Efficient downregulation of VEGF in retinal pigment epithelial cells by integrin ligand-labeled liposome-mediated siRNA delivery
Cheng-Wei Chen,1 Ming-Kung Yeh,2 Chia-Yang Shiau,3 Chiao-Hsi Chiang,4,* Da-Wen Lu5,*1Chengwei Biotechnology Co, Ltd, 2Bureau of Pharmaceutical Affairs, Military of National Defense Medical Affairs Bureau, 3Graduate Institute of Medical Sciences, 4School of Pharmacy, National Defense Medical Center,...
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| Autores principales: | , , , , |
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| Formato: | article |
| Lenguaje: | EN |
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Dove Medical Press
2013
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| Materias: | |
| Acceso en línea: | https://doaj.org/article/25e10bc290494bdb8ae2a6f765abd499 |
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| Sumario: | Cheng-Wei Chen,1 Ming-Kung Yeh,2 Chia-Yang Shiau,3 Chiao-Hsi Chiang,4,* Da-Wen Lu5,*1Chengwei Biotechnology Co, Ltd, 2Bureau of Pharmaceutical Affairs, Military of National Defense Medical Affairs Bureau, 3Graduate Institute of Medical Sciences, 4School of Pharmacy, National Defense Medical Center, 5Department of Ophthalmology, Tri-Service General Hospital, Taipei, Taiwan *These authors contributed equally to this workBackground: The purpose of this study was to demonstrate the effectiveness of an integrin peptide ligand-labeled liposomal delivery system loaded with vascular endothelial growth factor (VEGF)-siRNA in a model study of gene therapy for retinopathy using human retinal pigment epithelial cells.Methods: Arg(R)-Gly(G)-Asp(D) motif peptide conjugating polyethylene glycol modified (RGD-PEGylated) liposomes were prepared using a thin-film hydration method and optimized for surface charge, particle size, small interfering RNA (siRNA) load, and entrapment efficiency. Reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assays were used to determine VEGF levels in retinal pigment epithelial cells. Cytotoxicity was determined using the 3-[4, 5-dimethylthiazol-2-yl]-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay and flow cytometry.Results: Physicochemical properties, including particle size, zeta potential, and siRNA load, of the prepared RGD-PEGylated liposomes and their entrapment efficiency were determined to be within the following ranges: 123.8–234.1 nm, 17.31–40.09 mV, 5.27%–6.33%, and >97%, respectively. RGD-PEGylated liposome-mediated fluorescent-labeled siRNA delivery demonstrated significantly enhanced cellular uptake, and 3 mol% RGD-PEGylated liposomes (having 3β-[N-(N´, N´-dimethylaminoethane) carbamoyl] cholesterol (DC-cholesterol) DSPE and DSPE-PEG(2000)-RGD with molar ratio of 50/47/3) were shown to have better efficacy with regard to specificity for retinal pigment epithelial cells, reduced cytotoxicity, and knockdown of the target molecule.Conclusion: By integrin receptor-mediated endocytosis, 3 mol% RGD-PEGylated liposomes were shown to be a suitable vector when loaded with VEGF-siRNA for efficient downregulation of VEGF in retinal pigment epithelial cells at both the protein and gene levels. This integrin ligand-modified liposomal delivery system has therapeutic potential for ocular gene therapy.Keywords: vascular endothelial growth factor, siRNA delivery, liposome, retinal pigment epithelial cells |
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