VEGF receptor binding peptide-linked high mobility box group-1 box A as a targeting gene carrier for hypoxic endothelial cells.

High mobility group box-1 (HMGB-1) is a nuclear protein that can bind to and condense plasmid DNA. In this study, we developed a recombinant VEGF receptor binding peptide (VRBP) linked to HMGB-1 box A (VRBP-HMGB1A) as a targeting gene carrier to hypoxic endothelial cells. Hypoxic endothelial cells in ischemic tissues of solid tumors are important targets for gene therapy. A recombinant VRBP-HMGB1A expression vector, pET21a-VRBP-HMGB1A was constructed. VRBP-HMGB1A was over-expressed in BL21 strain and purified by nickel-chelate affinity chromatography. Complex formation between VRBP-HMGB1A and pCMV-Luc was confirmed by gel retardation assay. pCMV-Luc was retarded completely at a 2/1 weight ratio (peptide/plasmid). For transfection assays, calf pulmonary artery endothelial (CPAE) cells were incubated under hypoxia for 24 h, prior to transfection to induce the VEGF receptors on the cells. VRBP-HMGB1A/pCMV-Luc complexes were transfected to hypoxic CPAE cells. The highest transfection efficiency was at a 30/1 weight ratio (peptide/plasmid). In addition, VRBP-HMGB1A had higher efficiency than poly-L-lysine (PLL) specifically in hypoxic CPAE cells, However, VRBP-HMGB1A had lower efficiency than PLL in 293, H9C2, and normoxic CPAE cells. In MTT assay, VRBP-HMGB1A was less toxic than PLL to cells. In conclusion, VRBP-HMGB1A is a potential gene carrier for targeting hypoxic endothelial cells and thus, may be useful for cancer gene therapy.