Fabrication Of Hydroxyl-rich Branched Cationic Gene Vectors Based On Ring Opening Reactions Of Epoxides With Amines

Gene therapy provides a promising approach for the treatment of critical diseases,such as cancer,inherited diseases,and immunologically mediated diseases.The fundamental strategy of gene therapy based on delivery of therapeutic genes into targeted cells to cure or prevent diseases.However,the delivery of genes usually requires proper carriers to enhance the cellular uptake and prevent the degradation of DNA molecules.Nowadays,the gene carriers include viral and non-viral vectors.The viarl vectors usually suffer from immunogenicity and tumorigenicity,which hinder their wide application.As a result,the non-viral vectors,such as liposomes,peptides,polycations and organic/inorganic nanoparticles,have attracted much attention as an alternative approach.For example,the polycations such as poly(2-N,N-dimethylaminoethylmethacrylate)polyethyleneimine,are advantageous because of their low immunogenicity and flexibility for post-modification.However,these conventional polycations suffered from some drawbacks such as relatively low transfection efficacy,high cytotoxicity and non-degradability,which restricted their applications in gene therapy.Herein,we have demonstrated some recent achivements in the fabrication of polyhydroxyl-rich branched cationic gege gene vectors based on ring opening reactions of epoxides with amines to overcome the above limitations.The conventional polycationic gene vectors usually lack real-time imaging capability.In Chapter 2,we synthesized novel poly(glycidyl methacrylate)(PGMA)derivatives containing tetraphenylethene(TPE)moieties,namely TPE-PGEA/TPE,to enable real-time imaging capability via aggregation-induced emission(AIE)phenomena.In comparison with branched polyethyleneimine,TPE-PGEA/TPE nanoparticles exhibited lower cytotoxicity and higher transfection efficiency in HeLa and HepG2 cell lines.Moreover,TPE-PGEA/TPE nanoparticles retained the fluorescent capability,which facilitated real-time imaging of the polycaionic vectors in gene therapy.Although the PGMA derivatives exhibited enhanced transfection efficiency and real-time imaging capability,the fabrication of degradable polycationic gene vectors is still challenging.In Chapter 3,we synthesized polycationic gene vectors containing acetal groups(ARP)to enable biodegradability at acidic microenvironment of cancer cells(pH 5.0-5.5).In addition,the incorporation of fluorinated alkyl chains in the polycationic gene vectors(ARP-F)enhanced the transfection efficacy together with good biocompatibility and low cytotoxicity.Moreover,the application of ARP-F was demonstrated in ARP-F/pCas9-surv system,exhibiting enhanced tumor inhibition activities and increased sensitivity of cancer cells to anticancer drugs.Targeting capability is also important for polycationic gene vectors to improve the delivery efficacy of therapeutic genes into target cells and minimize the side effects to normal tissues.In Chapter 4,we synthesized a polyhydroxyl branched cationic gene vectors(LBP)containing lactose moieties to enable targeting capability against liver parenchymal cells with overexpressed asialoglycoprotein receptors.Aminoed-lactose(Lac-NH2)was synthesized with cyamine and lactose by the the excitation of dicarbonyl imidazole,and then Lac-NH2 was reacted with isocyanurate triglycidyl ester via ring-opening polymerization reaction to obtain branched polyhydroxy cation gene carriers(LBP).In vitro experiments showed that the disulfide bonds in LBP were susceptible to scission and led to degradation of LBP in reductive environments.Moreover,LBP also exhibited low cytotoxicity,high transfection efficacy and hepatic cancer cells targeting capability.More importantly,in vivo experiments showed that LBP were able to deliver pCas9-survivin gene into targeted cells to inhibit the growth of tumor tissues,and meanwhile improve the therapeutic effect sorafenib.In conclusion,a variety of hydroxyl-rich branched cationic gene vectors,TPE-PGEA/TPE,ARP-F and LBP,have been developed based on ring opening reactions of epoxides with amines.These new types of poly cationic gene vectors enabled real-time imaging,pH-responsive degradability,and targeting of cancer cells,which shed light on the design of next-generation multifunctional gene vectors for gene therapy.