Engineering Stimuli-Responsive Polymers For Gene Delivery Applications

Stimuli-responsive polymers could respond to external stimuli and this unique property has rendered stimuli-responsive polymers with wide applications in the fields of drug carriers,gene delivery,and tissue engineering.In this thesis,we focus on the applications of stimuli-responsive polymers in terms of gene delivery.Specifically,in the first chapter,we summarized the advancements of stimuli-responsive polymer-based gene carriers in recent years;in the second chapter,we synthesized?-cyclodextrin(?-CD)-based star polymers and engineered them as gene carriers and magnetic resonance imaging(MRI)contrast agent.Redox-responsive disulfide bonds were introduced into the design of cationic polymer brushes,which could be cleaved into low molecular weight species in cancer cells.This chemistry allowed the fabrication of thiol-responsive gene carriers with enhanced gene transfection efficiency in cancer cell lines.Lastly,to overcome the instability of gene carriers in serum,temperature-responsive polymers were incorporated into the fabrication of gene carriers with heterogeneous shells,extending blood circulation time and elevating gene transfection efficacy.The studies included in this thesis are as bellows:1.We report on the synthesis of star copolymers possessing dual functions of gene delivery vector and magnetic resonance(MR)imaging contrast enhancement.Starting from asymmetrically functionalized ?-cyclodextrin(?-CD)comprising of 7 azide moieties and 14 ?-bromopropionate functionalities at the upper and lower rim of rigid toroidal ?-CD core,(DOTA-Gd)7-CD-(PDMA)14 star copolymers were synthesized via the atom transfer radical polymerization(ATRP)of N,N-dimethylaminoethyl methacrylate(DMA)and subsequent click reaction with alkynyl-functionalized gadolinium(Gd3+)complex.DOTA-Gd,where DOTA is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid.The obtained Janus-type star copolymers,(DOTA-Gd)7-CD-(PDMA)14,could completely complex with anionic plasmid DNA(pDNA)via electrostatic interactions at N/P ratios equal or higher than 2 and exhibit optimal in vitro transfection efficiency at N/P ratio of 8.In addition.in vitro MR imaging experiment demonstrated considerably enhanced Ti relaxivity(r1?10.9 s-1mM-1)for the star copolymer compared to that of commercially available small molecular MR imaging contrast agents(2.4-3.2 s-1mM-1).The star-type topology of asymmetrically functionalized ?-CD based copolymers in combination with the integrated design of diagnostic and therapeutic functions augurs well for their potential applications in the field of image-guided gene therapy.2.Novel neutral-cationic brush block copolymer,poly[oligo(ethylene glycol)monomethyl ether methacrylate-co-folic acid methacrylate]-b-poly[2-(2-(2-(2-bromo-2-methylpropanoyloxy)-ethyl)disulfanyl)ethyl methacrylate-g-2-dimethylaminoethyl methacrylate].P(OEGMA-co-FAMA)-b-P(BSSMA-g-PDMAEMA)was synthesized via consecutive controlled radical polymerizations.Containing disulfide linkages bridging backbone and side chains in the cationic brush block and cancer cell-targeting ligands(folic acid)in the neutral hydrophilic block,the diblock copolymer was employed as tumor-targeted redox-responsive degradable nonviral gene delivery vector.P(OEGMA-co-FAMA)-b-P(BSSMA-g-PDMAEMA)brush block copolymers can condense plasmid DNA(pDNA)efficiently via forming electrostatic polyplex micelles.Under reductive milieu,pDNA can be released due to the cleavage of disulfide linkages and accordingly pDNA-binding cationic PDMAEMA side chains.In addition,the brush block copolymer exhibited low cytotoxicity and the corresponding polyplex micelles showed relatively high gene transfection efficiency.3.Adequate retention in blood circulation is the prerequisite for construction of gene delivery carriers towards systemic applications.The stability of gene carriers in bloodstream required them to effectively resist protein adsorption and maintain small size in bloodstream avoiding dissociation,aggregation.and nuclease digestion under salty and proteinous medium.Herein,a mixture of two block catiomers consisting of a same cationic block,poly{N-[N-(2-aminoethyl)-2-aminoethyl]aspartamide}(PAsp(DET)),but varying shell-forming blocks,poly[2-(2-methoxyethoxy)ethyl methacrylate](PMEO2MA)and poly[oligo(ethylene glycol)methyl ether methacrylate](POEGMA)was used to complex with plasmid DNA(pDNA)to fabricate polyplex micelles with mixed shells(MPMs)at 20 ?.The thermo-responsive property of PMEO2MA allows distinct phase transition from hydrophilic to hydrophobic by increasing incubation temperature from 20 ? to 37 ?,which resulted in distinct heterogeneous corona containing hydrophilic and hydrophobic regions at the surface of MPMs.The subsequent study verified this transition promoted further condensation of pDNA,thereby giving rise to improved complex and colloidal stabilities.The proposed system presented remarkable stability in salty and proteinous solution and superior tolerability to nuclease degradation.As compared with polyplex micelles formed from single POEGMA-b-PAsp(DET)block copolymer.in vivo circulation experiments in bloodstream further confirmed that the retention time of MPMs was prolonged significantly.Moreover,the proposed system exhibited remarkably high cell transfection activities especially at low N/P ratios and negligible cytotoxicity,thus portends promising utilities for systemic gene therapy applications.