Controlled gene delivery by DNA-gelatin nanospheres.

A novel system for gene delivery, based on the use of DNA-gelatin nanoparticles (nanospheres) formed by salt-induced complex coacervation of gelatin and plasmid DNA, has been developed. These particles were spherical, with a size range of 200-700 nm, contained 25-30% (w/w) DNA, and were stabilized by cross-linking of gelatin. As a consequence of being controlled by the cross-linking density of the gelatin matrix, the average release rate of DNA from nanospheres synthesized under standard conditions was 2.2%/day in serum. Nanosphere DNA incubated in bovine serum was more resistant to nuclease digestion than was naked DNA. Various bioactive agents could be encapsulated in the nanospheres by ionic interaction with the matrix components, physical entrapment, or covalent conjugation. Transfection of cultured cells with a luciferase plasmid was enhanced by conjugating human transferrin onto the nanosphere and coencapsulating the endolysolytic agent chloroquine. Under our experimental conditions, gene expression in mice subsequent to intramuscular injection of nanospheres containing 1 microg of a beta-galactosidase plasmid was greater and more prolonged than was observed after injection of an equal amount of naked DNA or DNA complexed with Lipofectamine.

DNA-polycation nanospheres as non-viral gene delivery vehicles.

Nanospheres synthesized by salt-induced complex coacervation of cDNA and polycations such as gelatin and chitosan were evaluated as gene delivery vehicles. DNA-nanospheres in the size range of 200-750 nm could transfect a variety of cell lines. Although the transfection efficiency of the nanospheres was typically lower than that of lipofectamine and calcium phosphate controls in cell culture, the beta-gal expression in muscle of BALB/c mice was higher and more sustained than that achieved by naked DNA and lipofectamine complexes. This gene delivery system has several attractive features: (1) ligands can be conjugated to the nanosphere for targeting or stimulating receptor-mediated endocytosis; (2) lysosomolytic agents can be incorporated to reduce degradation of the DNA in the endosomal and lysosomal compartments; (3) other bioactive agents or multiple plasmids can be co-encapsulated; (4) bioavailability of the DNA can be improved because of protection from serum nuclease degradation by the polymeric matrix; (5) the nanosphere can be lyophilized for storage without loss of bioactivity.

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.