Synthesis Of Hyperbranched Polyglycerol Derivatives And Their Application In Cancer Therapy

Cancer is one of the main diseases that threaten people's health and life,and chemotherapy is the main method for cancer therapy.However,there are several shortcomings of chemotherapy drugs,such as low water solubility,poor targeting,short half-life and serious side effects.Furthermore,multidrug resistance of tumor cells to chemotherapeutic agents is a major obstacle to the success of cancer chemotherapy.To solve these problems,many polymeric drug delivery systems have been paid extensive attentions by researchers in recent years.The application of hyperbranched polyglycerol(HPG)derivatives as drug carriers have favorable and wide application value and development prospect,due to its advantage of good water-solubility,stability,easy modification and excellent biocompatibility.In this paper,drug and gene co-delivery systems and drug carrier for reversion of multidrug resistance based on HPG derivative were prepared for tumor treatment.Includes the following three parts:(1)The drug/gene co-delivery method has emerged to yield promising results in cancer therapy,because the strategy could reduce doses of drug,reduce the adverse side effect decrease the occurrence and impact of drug resistance,and also enhance synergistic effects.However,how to design a drug/gene co-delivery carriers with high gene transfection efficiency and low cytotoxicity is still a challenge.To realize the co-delivery of docetaxel and pMMP-9 efficiently into tumor cells,a star-shaped amphiphilic copolymer consisting of hyperbranched polyglycerol derivative(HPG-C18)and dendritic poly(L-lysine)(PLLD)was synthesized by the click reaction between azido-modified HPG-C18 and propargyl focal point PLLD.The obtained HPG-C18-PLLD could form the nanocomplexes with docetaxel and pMMP-9.The gel electrophoresis assay was performed to test the gene binding ability of HPG-C18-PLLD,and the particle sizes and zeta potentials of the complex were characterized by DLS and transmission electron microscope(TEM).The complexes showed good gene delivery ability in vitro by inducing an obvious decrease in MMP-9 protein expression in MCF-7 cells.The apoptosis assay showed that the complex could induce a more significant apoptosis to breast cancer cells than that of docetaxel or pMMP-9 used alone.In vivo assay indicated that the codelivery strategy displayed a better effect on tumor inhibition.(2)In order to realize high transfection efficiency and low toxicity,and according to the characteristics of tumor microenvironment,a star cationic polymer is designed.The oligoethylenimine-conjugated?-cyclodextrin(?-CD-PEI600)and benzimidazole-modified 4-arms-polycaprolactone-initiated hyperbranched polyglycerol(PCL-HPG-BM)were synthesized as the host and guest molecules respectively,and then the co-delivery carrier of PCL-HPG-PEI600was formed from the pH-mediated inclusion interaction between?-CD and BM.The physical and chemical properties of PCL-HPG-PEI600 were confirmed by proton nuclear magnetic resonance(~1H NMR),gel permeation chromatography(GPC),fluorescence spectroscopy,dynamic light scattering(DLS)and isothermal titration calorimetry(ITC).For DOX delivery,PCL-HPG-PEI600/DOX displayed the pH-controlled DOX release and concentration-dependent inhibition effect on MCF-7 cells.For pMMP-9 delivery,the gel electrophoresis assay was performed to test the gene binding ability of PCL-HPG-PEI600,and the particle sizes and zeta potentials of the complex were characterized by DLS and transmission electron microscope(TEM).PCL-HPG-PEI600 showed excellent pMMP-9 delivery ability with significantly higher transfection efficiency than PEI25k in vitro and in vivo,especially in the presence of serum.It was also found that PCL-HPG-PEI600 could help pMMP-9 escape from the endosome due to its pH-sensitivity.For the co-delivery of PCL-HPG-PEI600/DOX/pMMP-9,it was found that the co-delivery strategy was much better than single DOX or pMMP-9 formulation used.PCL-HPG-PEI600/DOX/pMMP-9 resulted in the better inhibition effect on MCF-7 cells proliferation and migration in vitro.The cellular uptake pathway was also explored and found that the clathrin-dependent endocytosis was the main endocytosis pathway for PCL-HPG-PEI600 complexes into MCF-7 cells.The xenograft tumor model of breast cancer in BALB/C nude mice were established to study the in vivo antitumor efficacy.The result showed that the PCL-HPG-PEI600/DOX/pMMP-9 group exhibited the best antitumor efficacy.Moreover,PCL-HPG-PEI600displayed non-toxicity and excellent blood compatibility.(3)Multidrug resistance(MDR)is a significant impediment to the success of cancer chemotherapy.To overcome the multidrug resistance of tumors and improve the efficacy of chemotherapy,based on PCL-HPG,folate-targeted polymer(PCL-HPG-TPGS-FA)was prepared by introducing D-?-tocopheryl polyethylene glycol succinate(TPGS)and folic acid(FA).The physical and chemical properties of PCL-HPG-TPGS-FA were confirmed by ~1H NMR,DLS and TEM.DOX was selected as a model drug,The stability of the PCL-HPG-TPGS-FA/DOX was evaluated by dispersion in two commonly used biological media,pH 7.4 PBS and 10%FBS.The stability assay showed that the PCL-HPG-TPGS-FA/DOX remained stable with a slight size change during 7 days in both PBS and FBS.In vitro studies,Compared with free DOX and PCL-HPG-TPGS/DOX,PCL-HPG-TPGS-FA/DOX cause more cytotoxicity to MCF-7/ADR cells.The cellular uptake of PCL-HPG-TPGS-FA/DOX in MCF-7 and MCF-7/ADR cells was measured by flow cytometry and confocal laser scanning microscopy(CLSM).The results confirmed that PCL-HPG-TPGS-FA/DOX could enhance cellular uptake and increase the accumulation of DOX in MCF-7/ADR cells.Mitochondrial membrane potential change and ATP content have been measured,the results indicated blank PCL-HPG-TPGS-FA could inhibit the P-gp activity by reducing the mitochondrial membrane potential and depleting ATP content.The xenograft tumor model of drug-resistant breast cancer in BALB/C nude mice were established to study the in vivo antitumor efficacy.The result showed that the PCL-HPG-TPGS-FA/DOX group exhibited the best antitumor efficacy.Moreover,PCL-HPG-TPGS-FA displayed non-toxicity and excellent blood compatibility.