Cascade Chemo-photodynamic Therapy By A ROS-responsive Nanocarrier To Potentiate Checkpoint Blockade Immunotherapy For Cancer Treatment

Cancer remains one of the deadliest human diseases in the world.checkpoint blockade immunotherapy has become a first-line treatment option for multiple cancers(e.g.,anti-PD-1 antibody,anti-PD-L 1 antibody(aPD-Ll),anti-CTLA4 antibody).The relatively low response rates(ranging from 10%-40%for most tumor types)limit the scope of clinical application of checkpoint inhibitors.partial cancer treatments,such as certain chemotherapeutic agents(doxorubicin(DOX),oxaliplatin,paclitaxel,etc.),photodynamic therapy(PDT),hyperthermia,and radiotherapy can induce tumor cell death in an immunogenic manner,and this process is also known as immunogenic cell death(ICD).ICD-inducing modalities evoke antitumor immunological responses by promoting the generation of tumor-associated antigens from tumor cell residues,ultimately increasing the activation,proliferation,and infiltration of antigen-specific T cells to result in an immunogenic tumor microenvironment,which potentially increases response rates to checkpoint blockade immunotherapy.Therefore,the use of ICD-inducing modalities could be a promising strategy to potentiate the efficacy of checkpoint inhibitors.In this study,we prepared a lipid-polymer hybrid nanocarrier TKHNP-C/D,which enhances the immune response by stimulating two ICD-inducing modalities,namely,DOX-based chemotherapy and PDT of the photosensitizer Ce6.Potentiate the anti-tumor efficacy of the checkpoint inhibitors aPD-L1.Methods1.Preparation and characterization of nanoparticle TKHNP-C/DA reactive oxygen responsive polymer material,thioketal phosphate(TK-PPE),was synthesized.The drug-loaded nanoparticles TKHNP-C/D containing TK-PPE,hydrophobic doxorubicin(DOX)and photosensitizer Ce6 were prepared by simple nano-precipitation method.Furthermore,we studied the stability of TKHNP-C/D nanoparticles,UV absorption,size change of nanoparticle with illumination at different times,changes in DOX fluorescence after illumination,and compared the release behavior of nanoparticles with or without illumination in vitro.2.Evaluation of the anti-tumor effect and immune response of nanoparticle TKHNP-C/D in vitroFlow cytometric and CLSM were performed to compare the fluorescence changes of nanoparticles TKHNP-C/D with or without illumination.The anti-tumor effect of nanoparticle TKHNP-C/D cascade chemotherapy-photodynamic therapy was evaluated by MTT assay and apoptosis experiment.To investigate TKHNP-C/D mediated cascade-photodynamic induced immunogenic death,we examined the exposure of calreticulin(CRT)on the cell membrane by flow cytometric and confocal.3.In vivo evaluation of anti-tumor effect and immune response of nanoparticle TKHNP-C/DSmall animal imaging studies the distribution of nanoparticles in mice,taking out the main organs and tumors for quantitative analysis,and then performing tumor inhibition experiments,comparing the difference of nanoparticle illumination and non-light treatment,and analyzing the tumor volume changes after treatment.The proportion of cytotoxic T cells at the tumor site between different groups after the end of treatment was examined.After re-implantation,36 hours after administration,the mice were dissected,tumor lymph nodes were taken,and DC maturation ratio was detected by flow cytometric.4.Nanoparticle TKHNP-C/D mediated chemo-photodynamic therapy induced immune response,which combined with checkpoint inhibitors aPD-L1 for inhibition bilateral tumorsInspired by nanoparticle TKHNP-C/D cascade chemotherapy-photodynamic therapy to induce immune response,we hypothesized that this chemotherapy-photodynamic therapy has a synergistic effect with aPD-Ll checkpoint blockade.In the next study,we established bilateral breast cancer model by subcutaneously injecting 4T1 cells into the left and right regions of the mice.The tumor volume changes and the body weight changes of the mice were analyzed.The tumor tissues were sectioned and stained to analyze cell proliferation and apoptosis.To further explore the mechanism by which chemo-photodynamic therapy triggers immune response in combination with αPD-L1 checkpoint inhibitor,we examined cytotoxic T cells in bilateral tumor,an important indicator of anti-tumor system immune response tumor necrosis factor(TNF-α),interferon factor(IFN-y).Results1.Preparation and characterization of nanoparticle TKHNP-C/DReactive oxygen responsive polymer material,thioketal phosphate(TK-PPE).was synthesized.The successful synthesis of the material TK-PPE was characterized by H-NMR.The drug-loaded nanoparticles TKHNP-C/D containing TK-PPE,hydrophobic doxorubicin(DOX)and photosensitizer Ce6 were prepared by nanoprecipitation.TKHNP-C/D showed strong UV absorption at 405 nm and 490 nm,indicating that Ce6 and DOX were successfully entrapped into the nanoparticles.The nanoparticle TKHNP-C/D has good stability.Dynamic light scattering(DLS)and transmission electron microscopy(TEM)measurements revealed that the size of the nanoparticles shrinks with illumination.In the drug release experiment,the released DOX of TKHNP-C/D with illumination more than without.2.Evaluation of the anti-tumor effect and immune response of nanoparticle TKHNP-C/D in vitroCells incubated with TKHNP-C/D plus light irradiation(+L)exhibited stronger intracellular DOX fluorescence signals than cells without illumination.MTT and apoptosis experiments showed that TKHNP-C/D+L exhibited the highest anticancer activity,which was due to the cascade of chemo-photodynamic.The detection of calreticulin(CRT)by flow cytometric and CLSM demonstrated that the nanoparticle TKHNP-C/D can cause immunogenic death and enhance the immune response.3.In vivo evaluation of anti-tumor effect and immune response of nanoparticle TKHNP-C/DTKHNP-C/D has a significant enrichment at the tumor site.Due to the combination of chemo-photodynamic therapy,TKHNP-C/D with illumination can significantly inhibit tumors by a single administration,promote DC maturation and increase the proportion of T cells in tumor sites.4.Nanoparticle TKHNP-C/D mediated chemo-photodynamic therapy causes immune response combined with checkpoint inhibitor aPD-L1 for inhibition of bilateral tumorsThese cascade of chemotherapy-PDT can effectively promote the maturation of dendritic cells(DC),increase the infiltration of lymphocytes into tumor tissues,and trigger an effective anti-tumor immune response.Inspired by this result,the cascade chemo-PDT was further combined with PD-L1 immune checkpoint blockade,which not only efficiently inhibited primary tumor growth but also resulted in the regression of distant tumors in bilateral syngeneic mouse tumor models via a remarkable abscopal effect,demonstrating potentiated effectiveness of checkpoint blockade antibodies.ConclusionIn this paper,nanoparticle TKHNP-C/D encapsulated oxygen responsive material TK-PPE.DOX and photosensitizer Ce6 were prepared.The entrapped Ce6 produces reactive oxygen species at 660 nm,which not only kills tumor cells through the PDT effect,but also degrade TK-PPE to promote the release of DOX from the nanoparticles,achieving cascade inhibition of tumor growth.Futhermore,TKHNP-C/D can induce an immune response and synergize with aPD-L1 while inhibiting abscopal tumors.