Research On Intelligent Responsive Polymer-drug Conjugates Based On Doxorubicin For Synergistic Antitumor

Cancer,as one of the deadliest diseases in the world,remains a serious threat to human health.At present,the treatment of tumor is mainly surgical treatment,radiotherapy and drug treatment,drug treatment still plays a considerable role.However,drug resistance is easy to appear since patients use single chemotherapy drug,which causes the therapeutic effect remarkably reduce.Combined treatment is gradually becoming an effective new means of treating cancer.Photodynamic therapy(PDT)originated in 1900,Germany Raab first found that the combination of light and photosensitizer could produce cytotoxic effects.After decades of development,it has been proved that PDT can treat effectively non-small cell lung cancer and esophageal cancer.In order to reduce the appearance of drug resistance,the combination of a variety of chemotherapy drugs or chemotherapeutics and photosensitizers is employed to inhibit different metabolic pathways and thus increasing the apoptosis or necrosis of cancer cells.However,the rapid removal and non-specific distribution of chemotherapy drugs seriously reduce the effectiveness of chemotherapy,inevitably resulting in systemic toxicity.There are also many problems with photosensitizer,such as low water solubility,nontarget delivery and poor stability.Therefore,in order to maximize the effect of combined therapy and reduce its toxicity to normal tissues,it is a promising research direction to covalently graft the drugs into macromolecular polymers to form polymer-drug conjugates.Through the investigation of previous work,we choose anticancer drugs docetaxel(DTX),doxorubicin(DOX)and the second generation photosensitizer chlorin e6(Ce6)as model drugs to study tumor microenvironment responsive polymer-drug conjugates for synergistic cancer therapy.The study consists of two aspects:(1)Different chemotherapy drugs were coupled with amphiphilic block copolymer respectively,using the reduction-sensitive disulfide bond as a bridge.The conjugates could self-assemble into mixed micelle in water to realize the combination of various chemotherapy drugs.(2)We integrated both chemotherapeutic drug and photosensitizer on polymers with active targeting ability and good biocompatibility via pH-sensitive hydrazone bond,which achieve the combined application of chemotherapy and photodynamic therapy.The specific research contents and methods are as follows:1.Tumor reduction microenvironment responsive polymer-drug mixture micelles for co-delivery of drugs with synergistic cancer therapyDTX,as highly effective anticancer drug,has been approved to treat a variety of tumors by FDA.Tween 80 is often utilized to increase solubilization of DTX in clinic,but tween 80 will cause serious hypersensitivity and neurotoxicity in phase one clinical trial.In addition,DTX is limited in clinical application due to neutropenia and other immune system toxicity,which bring great damage to the body and mind of the patient.DOX is one of the most commonly used drugs in breast cancer chemotherapy.The mechanism of action is mainly that glycoside ligands of DOX are embedded in DNA,inhibiting the synthesis of DNA and RNA.There are also toxic side effects such as severe bone marrow inhibition and heart toxicity in clinical application of DOX.In order to solve the poor solubility and systemic toxicity of chemotherapy drugs,we designed an amphiphilic block copolymer mPEG2k-PCL2k-SS-COOH.The mPEG2k-PCL2k-SS-DOX and mPEG2k-PCL2k-SS-DTX conjugates were prepared via integrating DOX and DTX on the copolymer respectively.The successfully synthesis of the product was proved by 1H-NMR and FT-IR.The mixed micelles mPEG-PCL-SS-DOX/mPEG-PCL-SS-DTX(molar ratio =1:1)was prepared by dialysis method,and the critical micelle concentration(CMC)of mixed micelles was determined by pyrene probe method,which was 1.8×10-4mM.The mixed micelle showed spherical morphology and good dispersion with a size of 223.7 nm by TEM and DLS.The release behavior of mixed micelles in vitro was investigated by dynamic membrane dialysis,and the results showed that the formulation had obvious slow-release effect and had reduction-sensitive release.The hemolysis test displayed that the biocompatibility of mixed micelles was good,and the cellular uptake and cytotoxicity experiments in vitro indicated that the mixed micelle could accumulate effectively in MCF-7 cells and inhibit the growth of tumor cells.Therefore,the reduction-sensitive mixed micelles,which we designed from self-assembly of polymer-drug conjugates,can be used as a promising drugs co-delivery system for cancer treatment.2.Tumor pH-responsive polymer-drug nanoparticles for chemo-photodynamic therapyCe6 has been approved by the FDA and can be used for clinical photodynamic diagnosis.It has been characterized by high yield of singlet oxygen after light irradiation,fast elimination in vivo and small toxic side effects.However,Ce6 still needs to be improved for its non-specific distribution and hydrophobicity,the strong hydrophobicity of Ce6 affects its dispersion in physiological media,causing agglomeration and reducing the yield of ROS and limits its clinical application.HA,as a linear macromolecular mucopolysaccharide with good biocompatibility and hydrophilicity,can bind to overexpressed CD44 receptors on the surface of tumor cells to enhance the internalization of formulation.We use HA as the carrier material to construct the tumor target drug delivery system.HA-DOX-Ce6(HDC)conjugates were obtained by grafting DOX and Ce6 on HA via pH-sensitive bond.1H-NMR and FT-IR showed that the synthesis of conjugates was successful.HDC nanoparticles(HDC NPs)were prepared by direct dissolution,TEM and DLS showed that HDC NPs had good spherical structure,good dispersion and a size of 152.8 nm.In vitro release experiments,HDC NPs had the characteristics of pH-sensitive release.The assay of detection of singlet oxygen(1O2)in vitro demonstrated HDC NPs have stronger ability of singlet oxygen generation compared with free Ce6.In intracellular ROS detection experiments,the strongest fluorescence of HDC NPs under laser irradiation confirmed this conclusion again.HDC NPs could dramatically improve the cellular uptake of drugs due to its active target.Anticancer activities both in vitro and in vivo clearly exhibited that HDC NPs had the strongest antitumor efficacy,demonstrating the superiority of combined chemotherapy and PDT.HDC NPs have good targeting ability and fluorescence imaging ability from the biodistribution experiment in vivo.Our synthetic HDC NPs were capable of pH-response,self-targeting delivery and NIR fluorescence imaging-guided chemo-photodynamic therapy,which provide more possibilities for the study of multifunctional chemo-photodynamic therapy.In summary,we have carried out a detailed study on the use of tumor microenvironment responsive polymer-drug conjugates for synergistic cancer therapy,the results show that either the combination of chemotherapy drugs or chemo-photodynamic therapy can be as highly efficient nanoplatform to combat with cancers.The preparation of polymer-drug mixture micelles and polymer-drug nanoparticles can improve the dissolution of hydrophobic drugs,reduce systemic toxicity as well as enhance the therapeutic effect via EPR effect and active targeting.Our work provides more possibilities for the application of polymer-drug conjugates in the biomedical field.