Construction Of Multi-Stimuli Responsive Polymeric Prodrugs For Co-Delivery Of Anticancer Drugs And P53 Gene

Multifunctional stimuli-responsive polymer materials have been widely used in drug and gene delivery systems.This is due to the pH difference between healthy tissues(pH?7.4)and intracellular tumor environment,especially in the endosomal and lysosomal areas with a pH of about 4.5?6.5.In addition,the cytosolic concentration of GSH is 3 orders of magnitude higher than the extracellular concentration,which can provido a precondition for the construction of pH-and reduction-responsive drug delivery systems.It can rapidly respond in tumor cells and release the active drug,and inhibit the proliferation of cancer cells.Hence,the construction of multi-responsive polymeric prodrug has a good prospect in the treatment of cancer.The combination therapy of drugs and genes can not only avoid drug resistance,but also effectively improve the bioavailability of genes and drugs,reduce the systemic toxicity and dose of chemotherapy drugs,so as to reduce the pain of patients.This thesis is mainly focused on the design and synthesis of pH/reductiorn-responsive shell cross-linked polymeric prodrug,folic acid targeting and pH-responsive zwitterionic prodrug for the treatment of cancer via a combination of ring-opening polymerization(ROP),atom transfer radical polymerization(ATRP),reversible addition-fragmentation chain transfer(RAFT)and "click" chemistry.High efficiency gene vectors were designed,which can used to realize the combination therapy of drugs and genes.The present work mainly includes the following four parts:(1)One-Pot Synthesis of pH/Redox-Responsive Polymeric Prodrug and Fabrication of Shell Cross-Linked(SCL)Prodrug Micelles for Antitumor Drug Transportation.We report on a new kind of pH/redox-responsive dynamic covalent SCL micelles,which was fabricated by self-assembly of a multifunctional polymeric prodrug.Firstly,a macroinitiator PBYP-ss-iBuBr was prepared via ROP reaction,wherein BYP represents 2-(but-3-yn-1-yloxy)-2-oxo-1,3,2-dioxaphospholane.Subsequently,PBYP-hyd-DOX-ss-P(DMAEMA-co-FBEMA)prodrug was synthesized by one-pot method with a combination of ATRP and Cu(?)-catalyzed azide-alkyne cycloaddition(CuAAC)reaction using a doxorubicin(DOX)derivative containing azide group to react with the alkynyl group of the side chain in the PBYP block,while DMAEMA and FBEMA are the abbriviations of N,N-(2-dimethylamino)ethyl methacrylate and 2-(4-formylbenzoyloxy)ethyl methacrylate,respectively.The chemical structures of polymer precurcors and prodrugs have been fully characterized.The SCL prodrug micelles were obtained by self-assembly of the prodrug and adding cross-linker dithiol bis(propanoic dihydrazide)(DTP).Compared with the shell uncross-linked prodrug micelles,the SCL prodrug micelles can enhance the stability and prevent the drug from leaking in the body during blood circulation.The average size and morphology of the SCL prodrug micelles were measured by dynamic light scattering(DLS)and transmission electron microscopy(TEM).The SCL micelles can be dissociated under moderately acidic or/and reductive microenvironment,that is,endosomal/lysosomal pH medium or high GSH level in the tumorous cytosol.The results of DOX release also confirmed that the SCL prodrug micelles possessed pH/reduction-responsive properties.Cytotoxicity and cellular uptake analyses further revealed that the SCL prodrug micelles could be rapidly internalized into tumor cells through endocytosis and efficiently release DOX into the HeLa and HepG2 cells,which could efficiently inhibit the cell proliferation.This study provides a facile synthesis method for preparing multifunctional polymeric prodrugs,which hold great potential for optimal antitumor therapy.(2)Zwitterionic Shielded Polymeric Prodrug with Folate-Targeting and pH Responsiveness for Drug Delivery.FA-P(MPC-co-PEGMA-BZ)-g-DOX was synthesized via a combination of RAFT copolymerization,Schiff-base reaction,Click chemistry,and a reaction between the amine group of doxorubicin(DOX)and aldehyde functionalities of P(MPC-co-PEGMA-BZ)pendants,wherein MPC and PEGMA-BZ represent 2-(methacryloyloxy)ethyl phosphorylcholine and polyethylene glycol methacrylate ester benzaldehyde,respectively.The polymeric prodrug could self-assemble into nanoparticles in aqueous solution.The average particle size and morphologies of the prodrug nanoparticles were observed by DLS and TEM,respectively.We also investigated the in vitro drug release behavior and found that it displayed a fast dissociation of prodrug nanoparticles and released drug under mildly acidic microenvironment.The methyl thiazolyl tetrazolium(MTT)assay verified that P(MPC-co-PEGMA-BZ)copolymer possessed good biocompatibility and FA-P(MPC-co-PEGMA-BZ)-g-DOX prodrug nanoparticles showed higher cellular uptake than those prodrug nanoparticles without the FA moiety.The results of cytotoxicity and the intracellular uptake about non-folate/folate targeted prodrug nanoparticles further confirmed that FA-P(MPC-co-PEGMA-BZ)-g-DOX could be efficiently accumulated and rapidly internalized by HeLa cells due to the strong interaction between multivalent phosphorylcholine(PC)groups and cell membranes.This kind of multifunctional FA-P(MPC-co-PEGMA-BZ)-g-DOX prodrug nanoparticle with combined target-ability and pH responsiveness demonstrates promising potential for cancer chemotherapy.(3)Aminated Poly(glycidyl methacrylate)s-based Polymer for Constructing Efficient Gene Vectors.Firstly,we have prepared poly(glycidyl methacrylate)(PGMA)using RAFT polymerization,and then the ring-opening reaction of epoxy functional groups of PGMA pendants were performed by ethanol amine(EA),1-amino-2-propanol(AP)and N-(2-hydroxyethyl)ethylenediamine(HA),respectively,which were obtained there kinds of PGMA-based gene vectors containing hydroxyl groups(abbreviated as PGEA,PGAP and PGHA).The chemical structures and element contents of three kinds of polymers were characterized by IH NMR,GPC,XPS and FT-IR measurements.Afterwards,three kinds of polymers could be completely dissolved in aqueous solution and fully mixed with p53 gene to form polymers/gene complexes by electrostatic interaction.Agarose gel electrophoreses assay could evaluate that the ability of polymers/gene complexes to compress genes with different N/P ratios and the stability of complexes under negative charge environment.Meanwhile,the biological properties of polymers/gene complexes at different N/P ratios were evaluated by MTT assay and laser scanning confocal microscope.The results confirmed that the designed three kind of cationic polymers had better biocompatibility than that of PEI polymer at some concentration,the polymers/gene complexes had high transfection efficiency,which could inhibit lung cancer cells proliferation effectively.Moreover,the transfection efficiency of PGHA/p53 complex was higher than that of the other two polymers/gene complexes.Therefore,the constructed cationic polymer has a broad application prospect as a gene vectors(4)Construction of PCL-ss-P(GHA-co-PEGMA)Cationic Polymer for Co-delivery of DOX and p53 Gene.CPDB-ss-OH was first prepared via esterification reaction and PCL-ss-P(GMA-co-PEGMA)copolymers were then prepared by a combination of ROP and RAFT copolymerization.Finally,HA was used for ring opening reaction of epoxy groups of the polymer to obtain PCL-ss-P(GHA-co-PEGMA)cationic polymer.The chemical structure,molecular weight distribution and element content of CPDB-ss-OH,PCL-ss-P(GMA-co-PEGMA),and PCL-ss-P(GHA-co-PEGMA)were characterized by 1H NMR,GPC and XPS measurements.Amphiphilic PGHAP polymer could self-assemble into nanoparticles,which could encapsulate DOX and compress p53 gene to form DPGHAP/p53 complex.The fixation ability and stability of DPGHAP/p53 complexes were evaluated by agarose gel electrophoreses assay.The gel retardation assay showed that the p53 gene could be well immobilized and maintained stably under the electronegative conditions.Cell cytotoxicity assay showed the obvious antitumor effect of DPGHAP/p53 complexes against A549 and H1299 cells when compared to free drug or/and gene therapy applied alone,respectively.Furthermore,the results from laser scanning confocal microscope revealed that the DPGHAP/p53 complexes could effectively deliver and release DOX and the p53 gene into A549 cells.Therefore,the constructed cationic polymer has a potential application as vectors of anticancer drug and gene.