An intracranial tumor ablation synergistic immunotherapy system and preparation and application thereof

Abstract

The application discloses an intracranial tumor ablation synergistic immunotherapy system and preparation and application thereof, which comprises a hydrogel carrier and a co-loaded photothermal agent and an immunoadjuvant polymer vesicle loaded in the hydrogel carrier. In view of the location and extremely challenging characteristics of late GBM, and after photothermal therapy (PTT) ablation of the tumor, the tumor will cause recurrence and metastasis, and currently there is no similar treatment report on photothermal immunotherapy for glioblastoma (GBM) to realize physical ablation and immune activation of the tumor. The application first provides the dual advantages of photothermal immunotherapy to realize physical ablation and immune activation of the tumor, remodels the tumor microenvironment by inducing immunogenic cell death (ICD), activates antigen-presenting cells, promotes T cell proliferation and tumor infiltration, and shows high-efficiency treatment on in-situ late GBM mice.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a combined treatment system, specifically an intracranial tumor ablation and synergistic immunotherapy system, its preparation, and its application in the highly effective treatment of advanced glioblastoma. Background Technology

[0002] Glioblastoma (GBM) is the most common primary brain tumor in adults. The standard treatment is maximal resection combined with concurrent chemoradiotherapy (Stupp). However, early symptoms of central nervous system tumors (such as blurred vision and occasional seizures) are similar to those of benign lesions, leading to advanced stages at diagnosis. At this stage, effective treatments are scarce, and palliative care is the primary approach. Furthermore, compared to solid tumors like lung cancer, GBM not only has a large tumor burden but its super-invasive growth often results in deep invasion of brain tissue, making surgical resection impossible in approximately 60% of patients. Coupled with the strong immunosuppressive nature of GBM tumors, developing effective treatments for deeply invasive, advanced GBM without surgical indications remains a pressing clinical challenge.

[0003] GBM has a highly inhibitory immune microenvironment. This is mainly manifested in: (1) abnormal activation of immunosuppressive tumor-associated macrophages (TAMs) and regulatory T cells (Tregs) in the tumor microenvironment (TME) of GBM; (2) impaired dendritic cell (DC) migration, leading to insufficient infiltration and dysfunction of cytotoxic T lymphocytes (CTLs); and (3) high expression of immunosuppressive factors such as programmed cell death-ligand 1 (PD-L1) and indoleamine 2,3-dioxygenase (IDO) by GBM cells. Furthermore, due to the tight blood-brain barrier, most chemotherapy / targeted drugs cannot penetrate brain tumors, making brain drug administration far more difficult than for lung cancer or breast cancer.

[0004] With the development of precision medicine, EGFR-TKIs have become the standard treatment option for patients with EGFR-mutant advanced NSCLC. However, the Chinese Expert Consensus on Immunotherapy and Targeted Therapy for Central Nervous System Gliomas (Second Edition) does not recommend routine use of anti-EGFR drugs for patients with nGBM and rGBM. PD-1 and PD-L1 are important negative regulators in the tumor microenvironment, and blocking the interaction between these two molecules can enhance the anti-tumor ability of T cells. Currently, several PD-1 and PD-L1 inhibitors have been approved for the treatment of various solid tumors, including breast cancer. However, the Chinese Expert Consensus on Immunotherapy and Targeted Therapy for Central Nervous System Gliomas (Second Edition) does not recommend the use of anti-PD-1 therapy in nGBM patients with unmethylated MGMT promoters, nor does it recommend the use of anti-PD-1 therapy in rGBM patients. Immune checkpoint blockade (ICB) therapy has made significant breakthroughs in the treatment of solid tumors such as melanoma and lung cancer. However, GBM patients are not sensitive to immunotherapy, with a response rate of less than 10% and a short response time [see: Nivolumab with or without ipilimumab in patients with recurrent glioblastoma: Results from exploratory phase i cohorts of checkmate]. Moreover, GBM is a typical "cold tumor" with a highly inhibitory immune microenvironment. Summary of the Invention

[0005] Given the location and challenging nature of advanced GBM, and the fact that photothermal therapy (PTT) ablation of tumors can lead to recurrence and metastasis, effectively reshaping the immune microenvironment through local treatment and transforming "cold tumors" into "hot tumors" is crucial for improving the treatment efficacy of advanced GBM. Currently, there are no reports of similar treatments that achieve both physical ablation and immune activation of the tumor through combined photothermal and immunotherapy for glioblastoma (GBM).

[0006] The present invention adopts the following scheme.

[0007] A system for ablation and combined immunotherapy of intracranial tumors includes a hydrogel carrier and polymer vesicles loaded with a co-loaded photothermal agent and an immunoadjuvant.

[0008] This invention discloses a method for preparing the above-mentioned intracranial tumor ablation synergistic immunotherapy system, which includes the following steps: loading a hydrogel with co-loaded photothermal agent and immune adjuvant polymer vesicles to obtain the intracranial tumor ablation synergistic immunotherapy system.

[0009] In this invention, the photothermal agent includes one or more of metallic photothermal agents and inorganic non-metallic photothermal agents. For example, the photothermal agent is selected from one or more of noble metal photothermal agents (gold, platinum, palladium, etc.), metal compound photothermal agents (metal sulfides, metal oxides, etc.), and carbon photothermal agents (graphene / graphene oxide, carbon nanotubes, carbon dots, porous carbon). As an example, the photothermal agent is one or more of gold nanoclusters, gold nanorods, CuS, and graphene.

[0010] In this invention, the immune adjuvant includes one or more of the following: TLR agonists (TLR1 / 2, TLR3, TLR4, TLR7 / 8, TLR9), RLR agonists, and STING agonists; as examples, the immune adjuvants are one or more of the following: Poly(I:C), Poly(I:C-LC), ICGODN, LPS, MPL, E6020, imiquimod, R848 (Resiquimod), 5'-ppp-RNA, poly(dA:dT), SR-717, ADU-S100, and MK-1454.

[0011] In the intracranial tumor ablation and synergistic immunotherapy system of the present invention, the mass ratio of photothermal agent to immune adjuvant is 1:(0.01-50), preferably 1:(0.02-30), even more preferably 1:(0.03-20), further preferably 1:(0.04-10), further preferably 1:(0.05-5), and even more preferably 1:(0.05-2). For example, the mass ratio of photothermal agent to immune adjuvant is 0.1:1, 0.5:1, 1:1, 2:1, 5:1, 10:1, 20:1, 25:1, 30:1, 35:1, or any ratio within the range.

[0012] As an embodiment of the present invention, the polymer vesicles co-loaded with photothermal agent and immune adjuvant are polymer vesicles co-loaded with gold nanoclusters and poly(I:C). The present invention provides for the first time the dual advantages of photothermal immunotherapy in achieving physical ablation of tumors and immune activation. By inducing immunogenic cell death (ICD) to reshape the tumor microenvironment, activating antigen-presenting cells, and promoting T cell proliferation and tumor infiltration, it has shown highly effective treatment for orthotopic advanced GBM mice.

[0013] In this invention, the polymer is a hydrophilic segment -P(B-DTC)-cationic fragment, or the polymer is a hydrophilic segment -P(B-DTC)-cationic fragment and A-hydrophilic segment -P(B-DTC); wherein A is a target molecule and B is a cyclic ester or cyclic carbonate monomer unit; when the amphiphilic block polymer is a hydrophilic segment -P(B-DTC)-cationic fragment and A-hydrophilic segment -P(B-DTC), the molar percentage of A-hydrophilic segment -P(B-DTC) in the polymer is 0 to 40%, excluding 0.

[0014] In this invention, the hydrophilic segment is PEG, the cationic fragment is spermine or a low molecular weight PEI, DTC is dithiopentane trimethylene carbonate; B is a cyclic ester or cyclic carbonate monomer unit, such as trimethylene carbonate monomer TMC, lactide monomer LA, caprolactone monomer CL, etc.; wherein, the cationic fragment is preferably spermine.

[0015] The polymer of this invention is an amphiphilic block polymer, with a hydrophilic segment being a polyethylene glycol segment and a hydrophobic segment being a random copolymer P(B-DTC), where B is an ester monomer or carbonate monomer, and DTC is a disulfide five-membered ring carbonate unit; P indicates polymerization, and P(B-DTC) is a random copolymer of B-DTC. As an example, the polymer of this invention is PEG-P(TMC-DTC)-Sp.

[0016] Furthermore, in the above polymers, the molecular weight of the hydrophilic segment is 1000–15000 Da; the molecular weight of the hydrophobic segment is 1–20 times that of the hydrophilic segment; and the molecular weight of PDTC is 5%–50% of the total molecular weight of the hydrophobic segment. Preferably, the molecular weight of the hydrophilic segment is 2000–12000 Da; the molecular weight of the hydrophobic segment is 2–15 times that of the hydrophilic segment; and the molecular weight of the PDTC segment is 10%–40% of that of the hydrophobic segment. More preferably, the molecular weight of the hydrophilic segment is 3000–10000 Da; the molecular weight of the hydrophobic segment is 2–10 times that of the hydrophilic segment; and the molecular weight of PDTC is 10%–30% of the total molecular weight of the hydrophobic segment. Further preferred: the molecular weight of the hydrophilic segment is 4000-8000 Da, for example, the molecular weight of the hydrophilic chain segment is 4500 Da, 5000 Da, 5500 Da, 6000 Da, 6500 Da, 7000 Da, 7500 Da or any molecular weight within the range; the molecular weight of the hydrophobic chain segment is 2.5-8 times the molecular weight of the hydrophilic segment, for example, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 5.5 times, 6 times, 6.5 times, 7 times, 7.5 times or any multiple within the range; the molecular weight of PDTC is 10%-30% of the total molecular weight of the hydrophobic segment, for example, 15%, 20%, 25% or any data within the range.

[0017] This invention co-loads a photothermal therapeutic agent and a TLR agonist in vesicles, which are then embedded in a hydrogel (TIPs@Gel) to form an intracranial tumor ablation synergistic immunotherapy system. In practical applications, in situ injection is performed. The TIPs@Gel embedded in hyaluronic acid hydrogel can increase drug retention in the tumor, enhance efficacy and reduce toxicity, and reverse the immunosuppressive microenvironment, demonstrating highly effective treatment for in situ advanced GBM mice.

[0018] This invention discloses a combination drug, comprising the above-mentioned intracranial tumor ablation synergistic immunotherapy system and other anti-tumor drugs, wherein the other anti-tumor drugs are existing products, such as monoclonal antibodies.

[0019] This invention discloses the application of the above-mentioned intracranial tumor ablation synergistic immunotherapy system or combined drugs in the preparation of antitumor drugs. Preferably, solid tumors include brain tumors, such as glioblastoma, as well as other inoperable solid tumors.

[0020] In this invention, the anti-tumor drug can be an intracranial tumor ablation synergistic immunotherapy system as the main active ingredient, or the above-mentioned intracranial tumor ablation synergistic immunotherapy system can be used together with other drugs as active ingredients, or the above-mentioned intracranial tumor ablation synergistic immunotherapy system can be used in combination with other treatment methods, such as radiotherapy, thermotherapy, etc.

[0021] The main treatment deficiencies of glioblastoma (GBM) are: (1) abnormal activation of immunosuppressive tumor-associated macrophages (TAMs) and regulatory T cells (Tregs) in the tumor microenvironment (TME) of GBM; (2) impaired migration of dendritic cells (DCs), leading to insufficient infiltration and dysfunction of cytotoxic T lymphocytes (CTLs); and (3) high expression of immunosuppressive factors such as programmed cell death-ligand 1 (PD-L1) and indoleamine 2,3-dioxygenase (IDO) by GBM cells. This invention presents for the first time the dual advantages of photothermal immunotherapy in achieving physical ablation and immune activation of tumors. It can rapidly reduce tumor volume and achieve tumor ablation. By inducing immunogenic cell death (ICD), it remodels the tumor microenvironment, activates antigen-presenting cells, promotes T cell proliferation and tumor infiltration, and effectively remodels the immune microenvironment through local treatment. It also avoids the problem of tumor recurrence and metastasis after PTT ablation of tumors. It is a key method to improve the treatment effect of advanced GBM by transforming "cold tumors" into "hot tumors". Attached Figure Description

[0022] Figure 1 The diagram shows the preparation of (A) TIPs and (B) TIPs@Gel, and (C) GBM ablation combined with immunotherapy for the treatment of an in situ advanced GBM model.

[0023] Figure 2 Stability assessment of TIPs after treatment with GSH (10 mM GSH, 12 h), NIR irradiation and aCSF (12 h).

[0024] Figure 3 The 1H NMR spectra of HA, N3-HA, and DBCO-HA are shown (D2O, 400 MHz).

[0025] Figure 4 For the rheological properties of TIPs@Gel, (A) strain scan and (B) time scan of storage modulus (G′) and loss modulus (G″), (C) shear thinning behavior of TIPs@Gel (HA concentration: 5 mg / mL).

[0026] Figure 5 Photothermal activity of TIPs: heating kinetic curve.

[0027] Figure 6 For biocompatibility studies of TIPs@Gel, hemolysis tests of TIPs and gel components (n = 3), (A) representative images; (B) hemolysis rate.

[0028] Figure 7 To evaluate the in vitro antitumor effects of TIPs and TIPs@Gel; (A) cytotoxicity of TIPs, TIPs+NIR and TIPs@Gel (n = 3), (B) staining of tumor cells after 4 h of sample treatment (AuNCs: 200 μg / mL, poly(I:C): 10 μg / mL).

[0029] Figure 8 To study the in vivo residence and photothermal effects of TIPs@Gel, on day 16 post-inoculation, (A) colocalization of TIPs@Gel (fluorescence) with tumor sites (Evans blue) and (B) temperature of tumor sites in test mice.

[0030] Figure 9 A study on TIPs@Gel combined with ICB for the treatment of mice with mid-stage GL261 tumors in situ (n = 6; NIR: 808 nm, 1 W / cm², 5 min; AuNCs: 0.125 mg / kg; poly(I:C): 0.25 mg / kg; αCTLA-4: 1.0 mg / kg). (A) Schematic diagram of the treatment process for mid-stage GBM, on day 7 after inoculation. (B) Coronal H&E section of the brain of mice with mid-stage tumors (scale bar: 2 mm). (C) Temperature changes at the tumor site during treatment. (D) Mouse weight curve. (E) Mouse survival curve.

[0031] Figure 10For the study of TIPs@Gel combined with ICB for the treatment of advanced orthotopic GL261 tumor-bearing mice (n = 6; NIR: 808nm, 1 W / cm², 5 min; AuNCs: 0.2 mg / kg; αCTLA-4: 1.0 mg / kg), (A) Schematic diagram of the treatment process for advanced GBM, (B) H&E section of tumor in tumor-bearing mice on the 10th day after inoculation (scale bar: 2 mm), (C) Temperature changes at the tumor site during treatment, (D) Trend of mouse body weight change, and (E) Survival curve of mice (poly(I:C) at different doses). Detailed Implementation

[0032] Advanced GBM cannot be surgically removed, and unlike lung cancer and breast cancer, GBM is difficult to suppress. Existing techniques involve subcutaneously inoculating cured LLC mice with LLC cells or GBM (glioblastoma) cells again. Results showed that mice re-inoculated with LLC cells did not develop LLC tumors, while mice re-inoculated with GBM cells experienced slow tumor growth 10 days after GBM cell inoculation. This invention innovatively constructs a polymer vesicle nanomedicine co-loaded with a photothermal agent / immunoadjuvant and embeds it in a hydrogel (TIPs@Gel) for in situ injection; combined with NIR light irradiation, this therapy achieves the dual functions of surgical-like GBM ablation and immune activation. In this invention, the polymer vesicles (Thermo-ImmunoPolymersomes, TIPs) co-loaded with gold nanoclusters and poly(I:C) combined with NIR exhibit significant photothermal and ICD-inducing properties, and can also effectively activate the immune system; the TIPs@Gel embedded in hyaluronic acid hydrogel increases drug retention in the tumor, enhances efficacy while reducing toxicity, and reverses the immunosuppressive microenvironment. See [link to related documentation]. Figure 1 The invention illustrates the structure of the raw materials used and demonstrates highly effective treatment for orthotopic late GBM mice. This invention provides a novel strategy for treating late GBM.

[0033] The following specific experiments illustrate the technological advancements of this invention. The raw materials used are existing products, and the specific preparation procedures are as follows. Operation and performance testing are standard techniques.

[0034] The raw materials, including AuroVist (AuNCs, 1.9 nm), low molecular weight polyinosinic acid (LMW, poly(I:C), 0.2-1 kb, Invivogen), L-glutathione (GSH, Beyotime), sodium hyaluronate (Bloomage Biotech), 4-(4,6-dimethoxy[1,3,5]triazine-2-yl)-4-methylmorpholine chloride hydrate (DMTMM, J&K Scientific), DBCO-(CH2)2-NH2 (Ruixun Biotech), N3-(CH2)3-NH2 (Yisheng Biotech), and anti-CTLA-4 antibody (InVivoMAb, BE0164), are all commercially available products. The amphiphilic block copolymer PEG-P(TMC-DTC)-Sp (number average molecular weight Mn = 5.0-(15.0-2.0)-0.2 kg / mol) was synthesized according to our team's previous method and is an existing technology. GL261 mouse GBM cells, Bend.3 mouse brain microvascular endothelial cells, HT22 mouse hippocampal neurons, and dendritic cells (BMDCs) were existing products. GL261 cells were cultured in DMEM medium containing 10% FBS and 1% penicillin-dextrose antibody, while Bend.3 cells were cultured in a dedicated culture medium (Pronosel). When the cells reached 80% confluence, they were treated with 0.25% trypsin-EDTA digestion solution and collected for subsequent experiments. 7-8 week old female C57BL / 6 mice were purchased from Vital River Pharmaceuticals. All animal experiments were approved by the Laboratory Animal Ethics Committee of Soochow University, and the experimental procedures strictly followed the relevant provisions of the "Guidelines for the Care and Use of Laboratory Animals".

[0035] The particle size and distribution of polymer vesicles were determined using a Zetasizer Nano-ZS dynamic light scattering system (equipped with a 633 nm helium-neon laser, 25 °C backscatter detection mode). Drug loading (DLC) and encapsulation efficiency (DLE) within the polymer vesicles were determined using a Thermo NanoDrop micro-spectrophotometer. Vesicle morphology was characterized using a Hitachi HT7700 transmission electron microscope (accelerating voltage 100 kV). Cell apoptosis rate and immune cell phenotype were analyzed using a FACSverse (BD) flow cytometry system. Cell viability assays were performed using a Thermo Multiskan FC microplate reader, including CCK-8 assay (450 nm absorbance) and ATP chemiluminescence assay (chemiluminescence mode).

[0036] In the intracranial tumor ablation and synergistic immunotherapy system of the present invention, the preferred mass ratio of photothermal agent to immune adjuvant is 1:(0.04-10), for example 1:(0.05-5), according to the actual drug loading ratio.

[0037] This invention co-loads a photothermal therapeutic agent and a TLR agonist in vesicles, which are then embedded in a hydrogel (TIPs@Gel) to form an intracranial tumor ablation synergistic immunotherapy system. In practical applications, in situ injection is performed. The TIPs@Gel embedded in hyaluronic acid hydrogel can increase drug retention in the tumor, enhance efficacy and reduce toxicity, and reverse the immunosuppressive microenvironment, demonstrating highly effective treatment for in situ advanced GBM mice.

[0038] All data in this invention are expressed as mean ± standard deviation (SD) and analyzed using GraphPad Prism 8 software. Differences between groups were assessed using one-way ANOVA and Tukey's multiple comparison test. Unpaired t-tests were used for comparisons between two groups. Kaplan-Meier survival curves were estimated using the log-rank test. ns (no significance, p>0.05) indicates no significant difference, * p<0.05 indicates statistical significance, and ** p<0.01, *** p<0.001, and **** p<0.0001 indicate highly statistical significance.

[0039] Synthesis of DBCO-HA and N3-HA First, 400 mg of sodium hyaluronate (molecular weight: 1 MDa) was dissolved in deionized water to prepare a solution of 23 mg / mL. Then, 5 mL of DMTMM aqueous solution with a concentration of 54 mg / mL was added, and after stirring, 44 mL of DBCO-(CH2)2-NH2 solution with a concentration of 1 mg / mL (solvent: DMSO) was injected. The mixture was reacted at 37 °C for 24 h to obtain DBCO-HA.

[0040] The synthesis of N3-HA was carried out using a similar method: 1 mg / mL DBCO-(CH2)2-NH2 solution was replaced with 10.45 mg / mL N3-(CH2)3-NH2 solution (solvent: DMSO), the reaction was carried out at 37℃ for 24 h, and after dialyzing, the product N3-HA was obtained by freeze drying.

[0041] Example 1: Preparation and Characterization of TIPs and TIPs@Gel First, prepare a DMF stock solution (80 mg / mL) of PEG-P(TMC-DTC)-Sp, a poly(I:C) nuclease-free solution (10 mg / mL), and an AuNCs nuclease-free solution (20 mg / mL). Premix the AuNCs solution with 10 mM GSH solution at a 1:1 volume ratio, then add this mixture along with the poly(I:C) solution to phosphate buffer (PB, pH 6.0, 2 mM), maintaining a total volume of 900 μL. Subsequently, under stirring at 500 rpm, add 100 μL of the polymer solution and continue the reaction for 5 minutes. The reaction product is then purified by dialysis in PB (pH 6.0, 2 mM) and PB (pH 7.4, 10 mM) to obtain TIPs nanoparticles. By adjusting the drug formulation, TIPs with different ratios of co-loaded AuNCs and poly(I:C) can be obtained. For example, 50 μL of AuNCs solution was premixed with 50 μL of GSH solution, then added together with 5 μL of poly(I:C) solution to PB buffer, followed by the addition of 100 μL of polymer solution. This purified TIPs with a specific drug loading ratio were then used for in vitro cell experiments. Similarly, 25 μL of AuNCs solution was premixed with 25 μL of GSH solution, then added together with 50 / 100 / 150 μL of poly(I:C) solution to PB buffer, maintaining a total volume of 900 μL. This was followed by the addition of 100 μL of polymer solution, and purification yielded nanomedicines for GBM mouse treatment studies.

[0042] The particle size and particle size distribution of the above TIPs were characterized by dynamic light scattering (DLS), and the encapsulation stability of poly(I:C) in the TIPs was characterized by agarose gel electrophoresis (40V, 40 min).

[0043] TIPs were prepared by co-loading poly(I:C) and AuNCs in a PEG-P(TMC-DTC)-Sp copolymer. AuNCs are commercially available water-soluble gold nanoclusters. During self-assembly in buffer solution, the polymer can load poly(I:C) into the hydrophilic lumen while AuNCs are distributed in the hydrophobic membrane layer of the vesicles. Dynamic light scattering analysis showed that the average particle size of the TIPs was 60 nm (PDI < 0.15). TEM revealed that the TIPs were vesicle structures with a black membrane layer, indicating that the AuNCs were loaded into the hydrophobic membrane of the vesicles and maintained structural integrity under 808 nm laser irradiation. The PEG-P(TMC-DTC)-Sp support exhibited excellent loading capacity when co-loading poly(I:C) and AuNCs, with poly(I:C) being almost completely encapsulated. When the ratio of the two drugs was 15%, the encapsulation efficiencies of poly(I:C) and AuNCs exceeded 97% and 80%, respectively, and the results from multiple batches were stable (Table 1). Gel electrophoresis experiments showed that TIPs responded to intracellular GSH, triggering the responsive release of poly(I:C); simultaneously, no leakage of poly(I:C) occurred after treatment with light and artificial cerebrospinal fluid (aCSF). Therefore, the reduction-responsive characteristics of TIPs effectively prevent uncontrolled release of poly(I:C) that could cause nerve damage, while also achieving precise release under intracellular reduction conditions. Figure 2 ).

[0044] Table 1. Characterization of TIPs (n = 3)

[0045] a1 Determined by ICP-OES a2 Determined by nanodrop b Determined by DLS.

[0046] A TIPs@Gel composite hydrogel was prepared by vortexing a DBCO-HA and N3-HA aqueous solution (15 mg / mL) at a volume ratio of 1:1:1 with a TIPs solution (8 mg / mL, drug-to-material ratio of 15%). Its rheological properties were analyzed using a HAAKE MARS40 rheometer (equipped with a temperature-controlled platform and a 25.0 mm parallel stainless steel plate).

[0047] DBCO-HA and N3-HA aqueous solution (15 mg / mL) were vortexed at a volume ratio of 1:1 to prepare gel (hydrogel).

[0048] This invention designs an embedding system based on chemically cross-linked hyaluronic acid hydrogel, namely TIPs@Gel. The specific construction process is as follows: First, two functionalized hyaluronic acid derivatives, dibenzocyclooctylene-modified hyaluronic acid (DBCO-HA, abbreviated as DHA) and azidated hyaluronic acid (N3-HA, abbreviated as NHA), are efficiently synthesized via a one-pot method. The results are analyzed using proton nuclear magnetic resonance (NMR) spectroscopy. Figure 3 The degree of substitution of DHA and NHA was studied. Normalized calculations showed that the degree of substitution for N3-HA was 19.6%, and for DBCO-HA, it was 19.3%, ensuring both the efficiency of subsequent click chemistry reactions and maintaining the inherent biocompatibility of HA. Subsequently, equimolar amounts of DHA and NHA solutions were mixed under physiological pH conditions, and rapid in-situ crosslinking of the prepared N3-HA and DBCO-HA was achieved using copper-free click chemistry (DBCO-azidocycloaddition reaction). These crosslinked hydrogels can then be formed through click chemistry reactions. TIPs@Gel is prepared by mixing TIPs solution, DBCO-HA, and N3-HA. The two polymers form a chemically crosslinked hydrogel through click chemistry reactions. TIPs@Gel exhibits typical gel rheological characteristics, maintaining similar rheological properties to the gel itself even after loading with TIPs. Figure 4 A, B). Shear-thinning experiments showed that TIPs@Gel possesses shear-thinning properties, meaning that this gel can be used for injection (…). Figure 4 C).

[0049] Under irradiation with an 808 nm laser (1 W / cm², 5 min), the TIPs solution heated up to 18 °C. The introduction of TIPs endowed the gel with photothermal activity. Unexpectedly, the gel composite system had a stronger heating capacity than the free TIPs, while the blank hydrogel showed no photothermal effect. Figure 5 Fluorescence microscopy analysis revealed that TIPs@Gel combined with NIR irradiation significantly increased intracellular ROS levels in GL261 cells. This photothermal-oxidative stress synergistic effect provides a dual mechanism of action for tumor therapy.

[0050] The biocompatibility of medical materials is of paramount importance. Hemocompatibility of TIPs@Gel was assessed through a hemolysis assay, and its neurological safety was evaluated by co-culturing the material with Bend.3 cells and HT22 cells. The hemolysis assay showed that TIPs did not induce hemolysis even at a carrier concentration of 800 μg / mL. Furthermore, DHA and NHA did not cause damage to RBCs during incubation with RBC suspensions. Figure 6(A, B). Furthermore, different concentrations of DHA, NHA, and TIPs were co-incubated with Bend.3 and HT22 cells for 72 hours to assess their neurological safety. Cytotoxicity assays showed that the survival rates of Bend.3 and HT22 cells were >90% at any concentration, confirming that TIPs@Gel does not damage the blood-brain barrier or neurons, demonstrating neurological safety. In vitro biocompatibility experiments confirmed that the gel itself has no toxic effect on the aforementioned cells (survival rate >90%), meaning that TIPs@Gel can serve as a safe intracranial drug reservoir, enhancing local thermal ablation while reducing the risk of tissue damage.

[0051] Example 2 To determine the cell-killing effect of the formulation, GL261 cells were seeded in 96-well plates (5 × 10⁶ cells per well). 3 Cells were cultured for 24 h, followed by co-culturing with the reagent for another 4 h. For the "+NIR" group, cells were irradiated for 5 minutes per well at a power density of 1 W / cm² (this parameter will be used for subsequent in vitro experiments unless otherwise specified); after a further 24 h of culture, CCK-8 solution was added to each well, and incubation continued for 1 h. The absorbance at 450 nm was measured using a microplate reader, and cell viability (%) was calculated as the ratio of the absorbance of each sample to that of the PBS control group (n=4). For the Calcein / PI cell viability staining assay, GL261 cells were seeded in 24-well plates (2 × 10⁶ cells per well). 5 (100 cells) and cultured for 24 h. Then add the preparation and irradiate with light. After 4 h of irradiation, remove the culture medium and wash once with PBS. Then stain according to the kit instructions and take pictures with an inverted fluorescence microscope.

[0052] CCK-8 assay results showed that when AuNCs concentration was ≤ 200 μg / mL, TIPs and TIPs@Gel had no significant toxicity to tumor cells. However, under near-infrared irradiation, cell survival rate showed a significant concentration-dependent decrease, with the survival rate of the AuNCs concentration group at 200 μg / mL being only 10%. Figure 7 A). Further results from calcifero-green propidium iodide staining showed that, at the same AuNCs concentration, the temperature of TIPs+NIR was lower, and it could not completely kill tumor cells. Therefore, cells in the TIPs+NIR group detached from the well plate and were washed away, while the area around TIPs@Gel (upper right quadrant) showed a completely cell-free state. Figure 7 (B) This suggests that TIPs@Gel+NIR has a tumor-clearing effect similar to surgical resection. The TIPs@Gel of this invention directly ablates tumor cells through photothermal effects and simultaneously induces ICD. This dual mechanism of action of thermal ablation and immune activation provides an important foundation for photothermal-immunotherapy synergistic therapy.

[0053] Example 3: Establishing the GBM Model To establish a mid-to-late stage GBM mouse model, mice containing 1×10 6 Five μL of GL261 cell inoculum (containing 25% matrix gel) was injected into the left hemisphere of the mouse brain, with the injection site coordinates using the anterior fontanelle as the origin: (-0.5 mm, -2 mm, -2.5 mm). Treatment for the intermediate-stage model began on day 7 post-inoculation, using a Hamilton syringe for intratumoral injection of 5 μL of AuNCs (0.125 mg / kg, poly(I:C): 0.25 mg / kg). The "+NIR" group received near-infrared laser irradiation after injection, and additionally received intravenous injections of anti-CTLA-4 antibody (1 mg / kg) on ​​days 8, 10, and 13. Mouse weight and condition were monitored and recorded every two days. The inoculation method for the late-stage model was the same as that for the intermediate-stage model. Treatment began on day 10 after inoculation, with intratumoral injection of 8 μLTIPs@Gel (AuNCs: 0.2 mg / kg, poly(I:C): 0 / 0.2 / 0.4 / 0.6 mg / kg). The "+NIR" group received near-infrared laser irradiation after injection. Additionally, on days 11, 13, and 15, anti-CTLA-4 antibody (1 mg / kg) was administered intravenously. The weight and status of the mice were monitored and recorded every 2 days.

[0054] Example 4: Treatment Study of TIPs@Gel in the Intermediate GL261 Model Mid-stage GBM, due to its large tumor burden, not only affects key functional areas of the brain, but its highly invasive nature and blurred peritumoral boundaries also pose a severe challenge to surgical resection. This invention further discloses the therapeutic potential of the TIPs@Gel system in mid-to-late stage GBM. Fluorescently labeled TIPs@Gel was injected intratumorally on day 7, and Evans blue was intravenously injected on day 16 post-inoculation to label the tumor area. In vitro fluorescence imaging showed a high degree of overlap between the Cy5-labeled TIPs@Gel and Evans blue-traced areas. Figure 8 A); The in vivo photothermal effect of TIPs@Gel+NIR (AuNCs: 0.125 mg / kg) was tested, and the temperature at the tumor site in mice was around 50 ℃. Figure 8 B) can achieve the effect of thermal ablation of tumors.

[0055] like Figure 9 Treatment regimen A established an orthotopic GL261 mouse model. Seven days after tumor cell inoculation, the tumor planar area was approximately 2 mm². 2 Simulate the clinical characteristics of GBM in the mid-stage of disease with a large tumor burden that is difficult to completely resect. Figure 9B). By stereotactic injection of TIPs@Gel or TIPs combined with near-infrared irradiation (808 nm, 1 W / cm², 5 min), the intratumoral temperature gradient in the TIPs@Gel group increased to 53.0℃, significantly higher than the 48.7℃ in the TIPs group. Figure 9 C). It is worth noting that temperatures above 50°C have been proven to achieve effective photothermal ablation, with stronger killing ability against tumor tissue, but also stronger damage to normal tissue. Unexpectedly, the technical solution disclosed in this invention achieves effective tumor inhibition in high-temperature photothermal therapy while maintaining stable mouse weight. That is, this invention solves the problem of high-temperature killing of tumors and normal tissues in existing photothermal therapy, and achieves the goal of high-temperature targeted killing of tumors while avoiding damage to normal tissues.

[0056] In vivo treatment experiments showed that mice in the PBS group experienced significant weight loss starting from day 16 and all died within 20 days, with a median survival (MST) of only 19 days. Figure 9 D, E), MST was extended to 24.5 days in the TIPs@Gel treatment group and 26 days in the TIPs+NIR treatment group (***p, Figure 9 E), In addition, the therapeutic effect of intracranial injection of TIPs without hydrogel embedding alone was not as good as that of TIPs@Gel with gel, and the therapeutic effect of TIPs+NIR photothermal therapy was not significantly different from that of TIPs+NIR+ICB combination therapy (for brevity, the results of these two groups are not shown, but do not affect the understanding of the technical progress of the present invention by those skilled in the art); in particular, unlike the certain efficacy of injection of anti-CTLA-4 antibody in the treatment of other solid tumors, PBS+ICB and Gel+ICB had basically no therapeutic effect on the above-mentioned orthotopic GL261 mouse model, with a median survival (MST) of only 20.5 days and 21 days, respectively.

[0057] Notably, a single TIPs@Gel+NIR treatment delayed weight loss and increased MST to 28 days, fully validating the synergistic effect of photothermal ablation and immune activation. When the intratumoral temperature reached 53 °C, photothermal therapy not only induced tumor cell apoptosis and ICD (releasing DAMPs to enhance tumor immunogenicity), but also promoted the infiltration of immune cells into peripheral lymphoid tissues; while poly(I:C) could reverse the tumor immunosuppressive microenvironment and enhance antigen presentation to CTLs. ICB has a certain therapeutic effect on lung cancer; for example, in the orthotopic LLC model, immune checkpoint therapy ICB (αPD-1) increased the median survival of mice from 17 days in the PBS group to 26 days with statistical significance. However, existing technologies suggest that ICB treatment for GBM is ineffective (generally considered to have no therapeutic effect). Unexpectedly, the ICB combined treatment of this invention significantly prolonged MST to 34 days. Even more encouragingly, in such a late-stage model, TIPs@Gel+NIR combined with anti-CTLA-4 monotherapy (TIPs@Gel+NIR+ICB) achieved a complete response rate of 33.3% and no relapse within 90 days.

[0058] Example 5: Treatment Study of TIPs@Gel in an Advanced GL261 Model To further challenge the more aggressive GBM model, the tumor growth time was extended to 10 days. Figure 10 At this stage, the tumor becomes more aggressive, invading the ventricles and cortex and about to cross the midline of the brain. Figure 10 B). Temperature testing at the tumor site revealed that when the gold nanocluster dose was increased to 0.2 mg / kg, the temperature in the TIPs@Gel+NIR group reached 58 °C (Figure 10 C). Under triple therapy (TIPs@Gel+NIR+ICB), the MST of tumor-bearing mice reached 32.5 days, with 1 / 6 of the mice achieving complete tumor elimination and long-term survival, and stable body weight. Figure 10 (D, E). Furthermore, dose-response analysis revealed that at a fixed AuNCs dose (0.2 mg / kg), poly(I:C) exhibited the optimal synergistic effect in the 0.4 mg / kg group (1:2). This invention is the first to disclose a synergistic strategy based on local thermal ablation (TIPs@Gel+NIR) and ICB, which can overcome the limitations of the immunosuppressive TME in advanced GBM and provide a multimodal treatment option with transformative potential for patients with inoperable aggressive GBM.

[0059] Photothermal-immunotherapy has significant scientific value in improving the efficacy of tumor treatment. This invention develops an innovative anti-tumor strategy combining photothermal effects and immune activation. This strategy is based on TIPs nanocarriers, which have a stable and efficient ability to co-load poly(I:C) and AuNCs, with an average particle size of 60 nm and uniform particle size distribution. When loaded onto a hydrogel to form TIPs@Gel, it generates a strong photothermal effect under near-infrared light irradiation. The temperature at the tumor site can reach up to 53 °C under experimental conditions, significantly inducing thermal damage and apoptosis in tumor cells, resulting in 93% apoptosis. Simultaneously, it can induce immunogenic cell death and promote DC activation. This invention also utilizes a click-chemical cross-linked hydrogel system based on hyaluronic acid, enabling in situ injection into brain tumors. Unexpectedly, with the same photothermal agent, TIPs@Gel significantly enhances the photothermal effect with minimal damage to normal tissue. This hydrogel system exhibits good biocompatibility and enhances local therapeutic efficacy. In a mouse model of advanced GBM, TIPs@Gel significantly prolonged survival and achieved a tumor regression rate of 18.8%. Furthermore, this strategy effectively remodeled the local and systemic immune microenvironment, promoted the activation of tumor-derived digital cell networks (mDCs) and tumor-associated lymphoid tissue (CTLs), and significantly enhanced anti-tumor immune responses. By integrating local ablation with systemic immune activation, TIPs@Gel provides a new therapeutic paradigm for precision treatment of GBM and tumor immunotherapy, demonstrating significant clinical application potential.

Claims

1. An intracranial tumor ablation and synergistic immunotherapy system, comprising a hydrogel carrier and polymer vesicles loaded with a co-loaded photothermal agent and an immunoadjuvant on the hydrogel carrier.

2. The intracranial tumor ablation and immunotherapy system according to claim 1, characterized in that, Photothermal agents include one or more of metallic photothermal agents and inorganic non-metallic photothermal agents; immune adjuvants include one or more of TLR agonists, RLR agonists, and STING agonists.

3. The intracranial tumor ablation and synergistic immunotherapy system according to claim 1, characterized in that, The mass ratio of photothermal agent to immune adjuvant is 1: (0.01-50).

4. The intracranial tumor ablation and synergistic immunotherapy system according to claim 1, characterized in that, The polymer is a hydrophilic segment -P(B-DTC)-cationic fragment, or the polymer is a hydrophilic segment -P(B-DTC)-cationic fragment and A-hydrophilic segment -P(B-DTC); where A is the target molecule and B is a cyclic ester or cyclic carbonate monomer unit.

5. The intracranial tumor ablation and synergistic immunotherapy system according to claim 4, characterized in that, The molecular weight of the hydrophilic segment is 1000-15000 Da; the molecular weight of the hydrophobic segment is 1-20 times that of the hydrophilic segment; the molecular weight of PDTC is 5%-50% of the total molecular weight of the hydrophobic segment.

6. The preparation method of the intracranial tumor ablation synergistic immunotherapy system according to claim 1 includes the following steps: loading a hydrogel with co-loaded photothermal agent and immune adjuvant polymer vesicles to obtain the intracranial tumor ablation synergistic immunotherapy system.

7. A combination therapy comprising the above-mentioned intracranial tumor ablation synergistic immunotherapy system and other antitumor drugs.

8. The combined drug according to claim 7, characterized in that, Other anti-tumor drugs include monoclonal antibodies.

9. The use of the intracranial tumor ablation and immunotherapy system of claim 1 or the combined drug of claim 7 in the preparation of antitumor drugs.

10. The application according to claim 9, characterized in that, Tumors include brain tumors.

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