Gene transfer by DNA-gelatin nanospheres.
A DNA and gelatin nanoparticle coacervate containing chloroquine and calcium, and with the cell ligand transferrin covalently bound to the gelatin, has been developed as a gene delivery vehicle. In this study, the coacervation conditions which resulted in the formation of distinct nanoparticles are defined. Nanospheres formed within a narrow range of DNA concentrations and achieved incorporation of more than 98% of the DNA in the reaction. Crosslinking of gelatin to stabilize the particles does not effect the electrophoretic mobility of the DNA. DNA in the nanosphere is partially resistant to digestion with concentrations of DNase I that result in extensive degradation of free DNA but is completely degraded by high concentrations of DNase. Optimum cell transfection by nanosphere DNA required the presence of calcium and nanospheres containing transferrin. The biological integrity of the nanosphere DNA was demonstrated with a model system utilizing DNA encoding the cystic fibrosis transport regulator (CFTR). Transfection of cultured human tracheal epithelial cells (9HTEo) with nanospheres containing this plasmid resulted in CFTR expression in over 50% of the cells. Moreover, human bronchial epithelial cells (IB-3-1) defective in CFTR-mediated chloride transport were complemented with effective transport activity when transfected with nanospheres containing the CFTR transgene.
Truong-Le
V L
VL
Department of Pharmacology and Molecular Sciences, Johns Hopkins School of Medicine, Baltimore, Maryland, 21205, USA.
Walsh
S M
SM
Schweibert
E
E
Mao
H Q
HQ
Guggino
W B
WB
August
J T
JT
Leong
K W
KW
D002118:Calcium / Q000494:pharmacology; D002460:Cell Line; D002738:Chloroquine / Q000494:pharmacology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics* / Q000502:physiology; D004247:DNA / Q000235:genetics / Q000494:pharmacology; D015195:Drug Design; D005780:Gelatin / Q000235:genetics / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D018014:Gene Transfer Techniques; D006801:Humans; D008863:Microspheres; D014162:Transfection / Q000187:drug effects / Q000379:methods
C505032:CFTR protein, human; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D002738:Chloroquine; D005780:Gelatin; D004247:DNA; D002118:Calcium