Supramolecular Self-assembly Of Multifunctional Polymeric Gene Vectors Based On Host-guest Interaction

Gene therapy is considered to be a great potential approach to cure a series of disease,such as cancers and diabetes.Cationic polymer based polyplexes in general show relatively lower gene transfection efficiency with shorter circulation time in vivo,because there are some contradiction problems for polymeric gene vectors,such as suitable gene package and controllable realease and PEGylation dilemma.To overcome both extracellular and intracellular barriers in the gene delivery,a series of multifunctional cationic supramolecular gene delivery systems based on host-guest interaction were designed and studied in this work.In chapter 1,the common cationic polymers as gene vectors were introduced.A review on progress of the sensitive cationic polymeric gene vectors and the supramolecular assembly gene delivery system was presented.Our investigations for supranolecular self-assembly of multifunctional polymeric gene vectors based on host-guest interaction were explained.In chapter 2,this chapter describes the supramolecular host-guest polycationic gene delivery system based on poly(?-cyclodextrin)(PCD)and azobenzene-terminated polycations.The azobenzene-terminated linear(Az-LPDM)and branched(Az-BPDM)cationic polymers were synthesized by atom transfer radicalpolymerization(ATRP)of 2-dimethylamino ethyl methacrylate(DMAEMA).The formation and photo-sensitive behavior of the supramolecular polycations of azobenzene-terminated polycations Az-LPDM and Az-BPDM with PCD were confirmed by UV-vis is and NMR analysis.The supramolecular PCD/Az-BPDM/DNA and PCD/Az-LPDM/DNA polyplexes showed smaller size and were less positive than those of their corresponding polyplexes without PCD.Moreover,the UV irradiation may promote release of DNA from the photosensitive supramolecular polyplexes due to dissociation of supramolecular polyplexes.In vitro experiments revealed that the photosensitive supramolecular polycationic polyplexes(PCD/Az-LPDM/DNA and PCD/Az-BPDM/DNA)exhibited enhancement of cellular uptake,higher transfection efficiency,and lower cytoxicity compared to the azobenzene-terminated polycation/DNApolyplexes in the absence of PCD.Branched polycationic polyplexes showed higher transfection efficiency than its linear polycationic polyplexes.Furthermore,after UV irradiation,the transfection efficiency of photosensitive supramolecular polyplexes was improved resulting from more DNAs delivered and released inside of the cell nuclei.Thus this photo responsive supramolecular host-guest system containing azobenzene-terminated branched cationic polymers and PCD is a promising gene vector.In chapter 3,novel reduction degradable and photosensitive disulfide-containing azobenzene-terminated branched poly(2-(dimethylamino)ethyl methacrylate)s(Az-ss-BPDMs)and supramolecular host-guest self-assembly systems with poly(cyclodextrin)(PCD)were prepared and evaluated as non-viral gene delivery vectors.The reduction and light dual sensitive properties of the supramolecular polycations PCD/Az-ss-BPDMs and their polyplexes PCD/Az-ss-BPDMs/DNA were confirmed by UV-vis,SEC,DLS and zeta potential analyses,respectively.It was shown that the inclusion of PCD,introduction of disulfide bonds into branched polycations,increase of the branching degree of the branched Az-ss-BPDMs and use of UV irradiation could enhance the gene transfection efficiency and cellular internalization of the supramolecular disulfide containing azobenzene-terminated branched polycationic polyplexes(PCD/Az-ss-BPDM/DNA).Importantly,the transfection efficiency of the light and reduction dual-sensitive supramolecular PCD/Az-ss-BPDM/DNA polyplexes achieved almost 10 times higher value than that of 25 kDa PEI control;whereas the cytotoxicity of the supramolecular polyplexes was lower than that of PEI control.Thus this light and reduction dual responsive supramolecular host-guest system containing azobenzene-terminated branched cationic polymers with disulfide bonds and PCD is a promising gene vector.In chapter 4,first the disulfide-containing azobenzene-terminated branched polymer(Az-ss-BPDM-RhB)with the rhodamine-fluorescencnt functional groups was synthesized.The structure of Az-ss-BPDM-RhB was confirmed by UV-vis,NMR and HPLC analysis.The properties of the supramolecular PCD/Az-ss-BPDM-RhB/DNA polyplexes with diffent PEGylation degree were studied by DLS and zeta potential analyses.The inclusion of PEG could reduce the cytotoxicity of the supramolecular polyplexes.When the tranfection time was short(4 h),the gene transfection efficiency decreased with increasing the PEGylation degree for the PEGlytion supramolecular PCD/Az-ss-BPDM-RhB polyplexes.When the transfection time was long(above 24 h),the transfection efficiency and cellular uptake of PEGylation polyplexes was close to the supramolecular PCD/Az-ss-BPRM-RhB/DNA polyplexes without PEGylation.In addition,the supramolecular PCD/Az-ss-BPDM-RhB polyplexes were observed in cell by CLSM.In chapter 5,a targeted light and reduction dual sensitve multifunctional supramolecular assembly gene delivery system based on chapter 4 was designed and evaluated.The properties of the supramolecular PCD/Az-ss-BPDMs-RhB/DNA with folate-targeting group were characterized by ethidium bromide exclusion assay,DLS and zeta potential analyses.In vitro experiments revealed that the the transfection efficiency and cellular uptake of folate-targeting supramolecular PCD/Az-ss-BPDM-RhB/DNA polyplexes was enhanced using HeLa cells(folate receptor positive tumor cells)but the the transfection efficiency was not enhanced for receptor-negative COS7 cells.In vivo toxicity testing using the zebrafish embryo assay revealed that the supramolecular polycation PCD/Az-ss-BPDM-RhB and their polyplexes with and without PEGylation showed lower toxicity than PEI(25 kDa),which is in agreement with the in vitro cell cytoxicity result.Therefore,this light and reduction dual sensitve multifunctional supramolecular host-guest interaction.system containing folate-targeting group and rhodamine-fluorescencnt functional group might have potential application in gene therapy.