Synthesis, Characterization And Biological Evaluation Of New Polyamidoamine Dendrimers

Dendrimers are perfectly structured molecules with large numbers of cascade-branched units emerging from a central core, resulting in dense end groups at the molecular surface. The processes for the synthesis of dendrimers and the characteristics of dendrimers, both of which differ conspicuously from those of traditional polymers, greatly affect their physical and chemical properties. Dendrimers have therefore attracted considerable attention since the seminal studies first published on this topic. At the beginning of my present Ph.D thesis, the literature on dendrimers is reviewed, including the history of research on this topic, the methods used to synthesize dendrimers, and their molecular structure and characteristics. The special applications of dendrimers in host-guest recognition processes, as synthetic vectors for drug delivery and targeting purposes, and as active antibacterial and antitumor drugs are also described.Polyamine dendrimers were the first dendrimers to be developed, and many applications of these polymers are now being widely used. One of the most active fields of research on these polymers has been that of gene transfer. The PAMAM dendrimers have primary amine end groups which participate in DNA binding processes. Partially degraded dendrimers were reported to have more flexible structures and therefore to interact more efficiently with DNA than the intact ones. Such features of these dendrimers could be used to bind to biologically important RNA molecules, although very few efforts have been made so far on these lines. In order to develop flexible dendrimers for DNA and RNA binding applications, two series of PAMAM dendrimers were designed and synthesized, and their interactions with DNA/RNA molecules were assessed. These dendrimers have triethanolamine and triethyleneglycol cores, respectively, with various end groups, such as ester, primary amine, tertiary amine, quaternary ammonium and benzyl quaternary ammonium. The cores of both triethanolamine and triethyleneglycol are expected to give less densely packed branching units and end groups, while the various end groups could be used to assess the role and the impact of the interactions such as electrostatic interactions, H-bonding and â–¡-cation interactions between these dendrimers and DNA/RNA.I have synthesized these dendrimers from generations 1 to 8 using a conventional two-step iterative procedure: a) double alkylation of NH2 end groups with methyl acrylate (to generate the ester terminating dendrimers) followed by b) amidation of the terminal ester with the corresponding diamine. Microwave assisted reaction procedures were tested as a means of synthesizing PAMAM dendrimers with a triethanolamine core. Microwave irradiation was found to considerably improve both the alkylation and the amidatin reactions and to greatly reduce the reaction time. Furthermore, the dendrimers obtained in this way can be simply and conveniently purified.The gene transfer efficiency of the synthesized dendrimers of generations 1 to 8 were studied at Dr. Jean-Paul Behr's laboratory in France. The results will not be presented here. But we were pleased to learn that our dendrimers of generations higher than 5 were found to have a good level of transfection efficiency, and that the results obtained with some of our dendrimers were comparable to those obtained with the commercial PEI tranfection agent tested. Detailed functional studies are currently under way.In my Ph.D research project, a Candida ribozyme, Ca.LSU, was used as a model RNA to study the interactions between dendrimers and RNA molecules, because valuable information about ligand binding can be obtained by analyzing the inhibitory effects of ligands on the ribozyme activity. Ca.LSU is a self-splicing group I intron from the 26S rRNA of the opportunistic fungal pathogen Candida albicans. The inhibitory effects of our dendrimers on Ca.LSU ribozymes were studied in collaboration with Prof. Zhang Yi in the Life Science College at Wuhan University. The amine terminating dendrimers bind strongly to the ribozyme and efficiently inhibit the activity of the ribozyme, while the ester terminating dendrimers exert no inhibition on the ribozyme. It is the positively charged amine groups at the dendrimer surface that are responsible for the strong electrostatic interactions with the negatively charged phosphate groups of RNA. In addition, no significant differences were observed between the effects of the amine, tertiary amine, and quaternary amine terminating dendrimers on the activity of the RNA ribozymes, which suggests that the role of H-bonding is largely overshadowed by the strong electrostatic interactions occurring between the dendrimers and the ribozymes. Furthermore, these interactionswere found to increase with the dendrimer generation, as shown by the effects observed on both the gel mobility and the catalytic activity of the ribozymes. These data suggest that cooperative amplification processes may occur, depending on the size and the three-dimensional structure of the dendrimers.It has been reported that detergents with quaternary amine functions can disrupt cell membranes and thus have antimicrobial effects. We therefore studied the antimicrobial activities of our dendrimers on Escherichia coli and Bacillus subtilis, which are gram negative and gram positive bacteria, respectively. The antimicrobial activity of these dendrimers was found to depend on the functionalities of the dendrimer end group and on the specific types of microbe involved. Dendrimers with primary amine end groups inhibit the growth of E. coli and B. subtilis, while dendrimers with quaternary ammonium end groups clearly showed antimicrobial activities on B. subtilis. Dendrimers with benzyl quaternary ammonium end groups therefore have excellent antibacterial effects on both gram negative and gram positive bacteria. The antimicrobial activities of these dendrimers might be attributable to interactions between the positively charged amine end groups of the dendrimers and the negatively charged phosphate groups of the phospholipids present in the cell membranes.hi conclusion, two series of PAMAM dendrimers with triethanolamine and triethyleneglycol cores and various functional end groups at the surface were designed, synthesized and characterized. These dendrimers are good candidates for DNA delivery and RNA binding applications, as well as being active antimicrobial agents. Structure-controlled, size-tailored dendrimers should provide useful means of regulating many biological processes involving DNA and RNA, such as protein synthesis, mRNA splicing, RNA interference and RNA delivery processes. Further studies using these dendrimers for both DNA/RNA delivery and targeting purposes are currently under way.