{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,24]],"date-time":"2026-10-24T01:02:17Z","timestamp":1792803737024,"version":"4.5.0"},"reference-count":51,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2025,8,26]],"date-time":"2025-08-26T00:00:00Z","timestamp":1756166400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Basic Science Research Program through the National Research Foundation of Korea (NRF)","award":["NRF-2022R1F1A1073895"],"award-info":[{"award-number":["NRF-2022R1F1A1073895"]}],"id":[{"id":"https:\/\/ror.org\/013aysd81","id-type":"ROR","asserted-by":"crossref"}]},{"name":"Basic Science Research Program through the National Research Foundation of Korea (NRF)","award":["2025IL0063"],"award-info":[{"award-number":["2025IL0063"]}],"id":[{"id":"https:\/\/ror.org\/013aysd81","id-type":"ROR","asserted-by":"crossref"}]},{"name":"Asan Institute for Life Sciences, Seoul, Republic of Korea","award":["NRF-2022R1F1A1073895"],"award-info":[{"award-number":["NRF-2022R1F1A1073895"]}]},{"name":"Asan Institute for Life Sciences, Seoul, Republic of Korea","award":["2025IL0063"],"award-info":[{"award-number":["2025IL0063"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Bioengineering"],"abstract":"<jats:p>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.<\/jats:p>","DOI":"10.3390\/bioengineering12090919","type":"journal-article","created":{"date-parts":[[2025,8,26]],"date-time":"2025-08-26T14:43:18Z","timestamp":1756219398000},"page":"919","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["The Antiangiogenic Effect of VEGF-A siRNA-FAM-Loaded Exosomes"],"prefix":"10.3390","volume":"12","author":[{"given":"Woojune","family":"Hur","sequence":"first","affiliation":[{"name":"Department of Ophthalmology, Asan Medical Center, University of Ulsan College of Medicine, Seoul 05505, Republic of Korea"},{"name":"Department of Medical Science, University of Ulsan Graduate School, Seoul 05505, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0503-8900","authenticated-orcid":false,"given":"Basanta","family":"Bhujel","sequence":"additional","affiliation":[{"name":"Department of Ophthalmology, Asan Medical Center, University of Ulsan College of Medicine, Seoul 05505, Republic of Korea"},{"name":"Department of Medical Science, University of Ulsan Graduate School, Seoul 05505, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Seheon","family":"Oh","sequence":"additional","affiliation":[{"name":"Department of Ophthalmology, Asan Medical Center, University of Ulsan College of Medicine, Seoul 05505, Republic of Korea"},{"name":"Department of Medical Science, University of Ulsan Graduate School, Seoul 05505, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Seorin","family":"Lee","sequence":"additional","affiliation":[{"name":"Department of Ophthalmology, Asan Medical Center, University of Ulsan College of Medicine, Seoul 05505, Republic of Korea"},{"name":"Department of Medical Science, University of Ulsan Graduate School, Seoul 05505, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ho Seok","family":"Chung","sequence":"additional","affiliation":[{"name":"Department of Ophthalmology, Asan Medical Center, University of Ulsan College of Medicine, Seoul 05505, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jin Hyoung","family":"Park","sequence":"additional","affiliation":[{"name":"MS Eye Clinic, Seoul 06512, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8571-8350","authenticated-orcid":false,"given":"Jae Yong","family":"Kim","sequence":"additional","affiliation":[{"name":"Department of Ophthalmology, Asan Medical Center, University of Ulsan College of Medicine, Seoul 05505, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,8,26]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"289","DOI":"10.1136\/bjophthalmol-2014-305131","article-title":"Myopic choroidal neovascularization: Current concepts and update on clinical management","volume":"99","author":"Wong","year":"2015","journal-title":"Br. J. Ophthalmol."},{"key":"ref_2","first-page":"75","article-title":"Diabetic retinopathy: Early diagnosis and effective treatment","volume":"107","author":"Kollias","year":"2010","journal-title":"Dtsch. Arztebl. Int."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"495","DOI":"10.1159\/000517520","article-title":"Age-related macular degeneration: Pathophysiology, management, and future perspectives","volume":"244","author":"Flores","year":"2021","journal-title":"Ophthalmologica"},{"key":"ref_4","first-page":"40","article-title":"Retinal vein occlusion review","volume":"7","author":"Ip","year":"2018","journal-title":"Asia Pac. J. Ophthalmol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"15","DOI":"10.22336\/rjo.2019.4","article-title":"Corneal neovascularization: Updates on pathophysiology, investigations and management","volume":"63","author":"Sharif","year":"2019","journal-title":"Rom. J. Ophthalmol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"148","DOI":"10.1186\/s13045-021-01147-6","article-title":"KAI1 (CD82) is a key molecule to control angiogenesis and switch angiogenic milieu to quiescent state","volume":"14","author":"Lee","year":"2021","journal-title":"J. Hematol. Oncol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"14845","DOI":"10.3390\/ijms131114845","article-title":"The role of vascular endothelial growth factor A polymorphisms in breast cancer","volume":"13","year":"2012","journal-title":"Int. J. Mol. Sci."},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Rusciano, D., and Bagnoli, P. (2023). Pharmacotherapy and nutritional supplements for neovascular eye diseases. Medicina, 59.","DOI":"10.3390\/medicina59071334"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"100906","DOI":"10.1016\/j.preteyeres.2020.100906","article-title":"Incomplete response to anti-VEGF therapy in neovascular AMD: Exploring disease mechanisms and therapeutic opportunities","volume":"82","author":"Mettu","year":"2021","journal-title":"Prog. Retin. Eye Res."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"395","DOI":"10.7150\/thno\/v01p0395","article-title":"Lasting controversy on ranibizumab and bevacizumab","volume":"1","author":"Zou","year":"2011","journal-title":"Theranostics"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"321","DOI":"10.1097\/APO.0000000000000146","article-title":"Intravitreal injection\u2014Technique and safety","volume":"4","author":"Lai","year":"2015","journal-title":"Asia Pac. J. Ophthalmol."},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Wu, J., and Zhang, J. (2022). Neovascular remodeling and subretinal fibrosis as biomarkers for predicting incomplete response to anti-VEGF therapy in neovascular age-related macular degeneration. Front. Biosci., 27.","DOI":"10.31083\/j.fbl2704135"},{"key":"ref_13","first-page":"13","article-title":"Steroid-induced glaucoma and cataract","volume":"37","author":"Mohan","year":"1989","journal-title":"Indian J. Ophthalmol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"450","DOI":"10.1016\/j.optm.2005.06.013","article-title":"Complications of intravitreal steroid injections","volume":"76","author":"Reichle","year":"2005","journal-title":"Optometry"},{"key":"ref_15","first-page":"1989","article-title":"Recent advances in exosome-based drug delivery systems","volume":"16","author":"Kim","year":"2024","journal-title":"Cancers"},{"key":"ref_16","first-page":"1777","article-title":"RNAi-based therapeutics for ocular neovascularization","volume":"8","author":"Rossi","year":"2008","journal-title":"Expert Opin. Biol. Ther."},{"key":"ref_17","first-page":"284","article-title":"Biomimetic nanocomposites for corneal neovascularization treatment: From design to in vivo evaluation","volume":"38","author":"Kim","year":"2024","journal-title":"Bioact. Mater."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"eaau6977","DOI":"10.1126\/science.aau6977","article-title":"The biology, function, and biomedical applications of exosomes","volume":"367","author":"Kalluri","year":"2020","journal-title":"Science"},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Chen, Y.-F., Luh, F., Ho, Y.-S., and Yen, Y. (2024). Exosomes: A review of biologic function, diagnostic and targeted therapy applications, and clinical trials. J. Biomed. Sci., 31.","DOI":"10.1186\/s12929-024-01055-0"},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Wang, Y., Xiao, T., Zhao, C., and Li, G. (2023). The regulation of exosome generation and function in physiological and pathological processes. Int. J. Mol. Sci., 25.","DOI":"10.3390\/ijms25010255"},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Zeng, H., Guo, S., Ren, X., Wu, Z., Liu, S., and Yao, X. (2023). Current strategies for exosome cargo loading and targeting delivery. Cells, 12.","DOI":"10.3390\/cells12101416"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"226","DOI":"10.1016\/j.cca.2019.10.022","article-title":"Exosomes: Current use and future applications","volume":"500","author":"Chung","year":"2020","journal-title":"Clin. Chim. Acta"},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Zhang, Y., Liu, Y., Liu, H., and Tang, W.H. (2019). Exosomes: Biogenesis, biologic function and clinical potential. Cell Biosci., 9.","DOI":"10.1186\/s13578-019-0282-2"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"363","DOI":"10.31557\/APJCP.2023.24.2.363","article-title":"Exosome mediated cancer therapeutic approach: Present status and future prospectives","volume":"24","author":"Roy","year":"2023","journal-title":"Asian Pac. J. Cancer Prev."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"877","DOI":"10.1038\/s12276-024-01209-y","article-title":"Regulation of cargo selection in exosome biogenesis and its biomedical applications in cancer","volume":"56","author":"Lee","year":"2024","journal-title":"Exp. Mol. Med."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"836","DOI":"10.1038\/s12276-024-01201-6","article-title":"Recent advances in extracellular vesicles for therapeutic cargo delivery","volume":"56","author":"Kim","year":"2024","journal-title":"Exp. Mol. Med."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"145","DOI":"10.1186\/s12964-022-00959-4","article-title":"A review on exosomes application in clinical trials: Perspective, questions, and challenges","volume":"20","author":"Rezaie","year":"2022","journal-title":"Cell Commun. Signal."},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Jung, J.H., Back, W., Yoon, J., Han, H., Kang, K.W., Choi, B., Jeong, H., Park, J., Shin, H., and Hur, W. (2021). Dual size-exclusion chromatography for efficient isolation of extracellular vesicles from bone marrow derived human plasma. Sci. Rep., 11.","DOI":"10.1038\/s41598-020-80514-8"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"147","DOI":"10.1001\/jama.2023.26074","article-title":"Age-related macular degeneration: A review","volume":"331","author":"Fleckenstein","year":"2024","journal-title":"JAMA"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"106","DOI":"10.1016\/j.ophtha.2012.07.038","article-title":"Vascular endothelial growth factor A in intraocular vascular disease","volume":"120","author":"Miller","year":"2013","journal-title":"Ophthalmology"},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Moraru, A.D., Danielescu, C., Iorga, R.E., Moraru, R.L., Zemba, M., and Branisteanu, D.C. (2024). Review of guideline recommendations for optimal anti-VEGF therapy in age-related macular degeneration. Life, 14.","DOI":"10.3390\/life14101220"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"3241","DOI":"10.2147\/DDDT.S383101","article-title":"Progress and challenges of anti-VEGF agents and their sustained-release strategies for retinal angiogenesis","volume":"16","author":"Xu","year":"2022","journal-title":"Drug Des. Devel. Ther."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"3713","DOI":"10.2147\/IJN.S162939","article-title":"Doxorubicin and anti-VEGF siRNA co-delivery via nano-graphene oxide for enhanced cancer therapy in vitro and in vivo","volume":"13","author":"Sun","year":"2018","journal-title":"Int. J. Nanomed."},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Ubanako, P., Mirza, S., Ruff, P., and Penny, C. (2024). Exosome-mediated delivery of siRNA molecules in cancer therapy: Triumphs and challenges. Front. Mol. Biosci., 11.","DOI":"10.3389\/fmolb.2024.1447953"},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Iqbal, Z., Rehman, K., Mahmood, A., Shabbir, M., Liang, Y., Duan, L., and Zeng, H. (2024). Exosome for mRNA delivery: Strategies and therapeutic applications. J. Nanobiotechnol., 22.","DOI":"10.1186\/s12951-024-02634-x"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"494","DOI":"10.1038\/35078107","article-title":"Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells","volume":"411","author":"Elbashir","year":"2001","journal-title":"Nature"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"6328","DOI":"10.1073\/pnas.0914843107","article-title":"Functional delivery of viral miRNAs via exosomes","volume":"107","author":"Pegtel","year":"2010","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"654","DOI":"10.1038\/ncb1596","article-title":"Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells","volume":"9","author":"Valadi","year":"2007","journal-title":"Nat. Cell Biol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"3003","DOI":"10.4049\/jimmunol.1302177","article-title":"Interaction of mesenchymal stem cells with fibroblast-like synoviocytes via cadherin-11 promotes angiogenesis by enhanced secretion of placental growth factor","volume":"192","author":"Park","year":"2014","journal-title":"J. Immunol."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"1040","DOI":"10.1172\/JCI20465","article-title":"VEGF-A stimulates lymphangiogenesis and hemangiogenesis in inflammatory neovascularization via macrophage recruitment","volume":"113","author":"Cursiefen","year":"2004","journal-title":"J. Clin. Investig."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"899","DOI":"10.3892\/etm.2010.113","article-title":"siRNA-mediated knockdown of VEGF-A, VEGF-C and VEGFR-3 suppresses the growth and metastasis of mouse bladder carcinoma in vivo","volume":"1","author":"Wang","year":"2010","journal-title":"Exp. Ther. Med."},{"key":"ref_42","doi-asserted-by":"crossref","unstructured":"Wiszniak, S., and Schwarz, Q. (2021). Exploring the intracrine functions of VEGF-A. Biomolecules, 11.","DOI":"10.3390\/biom11010128"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"691","DOI":"10.1016\/j.cell.2007.06.054","article-title":"Autocrine VEGF signaling is required for vascular homeostasis","volume":"130","author":"Lee","year":"2007","journal-title":"Cell"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"611","DOI":"10.1038\/nrm.2016.87","article-title":"Mechanisms and regulation of endothelial VEGF receptor signalling","volume":"17","author":"Simons","year":"2016","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"ref_45","doi-asserted-by":"crossref","unstructured":"Valiulyte, I., Preitakaite, V., Tamasauskas, A., and Kazlauskas, A. (2018). Importance of the putative furin recognition site 742RNRR745 for antiangiogenic Sema3C activity in vitro. Braz. J. Med. Biol. Res., 51.","DOI":"10.1590\/1414-431x20187786"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"2705","DOI":"10.1093\/nar\/gkg393","article-title":"Structural variations and stabilizing modifications of synthetic siRNAs in mammalian cells","volume":"31","author":"Czauderna","year":"2003","journal-title":"Nucleic Acids Res."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"1097","DOI":"10.1177\/1947601911423031","article-title":"Vascular endothelial growth factor (VEGF) and its receptor (VEGFR) signaling in angiogenesis: A crucial target for anti- and pro-angiogenic therapies","volume":"2","author":"Shibuya","year":"2011","journal-title":"Genes Cancer"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"3029","DOI":"10.1074\/jbc.M111.293985","article-title":"Endogenous vascular endothelial growth factor-A (VEGF-A) maintains endothelial cell homeostasis by regulating VEGF receptor-2 transcription","volume":"287","author":"E","year":"2012","journal-title":"J. Biol. Chem."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"2093","DOI":"10.1242\/dev.126.10.2093","article-title":"Role of vascular endothelial-cadherin in vascular morphogenesis","volume":"126","author":"Prandini","year":"1999","journal-title":"Development"},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"50","DOI":"10.1016\/j.jconrel.2024.08.002","article-title":"Biomimetic nanocomplex based corneal neovascularization theranostics","volume":"374","author":"Ye","year":"2024","journal-title":"J. Control. Release"},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"102360","DOI":"10.1016\/j.nano.2021.102360","article-title":"Biomimetic nano drug delivery carriers for treating cardiovascular diseases","volume":"33","author":"Zinger","year":"2021","journal-title":"Nanomedicine"}],"container-title":["Bioengineering"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2306-5354\/12\/9\/919\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,9]],"date-time":"2025-10-09T18:33:04Z","timestamp":1760034784000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2306-5354\/12\/9\/919"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,8,26]]},"references-count":51,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2025,9]]}},"alternative-id":["bioengineering12090919"],"URL":"https:\/\/doi.org\/10.3390\/bioengineering12090919","relation":{},"ISSN":["2306-5354"],"issn-type":[{"value":"2306-5354","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,8,26]]}}}