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.
Han
Jee Seung
JS
Department of Bioengineering, College of Engineering, Hanyang University, Seoul 133-791, South Korea.
Kim
Hyun Ah
HA
Lee
Sanghyun
S
Lee
Minhyung
M
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D015687:Cell Hypoxia; D002460:Cell Line; D002470:Cell Survival; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D018014:Gene Transfer Techniques; D024243:HMGB1 Protein / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D040262:Receptors, Vascular Endothelial Growth Factor / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D024243:HMGB1 Protein; D010455:Peptides; D040262:Receptors, Vascular Endothelial Growth Factor