Immunoadjuvant Nanoparticles For Enhanced Photo-Immunotherapy In Triple-Negative Breast Cancer

Triple-negative breast cancer(TNBC)is the most aggressive subtype of breast cancer.Due to the high metastasis characteristics,its traditional clinical treatment is not effective.Therefore,it is urgent to find more effective treatment strategies.In recent years,the combination of photothermal therapy and immunotherapy has made great progress in cancer treatment.Photothermal therapy(PTT)can not only effectively kill solid tumors,but also stimulate the body’s immune response by releasing tumor-related inflammatory proteins,so that when combined with immunotherapy,it can achieve the synergistic effect of tumor treatment.Among them,Indocyanine green(ICG)mediated photothermal therapy combined with immune adjuvant glycated chitosan(GC)has been clinically tested for patients with advanced breast cancer,and it can not only remove solid tumors in situ,but also effectively inhibit distant metastases.However,ICG and GC used in clinical treatment are injected separately,and the rapid metabolism of ICG can easily affect the photothermal effect.At the same time,GC is difficult to effectively interact with cells due to its large molecule.Therefore,in view of these problems,we constructed an integrated nanoparticle GC@ICG for the treatment of triple-negative breast cancer combined with photothermal therapy.The specific research content is as follows:1.Preparation and characterization of GC@ICG nanoparticles.Firstly,5β-cholic acid(5βCA)was coupled to GC through amide bond to obtain polymer(5βCA-GC),and ICG was self-assembled to obtain GC@ICG nanoparticles.The chemical structure,morphology and photothermal conversion performance of the nanoparticles were evaluated by Fourier transform infrared spectrometer,scanning electron microscope(SEM)and other instruments,which laid a foundation for subsequent experiments.The results show that:the carboxyl group of 5βCA was successfully combined with the amino group of GC through the amide bond,and the synthesized GC@ICG nanoparticles exhibited spherical morphology with good uniformity,good stability and photothermal conversion performance.2.Cytological evaluation of GC@ICG nanoparticles in vitro.The CCK-8 assay was used to detect the toxicity of GC@ICG to cells,and the uptake of GC@ICG nanoparticles by cells was analyzed by confocal microscopy.In addition,the effects of cytotoxicity and photothermal killing induced by GC@ICG nanoparticles under near-infrared laser irradiation and the death of immunogenic tumor cells induced by photothermal therapy based on GC@ICG nanoparticles were also determined.The results show that:GC@ICG nanoparticles can enter into tumor cells with little toxicity;under laser irradiation,GC@ICG absorbs light energy to generate heat to kill tumor cells,and at the same time localizes in tumor cells.GC can bind to the antigen produced in the process of apoptosis,further enhancing the effect of immune response.3.GC@ICG in vivo animal evaluation of nanoparticles.First,a 4T1 mouse breast cancer model was constructed;the effect of the treatment was evaluated by monitoring the changes in the tumor size and body weight of the mice in each group after treatment;H&E staining and immunofluorescence staining were performed to analyze the biocompatibility of GC@ICG nanoparticles in vivo and the specific antitumor immune response.The results show that:GC@ICG combined with photothermal therapy can not only effectively kill tumor cells in situ,but also induce long-term anti-tumor immune effects and inhibit tumor metastasis;in addition,the results of immunofluorescence staining of distal tumor sections in the treatment group showed that after laser+GC@ICG treatment,it can effectively up-regulate the number of anti-tumor immune cells(CD8~+T cells)and down-regulate the number of M2 macrophages and regulatory T cells,exerting an excellent anti-tumor immune effect.And no obvious side effects on mice.In a word,we constructed an integrated GC@ICG nanoparticle,which can not only enter into the interior of tumor cells to effectively kill tumor cells,but also effectively inhibiting the growth of distant metastases.This strategy provides a new idea for the clinical treatment of triple-negative breast cancer.