Activated and non-activated PAMAM dendrimers for gene delivery in vitro and in vivo.
Nanotechnology, though not a new concept, has gained importance in medical breakthroughs. The preparation of nanosystems like polymeric nanoparticles can be used for drug and gene delivery. In this study dendrimeric nanoparticles prepared with generations 4 and 5 (G4, G5) polyamidoamine (PAMAM) dendrimers and plasmid DNA were characterized and their ability to transfect cells in vitro and in vivo evaluated. Additionally, the efficacy of these dendrimers on activation after heat treatment has been tested to attempt an enhancement in transfection activity over that of intact dendrimers. Measurements of the particle size and zeta potential as a function of the charge ratio and the generation of the polymer reveal that no significant differences were obtained in size by using G4 or G5 polymers in nonactivated dendriplexes prepared at different charge ratios. The zeta potentials of the dendriplexes are strongly positive and differ only slightly. Atomic force microscopy images of complexes showed that they are spherical, individualized, and homogeneously distributed. These vectors were also highly effective in protecting DNA from attack by DNase I and increased the efficiency of plasmid-mediated gene transfer in vitro to liver (HepG2) and colon (CT26) cancer cells as compared with naked DNA, even in the presence of 60% fetal bovine serum. Expression is enhanced at higher charge ratios with maximal values obtained at a charge ratio of 10:1 (+/-) and by increasing the dendrimer generation. Finally, the transfection activity of G4 and G5 dendriplexes was significantly enhanced in HepG2 and CT26 cells by activation of the dendrimers. In this respect we have optimized the time of activation to obtain the optimal levels of gene expression. Also, intravenously administered activated G4 and G5 dendrimer-DNA complexes are superior to the nonactivated ones in terms of gene transfer efficiency in vivo. In conclusion, our results showed that G4 and G5 PAMAM dendrimers are an effective nanosystem for gene delivery to colon and liver cancer cells in vitro, as well as for in vivo therapeutic applications.


BACKGROUND
This paper describes the synthesis and potential applications of mixed nanoparticles prepared with generations 4 and 5 (G4, G5) poly(amidoamine) (PAMAM) dendrimers and plasmid DNA. These mixed nanoparticles proved to be effective for gene delivery to colon and liver cancer cells in vitro, as well as in vivo.
Navarro
Gemma
G
Department of Pharmacy and Pharmaceutical Technology, University of Navarra, Pamplona, Spain.
Tros de Ilarduya
Conchita
C
D000818:Animals; D016923:Cell Death / Q000187:drug effects; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004247:DNA / Q000737:chemistry; D050091:Dendrimers; D003850:Deoxyribonuclease I / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D005260:Female; D015870:Gene Expression / Q000187:drug effects; D018014:Gene Transfer Techniques; D006358:Hot Temperature; D006801:Humans; D007275:Injections, Intravenous; D051379:Mice; D008807:Mice, Inbred BALB C; D018625:Microscopy, Atomic Force; D010316:Particle Size; D011073:Polyamines / Q000008:administration & dosage* / Q000737:chemistry / Q000494:pharmacology; D013499:Surface Properties / Q000187:drug effects; D013997:Time Factors; D014162:Transfection
D050091:Dendrimers; C104700:PAMAM Starburst; D011073:Polyamines; D004247:DNA; D003850:Deoxyribonuclease I