The Research Of Intelligent Nano-Drug Delivery System For The Dual-Drug Codelivery With A Synergistic Effect

Cancer is one of the major diseases endangering human health.Chemotherapy is often used clinically to catalyze the rapid suppression or killing of cancer cells,alleviating or eliminating various symptoms of the tumor.However,clinical practice shows that the existing chemotherapitic drugs also show some disadvantages,such as low drug utilization and serious side-effects.Single-drug therapy of single anti-tumor mechanism is also easy to stimulate or even enhance the other malignant proliferation mechanism of tumors.Hence,it's difficult to achieve good clinical results due to that it's easy for cancer cells to induce the drug resistance and tumor metastasis.The combination therapy of dual-drugs has attracted the medical scientists'attention and become a research hotspot owning to their action on the multiple targets of cancer cells at the same time.Considering these strategies,it's expected to improve clinical efficacy and reduce side-effects.Therefore,how to effectively reduce the toxic and side-effects and improve the antitumor efficacy has become the top priority of cancer treatment research.In order to achieve the above objectives,we constructed a series of nano-drug delivery systems based on charge-conversion combination cancer therapy in this dissertation.The block copolymers prepared by reversible addition-fragmentation chain transfer(RAFT)polymerization were used as nanocarriers.Simultaneously,the dual-drugs with different action mechanisms were grafted on the nanocarriers by chemical bonds which are sensitive to the microenvironment(such as pH,GSH or HAsc)of tumor tissues or extracellular of tumor cells,and then the charge-conversional group was induced onto the nanocarriers,which is sensitive to extracellular pH.These chemical bonds could be stable in the physiological circulation,which could inhibit the premature release of drugs,and reduce the toxic and side-effects.The surface charge of the nanoparticles could exhibit a charge-conversion from negative to postive,which could enhance the adsorption between the positively charged nanoparticles and the negatively charged tumor cell membrane.Depending on the nano-drug delivery system,the dual-drugs could concurrently reach the target and could kill or damage the cancer cells with distinct mechanisms and at different stages.As a result,cancer cells were eliminated with the synergistic effects by the dual-drugs,which could ont only improve the antitumor efficacy,but also could reverse multi-drug resistance.The main research results of this dissertation are as follows:(1)The polymeric prodrug m PPDPP-hyd-DOX was prepared by RAFT polymerization and"Click chemistry"reaction,then an intelligent dual-pH responsive nano-drug delivery system with a particle size at 81±1 nm(DLS method)was constructed.In the process of nanoparticles'transport from the mimicking systemic circulation(pH 7.4)to the extracellular in tumor tissues(pH 6.5),the surface charge of the polymeric micelles(M(DOX))exhibited a charge-conversion from-6.64±3.37 to 5.35±1.33 mV due to the protonation of the PDEA moieties.Compared with pH 7.4,the cytotoxicities,cellular uptake and apoptosis of cancer cells treated with M(DOX)at pH 6.5 were all significantly enhanced.(2)RAFT polymerization and"Click Chemistry"were utilized to prepare the polymeric prodrug PDPAO@imine-DOX/cis-6MP.6MP and DOX were coupled onto the PDPAO nanocarriers by GSH-sensitive Michael receptor and pH-sensitive imine bond,respectively,therefore,the intelligent nano-drug delivery system with dual-drugs for combination cancer therapy was constructed,which held an uniform particle size,narrow size distribution,high drug loadings with precise controllable drug ratios.The results of cytotoxicities showed that the synergistic effect of the dual-drugs against HeLa and HL-60 cells were DOX-dependent(higher synergistic effect with a higher DOX ratio),among which,free-(DOX₂/6MP)showed the highest synergistic effect against HeLa(CI=0.53)and HL-60(CI=0.32)cells,while M(DOX₂/6MP)also showed the strongest synergistic effect on HeLa(CI=0.62)and HL-60(CI=0.35)cells.Similarlly,the surface charge of the dual-drug conjugated polymeric micelles(M(DOX/6MP))could reversed from 9.31±1.11 to 7.29±0.76mV from the normal physiological environment(GSH 20 m M at pH 7.4)to the extracellular microenvironment of the tumor tissues,and the cytotoxicities and cellular uptake were significantly enhanced with the micellar form,hence,could improve the antitumor efficacy,this dual-drug based combination therapy could have great application in the treatment of acute myeloid leukemia.(3)The polymeric nanocarrier(PAIPO)was prepared by RAFT polymerization.PTX,DOX and Pt(II)(two drugs were randomly selected for the combination therapy,conjugated by GSH-sensitive disulfide bond,pH-sensitive imine bond and HAsc-sensitive platinum complex(Pt(IV)),respectively)were coupled onto PAIPO,and three kinds of dual-drug-conjugated nano-drug delivery systems(^DAM(PTX_n/Pt),^DAM(DOX_n/PTX),^DAM(DOX_n/Pt)and were constructed with the inducing pH-sensitive charge-reversal group(2,3-dimethyl maleic anhydride,DA).The particle size of the three combinatyional nano-drug delivery systems were ranging from 100-200 nm,which could not only be large enough to prevent premature leakage,but also small enough to avoid the capture of mononuclear phagocytosis system(MPS).During the process of transport from the mimicking physiological(pH7.4+GSH 20?M)to the extracellular microenvironment in tumor tissues,the surface charge of polymeric micelles could be reversed from approximately-10 to 5 mV due to the hydrolysis of the masking group DA molecule to expose the positively charged amino group on the surface.Simutaneously,The results of biochemical evaluation in vitro showed that this charge-conversional behavior could facilate the cellular uptake of micelles,enhance cytotoxicity and apoptosis rate of cancer cells.The cytoxic results further showed that ^DAM(PTX_0.2/Pt)(CI=0.37)and ^DAM(PTX₂/Pt)(CI=0.40)showed the strongest synergistic effect on HeLa and Skov-3 cell lines,respectively.For the combination of(DOX_n/PTX)group,^DAM(DOX₁/PTX)(CI=0.54)and ^DAM(DOX_0.5/PTX)(CI=0.46)showed the strongest synergistic effect on HeLa and Skov-3 cell lines,respectively.When it comes to the(DOX_n/Pt)group,^DAM(DOX_0.5/Pt)showed a strongest synergistic effect against Skov-3(CI=0.45),while exhibited a weaker synergistic effect against HeLa(CI=0.71-0.95).Taken together,except the(DOX_n/Pt)group of nano-drug delivery system for the combined therapy of cervical cancer,the other combined drug delivery systems echibit a strong synergistic effect in the treatment of cervical cancer and ovarian cancer,and could have important application prospects.