Construction of Efficient Polymeric Gene Delivery Systems and Their Antitumor Research

Author:Fang Hua Pan

Supervisor:tian hua yu


Degree Year:2019





Cancer,as a malignant tumor,seriously threatens people’s life and health.As a novel therapeutic method,gene therapy is expected to treat cancer fundamentally.However,efficient gene carriers are the key to successful gene therapy.Among them,polycationic gene carriers have been drawing increasing attention because of their low immunogenicity,widespread availabilities,and controllable structure.Conventional polymeric gene carriers including polyethylenimine(PEI),polylysine(PLL)and polyamide amine(PAMAM)exhibit poor transfection performance and high cytotoxicity.Constructing an efficient polymeric gene carrier with high transfection efficiency and low cytotoxicity has become a research hotspot.Meantime,the polymeric gene delivery systems tend to have short circulation time and easily adsorb negative proteins in blood.Therefore,how to improve the circulatory time and reduce protein adsorption in vivo have has been an urgent problem to be solved.To overcome these barriers,firstly,metal coordination interaction,eletrostatic interaction,hydrogen bonding interaction,and hydrophobic interaction are introduced into conventional polymeric gene carriers,resulting in enhanced transfection efficiency and lower cytotoxicity.Secondly,a tumor microenvironment responsive polymeric shielding system is developed for improving the circulation time and reducing negative protein adsorption in blood.To improve the transfection performance,zinc ion-containing bis(pyridin-2-methyl)amine compound is introduced into low molecular weight polyethyleneimine(PEI1.8k)via Schiff base bond.The synthesized carrier Zn-DPPA2 can form metal coordination interaction with cell membrane(or DNA),contributing to cellular uptake,ultimately achieving efficient gene transfection.In addition,the polymeric gene delivery system exhibits negligible cytotoxicity and excellent antiserum ability.Zn-DPPA2/pDNA nanocomplexes possess good size stability.After intravenous injection,nanocomplexes exhibits efficient gene transfection performance in tumor tissue.It is generally believed that the electrostatic interactions between gene carriers and cell membrane(or DNA)will influence the transfection efficiency of polymeric gene carriers.In general,appropriate electrostatic interactions contribute to DNA loading,condensation and endocytosis,which are beneficial to gene transfection.Nevertheless,too large charge density can result in deadly cytotoxicity,hindering the transfection performance.To overcome the bottleneck of electrostatic interaction between polymeric gene carrier and cell membrane(or DNA),"molecular string" RT(i.e.,p-toluylsulfonyl arginine)is introduced into PLL backbone,which contributes to the formation of multiple interactions(electrostatic interaction,hydrogen bonding interaction,and hydrophobic interaction)between gene carrier and cell membrane(or DNA),significantly promoting transfection efficiency.Additionally,the introduction of molecular string RT results in the formation of a-helix conformation,which is beneficial for the cellular uptake.The multiple interactions(electrostatic interaction,hydrogen bonding interaction,and hydrophobic interaction)and a-helix conformation synergistically improve the transfection efficiency of polymeric gene carrier.Additionally,the carrier PLL-RT exhibits excellent antiserum ability and outstanding RNA silencing efficiency.To verify the universality of molecular string RT in improving transfection performance,RT string are introduced into other polymers such as PEI and dendrimer PAMAM,the resulting carriers also showed enhanced transfection efficiency and reduced cytotoxicity.PLL-RT mediating therapeutic gene can effectively inhibited tumor growth after intratumoral administration.This work provided an idea for constructing efficient gene carriers by introducing multiple interactions(electrostatic interaction,hydrogen bonding interaction,and hydrophobic interaction)between carriers and cell membrane or a-helix characteristic.To improve the gene delivery efficiency of polymeric gene delivery system in vivo,we developed a tumor microenvironment responsive polymeric shielding system.PLL-RT was used as a carrier to load pDNA,H2O2-responsive thioketal dipropanedioic acid modified dextran(TDPAD)serves as shielding layer to coat PLL-RT/pDNA complexes.The H2O2 responsiveness of polymeic gene delivery system was verified by zeta potential,particle size,gene transfection and cellular uptake.After intravenous adminstration,this nanocomplexes showed extended circulatory half-life in vivo and could effectively accumulated in tumor tissue by EPR(enhanced permeability and retention)effect.After mediating therapeutic gene pDNA encoding shVEGF,this polymeric gene delivery system could achieve efficient and synergistic antitumor effect by utilizing a strategy of kill three birds with one stone.Firstly,this polymeric gene delivery system can significantly down-regulate the expression of VEGF in tumor tissue after mediating pDNA encoding shVEGF,possessing a pronounced antitumor effect.Secondly,the shielding material TDPAD can significantly consume the H2O2 in tumor tissue,which synergistically suppresses tumor growth.Mercaptopropionic acid(MPA)produced by the reaction of TDPAD with H2O2can induce cancer cell apoptosis and exert the antitumor efficacy.This polycationic gene delivery system shows good biocompatibility.The strategy of kill three birds with one stone,namely,polycation-mediated gene therapy,H2O2 consumption in tumor tissue,and MPA-induced cancer cell apoptosis has been confirmed in detail.This gene delivery system provides an ideal platform for constructing efficient in vivo polymeric carriers.In this thesis,many kinds of interactions(i.e.,metal coordination,electrostatic,hydrogen bonding,hydrophobic interaction)between polymeric gene carrier and cell membrane(or DNA)as well as a-helix characteristics were introduced,which significantly improved the transfection efficiency and lowered cytotoxicity of gene carriers.Moreover,a H2O2 responsive shielding system was constructed to overcome the multiple barriers during gene delivery,significantly promoting gene delivery efficiency in vivo.Such strategies provided theoretical basises for the construction of efficient polymeric gene delivery system and gene therapy.