Application of HP and ICT combined anti-PD-1 inhibitor hydrogel in residual cancer recurrence after IRFA

Abstract

The invention belongs to the technical field of anti-tumor, and discloses application of HP and ICT combined anti-PD-1 inhibitor hydrogel in residual cancer recurrence after IRFA. The excellent drug delivery capacity of the hydrogel system is utilized, and the in-vivo local slow release effect of ICT and BMS202 can be remarkably amplified. Through synergistic treatment of the TAN and the MDSC, local and whole body adaptive immunoreactions can be efficiently activated, TAN is effectively reprogrammed to be in a tumor suppression type, infiltration of PMN-MDSC is reduced, and the conclusion is verified in a plurality of mouse tumor models in the chapter. Therefore, the HP (at) ICT / BMS provides a promising delivery strategy for improving the sensitivity of anti-PD-L1 treatment and efficiently preventing and treating latent residual cancer and metastasis after IRFA operation.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of anti-tumor, more specifically, relates to the application of HP@ICT combined with anti-PD-1 inhibitor hydrogel in the recurrence of residual cancer after IRFA. BACKGROUND

[0002] In the treatment of HCC, immunotherapy is a promising strategy, where the research of immune modulators has made significant progress in both mechanism exploration and delivery technology, and the core is to enhance the anti-tumor effect by regulating the interaction between tumor microenvironment and immune system. Icaritin (ICT), as an innovative small molecule immunomodulator of traditional Chinese medicine in recent years, has played a significant role in suppressing tumors in multiple tumors. Although ICT capsules have been approved for oral administration for the treatment of advanced HCC in 2022, there are some limitations in the characteristics and administration route of ICT. ICT belongs to flavonoids, which has poor water solubility. During oral administration of ICT, problems such as instability of digestive tract lumen fluid, insolubility in the intestine, poor absorption of mucus and cell membranes, and loss of biological activity due to first-pass effect may occur. Therefore, compared with other anti-HCC drugs (gefitinib, sorafenib, etc.), a larger dose of ICT is often required in clinical practice, which may cause serious side effects.

[0003] The metastasis of residual tumor after RFA of liver cancer is also a major challenge in clinical treatment. In addition, studies have reported that RFA exhibits a unique "abscopal effect" in clinical treatment, that is, local ablation not only reduces the primary lesion, but also inhibits the growth of distant metastatic lesions by activating systemic immune response. However, the immune-inhibitory microenvironment caused by incomplete ablation can escape immune killing by recruiting MDSC and up-regulating PD-L1 expression, significantly weakening the systemic anti-tumor effect of RFA. In view of this key problem, the synergistic treatment strategy of RFA combined with immunomodulatory hydrogel has become a breakthrough research direction. Studies have reported that immune checkpoint inhibitors may be more effective for micrometastatic residual lesions, which is also applicable to HCC patients with residual cancer after ablation. Currently, there is no research on the synergistic treatment of ICT and anti-PD-L1 inhibitors to inhibit the progression and metastasis of residual cancer after IRFA. SUMMARY

[0004] Based on the above-mentioned defects existing in the prior art, the present application first provides a HP@ICT / BMS hydrogel loaded with poorly soluble traditional Chinese medicine small molecule Icaritin and PD-L1 inhibitor.

[0005] A second object of the present application is to provide a preparation method of the above-mentioned HP@ICT / BMS hydrogel.

[0006] A third object of the present application is to provide the application of HP@ICT / BMS hydrogel in the treatment of residual cancer after IRFA.

[0007] The object of the present application is achieved by the following technical solutions:

[0008] To solve the problems of solubility and systemic toxicity of ICT, the hydrogel drug delivery system used in the present application increases the solubility of ICT. Compared with free ICT and intragastric ICT groups, the treatment efficiency of HP@ICT is significantly improved, and there is no obvious side effect in local or systemic level.

[0009] We also explored the effect of ICT combined with anti-PD-L1 treatment. Studies have shown that ICT can down-regulate the expression of immune checkpoint ligand molecule PD-L1, which is expected to further enhance the sensitivity of ICT treatment, restore the anti-tumor activity of T cells, and enhance the immune response of the body to tumors. Therefore, we further added anti-PD-1 / PD-1-L1 small molecule inhibitors to synthesize HP@ICT / BMS combined immune hydrogel to evaluate the effect of reprogramming TAN in residual cancer to suppress tumor type and combined with anti-PD-L1 treatment to clear residual cancer after IRFA. Our results show that the complete remission rates of tumor in HP@ICT single drug hydrogel group and HP@ICT / BMS combined hydrogel treatment group are 33% and 83% respectively, and the HP@ICT / BMS combined treatment group enhances the anti-tumor response and more significantly inhibits the recurrence of residual cancer after IRFA. In summary, the above data shows that the HP@ICT / BMS combined treatment has more significant effect, can activate residual cancer immunity, significantly improve the sensitivity of residual cancer immunotherapy, and ultimately clear residual cancer.

[0010] Therefore, the present application first protects the application of HP@ICT / BMS hydrogel in the preparation of a drug for improving the sensitivity of residual cancer after liver cancer IRFA to anti-PD-L1 immunotherapy, wherein the HP@ICT / BMS hydrogel is prepared by mixing ICT, BMS202, HP-β-CD and pomerantz 407.

[0011] The present application also protects the preparation method of HP@ICT / BMS hydrogel, comprising the following steps:

[0012] S1, add the ICT solution to the HP-β-CD solution at room temperature, mix and dissolve to obtain an ICT / HP-β-CD solution;

[0013] S2, pre-cool the ICT / HP-β-CD solution of S1, then add BMS202, and finally slowly add pomerantz 407, stir and mix at 4℃ to ensure that pomerantz 407 is completely dissolved;

[0014] S3, after a period of time, HP@ICT / BMS hydrogel is obtained.

[0015] This invention revealed that the immunosuppressive response within the residual tumor tumor mesenchyme (TME) was further reduced in the HP@ICT / BMS hydrogel combined treatment group. Simultaneously, the HP@ICT / BMS combined treatment group effectively enhanced tumor-invasive CD8+. + T cell infiltration was significantly reduced, while the infiltration of immunosuppressive cells (Treg cells) and PMN-MDSCs was also significantly decreased.

[0016] Therefore, this invention also protects the use of the HP@ICT / BMS hydrogel in the preparation of drugs for antitumor immune responses, wherein the antitumor immune response refers to:

[0017] (1) HP@ICT hydrogel recruits residual effector T cells in liver cancer after RFA surgery, reduces Treg cell infiltration, and / or;

[0018] (2) HP@ICT hydrogel promotes the production of functional cytokines in residual tumor cells after RFA surgery for liver cancer, and / or;

[0019] (3) HP@ICT hydrogel promotes apoptosis of residual tumor cells in liver cancer after RFA surgery.

[0020] Preferably, the functional cytokines are Granzyme B, IFN-γ, and TNF-α.

[0021] Subsequent experiments showed that the HP@ICT / BMS combined treatment hydrogel group exhibited the best effect in inhibiting the growth of multiple liver metastases. Therefore, the present invention also protects the use of the HP@ICT / BMS hydrogel in the preparation of drugs to inhibit liver metastasis of residual cancer after IRFA in liver cancer.

[0022] In addition, the effect of HP@ICT / BMS combined treatment hydrogel group in preventing distant tumor growth was studied. The results showed that the signal of HP@ICT / BMS combined treatment hydrogel group was significantly weakened, indicating that it showed the best effect in inhibiting distant tumor growth. Therefore, the present invention also protects the application of HP@ICT / BMS hydrogel in the preparation of drugs for inhibiting the growth of distant tumors after IRFA of liver cancer.

[0023] Experiments show that HP@ICT utilizes local hydrogel drug delivery to achieve higher drug concentrations in residual cancer areas, thereby more effectively inhibiting tumor growth. When used in combination with BMS, the inhibitory effect is even more significant, with a complete elimination rate of residual cancer reaching 83%. Therefore, this invention also protects the application of HP@ICT / BMS hydrogel in the preparation of drugs for eradicating residual cancer after IRFA in liver cancer.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] This invention discloses the application of HP@ICT combined with an anti-PD-1 inhibitor hydrogel in the recurrence of residual cancer after IRFA. Utilizing the superior drug delivery capabilities of the hydrogel system, the in vivo local sustained-release effects of ICT and BMS202 can be significantly amplified. The synergistic treatment of these two drugs can efficiently activate local and systemic adaptive immune responses, effectively reprogramming TANs to a tumor-suppressive mode and reducing PMN-MDSC infiltration. This conclusion has been validated in multiple mouse tumor models described in this section. Therefore, HP@ICT / BMS provides a promising delivery strategy for improving sensitivity to anti-PD-L1 therapy and effectively preventing occult residual cancer and metastasis after IRFA. Attached Figure Description

[0026] Figure 1 The following are the characteristics of HP@ICT: (A) Schematic diagram of HP@ICT hydrogel synthesis; (B) Statistical graph of gelation time required when 10% HP-β-CD hydrogel is mixed with different mass fractions of P407 at 37℃; (C) Statistical graph of gelation time required when 10% HP-β-CD ICT hydrogel is mixed with different mass fractions of P407 at 37℃; (D) Statistical graph of gelation time required when 10% HP-β-CD ICT hydrogel is mixed with different mass fractions of P407 at room temperature; (E) Photographs of HP@ICT hydrogel at room temperature and physiological temperature; (F) Injectability of HP@ICT at 37℃; (G) Scanning electron microscope image of HP@ICT hydrogel. Scale bar: 20 μm; (H) Variation of viscoelastic parameters (η*, G, G) of HP@ICT at different temperatures; (I) G' and G'' of the hydrogel in strain amplitude scanning; (J) Viscosity changes showing the shear thinning characteristics of the hydrogel as the shear rate (0.1 - 100 s⁻¹) increases;

[0027] Figure 2 The graph shows the cellular uptake and biocompatibility of HP@ICT; (A) in vivo degradation of HP@ICT; (B) a representative immunofluorescence plot of HP@FITC uptake by neutrophils, scale bar: 50 μm; (C) a representative flow cytometry plot of drug uptake by neutrophils; and (D) quantitative analysis, ***representative. P ≤ 0.001. **** represents P ≤ 0.0001; (E) Fluorescence images at different time points after subcutaneous injection of ICG-PBS and HP@ICG in mice using small animal in vivo imaging monitoring; (F) Statistical graph of average fluorescence intensity in different groups (n = 3); (G) Representative photoacoustic images and (H) statistical graph showing the aggregation of HP@ICG in subcutaneous tumors of Balb / C mice (n = 3), *** representing P≤ 0.001; (IJ) Blood biochemical indicators of HP@ICT, etc. (AST, ALT, TBIL, Urea, Cre, UA) (n=6);

[0028] Figure 3 Biocompatibility assessment of HP@ICT and H&E staining of major organs in mice treated with different hydrogels are shown. Scale bar: 200 μm;

[0029] Figure 4 The experiment shows the anti-tumor effect of HP@ICT; (A) Schematic diagram of experimental design and grouping; (B) Growth curve of residual HCC tumors after HP@ICT treatment, with mice receiving I) control group, II) HPgel group, III) ICT group, and IV) HP@ICT group respectively; (C) Residual tumor inhibition rate after HP@ICT treatment and (D) Tumor weight; (E) Solid image of tumors after HP@ICT treatment; (F) Tumor volume curve of each mouse after HP@ICT treatment; (G) Statistical analysis of mouse body weight curves after HP@ICT treatment (n=5).

[0030] Figure 5 The diagram shows the TAN phenotype in residual tumor cells after HP@ICT reprogramming IRFA; (A) Flow cytometry and (B) statistical plot of N1 type TAN (CD86) in residual tumor cells after HP@ICT treatment; (C) Flow cytometry and (D) statistical plot of N2 type TAN (CD206) in residual tumor cells after HP@ICT treatment; (E) Flow cytometry and (F) statistical plot of PMN-MDSC type TAN in residual tumor cells after HP@ICT treatment (n=5); (G) Flow cytometry and (H) statistical plot of Treg cells in residual tumor cells after HP@ICT treatment; (I) Flow cytometry and (J) statistical plot of Treg cells in residual tumor cells after HP@ICT treatment (n=5).

[0031] Figure 6 HP@ICT enhances anti-tumor immunity in residual tumors after IRFA; (A) GrzmB in residual tumors after HP@ICT and other treatments. + CD8 + Flow cytometry and (B) statistical plot of T cells; (C) IFNγ in residual tumors after HP@ICT treatment. + CD8 + T cell flow cytometry and (D) statistical plot (n=5); (E) immunohistochemistry showing CD8+ in residual cancer tissue after IRFA surgery and HP@ICT treatment. + Representative images of T cell infiltration, scale bar: 100μm; (F) Representative TUNEL staining and H&E staining images of residual cancer tissue after HP@ICT treatment, scale bar: 100μm;

[0032] Figure 7 The diagram shows the residual cancer cells after HP@ICT / BMS synergistic eradication of IRFA; (A) Schematic diagram of experimental design and grouping; (B) Growth curves of residual HCC tumors after different treatments, with mice receiving I) control group, II) HPgel group, III) ICT group, and IV) HP@ICT group respectively; (C) Residual tumor inhibition rate under different treatments and (D) Tumor weight; (E) Photographs of residual cancer cells under different treatments; (F) Individual tumor volume curves of each group (n = 6); (G) Flow cytometry analysis statistics of CD45+ immune cells in the residual cancer microenvironment after different treatments; (H) Flow cytometry analysis statistics of TAN cells in the residual cancer microenvironment after different treatments; (I) Flow cytometry analysis statistics of N1 type TAN cells in the residual cancer microenvironment after different treatments; (J) Flow cytometry analysis statistics of N2 type TAN cells in the residual cancer microenvironment after different treatments; (M) Flow cytometry analysis statistics of Treg cells in the residual cancer microenvironment after different treatments; (K) Flow cytometry analysis statistics of CD8+ cells in the residual cancer microenvironment after different treatments. Flow cytometry and (L) statistical plot of T cells, (N) flow cytometry and (O) statistical plot of PMN-MDSC neutrophils in residual cancer microenvironment after different treatments (n=5).

[0033] Figure 8 The following diagram shows the effects of iRFA followed by HP@ICT / BMS combined therapy on intrahepatic metastases; (A) Schematic diagram of iRFA subcutaneous primary tumor and its liver metastases model construction and treatment grouping (control group, ICT gavage group, HP@ICT group, HP@BMS group and HP@ICT / BMS group); (B) Bioluminescence imaging of liver metastases in different treatment groups monitored by IVIS and (C) Statistical graph of their fluorescence intensity (n=5); (D) Mice were sacrificed after follow-up, and IVIS recorded in vitro bioluminescence imaging of liver metastases in different treatment groups and (E) Statistical graph of their fluorescence intensity; (F) Representative gross liver images of different treatment groups; (G) Representative H&E staining of the liver, with arrows pointing to metastases, scale bar: 100 µm; (H) Statistical graph of FCM of CD8+ T cells and (I) CD4+ T cells in the spleen of mice after different treatments such as HP@ICT / BMS; (J) Statistical graph of FCM of Treg cells and (K) PMN-MDSC cells; (L) GrzmB+ CD8+ FCM statistics of T cells, (M)IFNγ+CD8+ T cells, and (N)TNFα+CD8+ T cells (n=5);

[0034] Figure 9The following diagrams show the effects of HP@ICT / BMS combined therapy on distant metastases after iRFA: (A) Schematic diagram of the construction of the IRFA subcutaneous primary tumor and its distant (contralateral) metastases and treatment groupings (control group, ICT gavage group, HP@ICT group, HP@BMS group and HP@ICT / BMS group); (B) Bioimaging of distant (contralateral) metastases at different time points monitored by IVIS and (C) Statistical graph of their luminescence intensity (n=5); (D) Individual volume curves of distant tumors in each treatment group, with mice classified as I) control group, II) HPgel group, III) ICT group, and IV) HP@ICT group; (E) Growth curves of distant tumors in each treatment group; (F) Distant tumor inhibition rate and (G) Tumor weight in each treatment group; (H) Photographs of residual cancer under different treatments (n = 5); (I) Immunohistochemistry showing CD8+ in distant metastases after IRFA. Representative image of T cell infiltration; scale bar: 100 μm. (J) Representative TUNEL fluorescence staining and H&E staining images of distant metastases in different treatment groups after IRFA and HP@ICT / BMS hydrogel, scale bar: 100 μm. Detailed Implementation

[0035] To better illustrate the purpose, technical solution, and advantages of this invention, the invention will be further described below with reference to specific drawings and embodiments. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0036] Experimental cell lines: Mouse hepatocellular carcinoma cell line Hepa1-6 was purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences, and mouse hepatocellular carcinoma cell line (H22-Luc cells) was also purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. Neutrophils were extracted from the bone marrow of Balb / c mice.

[0037] Laboratory animals: Balb / c mice (female, 3-4 weeks old, weighing 17-21 g) were used in this study and purchased from Guangdong Yaokang Biotechnology Co., Ltd. All animals were approved by the Animal Experiment Ethics Committee of the Fifth Affiliated Hospital of Sun Yat-sen University (ethics number: 00139). Standardized husbandry and management were implemented throughout the experiment in a negative pressure isolator. The barrier system maintained a constant temperature of 22±2℃, relative humidity of 50±10%, and an air exchange rate of ≥15 times / h. An automatic lighting system maintained a 12-hour light-dark cycle, and adequate food and water were ensured. All animal experiments involved in this study complied with the relevant provisions of the "Regulations on the Management of Laboratory Animals" formulated by my country.

[0038] Cell culture: The Hepa1-6 hepatocellular carcinoma cell line was cultured in high-glucose DMEM complete medium; neutrophils and H22-Luc fluorescently labeled cell lines were maintained in RPMI 1640 medium. All media were supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution. Cells were placed in a tri-gas incubator to maintain a constant temperature of 37°C, a CO2 concentration of 5%, and a humidity of over 95%. The culture medium was replaced with fresh medium every 48 hours. When the cell confluence reached 80%-90%, the cells were passaged at a ratio of 1:3 for expansion.

[0039] Example 1: Preparation and characterization of HP@ICT

[0040] Materials and Methods:

[0041] 1. Preparation of HP@ICT

[0042] First, 10 mg of ICT was weighed and dissolved in 1 ml of DMSO solution. Then, at room temperature, the DMSO solution of ICT was added to 1 ml of 10% hydroxypropyl-β-cyclodextrin (HP-β-CD) solution, and the mixture was vortexed for 30 seconds to obtain an ICT / HP-β-CD solution. After complete dissolution, different mass fractions of pomoxarol 407 (P407) (18%, 20%, 22%, 25%, w / v) were slowly dissolved in the ICT / HP-β-CD solution, and the mixture was vortexed overnight in a refrigerator to ensure complete dissolution of P407. The HP@ICT hydrogel should be placed at 4°C for at least 24 hours before use. HP@BMS and HP@ICT / BMS hydrogels were prepared using the same procedure.

[0043] Gelation time determination: After the hydrogel was prepared, its gelation time was determined by shaking the vial. 10% (w / v) HP-β-CD in the presence of different concentrations (18%, 20%, 22%, and 25% w / v) in the presence of P407 was added to 2 ml vials. The sol-gel transition time was determined by inverting the vial until no flowability was observed, and this time was recorded as the "gelation time". Each data point is the average of three measurements.

[0044] Injectability assay: After transferring the hydrogel mixture to a 1 ml syringe, the injectability of HP@ICT was photographed using a digital camera.

[0045] Hydrogel morphology observation: HP@ICT hydrogels were prepared and freeze-dried overnight in a vacuum environment using a freeze dryer. The surface morphology and scaffold structure of the freeze-dried samples were observed using a HITACHI SU8010 scanning electron microscope (Japan).

[0046] Dynamic rheological determination: The dynamic rheological properties of HP@ICT hydrogel were tested using a Waters DHR-2 strain-controlled rheometer (USA). 1 ml of hydrogel was placed between parallel plates (40 mm plates, 1 mm gap). Dynamic viscosity was measured at 37°C with constant strain (1%) within a frequency range of 0.1 to 100 Hz.

[0047] 2. Characterization of HP@ICT

[0048] In vitro cell uptake assay: Confocal microscopy observation of drug uptake: Plating: Neutrophils extracted from bone marrow were seeded onto confocal dishes. Incubation: Incubated with HP@FITC hydrogel for 2 h, 4 h, and 6 h, respectively. Sample collection: Waste liquid was discarded, washed twice with PBS, fixed with ice-cold methanol for 15 min, and washed twice with PBS. Nucleus staining: Incubated with DAPI at room temperature for 5 min, washed twice with PBS, and finally observed the cell uptake ratio of HP@FITC using a laser confocal microscope. Flow cytometry determination of drug uptake: Plating: Neutrophils extracted from bone marrow were seeded onto confocal dishes. Incubation: Incubated with HP@FITC hydrogel for 2 h, 4 h, and 6 h, respectively. Sample collection: Neutrophils were collected by centrifugation (500 g, 5 min). The cell uptake ratio of fluorescent HP@FITC was analyzed by flow cytometry.

[0049] HP@ICG in vivo release behavior study: Experimental animals: Six Balb / c mice were used. Drug injection: Free ICG and HP@ICG (1 mg ICG / kg) were injected subcutaneously. IVIS in vivo imaging: Mice were anesthetized with isoflurane, and fluorescence images were collected daily for 8 days after injection at fixed time intervals. Excitation wavelength was 740 nm, emission wavelength was 845 nm, and exposure time was 1 s. IVIS imaging analysis and statistical analysis were performed.

[0050] HP@ICG Intratumoral Spread Behavior Study: Experimental Animals: Six H22 tumor-bearing Balb / c mice were used. The subcutaneous tumor formation process in mice is described below in the section on the establishment of the IRFA subcutaneous tumor model. Drug Injection: HP@ICG (1 mg ICG / kg) was injected intratumorally into both the control group (sham ablation) and the ablation group. Photoacoustic Imaging: To visualize the biodistribution and intensity ratio of photoacoustic (PA) signals in the tumor, anesthetized mice were placed on a heated monitoring platform, and the PA signals of these tumors were reconstructed using the Vevo LAZR-X system. Photoacoustic Imaging Analysis: All data were analyzed using Vevo LAB 3.1.1 software.

[0051] Biocompatibility testing of HP@ICT: Animal grouping: Healthy female mice (3-4 weeks old) were divided into Ctrl group, ICT gavage group, HP@BMS group, HP@ICT group, and HP@ICT / BMS group (n=6). Drug injection: The gavage group was injected with ICT solution via gavage needle, while the hydrogel group was injected intratumorally with HP@BMS, HP@ICT, and HP@ICT / BMS (ICT 50 mg / kg; BMS202 4 mg / kg) via ablation needle. The gavage group was administered the drug daily, while the hydrogel group was administered the drug only once. The total amount of ICT administered to each group was consistent. Blood collection by enucleation: Seven days after drug injection, peripheral blood was collected by enucleation into heparin anticoagulant tubes and subjected to blood biochemistry tests. Tissue sampling: Twenty-one days after drug injection, vital organs (heart, liver, spleen, lung, and kidney) of the mice were removed, placed in tissue fixation solution, and stained with H&E.

[0052] result:

[0053] 1. Preparation of HP@ICT

[0054] A hydrogel delivery system, named HP@ICT, was prepared using a simple physical mixing method. Figure 1 A). We measured the gelation time of P407 with different mass fractions in 10% HP-β-CD at physiological temperature (37℃) and customized it for subsequent experiments. The results showed that the gelation time at physiological temperature decreased significantly with increasing P407 hydrogel concentration (124 seconds at 18%, 90 seconds at 20%, 56 seconds at 22%, and 26 seconds at 25%). Figure 1 B). Considering gelation time, 22% HPgel gel should be the ideal concentration for a composite hydrogel delivery system, as it exists almost as a sol suspension at room temperature, allowing sufficient time for drug injection. Further addition of ICT did not significantly affect gelation time at room temperature or physiological temperature. Figure 1 C, D).

[0055] HP@ICT appears as a pale yellow microparticle solution at room temperature. When the temperature rises to 37°C, due to the rapid aggregation between enhanced microparticles, a three-dimensional microparticle network spontaneously forms, eventually transforming into a physical gel state. Figure 1 E). Furthermore, for hydrogels used in localized minimally invasive treatments, injectability is crucial for in vivo application. Therefore, we also tested the injectability of HP@ICT. At 37°C, this hydrogel could be continuously injected into PBS solution using a 24G syringe needle. Figure 1 F). Furthermore, scanning electron microscopy (SEM) images revealed that the lyophilized HP@ICT hydrogel possessed a rich porous microstructure, with ICT dispersed within the pores (F). Figure 1G). The results above demonstrate the thermosensitivity, injectability, and stable gel-forming ability of the HP@ICT hydrogel, enabling controlled and precise drug delivery within the local residual cavity after ablation.

[0056] The sol-gel transition and viscoelasticity of HP@ICT were observed through dynamic rheological measurements. Tests were conducted within a temperature range of 15–40 °C, with a fixed strain of 1% and a heating rate of 1 °C / min. Figure 1 As shown in Figure H, HP@ICT exhibits temperature-responsive gel properties, displaying low storage modulus (G′) and low loss modulus (G″) at low temperatures. As the temperature rises to 27°C, a point appears between G′ and G″, indicating gelation has occurred. The viscosity η changes with gelation temperature, revealing the material's temperature sensitivity. Strain scanning of HP@ICT shows that at 37°C, both G′ and G″ remain unchanged, with G′ being higher than G″, indicating that the hydrogel can stably maintain its gel state in bulk. Figure 1 I). Meanwhile, as the shear rate increased from 0.1 s⁻¹ to 100 s⁻¹, the viscosity of the hydrogel decreased significantly, indicating that the hydrogel possesses good shear strain resistance and injectability. Figure 1 J).

[0057] 2. Characterization of HP@ICT

[0058] The pale yellow HP@ICT was subcutaneously injected into mice. Degradation was observed at 1, 3, 8, and 14 days post-injection. Figure 2 As shown in Figure A, the gel underwent continuous and slow subcutaneous degradation on days 3 and 8 post-injection, with the hydrogel content significantly lower on day 8 compared to day 1. By day 14, no gel could be found under the skin, indicating that the gel had been completely metabolized and degraded.

[0059] HP@FITC hydrogels were prepared and co-incubated with neutrophils extracted from mouse bone marrow. Considering the short in vitro survival time of neutrophils, confocal imaging was performed at different time gradients of 2 h, 4 h, and 6 h. Immunofluorescence results showed that co-incubated neutrophils gradually phagocytosed HP@FITC (green fluorescence), and the fluorescence intensity increased with time. Figure 2 B). FCM analysis showed that drug uptake gradually increased, with a drug uptake rate of 76% in cells after 6 hours of incubation. Figure 2 C, D).

[0060] To further evaluate the in vivo sustained-release performance of HP@ICT, we used an in vivo imaging system (IVIS) to observe the release behavior of the hydrogel system in mice. Figure 2The EF results showed that HP@ICG remained at the subcutaneous tumor site for more than 8 days, while the ICG group was mostly metabolized within 4 days. This part of the experimental results indicates that HPgel hydrogel has the function of long-acting sustained-release drug delivery.

[0061] To further explore the formation and dispersion characteristics of hydrogels in the ablation model of this study, we used an ultra-high frequency photoacoustic multimodal imaging system to detect ICG signals within HP@ICG. Three-dimensional reconstructed images showed that, compared to the control group (sham ablation), the ablation group exhibited stronger photoacoustic signals within residual cancer tissue, indicating a higher hydrogel content. Figure 2 (GH). This is mainly because the tissue dehydrates and shrinks after ablation, allowing it to absorb and accommodate more hydrogel. In addition, the hydrogel can rapidly distribute to the ablation edge before gel formation, achieving continuous drug release, specifically targeting residual cancer tissue, and effectively increasing drug concentration.

[0062] Finally, we evaluated the short-term and long-term biocompatibility of the Ctrl group, ICT gavage group, HP@BMS, HP@ICT, and HP@ICT / BMS group in mice. First, on day 7 after drug administration, blood was collected from mice, and serum samples were prepared by centrifugation for blood biochemical analysis to detect the in vivo toxicity of the hydrogel drug. The results showed that, compared with the control group injected with PBS, the blood biochemical parameters such as AST, ALT, TBIL, Urea, Cre, and UA in the HP@BMS, HP@ICT, and HP@ICT / BMS groups were normal. Figure 2 (IJ) No significant liver or kidney function damage was observed in mice in any of the treatment groups, indicating that the hydrogel drug has good short-term biocompatibility.

[0063] Subsequently, to assess the long-term toxicity of the hydrogel drug, we administered the medication according to the above-mentioned groups (Ctrl group, ICT gavage group, HP@BMS group, HP@ICT group, HP@ICT / BMS group). The gavage group received the medication daily, while the hydrogel group received the medication only once post-surgery via ablation needle. The total ICT dosage was consistent across all groups. After 3 weeks, the heart, liver, spleen, lung, and kidney of each mouse were completely removed and fixed in 4% paraformaldehyde. H&E staining showed no significant pathological changes in these organs. Figure 3 This section of the results suggests that our synthesized HP@ICT hydrogel drug delivery systems exhibit good long-term biocompatibility.

[0064] Example 3

[0065] Materials and Methods:

[0066] Establishment of the IRFA subcutaneous tumor model: Cell preparation: H22 hepatocellular carcinoma cells were digested with 0.25% trypsin-EDTA (Gibco) and then adjusted to a density of 2×10⁻⁶. 6Cells / 100 μl PBS. Subcutaneous inoculation: BALB / c mice (female, 4 weeks old) were inoculated with cell suspension under the right axilla after a 7-day environmental acclimatization period. Tumor volume (V = major axis × minor axis² / 2) was monitored daily. Model validation: The model was confirmed to be successfully established when the tumor volume reached 50-70 mm³ (7-10 days post-inoculation). Equipment parameters: A 480 kHz radiofrequency generator and a 17G monopolar radiofrequency ablation needle (active end length 7 mm) were used. Ablation procedure: Mice were placed in an induction chamber containing 1.5% isoflurane. After the respiratory rate dropped to 40-50 breaths / minute, they were transferred to an anesthesia maintenance platform and continuously inhaled a 1.5-2% isoflurane gas mixture. After anesthesia, the mice were fixed in a prone position on VIVA electrode pads. Ultrasonic coupling agent was applied to the skin-electrode interface, and the ablation needle was precisely punctured to the center of the tumor under ultrasound guidance. Energy delivery: The ablation device was set to 5W output power, and ablation treatment was performed continuously for 15 seconds. During the procedure, the needle insertion position and depth needed to be controlled to prevent complete ablation of the tumor or excessive temperature at the base of the ablation needle causing carbonization of the tumor surface. After ablation, the ablation area was observed for active bleeding or other complications, and routine nutritional support was provided postoperatively. Grouping: IRFA-bearing Babl / c mice were randomly divided into 4 groups: Ctrl group, HP gel group, ICT group, and HP@ICT hydrogel group (n=5). Drug injection: Immediately after IRFA, drugs were administered through the ablation needle core. The control group was given PBS (50 µL / mouse), HP gel (50 µL / mouse), ICT solution (50 µL / mouse), and HP@ICT solution (50 µL / mouse) in the residual cavity (ICT 50 mg / kg). All groups received only this one administration. Monitoring: Tumor size and mouse weight were measured every two days using calipers, and residual tumor volume (mm³) was calculated using the following equation: Tumor long axis (a) × Short axis (b)² / 2; Average tumor inhibition rate (%) = (Average tumor weight in the normal PBS group - Average tumor weight in the treatment group) / Average tumor weight in the control group × 100%. Tissue Collection: After 3 weeks, mice were euthanized by cervical dislocation under anesthesia. Tumors were collected, photographed, and weighed. Tumor tissue was fixed in 4% paraformaldehyde for subsequent staining.

[0067] Flow cytometry detection of immune cell infiltration after IRFA: Dissection of tissues: Appropriate amounts of tumor tissue and spleen tissue (8 days after IRFA) were minced and added to cold PBS. Digestion of single-cell suspension: Tumor tissue was minced and digested with appropriate amounts of dissociation buffer (collagenase IV 1 mg / ml, DNase I 4 U / ml), and the resulting single-cell suspension was obtained through a 70 μm filter. Spleen tissue was crushed using a syringe plunger and the resulting single-cell suspension was obtained through a 70 μm filter. Lysis of red blood cells: The single-cell suspension was collected and lysed with red blood cell lysis buffer at room temperature for 5 min to remove red blood cells. Blocking: After culture, the supernatant was discarded by centrifugation, and 0.3% BSA was added for blocking for half an hour. Antibody staining: Gating of different cell types was achieved using the following fluorescently labeled specific antibodies: CD4+ T cells (CD3+CD4+), CD8+ T cells (CD45+CD8+), Treg cells (CD25+FOXP3+), GrzmB CD8+ T cells (CD8+GrzmB+), and IFN-γ CD8+ T cells (CD8+IFN-γ+). Staining was performed by incubation on ice in the dark for 30 min. Flow cytometry analysis was then performed.

[0068] Immunohistochemical detection of CD8 expression in residual tumor cells after IRFA: Tumor tissues from mice were collected, and the expression level of CD8 in the tumor tissues was detected by immunohistochemistry. Fixation, dehydration, and paraffin embedding: Subcutaneous tumor tissues were fixed with 4% paraformaldehyde, followed by graded alcohol dehydration and paraffin embedding. Sectioning and baking: Paraffin sections of tissue were cut to a thickness of 4-6 μm. The prepared paraffin sections were placed in a preheated 70 ℃ constant temperature oven for 2 h to completely melt the paraffin. Dewaxing and hydration: The sections were immersed sequentially in xylene I for 10 min, xylene II for 10 min, xylene III for 10 min, anhydrous ethanol for 5 min, 80% ethanol for 5 min, 50% ethanol for 5 min, and distilled water for 2 min; washed with PBS (5 min × 3 times). Antigen retrieval: EDTA / citric acid solution was boiled in an autoclave, and the hydrated sections were added. After autoclaving for 10 min, the sections were cooled to room temperature and washed three times with PBST for 10 min each time. Permeabilization: If the target protein is an intracellular antigen, treat with 0.1-0.3% Triton X-100 PBS solution for 10 min. Blocking endogenous peroxidase: Place tissue sections in a humidified chamber, circle with an immunohistochemical pen, and treat with 3% H2O2 for 10-30 min. Wash three times with PBST, 10 min each time. Blocking: Incubate with 10% goat serum in PBS solution at room temperature for 1 h. Primary antibody incubation: Dilute the primary antibody appropriately based on the target protein and antibody information; the dilution is the blocking solution. Incubate overnight in a humidified chamber at 4 ℃. Secondary antibody incubation: Remove the sections the next day, allow them to return to room temperature, and wash three times with PBST, 10 min each time. Dilute the secondary antibody (1:200) appropriately based on the target protein and antibody information. Incubate for 1-2 h in a humidified chamber at room temperature. DAB staining: Wash three times with PBST, 10 min each time. Develop the color using DAB staining solution (diluted according to kit requirements) and observe the staining under a microscope. Staining cell nuclei: Immerse in hematoxylin counterstaining solution for 10 min, rinse with running water until clear staining of cell nuclei is observed under a microscope. Mounting: Dehydrate with graded alcohols and mount (alcohol gradient opposite to hydration order). Dry at 37 ℃ and mount with neutral resin.

[0069] Immunofluorescence detection of apoptosis in residual tumor cells after IRFA: Fixation, dehydration, and paraffin embedding: Subcutaneous tumor tissue was fixed with 4% paraformaldehyde, followed by graded alcohol dehydration and paraffin embedding. Sectioning and baking: Paraffin sections of tissue were cut to a thickness of 4-6 μm. The prepared paraffin sections were placed in a preheated 70 ℃ oven for 2 h to completely melt the paraffin. Dewaxing and hydration: The sections were immersed sequentially in xylene I for 10 min, xylene II for 10 min, xylene III for 10 min, anhydrous ethanol for 5 min, 80% ethanol for 5 min, 50% ethanol for 5 min, and distilled water for 2 min; washed with PBS (5 min × 3 times). Antigen retrieval: EDTA / citric acid solution was boiled in an autoclave and then added to the hydrated sections. After autoclaving for 10 min, the sections were cooled to room temperature and washed three times with PBST for 10 min each time. Permeation: If the target protein is an intracellular antigen, it can be treated with 0.1-0.3% Triton X-100 PBS solution for 10 min. Blocking endogenous peroxidase: Place tissue sections in a humidified chamber, circle them with an immunohistochemical pen, and treat with 3% H2O2 for 10-30 min. Wash three times with PBST, 10 min each time. Blocking: Incubate with 10% goat serum in PBS solution at room temperature for 1 h. Staining: Stain mouse tumor tissue sections with one-step TUNEL staining solution. Mounting and observation: Mount the slides with an appropriate amount of anti-fluorescence quenching mounting medium containing DAPI, and observe the expression of TUNEL in the tumor tissue using a laser confocal microscope.

[0070] result:

[0071] 1. HP@ICT inhibits the growth of residual cancer after IRFA.

[0072] An iRFA subcutaneous tumor model was established and randomly divided into four treatment groups: control group, HPgel gel group, ICT group, and HP@ICT group. Figure 4 A). After completing the subcutaneous tumor iRFA procedure, a hydrogel was injected once into the residual cavity using an ablation needle. Tumor volume and body weight were then monitored every two days until the procedure was terminated. Figure 4 As shown in Figure B, tumors in the control group and the HPgel gel group grew rapidly, indicating that HPgel hydrogel itself has no anti-tumor effect, while tumor growth in the ICT and HP@ICT groups was significantly inhibited. Notably, HP@ICT showed superior efficacy, with a tumor inhibition rate as high as 90.1%, exceeding the 59.8% of ICT. Figure 4 C). The inhibitory effect on tumor volume is consistent with that on tumor weight ( Figure 4D). The tumor growth curves of the HP@ICT group showed a significant deceleration, with tumor volume remaining below 150 mm. Three-fifths of these mice achieved complete elimination of residual tumors. Figure 4 E, F). Furthermore, no significant changes in mouse body weight fluctuations were observed in any of the treatment groups (E, F). Figure 4 G). The above results suggest that the HP@ICT hydrogel group can significantly inhibit the progression of residual cancer after iRFA.

[0073] 2. HP@ICT reshapes the tumor immune microenvironment after IRFA

[0074] To further explore the antitumor mechanism of HP@ICT, we used FCM analysis to examine the immune landscape of residual cancer cells in different treatment groups 8 days later. First, we examined the phenotypic changes of TANs within the residual cancer cells. The results showed no significant difference in TAN phenotypic changes between the control group and the HPgel group, indicating that HPgel hydrogel itself has no antitumor effect or immune response. FCM analysis suggested that, compared with the free ICT group, the HP@ICT hydrogel group significantly increased the proportion of N1-like neutrophils in the residual cancer cells due to the sustained and slow release of ICT drugs from the hydrogel. Figure 5 A, B), and reduced the proportion of N2-like neutrophils ( Figure 5 (C, D), indicating that HP@ICT has a stronger TAN repolarization capacity. Meanwhile, the changes in the immunosuppressive cell population PMN-MDSCs within the residual tumor were similar to the results described in vitro. The free ICT group (14.0% ± 2.3%) showed a lower proportion of PMN-MDSCs than the control group (26.8% ± 6.0%) and the HPgel group (25.3% ± 4.7%). Figure 5 (E, F) The HP@ICT hydrogel group (7.1% ± 1.6%) showed a significantly lower proportion of PMN-MDSCs than the ICT group, significantly reducing the infiltration of PMN-MDSCs within the residual tumor TME. These results indicate that the sustained-release effect of HP@ICT hydrogel greatly increased the local ICT drug concentration and had a more significant effect on the TAN population. The increased N1-type TANs and decreased PMN-MDSCs are expected to further reverse the immunosuppressive microenvironment and activate residual tumor immunity.

[0075] To further verify the efficacy of ICT and HP@ICT in reversing immunosuppression in residual tumor cells after iRFA in liver cancer, we measured Treg cells (CD45+) in residual tumor cells 8 days after treatment in different treatment groups. + CD3 + CD4 + CD25 + FoxP3 +The study investigated the infiltration of Treg cells. Results showed that, compared to the control group and HPgel gel, free ICT inhibited Treg infiltration to some extent, reducing the percentage of Treg cells in residual tumor cells from 25.8% ± 2.5% to 18.4% ± 0.8% (P ≤ 0.05). The HP@ICT hydrogel group showed significantly fewer Treg cells than the free ICT group, further decreasing to 12% ± 3.1% (P ≤ 0.05). Figure 5 (G, H), indicating that HP@ICT can more significantly alter the number of Tregs. Simultaneously, HP@ICT significantly promotes CD8... + T cell infiltration was significantly higher in the HP@ICT hydrogel group compared to the control group (14.5% ± 1.0%). + The proportion of T cells increased to (25.6% ± 2.2%). Figure 5 (I, J). This indicates that HP@ICT has a stronger anti-tumor immune response and anti-tumor effect.

[0076] 3. HP@ICT promotes CD8+ T cell function in the post-iRFA TIME.

[0077] We further examined CD8 in residual cancer cells after HP@ICT hydrogel treatment. + The secretion of T cell effector molecules was monitored. The results were consistent with the aforementioned effects; HP@ICT hydrogel significantly increased CD8+ secretion. + Immunostimulatory molecules released by T cells, GrzmB in residual tumors of the free ICT group + CD8 + T increased from 2.8% to 15.7% ( P ≤ 0.05); IFN-γ + CD8 + T cell count increased from 2.9% to 16.2% in the Ctrl group. P ≤ 0.05) Figure 6 A, B). In the HP@ICT hydrogel group, CD8... + T cell effector molecules were significantly increased, including GrzmB + CD8 + T cells increased to 26.8%, IFNγ + CD8 + T cells increased to 21.2% ( Figure 6 (C, D). In summary, we can draw the following conclusions: HP@ICT hydrogel significantly increases local CD8 levels in residual tumor cells after IRFA. + It promotes the infiltration of T cells and enhances the function of effector T cells, thereby activating the immune system.

[0078] Consistent with the FCM results, immunohistochemical results confirmed that HP@ICT hydrogel significantly increased CD8 infiltration within residual cancer tissue. + T cells ( Figure 6 E), which can effectively activate local anti-tumor immune responses. Consistent with the anti-tumor effects observed in mouse models, TUNEL + Further staining revealed a significant increase in apoptosis of residual cancer cells in the HP@ICT hydrogel group. Figure 6 F). The above results suggest that HP@ICT hydrogel has the potential to enhance the role of ICT in reprogramming TANs, sustainably activating anti-tumor immunity, and exerting a strong inhibitory effect on residual tumors. This provides a promising approach for treating residual cancer after HCC surgery.

[0079] Example 4: HP@ICT combined with anti-PD-1 inhibitor to eradicate residual cancer after IRFA

[0080] Materials and Methods:

[0081] Establishment of a liver metastasis-bearing mouse model: H22 cell suspensions were obtained after intraperitoneal passage of mice, and the cell count was calculated. H22 cells were collected at a rate of 1×10⁶ cells per mouse. 6 A unilateral subcutaneous tumor model was established by subcutaneous injection of 100 μl of the drug into the right groin of Balb / c mice. Tumor formation was observed daily, and tumor size was measured using calipers, recording the major axis (a) and minor axis (b). The tumor size was then calculated using V=ab. 2 / 2 The tumor volume was obtained. Four days later, Lucifere-H22 cells in the logarithmic growth phase were collected. General anesthesia was induced in mice by intraperitoneal injection of sodium pentobarbital. Subsequently, isoflurane was continuously inhaled with the assistance of an anesthesia machine to ensure the Balb / c mice remained anesthetized. Under sterile conditions, the abdominal wall hair was removed, the abdominal skin was disinfected, the skin was incised with a scalpel, and the subcutaneous fat and fascia were bluntly separated with surgical forceps to expose the spleen. The spleen was then divided into 5 × 10⁻⁶ cells per mouse. 5 A 100 μl / mL concentration of Lucifere-H22 cells was injected into the spleen of mice to induce liver cancer cells to drain back into the liver via the spleen, splenic vein, and portal vein, thus establishing a liver metastasis model. Subcutaneous tissue and skin were sutured using sterile absorbable sutures. Mice were observed for anesthesia recovery and were monitored for 2 hours after recovery. If no abnormalities were observed, the mice were returned to the SPF-grade animal facility. After the metastasis model was established, IVIS imaging was performed daily to observe tumor formation.

[0082] Establishment of a bilateral subcutaneous tumor-bearing mouse model: H22 cell suspensions were obtained after intraperitoneal passage of mice, and the cell count was calculated. H22 cells were collected and inoculated subcutaneously into the right groin of Balb / c mice at a density of 1×10⁶ / 100 μl per mouse to establish a unilateral subcutaneous tumor model. Tumor formation was observed daily, and tumor size was measured using calipers. After 4 days, Lucifere-H22 cells in logarithmic growth phase were collected again, and 1×10⁶ H22 tumor cells were taken from each mouse and inoculated subcutaneously into the left groin of Balb / c mice to establish a contralateral subcutaneous tumor model. Tumor formation was observed daily, and tumor size was measured using calipers. The major axis (a) and minor axis (b) were recorded, and the tumor volume was calculated using V=ab² / 2.

[0083] Efficacy evaluation of HP@ICT / BMS for residual tumor after IRFA: After establishing the IRFA subcutaneous tumor model, IRFA-bearing Babl / c mice were randomly divided into 5 groups: Ctrl group, ICT gavage group, HP@BMS group, HP@ICT group, and HP@ICT / BMS group (n=6). Drug injection: The gavage group received ICT solution via gavage needle, while the hydrogel group received the drug via the ablation needle core immediately after IRFA surgery. HP@BMS, HP@ICT, and HP@ICT / BMS (ICT 50 mg / kg; BMS202 4 mg / kg) were injected intratumorally, respectively. The gavage group received the drug daily, while the hydrogel group received it only once. The total ICT dosage was consistent across all groups. Monitoring: Tumor size and mouse weight were measured every two days using calipers, and the residual tumor volume (mm) was calculated according to the following equation. 3 = Tumor long diameter (a) × short diameter (b) 2 / 2; Average tumor inhibition rate (%) = (average tumor weight in the normal PBS group - average tumor weight in the treatment group) / average tumor weight in the control group × 100%. Sampling: Three weeks later, the patient was euthanized by cervical dislocation under anesthesia. Tumors were harvested, photographed, and weighed. The tumor tissue was fixed in 4% paraformaldehyde for subsequent staining.

[0084] Efficacy evaluation of HP@ICT / BMS in IRFA-bearing liver metastasis models: After establishing liver metastasis and subcutaneous tumor IRFA models, IRFA-bearing Balb / c mice were randomly divided into 5 groups: Ctrl group, ICT gavage group, HP@BMS group, HP@ICT group, and HP@ICT / BMS group (n=6). Drug injection was the same as above. Two weeks after ablation administration, Balb / c mice underwent IVIS imaging. Specifically, after anesthetizing the mice, 100 mL of 150 mg / kg D-luciferase substrate was injected intraperitoneally. Ten minutes later, the mice were placed in an IVIS small animal in vivo imaging system for tumor bioluminescence imaging. Tissue collection: Two weeks later, the mice were euthanized by cervical dislocation after anesthesia, and the liver was excised for imaging. The liver tissue was fixed in 4% paraformaldehyde for subsequent H&E staining.

[0085] Efficacy evaluation of HP@ICT / BMS in distal tumor models after IRFA: After establishing distal tumor and subcutaneous tumor IRFA models, IRFA-bearing Balb / c mice were randomly divided into 5 groups: Ctrl group, ICT gavage group, HP@BMS group, HP@ICT group, and HP@ICT / BMS group (n=6). Drug injection was the same as above. Two weeks after ablation administration, Balb / c mice underwent IVIS imaging. Specifically, after anesthetizing the mice, 100 mL of 150 mg / kg D-luciferase substrate was injected intraperitoneally. Ten minutes later, the mice were placed in an IVIS small animal in vivo imaging system for tumor bioluminescence imaging. Tissue collection: Two weeks later, the mice were euthanized by cervical dislocation after anesthesia, and the distal tumor was excised for imaging. The tumor tissue was fixed in 4% paraformaldehyde for subsequent H&E staining.

[0086] result

[0087] 1. HP@ICT / BMS collaborative eradication of residual cancer after IRFA

[0088] The previous results indicated that ICT could increase the infiltration of CD8+ cytotoxic T cells in tumors. Therefore, we further investigated whether the combination of ICT and an anti-PD-L1 inhibitor could significantly enhance its anti-tumor effect. We encapsulated the small molecule PD-L1 inhibitor BMS202 onto an ICT gel to evaluate the combined therapeutic effect on residual tumor cells in vivo. An additional gavage group simulating routine oral administration of ICT was included for comparison. We randomly divided mice with iRFA subcutaneous tumor models into 5 groups, including a control group, an ICT gavage group (ig), an HP@ICT group, an HP@BMS group, and an HP@ICT / BMS hydrogel group (…). Figure 7A). Mice received either a single local gel injection or daily intragastric administration, with a consistent total ICT dose per mouse, for 3 weeks. Results indicated that, compared to HP@ICT, the HP@ICT / BMS combination therapy significantly inhibited residual cancer growth; within 21 days of administration, 5 out of 6 residual cancer cells completely disappeared, achieving complete remission, demonstrating significantly superior therapeutic efficacy compared to HP@ICT or HP@BMS alone. Figure 7 BF). This further validates that ICT improves the "cold tumor" state of hepatocellular carcinoma after iRFA and significantly enhances the sensitivity of residual cancer cells to anti-PD-L1 immunotherapy. Notably, the efficacy of HP@BMS is slightly inferior to that of HP@ICT, which may be attributed to the immune microenvironment after iRFA suppressing the efficacy of anti-PD-L1 therapy, while HP@ICT has the potential to effectively reprogram TANs, enhance cytotoxic lymphocyte function, activate anti-tumor immunity, and improve the response to anti-PD-L1 therapy. In addition, compared with the II) gavage group, the IV) hydrogel-loaded ICT group significantly inhibited the growth of residual cancer cells, with the tumor inhibition rate increasing from 17.0%±15.9% to 93.6%±0.3% (BF). Figure 7 C). Compared with the same dose of ICT, HP@ICT utilizes local hydrogel drug delivery to achieve higher drug concentrations in the residual cancer area, thereby more effectively inhibiting tumor growth. When used in combination with BMS, the inhibitory effect is even more significant, with a complete elimination rate of residual cancer reaching 83%.

[0089] Subsequently, the time interval (TIME) of residual cancer eight days after treatment was tracked using FCM to analyze changes in the infiltration of TAN, Treg, and CD8+ T cells after different treatments. Compared with the control group, the total number of CD45+ immune cells in the HP@ICT group increased by 1.3 times, while the total number of TAN cells decreased by 1.6 times. Figure 7 (G, H) indicates that HP@ICT effectively reduced neutrophil infiltration in the residual cancer microenvironment; while in the HP@ICT / BMS combined treatment group, the change in CD45+ immune cells increased by 2.8-fold, and the total number of TAN cells decreased by 2.9-fold. Further TAN phenotypic analysis showed that the proportion of N1-like TANs in the HP@ICT / BMS combined treatment group increased from 17.5%±4.5% to 48.7%±3.7%, while the proportion of N2-like TANs decreased from 25.6%±8.5% to 4.9%±2.1%. Figure 7 (I, J), indicating that the immunosuppressive response within the residual tumor tumor mesenchyme was further reduced in the combined treatment group. Simultaneously, the HP@ICT / BMS combined treatment group effectively enhanced the infiltration of tumor-invasive CD8+ T cells (I, J). Figure 7 K, L), while immunosuppressive cells Treg cells ( Figure 7 M) and PMN-MDSCs ( Figure 8 The infiltration of N and O decreased significantly.

[0090] 2. HP@ICT combined with anti-PD-1 inhibitors significantly inhibited residual liver metastasis after IRFA.

[0091] Recurrence and metastasis of residual cancer after iRFA are major causes of poor prognosis, posing a significant challenge to clinical practice. To further verify whether the combined use of ICT and anti-PD-L1 can prevent the metastasis of residual cancer cells, we first established a subcutaneous tumor model (primary tumor), and then established a liver metastasis model four days later by injecting luciferase-labeled H22 cells into the spleen. Figure 8 A demonstrates the process of establishing a liver metastasis model, with groupings as before, mainly including a control group, an ICT gavage group, an HP@ICT group, an HP@BMS group, and an HP@ICT / BMS group. Bioluminescent images of fLuc-H22 intrahepatic tumors were monitored using an IVIS imaging system, and the growth of liver metastases in different treatment groups was recorded. Results showed that the mice in the control group that received no treatment had the strongest in vivo imaging signal, indicating rapid metastatic growth; 40% (2 / 5) of the mice developed ascites within two weeks after iRFA of the primary tumor. The gavage group had a certain inhibitory effect on tumor metastasis, but the metastatic lesions continued to grow. Figure 8 B). The local gel-administered HP@ICT and HP@BMS groups showed relatively weak signal intensity, significantly enhancing their inhibitory effect on liver metastasis compared to the gavage group, thus improving the overall inhibitory effect. In contrast, the HP@ICT / BMS combined hydrogel group showed virtually no signal intensity, indicating that it exhibited the best effect in inhibiting the growth of multiple liver metastases, with most mice achieving complete remission. Figure 8 C).

[0092] After the treatment cycle, mouse liver tissue was collected for morphological observation and H&E staining to analyze tumor metastasis. Consistent with the above results, gross images and H&E staining showed multiple metastatic tumors in the livers of mice in the control and gavage groups. The number and extent of intrahepatic metastases were significantly reduced in the HP@ICT and HP@BMS groups, while the metastases in mice treated with HP@ICT / BMS after iRFA were almost non-existent (Figure 8D-G). The results indicate that the HP@ICT / BMS combined treatment hydrogel group can effectively inhibit the metastasis of residual cancer after IRFA.

[0093] To further verify the systemic immune effect activated by the HP@ICT / BMS combined treatment hydrogel group, we euthanized tumor-bearing mice 8 days after treatment and extracted immune cells from the spleen for flow cytometry analysis. The results showed, surprisingly, no significant difference in the number of CD8+ T cells, CD4+ T cells, and Treg cells in the spleen among different treatment groups. Figure 8H, I, J). However, compared with the control group (52.4% ± 3.4%), the HP@ICT / BMS combined treatment hydrogel group significantly reduced PMN-MDSCs in the spleen (2.4% ± 1.0%). Figure 8 K). Simultaneously, the HP@ICT / BMS group showed a significant increase in immunostimulatory molecules secreted by CD8+ T cells, such as GrzmB, IFN-γ, and TNF-α. Figure 9 L, M, N). The above results suggest that, despite local administration, HP@ICT / BMS can inhibit the production of PMN-MDSCs and, to some extent, activate systemic immune T-cell effector molecules. This combination therapy effectively stimulated a systemic anti-tumor immune response, ultimately inhibiting the growth of liver metastases.

[0094] 3. HP@ICT combined with anti-PD-1 inhibitors significantly inhibited distant tumor growth after IRFA.

[0095] In clinical practice, multiple liver lesions are more common than single lesions. Therefore, the goal of liver cancer treatment needs to be to more effectively inhibit the growth of multiple tumor lesions. Inspired by the excellent immune-activating effect of the HP@ICT / BMS combined hydrogel therapy group in inhibiting the progression of IRFA subcutaneous tumors and liver metastases, we further investigated its effect on preventing distant tumor growth. Experimental grouping and specific procedures are detailed below. Figure 9 A. IVIS bioluminescence imaging results showed that the mice in the control group that did not receive any treatment had the strongest imaging signal, indicating rapid growth of distant tumors. The gavage group had a certain inhibitory effect on tumor metastasis, but distant tumors were still growing. Figure 9 B). The signal intensity of HP@ICT and HP@BMS administered via local gel was significantly reduced compared to the gavage group, indicating a significantly enhanced effect in inhibiting distal tumor growth. The combined HP@ICT / BMS treatment with hydrogel showed a significant signal reduction, suggesting it exhibited the best effect in inhibiting distal tumor growth, with some mice achieving complete remission. Figure 9 C).

[0096] Compared with the control group and the gavage group, local injection of HP@ICT and HP@BMS monotherapy showed some inhibitory effect on secondary distal tumors. However, the HP@ICT / BMS combination therapy group showed the highest tumor inhibition rate and the smallest tumor volume. Figure 9 DH), of which 2 out of 5 mice achieved complete remission. Additionally, we detected a significant increase in CD8 expression in distal tumor tissue of the HP@ICT / BMS combined treatment hydrogel group, indicating that systemic immunity was activated after the combined use of ICT and BMS. Figure 9 I). H&E staining and TUNEL+ staining ( ​J) showed significant pathological damage in the distal tumor tissue of the HP@ICT / BMS group, further suggesting that the combined treatment group had the best tumor-suppressing effect.

[0097] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. The application of HP@ICT / BMS hydrogel in the preparation of drugs that enhance the sensitivity of residual hepatocellular carcinoma after IRFA to anti-PD-L1 immunotherapy, characterized in that, The HP@ICT hydrogel was prepared by mixing ICT, BMS202, HP-β-CD and Pomsarol 407.

2. A method for preparing HP@ICT / BMS hydrogel, characterized in that, Includes the following steps: S1. At room temperature, add the ICT solution to the HP-β-CD solution, mix and dissolve to obtain the ICT / HP-β-CD solution; S2. Pre-cool the ICT / HP-β-CD solution from S1, then add BMS202, and finally slowly add Pomozaxol 407. Stir and mix at 4°C to ensure that Pomozaxol 407 is completely dissolved. S3. After a period of time, the HP@ICT / BMS hydrogel is obtained.

3. The application of the HP@ICT / BMS hydrogel according to claim 2 in the preparation of drugs for antitumor immune responses, characterized in that, The aforementioned anti-tumor immune response refers to: (1) HP@ICT / BMS hydrogel recruits residual effector T cells in liver cancer after RFA surgery, reduces Treg cell infiltration, and / or; (2) HP@ICT / BMS hydrogel promotes the generation of functional cytokines in residual tumor cells after RFA surgery for liver cancer, and / or; (3) HP@ICT / BMS hydrogel promotes apoptosis of residual tumor cells in liver cancer after RFA surgery.

4. The application of the HP@ICT / BMS hydrogel according to claim 3 in the preparation of drugs for antitumor immune responses, characterized in that, The functional cytokines are Granzyme B, IFN-γ, and TNF-α.

5. The use of the HP@ICT / BMS hydrogel of claim 2 in the preparation of a drug for inhibiting liver metastasis of residual liver cancer after IRFA.

6. The use of the HP@ICT / BMS hydrogel of claim 2 in the preparation of a medicament for inhibiting the growth of distal tumors after IRFA in liver cancer.

7. The use of the HP@ICT / BMS hydrogel of claim 2 in the preparation of a drug for eradicating residual cancer cells after IRFA of liver cancer.

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