| Background:Colon cancer is a common malignant tumor of the digestive tract that occurs in the colon.It ranks second in the mortality rate of malignant tumors and has become one of the major public health problems that seriously threaten human health.The 5-year survival rate of most early-stage colon cancer patients after surgery,radiotherapy and chemotherapy can reach about 90%.However,the curative effect with large side effects cannot completely inhibit tumor growth and recurrence on advanced colon cancer patients.Immune checkpoint blocking therapy(ICB),a common immunotherapy,has shown a significant inhibitory effect on advanced colon cancer,but only a small number of patients were useful,and most colon cancer patients suffer from side effects that far outweigh their therapeutic effects.The reasons may be related to the hypoxia of the tumor microenvironment(TME),the increased infiltration of immunosuppressive lymphocytes and the decrease of cytotoxic T cells,and the lack of neoantigens on the surface of tumor cells that can be recognized by immune cells,etc.Therefore,how to improve the immunotherapy effect of advanced colon cancer and prolong the survival period by ameliorating the tumor immunosuppressive microenvironment is still an urgent problem to be solved.At present,it has been reported in relevant literature that photothermal therapy(PTT)can up-regulate the expression of PD-L1 on the surface of tumor cells,showing great advantages in inhibiting tumor growth and improving tumor microenvironment.Although its efficacy is still limited by factors such as deep tumor hypoxia and insufficient lymphocyte infiltration,and PTT combined with alkyl radicals to kill tumor cells is an effective way to solve the limitation of hypoxic environment.Ink,as an important part of traditional Chinese medicine,has good photothermal properties and can be used as photothermal agent.Sodium alginate(ALG),a by-product extracted from the traditional Chinese medicine kelp,can undergo ion exchange reaction with divalent cations to form a cross-linked network structure,thereby forming a hydrogel.It is often used as a carrier to avoid the inactivation of active substances such as sensitive drugs,proteins,cells and enzymes.On this basis,our study used ALG as the carrier to construct an ink-mediated All-in-one hydrogel for photothermal immunotherapy of advanced colon cancer,and evaluated its therapeutic effect on advanced colon cancer in cell lines and in vivo experiments.Objective:1.In this project,we used ALG as the carrier to construct a hydrogel drug delivery system with sustained drug release function that loaded the photothermal agent Ink,the alkyl radical initiator azobisisobutylimidazoline hydrochloride(2,2-azobis[2-(2imidazoline-2-acyl)propane]dihydrochloride,AIPH)and PD-L1 inhibitor(HY19991,HY).We have verified the inhibitory effect on proximal and distal subcutaneous tumors in Balb/c mouse model loaded with CT26 subcutaneous tumors and constructed a novel therapeutic strategy for advanced colon cancer.2.This study explored the molecular mechanism of the hydrogel drug delivery system in ameliorating tumor immune microenvironment and activating anti-tumor immunity under low-temperature photothermal therapy.Method:Part 1.Preparation and characterization of the All-in-one hydrogel in vitro.First,the mixed solution of All-in-one was prepared by mixing various ALG,Ink,AIPH and HY solutions,and then injected into an aqueous solution containing Ca2+ to obtain All-in-one hydrogel.Observe the properties of ALG reacting with ions to form gels throughout the process.The morphology of the freeze-dried All-in-one hydrogel was characterized in vitro by Scanning electron microscope(SEM).The weight of the freezedried All-in-one hydrogel immersed in the buffer were recorded,and the swelling rate of All-in-one hydrogel were calculated according to the weight change.The drug loading rate of the All-in-one hydrogel were calculated by the weight of different drugs loaded on the hydrogel.By comparing the gel-forming properties of ALG with different concentrations,the optimal ALG concentration was optimized for subsequent studies.The composition of All-in-one hydrogel and the drug release under different conditions were detected by UV spectrophotometer.The rheological analysis of All-in-one hydrogel were tested by rheometer.The photothermal effect and the photothermal stability of the All-in-one hydrogel after irradiated by 1064nm near infrared-Ⅱ(NIR-Ⅱ)laser were observed by the thermal infrared camera.Part 2.Study on the anti-tumor effect and immune activation mechanism of the Allin-one hydrogel in vitro.The alkyl radicals released by AIPH in vitro and in CT26 cells were detected by UV spectrophotometer,microplate reader,and confocal laser scanning microscopy(CLSM).The cytotoxicity of CT26 cells in different treatment groups and different temperatures were detected by CCK-8 testing kit.The apoptosis of CT26 cells induced by different treatment groups were evaluated by flow cytometry Annexin V/PI double staining and Calcein-AM/PI method.The expression of PD-L1 in CT26 cells at different temperatures were detected by western blot.The molecular mechanism of immunogenic cell death(ICD)in CT26 cells after different treatment groups were examined by ELISA kit and CLSM.The tibia and femur of Balb/c mice were used to extract bone marrow cells,then interleukin-4(IL-4)and granulocyte-macrophage colony-stimulating factor(GM-CSF)were added to stimulate differentiation into immature dendritic cells(DCs),and the differentiated DCs were co-cultured with CT26 cells that treated with different treatment groups,and the activation of DCs were detected by flow cytometry.Part 3.Study on the anti-tumor effect and immune activation mechanism of the Allin-one hydrogel in vivo.The Balb/c mouse models of primary and distant subcutaneous tuomor were constructed by inoculating CT26 cells subcutaneously on the buttocks of Balb/c mice,and thermal imaging images of each group were observed and recorded by the thermal infrared camera.The tumor volume and tumor inhibition rate of primary and distant subcutaneous tumor after treatment were calculated,then combined with histopathological changes,the therapeutic effect of colon cancer was evaluated.To assess biosafety across treatment groups,the body weights of the mice in each treatment group were recorded during the treatment process,and the vital organs and tumor tissues of the mice in each treatment group were taken for H&E and TUNEL staining on the 15th day of treatment.Then the tumor tissue were made into frozen sections and stained,immunohistochemistry was used to observe the expression of calretictdin(CALR)and high mobility group protein B1(HMGB-1)in tumor tissues after treatments.The expression of PD-L1 in the tumor tissue after PTT were observed by western blot.The cytokine levels of Balb/c mice in each treatment group were detected by ELISA after treatment.The activation of dendritic cells(DCs)in spleen and lymph nodes and the infiltration of T cells and Treg in tumor tissues aftere treatments were analyzed by flow cytometry.Result:Part 1.Preparation and characterization of the All-in-one hydrogel in vitro.The spherical Ink nanoparticles were observed to be effectively immobilized on the hydrogel surface under SEM.According to the results of ALG gelation,the hydrogel formed when the ALG concentration reached 5 mg/mL can effectively fix the contents and prevent them from escaping.In the absorption spectrum of the All-in-one hydrogel supernatant,the characteristic absorption peaks of each component can be seen,but no other absorption peaks were found,and the absorption spectrum curve at 44℃ was significantly higher than the curve at 37℃.The drug loading rates of All-in-one hydrogels in cell experiments were HY 48.31%,AIPH 2.42%and Ink 0.97%,and the drug loading rates in animal experiments were HY 20.51%,AIPH 12.82%and Ink 2.56%,respectively.Swelling ratio and rheological tests confirmed the properties of the All-in-one hydrogel,which was a non-Newtonian fluid state,and the elastic modulus was greater than the viscous modulus,the results were consistant with the characteristic of hydrogel.The thermal infrared imaging results showed that the photothermal effect of the All-in-one hydrogel was positive with Ink concentration.The higher the Ink concentration,the higher the temperature.And it also has thermal stability.Part 2.Study on the anti-tumor effect and immune activation mechanism of the Allin-one hydrogel in vitro.The results of the absorption spectrum of the supernatant of All-in-one hydrogel and 2,2’-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt(ABTS)solution after mixed with illumination showed that there was a characteristic absorption peak of ABTS+· at 736 nm,and the absorption spectrum curve at 44℃ was significantly higher than the curve at 37℃.The results of the microplate reader showed that the absorption value of ABTS+· at 736nm reached the highest peak after the mixed solution of All-in-one hydrogel solution and ABTS solution were illuminated for 10 minutes.At the same time,it was also observed that CT26 cells treated with All-in-one hydrogel group showed strong green fluorescence under CLSM,and the fluorescence intensity were significantly higher than that of other groups.The results of CCK-8 showed that the Allin-one hydrogel group had stronger cytotoxicity than the other groups after irradiation.The results of flow cytometry and CLSM also showed that the All-in-one hydrogel group induced massive apoptosis.The results of CCK-8 and western blot showed that the cytotoxicity rate of CT26 cells treated at 44℃ was only about 50%,and the expression of PD-L1 was up-regulated.ELISA and CLSM results showed that CT26 cells treated with All-in-one hydrogel group released more ATP and CALR,while the expression of HMGB-1 on the surface of CT26 cells increased.Flow cytometry results showed that the DCs co-incubated with the All-in-one hydrogel group had the highest activation ratio.Part 3.Study on the anti-tumor effect and immune activation mechanism of the Allin-one hydrogel in vivo.We have successfully constructed the model of CT26 subcutaneous tumor and contralateral distal subcutaneous tumor,and the thermal imaging images of each group were recorded under illumination.In the All-in-one hydrogel group,the tumor volume of the subcutaneous and distant subcutaneous tumor were the smallest,the tumor inhibition rate were the highest,and the HE and TUNEL staining of the tumor tissue showed extensive necrosis.During the whole treatment,the body weight of the mice in each treatment group did not decrease significantly,and no tissue necrosis was found in the H&E staining of each important organ.At the same time,both CALR fluorescence sections and HMGB-1 fluorescence sections of tumor tissue in the All-in-one hydrogel group showed large-area red fluorescence.The results of western blot showed that the high expression of PD-L1 in tumor tissues after photothermal treatment.ELISA results showed that the levels of IL-4,TNF-α and IFN-γ in mice in the All-in-one hydrogel group on the 7th day after treatmets were higher than those in other treatment groups.Flow cytometry analysis showed that the activation proportion of DCs in spleen and lymph nodes in the All-in-one hydrogel group were the highest,and the proportion of CD4+T cells and CD8+T cells in subcutaneous and distant subcutaneous tumor tissues were the highest,and the proportion of Treg cells were the lowest.Conclusion:1.For the first time,we constructed an All-in-one hydrogel drug delivery system by using ALG as a carrier and loading with photothermal agent Ink,alkyl radical initiator AIPH and PD-L1 inhibitor HY,which can immobilize the drug at the tumor site and controlled the release of different drugs in hydrogel according to temperature to avoid premature degradation.2.For the first time,it was found that the alkyl radicals released by AIPH have the effect of amplifying the photothermal-induced ICD,then enhance the anti-tumor immune response activated by low temperature photothermal.3.It was found that low-temperature PTT can induce the high expression of PD-L1 in tumor cells,and relieve the tumor immunosuppressive microenvironment,then combine with HY released by the All-in-one hydrogel to further enhance the anti-tumor immunotherapy effect.4.In vivo studies showed that All-in-one hydrogel reversed the immunosuppression of TME by up-regulating the expression of PD-L1 and enhancing ICD effect under lowtemperature PTT,and increased the recruitment of tumor-infiltrating lymphocytes against tumors,stimulate innate and adaptive immune responses to inhibite the growth and metastasis of colon cancer. |
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