Efficient Tumor Synergistic Chemoimmunotherapy By Self-Augmented ROS-Responsive Immunomodulatory Polymeric Nanodrug

Objective:Immunotherapy is a promising cancer treatment strategy.However,the insufficient immunogenicity of tumor cells and the effect of tumor immunosuppressive microenvironment affect its therapeutic effect.In this study,prodrug nanodelivery systems were constructed by synthesizing ROs-responsive polymers（polyacrylate derivatives）,encapsulated cellular immunogenic inducers（paclitaxel）,and indoleamine-2,3-dioxygenase（IDO-1）inhibitors（1-methyl-D,L-tryptophan）.The nano delivery system of prodrug,which can be stable in systemic circulation through intravenous administration,can realize passive aggregation of tumor sites by utilizing the high permeability and blocking effect（EPR effect）of solid tumors,and reduce the side effects caused by drug off-target.The ROS stimulation response and esterase sensitivity of the vector were utilized to form a"positive feedback loop"in tumor cells（ROS stimulated the release of PTX in tumor cells,and the released PTX further stimulated the production of ROS in cells）to promote drug release and improve drug concentration at the target site.While improving the immune response in vivo,it also regulates the tumor immunosuppressive microenvironment,inhibits tumor immune escape,and enhances the efficacy of immunotherapy.Then,it can cooperate with chemotherapy and immunotherapy to kill tumor cells with multiple targets.Experimental methods:1.Preparation of the nano-carrier:the carrier was composed of polyethylene glycol（PEG）as hydrophilic fragments,esterase-sensitive 1-MT ester and ROS-sensitive phenyl peroxalate as hydrophobic fragments,and finally each fragment was polymerized on the polymer with polyacrylate as the skeleton.Polyethylene glycol（PEG）constitutes the hydrophilic shell of the nanomaterial,1-MT ester and phenyl peroxalate constitute the hydrophobic core,and lipophilic drugs such as PTX can self-assemble with carrier materials in aqueous solution to form prodrug nanodelivery system through hydrophobic action and"π-π"conjugated interaction.2.Evaluation of physical and chemical properties of nanocarriers:（1）The biocompatibility and safety of intravenous administration of nanocarriers were evaluated by studying the destruction ability of different concentrations of nanocarriers on red blood cells.（2）The particle size,zeta potential and morphological characteristics of prodrug nanocarriers were observed by using Malvern nanoparticle size-Zeta potential tester and transmission electron microscope.（3）The encapsulation rate and drug loading of prodrug nanocarriers were studied by HPLC.（4）The release behavior of PTX and1-MT in prodrug nanocarriers under esterase and H₂O₂ conditions was studied by thin film dialysis method,and their concentrations were determined by high performance liquid chromatography.3.In vitro cell experiments:（1）The uptake of prodrug nanocarriers by cells at different time periods was studied by flow cytometry and confocal microscopy using the fluorescent dye Nile Red（NR）encapsulated in nanocarriers.（2）After the cells were treated with different formulations,the cytotoxicity of different formulations was determined by MTT method.（3）The cell cycle arrest of prodrug nanocarriers was observed by PI staining,and cell apoptosis was observed by Annexin V-PI staining.Finally,the results were analyzed by flow cytometry.（4）After 0.5 h incubation with the medium containing 10μg/m L DCFH-DA,the ROS content of the cells treated with different preparations was evaluated by flow cytometry.（5）After the cells were treated with different preparations,the cell supernatant was collected and the content of Kyn in the cell environment was determined by HPLC.（6）The cells treated with different preparations were treated with 4%paraformaldehyde and stained overnight with CRT or HMGB1 antibodies at 4℃.After incubation with fluorescentially labeled secondary antibodies,the cells were analyzed by flow cytometry.Meanwhile,the culture-medium was collected according to the manufacturer’s instructions and ATP analysis was performed using ELISA kit.Together with the above indicators,the ability of the nanocarriers to induce ICDs in vitro was evaluated.4.In vivo experiments:（1）The distribution of the carrier in different parts of the body at different times was studied after the nano preparation containing fluorescent dye Di R was applied to the tumor bearing mice.（2）The results of tumor inhibition experiment of different preparations were analyzed.（3）Tumor tissues were treated with antibodies of CRT,IDO-1 and Ki67,and then incubated and stained with secondary antibodies.Histopathological changes were observed to evaluate the ability of nanodelivery to induce tumor cells ICD in vivo.（4）Mouse tumor cell suspension was prepared by multi-antibody staining and analyzed by flow cytometry to observe DC_S and tumor-associated macrophages in tumor.The expression of CD8⁺T cells and Treg _S was evaluated to evaluate the ability of nanocarriers to activate immune response in vivo.Results:The nano delivery system of prodrug could decrease the concentration of Kyn and increase the expression of CRT,HMGB1 and ATP in tumor cells.It showed high efficiency in inducing cell immunogenic death（ICD）and inhibiting IDO-1.In vivo,the prodrug nanodelivery system was mainly distributed in liver,spleen and tumor,and showed obvious tumor targeting effect.It can significantly reduce the expression of cell proliferation signal ki67in solid tumors,and inhibit the growth of tumor weight and volume.Increase CRT concentration in tumor and induce ICD effect.It can enhance the expression of CD11c⁺,CD80⁺and CD86⁺（DC_S）,and CD8⁺（CD8⁺T cells）signal,and inhibit the expression of F4/80⁺and CD206⁺（M₂TAM_S）,and CD4⁺,CD25⁺,Fox P3⁺（Tregs）signal.Promote the infiltration of effector T cells in tumor tissue and enhance anti-tumor immune response.Inhibit the invasion of regulatory T cells（Treg _S）and M₂ tumor-associated macrophages（M₂TAM_S）to regulate the tumor immunosuppressive microenvironment.Nano-carriers combined with PTX and 1-MT in4T1 tumor bearing mice can significantly improve the targeting ability of drugs,have better tumor inhibition effect than other preparation groups,and reduce the occurrence of lung metastasis.At the same time,the nano delivery system can release PTX in response to ROS stimulation,which induces intracellular ROS generation and further promotes the release of PTX to form a positive feedback loop,thus enhancing drug release at the target site and improving drug concentration at the target site through ROS stimulation response in tumor cells.Conclusions:The co-delivery of cellular immunogenic inducer（PTX）and IDO-1inhibitor（1-MT）by ROS stimulated responsive nanocallers can effectively improve the targeting ability of therapeutic drugs and reduce the side effects caused by off-target.While enhancing the immunogenicity of tumor cells,it can change the tumor immunosuppressive microenvironment and activate the anti-tumor immune response of the body,which can cooperate with chemotherapy and immunotherapy and provide a promising treatment plan for tumor.