Structurally Flexible PAMAM Dendrimers As Novel Nanovectors For Nucleic Acid Delivery

DNA-based gene therapy and siRNA-based gene silencing technique show great promise as therapeutic approaches for treating and controlling inherited or acquired diseases. The lack of safe and effective vectors for nucleic acid delivery, however, represents the main obstacle to a successful gene therapy. To date, the most efficient vectors are viruses; however, viruses have fatal drawbacks relating to immunogenicity and toxicity. Therefore, developing safe and efficient non-viral vectors for nucleic acid delivery is urgently needed and of paramount importance for gene therapy. Among the non-viral vectors, cationic dendrimers, characterized by a unique nanoscale globular architecture, regular dendritic branching and radial symmetry, are a special family of polymeric vectors. Due to their unique architectures and properties, dendrimers are expected to be ideal non-viral vectors for nucleic acid delivery. Up to now, the most extensively studied dendrimers for nucleic acid delivery are poly(amidoamine) (PAMAM) dendrimers, with partially degraded and structurally fractured PAMAM dendrimers being two orders of magnitude more efficient as compared to non-degraded ones. This could reasonably be ascribed to the fact that partially degraded dendrimers are endowed with a more flexible and open structure compared to their intact counterparts, and thus the dendrimer interior has easier accessibility to be protonated, offering greater proton sponge effect for the endosome release of nucleic acids. My Ph.D work mainly focused on the development of structurally flexible and precisely controlled dendrimers as efficient nanovectors for nucleic acid delivery in gene therapy.We synthesized triethanolamine (TEA) core PAMAM dendrimers from generation 1 to 7 by using the conventional synthetic strategy, namely, iterative amidation and Michael addition. The synthesized dendrimers were characterized by IR, NMR, MS, HPLC and GPC. The obtained results demonstrated that our dendrimers had well-defined structure and narrow polydispersity. We also studied the structural confirmation of dendrimers and the DNA/dendrimers interactions by computer modeling, which further confirmed that our dendrimers had more flexible and open structures compared to the traditional NH3-core dendrimers.We further studied the dendrimers/nucleic acid complexes formation by gel retardation, TEM and DLS. Our dendrimers can form stable nanoparticles with DNA or siRNA, effectively protect them from enzymatic degradation. Further investigation on cell uptake used confocal microscopy and specific endocytic inhibitors and biomarkers demonstrated that our dendrimers can promote efficient cell uptake of nucleic acid mainly via macropinocytosis.We then assessed our dendrimers for DNA transfection in vitro and in vivo. We chose luciferase and enhanced green fluorescent protein (EGFP) models to test the DNA delivery ability of our dendrimers on epithelial HeLa cells and fibroblast LMTK- cells. High-generation dendrimers (G≥5) were shown to be efficient vectors for in vitro transfection on both HeLa cells and LMTK- cells. Most importantly, these dendrimers were also excellent vectors for gene delivery in vivo to the mouse thymus, a challenging organ for synthetic vectors. Altogether, these findings validate our rationally designed approach of structurally flexible dendrimers with a chemically defined structure for DNA transfection, and demonstrate the potential of these dendrimers as effective vectors for DNA delivery in gene therapy applications.We further tested our dendrimers to deliver siRNA for targeting heat-shock protein 27 (Hsp27) in prostate cancer PC-3 cells. Heat-shock protein 27 is an attractive therapeutic target in castrate-resistant prostate cancer. Our dendrimers could effectively deliver Hsp27 siRNA, induce potent and specific gene silencing of Hsp27, leading to caspase-dependent apoptosis-induced anticancer activity. In addition, the siRNA/dendrimer complexes are non-cytotoxic under the conditions for siRNA gene silencing. Altogether, the TEA-core PAMAM dendrimer mediated siRNA delivery combined with RNA interference targeting specifically Hsp27 may constitute a promising approach to combat castrate-resistant prostate cancer, for which there is no efficacious treatment.In conclusion, we have successfully designed and synthesized structurally flexible dendrimers. And these dendrimers had well-defined structure and narrow polydispersity. They can self-assemble with nucleic acid to form nanoscale complexes, which can effectively protect nucleic acid from enzymatic degradation and promote efficient cell uptake. Most importantly, these dendrimes can mediate efficient DNA delivery for gene transfection and siRNA delivery for gene silencing. So they are promising nanovectors for DNA and siRNA-based gene therapy.