A photothermal synergistic tumor stem cell stemness inhibition system and preparation and application thereof

Abstract

This invention discloses a photothermal synergistic tumor stem cell inhibition system, its preparation method, and its applications. Addressing the current lack of effective treatments for large / unresectable GBM and its strongly immunosuppressive microenvironment, this invention constructs an injectable hydrogel platform (MIN-PPIC@iGel) integrating multiple key functions. This platform achieves a synergistic therapeutic model involving photothermal ablation for local tumor reduction, inhibition of residual GBM stem cells (GSCs), and activation of dendritic cells (DCs)-mediated anti-tumor immunity. Cellular experiments show that this invention can synergistically kill GL261 cells, significantly inhibit tumor stem cells and malignant phenotypes, induce significant ICD, and enhance DC maturation and activation effects. In a large-volume orthotopic GBM mouse model, a single intratumoral injection of MIN-PPIC@iGel combined with short-term near-infrared light irradiation doubled survival; further combination with antibody therapy unexpectedly achieved long-term tumor-free survival in 50% of mice, providing a new strategy for the local treatment of unresectable GBM.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a locally injectable drug, specifically a photothermal synergistic tumor stem cell stemness inhibition system and its preparation method and application. It promotes durable immunotherapy for large-volume glioblastoma through a strategy of local photothermal tumor reduction, tumor stem cell stemness inhibition and dendritic cell activation. Background Technology

[0002] Glioblastoma (GBM) is the most aggressive primary brain tumor, with an extremely poor prognosis and high recurrence rate. A significant proportion of patients cannot achieve complete resection due to large tumor burden, deep lesion location (e.g., in the thalamus or brainstem), multiple lesions, or adjacent functional cortical areas [Updated clinical practice guidelines for the management of adult diffuse gliomas; A road map for the treatment of pediatric diffuse midline glioma; EANO guidelines on the diagnosis and treatment of diffuse gliomas of adulthood]. For these unresectable cases, standard chemoradiotherapy combined with triglyceride tumour (TTF) offers only limited clinical benefit. The efficacy of systemic drugs and intracellular chemotherapy (ICB) is severely hampered by tumor-to-tumor heterogeneity (BBB), the persistent presence of glioblastoma cells (GSCs), and the immunosuppressive tumor microenvironment (TME).

[0003] Glioblastoma is the most aggressive primary malignant brain tumor of the central nervous system, clinically characterized by invasive growth, rapid progression, and a high recurrence rate. Even with the current first-line standard of care (Stupp regimen), namely maximal surgical resection combined with radiotherapy and temozolomide (TMZ) chemotherapy, the median survival is less than 15 months, and the five-year survival rate is less than 10%. Treatment failure is mainly due to the unique physiological and pathological characteristics of GBM: the blood-brain barrier (BBB) ​​restricts drug entry into the brain, glioblastoma stem cells (GSCs) drive recurrence and drug resistance, the immunosuppressive tumor microenvironment (TME) increases immune escape, and the hypoxic environment weakens treatment efficacy. Although innovative strategies such as targeted therapy and immunotherapy have achieved breakthroughs in various solid tumors, they are difficult to implement effectively in GBM due to low delivery efficiency. For example, 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.

[0004] Therefore, in cases where surgery is not feasible, there is an urgent clinical need for treatment strategies that can be implemented locally: to both reduce and eliminate large tumors and simultaneously eradicate the biological root causes of recurrence. Summary of the Invention

[0005] GBM is a typical immunologically cold tumor, characterized by low immunogenicity, abundant infiltration of immunosuppressive cells, weak antigen presentation ability, and insufficient T cell infiltration, making it difficult for immune adjuvants, immune checkpoint inhibitors, and immune cell chemokines to exert their due effects. Addressing the lack of effective treatments for inoperable large-volume GBM and its strongly immunosuppressive microenvironment, this invention constructs an injectable hydrogel platform, MIN-PPIC@iGel, integrating three major functions: surgical-like tumor reduction, inhibition of GSCs, and activation of dendritic cells. Combined with near-infrared light irradiation, it achieves spatiotemporally coordinated drug release. This platform, combined with immune checkpoint therapy, effectively mobilizes immune cells in cervical lymph nodes, improves the immunosuppressive TME, achieves 50% tumor-free survival in a large-volume orthotopic GBM mouse model, and forms durable anti-tumor immune memory, effectively preventing tumor recurrence.

[0006] The present invention adopts the following technical solution.

[0007] A photothermal synergistic tumor stem cell inhibition system includes a hydrogel and a photothermal agent, a tumor stem cell inhibitor, and an immune adjuvant loaded in the hydrogel.

[0008] In this invention, a photothermal agent, a tumor stem cell inhibitor, and an immune adjuvant are loaded onto a hydrogel via a polymer carrier. Specifically, the photothermal agent, tumor stem cell inhibitor, and immune adjuvant can be co-loaded in the polymer carrier before being loaded onto the hydrogel; alternatively, multiple photothermal agents, tumor stem cell inhibitors, and immune adjuvants can be loaded onto different polymer carriers before being loaded onto the hydrogel; or, alternatively, one of the photothermal agent, tumor stem cell inhibitor, and immune adjuvant can be loaded onto different polymer carriers before being loaded onto the hydrogel.

[0009] In this invention, the polymer carrier can be either polymer micelles or polymer vesicles.

[0010] This invention discloses a method for preparing the above-mentioned photothermal synergistic tumor stem cell stemness inhibition system, which includes the following steps: loading a photothermal agent, a tumor stem cell inhibitor, and an immune adjuvant onto a hydrogel to obtain the photothermal synergistic tumor stem cell stemness inhibition system.

[0011] As an example, the photothermal synergistic tumor stem cell stemness inhibition system of the present invention includes a hydrogel carrier, and polymer micelles co-loaded with a photothermal agent and a tumor stem cell inhibitor, as well as polymer vesicles loaded with an immune adjuvant loaded on the hydrogel carrier. The preparation method is as follows: the photothermal synergistic tumor stem cell stemness inhibition system is obtained by loading the hydrogel with the polymer micelles co-loaded with the photothermal agent and the tumor stem cell inhibitor, as well as the polymer vesicles loaded with an immune adjuvant.

[0012] In this invention, the photothermal agent includes one or more of the following: metallic photothermal agents, inorganic non-metallic photothermal agents, and organic photothermal agents. For example, the photothermal agent is selected from one or more of the following: noble metal photothermal agents (gold, platinum, palladium, etc.), metal compound photothermal agents (metal sulfides, metal oxides, etc.), carbon photothermal agents (graphene / graphene oxide, carbon nanotubes, carbon dots, porous carbon), dye photothermal agents (indocyanine green or similar), and polymer (polythiophene, polypyrrole, polydopamine) photothermal agents; preferably, the photothermal agent is one or more of the following: indocyanine green or similar.

[0013] 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), ICG ODN, Poly(I:C-LC), LPS, MPL, E6020, imiquimod, R848 (Resiquimod), 5'-ppp-RNA, poly(dA:dT), SR-717, ADU-S100, and MK-1454.

[0014] In this invention, the tumor stem cell inhibitor is preferably a tumor stem cell pathway inhibitor; such as STAT3 pathway inhibitors, Hedgehog pathway inhibitors, Wnt pathway inhibitors, Notch pathway inhibitors, NF-κB pathway inhibitors, and preferably small molecule inhibitors of the STAT3 pathway, such as any one or more of Static, S3I-201, BP-1-102, WP1066, and Napabucasin.

[0015] In the photothermal synergistic tumor stem cell inhibition system of the present invention, the mass ratio of photothermal agent, tumor stem cell inhibitor, and immune adjuvant is (10-1000):(0.1-100):1000, preferably (20-800):(1-80):1000, further preferably (50-600):(2-60):1000, and even more preferably (70-500):(5-45):1000. For example, the mass ratio of photothermal agent, tumor stem cell inhibitor, and immune adjuvant is 75:37:1000, 110:37:1000, 125:37:1000, 500:15:1000, 500:5:1000, or any ratio within the range.

[0016] Glioblastoma (GBM) is the most common and aggressive primary brain tumor with an extremely poor prognosis. Its treatment faces multiple challenges, including the blood-brain barrier (BBB) ​​limiting drug entry into the brain, the hypoxic and immunosuppressive tumor microenvironment (TME) weakening efficacy, and relapse and drug resistance driven by glioblastoma stem cells (GSCs). Clinical treatment options are particularly limited for large-volume GBMs or those that are inoperable due to their location. To address these issues, this invention focuses on key problems in GBM treatment, designing nanomedicines with a synergistic mechanism to promote immune activation, providing a new approach to improving the therapeutic effect of GBM.

[0017] In this invention, the raw materials for preparing the polymer carrier include polymers, and further, the polymer carrier is assembled from polymers. The polymer includes one or more of the hydrophilic segment -P(A-DTC) and the hydrophilic segment -P(B-DTC)-cationic segment, wherein A is a cyclic ester or cyclic carbonate monomer unit, B is a cyclic ester or cyclic carbonate monomer unit, and A and B may be the same or different.

[0018] In this invention, the hydrophilic segment is polyethylene glycol (PEG), the cationic segment is spermine or a low molecular weight PEI, and DTC is dithiopentane trimethylene carbonate; the cyclic ester or cyclic carbonate monomer unit is selected from trimethylene carbonate monomer TMC, lactide monomer LA, caprolactone monomer CL, etc.; wherein, the cationic segment is preferably spermine.

[0019] The polymers of this invention are amphiphilic block polymers, with hydrophilic segments being polyethylene glycol segments and hydrophobic segments being random copolymers P(A-DTC) or P(B-DTC). A / B represents ester monomers or carbonate monomers, and DTC represents disulfide five-membered ring carbonate units. P indicates polymerization; P(A-DTC) is a random copolymer of A-DTC, and P(B-DTC) is a random copolymer of B-DTC. As examples, the polymers of this invention are PEG-P(CL-DTC) and PEG-P(TMC-DTC)-Sp.

[0020] Furthermore, in the above polymers, the molecular weight of the hydrophilic segment is 1000–15000 Da; the molecular weight of the hydrophobic segment is 0.5–20 times that of the hydrophilic segment; and the molecular weight of PDTC is 5%–80% of the total molecular weight of the hydrophobic segment. Preferably, the molecular weight of the hydrophilic segment is 1500–12000 Da; the molecular weight of the hydrophobic segment is 0.7–15 times that of the hydrophilic segment; and the molecular weight of the PDTC segment is 8%–70% of that of the hydrophobic segment. More preferably, the molecular weight of the hydrophilic segment is 1700–10000 Da; the molecular weight of the hydrophobic segment is 0.8–10 times that of the hydrophilic segment; and the molecular weight of PDTC is 10%–60% of the total molecular weight of the hydrophobic segment. Further selection: The molecular weight of the hydrophilic segment is 1800-8000 Da, for example, the molecular weight of the hydrophilic chain segment is 2000 Da, 2500 Da, 3000 Da, 3500 Da, 4000 Da, 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 segment is 0.9 to 8 times that of the hydrophilic segment, such as 0.92 times, 1 times, 1.5 times, 2 times, 2.5 times, 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% to 60% of the total molecular weight of the hydrophobic segment, such as 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or any data within the range.

[0021] As specific examples, when the polymer carrier is a polymer micelle, the molecular weight of the hydrophilic segment is 1700–3000 Da; the molecular weight of the hydrophobic segment is 0.8–3 times that of the hydrophilic segment; and the molecular weight of PDTC is 30%–60% of the total molecular weight of the hydrophobic segment. When the polymer carrier is a polymer vesicle, the molecular weight of the hydrophilic segment is 3000–10000 Da; the molecular weight of the hydrophobic segment is 3–10 times that of the hydrophilic segment; and the molecular weight of PDTC is 10%–30% of the total molecular weight of the hydrophobic segment.

[0022] Addressing the current lack of effective treatments for large / unresectable GBM and its strongly immunosuppressive microenvironment, this invention constructs an injectable hydrogel platform (MIN-PPIC@iGel) integrating multiple key functions. This platform achieves a synergistic three-tiered therapeutic approach: localized photothermal ablation for tumor reduction, inhibition of residual GBM stem cells (GSCs), and activation of dendritic cell (DC)-mediated anti-tumor immunity. Cellular experiments show that the drug disclosed in this invention can synergistically kill GL261 cells, significantly inhibit tumor stem cells and malignant phenotypes, induce significant ICD, and further amplify the maturation and activation effects of DCs. In a mouse model of large-volume orthotopic GBM, a single intratumoral injection of MIN-PPIC@iGel combined with short-duration near-infrared light irradiation doubled the survival time. Further combination with CTLA-4 antibody therapy significantly inhibited stem cells, effectively mobilized the immune response in cervical lymph nodes and the tumor site, activated local and systemic anti-tumor immunity, induced strong immune memory, and achieved long-term tumor-free survival in 50% of mice. This invention provides a novel strategy for the local treatment of unresectable GBM. In this invention, the assembly material of the polymer carrier includes polymers, preferably assembled from polymers.

[0023] This invention discloses a combination drug comprising the above-mentioned photothermal synergistic tumor stem cell stemness inhibition system and other anti-tumor drugs, wherein the other anti-tumor drugs are existing products, such as monoclonal antibodies.

[0024] This invention discloses the application of the above-mentioned photothermal synergistic tumor stem cell stemness inhibition system or its combination with drugs in the preparation of antitumor drugs. Preferably, the tumor includes brain tumors, such as glioblastoma, as well as other inoperable solid tumors.

[0025] In this invention, the anti-tumor drug can be mainly composed of a locally injected tumor ablation synergistic immunotherapy system, or the above-mentioned locally injected tumor ablation synergistic immunotherapy system can be used together with other drugs as active ingredients, or the above-mentioned locally injected tumor ablation synergistic immunotherapy system can be used in combination with other treatment methods, such as radiotherapy and thermotherapy.

[0026] Existing technologies that improve the efficacy of first-line TMZ by enhancing the hypoxic microenvironment of the tumor site are largely ineffective in the treatment of large or unresectable GBM. To address this serious challenge, this invention utilizes an injectable hydrogel to design a local treatment regimen integrating three major functions: PTT ablation, GSC inhibition, and DC activation, termed MIN-PPIC@iGel. A single intratumoral injection delivers all three active components into the GBM, combined with a single near-infrared (NIR) irradiation, effectively treating large GBM. The regimen comprises: (i) micelles loaded with indocyanine green (ICG), (ii) continuously released napabucacin (Nap), and (iii) polymer vesicles loaded with poly(I:C) (PPIC). The synergistic action of these three mechanisms achieves NIR-triggered tumor photothermal ablation, achieving the cytoreductive effect of LITT, reducing the stemness and invasiveness of GSCs, amplifying antigen cross-presentation, and activating cytotoxic T lymphocytes (CTLs). More importantly, the constructed hyaluronic acid (HA)-based hydrogel possesses mechanical properties compatible with brain tissue and adhesion to tumor tissue. As a local drug reservoir, it enables phased, controlled drug release for treatment: combined with NIR, it initiates early photothermal effects to induce tumor cell ablation, thereby achieving sustained suppression of the glomerular sclerosis (GSC) population and immune initiation in cervical lymph nodes (CLNs). Experimental results show that in a mouse model bearing large orthotopic GL261 tumors, a single stereotactic injection of MIN-PPIC@iGel followed by short-term NIR irradiation significantly prolonged the survival of mice; further combination with αCTLA-4 significantly reduced the GSC population, alleviated T-cell myeloisolysis, and established durable immune memory, ultimately resulting in a significant extension of survival in tumor-bearing mice, with 50% of mice achieving tumor-free survival. In summary, this invention provides a novel, localized, programmed immunotherapy strategy for patients with large or unresectable GBM. Attached Figure Description

[0027] Figure 1 Characterization of the stability and photothermal properties of MIN. (A) Schematic diagram of MIN structure. (B) Particle size of MIN stored at different temperatures for 14 days; (C) Particle size after incubation in PBS containing 10% fetal bovine serum for 24 h; (D) Particle size change of MIN after NIR irradiation; (E) Temperature rise curves of MIN, MI, or free ICG under NIR irradiation (808 nm, 1 W / cm²). 2 (5 min).

[0028] Figure 2 Characterization of PPIC. (A) Schematic diagram of PPIC structure. Particle size distribution of PPIC after 30 days of storage at different temperatures (B) or 24 hours in the presence of 10% FBS (C).

[0029] Figure 3 Synthesis and characterization of hydrogel components. (A) Synthetic route, (B) FTIR, and (C) FTIR of HA-ADH and OHA. 1 1H NMR spectrum (400 MHz, D2O).

[0030] Figure 4 Characterization of the hydrogel's mechanical properties, injectability, self-healing properties, and adhesion. (A) Schematic diagram of the hydrogel's gelation process and (B) Schematic diagram of its composition. Rheological properties of the hydrogel: (C) Time-scan curves, (D) Temperature-scan curves, (E) Shear-thinning behavior, and (F) Cyclic strain scanning and macroscopic self-healing demonstration. (G) Demonstration of the hydrogel's tight adhesion to tissue. (H) Scanning electron microscopy image of the lyophilized hydrogel.

[0031] Figure 5 For cytotoxicity and hemolysis experiments. (A) Toxicity of simulated gel-shedded HA-ADH and OHA to neuronal HT22 cells (n = 6). (B) Hemolysis experiment of hydrogel and its components (n = 3).

[0032] Figure 6 The cumulative release curves of MIN and PPIC from the hydrogel in PB (pH 6.8) with and without HAase, and the linear fit of the release in the first 6 days (n = 3).

[0033] Figure 7Uptake and toxicity of MIN in GL261 cells. (A) Uptake of MIN after 2 h and 6 h of incubation with cells (n = 3). (B) Uptake after 48 h of incubation with MIN or IN (n = 6) and (C) Cell viability (808 nm, 1 W / cm²) with or without NIR irradiation after treatment with MIN, MI (ICG concentration: 0.5, 5 μg / mL; Nap concentration: 0.15 μg / mL). 2 (5 min, n=4).

[0034] Figure 8 (A) Representative FC plot and (B) quantitative analysis (n = 3) of the apoptosis rate of GL261 cells induced by MIN combined with NIR light irradiation 48 h after treatment (ICG: 5 μg / mL; Nap: 0.15 μg / mL; NIR: 808 nm, 1 W / cm²) 2 (5 min).

[0035] Figure 9 ICD was generated in GL261 cells (n = 3) induced by MIN combined with NIR incubation for 12 h. CRT + (A) Representative FC plot of cell proportions, (B) quantitative analysis, and (C) ATP concentration in the culture medium (ICG: 5 μg / mL; Nap: 0.15 μg / mL; NIR: 808 nm, 1 W / cm²). 2 (5 min).

[0036] Figure 10 To induce DC maturation, cytokine secretion, and T cell chemokine upregulation in MIN-PPIC / L therapy (n=3). (A) PPIC uptake by BMDCs. (B) Experimental procedure of Figure C1. (C) Ratio of CD80⁺CD86⁺ mDCs and (D) MHC-I expression. Concentrations of (E) TNF-α, (F) IL-12, and (G) IL-6 in BMDC culture medium. mRNA expression of (H) CCL5 and (I) CXCL10 in DCs (ICG: 5 μg / mL, Nap: 0.15 μg / mL; poly(I:C): 10 μg / mL; NIR: 808 nm, 1W / cm²). 2 (5 min).

[0037] Figure 11In vivo release of (CF-labeled) MIN and (Cy5-labeled) PPIC after intratumoral injection of MIN-PPIC@iGel into orthotopic GL261 tumors in mice. (A) Experimental procedure. (B) Ex vivo fluorescence imaging and (C) semi-quantitative analysis of MIN and PPIC in brain tissue and CLN (n = 4). (D) CLSM map of the distribution of released MIN (green) and PPIC (red) in brain tissue sections. (E) Compositional analysis of CD45⁺ immune cells in the tumor, and (F) analysis of uptake of MIN and (G) PPIC by different immune cell subsets (n = 3) (ICG: 0.05 mg / kg (125 μg / mL), Nap: 0.015 mg / kg (37 μg / mL); PPIC: 0.4 mg / kg (1 mg / mL); Cy5: 0.05 mg / kg; CF: 0.1 mg / kg; NIR: 808 nm, 1 W / cm²) 2 (5 min).

[0038] Figure 12 In vivo photothermal performance of MIN@iGel / L on day 7 of GBM-bearing tumor inoculation via a single injection (n=3). (A) Infrared thermographic images of tumors at different time points under NIR irradiation with different ICG concentrations of MIN@iGel; (B) Quantitative analysis of the highest temperature; (C) Changes in body weight (NIR: 808 nm, 1 W / cm²). 2 (5 min).

[0039] Figure 13 To investigate the efficacy of MIN-PPIC@iGel / L+ICB on large-volume orthotopic GL261 mice. (A) Experimental procedure. (B) Body weight change curve and (C) Survival curve of mice (n = 6).

[0040] Figure 14 To investigate the efficacy of different ICG doses of MIN-PPIC@iGel / L+ICB in orthotopic GL261 mice, with ICB and iGel+ICB groups as controls. Mouse (A) weight change and (B) survival curves (n = 6).

[0041] Figure 15 This study compares the efficacy of the MIN-PPIC@iGel / L+ICB strategy with GSC inhibition alone (MIN@iGel+ICB) and GSC inhibition combined with PTT (MIN@iGel / L+ICB) in large-volume orthotopic GL261 mice. (A) Body weight change and (B) survival curves of the mice (n = 6).

[0042] Figure 16MIN-PPIC@iGel / L+ICB treatment in orthotopic GBM mice suppressed T-cell myeloisolation (n = 5). The content of (A) T cells in total CD45 cells and (B) CD45 cells in the bone marrow... + T and (C)CD8 + The proportion of T cells.

[0043] Figure 17 Analysis of memory T cells in mice surviving MIN-PPIC@iGel / L+ICB treatment (day 70) (n = 3, healthy mice as controls). (A) Experimental procedure. (B) Content of CD8⁺T and CD4⁺T cells in the brain and spleen. (C) T CM and T EM The gate scheme. (D) T in the brain and spleen CM and T EM The proportion of cells.

[0044] Figure 18 Immunological analysis of orthotopic GBM mouse tumors treated with MIN-PPIC@iGel / L+ICB (n = 5). (A) Experimental procedure. (B) Proportion of mDCs and (C) MHC-I molecule expression levels in tumor tissue. (D) Proportion of CD69⁺CD8⁺T, (E) CD69⁺CD4⁺T, (F) IFNγ⁺CD8⁺T, (G) IFNγ⁺CD4⁺T, (H) Treg and (I) GSCs cells (ICG: 0.05 mg / kg, Nap: 0.015 mg / kg; PPIC: 0.4 mg / kg; L (NIR): 808 nm, 1 W / cm² 2 (5 min).

[0045] Figure 19 Immunological analysis of lymphoid organs in orthotopic GBM mice treated with the disease (n = 5). (A) mDCs, (B) CD8 in the CLN. + T、(C)CD69 + CD8 + T、(D)CD4 + T and (E)CD69 + CD4 + T cell content. (F) mDCs, (G) CD107a in the spleen. + NK、(H)IFNγ + CD8 + T and (I)IFNγ + CD4 + The content of T cells and the (J)M2 / M1 ratio.

[0046] Figure 20MIN-PPIC@iGel / L+ICB can reshape the immunosuppressive state of the tumor microenvironment and construct a long-term immune regulation system.

[0047] Figure 21 Press MIN-PPIC@iGel / L+ICB Figure 3 H&E staining of major organs in mice that survived 70 days after treatment with regimen .22A and healthy mice (scale bar: 100 μm).

[0048] Figure 22 This is a schematic diagram of a local treatment strategy for large-volume GBM based on a single intratumoral injection of a hydrogel encapsulating co-loaded ICG and Nap micelles (MIN) and poly(I:C) vesicles (PPIC) combined with a single NIR irradiation. Detailed Implementation

[0049] Despite the challenges in effectively treating GBM, multiple obstacles remain, including BBB-restricted drug entry into the brain, hypoxia and an immunosuppressive microenvironment weakening treatment response, and GSCs-driven relapse and drug resistance. This is particularly true for large-volume or invasive, unresectable GBM, where current treatment options are extremely limited. Although innovative strategies such as targeted therapy, immunotherapy, and oncolytic viruses are emerging, and nanodelivery systems offer new tools to overcome these obstacles, the current 5-year survival rate remains below 10%. Existing treatments for GBM are mostly effective for small tumors, administered within hours to three days after tumor cell injection, achieving some efficacy. However, they are largely ineffective for large-volume or unresectable GBM. Photothermal therapy (PTT) converts near-infrared light into heat at the tumor site, achieving a cell-reducing effect similar to surgical resection. However, this method cannot effectively eliminate glioma stem cell-like cells (GSCs-like cells) remaining in the cooler peritumoral region and struggles to reverse the "cold" tumor microenvironment and stimulate a systemic immune response, ultimately leading to inevitable tumor recurrence. Other local treatment strategies for the brain, such as rigid chemotherapy implants (Gliadel chips), are often limited by problems such as incompatibility with the mechanical properties of brain tissue, limited diffusion range, and poor applicability to non-cavitary solid lesions.

[0050] Therefore, developing novel treatment strategies that combine efficient delivery, microenvironment regulation, and multi-mechanism synergistic effects to address key obstacles in GBM treatment is of significant scientific importance and practical value.

[0051] This invention constructs an injectable hydrogel platform (MIN-PPIC@iGel) integrating multiple key functions to achieve a synergistic therapeutic modality involving three levels of linkage: photothermal ablation for local tumor reduction, inhibition of residual GBM stem cells (GSCs), and activation of dendritic cell (DC)-mediated anti-tumor immunity. As an example, the photothermal synergistic tumor stem cell stemness inhibition system of this invention includes a hydrogel carrier and polymer micelles co-loaded with a photothermal agent and a tumor stem cell inhibitor, as well as polymer vesicles loaded with an immune adjuvant loaded on the hydrogel carrier. The preparation method involves loading the hydrogel with co-loaded photothermal agent and tumor stem cell inhibitor polymer micelles, as well as polymer vesicles loaded with an immune adjuvant, to obtain the photothermal synergistic tumor stem cell stemness inhibition system. The polymer includes one or more of the hydrophilic segment -P(A-DTC) and the hydrophilic segment -P(B-DTC)-cationic fragment; wherein A is a cyclic ester or cyclic carbonate monomer unit, and B is a cyclic ester or cyclic carbonate monomer unit; A and B can be the same or different.

[0052] The following specific experiments illustrate the technological advancements of this invention. The raw materials used are existing products, and the specific preparation operations and performance tests are conventional techniques. Napabucacin (Nap), indocyanine green (ICG), polyethylene glycol 350 (PEG350), sodium hyaluronate (HA, 953 kDa, Bloomage Biotech), polyinosinic acid (poly(I:C), 0.2-1 kb), sodium periodate (NaIO4), adipic acid dihydrazide (ADH), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), N-hydroxysuccinimide (NHS), and 5,6-hydroxyfluorescein (CF) were purchased and used directly. Polyethylene glycol-poly(caprolactone-dithiopentane-trimethylene carbonate) (PEG-P(CL-DTC), number average molecular weight 2-0.8-1.1 kg / mol), polyethylene glycol-poly(trimethylene carbonate-dithiopentane-trimethylene carbonate)-spermine (PEG-P(TMC-DTC)-Spe, number average molecular weight 5.0-(13.2-1.8)-0.2 kg / mol), and Cy5-labeled PEG-P(TMC-DTC) were all synthesized according to previously reported methods and are existing technologies.

[0053] 7-8 week old female C57BL / 6 mice, GL261 mouse GBM cells, and mouse neuronal cell line HT22 are existing products. Bone marrow-derived dendritic cells (BMDCs) were prepared from the femur of C57BL / 6 mice. The specific operation was carried out using standard techniques, and the cells were cultured in DMEM medium containing 10% FBS and 1% penicillin-dextrose antibodies. All animal experiments of this invention were approved by the Experimental Animal Ethics Committee of Soochow University, and the experimental process strictly followed the relevant provisions of the "Guidelines for the Care and Use of Experimental Animals".

[0054] The proton nuclear magnetic resonance spectrum of polymers (NMR)1 H NMR was measured using an AVANCEIII HD-400 400 MHz NMR spectrometer (Bruker), with deuterated water (D2O) and CDCl3 as solvents. The residual solvent signals H2O (δ 4.80 ppm) and CHCl3 (δ 7.26 ppm) were used as calibration chemical shifts. Fourier transform infrared spectra of the polymer were acquired using an IRTracer-100 infrared spectrometer (SHIMADZU). An 808 nm near-infrared laser was purchased from Ningbo Fengke Optoelectronics Co., Ltd. Changes in solution and cell temperature induced by the ICG nanoparticles combined with NIR were monitored using a thermocouple thermometer (TASI). In vivo thermal imaging was acquired using a handheld infrared thermal imager (HIKMICRO). Enzyme-linked immunosorbent assay (ELISA) and ATP chemiluminescence assay were performed using a Multiskan FC microplate reader (Thermo). The hydrogels were lyophilized using an Alpha 1-2 LDplus freeze dryer (CHRIST), and their microstructure was observed using a Regulus 8230 scanning electron microscope (Hitachi). Rheological properties were determined using a HAAKEMARS40 rheometer (Thermo Scientific). The absorption spectra of the ICG formulation were obtained using a UH-5300 double-beam UV-Vis spectrophotometer (Hitachi). The emission spectra of Cy5-PPIC were measured using a Cary Eclipse G9800A fluorescence spectrophotometer (Agilent). Tissue sections were photographed using a confocal laser scanning microscope (CLSM, TCS SP5, Leica).

[0055] An orthotopic GL261 tumor mouse model was established using conventional methods. 5 × 10 4 One GL261 cell was suspended in 5 μL of cold PBS containing 25 vol% matrix gel and slowly injected into the left striatum of 6-8 week old female C57BL / 6J mice using a stereotaxic instrument (injection site: origin at the anterior fontanelle, 1.9 mm to the left, 0.5 mm superior, and 3.1 mm deep). The injection time was 3 min, and the cells were left in place for 5 min. The in situ GBM model was successfully established. The tumor progressed rapidly, and the mice experienced rapid weight loss around days 16-20. The mice died within 3-5 days of the onset of weight loss. The treatment effect was assessed based on the mice's weight.

[0056] All data in this invention are expressed as mean ± standard deviation and analyzed using GraphPad Prism 9.5 and Origin 2021 software. Significant differences between two groups were assessed using t-tests, and significant differences between three or more groups were assessed using one-way ANOVA combined with Tukey's multiple comparison test. Mouse survival curves were plotted using the Kaplan-Meier method, and differences between groups were compared using the log-rank test. Statistical significance was set as: * p <0.05,** p <0.01, *** p <0.001, **** p <0.0001.

[0057] Example 1: Preparation and Characterization of the Injectable Hydrogel Platform (MIN-PPIC@iGel) 1. Preparation and characterization of MIN The preparation of polymer micelles co-loaded with ICG and Nap (referred to as MIN) is as follows: ICG and Nap are mixed in a 1:1 molar ratio and added to a PEG-P (CL-DTC) polymer solution (using PEG350 as solvent, polymer concentration 200 mg / mL); then the mixture is added dropwise to PB buffer (pH 7.4, 10 mM) under stirring at 37°C, and the core-crosslinked micelle drug spontaneously forms.

[0058] Cy5-labeled MIN was prepared by incorporating 0.5 wt.% of a Cy5-labeled polymer into a polymer and then using the same method.

[0059] Following the method described above, micelles containing only ICG (referred to as MI) and a mixture of free ICG and Nap (referred to as IN) were used as controls.

[0060] The particle size and PDI of the nanomedicine were determined using DLS. To assess its stability, the particle size of the samples was monitored after storage at 37℃, 4℃, and -20℃ for 14 days, and after incubation at 37℃ with 10% serum for 1 day. The photothermal properties of different ICG formulations (ICG, MI, MIN) and different MIN concentrations (ICG concentrations: 5, 10, 20 μg / mL) were measured in 96-well plates using a NIR laser (808 nm, 1 W / cm²). 2 Irradiate for 5 minutes, and record temperature changes every 30 seconds.

[0061] PEG-P (CL-DTC), photosensitizer ICG, and stem cell inhibitor Nap can be readily self-assembled in aqueous phase to form micelles co-loaded with ICG and Nap, called MIN, as shown in the schematic diagram below. Figure 1As shown in Figure A. DLS test results show that the particle size is approximately 25 nm (PDI < 0.1), and the reproducibility is excellent. The particle size after 14 days of storage at 37, 4, and -20°C and after 24 hours in PB containing 10% FBS is basically the same as that of the freshly prepared particles. Figure 1 (B, C), indicating that MIN exhibits good storage and serum stability. After NIR irradiation (808 nm, 1 W / cm²), the serum stability was further improved. 2 (Unless otherwise specified, this illumination condition is used for 5 min), the temperature is raised to 55℃ and maintained for 3 min, and the particle size remains unchanged at MIN. Figure 1 (D) indicates that MIN did not aggregate or dissociate after exerting its PTT effect, and could still deliver Nap to cells. Interestingly, the temperature generated by MIN micelles under NIR light was significantly higher than that of free ICG ( Figure 1 E). Furthermore, the warming effect of MIN induced by NIR light is ICG concentration-dependent (E). Figure 1 F).

[0062] 2. Preparation and characterization of PPIC The specific preparation steps of poly(I:C)-loaded polymer vesicles (PPIC) are as follows: Take 100 µL of PEG-P(TMC-DTC)-sp polymer DMF solution (50 mg / mL) and add it to 900 µL of HEPES buffer (pH 6.8, 5 mM) containing poly(I:C) with stirring. The amount of poly(I:C) added is 10 wt% of the polymer mass. After the addition is complete, stir as usual, and then dialyze the solution sequentially in HEPES pH 6.8, a mixture of HEPES pH 6.8 and pH 7.4 (v / v = 1 / 1), and HEPES pH 7.4 using a 1000 kDa dialysis bag to obtain the product. The particle size, particle size distribution, and stability evaluation methods of PPIC are the same as those of MIN.

[0063] The polymeric vesicles (PPICs) loaded with poly(I:C) are synthesized from the polymer PEG-P(TMC-DTC)-Spe via a two-step reaction, which is a current technology: First, PEG-P(TMC-DTC) is obtained by active ring-opening polymerization of trimethylene carbonate (TMC) and DTC; then, the terminal hydroxyl groups are activated and reacted with the amino groups of spermine. PPICs are prepared by self-assembly of PEG-P(TMC-DTC)-Spe and poly(I:C) in aqueous solution. The resulting polymeric vesicles have a reduction-sensitive disulfide-crosslinked vesicle membrane. Poly(I:C), due to its negative charge, is incorporated into the vesicle interior by combining with the spermine in the vesicle shell. Figure 2A). DLS test results showed that PPIC exhibited a uniform particle size distribution, with an average hydrated particle size of approximately 55 nm and a PDI < 0.1, demonstrating excellent storage stability and serum stability. Figure 2 B, C).

[0064] 3. Preparation and characterization of HA-ADH and OHA This invention synthesizes two functionally modified hyaluronic acid (HA) compounds for preparing injectable hydrogels with mechanical properties matching those of brain tissue, enabling rapid photothermal ablation and long-term immune activation at the site of brain tumors. One of the hydrogel raw materials is adipic acid dihydrazide-modified hyaluronic acid (HA-ADH). Its synthesis involves dissolving HA (1.0 g, -COOH equivalent: 2.64 mmol) in deionized water (100 mL, pH 5.4), adding EDC (1.0 g, 5.28 mmol) and NHS (0.75 g, 5.28 mmol), and activating the mixture by stirring at 1000 rpm for 2 h. Subsequently, ADH (2.529 g, 14.5 mmol) is added, and the reaction is stirred at room temperature for 24 h. The product is dialyzed against deionized water (MWCO 3500 Da) for 3 days, and then freeze-dried to obtain HA-ADH. 1 The structure was characterized by 1H NMR and FTIR. Another raw material for forming the hydrogel was oxidized hyaluronic acid (OHA), which was synthesized by dissolving HA (1 g, -COOH equivalent: 2.64 mmol) in deionized water (80 mL, pH 5.4), adding NaIO4 (0.65 g, 3 mmol), stirring at 1000 rpm for 24 h at room temperature in the dark, and finally adding 1 mL of ethylene glycol and stirring for 1 h to terminate the reaction. The product was dialyzed against deionized water in the dark (MWCO 3500 Da) for 3 days and then freeze-dried to obtain OHA.

[0065] This invention designs a hydrogel based on modified hyaluronic acid (HA) crosslinked with a Schiff base for encapsulating two nanomedicines to form a drug reservoir. The hydrazide-modified polymer HA-ADH is obtained by activating the HA carboxyl group with EDC and NHS, followed by coupling with adipic acid dihydrazide (ADH); oxidized HA (OHA) is obtained by partially oxidizing and ring-opening HA with sodium periodate. Figure 3 A). FTIR test results show that HA-ADH is effective at 1600-1550 cm⁻¹. -1 The characteristic peaks of amide-CO-NH- stretching vibrations are present at 1750-1700 cm⁻¹. -1 The presence of characteristic peaks for the C=O bending vibration of aldehyde groups confirms the structures of the two polymers; however, the hydrogel obtained after mixing not only shows the disappearance of characteristic peaks for -CO-NH- and C=O, but also shows peaks in the 1690-650 cm⁻¹ region. -1The presence of a characteristic peak of imine C=N bending vibration at this point proves that a Schiff base bond has formed in the hydrogel. Figure 3 B). 1 The 1H NMR spectrum showed characteristic peaks for HA-ADH and OHA, further confirming its successful synthesis. Figure 3 C). Furthermore, by comparing HA... 1 The degree of substitution of ADH in HA-ADH can be calculated by integrating the methyl peak of N-acetylglucosamine (δ 2.0 ppm) and the α-methylene group adjacent to the carbonyl group in the coupled ADH (δ 1.6 ppm). Similarly, by comparing the integral sum of the three characteristic peaks of N-acetylglucosamine in HA (δ 2.0 ppm) and the three characteristic peaks of OHA (δ 5.0-5.2 ppm), the oxidation degree of OHA can be calculated to be 11%.

[0066] 4. Preparation and characterization of hydrogels A hydrogel based on the Schiff base reaction can be rapidly formed by mixing an aqueous solution of HA-ADH with OHA. Figure 4 A). Take 80 μL of HA-ADH aqueous solution (20 mg / mL), mix it with MIN solution and PPIC solution, then add PBS to make up the total volume to 280 μL. Add 40 μL of OHA (40 mg / mL) under normal vortex stirring and mix well. Let it stand to form a gel (final HA concentration is 10 mg / mL), obtaining a hydrogel containing MIN and PPIC (MIN-PPIC@iGel). Following the above method, omit the MIN and PPIC solutions, use only the MIN solution or the PPIC solution, and obtain a blank hydrogel (iGel), a hydrogel containing MIN (MIN@iGel), and a hydrogel containing PPIC (PPIC@iGel), respectively. Figure 4 B).

[0067] The rheological properties of the hydrogel were measured at 37℃ using a rheometer (equipped with a 25 mm parallel plate). The storage modulus of the hydrogel was concentration-dependent, with storage moduli of 120, 440, and 760 Pa corresponding to HA concentrations of 5, 10, and 20 mg / mL, respectively. The storage modulus of 10 mg / mL HA (i.e., 1% iGel) was similar to that of mouse brain tissue; therefore, hydrogels prepared at this concentration were selected for subsequent experiments. Furthermore, the modulus of MIN-PPIC@iGel obtained by incorporating MIN and PPIC into the 1% iGel hydrogel was similar to that of iGel, indicating that the addition of MIN and PPIC did not significantly affect the mechanical properties of the hydrogel. Figure 4 C). The hydrogel maintains stable mechanical properties within the temperature range of 25-58℃. Figure 4D). In addition, the hydrogel exhibits significant shear-thinning properties ( Figure 4 E) provides a rheological basis for its in-situ injection, and the hydrogel can recover to its initial mechanical properties after injection, demonstrating good self-healing ability. Figure 4 F), the hydrogel exhibits significant adhesion to brain tissue ( Figure 4 G). SEM images show that the hydrogel has a well-connected three-dimensional porous structure with pore sizes of approximately 70 μm, which is beneficial for the loading and release of nanomedicines. Figure 4 H).

[0068] 5. Safety assessment To evaluate the potential cytotoxicity of the hydrogel components to normal neurons, HT22 cells were seeded in 96-well plates (1×10³ / well, 90 μL) and cultured overnight. After 48 h of incubation with OHA and HA-ADH (10 μL, 10 mg / mL), cell viability was assessed using the CCK-8 assay (n = 6). After incubation of HT22 neurons with HA-ADH and OHA at a concentration of 10 mg / mL for 48 h, CCK-8 assay results showed that cell viability was close to 100%. Figure 5 A). The hemolysis test was used to detect the blood compatibility of each component of MIN-PPIC@iGel. The results of the hemolysis test showed that each component of MIN-PPIC@iGel did not cause extensive red blood cell damage and did not cause hemolysis. Figure 5 (B) showed excellent blood compatibility. The results indicate that MIN-PPIC@iGel can serve as a safe local injection drug reservoir, enhancing local thermal ablation while maintaining good tissue safety.

[0069] Example 2: In vitro release and cell experiments of MIN-PPIC@iGel A hydrogel, MIN-PPIC@iGel (1 mL, Cy5 concentration: 5 µg / mL, ICG concentration: 125 µg / mL, HA concentration: 10 mg / mL), containing Cy5-PPIC and MIN, was prepared in 5 mL centrifuge tubes. 0.5 mL of release medium, namely PB (pH 6.8, 10 mM) and PB containing 50 U / mL hyaluronidase (n = 3), was added, and the mixture was incubated at 37°C in a shaker. At preset time points (days 0.5, 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, and 20), the entire release solution was collected after centrifugation (100×g, 3 min), and 0.5 mL of the corresponding fresh medium was added. The concentrations of MIN and Cy5-PPIC in the collected release solutions were determined using UV-Vis and fluorescence spectroscopy, respectively. The release kinetics of two nanomedicines in MIN-PPIC@iGel were investigated in PB (pH 6.8, mimicking the pH of tumor tissue) and PB containing hyaluronidase (HAase) (pH 6.8): HAase degrades HA, resulting in faster release of MIN and PPIC, with nearly 100% release after 18 days. Figure 6 Magnified analysis of the release of MIN and PPIC from the hydrogel within the first 6 days revealed that the release of both drugs was linear with time, approaching zero-order kinetics, primarily due to hydrogel degradation. The release rate in the HAase-containing group was significantly higher than in the enzyme-free medium. Notably, in both cases, the release rate of MIN was significantly faster than that of PPIC, indicating a faster diffusion rate within the hydrogel. Therefore, this time-sequential release characteristic of the two nanomedicines, MIN and PPIC, in MIN-PPIC@iGel ensures a favorable time window for the coordinated action of each component.

[0070] The following examples illustrate the role of the two nanomedicines, MIN and PPIC, in MIN-PPIC@iGel.

[0071] Cells were seeded in 24-well plates (5 × 10⁴ / well), and Cy5-MIN (Cy5 concentration 0.1 μg / mL, n=3) was added and incubated for 2 h or 6 h. Cells were then collected, washed with PBS, resuspended, and fluorescence intensity was detected using FC. Analysis was performed using FlowJo V10 software. Flow cytometry results showed that a significant fluorescence signal was detected after 2 h of incubation with Cy5-MIN. After 6 h, the fluorescence intensity continued to increase, but the trend slowed down, indicating that MIN could be rapidly internalized by GL261 cells. Figure 7 A).

[0072] The cytotoxicity of MIN and IN to GL261 cells was assessed using the CCK-8 assay. GL261 cells were seeded in 96-well plates (90 μL, 5 × 10³ / well) and cultured overnight. 10 μL of MIN or IN (ICG:Nap = 5:0.15; Nap concentration: 0.01–1 μg / mL) was added. After incubation for 48 h, 10 μL of CCK-8 reagent was added, and the cells were incubated at 37°C for 0.5 h. Cell viability was calculated by measuring absorbance at 450 nm using a microplate reader (n=6). To investigate the cytotoxic effect of MIN combined with NIR irradiation on GL261 cells, 10 μL of MI or MIN (ICG concentration: 0.5, 5 μg / mL; Nap concentration: 0.15 μg / mL) was added to cells seeded in 96-well plates. The plates were then placed on a heating plate to maintain the temperature at 37℃ before light irradiation, followed by NIR irradiation (808 nm, 1 W / cm², 5 min). After incubation for 48 h, cell viability was assessed using the CCK-8 assay (n=4). The cytotoxic effect of MIN on GL261 cells was evaluated using the CCK-8 assay. The results showed that MIN exhibited significantly higher cytotoxicity than the free drug mixture (IN), with a half-maximal inhibitory concentration (IC50) of 0.37 μg / mL, approximately 2.5 times lower than the IN group (0.91 μg / mL). Figure 7 B). Notably, MIN combined with NIR laser irradiation (MIN+L) exhibited a significant chemo-photothermal synergistic effect, and the cell death rate induced by MIN+L was significantly higher than that of photothermal therapy alone (MI+L) and chemotherapy alone (MIN). Figure 7 (C) The comparison results showed that both concentrations of Nap could synergistically kill residual cancer cells after photothermal treatment.

[0073] Assay for MIN-induced apoptosis and immunogenic cell death in GL261 cells. GL261 cells were seeded in 24-well plates (5 × 10⁴ / well) and cultured overnight. Cells were then divided into 8 groups: PBS, MI, IN, MIN, PBS+L, MI+L, IN+L, and MIN+L (ICG concentration: 5 μg / mL, Nap concentration: 0.15 μg / mL, n = 3). After adding the appropriate reagents, the wells were placed on a heated platform to maintain an initial temperature of 37°C and irradiated with NIR. After 48 h of further culture, the cells were digested with Accutase, centrifuged (1000 rpm, 3 min), washed with PBS, and stained with Annexin V-APC / 7-AAD according to the manufacturer's instructions. The apoptosis rate was detected by flow cytometry. The results showed that compared with the PBS, MI, or IN groups, MIN treatment induced a significantly greater apoptosis in GL261 cells (**** p, Figure 8In particular, when combined with NIR irradiation, MIN+L-induced apoptosis was not only nearly 3 times greater than the MIN group, but also approximately 4 times greater than the MI+L and IN+L groups. In contrast, there was no significant difference in the MI+L and IN+L groups with or without NIR irradiation. Flow cytometry analysis showed ( Figure 9 (A, B) Without combined NIR treatment, MIN treatment caused almost no CRT exposure in cells, while combined NIR irradiation significantly increased the CRT exposure level on the surface of GL261 cells in all three groups: approximately 7-fold in the MI+L group, and approximately 6-fold in the IN+L and MIN+L groups. In particular, the proportion of CRT+ cells in the MIN+L group was twice that of the MI+L group and 1.5 times that of the IN+L group. The results of soluble ATP release showed a similar trend. Figure 9 In group C, the ATP content in the cell culture medium of the MIN+L group was significantly higher than that of the other groups.

[0074] Uptake and activation of PPIC by BMDCs. BMDCs were seeded in 12-well plates (5 × 10⁵ / well), and 100 μL of Cy5-PPIC (Cy5 concentration: 0.1 μg / mL) was added. After incubation for 2 h and 6 h, cells were collected and washed, stained with FITC-anti-CD11c antibody, and the fluorescence intensity of Cy5 in CD11c-positive cells was detected by flow cytometry (n = 3). PPICs were efficiently internalized by BMDCs within 2 h, and the amount of internalization further increased after 6 h. Figure 10 A). A Transwell chamber immune activation model was conventionally constructed: GL261 cells were seeded in the upper chamber and treated with PBS, MIN, PPIC, and MIN-PPIC (some groups were irradiated with NIR, denoted as / L). BMDCs were then added to the lower chamber to study the activation of DCs after photothermal immunotherapy. Figure 10 B). The results showed that the proportion of CD80+CD86+ mature DCs (mDCs) in the MIN / L group was significantly higher than that in the PBS and MIN control groups (**** p). The PBS combined with NIR group had almost no activating effect. Unexpectedly, the proportion of mDCs in the MIN-PPIC / L group was further increased to 30% compared with the MIN / L group, which reflects the synergistic effect of TAA and poly(I:C) on DC maturation. Figure 10 C). Notably, the expression level of MHC-I molecules on the surface of DCs in the MIN-PPIC / L group was significantly increased, reaching approximately 2.4 times that of the MIN / L group. Figure 10(D) This means that the drug system of the present invention helps to amplify the ability of DC antigen presentation and facilitates further T cell activation. Meanwhile, the secretion levels of anti-tumor inflammatory cytokines such as TNF-α, IL-12, and IL-6 in the MIN-PPIC / L group culture medium were the highest among all groups. Figure 10 EG).

[0075] Furthermore, compared with the MIN / L group, MIN-PPIC / L significantly promoted the expression of chemokine CCL5 (** p) and CXCL10 (*** p) mRNA secreted by DCs. Figure 10 H, I), which helps recruit CD8+ T cells, Th1 cells, and NK cells to the GBM site, is beneficial for alleviating the highly immunosuppressive TME of GBM. These results reveal that the drug system of this invention has the characteristics of an in situ vaccine, with a dual regulatory mechanism: on the one hand, it enhances the recruitment capacity of immune cells by promoting the transcription of key chemokines (CXCL10, CCL5); on the other hand, it remodels the mature phenotype by inducing high expression of MHC-I molecules and co-stimulatory molecules (CD80, CD86) on the surface of DCs, thereby enhancing the cross-presentation efficiency of tumor antigens. In summary, this invention remodels the immune-cold TME into an inflammatory state with T cell infiltration, completing the function of amplifying antigen cross-presentation and activating cytotoxic T lymphocytes in the third stage of this local treatment regimen.

[0076] Example 3: Release of nanomedicines from MIN-PPIC@iGel in in situ GBM tumors The in vivo release behavior and distribution of nanomedicines were investigated using carboxyfluorescein (CF)-labeled MIN (CF-MIN) and Cy5-PPIC-embedded hydrogel MIN-PPIC@iGel. An orthotopic GBM mouse model was established. On day 7 post-tumor inoculation, 8 µL of MIN-PPIC@iGel (containing ICG concentration: 125 µg / mL, Nap concentration: 37 µg / mL; PPIC concentration: 1 mg / mL) was injected into the tumor using a brain-guided localization device. 5–10 min post-injection, the tumor was irradiated with NIR (808 nm, 1 W / cm², 5 min, n = 3). Mice were sacrificed at 4 h, 12 h, 2 d, and 5 d, and in vitro fluorescence imaging and semi-quantitative analysis of the heart, liver, spleen, lung, kidney, brain, and cervical lymph nodes were collected. Brain tissue was then fixed with 4% paraformaldehyde, frozen sectioned, stained with DAPI, and the distribution of nanomedicines in the tumor tissue was observed using CLSM. The uptake of CF-MIN and Cy5-PPIC by various cells in the tumor microenvironment was measured by flow cytometry (n = 3).

[0077] The above research results have confirmed that MIN-PPIC@iGel has good biocompatibility and the ability to release two nanomedicines in a time sequence. Figure 11 A shows the in vivo release and intracellular distribution of hydrogels encapsulating CF-MIN and Cy5-PPIC in an orthotopic GBM mouse model. In vitro imaging analysis using IVIS revealed that MIN-PPIC@iGel could store and continuously release the drug for more than 5 days. The signal decay of CF-MIN was faster than that of Cy5-PPIC, confirming that the release rate of MIN from tumors in vivo was faster than that of PPIC. This conclusion was also reached from brain tissue sections. Figure 11 (BD), this release timing is unexpected by those skilled in the art, and is particularly beneficial for the synergistic effect.

[0078] It is noteworthy that, over time, the fluorescence signal of Cy5-PPIC becomes increasingly enriched at the CLN ( Figure 11 B, C) help mobilize the body's anti-tumor immune response and enhance the anti-tumor effect. Flow cytometry analysis of single-cell suspensions showed that Cy5-PPICs were first taken up by tumor-resident immune cells and migrated to CLNs via DC-mediated migration (Cy5-PPICs). Figure 11 G). Furthermore, the number of neutrophils in brain tissue showed a trend of first increasing and then decreasing after treatment. Figure 11 E) indicates that the acute inflammatory response triggered by the treatment is manageable and will not lead to persistent tissue damage.

[0079] The above results confirm the feasibility of MIN-PPIC@iGel as a drug reservoir for local injection. By releasing different therapeutic components in a sequential manner, it is expected to coordinate the treatment process, enabling immune cells to take up PPIC and migrate to the CLN to mobilize the peripheral immune response, thereby improving the GBM immunosuppressive TME and achieving highly efficient anti-tumor immunity.

[0080] Example 4: Photothermal properties of MIN@iGel+NIR in mice An orthotopic GBM mouse model was constructed. On day 7 after GL261 cell inoculation, 8 µL of MIN@iGel hydrogel (ICG concentrations of 75, 110, and 125 μg / mL) was injected intratumorally using a stereotaxic device, with the needle left in place for 5 min. 5–10 min after injection, the tumor was irradiated with NIR, and the tumor temperature was monitored using an infrared thermal imager (n = 3). Mouse body weight was recorded every two days for 20 consecutive days until it returned to baseline. To assess changes in tumor tissue after photothermal therapy, in a separate experiment, mice injected intratumorally with MIN@iGel (125 μg / mL) and irradiated with NIR were sacrificed at 0.5, 4, 12, and 48 h. Brain tissue was collected, sectioned, stained with H&E, and observed under a microscope. A single stereotactic intratumoral injection of 8 μL of MIN@iGel followed by NIR irradiation (808 nm, 1 W / cm², 5 min) was recorded as MIN@iGel / L, and changes in tumor temperature and body weight were recorded. The results showed that when the ICG concentration in the hydrogel was 75, 100, and 125 μg / mL (equivalent to 0.03, 0.04, and 0.05 mg / kg), the temperature at the tumor site rapidly reached a plateau of approximately 49, 53, and 58 °C within 2 minutes. Figure 12 A, B) Figure 12 C represents the change in body weight of the mice treated with this procedure.

[0081] Example 5: Therapeutic Experiment of MIN-PPIC@iGel in Mice Bearing Large-Volume In Situ GL261 Tumors On day 7 after GL261 tumor inoculation, 8 µL of MIN-PPIC@iGel was injected intratumorally, containing ICG at a concentration of 125 µg / mL (0.05 mg / kg), Nap at a concentration of 37 µg / mL (0.015 mg / kg), and poly(I:C) at a concentration of 1 mg / mL (0.25 mg / kg). Five minutes after injection, the tumor was irradiated with NIR. In the combined immune checkpoint blockade therapy (ICB) group, anti-CTLA-4 antibody (1 mg / kg) was injected via tail vein on days 8, 11, and 14 (n = 6). Mouse status, body weight, and survival curves were monitored. The following control experiments were conducted simultaneously: PBS group, MIN-PPIC@iGel+ICB group and MIN and PPIC mixed with NIR plus ICB group (MIN-PPIC / L+ICB); ICB group, iGel+ICB group and MIN-PPIC@iGel / L(110)+ICB group were set up; hydrogel containing only anti-stem cell effect was set up (MIN@iGel+ICB) and photothermal and anti-stem cell effect combined group (MIN@iGel / L+ICB).

[0082] Three mice that survived the above treatment were euthanized on day 70 (n = 3). Brain and spleen tissues were collected to prepare single-cell suspensions. The proportions of effector memory T cells (TEM, CD44+CD62L-) and central memory T cells (TCM, CD44+CD62L+) subsets were determined by flow cytometry.

[0083] In the treatment simulation of an unresectable, large-volume GL261 tumor model in situ ( Figure 13 In A), a single injection of MIN-PPIC@iGel (ICG dose of 0.05 mg / kg) combined with NIR irradiation (MIN-PPIC@iGel / L) significantly improved efficacy, more than doubling the median survival (MST) (MST: 46 days, PBS group MST: 19.5 days). When combined with systemic anti-CTLA-4 immune checkpoint blockade therapy (MIN-PPIC@iGel / L + ICB), the therapeutic effect was further enhanced, increasing the MST to 64.5 days and achieving tumor-free cure in 50% of the animals. Both groups were well-tolerated, and mouse body weight remained stable within 40 days. Figure 13 (B, C, D). In contrast, the mixture of gel-free nanomedicines plus CTLA-4 treatment (MIN-PPIC / L+ICB) showed only weaker efficacy, especially with a significant decrease in mouse body weight on day 25.

[0084] Following the above administration strategy, the effects of ICG concentration (i.e., photothermal temperature) and the individual effects of the three components on the overall therapeutic effect were further investigated. The results confirmed that single ICB treatment and iGel+ICB treatment had negligible effects on prolonging MST in mice, and the mice showed a significant decrease in body weight. The MIN-PPIC@iGel / L (110 μg / mL)+ICB group resulted in a tumor local temperature of 53 degrees Celsius, indicating that it only prolonged the MST to 45 days in mice, with no cases of cure. Figure 14 A, B).

[0085] Following the above dosing strategy, for this large in situ brain tumor, GSC inhibition and ICB treatment alone (MIN@iGel+ICB) only slightly prolonged the MST (31.5 days) in mice; while anti-GSC treatment combined with NIR-induced photothermal and ICB treatment (MIN@iGel / L+ICB), although prolonging the MST to 39 days compared to MIN@iGel+ICB, did not achieve complete cure in mice, and the mice showed a significant decrease in body weight, indicating that it led to GBM cell escape and subsequent tumor recurrence. Figure 15(A, B). In contrast, while the treatments in the PPIC@iGel / L and PPIC@iGel / L+ICB groups prolonged the lifespan of mice to some extent (MST of 27 and 30 days respectively), no mice were cured. These results confirm that the MIN-PPIC@iGel / L+ICB regimen of this invention achieves highly efficient treatment and long-term control of large-volume orthotopic brain tumors by coordinating photothermal tumor reduction, GSC cell inhibition, and immune activation. Furthermore, observation of changes in the experimental group at different time points after treatment using H&E staining of brain tissue sections revealed that the extent of PTT damage was precisely limited to the tumor region. This was manifested in the gradual decrease in tumor cell density and the gradual loosening of tissue structure due to cell death as the time after PTT increased from 0.5 h to 48 h, while adjacent healthy brain parenchyma remained largely undamaged. These results indicate that this invention achieves a thermal ablation tumor reduction effect comparable to LITT, but its unique advantage lies in its ability to target residual GSC cells and simultaneously and powerfully reverse immunosuppressive TME through continuously acting bioactive components.

[0086] Example 6: Detection of immune response in large-volume orthotopic GL261 mice treated with MIN-PPIC@iGel On day 7 after tumor inoculation, 8 µL of MIN@iGel or MIN-PPIC@iGel was injected intratumorally, with ICG concentration of 125 µg / mL (0.05 mg / kg), Nap concentration of 37 µg / mL (0.015 mg / kg), and poly(I:C) concentration of 1 mg / mL (0.25 mg / kg). Five minutes after injection, the tumor was irradiated with NIR light. On days 8 and 11, anti-CTLA-4 antibody (1 mg / kg) was injected via the tail vein (n = 5). On day 13, mice were sacrificed, and cervical lymph nodes (CLNs), spleen, and brain tumor tissue were collected to prepare single-cell suspensions for flow cytometry analysis.

[0087] The bone marrow sequestration of T cells, a common phenomenon in GBM patients, is a significant reason for insufficient T cell infiltration at the tumor site and subsequent immunosuppression. Surprisingly, two days after treatment (day 13 post-vaccination), the MIN-PPIC@iGel / L+ICB treatment strategy reduced T cell retention in mouse bone marrow by more than two-fold compared to the PBS group, particularly reducing CD8+ T cells by three-fold, reaching levels comparable to healthy mice. Figure 16 In summary, the MIN-PPIC@iGel / L combined with ICB strategy not only improved the tumor immunosuppressive microenvironment in GBM mice, but also successfully activated the systemic innate and adaptive immune responses against GBM, reversing the bone marrow segregation phenomenon of T cells, thereby significantly inhibiting the progression of in situ GBM from an overall perspective.

[0088] Developing long-term anti-tumor immune memory is crucial for preventing tumor recurrence, and memory T cells play a key role in this process. In particular, GBM, unlike lung cancer and breast cancer, is often difficult to suppress; and surgically inoperable advanced GBM is even more challenging. Furthermore, existing techniques have shown that mice re-inoculated with LLC cells (lung cancer) or GBM cells do not develop LLC tumors, while those re-inoculated with GBM cells show slow tumor growth 10 days after inoculation. This invention detected and analyzed the changes in the content of immune memory T cells in the brain tissue and spleen of GBM mice cured and surviving after MIN-PPIC@iGel / L+ICB treatment compared to untreated healthy mice (naive). Figure 17 A). The results showed that the number of CD4+ T and CD8+ T cells in the brain tissue of cured mice increased significantly by 5-7 times, and the number of CD4+ T and CD8+ T cells in the spleen also increased by 1.5 times. Figure 17 B). Correspondingly, the number of central memory T cells (TCM) and effector memory T cells (TEM) in brain tissue also increased significantly by about 5 times, indicating the formation of local immune memory in brain tissue; the number of TCM in the spleen also increased by 1.5 times, indicating the formation of systemic immune memory. Figure 17 (C, D); these all effectively prevented tumor recurrence.

[0089] The tumor, spleen, and CLNs were examined and studied two days after the end of treatment (day 13 post-vaccination). Figure 18 A). Immunoassay of TME showed that the MIN-PPIC@iGel / L+ICB group significantly increased the proportion of mDCs compared with MIN@iGel (** p, Figure 18 B) and the expression level of its surface MHC-I molecules (**** p, Figure 18 C), at the same time, activated T cell subsets (CD69+CD8+ T cells and CD69+CD4+ T cells) significantly increased ( Figure 18 D, E), increased IFNγ+CD8+T and IFNγ+CD4+T cells ( Figure 18 (F, G) indicates that the treatment produced T cells with strong tumor-killing capabilities. IFNγ secreted by IFNγ+ T cells can also directly inhibit tumor proliferation and upregulate MHC-I molecule expression in tumor cells, making them more easily recognized by the immune system and activating macrophages. Simultaneously, the number of immunosuppressive T cells (Tregs) was significantly reduced. Figure 18 H). More importantly, the proportion of GSCs, the main culprit for tumor recurrence after treatment, was significantly reduced after MIN@iGel+ICB treatment, and MIN-PPIC@iGel / L+ICB further reduced it (H). Figure 18 (I) This demonstrates that the antigens generated by killing GSCs in the technical solution of this invention can be more effectively presented to T cells, thereby activating immune cells to kill residual GSCs. These changes collectively create a local microenvironment conducive to tumor clearance.

[0090] The lymphoid tissue nucleus (CLN) plays a crucial monitoring role in the brain's immune microenvironment and is the most critical draining lymphoid organ for GBM. Results showed that MIN-PPIC@iGel / L+ICB treatment in the CLN resulted in the most significant increases in mDCs, CD8+ T cells, and early activated CD69+ T cell subsets, far exceeding the effects of MIN@iGel+ICB (single stem cell inhibition, G2) and MIN@iGel / L+ICB (single photothermal therapy, G3). This confirms the effective migration, cross-activation, and T cell activation of tumor antigens from the primary site to the CLN. Figure 19 AE). At the systemic immune level, a similar phenomenon can be observed in the spleen: the proportions of mDCs, CD107a+NK cells, and IFNγ+T cells in the spleen of mice in the MIN-PPIC@iGel / L+ICB group were significantly increased compared to the control group. Figure 19 In addition, the proportion of M2 / M1 macrophages was significantly reduced (FI). Figure 19 J). The results confirmed that the technical solution of the present invention has the ability to mobilize the body's innate immunity, reduce the state of systemic immunosuppression, and successfully enable the anti-tumor immune response to spread from the local GBM tumor to the systemic immune organs.

[0091] These data demonstrate that MIN-PPIC@iGel / L+ICB treatment successfully transforms local ablation and GSC-like characterization into an in situ vaccine-like event, thereby establishing durable immune surveillance against glioblastoma (GBM) recurrence. Figure 20A significant challenge in GBM treatment is that tumors can evade immune surveillance through mechanisms such as immune checkpoint activation, myeloid-derived suppressor cells (MDSCs), and regulatory T cells (Tregs). Although thermal ablation can release tumor antigens, these antigens are often difficult to effectively recognize and utilize within the immunosuppressive tumor microenvironment (TME) of GBM. This invention, MIN-PPIC@iGel / L, fills this gap by promoting dendritic cell (DC) maturation, thereby enhancing the activation and presentation of tumor antigens. Research results show that MIN-PPIC@iGel / L+ICB can effectively transform tumor fragments generated by thermal ablation into a potent in situ tumor vaccine, manifested by elevated levels of pro-inflammatory cytokines (TNF-α, IL-12) and the induction of expression of immune cell recruitment-related chemokines such as CCL5 and CXCL10. It also drives the recruitment and infiltration of CD8⁺ T cells throughout the body and establishes long-term anti-GBM immune memory—an effect that is typically difficult to achieve with thermotherapy alone.

[0092] To investigate the safety of MIN-PPIC@iGel / L+ICB treatment, sections of the major organs (heart, liver, spleen, lung, and kidney) from the cured mice were collected and prepared for H&E staining analysis. The results showed that no significant damage was observed in the major organs of the mice treated with this strategy. Figure 21 The results, combined with the normal body weight of the mice, indicate that this combination therapy has good safety. In conclusion, the treatment strategy of MIN-PPIC@iGel / L combined with ICB can not only eliminate tumors and reduce tumor burden, improve immunosuppressive TME, and mobilize immune cells throughout the body, but also does not cause systemic toxic side effects, and has good potential for clinical application.

[0093] Therefore, the significance of this invention lies in the fact that it does not propose a completely new independent molecular mechanism, but rather establishes a technical solution specifically for GBM, especially by integrating functional modules into a clinically relevant treatment sequence and achieving functional synergy. For large-volume or unresectable in situ GBM, this invention designs a locally injectable hydrogel platform (MIN-PPIC@iGel) with spatiotemporally programmed drug release, which synergistically performs three major functions: photothermal tumor reduction, GSC inhibition, and sustained immune activation. Figure 22This approach improves drug bioavailability, induces more cell death and significant ICD, and significantly enhances DC activation and antigen presentation capabilities. In large-volume GBM tumors in situ, a single stereotactic injection of MIN-PPIC@iGel combined with short-duration NIR irradiation achieves surgical-like tumor cell reduction, while preventing recurrence and remodeling the immunosuppressive tumor microenvironment. When combined with anti-CTLA-4 immune checkpoint blockade, this strategy successfully transforms the immune-cold GBM microenvironment into a T-cell-rich inflammatory state, improves T-cell myeloisolation, establishes durable anti-tumor immune memory, and enables 50% of tumor-bearing mice to achieve tumor-free cure. Importantly, MIN-PPIC@iGel functions similarly to a flexible local interventional device, its administration method is compatible with existing neurosurgical workflows, and its components (ICG, Nap, poly(I:C), HA) all have promising clinical translation prospects. Therefore, this treatment strategy overcomes the limitations of simple physical ablation or systemic immunotherapy. In summary, the MIN-PPIC@iGel / L therapy designed in this invention achieves a temporal synergy of tumor reduction, glioma stem cell inhibition, and immune activation through material programming, providing a promising new strategy for patients with large-volume or unresectable GBM who currently have almost no treatment options.

Claims

1. A photothermal synergistic tumor stem cell stemness inhibition system, comprising a hydrogel and a photothermal agent, a tumor stem cell inhibitor, and an immune adjuvant loaded in the hydrogel.

2. The photothermal synergistic tumor stemness inhibition system according to claim 1, characterized in that, Photothermal agents, tumor stem cell inhibitors, and immune adjuvants are loaded onto hydrogels via polymer carriers; the polymer carriers include one or more of polymer micelles and polymer vesicles.

3. The photothermal synergistic tumor stem cell stemness inhibition system according to claim 2, characterized in that, Photothermal agents include one or more of metallic photothermal agents, inorganic non-metallic photothermal agents, and organic photothermal agents; immune adjuvants include one or more of TLR agonists, RLR agonists, and STING agonists; tumor stem cell inhibitors are tumor stem cell pathway inhibitors; the raw materials for preparing polymer carriers include polymers; the polymers include one or more of hydrophilic segment -P(A-DTC) and hydrophilic segment -P(B-DTC)-cationic fragments, wherein A is a cyclic ester or cyclic carbonate monomer unit, B is a cyclic ester or cyclic carbonate monomer unit, and A and B may be the same or different.

4. The photothermal synergistic tumor stem cell stemness inhibition system according to claim 3, characterized in that, The hydrophilic segment is polyethylene glycol, the cationic segment is spermine or a low molecular weight PEI, and DTC is dithiopentane trimethylene carbonate; the cyclic ester or cyclic carbonate monomer unit is selected from one or more of trimethylene carbonate monomer, lactide monomer, and caprolactone monomer.

5. The photothermal synergistic tumor stem cell stemness inhibition 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 0.5-20 times that of the hydrophilic segment; the molecular weight of PDTC is 5%-80% of the total molecular weight of the hydrophobic segment.

6. The preparation method of the photothermal synergistic tumor stem cell inhibition system according to claim 1 includes the following steps: loading a photothermal agent, a tumor stem cell inhibitor, and an immune adjuvant onto a hydrogel to obtain the photothermal synergistic tumor stem cell inhibition system.

7. The method for preparing the photothermal synergistic tumor stem cell stemness inhibition system according to claim 6, characterized in that, A photothermal synergistic tumor stem cell inhibition system was obtained by loading hydrogels with co-loaded photothermal agents and tumor stem cell inhibitor polymer micelles, as well as polymer vesicles loaded with immune adjuvants.

8. The method for preparing the photothermal synergistic tumor stem cell stemness inhibition system according to claim 6, characterized in that, In the photothermal synergistic tumor stem cell inhibition system, the mass ratio of photothermal agent, tumor stem cell inhibitor, and immune adjuvant is (10-1000):(0.1-100):1000.

9. A combination drug comprising the photothermal synergistic tumor stem cell stemness inhibition system of claim 1 and other antitumor drugs.

10. The use of the photothermal synergistic tumor stem cell stemness inhibition system of claim 1 or the combined drug of claim 9 in the preparation of antitumor drugs.

Images