Controlled Gene Delivery by DNA–Gelatin Nanospheres

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Abstract
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 μg of a β-galactosidase plasmid was greater and more prolonged than was observed after injection of an equal amount of naked DNA or DNA complexed with Lipofectamine. Polymeric controlled delivery of genes is demonstrated by the use of gelatin–DNA nanospheres. The nanospheres, with a size range of 200–700 nm, were synthesized by sodium sulfate-induced complex coacervation between gelatin and plasmid DNA. The DNA was released at different rates depending on the degree of cross-linking of the gelatin matrix. The nanospheres were capable of multiple plasmid coencapsulation and provided partial protection of the embedded DNA against nuclease degradation. In vitro transfection of cultured cells was enhanced by conjugating human transferrin onto the nanospheres and coencapsulating the endolysolytic agent chloroquine. Injection of nanospheres in mouse footpad and tibialis muscle resulted in greater and more prolonged expression of the lacZ gene product than did an equivalent dose of naked DNA or Lipofectamine–DNA.