Research On Liposomes Containing Capsaicin And Palladium Nano-enzyme Modified By Targeted Peptide RGD Enhanced Radiotherapy To Induce ICD

Objective:Recent years,radiotherapy(RT)has been shown to induce immunogenic cell death(ICD)to reprogram the tumor immune microenvironment,thereby activating the antitumor adaptive immune responses.However,RT resistance factors such as poor X-ray uptake by tumor cells,hypoxia and overexpressing glutathione(GSH)cause insufficient RT-induced ICD.Therefore,there is an urgent need to construct a multimodal nanoplatform to amplify the activation of systemic immune responses by inducing more ICDs through enhanced radiotherapy sensitivity.Methods:The RGD-CapLip@Pd@Cu-MOF liposomes were prepared by Palladium(Pd)nanozymes,2-methylimidazole,Cu(NO3)2,1,2-dioleyl-sn-glycerol-3-phosphatecholine,cholesterol,capsaicin,DSPE-PEG-RGD.Using transmission electron microscopy(TEM),scanning electron microscope(SEM),dynamic light scatting(DLS),energy-dispersive spectrometer(EDS),common X-ray diffractometry(XDR),X-ray photoelectron spectrometry(XPS)and other instruments to test and characterize the materials.The effects of nano-liposomes on the viability and proliferation of colon cancer cells were detected by CCK8,clonal formation assay and CFDA SE labeled tumor cells.Transwell migration and invasion experiments were conducted to investigate the effect of RGD-CapLip@Pd@CuMOF combined RT on the migration and invasion ability of colon cancer cells.Flow cytometry was used to detect and evaluate the proportion of apoptosis,cell cycle distribution and oxidative stress level in different treatment groups.Detection of mitochondrial membrane potential in colon cancer cells by JC-1 fluorescent probe.The cells were stained with Calcein-AM/PI double staining to distinguish the proportion of living and dead cells after different treatment groups.The Seahorse XF-96 analyzer measured mitochondrial O2 consumption rate(OCR)to assess the effects of RGD-CapLip@Pd@Cu-MOF on mitochondrial function.The level of y-H2AX was detected by immunofluorescence assay and the length of comet tail was measured by comet assay to evaluate the degree of DNA damage.To assess the level of ICD induced by different treatment groups,adenosine triphosphate(ATP)and high mobility group box 1(HMGB1)in the supernatant of cell culture medium were detected by ELISA kit.The expression levels of Calreticulin(CRT),Heat shock protein(HSP)90 and HMGB1 were determined by immunofluorescence.Western Blot experiments were performed to detect the effects of RGD-CapLip@Pd@CuMOF liposomes on the apoptosis and cuproptosis levels of colon cancer cells.A C57BL/6 subcutaneous graft tumor model was constructed,and RGD-CapLip@Pd@Cu-MOF liposomes were injected into the tail vein and RT were given to investigate its effects on colon cancer proliferation and tumor microenvironment.In vivo imaging was performed to verify the targeting of RGD-CapLip@Pd@Cu-MOF liposomes on tumors.Blood biochemical assays were performed to verify the biosafety of this nanoplatform.Results:The results of TEM,SEM and DLS showed that RGD-CapLip@Pd@Cu-MOF liposomes were regular cubes with average particle size of 130 nm and surface potential of 12 mV Cell viability,proliferation,migration and invasion ability and mitochondrial membrane potential were significantly decreased in RGD-CapLip@Pd@Cu-MOF liposome combined RT group.The level of apoptosis,oxidative stress and the ratio of dead/alive cells were significantly increased.OCR results showed that cell oxygen consumption rate of RGD-CapLip@Pd@Cu-MOF group was the lowest,which could improve the oxygen depletion level in tumor microenvironment(TME).The results of r-H2AX immunofluorescence and comet experiments showed that RGD-CapLip@Pd@Cu-MOF had the greatest degree of DNA damage and could enhance DNA double strand breaks when combined with RT.ELISA(ATP,HMGB1)and immunofluorescence(CRT,HMGB1,HSP90)confirmed that RGD-CapLip@Pd@Cu-MOF+RT can cause more ICDs,and thus release or expose more DAMPs.Western Blot results showed that RGD-CapLip@Pd@CuMOF could promote cuproptosis and apoptosis.Animal experiments show that RGDCapLip@Pd@Cu-MOF+RT can specifically target tumor and inhibit the proliferation of colon cancer.In addition,the infiltration levels of CD86+DCs and CD8+T cells were significantly increased in tumor tissues treated with RGD-CapLip@Pd@Cu-MOF+RT.Conclusions:In this study,an efficient and safe multi-mode nanotherapy platform was developed,which can accurately target tumor tissues,enhance radiotherapy sensitivity by improving oxygen deficiency in TME,promoting cuproptosis,depleting GSH,amplifying oxidative stress,induce amplification of ICD,activate anti-tumor immune response,and transform immunosuppressed "cold tumor" into immune response "hot tumor".This multimodal nanotherapy platform provides a promising design strategy for antitumor immunotherapy.Part Ⅰ:Preparation and characterization of RGD-CapLip@Pd@CuMOF liposomesObjective:Radiotherapy can induce ICDs to enhance tumor immunogenicity and thus activate adaptive immune responses.The purpose of this part is to synthesize RGDCapLip@Pd@Cu-MOF liposomes with tumor targeting and radiotherapy sensitization and characterize its properties.Methods:The RDGCapPd@Cu-MOF liposomes were prepared and synthesized by biomimetic mineralization method.Transmission electron microscopy(TEM)was used to observe the morphology,nanoparticle size and Zeta potential analyzer(DLS)to measure the particle size surface potential,energy dispersion spectroscopy(EDS)elemental analysis,Xray diffraction(XRD)structural integrity characterization,X-ray photoelectron spectroscopy(XPS)to measure the surface chemical spectrum.POD activity was detected by TMB(tetramethylbenzidine)method.The GSH kit was used to measure the GSH content and evaluate the GSH consumption capacity of the material.Results:TEM results showed that RGD-CapLip@Pd@Cu-MOF showed a regular cube structure,and Pd nanozyme wrapped in a cube showed an irregular spherical shape.EDS analysis showed that the nanoparticles were mainly composed of Cu,Pd and Zn elements.The XRD pattern confirmed the formation of ZIF-8 crystal phase and the ZIF-8 crystal structure with Cu and Pd nanozyme did not change.The surface chemistry of XPS spectra confirms the presence of Cu and Pd.In addition,RGD-CapLip@Pd@Cu-MOF showed high POD activity and GSH depletion ability.Conclusions:A multi-modal tumor treatment liposomes were successfully synthesized.Characterization tests showed that this nano-liposome had regular cube structure,small particle size,large specific surface area and high enzyme activity.Part Ⅱ:Evaluation of antitumor efficacy of RGD-CapLip@Pd@CuMOF liposome in vitroObjective:This part aims to explore the effects of RGD-CapLip@Pd@Cu-MOF on tumor cell proliferation,migration and invasion,apoptosis and cycle distribution at the cellular level.The mechanism of promoting radiotherapy sensitization and whether ICDs can be amplified and induced.Methods:The effects of RGD-CapLip@Pd@Cu-MOF on GSH level and mitochondrial function of tumor cells were detected by GSH kit and Seahorse method.The effects of RGDCapLip@Pd@Cu-MOF on tumor cell viability and proliferation were detected by CCK8,clonal formation and CFDA SE methods.Transell migration and invasion assay to evaluate the effect of RGD-CapLip@Pd@Cu-MOF on the migration and invasion ability of tumor cells.Annexin V-FITC/PI double staining,JC-1 fluorescent probe and Calcein-AM/PI double staining were used to evaluate the effects of RGD-CapLip@Pd@Cu-MOF on apoptosis,mitochondrial membrane potential and the ratio of death to death of tumor cells.DCFH-DA fluorescent probe and PI single stain were used to detect the effects of RGDCapLip@Pd@Cu-MOF on cellular oxidative stress and cell cycle distribution.DNA damage was evaluated by γ-H2AX immunofluorescence and single cell gel electrophoresis(comet assay).ICD levels were evaluated by ELISA and immunofluorescence of DAMPs characteristic signaling molecules.qRT-PCR were used to detect the expression of immunostimulation-related genes and WB was used to detect the expression of cuproptosis and apoptosis related proteins.Results:RGD-CapLip@Pd@Cu-MOF reduced GSH levels and inhibited maximum mitochondrial respiration.RGD-CapLip@Pd@Cu-MOF combined with RT inhibited cell proliferation,migration and invasion,increased cell apoptosis,decreased mitochondrial membrane potential,and increased the ratio of dead/live cells.Compared with RT alone group,RGD-CapLip@Pd@Cu-MOF combined with RT can enlarge oxidative stress and arrest cell cycle in G2/M phase,with higher degree of DNA damage.The HMGB1 and ATP levels in the supernatant of RGD-CapLip@Pd@Cu-MOF+RT were higher than those in the RT alone group,and immunofluorescence results showed higher HMGB1 expression levels,more CRT and HSP90 were exposed to the cell surface.qRT-PCR results showed that RGDCapLip@Pd@Cu-MOF promoted more immunostimulation-related genes expression.WB results showed that RGD-CapLip@Pd@Cu-MOF could promote cuproptosis and apoptosis.Conclusions:RGD-CapLip@Pd@Cu-MOF can promote radiation sensitivity by depleting GSH,inhibiting mitochondrial respiration,enlarging oxidative stress,blocking cell cycle,and promoting cuproptosis,then thus induce elevated ICD levels.Part Ⅲ:Evaluation of antitumor effect of RGD-CapLip@Pd@Cu-MOF liposome in vivo and its effect on tumor microenvironmentObjective:This part aims to explore the biosafety,targeting,effect on tumor proliferation and tumor microenvironment of RGD-CapLip@Pd@Cu-MOF in vivo,as well as the antitumor effect of combination with PD-1 antibody.Methods:Rhodamine fluorescent dye was used to label RGD-CapLip@Pd@Cu-MOF and injected into caudal vein.Fluorescence imaging of subcutaneous tumor-bearing mice at different time points was recorded by small animals live imaging system,and tumor targeting of this liposome was evaluated.Normal mice were injected with different nanoparticles through the tail vein,and orbital blood was taken two weeks later,centrifuged,and supernatant was taken to detect the levels of alanine aminotransferase(ALT),aspartate aminotransferase(AST),blood urea nitrogen(BUN)and creatinine(CRE),so as to evaluate the biocompatibility and safety of RGD-CapLip@Pd@Cu-MOF.The 6-week-old C57BL/6 mice were inoculated with MC38 murine colon cells,and were randomly divided when the tumor volume reached 100 mm3.The mice were given different treatments according to the experimental design.The tumor volume and body weight of the mice were monitored every 1 day,and the mice were excluded after 14 days.The tumor tissues were prepared into paraffin sections or frozen sections.The antitumor effect of RGD-CapLip@Pd@Cu-MOF combined RT and its effect on TME were evaluated by HE staining,Ki-67,CD4,CD8 immunohistochemistry and TUNEL,ROS,CRT,HMGB1 immunofluorescence.Results:After injecting rhodamine fluorescent dye labeled RGD-CapLip@Pd@Cu-MOF into the tail vein of mice,the fluorescence mainly accumulated in the tumor site,but no fluorescence distribution was observed in other sites.The levels of ALT,AST,BUN and CRE were all within the normal range,and there were no obvious abnormalities in liver and kidney function.Compared with RT group alone,RGD-CapLip@Pd@Cu-MOF combined RT group had the maximum reduction in tumor burden and weight,the lowest tumor cell proliferation activity and the highest apoptosis level,which promoted more HMGB1 and CRT expression and translocation.The proportion of CD86+CD11c+DC and CD8+CD3+T cells increased,and the serum levels of IFN-γ and TNF-α increased significantly.When combined with PD-1 antibody,compared with RT+PD-1 antibody,the RGDCapLip@Pd@Cu-MOF+RT+PD-1 antibody group had the most necrosis and apoptosis of tumor cells,lowest proliferation ability,and higher immunohistochemical positive rate of CD4 and CD8.Conclusions:RGD-CapLip@Pd@Cu-MOF has specific tumor-targeting properties,slow release effect,good biocompatibility and safety,which can enhance RT sensitivity and induce more CTLs infiltration in the TME.