Study On Redox-responsive Prodrugs For Combination Cancer Chemotherapy

Cancer is one of the leading causes of death in the world and chemotherapy is the main treatment in cancer therapy,especially for cancers that could not be treated by topical therapy and metastatic cancers.However,the non-specific distribution of chemotherapy drugs causes lower bioavailability and associated severe side effects.Besides,the multidrug resistance of cancer cells also limits the therapeutic effect of single drug therapy.Combination chemotherapy is common in clinic practice for the treatment of cancer to enhance antitumor efficacy,reduce multi-drug resistance and decrease side effects.The taxane,paclitaxel(PTX),acts against a wide range of tumors by stabilizing microtubules and inhibiting the depolymerization of microtubules.Therefore,PTX disrupts normal cell mitosis and cell proliferation,and causes cell apoptosis.Doxorubicin(DOX)is an anthracycline antibiotic which could intercalate DNA and damage the structure of DNA,then induce apoptosis in tumor cells.PTX and DOX have different mechanisms of action for their antitumor effects.Besides,PTX block the cell cycle progression in late G2/M phases,while DOX is cell-cycle non-specific.Therefore,the combination of the two drugs may achieve combinative effect in cancer treatment.Many researches have shown that the combination of PTX and DOX significantly improved tumor response of breast cancer compared with PTX or DOX alone.However,the current cocktail administration still accompany with unexpected adverse effects due to the non-specific drug distribution.For example,the combination of DOX and PTX was limited by the dose-dependent side effects of neutropenia,neuropathy and cardiotoxicity.And the clinical pharmacokinetic studies indicated that DOX and PTX had a noncoordinated plasma distribution when administration in combination.Therefore,the major challenge is how to deliver the combined drugs of correct ratio to the target,as the drugs have dissimilar pharmacokinetics and biodistributions.D-?-tocopherol polyethylene glycol 1000 succinate(TPGS)is the biocompatible material that widely used as pharmaceutical nano-vehicles.In this work,we synthesized reduction responsive polymer-drug conjugates of TPGS-S-S-DOX and TPGS-S-S-PTX through a disulfide bond and prepared micelles for the co-delivery of DOX and PTX.The research included two main parts:(1)The preparation and characterization of TPGS-S-S-DOX/TPGS-S-S-PTX micelles,(2)the in vitro and in vivo evaluation of TPGS-S-S-DOX/TPGS-S-S-PTX micelles.The main researches were as follows:1.The preparation and characterization of TPGS-S-S-DOX/TPGS-S-S-PTX micellesFirst,dithiodipropionic anhydride(DTDPA)was obtained by the conversion of 3,3'-dithiodipropionic acid(DTDP),a disulfide donor,to increase the reactivity towards hydroxyl groups.Then TPGS with a terminated hydroxyl was activated with DTDPA,thus forming the TPGS-S-S-COOH,which is then further interacted with primary amine group of DOX and hydroxyl group of PTX to obtain the redox responsive TPGS-S-S-DOX and TPGS-S-S-PTX,respectively.The structures of the products were confirmed by measurement of melting point,1H NMR spectrum,FT-IR spectra and HPLC.The PTX content in TPGS-S-S-PTX was about 20%calculated with 1H NMR spectrum.The DOX content of the TPGS-S-S-DOX was about 24%determined by UV.The TPGS-S-S-DOX and TPGS-S-S-PTX can self-assemble into micelles in water.The TEM images revealed that the micelles were spherical in morphology.The mean diameters and zeta potential is 98.5±1.18nm and-9.89 ± 0.76m V,respectively.2.The in vitro and in vivo evaluation of TPGS-S-S-DOX/TPGS-S-S-PTX micellesIn vitro drug release experiments were performed by the dialysis method.The release of DOX and PTX was sustained in release medium without DTT.When adding 10mM DTT to the release medium,faster release rate and increased cumulative released amount of DOX and PTX were observed.The results revealed that the micelles have a reduction sensitive drug release property.The in vitro cytotoxicities of PTX?M(PTX)?DOX ? M(DOX)were evaluated against B16 cells by MTT assay.The results showed that all samples exhibited a time-and dose-dependent cell proliferation inhibition behavior.The effect of the drug ratio on the anti-tumor activity in B16 cells was also evaluated.The results indicated that the micelles with DOX/PTX ratio of 1:1 and 4:1(w/w)showed higher proliferative inhibition of B16 cells and synergistic effect.The cellular uptake and cell apoptosis was observed under fluorescent microscopy.The cellular uptake experiment indicated that the micelles can be taken up by the cells efficiently and a time dependent cellular accumulation was observed.The results of cell apoptosis showed that the micellar formulations of DOX/PTX can effectively inhibit the proliferative of B16 cells and the number of cells was decreased significantly.Chromatin condensation and nuclear fragmentation were also observed.In vivo anti-tumor efficiency was evaluated in B 16-bearing Kunming mice built by subcutaneous injection of B16 cells.The tumor size and body weight were monitored to evaluate the antitumor effect and the systematic safety,compared with free DOX/PTX combination.After in vivo antitumor experiment,the mice were sacrificed and the excised tumor and normal tissues were stained with hematoxylin and eosin(H&E)to observe the physiological change of each tissue.The results indicated that both micellar formulations of DOX/PTX and free DOX/PTX combination showed an antitumor effect,and higher tumor suppression was found in micellar formulations of DOX/PTX.Anorexia and body weight loss was observed in mice treated with free DOX/PTX combination,while body weight of the mice treated with micellar form was similar to those treated with NS.The results of H&E staining showed that the tumor of mice treated with micellar formulations of DOX/PTX has a greater necrosis level than that of mice treated with free DOX/PTX combination,and no obvious systematic toxicity was found.