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

ABSTRACT: 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.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.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.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.

SUBMITTER: Chen CW

PROVIDER: S-EPMC3225218 | biostudies-other | 2011

REPOSITORIES: biostudies-other

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Project description:AimTo demonstrate the feasibility of mesenchymal stem cell (MSC)-mediated nano drug delivery, which was characterized by the "Trojan horse"-like transport of hypoxia-inducible factor-1α small interfering RNA (HIF-1α siRNA) between MSCs and retinal pigment epithelial cells (RPE) under hypoxia environment.MethodsPlasmid and lentivirus targeting the human HIF-1α gene were designed and constructed. HIF-1α siRNA was encapsulated into poly(lactic-co-glycolic acid) nanoparticles (PLGA-NPs) through the water-in-oil-in-water (w/o/w) multiple emulsion technique. The effect of PLGA-NPs uptake on the expression of HIF-1α mRNA was tested in RPE cells by real-time quantitative polymerase chain reaction (qPCR) and additional transfected conditions were used as control, including lentivirus group, nude plasmid group and blank PLGA group. MSCs were transfected with the NPs and the transfection efficacy was evaluated by flow cytometry. Transwell co-culture system of transfected MSCs and RPE cells was constructed under hypoxia environment. The effects of MSC-loaded HIF-1α siRNA PLGA-NPs on proliferation, apoptosis, and migration of RPE cells were then evaluated. The effect of transfected MSCs on HIF-1α expression of RPE cells was analyzed by using qPCR at the time points 24h, 3d, and 7d.ResultsThe average diameter of PLGA-NPs loaded with HIF siRNA was 314.1 nm and the zeta potential was -0.36 mV. The transfection efficiency of PLGA-NPs was 67.3%±5.2% into MSCs by using flow cytometry. Compared with the lentivirus group, the PLGA-NPs loaded with HIF-1α siRNA can effectively reduce the expression of HIF-1α mRNA up to 7d in RPE (0.63±0.05 at 7d, P<0.001). In the Transwell co-culture system of transfected MSCs and RPE, the abilities of proliferation (2.34±0.17, 2.40±0.28, 2.47±0.24 at 48h, F=0.23, P=0.80), apoptosis (14.83%±2.43%, 12.94%±2.19%, 12.39%±3.21%; F=0.70, P=0.53) and migration (124.5±7.78, 119.5±5.32, 130±9.89, F=1.33, P=0.33) of the RPE cells had no differences between MSC-loaded HIF-1α siRNA PLGA-NPs and other groups. The inhibition of PLGA on the HIF-1α mRNA expression in RPE cells could continue until the 7th day, the level of HIF-1α mRNA was lower than that of other groups (F=171.98, P<0.001).ConclusionThe delivery of PLGA-NPs loaded with HIF-1α siRNA carried by MSCs is found to be beneficial temporally for HIF-1α mRNA inhibition in RPE cells under hypoxia environment. The MSC-based bio-mimetic delivery of HIF-1α siRNA nanoparticles is a potential method for therapy against choroidal neovascularization.

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

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