| There are high expectations that gene therapy with DNA or RNA can cure some human diseases caused by genetic disorders, such as cancer, heart cerebrovascular disease, nervous system disease and viral infection. However, due to the poor stability of naked nucleic acids in the bloodstream and its poor cellular internalization efficiency, appropriate vectors for gene delivery are necessary to push them into the clinic. Although viral vectors showed evolutionarily high gene transfection efficiency in clinic trials, safety concerns on the random recombination immunogenicity, limited nucleic acid loading capacity and high cost limited their wide clinical application. Therefore, much attention has been shifted to non-viral vectors. Polycations, a major class of non-viral gene carriers, have been reported to have good potential for gene delivery, because of their low immunogenicity and their flexible potential for structure modification.In this paper, we studied on structural modification of several biodegradable and amphiphilic cationic copolymers based on PEG, polyester and poly(2-dimethylaminoethyl methacrylate)(PDMAEMA), and investigated their properties as DNA or siRNA delivery vectors both in vitro and in vivo.By this, structure-function relationship of gene nanocarrier was revealed and may provide some guidance in gene vector design.Firstly, we modified the cationic PDMAEMA side-chains of amphiphilic PCL-g-PDMAEMA(PCD) by copolymerization of two electroneutral monomers with different hydrophobicities, 2-hydroxyethyl methacrylate(HEMA) and 2-hydroxyethyl acrylate(HEA), to obtain PCD-HEMA and PCD-HEA. It was found that incorporations of minimal HEMA or HEA moieties in PDMAEMA led to reductions and increases, respectively, in the surface hydrophilicity of the naked NPs and NPs/DNA complexes, which significantly affected the gene transfection efficiency on HeLa cells in vitro: comparing to PCD, PCD-HEMA showed a much higher transfection efficiency while PCD-HEA showed a lower transfection efficiency. Further research indicated that the incorporation of HEMA moieties facilitated an enhancement in both cellular uptake and endosomal/lysosomal escape, leading to a higher transfection efficiency. Moreover, the process of endosomal/lysosomal escape confirmed in our research that PCD and its derivatives did not just rely on the proton sponge mechanism, but also on membrane damage due to the polycation chains, especially hydrophobic modified ones. Hence, it was proved that hydrophobic modification of cationic side-chains was a crucial route to improve gene transfection mediated by polycation NPs.Secondly, we investigated the contribution of polyester segments in siRNA delivery in vitro by introducing different ratio of DLLA moieties in PCL segments of m PEG-b-PCL-g-PDMAEMA(PECD) to obtain mPEG-b-p(CL-co-DLLA)-g-PDMAEMA(PECLD). It was noticed that compared with the other ratios of DLLA moieties, a certain molar ratio(about 70%) of the NPs, named PECLD-70, showed the highest gene knockdown efficiency but poorest cellular uptake ability in vitro. Further research revealed that NPs with various compositions of the polyester cores showed not only different different endocytosis mechanisms thus influencing the cellular uptake efficiency, but also different capabilities in escaping from endosome/lysosome due to various degradation rate of polyester segments.At last, we designed a ternary siRNA delivery system via layer-by-layer assembly by PECD NPs, siRNA and HA-g-mPEG(HgP). These ternary complexes could stably combine si RNA into a negative surface charged nanocomplexes and efficiently deliver siRNA into CD44 overexpression cells with low cytotoxicity, which was contributed by anionic HgP coating. Further, these ternary complexes showed a prolonged blood circulation and reduced liver/spleen accumulation. So it was proved that by coating with HgP to form siRNA ternary formulation is available for siRNA delivery in vivo. |
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