Amphiphilic Biodegradable Cationic Polymers As Nuclein Carriers And Layer-by-Layer Assembled Gene Delivery Systems

Gene therapy holds great promise to treat various genetic diseases, such as cancers, viral infection and cardiovascular diseases. Developing safe and effective delivery carriers is a prerequisite for gene therapy in clinic. Viral vector systems have generally been used, owing to their superior ability to deliver and express genes to target cells. However, the shortcomings (such as safety, immunogenicity, low transgene size and high cost) limit the application of viruses for gene delivery. In order to overcome the drawbacks of viral carriers, non-viral carriers have been designed as alternative systems. The advantages associated with non-viral carriers include large scale manufacture, low immunogenic response, versatile modifications and the capacity to carry large inserts.Firstly, a kind of amphiphilic copolymer, Poly(ε-caprolactone)-graft- poly(2-(dimethylamino) ethyl methacrylate) (PCL-g-PDMAEMA) was synthesized by combination of ROP and ATRP polymerization in our lab. PCL-g-PDMAEMA can self-assemble into core-shell nanoparticles (NPs) with ultralow critical association concentration at about 8.1×10-4 g/L, which present pH dependent temperature-sensitivity. It was found that PCL-g-PDMAEMA nanoparticles were able to entrap hydrophobic paclitaxel and load DNA simultaneously. Hydrophobic drug paclitaxel loaded by PCL-g-PDMAEMA NPs could be released faster in acidic environment than that in neutral environment, and PCL-g-PDMAEMA NPs showed higher gene transfection efficiency in 293T,HepG2 and PC-3 cells than Lipofectamine 2000 in vitro. In addition, gene transfection efficiency was enhanced by the addition of 5% serum. Besides, confocal microscopic measurements indicated that PCL-g-PDMAEMA NPs/DNA complexes could escape from the endosome and release the payloads effectively in cytoplasm. These results suggest PCL-g-PDMAEMA has great potential for achieving the synergistic effect of drug and gene therapies in vivo.Secondly, to improve the biocompatibility and transfection efficiency of PCL-g-PDMAEMA NPs, its PEGylation derivation, mPEG-(PCL-g-PDMAEMA) (PECD), was synthesized. Agarose gel electrophoresis assay show that DNA was completely retarded at N/P ratio of 4 for PECD NPs. PEGlation can reduce the net positive charge to 10-18 mv, but the results of CCK-8 assay show that PECD NPs/DNA complexes were toxic to HeLa and HepG2 cells at higher N/P. Whereas, PECD NPs/DNA complexes present better gene transfection efficiency than PEI/DNA and Lipofectamine 2000/DNA in HeLa, HepG2 and dorsal root ganglion (DRG) cells even at low dose as N/P of 5, at which PECD/DNA NPs complexes show comparable toxicity with PEI and Lipofectamine. CLSM results show that PECD NPs/DNA complexes were more effective to escape from late endosomes/lysosomes. In addition, PECD NPs/DNA complexes were stable during the transfection assay and not aggregated into large particles as PCL-g-PDMAEMA/DNA complexes. Therefore, PEGylation of PCL-g-PDMAEMA can improve the DNA delivery properties.Thirdly, to develop a more effective and safe carrier for gene and drug delivery, ternary complexes constructed with amphiphilic cationic, DNA and anionic polymer polyglutamic-graft-poly(ethylene glycol) (PGA-g-mPEG) were prepared. The coating of PGA-g-mPEG onto binary complexes of polycaprolactone-graft-poly (N,N-dimethylaminoethyl methacrylate) (PCL-g-PDMAEMA)/DNA was able to decreases the zeta potential of nano-sized ternary complexes to near neural charge. As a result, the stability of complexes in endosomes and escape ability from endosomes were enhanced without weakening the internalization efficiency of ternary complexes into cells. In vitro cytotocxicity using CCK-8 and transfection experiment show that these ternary complexes present lower cytotoxicity and higher gene transfection efficiency tahn binary complexes. Besides, Lactate dehydrogenase (LDH) assays were performed to quantify the membrane-damaging effects of complexes, which is in line with the conclusion drawn from CCK-8 assay. Intravenous administration of ternary complexes coated by PGA-g-mPEG resulted in RFP expression in tumor, which was further enhanced by the coating of PGA-g-PEG-folate. Therefore, it is concluded that the ternary complexes delivery system is a promising gene delivery system for in vivo application to achieve the synergistic/combined effect of drug and gene therapy.Finally, we prepared charge-reversal functionalized complex systems, PEI/PAH-Cit/PEI/MUA-Au NPs for siRNA delivery by deposition of PEI, and PAH-Cit on MUA-Au NPs. The charge reversion of PAH-Cit in the siRNA/PEI/PAH-Cit/PEI/MUA-Au NPs system was confirmed by polyacrylamide gel electrophoresis (PAGE). siRNA/PEI/PAH-Cit/PEI/MUA-Au NPs presents good gene transfection efficiency in HeLa and 293T cells, which is better than that of PEI and Lipofectamine. At the ratio of Au/DNA 10, PEI/PAH-Cit/PEI/MUA-Au NPs present highest inhibition effect with 80.0% knockdown efficiency, which is higher than that of Lipofectamine 2000 about 66.0%. And negative controls PEI/Au NPs and PEI/PSS/PEI/Au NPs are less effective in knockdown of lamin A/C expression. Therefore, it is believed that the charge-reversal PAH-Cit may play an important role in releasing siRNA to improve siRNA silencing. Another evidence of charge reversion facilitating in releasing siRNA is confirmed by CLSM. It was found that more cy5-siRNA was distributed over the cytoplasm for cy5-siRNA/PEI/PAH-Cit/PEI/MUA-Au NPs complexes. However, the formed complexes for siRNA and Lipofectamine 2000 or PEI were likely to be tightly clustered in punctuate structures rather than distributed over the cytoplasm. In addition, cell viabilities by MTT assay indicated that gold nanoparticles were nontoxic to cells.