Structural Impact Of Graft And Block Copolymers Based On Poly(N-vinylpyrrolidone) And Poly(2-dimethylaminoethyl Methacrylate) In Gene Delivery

Gene therapy has attracted much attention for its promising application in the treatment of inherited and acquired diseases. The principle of gene therapy is that therapeutic genes are delivered into patient's host cells to produce or silence functional proteins to cure diseases. The key factor for successful application in gene therapy is gene vector with high transfection efficiency and low cytotoxicity. Gene vectors can be divided into non-viral vectors and viral vectors. Polycations as an important kind of non-viral vectors can load genes effectively and deliver genes into cells. Among those polycations, PDMAEMA has been widedly used as a model for polycation research. Most studies concentrated on the influences of different PDMAEMA structures such as graft and block structures on their abilities of gene delivery. However,the characteristics of graft copolymer and block copolymer in gene compaction, polyplexes stability, cytotoxicity, transfection efficiency, internalization and intracellular distribution of polyplexes were few systematically investigated.In this work, PgP and PbP with equal PDMAEMA segments and PVP as the starting parts were prepared via the living radical polymerization of ATRP and RAFT, respectively. PDMAEMA with pendant tertiary amine groups can facilitate the endosomal escape of the polyplexes. PVP can shield the excess positive charge of PDMAEMA to reduce the cytotoxicity without sacrificing gene transfection efficiency of the polyplexes due to its synergistic role in gene delivery. Compared with PbP, PgP could condense pDNA more effectively into polyplexes with smaller size, higher zeta potential and better stability. The transfection efficiency of PgP at low N/P ratio of 4:1 was not only higher than that of PbP, but also much higher than that of commercial transfection reagent Lipofectamine and PEI, the gold standard of tranfection reagent. Further, we compared PgP, PbP and PEI on protein absorption ability. Both PgP and PbP had less BSA absorption compared with PEI, indicating that the PVP could resist BSA absorption. The cytotoxicity of PgP and PbP evaluated on HepG2 cells through MTT method indidcated that PbP/pDNA and PgP/pDNA polyplexes had well compatibility, transfection in HepG2 cells reveals that the efficiency of PgP/pDNA is obvious higher than that of PbP/pDNA. Internalization of PgP/pDNA complexes measured by flow cytometry was more than that of PgP/pDNA complexes. The intracellular distributions of these polyplexes were studied by confocal laser scanning microscopy(CLSM), it showed that the endosomal escape ability of PgP/pDNA was stronger than PbP/pDNA, and the colocalization ratio of the DNA and Lysotracker Green(endosomes/lysosomes) was also calculated and it was in accordance with the confocal results. Therefore, the high transfection efficiency of PgP originated from the higher cellular uptake efficiency and better endosomal escape compared with PbP. All the results clarified the relationship between structures and properties of the polycations based on PDMAEMA for gene delivery.