Tumor Microenvironment-responsive Polymer Drug Delivery System For Delivering DTX And YC-1 To Synergistically Treat Triple-negative Breast Cancer

Objective:Triple negative breast cancer（TNBC）has malignant biological behavior with high invasiveness and high metastasis.Compared with other subtypes of breast cancer,TNBC is characterized by high heterogeneity,young onset age,high recurrence and metastasis rate,rapid progression and poor prognosis,which has become the hot and difficult issue of breast cancer.At present,the research on signal transduction pathway and regulatory mechanism related to TNBC is still incomplete,and the occurrence and development of TNBC is a multi-factor,multi-step complex process.Therefore,in order to achieve a better efficacy,therapy targeting multiple signaling pathways and multiple gene targets should be combined on the basis of chemotherapy and other therapeutic methods.In recent years,FDA has been actively advocating the development of new drug combination therapy of TNBC.Hypoxia inducible factor 1-α（HIF-1α）and its target gene products are highly active in TNBC and play an important role in tumorigenesis,angiogenesis,invasion,immune escape,drug resistance and other physiological activities.Therefore,it is necessary to study whether cytotoxic chemotherapeutic drugs combined with HIF-1αinhibitors can improve the therapeutic effect of TNBC.In addition,how to overcome the complex physiological environment of human body and deliver the combined therapeutic drugs to the tumor site is a hot and difficult research topic at present.Polymer drug delivery system provides a paradigm shift for the treatment of tumors.The construction of new anti-tumor drugs with high biosafety polymers will significantly improve the defects of traditional drug dosage forms,such as improving water solubility,bioavailability and tumor targeting.A variety of polymer drug delivery systems are in clinical trials.For example,Xyotax^?,a conjugate of polyglutamate and paclitaxel,has shown good pharmacokinetics and high anti-tumor activity in clinical trials,showing great application value and development prospect.In various types of polymer drug delivery systems,tumor microenvironment-responsive polymer drug delivery systems can remain stable in the blood circulation,selectively enrich in tumor tissues and release drugs in response to tumor microenvironment.Thus,it can achieve efficient targeted drug delivery to tumor tissue,while reducing adverse reactions to normal tissue,thus improving the therapeutic index.Based on the above research background,in this study,Docetaxel（DTX）was combined with Lificiguat（YC-1）,a HIF-1αinhibitor,to investigate the feasibility of cytotoxic chemotherapy combined with HIF-1αinhibitor to improve the therapeutic effect of TNBC.At the same time,this study explored how to construct a tumor microenvironment-responsive polymer drug co-delivery system for delivering DTX and YC-1,and studied their synergistic treatment of TNBC with the help of image means such as fluorescence imaging,microcomputer tomography（Micro CT）.Materials and Method:Human MDA-MB-231 cell line was used as the anti-tumor model of TNBC in vitro,and MDA-MB-231 cell xenograft was established in Balb/c nude mice as the anti-tumor model of TNBC in vivo.Fluorescence imaging technology was used to monitor and evaluate the behavior of polymer drug delivery system at the cellular level and in animals.Pathological analysis and Micro CT were used to study the biosafety of polymer drug delivery system and evaluate the metastasis of tumor.The specific research methods are as follows:1.The combination index（CI）of DTX and YC-1 was investigated to judge whether the two drugs had synergistic anti-TNBC effect.The anti-TNBC mechanism of YC-1 was studied.On this basis,the synergistic anti-tumor mechanism of YC-1 and DTX was explored.2.By constructing two kinds of tumor microenvironment-responsive polymer drug delivery systems,the effects of tumor high enrichment and responsive drug release on the anti-tumor effect of polymer drug delivery system were studied,respectively.The polymer drug delivery systems were characterized by ¹H nuclear magnetic resonance spectra（¹H NMR）,gel permeation chromatography（GPC）,and dynamic light scattering（DLS）.The cellular uptake of polymer drug delivery systems and their distribution in vivo were studied by fluorescence imaging.The effects of tumor enrichment and responsive drug release on the anti-TNBC effect of polymer drug delivery system were studied in vitro and in vivo.3.On the basis of previous research,a tumor microenvironment-responsive polymer drug co-delivery system with tumor enrichment performance and responsive drug release performance was designed and constructed for delivering DTX and YC-1.The anti-TNBC effect of the polymer drug co-delivery system in vivo and in vitro was investigated.Results:1.YC-1 inhibited the proliferation of MDA-MB-231 cells in a concentration-dependent manner.YC-1 inhibited the expression of HIF-1αand the invasion of MDA-MB-231 cells.CI value of YC-1 combined with DTX was basically within the range of<1.Compared with the single drug group,the mitochondrial membrane potential of YC-1 combined with DTX decreased significantly,and the apoptosis rate and G2/M phase cell ratio increased significantly,while the G2/M checkpoint protein changed abnormally.2.Two kinds of tumor microenvironment-responsive polymer drug delivery systems were successfully constructed.Both of them could self-assemble into uniform spherical particles with size of about 110～120nm.Fluorescence imaging results showed that the polymer drug delivery system had long retention time and high enrichment at tumor site,and it had higher anti-tumor efficiency in vivo than free drugs.The tetrapeptide Gly-Phe-Leu-Gly（GFLG）junction in the polymer drug delivery system was broken under the action of papain,which has a similar effect to cathepsin B,releasing the drug DTX.The results of anti-tumor experiments in vivo and in vitro confirmed that the performance of responsive drug release improved the anti-TNBC effect of polymer drug delivery system.3.Based on the above polymer drug delivery system,a tumor microenvironment-responsive polymer drug co-delivery system was designed and constructed.The polymer drug co-delivery system could self-assemble into regular and uniform spherical particles with a particle size of about 60nm.The polymer drug co-delivery system was capable of responsive drug release in response to papain.The results of in vitro anti-tumor experiments showed that the co-delivery system had the same synergistic anti-tumor mechanism as free drugs.In addition,the fluorescence imaging results showed that the drug co-delivery system had a long retention time and high concentration in the tumor site.The drug co-delivery system reduced the toxic and side effects of free drugs and had a higher anti-tumor effect in vivo.Conclusion1.Cytotoxic chemotherapy drug DTX combined with HIF-1αinhibitor YC-1 has synergistic anti-TNBC effect.The combination of DTX and YC-1 inhibited the function of G2/M checkpoint and promoted the apoptosis of tumor cells.Compared with monotherapy,DTX combined with YC-1 can improve the therapeutic effect of TNBC in situ tumor and inhibit tumor metastasis.Our study provides the studying foundation and reference for clinical screening of TNBC therapy drugs.2.The molecular structure and physicochemical properties of polymer drug delivery system affect its biological effect.Tumor enrichment performance and responsive drug release performance are important factors affecting the anti-tumor effect of polymer drug delivery system.3.By constructing a tumor microenvironment-responsive polymer drug co-delivery system,the enrichment of the combined drugs in the tumor site was increased,and the drugs were selectively released in the tumor cells,which reduced the toxicity of the free combined drugs and improved the therapeutic effect of the combined drugs on TNBC.Our study provides design idea,studying foundation and technical support for the construction of polymer drug co-delivery system.4.Imaging technology plays an important guiding role in the process of treatment.Fluorescence imaging technology can observe the distribution of polymer drug delivery system in vivo and realize the real-time monitoring of drugs in vivo.Confocal imaging can be used to study the behavior of therapeutic drugs in vitro,especially at the cellular level,and to explore the anti-tumor mechanism of drugs.Micro CT technology has the reference value for evaluating the health status of tumor-bearing mice.