A cholesterol-targeted catalytic hydrogel for enhancing microwave ablation therapy and anti-tumor immune response and its application

Abstract

The present invention provides a cholesterol-targeted catalytic hydrogel for enhancing microwave ablation therapy and anti-tumor immune response and its application. Aiming at the high correlation between cholesterol and tumor immunosuppression, the present invention takes the cholesterol-rich tumor fragments after ablation as the starting point, proposes a solution strategy of introducing COD and Hemin at the same time, and enables cholesterol to activate the body's immune response in a "waste-to-treasure" manner, creating the best conditions for MWA sequential ICIs treatment. The enhanced MWA sequential immunotherapy strategy based on DA‑COD‑OD‑HCS hydrogel achieves the synergistic effects of MWA, ferroptosis, immunosuppressive microenvironment remodeling and immunotherapy of liver cancer, effectively reduces the recurrence and metastasis rate of liver cancer, and provides new methods and new ideas for the safe and efficient interventional treatment of unresectable liver cancer.

Description

Technical Field

[0001] The invention relates to the field of polymer materials, and in particular to a cholesterol-targeted catalytic hydrogel for enhancing microwave ablation therapy and anti-tumor immune response and an application thereof. Background Art

[0002] Hepatocellular carcinoma (HCC) is a malignant tumor with high morbidity and mortality. More than 80% of patients have lost the opportunity for surgical resection when they seek medical treatment. Microwave ablation (MWA) has become the basic treatment strategy for clinically inoperable HCC, but unfortunately, the recurrence and metastasis rates of liver cancer after MWA are still high. There is a lot of evidence that immune checkpoint blockade therapy has brought new opportunities for the prevention and treatment of recurrence and metastasis of liver cancer after MWA. However, due to the existence of the tumor's own immunosuppressive microenvironment, only a few HCC patients have a good response to immune checkpoint inhibitors (ICIs). Many authoritative reports have confirmed that cholesterol plays an important role in the regulation of tumor immunosuppression and is highly correlated with the low response rate of immune checkpoint blockade therapy. A large amount of cholesterol will be released from the tumor cell fragments in the ablation lesion after MWA, which may further aggravate the immunosuppression of the tumor. The immunosuppressive residual tumor microenvironment (IRTM) is a key factor leading to the high recurrence and metastasis rate of HCC after microwave ablation, and the cholesterol-rich tumor fragments after microwave ablation play an important role in inducing the deterioration of IRTM. Summary of the invention

[0003] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide a cholesterol-targeted catalytic hydrogel that enhances microwave ablation therapy and anti-tumor immune response.

[0004] Another object of the present invention is to provide a method for preparing the cholesterol-targeted catalytic hydrogel for enhancing microwave ablation therapy and anti-tumor immune response.

[0005] Another object of the present invention is to provide an application of the cholesterol-targeted catalytic hydrogel for enhancing microwave ablation therapy and anti-tumor immune response.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] A method for preparing a cholesterol-targeted catalytic hydrogel for enhancing microwave ablation therapy and anti-tumor immune response comprises the following steps:

[0008] (1) cholesterol oxidase (COD) and dimethylmaleic anhydride (DA) are dissolved in Hepes buffer and mixed, pH is controlled and stirred for reaction, and after the reaction is completed, the mixture is filtered to obtain DA-COD;

[0009] (2) adding hemin, 1-ethyl-3-(3-dimethylaminopropyl) semicarbazide (EDC), and N-hydroxysuccinimide (NHS) to dimethyl sulfoxide (DMSO) for activation, then adding carboxymethyl chitosan (CS) for stirring reaction, and after the reaction is completed, dialyzing and freeze-drying to obtain hemin-carboxymethyl chitosan conjugate (HCS);

[0010] (3) adding dextran and sodium periodate to water, stirring to react in the dark, dialyzing, and freeze-drying to obtain oxidized dextran (OD);

[0011] (4) After adding DA-COD, HCS conjugate and oxidized dextran into water and mixing, the mixture is allowed to gel at room temperature to obtain a cholesterol-targeted catalytic hydrogel that enhances microwave ablation therapy and anti-tumor immune response.

[0012] The ratio of cholesterol oxidase to dimethylmaleic anhydride in step (1) is 100 U: 1-3 mg, preferably 100 U: 2.5 mg.

[0013] The pH of the Hepes buffer in step (1) is 8.5 to 9.5, preferably 9.

[0014] The pH controlled in step (1) is controlled to be greater than or equal to 7.4.

[0015] The stirring reaction time in step (1) is 3 to 4 hours.

[0016] The filtration in step (1) is performed by using a filter device with a molecular weight cutoff of 8 to 12 kDa; preferably, ultrafiltration is performed by using a centrifugal filter device with a molecular weight cutoff of 10 kDa.

[0017] The molar ratio of hemin, EDC, NHS and carboxymethyl chitosan in step (2) is 1-2:1-2:1-2:4-6; preferably 1:1:1:5.

[0018] The activation time in step (2) is 1 to 3 hours, preferably 2 hours.

[0019] The stirring reaction time in step (2) is 6 to 12 hours, preferably 8 hours.

[0020] The dialysis conditions of step (2) are to use a dialysis bag with a molecular weight cutoff of 8000 to 14000 Da to dialyze in a sodium bicarbonate solution with a pH of 8 to 9 for 20 to 28 hours.

[0021] The molar ratio of dextran to sodium periodate in step (3) is 1-2:10-20; preferably 1:18.5.

[0022] The molecular weight of the dextran described in step (3) is 4000.

[0023] The time of the light-shielded stirring reaction in step (3) is 64 to 80 hours, preferably 72 hours.

[0024] The dialysis conditions in step (3) are to use a dialysis tube with a molecular weight cut-off of 3000 to 4000 Da for dialysis in deionized water.

[0025] The mass ratio of the HCS conjugate, oxidized dextran and water in step (4) is 1-10:10-30:60-90.

[0026] The final concentration of DA-COD in step (4) is 40-60 U / mL, calculated as the enzyme activity unit of COD before modification.

[0027] The placement time described in step (4) is 3 to 10 minutes.

[0028] A cholesterol-targeted catalytic hydrogel for enhancing microwave ablation therapy and anti-tumor immune response is prepared according to the above preparation method.

[0029] The cholesterol-targeted catalytic hydrogel for enhancing microwave ablation therapy and anti-tumor immune response is used in the preparation of anti-tumor drugs.

[0030] The tumor is hepatocellular carcinoma.

[0031] The cholesterol-targeted catalytic hydrogel for enhancing microwave ablation therapy and anti-tumor immune response is used in the preparation of microwave ablation synergistic enhancement drugs.

[0032] A drug for enhancing anti-tumor immune response after microwave ablation therapy, comprising the cholesterol-targeted catalytic hydrogel for enhancing microwave ablation therapy and anti-tumor immune response.

[0033] The drug for enhancing the anti-tumor immune response after microwave ablation therapy also includes anti-PD-L1.

[0034] Compared with the prior art, the present invention has the following advantages and effects:

[0035] The present invention aims at the high correlation between cholesterol and tumor immunosuppression, takes the cholesterol-rich tumor fragments after ablation as the starting point, and proposes a solution strategy of introducing COD and Hemin at the same time, so that cholesterol can activate the body's immune response in a "waste-to-treasure" way, creating the best conditions for sequential ICIs treatment with MWA. In this process, COD can efficiently decompose the cholesterol in the tumor fragments in the ablation focus, thereby effectively alleviating the immunosuppressive effect of cholesterol on the tumor; at the same time, Hemin can react with the decomposition product of cholesterol, hydrogen peroxide, and further improve the immunosuppressive microenvironment while inducing ferroptosis of residual cancer cells; through the synergistic immune microenvironment regulation of the two, a favorable microenvironment is created for MWA combined immunotherapy, which is expected to solve the clinical problem of easy recurrence and metastasis of tumors after conventional MWA surgery.

[0036] In addition, the enhanced MWA sequential immunotherapy strategy based on DA-COD-OD-HCS hydrogel achieves the synergistic effects of liver cancer MWA, ferroptosis, immunosuppressive microenvironment remodeling and immunotherapy, effectively reducing the recurrence and metastasis rate of liver cancer, and providing new methods and new ideas for the safe and efficient interventional treatment of unresectable liver cancer. In order to simulate the residual cancer microenvironment after MWA surgery, the subsequent experiments of this study were conducted using the incomplete microwave ablation (iMWA) method. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is a diagram of the characterization experiment results related to OD-HCS hydrogel.

[0038] Figure 2 This is a graph showing the results of the cell experiment in Example 7 to verify the effect of the hydrogel in the tumor microenvironment.

[0039] Figure 3 This is a graph showing the results of the experiment in Example 7 to evaluate the ability of catalytic hydrogels to induce cell death of different types.

[0040] Figure 4 This is a graph showing the results of the anti-tumor effect experiment of the cholesterol-catalyzed hydrogel combined with iMWA in vivo in Example 8.

[0041] Figure 5 This is a graph showing the relevant results of the therapeutic effect verification experiment of iMWA combined with DA-COD-OD-HCS in Example 8.

[0042] Figure 6 This is a graph showing the results of an experiment to verify the anti-tumor effect of iMWA combined with cholesterol-catalyzed hydrogel in a highly malignant N1S1 HCC rat tumor model in Example 8.

[0043] Figure 7This is a graph showing the results of the experiment in Example 9 to verify the effect of cholesterol-targeted catalytic hydrogel in enhancing anti-tumor immunotherapy in vivo.

[0044] Figure 8 This is a graph showing the experimental results of safety assessment of cholesterol-targeted catalytic hydrogel-enhanced in vivo anti-tumor immunotherapy in Example 9. DETAILED DESCRIPTION

[0045] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0046] If no specific experimental conditions are specified in the following embodiments, conventional experimental conditions or experimental conditions recommended by the reagent company are generally followed. The materials and reagents used, unless otherwise specified, are all reagents and materials obtained from commercial sources.

[0047] Example 1 Modification of cholesterol oxidase (COD)

[0048] (1) COD was dissolved in Hepes buffer at pH 9.0 at a concentration of 100 U / ml, and then dimethylmaleic anhydride (DA) was added at a concentration of 2.5 mg / ml and stirred; during the reaction, the pH value of the reaction solution was continuously monitored with a pH test paper. When the pH value was less than 7.4, a certain amount of Na2CO3 solution was added to the reaction solution to adjust the pH value, and the reaction solution was stirred at room temperature for 3 to 4 hours;

[0049] (2) Use a centrifugal filter device (molecular weight cutoff, MWCO = 10 kDa) to ultrafilter to remove excess DA to obtain purified DA-COD. The resulting product is stored at -20°C for future use.

[0050] Referring to the above method, dimethylmaleic anhydride was replaced by butyric anhydride (SA) to prepare SA-COD.

[0051] Unless otherwise specified, the enzyme activity units U described below are all calculated based on the COD before modification.

[0052] Example 2 Preparation of HCS Conjugate

[0053] (1) Hemin, EDC, and NHS were added to anhydrous DMSO at a molar ratio of 1:1:1 to activate the carboxyl group of hemin for 2 hours. For every 1 mmol of hemin, 10 mL of DMSO was required;

[0054] (2) Carboxymethyl chitosan (CS) was added to the activated Hemin in step (1) and mixed at a molar ratio of CS: Hemin of 5:1. The mixture was stirred for overnight reaction (8 h). The mixture was then purified by soaking in a sodium bicarbonate solution (pH 8-9) for 24 h using a dialysis bag with a MWCO value of 8000-14000 Da, and then freeze-dried for 3 days to obtain an HCS conjugate. The obtained product was stored at -20°C for future use.

[0055] Example 3 Preparation of Oxidized Dextran (OD)

[0056] 111.7 mg / mL of dextran (Mw 4000) and 110.6 mg / mL of sodium periodate (molar ratio of dextran: sodium periodate = 1:18.5) were added to deionized water, stirred for 72 h at room temperature in the dark, then purified using a dialysis tube (molecular weight cutoff = 3500 Da) for 48 hours (dialysis against deionized water), and lyophilized to obtain oxidized dextran, which was stored at -20°C for future use.

[0057] Example 4 Preparation and Effect Verification of OD-HCS Hydrogel Matrix

[0058] The HCS conjugate prepared in Example 2 (1, 5, 10 wt%) and the oxidized dextran prepared in Example 3 (10, 20, 30 wt%) were mixed to prepare a hydrogel matrix with the remainder being water, and the gelation was then observed at 0.5, 5 and 20 min.

[0059] The experimental results are as follows Figure 1 As shown in Figure A, after 5 wt% HCS conjugate is mixed with 20 wt% oxidized dextran, it can form a gel within 5 minutes, which is more conducive to the fixation of drugs on the residual tumor after iMWA.

[0060] Example 5 Preparation of cholesterol targeted catalytic hydrogel

[0061] The HCS conjugate prepared in Example 2 (5 wt%) and the oxidized dextran prepared in Example 3 (20 wt%) were mixed with cholesterol oxidase (50 U / mL), the balance being water, and placed at room temperature for 5 min to prepare COD-OD-HCS hydrogel. Unless otherwise specified, the hydrogels used in subsequent experiments were all prepared with this ratio, and if there were missing components, deionized water of the same mass was used instead.

[0062] Referring to the above steps, cholesterol oxidase was replaced with SA-COD and DA-COD prepared in Example 1 to prepare SA-COD-OD-HCS / DA-COD-OD-HCS hydrogel; the structure of the OD-HCS surface was observed using a scanning electron microscope, and the results were as follows: Figure 1As shown in B; the elastic modulus and viscous modulus of OD-HCS hydrogel were tested using a rotational rheometer, and the results are shown in Figure 1 As shown in C.

[0063] Example 6 Verification of the catalytic effect of cholesterol-targeted catalytic hydrogel

[0064] 6.1 Preparation of standard curve

[0065] The generated H2O2 was quantitatively detected using ABTS-HRP enzyme activity assay to evaluate the effect of DA-COD-OD-HCS hydrogel-mediated hydrogen peroxide generation.

[0066] First, 50 μL of hydrogen peroxide solution with different concentrations (0, 100, 200, 300 and 400 μM) was added to each well of a 96-well plate, and then 150 μL of ABTS-HRP detection solution was added and reacted for 5 minutes. The OD value was measured at 800 nm using a UV-visible spectrophotometer, and a H2O2 standard curve was drawn.

[0067] 6.2 ABTS-HRP enzyme activity assay to quantitatively detect cholesterol catalytic activity

[0068] Next, 5 μL of cholesterol (CHOL, 50 μM), linoleic acid (LA, 450 μg / mL), or cancer cell lysate (1×10 6 cells / mL) (10 μL PMSF (phenylmethylsulfonyl fluoride) was added to each 1 ml RIPA cell lysate to make the final concentration of PMSF 1 mM to prepare the lysate; 1×10 cells / well of a 6-well plate 6 The amount of H22 cells was added with 150-250 μL lysate to obtain cancer cell lysate), and reacted for 5 minutes at pH 6.8 or 7.4. Then, 50 μL of each reaction mixture was added to each well of a 96-well plate, and 150 μL of ABTS-HRP detection solution was added to react for 5 minutes. Finally, the OD value of the mixture was measured at 800 nm by a UV-visible spectrophotometer, and the cholesterol catalytic activity was determined according to the above H2O2 standard curve.

[0069] To evaluate the effect of different pH values ​​on the catalytic activity of COD, DA-COD or SA-COD enzymes, each sample (1 U / mL) was incubated at pH 6.8 or pH 7.4 for 2, 4, 8, 12 or 24 h. The cholesterol catalytic capacity of each sample at different pH values ​​was determined using the ABTS-HRP assay.

[0070] 6.3 TMB colorimetric method to evaluate the ability of hydrogel to generate hydroxyl groups

[0071] COD / DA-COD / SA-COD (5 U / mL) mixed with TMB (tetramethylbenzidine, 1 mM) or the hydrogel prepared by the method of Example 5 ([hemin] = 20 μg / mL) was injected into the lysate containing cholesterol (5 mg / mL) or cancer cell lysate (1×10 6 cells / mL) to evaluate the ability of catalyzing the hydrogel to generate ·OH.

[0072] 6.4 Experimental Results

[0073] The catalytic activity of COD under physiological conditions was evaluated by measuring the amount of H2O2 generated from the mixture of COD and cholesterol at different pH values ​​and temperatures.

[0074] COD showed high catalytic activity at pH 6-8 and 37-45°C, indicating that it has effective catalytic performance under physiological conditions. To determine the acid response and release ability of DA-COD, we studied the cholesterol catalytic activity of DA-COD at pH 6.8 and 7.4. Compared with COD, DA-COD showed about 81.14% of cholesterol catalytic activity at pH 6.8, but no activity at pH 7.4, indicating that DA-COD has a good response to the acidic tumor microenvironment and cholesterol catalytic ability ( Figure 1 Next, we investigated the acid response and cholesterol catalytic ability of DA-COD after mixing with OD-HCS hydrogel. DA-COD-OD-HCS hydrogel showed better acid response and cholesterol catalytic activity than SA group at pH 6.8 ( Figure 1 Interestingly, DA-COD-OD-HCS hydrogel exhibited significant catalytic ability at pH 6.8, promoting the peroxidation of linoleic acid ( Figure 1 (G) When using tumor cell fragments as substrates, we demonstrated that DA-COD-OD-HCS hydrogels

[0075] 6.8 also showed catalytic activity and ·OH generation ( Figure 1 In conclusion, DA-COD-OD-HCS hydrogel promoted pH-responsive lipid peroxidation more effectively than COD / SA-COD-OD-HCS hydrogel.

[0076] Example 7 Cell experiment verifies the effect of hydrogel in tumor microenvironment

[0077] 7.1 Evaluation of the ability of cholesterol-catalyzed hydrogels to induce intracellular lipid peroxidation

[0078] H22 cells (1×105 cells per well) were preseeded in 12-well plates and incubated with PBS, COD, DA-COD, or SA-COD in the presence or absence of cancer cell lysate (1×10 6 The cells were co-incubated for 6 hours in the presence of 100 dead cells (400 dead cells) to evaluate the ability of cholesterol-catalyzed hydrogels to induce intracellular lipid peroxidation. The pH value was adjusted to 7.4 or 6.8. Subsequently, each group of cells was washed with PBS and incubated in a fresh culture medium containing BODIPY-C11 (1.5 μM) or DCFH-DA dye (1.5 μM) for 30 minutes. The cells were then fixed with a 4 wt.% paraformaldehyde solution, stained with 1 μg / mL of DAPI, and imaged using a laser confocal microscope (Leica, STELLARIS5). In addition, each group of H22 cells was incubated in a fresh culture medium containing BODIPY-C11 (20 μM) or DCFH-DA (20 μM) for 4 hours, detected using a flow cytometer (BD, FACSCanto II), and analyzed using FlowJo_v10.8.1 (FlowJoTM software).

[0079] 7.2 Verification of the effect of cholesterol-catalyzed hydrogel in inducing cell ferroptosis

[0080] H22 cells were pre-seeded in 12-well plates (1×105 cells per well) and cancer cell lysate (1×10 6 Dead cells) were co-incubated with or without Fer-1 (10 μM) and GSH (1 mM) for 6 h to evaluate the rescue effect of Fer-1 and GSH. Then, the cells were stained with BODIPY-C11 (1.5 μM) dye and observed by confocal microscopy.

[0081] H22 cells were treated as described above for 24 h to assess HMGB1 release. Then, cells were washed twice with PBS, fixed with 4 wt.% paraformaldehyde solution for 20 min, permeabilized with 0.1 wt.% Triton X-100 for 10 min, blocked with 5% FBS for 30 min, and incubated with anti-HMGB1 primary antibody (1:1000) for 1 h, and then incubated with Alexa-525 labeled secondary antibody (1:500) for 30 min, following the manufacturer's procedure. Finally, cells were stained with DAPI for 10 min and observed by confocal microscopy. HMGB1 release from H22 cells after various treatments was assessed by flow cytometry.

[0082] 7.3 Evaluation of the effect of hydrogels on cellular calreticulin expression

[0083] H22 cells were pre-seeded in 12-well plates (1×10 5After receiving the same treatment as described above, the cells were incubated with anti-calreticulin (CRT) primary antibody (1:1000) for 1 hour, incubated with Alexa-488 labeled secondary antibody (1:500) for 30 minutes, and then stained with DAPI for 10 minutes to evaluate the expression of calreticulin (CRT). The stained cells were observed by confocal microscopy and analyzed by flow cytometry.

[0084] 7.4 Evaluation of the ability of catalytic hydrogels to induce cell death in different cell types

[0085] To evaluate the ability of catalytic hydrogels to induce cell death, H22, 4T1, HeLa, and A549 cells (1×105 cells per well) pre-seeded in 12-well plates were incubated with COD-OD, SA-COD-OD, or DA-COD-OD in the presence or absence of the corresponding cancer cell lysate (1×10 6 The cells were incubated in the presence of 40 dead cells for 24 h, and then the cell viability was detected using the standard MTT assay.

[0086] 7.5 Experimental Results

[0087] After applying COD and HCS to convert cholesterol into ·OH through an efficient cascade reaction, we investigated the antitumor effects of these compounds on tumor cell ferroptosis induced by tumor cell fragments. Using commercial BODIPY-C11 or DCFH-DA as specific probes, the effects of COD, DA-COD or SA-COD on lipid peroxidation in cancer cells under the action of cancer cell lysates, especially at pH 6.8 and 7.4, were studied and analyzed by confocal microscopy and flow cytometry. The results showed that in the presence of cell lysate, the intracellular lipid peroxidation capacity caused by DA-COD (DA = 2 mg / mL, COD = 5 U / mL) was significantly stronger than that in the absence of lysate ( Figure 2 In addition, H22 cells were treated with cell lysate (1×10 6 In the case of treatment with 5 dead cells (100 U / mL), DA-COD induced intracellular lipid peroxidation more effectively than COD (5 U / mL) in an acidic environment of pH 6.8. Therefore, DA-COD can effectively induce intracellular lipid peroxidation.

[0088] Next, we investigated the rescue effects of two potent ferroptosis inhibitors, ferroptosis inhibitor 1 (Fer-1) and glutathione (GSH), on cell death induced by DA-COD plus cell lysate. Interestingly, confocal microscopy and BODIPY-C11 analysis revealed that the addition of Fer-1 and GSH could effectively inhibit the cell death of H22 cells in the presence of DA-COD and cell lysate (1×106 In line with this, Fer-1 or GSH treatment could effectively reverse the cytotoxicity of H22 cells treated with DA-COD and cell lysate ( Figure 2 CE), indicating that DA-COD and tumor cell lysate are potent inducers of cell death via the ferroptosis pathway.

[0089] Confocal microscopy showed that when H22 cells were treated with DA-COD-HCS in an acidic environment of pH 6.8 for 24 h, the release of HMGB1 in the nucleus was significantly increased, and the expression of CRT was increased, which was in sharp contrast to the results of the other treatment groups, which had little effect on DAMPs ( Figure 2 In addition, the excellent cytotoxicity of this combination treatment was confirmed in MCF-7, 4T1, HeLa, and A549 cells ( Figure 3 ). The above results indicate that DA-COD can gradually release COD in the acidic tumor environment. DA-COD-HCS utilizes cell lysis products as a source of cholesterol, leading to a lipid peroxidation chain reaction, which ultimately leads to cancer cell death through the ferroptosis pathway.

[0090] Example 8 Animal experiment to verify the treatment effect of hydrogel after microwave ablation

[0091] 8.1 Experimental animals and modeling

[0092] Female BALB / c mice and male SD rats were purchased from Hangzhou Hangsi Biotechnology Co., Ltd. All experiments were performed according to the protocols approved by the Experimental Animal Center of Lishui Central Hospital.

[0093] H22 and 4T1 cells (2×10 6 The cells were suspended in 50 μL PBS and injected subcutaneously into the right abdomen of each BALB / c mouse to establish subcutaneous H22 and 4T1 tumor models.

[0094] N1S1 cells (6×10 6 The 100 mL PBS containing 30% Matrigel (Corning) was mixed with 100 mL PBS and injected into the right lower liver lobe of each SD rat to establish an orthotopic liver N1S1 tumor model.

[0095] 8.2 Incomplete microwave ablation (iMWA) treatment method

[0096] iMWA was performed by inserting a microwave ablation probe disinfected with 75% ethanol into the mouse tumor to a depth of 0.5 cm, adjusting the microwave ablation output power to 5 W, and performing the procedure for 2 minutes. An infrared thermal imager (Fortric225) was used to monitor the tumor surface temperature to ensure that the tumor temperature remained below 60°C.

[0097] 8.3 Retention effect of DA-COD-OD-HCS hydrogel in tumor after iMWA.

[0098] To evaluate the retention effect of DA-COD-OD-HCS hydrogel in tumors after iMWA, we carefully recorded the retention time of Cy5.5-labeled DA-COD-OD-HCS hydrogel (10 mg / mL) in tumors. Six H22 tumor-bearing mice were randomly divided into two groups and subjected to microwave irradiation at 5W power for 2 minutes. Subsequently, three mice were injected with Cy5.5-labeled DA-COD-OD-HCS hydrogel (10 mg / mL; colloid group) and three were injected with Cy5.5-labeled (2 mg / mL Cy5.5 fluorescent dye was added) DA-COD (10 mg / mL; no-colloid group). Afterwards, the in vivo imaging system (IVIS, PerkinElmer) was used for evaluation at different time points (0.5, 24, 48, 72, 120 hours), and the Cy5.5 fluorescence intensity of each group was recorded. Twenty-four H22 tumor-bearing mice were divided into four groups (n=6) and injected with DA-COD+OD-HCS (group I), DA-COD+OD (group II), DA-COD+HCS (group III) or DA-COD (group IV). Finally, tumor tissues were collected at 24 hours and 72 hours after injection and observed under a laser confocal microscope using frozen sections.

[0099] 8.4 Evaluation of the ability of cholesterol-catalyzed hydrogels to induce intracellular lipid peroxidation in vivo

[0100] To evaluate the ability of cholesterol-catalyzed hydrogels to induce intracellular lipid peroxidation in vivo, we evaluated HMGB1 release and lipid peroxidation in tissues of H22 tumor-bearing mice after different treatments. Twelve H22 tumor-bearing mice were randomly divided into four groups (n=3): Group I, iMWA; Group II, iMWA+colloid; Group III, iMWA+DA-COD+colloid; Group IV, iMWA+SA-COD+colloid, where colloid refers to the OD-HCS hydrogel prepared in Example 4. All groups received iMWA treatment, and the residual tumors of Groups II, III, and IV were injected with OD-HCS, DA-COD-OD-HCS, and SA-COD-OD-HCS hydrogels, respectively. After 24 hours, tumor sections of each group were taken and incubated for 1 hour with anti-HMGB1 primary antibody (1:200, MultiSciences, catalog number: 70-ab40050-100), and then incubated for 30 minutes with Alexa-525 labeled secondary antibody (1:500, Jackson, catalog number: 111-545-003). Finally, the sections were counterstained with DAPI and observed under a laser confocal microscope. In addition, tumor tissue sections were incubated with BODIPY-C11 dye (1.5 μM) for 30 minutes, counterstained with DAPI for 10 minutes, and observed under a confocal microscope.

[0101] 8.5 Study on the anti-tumor effects of different treatments on H22 tumor-bearing mice

[0102] H22 tumor-bearing mice (tumor volume approximately 80 mm 3) were randomly divided into six groups (n=6 in each group) to study the anti-tumor effects of different treatments on tumor-bearing mice (Group I, Ctrl group; Group II, iMWA group; Group III, iMWA+colloid+SA-COD group; Group IV, DA-COD group; Group V, DA-COD+colloid group; Group VI, iMWA+colloid+DA-COD group. Colloid refers to OD-HCSs). In Group I, H22 or 4T1 tumor-bearing mice were directly injected with 100 μM BBT-2FT probe. Mice in Groups II and VI were first treated with iMWA (5W, 2 minutes), and then the residual tumors in Groups III and VI were injected with BBT-2FT-labeled SA-COD-OD-HCS (10 mg / mL) and BBT-2FT-labeled DA-COD-OD-HCS (10 mg / mL), respectively. Group IV and Group V were directly injected with BBT-2FT-labeled DA-COD (10 mg / mL) and BBT-2FT-labeled (BBT-2FT is a near-infrared zone II dye, CAS registration number: 1070910-84-7) DA-COD-OD-HCS (10 mg / mL). Subsequently, the mice were observed at different time points (0, 7, 14 and 21 days) using a near-infrared II in vivo imaging system (NIR-II, MARS, Artemis Intelligent Imaging), and the NIR-II fluorescence intensity of BBT-2FT in each group was recorded. At the same time, at the beginning of treatment, a digital caliper was used to record the length and width of the tumor in each group every other day, and the tumor volume was calculated according to the following formula: Tumor volume = L×W×W / 2. Tumor volume greater than 1000mm 3 Death was defined as the day of death, and the survival time of each mouse was recorded. During the treatment, the weight of each mouse was recorded using a digital balance. After the death of the mice, the tumors of each group were removed, embedded in paraffin, sliced, and the anti-tumor effects of each group were evaluated by hematoxylin and eosin (H&E) staining and TUNEL staining.

[0103] 8.6 Study on the anti-tumor effects of different treatments on 4T1 tumor-bearing mice

[0104] Referring to the method in 8.5, 4T1 tumor-bearing mice (tumor volume about 80 mm) were used. 3 ) were used for the experiment and the treatment effects were evaluated in the same sections.

[0105] 8.7 Validation of the efficacy of iMWA combined with cholesterol catalytic hydrogel in the treatment of orthotopic N1S1 tumor model

[0106] In addition, twenty-four SD rats with N1S1 tumor burden were randomly divided into four groups (n = 6 in each group) to further verify the efficacy of iMWA combined with cholesterol catalytic hydrogel in the treatment of orthotopic N1S1 tumor model: Group I, Ctrl group; Group II, iMWA + SA-COD + colloid group; Group III, iMWA + COD + colloid group; Group IV, iMWA + DA-COD + colloid group. On day 0, groups II, III, and IV were first treated with iMWA. The MWA probe was disinfected with 75% ethanol and inserted into the orthotopic N1S1 liver tumor. The MWA output power was set to 5W for 2 minutes. At the same time, an infrared thermal imager (Fortric225) was used to monitor the tumor surface temperature to ensure that the tumor temperature remained below 60°C. Within ten minutes after iMWA treatment, groups II, III, and IV were injected with SA-COD + colloid, COD + colloid, and DA-COD + colloid, respectively. Colloid refers to OD-HCSs. Subsequently, 3.0-T MRI was used to image the SD rats in each group on days 0, 7, and 14, and the tumor volume of each group was recorded and quantified. Finally, after sacrificing the rats in each group, the orthotopic liver tumors were removed and H&E and Ki67 immunohistochemical staining were performed.

[0107] 8.8 Experimental Results

[0108] To effectively ensure the antitumor ability of cholesterol-catalyzed hydrogel after iMWA, we carefully evaluated the retention and therapeutic effect of DA-COD-OD-HCS hydrogel on residual tumors after iMWA. 3 ) were inoculated subcutaneously into BALB / c mice, and the tumor area was heated using a commercial MWA system. The temperature of the tumor area was controlled at about 60°C by a thermal imager for 2 minutes to establish a residual tumor model after iMWA ( Figure 4 Figure 2 (A). We used the IVIS in vivo fluorescence imaging system to monitor the fluorescence of covalently labeled Cy5.5 on COD to evaluate the retention efficiency of cholesterol-catalyzed hydrogels within the tumor. At 5 days post-injection (pi), DA-COD-OD-HCS hydrogels retained approximately 50% of COD at the residual tumor site. In contrast, without the use of cholesterol-catalyzed hydrogels, COD retention at the residual tumor site was only approximately 5% ( Figure 4 In addition, through laser confocal microscopy, we found that cholesterol-catalyzed hydrogels could still effectively promote the retention and lateral diffusion of Cy5.5-labeled DA-COD in residual tumors within 72 hours after iMWA, while there was almost no Cy5.5 fluorescence in tumor sections of mice injected with DA-COD ( Figure 4 (D) These results suggest that DA-COD-OD-HCS hydrogel can promote the long-term retention and lateral diffusion of COD in residual tumors after iMWA treatment.

[0109] To determine the antitumor effect of cholesterol-catalyzed hydrogels combined with iMWA in vivo, we carefully evaluated the ability of DA-COD-OD-HCS hydrogels to induce intratumoral lipid peroxidation and immunogenic cell death (ICD). 3 ) were randomly divided into four groups and received the following treatments: Group I, DA-COD; Group II, iMWA+COD+OD-HCS; Group III, iMWA+DA-COD+OD-HCS; Group IV, iMWA+SA-COD+OD-HCS. First, immunofluorescence staining was performed using anti-HMGB1 primary antibodies to evaluate the effectiveness of these treatments in inducing ICD of residual cancer cells in vivo. Semi-quantitative analysis showed that the reduction efficiency of HMGB1 signals in tumor sections treated with iMWA combined with DA-COD-OD-HCS (Group III) was the greatest, at only 18.6%, compared with Group I ( Figure 4 In addition, we found that the tumor sections of group III showed significantly stronger BODIPY-C11 fluorescence than those of other groups ( Figure 4 These results indicate that cholesterol-catalyzed hydrogels can effectively induce immunogenic cell death of cancer cells by continuously initiating lipid peroxidation after local injection of iMWA.

[0110] To evaluate the therapeutic effect of iMWA combined with DA-COD-OD-HCS, 36 H22 tumor-bearing mice transfected with the NIR-II fluorescent dye BBT-2FT were randomly divided into six groups and received the following treatments: Group I, Ctrl; Group II, iMWA; Group III, iMWA+Glue+SA-COD; Group IV, DA-COD; Group V, DA-COD+Glue; Group VI, iMWA+Glue+DA-COD. Glue refers to OD-HCS hydrogel. By recording the NIR-II bioluminescent signal, it was semi-quantitatively determined that approximately 50% of the tumor tissue was not destroyed after iMWA treatment, and mice in Group VI (iMWA+Glue+DA-COD) showed the most effective tumor inhibitory effect. In addition, by H&E staining, we found that tumor sections from mice in Group VI showed the most severe histological damage. Microscopic examination by TUNEL staining further confirmed the superior therapeutic effect of this combined treatment in promoting apoptosis of tumor tissue ( Figure 5 Middle A). The tumor growth rate of mice treated with iMWA sequential cholesterol-catalyzed hydrogel was significantly reduced ( Figure 5 The median survival of mice treated with iMWA combined with cholesterol-catalyzed hydrogel was 45 days, significantly longer than that of group I (21 days) and other treatment groups ( Figure 5In addition, the combined therapy did not affect the average body weight of any mice during the treatment period, indicating that the cholesterol-catalyzed hydrogel has good biocompatibility ( Figure 5 (middle D).

[0111] To further elucidate the synergistic effect of cholesterol-catalyzed hydrogel in iMWA treatment of liver cancer, we applied the combined treatment of iMWA and cholesterol-catalyzed hydrogel to the 4T1 tumor mouse model. Thirty-six 4T1 tumor-bearing mice transfected with the NIR-II fluorescent dye BBT-2FT were randomly divided into six groups and received the following treatments: Group I, Ctrl; Group II, iMWA; Group III, iMWA+Glue+SA-COD; Group IV, DA-COD; Group V, DA-COD+Glue; Group VI, iMWA+Glue+DA-COD. Glue refers to OD-HCS hydrogel. By recording NIR-II bioluminescent signals, H&E staining, TUNEL staining, and measurement of tumor volume, we found that the combined treatment of iMWA and cholesterol-catalyzed hydrogel had a greater tumor inhibitory effect than intratumoral injection of iMWA or DA-COD alone, and significantly prolonged the median survival after iMWA ( Figure 5 In addition, the body weight of all mice did not change significantly during the treatment ( Figure 5 The above results indicate that cholesterol-catalyzed hydrogels for tumor fixation is a promising strategy to improve the efficacy of conventional MWA for tumor treatment.

[0112] Subsequently, the antitumor effect of iMWA combined with cholesterol-catalyzed hydrogel was further confirmed in the highly malignant N1S1 HCC rat tumor model. Twenty-four rats bearing N1S1 liver tumors were randomly divided into four groups and received the following treatments: Group I, Ctrl; Group II, iMWA+SA-COD+Glue; Group III, iMWA+COD+Glue; Group IV, iMWA+DA-COD+Glue. iMWA treatment was performed 8 days after the establishment of the N1S1 orthotopic liver tumor model, and the tumor volume was approximately 350mm 3 . The hydrogel in each group was injected into the tumor on the day of treatment. The rats were intraperitoneally injected with a commercial gadolinium contrast agent one day before treatment and 7 and 14 days after treatment, and 3.0-T magnetic resonance imaging was performed to record the tumor volume. Group IV (iMWA+DA-COD+Glue) showed a strong antitumor effect in liver cancer treatment, and after 14 days, 4 of 5 rats were cured, while group II (iMWA+SA-COD+Glue) and group III (iMWA+COD+Glue) treatment only partially inhibited the in situ HCC growth ( Figure 6Finally, liver residual tumor observation, H&E staining, and Ki67 staining further confirmed that iMWA combined with cholesterol-catalyzed hydrogel therapy could significantly inhibit the in situ proliferation of HCC cells by killing tumor tissue and had no significant effect on the health status of rats, such as body weight ( Figure 6 In summary, the experimental results show that MWA combined with cholesterol catalytic hydrogel can effectively inhibit tumor growth without causing obvious side effects.

[0113] Example 9 Verification of the effect of cholesterol-targeted catalytic hydrogel on enhancing anti-tumor immunotherapy in vivo

[0114] 9.1 Combining in situ cholesterol-catalyzed hydrogel immobilization and iMWA treatment to enhance antitumor immune responses

[0115] To explore the mechanism of iMWA combined with DA-COD-OD-HCS hydrogel to enhance anti-tumor immunotherapy, a dual tumor mouse model including primary tumor and distant tumor was established. 4T1 cells (2×10 6 ) were suspended in 50 μL PBS and subcutaneously inoculated into the right and left abdomens of mice on days 0 and 7, representing primary tumors and distant tumors, respectively. On day 8, 30 mice were randomly divided into six groups (5 in each group) and treated as follows: Group I, Ctrl group; Group II, anti-PD-L1 group; Group III, iMWA+Glue group; Group IV, iMWA+Glue+anti-PD-L1 group; Group V, iMWA+Glue+DA-COD group; Group VI, iMWA+Glue+DA-COD+anti-PD-L1 group. Glue represents OD-HCS hydrogel. Each mouse in Group II was injected with anti-PD-L1 antibody (20 μg / time) through the tail vein. The right primary tumors of mice in Groups III, IV, V, and VI were first treated with iMWA. Subsequently, the residual tumors on the right side of mice in Group III were injected with OD-HCS hydrogel (10 mg / mL). In group IV, the right residual tumor was first injected with OD-HCS hydrogel (10 mg / mL), and then anti-PD-L1 (20 μg / time) was injected through the tail vein. In group V, the right residual tumor was injected with DA-COD-OD-HCS hydrogel (10 mg / mL). In group VI, the right residual tumor was injected with DA-COD-OD-HCS hydrogel (10 mg / mL), followed by anti-PD-L1 (20 μg / time) through the tail vein. In addition, all mice in groups II, IV and VI were injected with anti-PD-L1 (20 μg / time) through the tail vein on days 9, 11 and 15, respectively.

[0116] During the treatment period, the length and width of each tumor were recorded every two days with a digital caliper, and the tumor volume was calculated according to the following formula: tumor volume = L × W × W / 2. Tumor volume exceeded 1000 mm 3 The death was defined as 1.5 d, and the survival time of each group of mice was recorded. The weight of each mouse during the treatment was recorded using a digital balance.

[0117] Mice in each group were sacrificed on day 19, and lymph nodes close to the primary tumor and distant tumors were collected, and single-cell suspensions and homogenates were prepared according to established protocols for subsequent antibody labeling. Then, dendritic cells (CD80 + CD86 + ) and CD8 + T cells (CD3 + CD4 - CD8 + ) and Tregs (CD3 + CD4 + Foxp3 + ) were stained and analyzed by flow cytometry. In addition, after obtaining distant tumors of each group of mice and preparing paraffin sections, anti-CD86 (BD Biosciences, catalog number: 553689), anti-CD8 (Invitrogen Antibodies, catalog number: 14-0081-82), anti-Foxp3 (Invitrogen Antibodies, catalog number: 14-5773-82), anti-Ki67 (Abcam, catalog number: ab15580) and TUNEL (Abcam, catalog number: ab66108) antibodies were used to measure protein expression and localization after different treatments. The supernatant of the tumor homogenate was collected and the concentrations of tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) were determined using TNF-α (Invitrogen, catalog number: 88-7324-88) and IFN-γ (Invitrogen, catalog number: 88-7314-88) ELISA kits.

[0118] 9.2 Experimental Results

[0119] To explore the ability of iMWA combined with cholesterol-catalyzed hydrogel to enhance antitumor immune responses, we combined sequential iMWA treatment and intratumoral cholesterol-catalyzed hydrogel immobilization with anti-PD-L1 immunotherapy ( Figure 7Thirty mice with two H22 tumors on each side were randomly divided into six groups (5 mice in each group) and received the following treatments: Group I, Ctrl group; Group II, anti-PD-L1 group; Group III, iMWA+Glue group; Group IV, iMWA+Glue+anti-PD-L1 group; Group V, iMWA+Glue+DA-COD group; Group VI, iMWA+Glue+DA-COD+anti-PD-L1 group.

[0120] Tumor size measurements showed that intratumoral injection of cholesterol-catalyzed hydrogel combined with anti-PD-L1 antibody after iMWA treatment more effectively inhibited the growth of primary and distant tumors than iMWA or anti-PD-L1 immunotherapy alone ( Figure 7 (B, C). Four of the five mice treated with iMWA combined with sequential cholesterol-catalyzed hydrogel fixation and anti-PD-L1 injection were cured, while two of the mice treated with sequential iMWA and cholesterol-catalyzed hydrogel were cured, and the cured mice in these two groups showed no obvious recurrence within 60 days. Compared with the median survival of 20 days in group I, the median survival of mice treated with anti-PD-L1 (group II), iMWA+Glue (group III), and iMWA+Glue+anti-PD-L1 (group IV) was 28 days, 26 days, and 30 days, respectively ( Figure 7 Middle D). Overall, the novel combination therapy of cholesterol-catalyzed hydrogel injected into residual tumors and anti-PD-L1 antibody effectively inhibited the growth of primary and distant tumors.

[0121] To further investigate the mechanism of how this cholesterol-catalyzed hydrogel effectively enhances anti-PD-L1 immunotherapy, we carefully evaluated the effect of this combination therapy on the immune system 4 days after the last anti-PD-L1 injection. The results showed that intratumoral injection of cholesterol-catalyzed hydrogel (with or without combined anti-PD-L1 injection) significantly promoted the maturation of dendritic cells (DCs) in lymph nodes near the primary tumor after iMWA treatment ( Figure 7 In E), this is consistent with its ability to induce HMGB1 release and CRT expression. In addition, the combination therapy of cholesterol-catalyzed hydrogel and anti-PD-L1 antibody after iMWA significantly increased the frequency of tumor-infiltrating CD3+CD8+ cells in distant tumors ( Figure 7 F) and reduced the infiltration of immunosuppressive T regulatory cells (Tregs) ( Figure 7 Middle G), significantly increased the proportion of CD3+CD8+ / Tregs in distant tumors ( Figure 7 In addition, the combination therapy significantly increased the secretion levels of cytotoxic cytokines (including TNF-α and IFN-γ) in distant tumors ( Figure 7I, J), thereby effectively inhibiting the proliferation of distant tumors and enhancing their apoptosis. In summary, the combination therapy of cholesterol-catalyzed hydrogel fixation and anti-PD-L1 injection in residual tumors after iMWA treatment enhanced the host's antitumor immune response.

[0122] 9.3 Safety evaluation of cholesterol-targeted catalytic hydrogels to enhance in vivo anti-tumor immunotherapy

[0123] We carefully evaluated the biosafety of the combination therapy using standard serum biochemical assays, routine whole blood analysis, and histological examination. Mice were sacrificed at 0, 12, 72, and 68 h after continuous treatment with iMWA or cholesterol-catalyzed hydrogels and then subjected to the corresponding tests ( Figure 8 First, H&E-stained sections of major organs collected from mice at 0, 12, 72, and 168 hours after combined treatment were observed under a microscope, and no obvious histological damage was found ( Figure 8 (B). Secondly, after 12 hours of combined treatment, the white blood cell (WBC) count increased significantly. Platelet (PLT), hemoglobin (HGB), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and lymphocyte (LYM) counts increased slightly, and mean corpuscular volume (MCV) and monocyte (MON) and neutrophil (NEU) counts decreased slightly; however, these physiological indices gradually returned to the corresponding normal levels after 168 hours of combined treatment. In addition, the effects of combined treatment on red blood cell (RBC), hematocrit (HCT), and eosinophil (EOS) levels were negligible ( Figure 8 (middle C~L).

[0124] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for preparing a cholesterol-targeted catalytic hydrogel for enhancing microwave ablation therapy and anti-tumor immune response, characterized in that The steps include: (1) Cholesterol oxidase and dimethylmaleic anhydride are dissolved in Hepes buffer and mixed, pH is controlled and stirred for reaction, and after the reaction is completed, filtration is performed to obtain DA-COD; (2) adding hemin, EDC, and NHS to DMSO for activation, then adding carboxymethyl chitosan and stirring for reaction, dialyzing and freeze-drying after the reaction to obtain HCS conjugate; (3) adding dextran and sodium periodate to water, stirring to react in the dark, dialyzing, and freeze-drying to obtain oxidized dextran; (4) After adding DA-COD, HCS conjugate and oxidized dextran into water and mixing, the mixture is allowed to gel at room temperature to obtain a cholesterol-targeted catalytic hydrogel that enhances microwave ablation therapy and anti-tumor immune response.

2. The preparation method according to claim 1, characterized in that: The ratio of cholesterol oxidase to dimethylmaleic anhydride in step (1) is 100 U: 1-3 mg; The pH of the Hepes buffer in step (1) is 8.5 to 9.5; The controlled pH in step (1) is greater than or equal to 7.4; The stirring reaction time in step (1) is 3 to 4 hours; The filtration in step (1) is carried out using a filtration device with a molecular weight cut-off of 8 to 12 kDa.

3. The preparation method according to claim 1, characterized in that: The molar ratio of hemin, EDC, NHS and carboxymethyl chitosan in step (2) is 1-2:1-2:1-2:4-6; The activation time in step (2) is 1 to 3 hours; The stirring reaction time in step (2) is 6 to 12 hours; The dialysis conditions of step (2) are to use a dialysis bag with a molecular weight cutoff of 8000 to 14000 Da to dialyze in a sodium bicarbonate solution with a pH of 8 to 9 for 20 to 28 hours.

4. The preparation method according to claim 1, characterized in that: The molar ratio of dextran to sodium periodate in step (3) is 1-2:10-20; The time of the light-proof stirring reaction in step (3) is 64 to 80 hours; The dialysis conditions in step (3) are to use a dialysis tube with a molecular weight cut-off of 3000 to 4000 Da for dialysis in deionized water.

5. The preparation method according to claim 1, characterized in that: The mass ratio of the HCS conjugate, oxidized dextran and water in step (4) is 1-10:10-30:60-90; The final concentration of DA-COD in step (4) is 40-60 U / mL, calculated as the enzyme activity unit of COD before modification; The placement time described in step (4) is 3 to 10 minutes.

6. A cholesterol-targeted catalytic hydrogel for enhancing microwave ablation therapy and anti-tumor immune response, prepared according to the preparation method according to any one of claims 1 to 5.

7. Use of the cholesterol-targeted catalytic hydrogel for enhancing microwave ablation therapy and anti-tumor immune response as claimed in claim 6 in the preparation of anti-tumor drugs.

8. Use of the cholesterol-targeted catalytic hydrogel for enhancing microwave ablation therapy and anti-tumor immune response as claimed in claim 6 in the preparation of microwave ablation synergist-enhancing drugs.

9. A drug for enhancing anti-tumor immune response after microwave ablation therapy, characterized in that: The cholesterol-targeted catalytic hydrogel for enhancing microwave ablation therapy and anti-tumor immune response as described in claim 6.

10. The drug according to claim 9, characterized in that: The drug for enhancing the anti-tumor immune response after microwave ablation therapy also includes anti-PD-L1.

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