Construction Of Responsive Polyprodrug Micelles Of Drug Self-Delivery And Antitumor Activity

Cancer is the most difficult disease to cure in the world.Generally,surgery,chemotherapy and radiotherapy are common methods for curing cancers.Surgery and radiotherapy have great side effect to normal tissues and the metastasis and recurrence of cancers make it incurable.Chemotherapy is most common method for cancer therapy in clinic,but the drawbacks of poor water solubility,side effect to normal tissue and rapily metabolism and excretion of small molecule anticancer drugs limit the further application in clinic.Drug delivery system for cancer chemotherapy has been widely developed as an emerging and cross-functional field.It can increase the water solubility and change the distribution of anticancer drugs in vivo,deliver anticancer drugs to tumor tissues through enhanced permeability and retention（EPR）effect,improve the efficiency of cancer treatment and decrease the side effect to normal tissues.However,the most important factors for the new drugs or drug delivery systems in clinical application are efficacy,safety,economics and convenience.For most of drug delivery systems,its complex preparation processes and un-approved components of organic polymers or molecules by food and drug administration（FDA）limit the further application in clinic.Therefore,in order to accelerate the process of application in clinic of traditional drug delivery systems,the effective,safe,economic and simple drug self-delivery system need to be developed for cancer chemotherapy.The drug self-delivery system of polyprodrug micelles were polymerized by drugs and reversible covalent bonds.The polyprodrugs were prepared by simple method and avoided the usage of un-approved components by FDA,which make it a perfect method for addressing the problems of traditional drug delivery systems.This dissertation focus on the construction of self-delivery polyprodrug micelles and evaluation of anticancer drug activity to tumor cells in vitro and in vivo.This dissertation employ antitumor drugs of doxorubicin and methotrexate as model drugs to synthetize amphiphilic linear polyprodrug and hyperbranched polyprodrug micelles for cancer chemotherapy,meanwhile,evaluation of the cell viability and tumor inhibitation of the polyprodrug micelles.In chapter 2,we present a novel glutathione-responsive amphiphilic linear polyprodrug micelles with one-pot synthesis method and evaluate the drug release properties and tumor inhibitation in vitro.The anticancer drug doxorubicin（DOX）,disulfide-based diacrylate（DSDA）and amino-polyethylene glycol monomethyl ether were linked by Michael addition in one-pot synthesis.The glutathione（GSH）-responsive amphiphilic linear polyprodrug micelles were completely disrupted with the concentration of GSH=1 mg mL^-1.The accumulative release rate of DOX analogues with drug activity from micelle was 67.9%under pH 7.4 and GSH=1 mg mL^-1 conditions after 72 h.The cell uptake experiment showed that the micelle of DOX-DSDA-PEG was indeed taken up by A549 cells and distributed to cell nuclei.The in vitro cell viabilities of A549 cells between DOX-DSDA-PEG and DOX were no significant difference.The results illustrated that the completely biodegradable linear polyprodrug micelle with glutathione-responsive bonds in backbone can be disrupted at high concentration of GSH in tumors,releasing DOX analogues for cancer therapy.In chapter 3,we develop an amphiphilic glutathione-responsive hyperbranched polyprodrug（DOX-S-S-PEG）micelle with drug repeat units in hydrophobic cores linked by disulfide bonds.The hyperbranched polyprodrug micelles were stable without GSH in pure water.The accumulative release rate of DOX analogues with drug activity from micelle was82%under pH 7.4 and GSH=1 mg mL^-1 conditions after 72 h.Cell viabilities of A549 cells and 293T cells were evaluated by CCK-8 and Muse Annexin V&Dead Cell Kit and illustrated that the disrupted polyprodrugs in tumor cells can release DOX analogues with drug activity for killing tumor cells.Meanwhile,the un-disrupted polyprodrugs possessed low cytotoxicity to normal cells.The cell uptake experiments showed that the micelles of DOX-S-S-PEG were totally taken up by A549 cells and distributed to cell nuclei after 10 h.In chapter 4,we prepared an amphiphilic and completely degradable polymer–drug conjugates based on acid-sensitive 100%hyperbranched polyacetals for cancer therapy.The uptake process of DOX-HBPA-PEG by A549 cells showed that the micelle was totally taken up by A549 cells after 1 h.The A549 cell viability of HBPA-PEG was 100%after 24 h at the concentration of 280μg mL^-1 and the A549 cell viability of DOX-HBPA-PEG was 37%after24 h at the concentration of 83.3μg mL^-1(DOX concentration is 10μg mL^-1).The results of cell viability of DOX-HBPA-PEG and HBPA-PEG showed that the completely degradable polymer–DOX conjugates were effective drug delivery systems and possess good biocompatibility.In chapter 5,we presented a novel linear polyprodrug micelle with pH and glutathione dual responsiveness based on methotrexate.The accumulative release rate of MTX from micelle was 89%under pH 6.0 and GSH=1 mg mL^-1 conditions after 72 h.The cell viabilities results evaluated by flow cytometry showed that the drug active site of MTX did not change in polyprodrugs.The micelles overlapped with lysosome by cellular uptake and the distribution in vivo was higher in tumor tissues.The biological evaluation and histological analysis confirmed that the linear polyprodrug micelles were more effective to kill tumor cells than free MTX.At the same time,their side effect was less in normal tissues.As a result,the tumor growth could be effectively inhibited in vivo.In chapter 6,we prepared a high water soluble polyethylene glycol monomethyl ether modified methotrexate prodrug for cancer therapy.The cell viabilities of MTX and MTX-mPEG were similar evaluated by CCK-8 experiment.The distribution of prodrug MTX-PEG-Rhodamine in vivo was higher in tumor tissues compared with MTX.The tumor volume（16 days later）of group saline,MTX and MTX-PEG-Rhodamine were increased3.7-fold,2.8-fold and 1.4-fold compared with initial tumor volume,respectively.The TUNEL and tumor volume results further confirmed that the tumor growth could be effectively inhibited in vivo by prodrug.