A novel synthetic peptide vector system for optimal gene delivery to bone marrow stromal cells.
A 23-amino acid, bifunctional, integrin-targeted synthetic peptide was evaluated for ex vivo gene delivery to rabbit bone marrow stromal cells (BMSCs). The peptide (K)(16)GRGDSPC consists of an amino terminal domain of 16 lysines for electrostatic binding of DNA, and a 7-amino acid integrin-binding domain at the carboxyl terminal. PcDNA3-EGFP plasmids were transfected into BMSCs by (K)(16)GRGDSPC and the positive cells gave out a bright green fluorescence. High levels of gene delivery of pcDNA3-TGF-beta1 plasmids were obtained with 2 to 4 microg/ml DNA concentration, with (K)(16)GRGDSPC at an optimal peptide: DNA w/w ratio of 3:1, with a required exposure time of more than 4 h but shorter than 24 h for BMSC exposure to the peptide/DNA complexes with completely absent serum in the initial stage; with 100 microM chloroquine and at least 8 h exposure for BMSC exposure to chloroquine; with a fusogenic peptide at an optimal (K)(16)GRGDSPC/DNA/fusogenic peptide w/w ratio of 3:1:5; and with Lipofectamine 2000 at an optimal (K)(16)GRGDSPC/DNA/Lipofectamine 2000 w/w ratio of 3:1:2 at a constant DNA concentration of 2 microg/ml. Chloroquine, the fusogenic peptide and Lipofectamine 2000 all significantly promoted gene delivery, but chloroquine was more effective than the fusogenic peptide and had obvious synergistic effects with Lipofectamine 2000. Under optimal conditions, TGF-beta1 gene was transfected into BMSCs without observable toxicity, and the stable expression was examined by RT-PCR and Western blot analysis. The stable transgenic cells showed obvious bands. This novel synthetic peptide, providing a new way for the use of polylysine and RGD motif in DNA vector system, is potentially well suited to ex vivo gene delivery to BMSCs for experimental and clinical applications in the field of bone tissue engineering.
Haitao
Pan
P
Department of Orthopedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China.
Qixin
Zheng
Z
Xiaodong
Guo
G
D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology* / Q000378:metabolism; D002738:Chloroquine / Q000494:pharmacology; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000187:drug effects; D049452:Green Fluorescent Proteins / Q000235:genetics; D016023:Integrins; D008055:Lipids / Q000494:pharmacology; D010455:Peptides / Q000235:genetics*; D011817:Rabbits; D017154:Stromal Cells / Q000166:cytology* / Q000378:metabolism; D053773:Transforming Growth Factor beta1 / Q000235:genetics
D016023:Integrins; D008055:Lipids; C086724:Lipofectamine; D010455:Peptides; D053773:Transforming Growth Factor beta1; C485184:enhanced green fluorescent protein; C519205:hexadecalysyl-glycyl-arginyl-glycyl-aspartyl-serinyl-prolyl-cysteine; D049452:Green Fluorescent Proteins; D002738:Chloroquine