体内新生血管形成模型（角膜炎、结膜炎或糖尿病性视网膜病变模型）的开发。
