The Effects Of Hydrophilicity And Hydrophobicity Of Cationic Polymethacrylates On Gene Delivery

Gene therapy has been proven to be a promising cancer treatment by delivering therapeutic nucleic acids into trgeted cells to correct genetic disorders featuring high efficacy and limited side effects.Cationic lipids and polymers based non-viral vectors have many advantages such as low immunogenicity,not being integrated into chromosomes,convenience in modification when compared with viral vectors,but their clinical translations were limited by low transfection efficiencies.Non-viral gene vectors,cationic lipids and polymers,both form complexes with nucleic acids through electrostatic interactions,which protects nucleic acids from emzymolysis and improves cell uptake.After entering cells,lysosomal escape,nuclear localization and DNA/polymer disassociation are main barriers to efficient gene transfection or silencing.These actions are strongly influenced by electrostatic interaction and the hydrophilicity/hydrophobicity of the polymer carriers.However,it remains unclear how the hydrophilicity/hydrophobicity of polymers affects lysosomal escape,nuclear localization and DNA/polymer disassociation of polyplexes.The first charpter summarizes the most recent studies in these aspects.According to the previous research work on polymers’ hydrophilicity and hydrophobicity,we proposed that polyplexes need excellent ability both in lysosome escaping and DNA dissociation for efficient gene transfection.Along this line,in the second chapter,we designed three methacrylate monomers with different degrees of hydrophilicity and prepared a series of homopolymers and copolymers through(co)polymerization of these three monomers.Their interactions with DNA and the conditions for forming stable polyplex were explored.The luciferase and EGFP transfection in HeLa,A549 and SW480 cell lines of the corresponding polyplexes as well as bPEI 25k and PDMAEMA control polyplexes were measured.Their relationship between transfection efficacy and cationic polymers’ hydrophilicity/hydrophobicity was inferred.We then used confocal laser scanning microscopy to observe the disassociation of DNA with different polymers through fluorescence resonance energy transfer(FRET),and found that the quick disassociation of DNA and B75D25 polymer contributed most to its high gene transfection efficiency.Substantially decreased gene transfection or silencing efficiency at low neucleic acid doses is another major limitation to non-viral gene vectors’ clinical translation because the resulting concentrations of intravaneously administered non-viral vectors/nucleic acid enriched in a targeted location(i.e.solid tumor)are inherently low,far below one microgram per gram tissue.The substantially reduced transfection efficiency at low tissue polyplex concetrations leads to low in vivo therapeutic efficacy of non-viral vectors.Therefore,in the third chapter,we investigated the luciferase and EGFP gene transfection of the B75D25 polyplex in different DNA doses in HeLa cell line,and found that the B75D25 polyplex was able to retain the same gene transfection efficiency even at very low DNA doses,while the transfection efficiencies of PEI and PDMAEMA polyplexes decreased sharply as the polyplex concentration decreased.By investigating endocytosis pathways of the B75D25 polyplex,we found that B75D25 polyplex entered cells via macropinocytosis rather than the digestive endocytosis,which was the pathway that PEI and PDMAEMA polyplexes used.We further investigated the luciferase gene transfection efficiencies of polyplexes of the polymers with different hydrophilicity/hydrophobicity at low DNA doses,and used molinspiration to quantify the hydrophilicity/hydrophobicity of the polymers.The relationship between polymers’ hydrophilicity/hydrophobicity and gene transfection efficiency at low DNA doses were inferred.It was concluded that the polymer with right hydrophobicity/hydrophilicity balance formed polyplex capable of endocytosis via macropinosis,which in turn bypassed the digestive lyposomes,while such polyplexes as PEI and PDMAEMA which enter cells via digestive endocytosis are trapped into lysosomes due to the weakened lysosomal escape at low doses.In the fourth chapter,to reduce toxicity and improve blood circulation of the polyplexes,we used anionic polymers y-polyglutamic acid(y-PGA),sodium hyaluronate(HANa),and heparin sodium to shield the positive charge of the B75D25 polyplex.We found that y-PGA coated polyplex kept high gene transfection efficiency at low DNA doses even in GGT(glutamic acid receptor)negative HeLa cell line.γ-PGA coated polyplexes also entered cell via macropinocytosis and kept the high gene transfection efficacy at low DNA doses.Moreover,y-PGA coated polyplexes loaded with tumor suicide gene pTRAIL showed excellent therapeutic effect on nude mice bearing HeLa xenograft tumor through i.v.injection comparing to PEI/pTRAIL(p<0.01).In the fifth chapter,as the first work of my PhD study,a novel dendrimer containing phosphoramidate structure was designed and synthesized from a monomer pair of methylacrylolethyl phosphite(MA-PP)and cysteamine(CA).After periferial modification with methylacrylic phosphorylcholine(MPC),the dendrimer became water-soluble and degradable by phospholipase C(PLC)overexpressed in cancer cells.The DOX-loaded dendrimer,PAD-MPC/DOX,was significantly enriched in MCF-7/ADR cells compared to free DOX.The in vivo antitumor effect of PAD-MPC/DOX was tested in nude mice bearing MCF-7/ADR xenografted tumor with i.v.injection.The PAD-MPC/DOX showed excellent therapeutic effect with no toxicity to hearts and no weight loss even at high DOX doses.