| The application of gene therapy in clinical trails relies on the development of efficient and safe non-viral gene delivery system. Chemical modification is the common method to optimize the gene delivery at present. For example, based on the huge difference between the intracellular and extracellular environment, scientists designed stimuli-responsive gene delivery system to realize gene vectors with extracellular stability, intracellular responsive disassembly and efficient transfection. Spherical self-assembly aggregates can usually be achieved through this kind of method. However, recent studies have found that the topological structure plays a critical role in the gene delivery process such as cellular internalization and biodistribution as well as efficient transfection. The main work consisted of the following two parts:First, the pH-sensitive PEGylated gene delivery system was obtained based on the lower pH of endosome, which relized the excellent extracellular stability and easy intracellular gene release capability. The pH-sensitive polycation PEI-N=CH-mPEG had been synthesized via benzoic imine linker according to the acidic environment of endosome to study the influence of PEG-detachment. The corresponding stable PEI-NH-CH2-mPEG without pH-sensitivity was synthesized as control. The results showed that both PEGylated polycations could condense DNA into tight spherical nanoparticles about 80 nm in size and 10 mV in zeta-potential, which showed excellent stability under physiological conditions. The benzoic imine linkers were easily cleaved under the pH of 5, which was similar with the endosomal pH. Human hepatoblastoma cell line (HepG2) cell culture results indicated that the cytotoxicity and cellular uptake efficiency of both PEGylated gene nanoparticles decreased remarkably because of the shielding effect of PEG shell. Due to the cleavage of benzoic imine linker at acidic environment of endosome, the PEG shell of PEI-N=CH-mPEG/DNA polyplexes was detachable and led to quick endosomal escape. So PEI-N=CH-mPEG/DNA polyplexes showed higher gene expression than the PEI-NH-CH2-mPEG/DNA polyplexes. Based on these results, we concluded that PEGylated polyplexes via benzoic imine linker showed high extracellular stability, intracellular DNA release and effective gene transfection.Second, we realize the morphology regulation of vector by supramolecular self-assembly based on host-guest interaction and further study the influence of morphology on the gene delivery system. Two kinds of PEGylated polycations PEI-SeSe-mPEG5k and PEI-mPEG2k were synthesized. At the acidic environment, a-CD should exclusively thread on the PEG block in the polymers to form rigid block because of the protonated PEI The rigid block fraction can be regulated by varying the addition of α-CD. Aggregates evolved from predominant nanospheres (PEG and PEG/a-CD blocks as the core and the protonated PEI as the shell) to nanoplatelets (PEG/a-CD blocks parallelly arranged as the core and the protonated PEI as the shell) with the increase of the rigid block fraction by increasing the addition of a-CD. Using the nanospheres and nanoplatelets obtained by PEI-mPEG2k/a-CD, we further investigated the DNA binding ability and influence of cellular internalization of two kinds of supramolecular assemblies. The results showed that nanoplatelets could better bind DNA and the resulting DNA/PEI-mPEG2k/a-CD nanoplatelets showed less cellular uptake, which maybe due to the different endocytosis. Different shapes of complexes were formed by supramolecular self-assembly based on host-guest interaction, and were further used to study the influence of morphology on the gene delivery system. |
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