Immunosuppression Improvement-Innate Immunity Activation By Nanosystem For Boosting Antitumor Chemo-Immunotherapy

Due to the complexity of tumor pathogenesis,chemo-immunotherapy is one of the most promising treatments for malignancies.However,the immunosuppressive tumor microenvironment（TME）not only prevents the formation of an effective anti-tumor innate immune response at the tumor site,but also further hinders the efficacy of chemotherapy.Therefore,improving immunosuppressive TME and maximizing the induction of effective innate immune responses is an urgent problem for chemotherapy combined with immunotherapy.Based on that,this study constructed a differential delivery nanosystem that improved immunosuppression and activated innate immunity simultaneously to achieve synergistic effects of chemo-immunotherapy.The main research of this paper included the following aspects:1.Synthesis and characterization of CA-1@HMnO₂/HA-P-mAbFirstly,manganese dioxide shells were encapsulated by redox method using silica as a template,and then the silica core was removed by etching with sodium carbonate to obtain hollow mesoporous manganese dioxide（HMnO₂）nanoparticles.And HMnO₂was loaded with hydrophobic chemotherapeutic agent curcumin analog（CA-1）（homemade）.Next,it was modified with hyaluronic acid（HA）layer by layer.The obtained nanoparticles were coupled with matrix metalloproteinase-responsive peptide（P）and monoclonal antibody（mAb）specifically targeting regulatory T cells（Treg）sequentially by amide reaction.Finally,the successful preparation of CA-1@HMnO₂/HA-P-mAb nanosystems were characterized using XPS analysis,BET analysis,UV-Vis full wavelength scanning,laser nanoparticle sizer,transmission electron microscopy,elemental mapping analysis and drug loading.The results showed that the HMnO₂ nanoparticles had a clear hollow structure with an average pore radius of 5.1±0.3 nm,and the CA-1@HMnO₂/HA-P-mAb nanoparticles have a uniformly dispersed spherical shape with a particle size of 257.7±15.7 nm,a potential of-11.5±0.6 m V,an encapsulation efficiency of 93.4±1.8%,and a drug loading efficiency of23.7±0.3%.2.In vitro characterization of CA-1@HMnO₂/HA-P-mAbTo examine the enzymatic responsiveness of peptide（P）,the changes of P before and after warming by matrix metalloproteinase-2（MMP-2）were analyzed by HPLC;the glutathione responsive properties of the nanosystem were examined by TEM results to reveal the intelligent responsive CA-1 release pattern.The results showed that the P in the CA-1@HMnO₂/HA-P-mAb nanosystem underwent breakage under the condition of MMP-2;the HMnO₂ nanoparticles collapsed rapidly to release CA-1 in response to the high GSH in tumor cells,and the cumulative release rate could reach75.9±2.5%at 72 h.3.In vitro study of the antitumor activity of CA-1@HMnO₂/HA-P-mAbIn vitro studies were performed using mouse breast cancer 4T1 cells.First,the CD44 receptors on the surface of 4T1 cells were blocked using HA pretreatment,and the uptake of FITC@HMnO₂/HA-P-mAb nanosystem was investigated by CLSM and flow cytometry;the mechanism of CA-1-induced apoptosis was elucidated by JC-1fluorescence staining and western blotting assay.The results revealed that CA-1@HMnO₂/HA formed by pretreating nanosystem with MMP-2 effectively targeted tumor cells through a CD44 receptor-mediated pathway with a 6-h uptake rate of 79.6±3.2%,and CA-1 mediated apoptosis in a mitochondria-dependent pathway.In addition,to investigate the in vitro immune activation of the nanosystem,CLSM was used to examine the expression of calreticulin（CRT）,an indicator of immunogenic death,after drug action on tumor cells;the activation of the interferon-stimulated gene（STING）pathway and the expression of interferon-related genes（Ifnb and Cxcl10m RNA）were examined by western blotting and real-time PCR assays,respectively;and the expression of DC maturation marker protein（CD80 and CD86）was determined by flow cytometry.The results showed that CA-1 could increase CRT expression and induce immunogenic death of tumor cells;Mn²⁺released from HMnO₂ was an effective immunostimulant,which significantly up-regulated STING protein,Ifnb m RNA and Cxcl10 m RNA expression,promoted IFN-βproduction,and consequently enhanced DC maturation and achieved effective immune activation.4.In vivo study of antitumor activity of CA-1@HMnO₂/HA-P-mAbIn vivo studies were performed in BALB/c mice with 4T1 tumors.Small animal live imaging experiments demonstrated the good tumor targeting ability of the nanosystem.After 14 days of treatment,the tumor suppression rate of CA-1@HMnO₂/HA-P-mAb group mice reached 87.7±4.5%;the results of H&E staining and TUNEL staining of tumor tissue sections showed that the CA-1@HMnO₂/HA-P-mAb nanosystem had superior anti-tumor activity.In addition,the body weight,blood routine（leukocytes,erythrocytes,hemoglobin and platelets）and blood biochemical（alanine aminotransferase,aspartate aminotransferase,creatinine and urea nitrogen）were in the normal range,and the constructed nanosystem was safe in vivo.5.In vivo immunological study of CA-1@HMnO₂/HA-P-mAbBALB/c mice with 4T1 tumors were used to establish an animal model to study the in vivo immune response of mice.The related cytokines was tested by ELISA kit,and intratumoral immune cells infiltration was examined by flow cytometry and immunofluorescence staining.The results showed that the nanosystem could effectively inhibit Treg cell activity and subsequently reduce the infiltration of M2-type macrophages and myeloid-derived suppressor cells to alleviate immunosuppression.In the meantime,the nanosystem activated the innate immune response by increasing production of type I IFN and secretion of immune promoting factors（IL-12,IFN-γ）.Also,the nanosystem enhanced DC cell and cytotoxic T cell infiltration.In conclusion,this study successfully constructed a differential delivery drug delivery system,which effectively improved the immune microenvironment of tumors by simultaneous immunosuppression improvement and innate immunity activation,and realized the enhancement of chemotherapy and immunotherapy.It provided theoretical and experimental basis for the comprehensive treatment of tumors.