Preparation And Characterization Of PEG-based Polymeric Prodrug And Co-carrier Of Gene And Anticancer Drug

In recent years,nano drug delivery systems have been widely studied by researchers because of its active targeting effect to tumors by enhanced permeability and retention(EPR)effect of solid tumors.The nanoparticles(NPs)can effectively optimize the water solubility of drugs,weaken its cytotoxicity,and prolong the circulation time in the body.Moreover,stimuli-responsive drug carriers further reduce the side effect and improve the drug utilization rate,which are designed by the difference physiological environments of tumor cells and normal cells1.Polymer-doxorubicin prodrug with biocompatibility,pH response,and main chain breakability prepared by catalyst-free click reactionAmino-yne click polymerization is a novel efficient and mild polymerization reaction,and the enamine bond(-ena-)gained from the reaction is sensitive to acid,which can be used to prepare stimulus-responsive polymeric prodrug.Herein,an alkynyl-terminated polymer containing alternately distributed low molecular weight polyethylene glycol(PEG)and hexamethylenediamino(HMDA)linked by enamine bonds,abbreviated as A-P(PEG-alt-HMDA)-A,was synthesized within 3 h at 35? without catalyst.And then a pH-responsive polymeric prodrug(DOX-ena-PPEG-ena-DOX)was further prepared through the amino-yne click reaction between the alkynyl groups of A-P(PEG-alt-HMDA)-A and the amino group of doxorubicin hydrochloride(DOX·HCl).The chemical structures were characterized by nuclear magnetic resonance spectroscopy(NMR),matrix assisted laser desorption/ionization-time of flight mass spectrometry(MALDI-TOF MS),gel permeation chromatography(GPC),Fourier transform infrared spectroscopy(FT-IR),and ultraviolet-visible absorption spectroscopy(UV-vis).The resulting prodrug can self-assemble into nanoparticles(NPs)in aqueous solution.The self-assembly behavior and stability experiment of NPs were measured by dynamic light scattering(DLS)and transmission electron microscopy(TEM).The accumulative release of doxorubicin(DOX)was tested with different pH media.Subsequently,we investigated cell cytotoxicity and intracellular uptake of the prodrug.It turned out that the prodrug nanoparticles could be internalized into cancer cells,release original DOX and efficiently inhibit the proliferation of cancer cells.These results show that the pH-responsive DOX-ena-PPEG-ena-DOX has the potential for using in cancer therapy.2.PEG-based co-delivery system of p53 gene and doxorubicin with pH-response prepared via atom transfer radical polymerizationAtom transfer radical polymerization(ATRP)is a commonly used controlled/living radical polymerization with abundant monomers and is one of the effective tools for synthesizing nano drug carriers.The p53 gene is one of the most important tumor suppressor genes and is widely used in cancer therapy.The p53 protein encoded by p53 gene can induce.apoptosis or cell senescence,inhibit tumor angiogenesis,and improve chemotherapy sensitivity of tumor cells.It has been applied in basic research and clinical treatment.The combination of p53 gene and chemotherapeutic drugs can effectively inhibit the emergence of multidrug resistance and improve the therapeutic effect.In this study,an acid-sensitive gene and drug co-carrier were synthesized by ATRP.First,the random copolymer of glycidyl methacrylate(GMA)and alcohol methyl ether methacrylate(PEGMA),abbreviated as P(GMA-PEGMA),was synthesized via ATRP with ethyl-2-bromoisobutanoate(EBiB)as the initiator,and CuBr/2,2'-bipyridyl(bpy)as catalyst.After the ring-opening reaction and oxidizing reaction,the random copolymer P(hOGMA-PEGMA)containing active aldehyde groups was synthesized.And then the pH-responsive prodrug P(hOGMA-g-(DOX/HEMA)-PEGMA)further prepared linked by Schiff base between the aldehyde groups of P(hOGMA-PEGMA)and the amino group of doxorubicin hydrochloride(DOX·HCl)and hydroxyethylethylenediamine(HEMA).The chemical structures were characterized by nuclear magnetic resonance spectroscopy(NMR),gel permeation chromatography(GPC)and ultraviolet-visible absorption spectroscopy(UV-vis).The cationic prodrugs can self-assemble into nanoparticles,abbreviated as DOX/N NPs,in aqueous solution.The particle size and particle size distribution of the nanoparticles were characterized by dynamic light scattering(DLS).The in vitro release behavior of the drug was characterized by fluorophotometer.The in vitro release behavior of DOX was explored under different pH media.And we used zeta potential measurement to verify the ability of the nanoparticles condensd gene.The biocompatibility of the polymer P(hOGMA-PEGMA)and the tumor cells cytotoxicity of the gene-loaded nanoparticles were verified by MTT assay.At the same time,we observed endocytosis and gene transfection of the gene-loaded nanoparticles(DOX/DNANPs)by a live cell imaging system.These results indicate that the nanoparticles containing DOX and p53 genes have broad application prospects in the treatment of cancer.