{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,2]],"date-time":"2026-10-02T09:09:35Z","timestamp":1790932175036,"version":"4.1.0"},"reference-count":51,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2019,4,18]],"date-time":"2019-04-18T00:00:00Z","timestamp":1555545600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100003329","name":"Ministerio de Econom\u00eda y Competitividad","doi-asserted-by":"publisher","award":["SAF2014\u201353092-R"],"award-info":[{"award-number":["SAF2014\u201353092-R"]}],"id":[{"id":"10.13039\/501100003329","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100003451","name":"UPV\/EHU","doi-asserted-by":"publisher","award":["PPG17\/65, GIU17\/032"],"award-info":[{"award-number":["PPG17\/65, GIU17\/032"]}],"id":[{"id":"10.13039\/501100003451","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100003451","name":"UPV\/EHU","doi-asserted-by":"publisher","award":["Fellowship to J Torrecilla and I G\u00f3mez-Aguado"],"award-info":[{"award-number":["Fellowship to J Torrecilla and I G\u00f3mez-Aguado"]}],"id":[{"id":"10.13039\/501100003451","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Nanomaterials"],"abstract":"<jats:p>Gene silencing targeting proangiogenic factors have been shown to be a useful strategy in the treatment of corneal neovascularization (CNV). Among interference RNA (RNAi) molecules, short-hairpin RNA (shRNA) is a plasmid-coded RNA able to down-regulate the expression of the desired gene. It is continuously produced in the host cell, inducing a durable gene silencing effect. The aim of this work was to develop a solid lipid nanoparticle (SLN)-based shRNA delivery system to downregulate metalloproteinase 9 (MMP-9), a proangiogenic factor, in corneal cells for the treatment of CNV associated with inflammation. The nanovectors were prepared using a solvent emulsification-evaporation technique, and after physicochemical evaluation, they were evaluated in different culture cell models. Transfection efficacy, cell internalization, cell viability, the effect on MMP-9 expression, and cell migration were evaluated in human corneal epithelial cells (HCE-2). The inhibition of tube formation using human umbilical vein endothelial cells (HUVEC) was also assayed. The non-viral vectors based on SLN were able to downregulate the MMP-9 expression in HCE-2 cells via gene silencing, and, consequently, to inhibit cell migration and tube formation. These results demonstrate the potential of lipid nanoparticles as gene delivery systems for the treatment of CNV-associated inflammation by RNAi technology.<\/jats:p>","DOI":"10.3390\/nano9040631","type":"journal-article","created":{"date-parts":[[2019,4,18]],"date-time":"2019-04-18T11:58:21Z","timestamp":1555588701000},"page":"631","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":25,"title":["MMP-9 Downregulation with Lipid Nanoparticles for Inhibiting Corneal Neovascularization by Gene Silencing"],"prefix":"10.3390","volume":"9","author":[{"given":"Josune","family":"Torrecilla","sequence":"first","affiliation":[{"name":"Pharmacokinetic, Nanotechnology &amp; Gene Therapy Group (PharmaNanoGene), Faculty of Pharmacy, Centro de investigaci\u00f3n Lascaray ikergunea, University of the Basque Country UPV\/EHU, Paseo de la Universidad 7, 01015 Vitoria-Gasteiz, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4189-5085","authenticated-orcid":false,"given":"Itziar","family":"G\u00f3mez-Aguado","sequence":"additional","affiliation":[{"name":"Pharmacokinetic, Nanotechnology &amp; Gene Therapy Group (PharmaNanoGene), Faculty of Pharmacy, Centro de investigaci\u00f3n Lascaray ikergunea, University of the Basque Country UPV\/EHU, Paseo de la Universidad 7, 01015 Vitoria-Gasteiz, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"M\u00f3nica","family":"Vicente-Pascual","sequence":"additional","affiliation":[{"name":"Pharmacokinetic, Nanotechnology &amp; Gene Therapy Group (PharmaNanoGene), Faculty of Pharmacy, Centro de investigaci\u00f3n Lascaray ikergunea, University of the Basque Country UPV\/EHU, Paseo de la Universidad 7, 01015 Vitoria-Gasteiz, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2478-0884","authenticated-orcid":false,"given":"Ana","family":"del Pozo-Rodr\u00edguez","sequence":"additional","affiliation":[{"name":"Pharmacokinetic, Nanotechnology &amp; Gene Therapy Group (PharmaNanoGene), Faculty of Pharmacy, Centro de investigaci\u00f3n Lascaray ikergunea, University of the Basque Country UPV\/EHU, Paseo de la Universidad 7, 01015 Vitoria-Gasteiz, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mar\u00eda \u00c1ngeles","family":"Solin\u00eds","sequence":"additional","affiliation":[{"name":"Pharmacokinetic, Nanotechnology &amp; Gene Therapy Group (PharmaNanoGene), Faculty of Pharmacy, Centro de investigaci\u00f3n Lascaray ikergunea, University of the Basque Country UPV\/EHU, Paseo de la Universidad 7, 01015 Vitoria-Gasteiz, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3195-6385","authenticated-orcid":false,"given":"Alicia","family":"Rodr\u00edguez-Gasc\u00f3n","sequence":"additional","affiliation":[{"name":"Pharmacokinetic, Nanotechnology &amp; Gene Therapy Group (PharmaNanoGene), Faculty of Pharmacy, Centro de investigaci\u00f3n Lascaray ikergunea, University of the Basque Country UPV\/EHU, Paseo de la Universidad 7, 01015 Vitoria-Gasteiz, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2019,4,18]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"130","DOI":"10.1016\/j.exer.2018.07.006","article-title":"Targeting corneal inflammation by gene therapy: Emerging strategies for keratitis","volume":"176","author":"Torrecilla","year":"2018","journal-title":"Exp. Eye Res."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"565","DOI":"10.1016\/j.survophthal.2017.11.001","article-title":"Dendritic cells in the cornea during Herpes simplex viral infection and inflammation","volume":"63","author":"Kwon","year":"2018","journal-title":"Surv. Ophthalmol."},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Lee, S.H., Kim, K.W., Joo, K., and Kim, J.C. (2016). Angiogenin ameliorates corneal opacity and neovascularization via regulating immune response in corneal fibroblasts. BMC Ophthalmol., 16.","DOI":"10.1186\/s12886-016-0235-z"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/S1350-9462(01)00015-5","article-title":"MMPs in the eye: Emerging roles for matrix metalloproteinases in ocular physiology","volume":"21","author":"Sivak","year":"2002","journal-title":"Prog. Retin. Eye Res."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"2065","DOI":"10.1074\/jbc.M107611200","article-title":"Matrix metalloproteinase gelatinase B (MMP-9) coordinates and effects epithelial regeneration","volume":"277","author":"Mohan","year":"2002","journal-title":"J. Biol. Chem."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"165","DOI":"10.1159\/000338819","article-title":"TNF-\u03b1 stimulates MMP-2 and MMP-9 activities in human corneal epithelial cells via the activation of FAK\/ERK signaling","volume":"48","author":"Yang","year":"2012","journal-title":"Ophthalmic Res."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"28","DOI":"10.1186\/s40662-017-0094-6","article-title":"Therapeutic approaches for corneal neovascularization","volume":"4","author":"Feizi","year":"2017","journal-title":"Eye Vis."},{"key":"ref_8","unstructured":"Lu, X.X., and Zhao, S.Z. (2018). Gene-based therapeutic tools in the treatment of Cornea Disease. Curr. Gene Ther."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"193","DOI":"10.1016\/j.survophthal.2017.10.006","article-title":"Gene-based antiangiogenic applications for corneal neovascularization","volume":"63","author":"Liu","year":"2018","journal-title":"Surv. Ophthalmol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"407","DOI":"10.1038\/sj.eye.6701895","article-title":"Corticosteroid-induced glaucoma: A review of the literature","volume":"20","author":"Kersey","year":"2006","journal-title":"Eye"},{"key":"ref_11","first-page":"113","article-title":"Update on immunosuppressive therapy for corneal transplantation","volume":"5","author":"Murphy","year":"2010","journal-title":"Int. Ophthalmol. Clin."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"730","DOI":"10.1111\/bph.12330","article-title":"Small-interfering RNAs (siRNAs) as a promising tool for ocular therapy","volume":"170","author":"Loma","year":"2013","journal-title":"Br. J. Pharmacol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"161794","DOI":"10.1155\/2014\/161794","article-title":"Lipid nanoparticles as carriers for RNAi against viral infections: Current status and future perspectives","volume":"2014","author":"Torrecilla","year":"2014","journal-title":"BioMed Res. Int."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1797","DOI":"10.1093\/nar\/gkr927","article-title":"Strand antagonism in RNAi: An explanation of differences in potency between intracellularly expressed siRNA and shRNA","volume":"40","author":"Jin","year":"2012","journal-title":"Nucleic Acids Res."},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Rodr\u00edguez-Gasc\u00f3n, A., del Pozo-Rodr\u00edguez, A., and Isla, A. (2016). Gene Therapy in the Cornea, John Wiley Sons, Ltd.. Available online: https:\/\/onlinelibrary.wiley.com\/doi\/abs\/10.1002\/9780470015902.a0024274.","DOI":"10.1002\/9780470015902.a0024274"},{"key":"ref_16","unstructured":"(2019, March 08). ClinicalTrials.gov, Available online: https:\/\/clinicaltrials.gov\/."},{"key":"ref_17","unstructured":"(2019, March 08). Gene Therapy Clinical Trials Worldwide. Available online: http:\/\/www.abedia.com\/wiley\/."},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Mohan, R.R., Sinha, S., Tandon, A., Gupta, R., Tovey, J.C., and Sharma, A. (2011). Efficacious and safe tissue-selective controlled gene therapy approaches for the cornea. PLoS ONE, 6.","DOI":"10.1371\/journal.pone.0018771"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"23","DOI":"10.1089\/jop.2018.0024","article-title":"Linear Polyethylenimine-DNA Nanoconstruct for Corneal Gene Delivery","volume":"35","author":"Rodier","year":"2019","journal-title":"J. Ocul. Pharmacol. Ther."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1847","DOI":"10.2217\/nnm-2018-0112","article-title":"Gene delivery in the cornea: In vitro & ex vivo evaluation of solid lipid nanoparticle-based vectors","volume":"13","author":"Albano","year":"2018","journal-title":"Nanomedicine"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"345","DOI":"10.1089\/hum.2011.115","article-title":"Dextran and Protamine-Based Solid Lipid Nanoparticles as Potential Vectors for the Treatment of X-Linked Juvenile Retinoschisis","volume":"23","author":"Delgado","year":"2012","journal-title":"Hum. Gene Ther."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"40","DOI":"10.1016\/j.biomaterials.2016.03.004","article-title":"Structural recovery of the retina in a retinoschisin-deficient mouse after gene replacement therapy by solid lipid nanoparticles","volume":"90","author":"Apaolaza","year":"2016","journal-title":"Biomaterials"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"72","DOI":"10.1039\/C8NR05520C","article-title":"Stimuli-responsive lipid-based magnetic nanovectors increase apoptosis in glioblastoma cells through synergic intracellular hyperthermia and chemotherapy","volume":"11","author":"Tapeinos","year":"2019","journal-title":"Nanoscale"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"349","DOI":"10.4103\/0250-474X.57282","article-title":"Solid lipid nanoparticles: A modern formulation approach in drug delivery system","volume":"71","author":"Mukherjee","year":"2009","journal-title":"Indian J. Pharm. Sci."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"184","DOI":"10.1016\/j.ejpb.2016.10.016","article-title":"Applications of lipid nanoparticles in gene therapy","volume":"109","year":"2016","journal-title":"Eur. J. Pharm. Biopharm."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1743","DOI":"10.1080\/17425247.2016.1201059","article-title":"Application of lipid nanoparticles to ocular drug delivery","volume":"13","author":"Battaglia","year":"2016","journal-title":"Expert Opin. Drug Deliv."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"808","DOI":"10.1016\/j.colsurfb.2016.07.026","article-title":"Silencing of hepatitis C virus replication by a non-viral vector based on solid lipid nanoparticles containing a shRNA targeted to the internal ribosome entry site (IRES)","volume":"146","author":"Torrecilla","year":"2016","journal-title":"Colloid Surf. B Biointerfaces"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"181","DOI":"10.1016\/j.ijpharm.2014.12.047","article-title":"Solid lipid nanoparticles as non-viral vector for the treatment of chronic hepatitis C by RNA interference","volume":"479","author":"Torrecilla","year":"2015","journal-title":"Int. J. Pharm."},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Ke, Y., Wu, Y., Cui, X., Liu, X., Yu, M., Yang, C., and Li, X. (2015). Polysaccharide hydrogel combined with mesenchymal stem cells promotes the healing of corneal alkali burn in rats. PLoS ONE, 10.","DOI":"10.1371\/journal.pone.0119725"},{"key":"ref_30","unstructured":"Carpentier, G. (2019, March 25). Angiogenesis Analyzer for Image J, Available online: http:\/\/rsb.info.nih.gov\/ij\/macros\/toolsets\/Analogenesis%20Analyzer.txt."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"72","DOI":"10.1089\/oli.2005.15.72","article-title":"Blocking mouse MMP-9 production in tumor cells and mouse cornea by short hairpin (sh) RNA encoding plasmids","volume":"15","author":"Azkur","year":"2005","journal-title":"Oligonucleotides"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"500","DOI":"10.1166\/jbn.2015.1968","article-title":"Solid lipid nanoparticles as non-viral vectors for gene transfection in a cell model of Fabry disease","volume":"11","author":"Apaolaza","year":"2015","journal-title":"J. Biomed. Nanotechnol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"413","DOI":"10.1016\/j.ijpharm.2014.02.038","article-title":"A novel gene therapy vector based on hyaluronic acid and solid lipid nanoparticles for ocular diseases","volume":"465","author":"Apaolaza","year":"2014","journal-title":"Int. J. Pharm."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"495","DOI":"10.1016\/j.ejpb.2011.06.005","article-title":"Understanding the mechanism of protamine in solid lipid nanoparticle-based lipofection: The importance of the entry pathway","volume":"79","author":"Delgado","year":"2011","journal-title":"Eur. J. Pharm. Biopharm."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"35","DOI":"10.1016\/j.ijpharm.2011.12.052","article-title":"Dextran-protamine-solid lipid nanoparticles as a non-viral vector for gene therapy: In vitro characterization and in vivo transfection after intravenous administration to mice","volume":"425","author":"Delgado","year":"2012","journal-title":"Int. J. Pharm."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"273","DOI":"10.1016\/j.jconrel.2015.09.033","article-title":"Solid lipid nanoparticle-based vectors intended for the treatment of X-linked juvenile retinoschisis by gene therapy: In vivo approaches in Rs1h-deficient mouse model","volume":"217","author":"Apaolaza","year":"2015","journal-title":"J. Control. Release"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"261","DOI":"10.1016\/j.ijpharm.2007.03.015","article-title":"Solid lipid nanoparticles: Formulation factors affecting cell transfection capacity","volume":"339","author":"Delgado","year":"2007","journal-title":"Int. J. Pharm."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"551","DOI":"10.1016\/j.preteyeres.2012.05.003","article-title":"Ocular surface inflammation is regulated by innate immunity","volume":"31","author":"Ueta","year":"2012","journal-title":"Prog. Retin. Eye Res."},{"key":"ref_39","first-page":"935","article-title":"Molecular mechanism of the disruption of barrier function in cultured human corneal epithelial cells induced by tumor necrosis factor-alpha, a proinflammatory cytokine. Nihon","volume":"114","author":"Kimura","year":"2010","journal-title":"Ganka Gakkai Zasshi"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"2752","DOI":"10.1167\/iovs.12-11294","article-title":"Protection of human corneal epithelial cells from TNF-\u03b1- induced disruption of barrier function by rebamipide","volume":"54","author":"Kimura","year":"2013","journal-title":"Investig. Ophthalmol. Vis. Sci."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"183","DOI":"10.1002\/cbf.2991","article-title":"Tryptase compromises corneal epithelial barrier function","volume":"32","author":"Li","year":"2014","journal-title":"Cell Biochem. Funct."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"452685","DOI":"10.1155\/2014\/452685","article-title":"Inhibition of corneal neovascularization by topical and subconjunctival tigecycline","volume":"2014","author":"Goktas","year":"2014","journal-title":"J. Ophthalmol."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"402","DOI":"10.1002\/jcp.21869","article-title":"Cytokines and signaling pathways regulating matrix metalloproteinase-9 (MMP-9) expression in corneal epithelial cellsy","volume":"221","author":"Gordon","year":"2009","journal-title":"J. Cell. Physiol."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"43","DOI":"10.1016\/j.preteyeres.2011.09.001","article-title":"Gene Therapy in the Cornea: 2005-present","volume":"31","author":"Mohan","year":"2012","journal-title":"Prog. Retin. Eye Res."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"235","DOI":"10.3109\/08820538.2011.588652","article-title":"Inflammatory corneal neovascularization: Etiopathogenesis","volume":"26","author":"Clements","year":"2011","journal-title":"Semin. Ophthalmol."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1139\/y04-120","article-title":"Endothelial cell regulation of matrix metalloproteinases","volume":"83","author":"Haas","year":"2005","journal-title":"Can. J. Physiol. Pharmacol."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"1138","DOI":"10.1038\/eye.2012.135","article-title":"Effects of N-acetylcysteine on matrix metalloproteinase-9 secretion and cell migration of human corneal epithelial cells","volume":"26","author":"Ramaesh","year":"2012","journal-title":"Eye"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"6056","DOI":"10.1167\/iovs.17-22852","article-title":"Topical application of mizoribine suppresses CD4+ T-cell-mediated pathogenesis in murine dry eye","volume":"58","author":"Zhang","year":"2017","journal-title":"Investig. Ophthalmol. Vis. Sci."},{"key":"ref_49","doi-asserted-by":"crossref","unstructured":"Webb, A.H., Gao, B.T., Goldsmith, Z.K., Irvine, A.S., Saleh, N., Lee, R.P., Lendermon, J.B., Bheemreddy, R., Zhang, Q., and Brennan, R.C. (2017). Inhibition of MMP-2 and MMP-9 decreases cellular migration, and angiogenesis in in vitro models of retinoblastoma. BMC Cancer, 17.","DOI":"10.1186\/s12885-017-3418-y"},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"1105","DOI":"10.1172\/JCI0215755","article-title":"Role of matrix metalloproteinase-9 in angiogenesis caused by ocular infection with herpes simplex virus","volume":"110","author":"Lee","year":"2002","journal-title":"J. Clin. Investig."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"118","DOI":"10.1016\/j.exer.2017.08.007","article-title":"Inhibition of HUVEC tube formation via suppression of NF\u03baB suggests an anti-angiogenic role for SLURP1 in the transparent cornea","volume":"164","author":"Swamynathan","year":"2017","journal-title":"Exp. Eye Res."}],"container-title":["Nanomaterials"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2079-4991\/9\/4\/631\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T12:46:28Z","timestamp":1760186788000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2079-4991\/9\/4\/631"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2019,4,18]]},"references-count":51,"journal-issue":{"issue":"4","published-online":{"date-parts":[[2019,4]]}},"alternative-id":["nano9040631"],"URL":"https:\/\/doi.org\/10.3390\/nano9040631","relation":{},"ISSN":["2079-4991"],"issn-type":[{"value":"2079-4991","type":"electronic"}],"subject":[],"published":{"date-parts":[[2019,4,18]]}}}