Synthesis Of NK Cell-mimetic Multifunctional Nanohybrids And Application In Breast Cancer Immunotherapy

Cancer is currently one of the main causes of human death,and breast cancer is the most frequently diagnosed malignant tumor in women.Immunotherapy has brought groundbreaking changes to cancer treatment,especially the immune checkpoint blockade(ICB),as one of the most attractive strategies,however,clinical application of ICB was limited by insufficient immunogenicity in tumor sites.Breast cancer known as a kind of immune "cold" tumor,which is lack of immunogenicity,with low lymphocyte infiltration efficiency,harbors an immunosuppressive tumor microenvironment.Therefore,transferring the "cold"microenvironment into"hot" one is the key to breast cancer treatmentCurrently,chemotherapy as the cornerstone of modern cancer treatment,also plays a paramount role in the process of breast cancer treatment.Unfortunately,the alone use of chemotherapeutics mostly leads to serious adverse reactions and drug resistance.Certain chemotherapeutics such as oxaliplatin(OXA)could induce immunogenic cell death(ICD)in cancerous cells.Antigen molecules induced by ICD can promote the infiltration of toxic T lymphocytes(CTL)and accelerate tumor regression.Therefore,we adopt chemotherapy combined with immune checkpoints for breast cancer treatment.Indoleamine 2,3 dioxygenase(IDO)is an important immune checkpoint.IDO can decompose tryptophan(Trp)into kynurenine(Kyn).Deficiency of Trp will cause the immune cells stagnate of growth even apoptosis.The accumulation of Kyn leads to the production of regulatory T(Treg)cells,inhibits the activation of cytotoxic lymphocytes,resulting in immune escape.Preclinical IDO inhibitors,such as 1-methyl-d-tryptophan(1-MT),have shown potential to enhance the efficacy of immunotherapy.However,due to the complex immune environment in tumors and other inhibitory factors,the clinical effect of a single IDO inhibitor is not well.At the same time,insufficient tumor targeting and poor water solubility also hinder its clinical application.For as such,the co-delivery of multiple drugs to the tumor site is the main difficulty that needs to be dealt urgently.In most malignant tumors,including breast cancer,tumor-associated macrophages(TAM),play a key role in tumor tissue remodeling,angiogenesis,and immune resistance,as the most abundant stromal cells.TAM can be divided into pro-inflammatory M1 type and anti-inflammatory M2 type.M1 type macrophages can kill tumor cells by secreting a large number of cytokines,while M2 type macrophages can promote angiogenesis and tumor growth.In the immunosuppressive environment of tumors,the proportion of M2 type is higher than that of M1 type.Therefore,converting TAM into M1 type as much as possible will be beneficial to the treatment of tumors.Biomimetic nanoparticles are emerging delivery vehicles,mainly through the masking effect of the cell membrane on the nanoparticles to improve the efficiency of drug delivery.This can endow the nanoparticles with targeting functions,and even improve the effect of the loaded drugs.Therefore,cell membrane of natural killer cells(NK)was been used to mask the nanoparticles to improve tumor homing ability and reduce side effects in our project.In addition,the specific membrane proteins of NK cells can induce anti-tumor M1 polarization of tumor-associated macrophages(TAM),reduce immunosuppressive M2 polarization,and further reverse the tumor’s immunosuppressive microenvironmentIn response to the above problems,we designed an NK cell-mimetic nanohybrids,co-loaded with 1-MT and OX A.The nano-drug delivery system utilizes the targeting properties of NK cell membranes,which is enriched in tumor tissues,induces TAM polarization,and the drugs were released under the acidic and reducibility environment catalysis.OXA induces ICD in tumor cells and accelerates the maturation of dendritic cells,which leads to CTL infiltration in tumors;1-MT up-regulates the ratio of Trp to Kyn to relieve immunosuppression caused by Treg and transform the immune environment from "cold" to "hot".This chemoimmunotherapeutic delivery system could synergistically inhibit breast cancer,according to research content as follows:1.Preparation and characterization of NK cell-mimetic nanohybridsThe prodrug monomer of PEG-modified OXA-1-MT is prepared by chemical bonding,the drug-based hybrids is prepared through the dialysis method,and NK cell-mimetic modification is performed to prepare NK-DNH.TEM observations showed that the nano-formulations were uniformly rounded,and DLS inspections showed that the particle sizes of DNH and NK-DNH were 69.3 nm and 83.4 nm,respectively.SDS-PAGE proved that the NK membrane was successfully wrapped,and WB results further confirmed that the surface of NK-DNH contained NK cell membrane proteins.It has been verified by experiments that it has good stability in vitro and can release drugs responsively.2.In vitro uptake and efficacy evaluation of NK cell-mimetic nanohybridsThe 4T1 cell line was used to investigate the performance in vitro.The results show that NK-DNH can be taken by tumor cells effectively.The in vitro tumor killing ability was investigated by MTT method,which proved that it can effectively inhibit the viability of tumor cells.It can also induce the immunogenic death of tumor cells,which marked by CRT efflux,ATP secretion,and HMGB1 release,and the results of flow cytometry showed that the treated tumor cells contributed to the maturation of dendritic cells.3.Evaluation of the efficacy and safety of NK cell-mimetic nanohybrids in vivoIn vivo experimental evaluation utilized mouse breast cancer model showed that NK-DNH has good tumor targeting ability and can inhibit tumor growth significantly.The mechanism was studied through flow cytometry and ELISA,and various tumor tissues were analyzed.The proportion of immune cells indicates that NK-DNH can increase the infiltration of effector T cells,promote the conversion of macrophages to the M1 phenotype,and down-regulate the number of Treg cells.Based on the above results,the combined treatment method can effectively improve the immune microenvironment of tumors,and recruit immune cells in the body to jointly destroy cancer cells.This object providing new ideas for the development and clinical application of tumor treatment preparations.