Fluorescent Probes Based On Salicylaldehyde Nitrogen Mustard Derivatives Target The Endoplasmic Reticulum And Induce Immunogenic Cell Death Of Tumor Cells

In recent years,tumor immunotherapy has received extensive attention due to its advantages of fast onset,low side effects,prolonging the life of patients and even clinical cure compared with traditional methods such as chemotherapy and radiotherapy.The efficacy of tumor immunotherapy is determined by the ability to induce tumor cells,and the induction of immunogenic cell death(ICD)of tumor cells can significantly improve the body’s anti-tumor immune response.ICD is closely related to endoplasmic reticulum(ER)stress,and type II ICD inducers that act directly on the ER have been shown to be more effective than type I ICD inducers that act indirectly on the ER.A series of ER-targeted photodynamic/photothermal or metal-complex ICD inducers have been reported in recent years,however,these ER-targeted ICD inducers suffer from the disadvantages of complex synthesis or heavy metal toxicity.On the other hand,because small-molecule drugs account for 75%of FDA-approved drugs,the synthesis of type II ICD small-molecule inducers has good clinical application prospects.Therefore,in this paper,we used p H-and polarity-sensitive,endoplasmic reticulum-targeting salicylaldehyde(SA)and chemotherapeutic drug chlorambucil(Cbl)to prepare an ER-targeted ICD inducer SA-Cbl with fluorescent self-reporting ability through a simple one-step synthesis method.This study systematically verified whether SA-Cbl could induce immunogenic cell death of tumor cells,and also examined the ability of the prepared tumor vaccine to induce anti-tumor immune response in mice.The specific research is as follows:1.Synthesis of Salicylaldehyde-Nitrogen Mustard DerivativesSA-Cbl was synthesized by one-step esterification of salicylaldehyde(SA)and chlorambucil(Cbl)in an alkaline environment.Then,the photophysical properties of SA-Cbl in different p H,polarity,viscosity or protein solutions were investigated,and it was found that SA-Cbl could emit light at similar p H and polarity in the endoplasmic reticulum,and at the same time,it could specifically bind to endoplasmic reticulum proteins.Because SA-Cbl fluoresces due to restriction of intramolecular motion(RIM)after binding to proteins,SA-Cbl has the ability to image organelles.2.Validation of SA-Cbl targeting the built-in net and inducing immunogenic cell death in tumor cellsUsing confocal microscopy,it can be observed that SA-Cbl can quickly enter cells and target the endoplasmic reticulum,efficiently induce the production of ROS and the efflux of calcium ions in the endoplasmic reticulum,and cause oxidative stress in the endoplasmic reticulum.At the same time,SA-Cbl can effectively induce the expression of immunogenic cell death-related damage-associated molecular patterns(DAMPs)in tumor cells,including calreticulin(CRT)exposed on the cell membrane surface,release of high mobility group box1(HMGB1),heat shock protein 90(HSP90)and adenosine triphosphate(ATP).3.Verification of the ability of tumor vaccine prepared by SA-Cbl to induce immune activationThe co-incubation of mouse bone marrow-derived dendritic cells(BMDC)and SA-Cbl-treated B16F10 melanoma cells showed that the phagocytosis of melanoma cells by BMDC was stronger with the increase of dose and time.At the same time,the maturation markers on the surface of BMDC cells were up-regulated,indicating that SA-Cbl-treated tumor cells produced ICD effect and effectively induced DC cell activation.Next,we carried out preventive tumor vaccination experiments on animals,and observed that the tumor growth of mice vaccinated with SA-Cbl-prepared tumor vaccine was inhibited.Population analysis showed that the level of maturation of DC cells increased in the draining lymph nodes,the infiltration of DC cells and CD8~+cells with the function of killing tumor cells in the tumor microenvironment increased,and the infiltration of myeloid-derived suppressor cells(MDSC)regulatory T cells(Treg)with immunosuppressive ability decreased,these results suggest that SA-Cbl exhibits superior efficacy in inducing antitumor immunity and immune memory effects.In summary,the SA-Cbl synthesized in this study can target the endoplasmic reticulum of tumor cells,cause endoplasmic reticulum stress,induce tumor immunogenic cell death and produce a large amount of DAMP,thereby significantly activating the body’s anti-tumor immunity.This study has the following highlights:First,SA-Cbl has the property of targeting and binding to the endoplasmic reticulum to generate fluorescence,and can be used as an endoplasmic reticulum fluorescent probe to monitor the ICD process.Second,the cytotoxicity of SA-Cbl was greatly reduced after binding to serum proteins,demonstrating good biological safety in animal experiments.Third,the design idea of this study is to synthesize a fluorescent group targeting the endoplasmic reticulum and a type I ICD inducer into a new type II ICD inducer with the characteristics of simple synthesis and significant immune efficacy.Our study provides new ideas for the design and synthesis of type II ICD inducers in the future.