Studies On Gene Delivery Mediated By Polyamidoamine Dendrimer

The development of efficient and safe gene delivery carriers that can transfer DNA into the nuclei of target cells is a key factor for success of gene therapy. Polyamidoamine (PAMAM) dendrimers are a class of nanoscopic polymers with highly branched spherical structure and a unique surface of primary amines as well as tertiary amines at branch points. PAMAM can transport DNA into a large variety of cell types and have emerged as a promising non-viral gene carrier for genetic medicines due to their safety and efficiency. In the present study, the interactions between plasmid DNA and PAMAM were characterized by physicochemical study and kinetic analysis. And nanoparticulate formuations of PAMAM were prepared with poly(lactide-co-glycolide) (PLGA) and poly(lactic acid) (PLA). These cationic nanoparticles were then evaluated as gene delivery carriers. In addition, cationic liposome/PAMAM/DNA ternary complexes were prepared and the application in gene transfection was examined. The main results are as follows:(1) Agarose gel electrophoresis, [Ru(phen)2dppz]2+ displacement and exclusion assays, particle size and zeta potential measurements were used to characterize the interactions of DNA with PAMAM. And DNA conformational changes were studied by circular dichroism (CD) and FTIR spectroscopy techniques. The results showed that PAMAM and DNA formed stable complexes at N/P ratios above 1.5. The mean particle size of the compact complexes was in the range of 150-180 nm. The zeta potentials were positive and increased with the increase of N/P ratios. Compared to naked DNA, CD spectra of complexed DNA was changed and had a decreased intensity at 270nm. High salts and excessively low or high pH values could also result in decreased CD signals of complexed DNA. FTIR spectroscopy indicated that complexed DNA remained in characteristic B form conformations but with base stacking interactions strongly disturbed.(2) We studied the kinetics of PAMAM binding to DNA and the reverse process of DNA dissociation from the complexes by fluorescence stopped-flow technique. The results revealed that DNA condensation was the rate determining step during the complexation process, while DNA unfolding and expansion was the rate determining step during the DNA dissociation process. At N/P ratios before reaching the thermodynamically most stable state, the complexes of DNA and PAMAM were incompact and could dissociate to some extent. And at N/P ratios above 2.0, DNA was fully condensed by PAMAM and dissociation was increasingly difficult.(3) PLGA nanoparticles containing PAMAM were prepared by emulsion-diffusion-evaporation technique and then complexed with DNA. Agarose gel electrophoresis, fluorescence displacement, particle size and zeta potential measurements were carried out to study the physicochemical properties of the complexes, nuclease digestion assay was used to investigate the protection effect of complexes to DNA, MTT assay was employed to assess the cytotoxicities of the complexes, and transfection experiment was determined to search for the transfection activities of the complexes. The results showed that DNA formed stable and compact complexes with cationic nanoparticles at N/P ratios above 2. The zeta potential of the complexes increased with the increase of charge ratios and became positive at N/P ratios above 2. Nuclease digestion assay showed that the complexes were efficient to provide protection against enzymatic cleavage. The cytotoxicities of the complexes in HepG2 and NIH-3T3 cells were comparatively slight. In serum-free medium, the complexes of DNA with cationic nanoparticles were efficiently delivered into the two kinds of cells and showed higher transfection efficiency than PAMAM/DNA complexes. Furthermore, the optimum N/P ratios for transfection (0.9 to 1.5) were below the fully condensed point, which preferred to dissociate before nuclear entry.(4) We prepared PLA nanoparticles containing PAMAM by emulsion-diffusion-evaporation technique. Physicochemical properties, cytotoxicity and transfection efficiency of the complexes prepared by DNA and the cationic nanoparticles were determined to search for the relationships between structural properties and transfection activities. DNA was shown to form stable and positively charged complexes with cationic nanoparticles at N/P ratios above 2 by agarose gel electrophoresis, fluorescence displacement, particle size and zeta potential measurements. Nuclease digestion assay showed that DNA was protected from enzymatic digestion by the complexes. MTT assay indicated that cytotoxicities of the complexes in HepG2 and NIH-3T3 cells were relatively slight. In the absence of serum, DNA was efficiently delivered into the two kinds of cells by cationic nanoparticles and the transfection efficiency was much higher than PAMAM. In addition, the optimum N/P ratios for transfection were between 2 to 5.(5) DNA was firstly mixed with PAMAM, and DOTAP/DOPE cationic liposome was then added to form ternary complexes. The physicochemical properties, cytotoxicities and transfection efficiencies of the ternary complexes were examined. It was found that the three components formed stable complexes at PAMAM/DNA ratios above 0.5 (the ratio of cationic liposome to DNA was fixed at 3.0). The ternary complexes could protect DNA from nuclease digestion, but resulted in rather low cell viability. In the presence of serum, the ternary complexes were delivered into the HepG2 cells more efficiently than binary liposome/DNA complexes, and the optimum charge ratios of PAMAM to DNA were from 1.2 to 2.0.In conclusion, PAMAM and DNA self-assembled through electrostatic interactions to form complexes that could dissociate to some extent. While at N/P ratios above 2.0, complex dissociation became increasingly irreversible. In addition, PLGA, PLA nanoparticles and cationic liposome had some enhancement effects to transfection efficiencies mediated by PAMAM.