Preparation and Characterization of RGDS/Nanodiamond as a Vector for VEGF-siRNA Delivery.

Small interfering RNA (siRNA) has great potential for treating or preventing diseases. Safe and efficient vectors are extremely needed for specific delivery of siRNA. Here VEGF-siRNA/RGDS/nanodiamond was prepared by conjugating Arg-Gly-Asp-Ser (RGDS) and VEGF-siRNA to nanodiamond delivery particles. VEGF-siRNA could be clinically used in anti-angiogenic gene therapy to inhibit tumor growth via the down-regulation of the expression of vascular endothelial growth factor (VEGF). The differential scanning calorimeter (DSC) analysis evidenced that RGDS and/or VEGF-siRNA were effectively linked to nanodiamond. The release assays indicated that in the presence of RGDS the release time of VEGF-siRNA was prolonged by 6 folds. This enabled VEGF-siRNA/RGDS/nanodiamond to release and transfer VEGF-siRNA in a long-acting manner, and thereby to significantly decrease the expression of VEGF mRNA and protein. Real-Time PCR analysis revealed that the expression of VEGF mRNA was decreased 87.56 ± 1.6% by VEGF-siRNA/RGDS/nanodiamond. Enzyme-Linked Immunosorbent Assay (ELISA) indicated that the expression of VEGF protein was down-regulated to 39.8 ± 1.8%. The down-regulation of VEGF protein expression was also observed in Western blotting experiments. In human umbilical vein endothelial cells (HUVEC) test, VEGF-siRNA/RGDS/nanodiamond decreased the formation of the tubes and exhibited no testable cytotoxicty. All the results consistently suggested that RGDS/nanodiamond is an ideal non-viral tumor-targeting vector for siRNA transfer, and VEGF-siRNA/RGDS/nanodiamond may be a promising regimen of gene therapy in carcinoma.