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 Lu^5,*

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 work

Background: 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

A Letter to the Editor has been received and published for this article.