Novel RGD-lipid conjugate-modified liposomes for enhancing siRNA delivery in human retinal pigment epithelial cells

Abstract

Background Human retinal pigment epithelial cells are promising target sites for small interfering RNA (siRNA) that might be used for the prevention and/or treatment of choroidal neovascularization by inhibiting the expression of angiogenic factor; for example, by downregulating expression of the vascular endothelial growth factor gene. Methods A novel functional lipid, DSPE-PEG-RGD, a Arg(R)-Gly(G)-Asp(D) motif peptide conjugated to 1, 2-distearoyl- sn -glycero-3-phosphoethanolamine- N-[maleimide (polyethylene glycol)-2000], was synthesized for the preparation of siRNA-loaded RGD-PEGylated liposomes to enhance uptake of encapsulated siRNA in retinal pigment epithelial cells. Various liposomes, with 1 mol% and 5 mol% PEGylated lipid or 1 mol% and 5 mol% RGD-PEGylated lipid, were fabricated. Results Characterization of the liposomes, including siRNA entrapment efficiency, average particle size and ζ-potential, were determined to be as follows: >96%, 129.7 ± 51 to 230.7 ± 60.7 nm, and 17.3 ± 0.6 to 32 ± 1.3 mV, respectively. For the in vitro retinal pigment epithelial cell studies, the RGD-PEGylated liposomes had high delivery efficiency with siRNA delivery, about a four-fold increase compared with the PEGylated liposomes. Comparison of the various liposomes showed that the 1 mol% RGD-modified liposome had less cytotoxicity and higher siRNA delivery efficiency than the other liposomes. The antibody blocking assay confirmed that uptake of the 1 mol% RGD-PEGylated liposome was via integrin receptor- mediated endocytosis in retinal pigment epithelial cells. Conclusion The results of this study suggest that RGD-PEGylated liposomes might be useful for siRNA delivery into retinal pigment epithelial cells by integrin receptor-medicated endocytosis.

Influence of charge on FITC-BSA-loaded chondroitin sulfate-chitosan nanoparticles upon cell uptake in human Caco-2 cell monolayers

“…Caco-2 cells were routinely grown in cultured Dulbecco's Modified Eagle Medium supplemented with 100 IU/mL penicillin, 100 mg/mL streptomycin, 1% nonessential amino acid, and 10% fetal bovine serum in 5% CO 2 at 37°C. The cells were seeded on flat-bottomed 96-well tissue culture plates (Greiner Labortechnik, Frickenhausen, Germany) for cell toxicity assays, on 6-well tissue culture plates (Nalge Nunc, Elkhart, IN) for flow cytometry (Becton Dickinson, FACScan flow cytometer, Heidelberg, Germany) and confocal microscopy (Carl Zeiss, Göttingen, Germany), 17,18 or on 6-Transwell tissue culture plates (Corning) for cell transmembrane assays.…”

“…17 The mean fluorescence channel and mean number of positive cells were derived using CellQuest software. To determine the levels of FITC-BSA, Caco-2 cells were analyzed with excitation at 488 nm and a 530 nm band-pass filter in the emission path.…”

Efficient downregulation of VEGF in retinal pigment epithelial cells by integrin ligand-labeled liposome-mediated siRNA delivery

“…These results are consistent with our previous report. 26 The particle size of the liposomes was measured at different time intervals, showing that the 3 mol% PEGylated liposomes and RGD-PEGylated liposomes maintained a stable particle size for 16 days, while the size of the non-PEGylated liposomes was significantly increased ( Figure 1B).…”

“…RGD oligopeptide-conjugated 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE)-PEG2000-RGD (molecular weight of PEG, 2000 Da) was synthesized using our previously reported method. 26 …”

“…After 4 hours, the cells were washed three times with phosphatebuffered solution and then detached using 0.05% trypsin and 0.02% EDTA, washed with phosphate-buffered solution, and resuspended in 0.5 mL of phosphate-buffered solution for flow cytometric assay, as described elsewhere. 26,31 Cell integrity was measured using a flow cytometer (Becton Dickinson, Heidelberg, Germany). Forward and side light scatter was used to gate the desired scattered events of the normal cells and dead cells.…”