Regulate The Immune Microenvironment Of Breast Cancer By Dual-drug Co-loaded Hydrogel

Objective:Breast cancer is the malignant tumor with the highest incidence among women and poses a serious threat to women’s health.The existing therapeutic methods have some problems,such as poor selectivity,toxicity and side effects,and easy to produce drug resistance,which restrict the therapeutic effect of breast cancer.In recent years,immunotherapy has shown great promise,but the complex tumor microenvironment enables tumor cells to achieve immune escape through a variety of mechanisms,so the single application of immunotherapy is limited.Some chemotherapy drugs can induce a certain degree of anti-tumor immune response while eliminating tumor cells,improving patients’response rate to immunotherapy.Therefore,combination of chemotherapy and immunotherapy is expected to improve the therapeutic effect of breast cancer.Previous studies have found that the chemotherapy drug gambogic acid（GA）can not only effectively kill tumor cells,but also induce immunogenic cell death（ICD）,which is expected to play a synergistic anti-tumor effect with immunotherapy.It has been reported that tumor will acquire immune resistance by driving the expression indoleamine2,3-dioxygenase（IDO）,making the immune stiffness induced by chemotherapy weaker.Appling of IDO inhibitors can overcome the related immune resistance and enhance the anti-tumor immune response.Therefore,this thesis intends to construct a dual-drug co-loaded hydrogel local delivery system GM-1MT@Gel with good biocompatibility for co-delivery of gambogic acid and IDO inhibitor（1MT）,to study the synergic effect of gambogic acid and 1MT on breast cancer through chemotherapy and immunotherapy,and to explore the synergic effect and mechanism of the two in regulating breast cancer immune microenvironment.To provide a new treatment strategy for breast cancer treatment.Methods:Firstly,the prodrug GM was synthesized by esterification reaction of GA and mPEG₂₀₀₀,and the porous hydrogel carrier was formed by cross-linking chitosan andβ-glycerol phosphate disodium salt.The microstructures and rheological properties of hydrogels were investigated by scanning electron microscopy（SEM）and rheology,and the drug release characteristics in vitro and degradation characteristics in vivo were also studied.Then,the synergistic effect of GA/GM with 1MT and its ability to induce ICDs were investigated at the cellular level.Further,the in vivo antitumor effect and immunoregulatory mechanism of GM-1MT@Gel were studied by establishing a mouse breast cancer model in situ.Finally,the biodistribution and biosafety of GM-1MT@Gel were investigated.Results:First,¹H NMR spectra proved that GM synthesis was successful,SEM showed that the inside of the hydrogel was three-dimensional network structure,rheological test results showed that the hydrogel could form stable gel at 37℃.The results of drug release and distribution in vivo indicate that GM-1MT@Gel can accumulate in the tumor site for a long time.Secondly,1MT could enhance the antitumor activity of GA/GM,inhibit cell migration and promote cell apoptosis,which had a synergistic effect.GA/GM could induce ICD,and the ICD effect was enhanced after combined with 1MT.Finally,in vivo antitumor studies,GM-1MT@Gel was more effective in inhibiting tumor growth,inducing ICDs,calreticulin（CRT）exposure,and High-mobility group box1（HMGB1）secretion than free（GM+1MT）,monotherapy（1MT@Gel,GM@Gel）,and positive paclitaxel（albumin therapy）,thus promoting dendritic cell maturation.Increase CD8⁺T lymphocyte infiltration in tumor microenvironment.At the same time,GM-1MT@Gel could inhibit the degradation of tryptophan,down-regulate regulatory T cells,and up-regulate the levels of cytokines IFN-γ,TNF-αand IL-6,thus reshaping the breast cancer immune microenvironment and activating the anti-tumor immune response.Conclusion:In summary,a dual-drug co-loaded hydrogel local delivery system GM-1MT@Gel was successfully constructed,which increased drug accumulation at the tumor site and could slow drug release,significantly enhancing the anti-breast cancer effect at the cellular level and in model mice.GA combined with 1MT can activate anti-tumor immune response by inducing ICD,increase cytotoxic T lymphocyte infiltration,interfere with IDO activity,reverse breast cancer immunosuppressive microenvironment,transform"cold"tumor（non-T cell infiltrating tumor）into"hot"tumor（T cell inflammatory tumor）,and achieve synergistic chemotherapy and immune treatment of breast cancer.