SYNTHESIS AND PHYSICO-CHEMICAL CHARACTERIZATION OF NANOCOMPLEXES CHITOSAN/DNA FOR GENE DELIVERY APPLICATIONS

Chitosan-DNA nanoparticles were prepared using a recently patented methodology (PCT n° RM2004A000555). The most important parameters for nanoparticle synthesis were investigated, including the N/P ratio of chitosan and DNA, pH of the buffer, and molecular weights of chitosan and DNA. At an amino group to phosphate group ratio (N/P ratio) of 5 and 10 and chitosan concentration of 1 mg/ml (molecular weight 50 kDa), particles size was optimized to 80–120 nm with a narrow distribution. The size of chitosan-DNA complexes depends on the molecular weight, that in our experiments was varied from 3.4 to 50 kDa. The charge of these particles was positive with a zeta potential of +11 mV(with N/P ratio equal to 10) at pH lower than 6.0, and almost zero close to pH 7.0. The transfection efficiency of chitosan-DNA nanoparticles was cell-type dependent. We analysed the transfection efficiency on different mammalian cell lines by using 80 nm chitosan nanoparticles. In particular in the cell line HEK293 we obtained 41% of plasmidic DNA transfection against a positive control of 31% with liposome ESCORT IV, achieving only 2% with 16HBE and 15% with H441, being such cell lines very difficult to transfect.