Temperature-sensitive dual-drug co-loaded hydrogel, preparation method and application thereof

Abstract

The application discloses a temperature-sensitive double-drug-loaded hydrogel, a preparation method and application thereof, and relates to the technical field of antitumor drugs. The double-drug-loaded hydrogel comprises an esterification reaction product of polyethylene glycol and gambogic acid, an IDO inhibitor and a temperature-sensitive gel, and is prepared by loading the gambogic acid and the IDO inhibitor in the temperature-sensitive gel. The IDO inhibitor and the gambogic acid are delivered together, and the tumor growth is synergistically inhibited, and the therapeutic effect is greater than that of single drug treatment. The temperature-sensitive double-drug-loaded hydrogel is in a solution state at room temperature and is in a gel state at body temperature, and can meet the injection requirement.

Description

Technical Field

[0001] This invention relates to the field of antitumor drug technology, and more specifically, to a temperature-sensitive dual-drug co-loaded hydrogel, its preparation method, and its application. Background Technology

[0002] Breast cancer is the most common malignant tumor among women, posing a serious threat to their health. In 2020, the incidence of breast cancer surpassed that of lung cancer, with 2.3 million new cases (11.7%). Currently, breast cancer treatment still primarily relies on traditional methods such as surgical resection, radiotherapy, and chemotherapy, especially for triple-negative breast cancer, where efficacy remains unsatisfactory and adverse reactions are numerous. Therefore, there is an urgent need to explore new breast cancer treatment strategies to improve treatment outcomes.

[0003] With the development of cell biology and tumor immunology, research on tumor immunotherapy has been translated into clinical practice. Immunotherapy has shown good anti-tumor effects in the clinical practice of some cancers, but the beneficiary group remains very limited (~20%). Breast cancer, as a "cold" tumor (non-T cell infiltration), has an immunosuppressive microenvironment that limits the application of immunotherapy. Chemotherapy-immunotherapy combination therapy provides a new method to improve treatment efficacy, but it still suffers from problems such as low drug concentration at the lesion site and significant toxic side effects. Developing innovative drugs that are highly effective and low in toxicity and can be specifically delivered to the lesion site using advanced pharmaceutical technology is an effective strategy to overcome the limitations of current clinical treatments.

[0004] In recent years, pharmacological studies have revealed that gambogic acid (GA), an active monomer extracted from natural products, exhibits strong antitumor activity against various tumors, including leukemia, liver cancer, melanoma, breast cancer, gastric cancer, pancreatic cancer, prostate cancer, and lung cancer. Its mechanisms of action primarily include inducing tumor cell apoptosis and inhibiting tumor cell proliferation. Recent research has also found that GA can not only directly kill tumor cells but also induce immunogenic cell death (ICD). The development of ICD involves tumor cells secreting adenosine triphosphate (ATP) as a "find me" signal, recruiting antigen-presenting cells (APCs) into the tumor immune microenvironment (TME); calreticulin (CRT) extravasates from the endoplasmic reticulum to the cell membrane surface as an "eat me" signal, promoting the phagocytosis of dead tumor cells by dendritic cells (DCs); high-mobility group box 1 (HMGB1) released from tumor cells binds to the TLR-4 receptor on the surface of DCs, promoting antigen presentation to T cells, thereby activating the anti-tumor immune response; and further promoting the infiltration of cytotoxic T lymphocytes (CTLs) into the TME, secreting interferon-γ (IFN-γ). However, after ICD occurs, the upregulated IFN-γ in the TME induces the synthesis of an inhibitory immune checkpoint—indoleamine 2,3-dioxygenase (IDO). IDO is highly expressed in various tumor cells (such as breast cancer, cervical cancer, bladder cancer, and lung cancer) and catalyzes the conversion of the essential amino acid tryptophan (Trp) to kynurenine (Kyn).On the one hand, Trp is a fundamental amino acid for T cell activation, proliferation, and catabolism. With the participation of IDO, Trp is degraded, thereby mediating T cell immunosuppression by upregulating general control nonderepressible protein kinase 2 (GCN2) and downregulating mammalian target of rapamycin complex 1 (mTORC1). On the other hand, overexpression of IDO and accumulation of Kyn in tumor tissues can activate regulatory T cells (Tregs) by binding to the aryl hydrocarbon receptor (AHR), thereby activating downstream gene expression and myeloid-derived suppressor cells (MDSCs), inhibiting the function of effector T cells and natural killer cells, and promoting angiogenesis in solid tumors. In recent years, researchers have developed various IDO inhibitors (such as Epacadostat from Incyte, Indoximod from Newlink, and BMS-986205 from BMS), all of which can inactivate the enzyme IDO through specific binding, thereby restoring T cell proliferation. Although the use of IDO inhibitors can activate the human immune system, tumor tissues can also evade the immune system through other means, and the therapeutic effect of using IDO inhibitors alone is limited.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The purpose of this invention is to provide a temperature-sensitive dual-drug co-loaded hydrogel, its preparation method, and its application to solve the above-mentioned technical problems.

[0007] This invention is implemented as follows:

[0008] In a first aspect, the present invention provides a temperature-sensitive dual-drug co-loaded hydrogel, comprising: an esterification reaction product of polyethylene glycol and gambogeylic acid, an IDO inhibitor, and a temperature-sensitive gel, wherein gambogeylic acid and the IDO inhibitor are encapsulated in the temperature-sensitive gel; the temperature-sensitive gel contains 0.006-0.01M gambogeylic acid and 0.01-0.02M IDO inhibitor; the IDO inhibitor is 1-methyl-D-tryptophan (1MT), pacadostat (INCB24360), navoximod (GDC-0919, NLG919), Linrodostat (BMS-986205), DO-IN-2, PF-06840003, INCB024360, Exiguamine A, or benzimidazole.

[0009] The inventors co-delivered an IDO inhibitor with the chemotherapeutic drug gamboge. Based on the IDO inhibitor's ability to relieve the immunosuppressive activity of effector T cells and natural killer cells induced by IDO in tumor tissue, the resulting dual-drug co-loaded hydrogel possesses both the function of reversing the tumor immunosuppressive microenvironment and the antitumor activity of gamboge. The inventors' research shows that the IDO inhibitor and the chemotherapeutic drug gamboge synergistically inhibit tumor growth, with efficacy greater than that of a single drug and superior to the positive control drug paclitaxel-albumin.

[0010] Furthermore, the temperature-sensitive dual-drug co-loaded hydrogel exists as a solution at room temperature and as a gel at body temperature, meeting the requirements for injection. It also exhibits good biocompatibility. In vivo and in vitro, it allows drugs to remain at the tumor site for extended periods, acting as a reservoir effect.

[0011] Therefore, the temperature-sensitive dual-drug co-loaded hydrogel provided by the present invention has good application prospects for anti-tumor drugs.

[0012] Linking polyethylene glycol to gamboge acid helps improve the water dispersibility of gamboge acid, increase its utilization rate, prolong the drug's half-life, and facilitate the preparation of temperature-sensitive dual-drug co-loaded hydrogels.

[0013] In a preferred embodiment of the present invention, the thermosensitive gel is a hydrogel with a three-dimensional network structure formed by a natural cationic polymer and a crosslinking agent.

[0014] In one alternative embodiment, the natural cationic polymer is selected from galactomannan or chitosan; the crosslinking agent is selected from sodium β-glycerophosphate.

[0015] Chitosan (CS) is a natural cationic polymer with advantages such as antibacterial activity, good biocompatibility, and biodegradability. Its molecular structure contains a large number of amino and hydroxyl groups, allowing it to form covalently cross-linked, injectable hydrogels with a three-dimensional network structure with the cross-linking agent β-glycerol phosphate disodium salt pentahydrate (β-GP).

[0016] In a preferred embodiment of the present invention, the volume ratio of chitosan to sodium β-glycerophosphate in the thermosensitive gel is 10:1 to 1:1. The inventors have found that this volume ratio is particularly critical for the gelation time and the stability of the hydrogel.

[0017] In one optional embodiment, the volume ratio of chitosan to sodium β-glycerophosphate in the thermosensitive gel is 3:1. When the volume ratio of chitosan to sodium β-glycerophosphate is 3:1, the gelation time is 1-2 minutes, and the stability is better. It can maintain a clear and transparent solution state for a long time at 4°C.

[0018] In a preferred embodiment of the present invention, the relative molecular mass of polyethylene glycol in the esterification reaction product is 1000-4000 Da.

[0019] In one alternative embodiment, the temperature-sensitive dual-drug co-loaded hydrogel is in a solution state at 0°C-10°C and in a gel state above 36°C. This facilitates in-situ injection.

[0020] Secondly, the present invention also provides a method for preparing a temperature-sensitive dual-drug co-loaded hydrogel, comprising the following steps:

[0021] A temperature-sensitive dual-drug co-loaded hydrogel was prepared by mixing a mixed solution of a product synthesized from the esterification reaction of polyethylene glycol and gambogeylic acid and an IDO inhibitor with a solution of raw materials used to prepare a temperature-sensitive gel.

[0022] The above preparation method is simple and easy to implement. The temperature-sensitive dual-drug co-loaded hydrogel has good stability, can be stored for a long time, and is easy to promote and apply.

[0023] In a preferred embodiment of the present invention, the above preparation method includes: blending a mixed solution of esterification reaction product and IDO inhibitor with an unloaded hydrogel solution composed of a natural cationic polymer and a crosslinking agent to obtain a temperature-sensitive dual-drug co-loaded hydrogel.

[0024] In a preferred embodiment of the present invention, the above preparation method includes: first, mixing a mixed solution of esterification reaction product and IDO inhibitor with a natural cationic polymer solution to obtain a drug-loaded hydrogel precursor, and then mixing the drug-loaded hydrogel precursor with a crosslinking agent solution to obtain a temperature-sensitive dual-drug co-loaded hydrogel.

[0025] In a preferred embodiment of the present invention, the mass-volume concentration of the above-mentioned natural cationic polymer is 2-3% (2-3g per 100mL), and the mass-volume concentration of the crosslinking agent solution is 40%-60%.

[0026] In thermosensitive gels, the volume ratio of chitosan to sodium β-glycerophosphate is 10:1 to 1:1.

[0027] Thirdly, the present invention also provides a composition comprising: a temperature-sensitive dual-drug co-loaded hydrogel or a temperature-sensitive dual-drug co-loaded hydrogel prepared by the above-described method for preparing the temperature-sensitive dual-drug co-loaded hydrogel.

[0028] The composition also includes pharmaceutically acceptable excipients.

[0029] In other embodiments, the pharmaceutical composition may further include pharmaceutically feasible excipients, including but not limited to fillers, lubricants, disintegrants, binders, and flow aids.

[0030] In the preferred embodiment of the present invention, pharmaceutically acceptable excipients include, but are not limited to, one or a combination of polyvinylpyrrolidone and its derivatives, polyvinyl alcohol and its derivatives, methylcellulose and its derivatives, ethylcellulose and its derivatives, hydroxypropylcellulose and its derivatives, starch and its derivatives, polyethylene glycol and its derivatives, lactose, sucrose, mannitol, trehalose, sorbitol, dextrin, microcrystalline cellulose, acrylic resin, dicalcium phosphate, calcium stearate, sodium stearoyl fumarate, silicon dioxide, titanium dioxide, talc, and indigo.

[0031] In a preferred embodiment of the present invention, the composition is an injection.

[0032] Fourthly, the present invention also provides the use of temperature-sensitive dual-drug co-loaded hydrogels or temperature-sensitive dual-drug co-loaded hydrogels prepared by the above-mentioned method for preparing temperature-sensitive dual-drug co-loaded hydrogels in the preparation of antitumor drugs or reagents, wherein the tumor is selected from any one of oral cancer, lung cancer, liver cancer, colon cancer, breast cancer, osteosarcoma, brain tumor, melanoma, gastric cancer, pancreatic cancer and prostate cancer.

[0033] In one alternative embodiment, the antitumor drug or reagent has at least one of the following uses:

[0034] (1) Inducing ICD in tumorigenesis;

[0035] (2) Promotes the maturation of dendritic cells (DCs);

[0036] (3) Increases T cell infiltration in the tumor microenvironment;

[0037] (4) Inhibits the degradation of tryptophan;

[0038] (5) Downregulation of Tregs cell proportion;

[0039] (6) Upregulates the proportion of cytotoxic T lymphocytes;

[0040] (7) Reversing the tumor immunosuppressive microenvironment;

[0041] (8) Increase the level of at least one of the following cytokines in tumor cells: TNF-α, IFN-γ and IL-6.

[0042] The present invention has the following beneficial effects:

[0043] (1) The inventors co-delivered an IDO inhibitor and the chemotherapeutic drug gamboge. Based on the fact that the IDO inhibitor can relieve the immunosuppressive activity of effector T cells and natural killer cells induced by IDO in tumor tissue, the resulting dual-drug co-loaded hydrogel possesses both the function of reversing the tumor immunosuppressive microenvironment and the antitumor activity of gamboge. The inventors' research shows that the synergistic effect of the IDO inhibitor and the chemotherapeutic drug gamboge inhibits tumor growth, with an efficacy greater than that of a single drug and superior to the positive control drug paclitaxel albumin.

[0044] (2) The temperature-sensitive dual-drug co-loaded hydrogel is in a solution state at room temperature and a gel state at body temperature, which can meet the needs of injection. Moreover, the temperature-sensitive dual-drug co-loaded hydrogel has good biocompatibility. In vivo and in vitro, it can allow drugs to remain at the tumor site for a long time, playing a reservoir effect.

[0045] (3) The preparation method of temperature-sensitive dual-drug co-loaded hydrogel is simple and easy to implement. The dual-drug co-loaded hydrogel has good stability, can be stored for a long time, and is easy to promote and apply. Attached Figure Description

[0046] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0047] Figure 1The images show the two states of the blank hydrogel and the temperature-sensitive dual-drug co-loaded hydrogel prepared in Example 1 of this invention at different temperatures.

[0048] Figure 2 To examine the microstructure of the empty hydrogel gel (a) and the dual-drug co-loaded hydrogel GM-1MT@gel (b) using scanning electron microscopy;

[0049] Figure 3 Figure showing the results of hydrogel rheological analysis;

[0050] Figure 4 Figure showing the volume distribution and weight analysis of residual hydrogel in mice;

[0051] Figure 5 This is a tissue distribution diagram of the drug in mice;

[0052] Figure 6 The tumor cell apoptosis induction rate of GM, a compound synthesized from the esterification reaction of gambogeylic acid (GA) and polyethylene glycol with gambogeylic acid.

[0053] Figure 7 A statistical graph of tumor volume in a mouse breast cancer model from 0 to 18 days under different treatment conditions;

[0054] Figure 8 A statistical graph showing the tumor quality of mice in different drug administration groups;

[0055] Figure 9 Flow cytometry image of T lymphocytes (CD3+CD8+) in the tumor microenvironment;

[0056] Figure 10 for Figure 9 A bar chart for statistical analysis;

[0057] Figure 11 Flow cytometry image of Tregs in the tumor microenvironment;

[0058] Figure 12 for Figure 11 A bar chart for statistical analysis;

[0059] Figure 13 The figure shows the statistical results of IFN-γ, TNF-α and IL-6 levels in mouse tumor tissue. Detailed Implementation

[0060] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0061] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0062] Example 1

[0063] The preparation method of temperature-sensitive dual-drug co-loaded hydrogel is as follows:

[0064] Weigh out a certain amount of CS (chitosan) and β-GP (sodium β-glycerophosphate). Dissolve CS in 0.1 mol / L acetic acid to prepare a 2.2% (w / v) CS solution. Dissolve β-GP in water to prepare a 56% (w / v, g / 100 mL) β-GP solution and incubate at 4°C overnight. Mix the CS solution and β-GP solution in different proportions, and add the β-GP solution dropwise to the CS solution. Stir for 15 min to obtain a blank hydrogel.

[0065] In this embodiment, a stepwise mixing method is used to prepare the temperature-sensitive dual-drug co-loaded hydrogel. In other embodiments, the mixed solution of gambogeylic acid (GM) covalently linked to polyethylene glycol and 1MT (1-methyl-D-tryptophan) can also be directly mixed with the above-mentioned blank hydrogel.

[0066] Specifically, gambogeylic acid and IDO inhibitor are first dissolved in 2.2% CS solution, and β-GP solution is added dropwise to the above solution. After stirring for 15 minutes, the drug-loaded hydrogel (GM-1MT@gel) is obtained.

[0067] First, 12 mg GM and 9 mg 1 MT were dissolved in 3 mL of 2.2% (w / v) CS solution to prepare a 2.2% drug-containing CS solution. Then, 56% β-GP solution was added to make the total mixture of 2.2% CS solution and 56% β-GP solution in a 3:1 ratio. The mixture was stirred for 15 min to obtain the drug-loaded hydrogel (GM-1MT@gel).

[0068] Reference Figure 1 As shown, both the prepared gel and GM-1MT@gel are free-flowing liquids at room temperature and are injectable. Upon heating to 37°C, they form stable gels, achieving a sol-gel transition. They exhibit good temperature sensitivity.

[0069] Scanning electron microscopy (SEM) was used to observe the prepared gel and GM-1MT@gel, respectively. Specifically, the surface morphology of the two hydrogel samples was observed under a cryo-SEM after freeze-fixation, freeze-fracture, and conductive spraying. (Refer to...) Figure 2 As shown, the results indicate that the hydrogel has an interconnected three-dimensional network structure, providing a basis for loading diverse drugs. After loading drugs onto the hydrogel ( Figure 2In (b) of the sample, the surface becomes rougher, but its internal microstructure remains basically unchanged, indicating that the drug loading has no significant effect on the formation of the hydrogel skeleton.

[0070] Example 2

[0071] This embodiment investigates the rheological properties of the GM-1MT@gel hydrogel in Example 1: the rheological behavior of the hydrogel at different temperatures (10-60℃) and 37℃ was studied using a rheometer.

[0072] (1) The heating rate during the test was 1℃ / min, the temperature range was 10-60℃, and data were collected at a constant frequency of 1.0Hz and a strain amplitude of 1%. The changes in storage modulus (G') and loss modulus (G") with temperature were observed.

[0073] (2) The temperature was set to 37℃, and data were collected at a constant frequency of 1.0Hz and a strain amplitude of 1% to observe the changes in energy storage modulus (G') and loss modulus (G") over time.

[0074] Rheological analysis of the hydrogel was performed, and the results were referenced. Figure 3 As shown, a G″ / G′ value close to 0 indicates that the gel state is similar to a solid, while a value close to 1 indicates that the gel state is similar to a liquid. The hydrogel exhibits solid-like behavior; as the temperature increases, the G′ of the hydrogel is greater than G″. At 37℃, G′ remains greater than G″ over time, indicating that the hydrogel can rapidly gel at physiological temperatures and form a stable gel system at 37℃.

[0075] Example 3

[0076] This embodiment studies the in vivo degradation of hydrogels.

[0077] To study the in vivo degradation properties of hydrogel, 6-8 week old female Balb / c mice were selected, and 100 μL of hydrogel was injected subcutaneously into the right back of the mice. Three mice were sacrificed at timed intervals on days 0, 3, 7, 14, and 21. The skin at the injection site was dissected to observe the degradation of the hydrogel and the condition of the surrounding skin tissue. The data were recorded by taking pictures and weighing the residual gel.

[0078] Results reference Figure 4 As shown, the experimental results indicate that the volume and weight of the hydrogel in mice gradually decreased over time, and the hydrogel (GM-1MT@gel) was completely degraded after 21 days. Furthermore, no redness, swelling, ulceration, or other lesions appeared on the skin around the hydrogel injection site, demonstrating good biocompatibility.

[0079] Example 4

[0080] In vivo distribution experiment of GM-1MT@gel.

[0081] A hydrogel, ICG@gel, loaded with a fluorescent dye was prepared using the fluorescent molecule ICG instead of a drug. The tumor volume was increased to approximately 200 mm². 3 Subsequently, mice were randomly divided into two groups. One group received 100 μL of free ICG (diluted and dissolved in physiological saline), and the other received ICG@gel, both injected into the tumor site. In vivo imaging was performed at 4 h, 8 h, 12 h, 24 h, 48 h, and 72 h post-administration to detect the fluorescence intensity of ICG in the tumor site. Immediately after the in vivo imaging at 72 h, the mice were euthanized, and their heart, liver, spleen, lungs, kidneys, and tumors were dissected and fixed in 4% paraformaldehyde for in vitro imaging to detect the fluorescence intensity of ICG in each tissue.

[0082] Experimental results refer to Figure 5 As shown, the results indicated that over time, the ICG fluorescence intensity at the tumor site in the ICG group gradually decreased and the rate of decline was relatively rapid, while the rate of decline in ICG fluorescence intensity in the ICG@gel group was relatively slow. From 24 hours onwards, the fluorescence intensity of the ICG@gel group began to show a significant difference from that of the free ICG group (P<0.05). After 72 hours, the heart, liver, spleen, lungs, kidneys, and tumors of the mice were dissected for in vivo imaging. The free ICG group no longer exhibited ICG fluorescence at the tumor site, while the ICG@gel group still showed high ICG fluorescence intensity. These data suggest that the drug can accumulate at the tumor site for a relatively long period and, through the "reservoir effect," prolong the drug's release time in vivo.

[0083] Example 5

[0084] The effect of esterification reaction of gambogeylic acid (GA) and polyethylene glycol with gambogeylic acid (GM) on tumor cell apoptosis. Figure 6 Both GA and GM can effectively induce tumor cell apoptosis, and there is no significant difference between the two. This indicates that after GM is covalently linked to GA by polyethylene glycol, it not only improves the water dispersibility of GA, but also does not affect its tumor killing effect. This is different from the method of improving water solubility by preparing liposomes in the existing patent 202110671673X.

[0085] Example 6

[0086] In vivo antitumor experiment of GM-1MT@gel.

[0087] Establishment of a breast cancer model: 4T1 cells in the logarithmic growth phase and in good growth condition were digested and collected. The cells were washed once with serum-free and antibiotic-free 1640 medium, then resuspended in serum-free and antibiotic-free 1640 medium. The cell concentration was counted and adjusted to 3 × 10⁶ cells / mL. 100 μL of the cell suspension was injected subcutaneously into the second mammary pad of mice to establish a mouse breast cancer model.

[0088] Grouping: When the tumor volume reaches approximately 100mm 3 Mice were randomly divided into 7 groups of 5 mice each (n=5). The groups were as follows: ① saline, ② gel, ③ free drug (GM+1MT), ④ GM@gel, ⑤ 1MT@gel, ⑥ GM-1MT@gel, ⑦ positive control drug paclitaxel albumin (PTX).

[0089] Treatment: The drug was administered via peritumoral injection. Starting from day 0, the major axis (a) and minor axis (b) of the mouse tumor were measured every two days using calipers, and the tumor volume was calculated. Treatment ended on day 18, and the mice were sacrificed. The tumors of each group were collected, weighed, and the tumor inhibition rate was calculated.

[0090] Tumor volume in mice was measured every two days, and tumor growth curves for each group of mice were referenced. Figure 7 As shown, the Saline group exhibited the fastest tumor growth. Compared to the Saline group, all other groups showed varying degrees of inhibitory effects. The gel, GM+1MT, and 1MT@gel groups showed mild tumor growth inhibition, while the GM@gel and PTX groups showed moderate tumor growth inhibition. The positive control drug PTX, administered twice via tail vein injection, showed weak inhibition of tumor growth. The combined administration of the two drugs, GM-1MT@gel, demonstrated the strongest tumor growth inhibition effect, with an average tumor volume as low as 198 mm. 3 This indicates that the GM-1MT@gel provided by this invention has a superior in vivo antitumor effect compared to positive control drugs.

[0091] After treatment, tumors were collected from each group of mice for further analysis. First, the tumor weight was measured and the tumor inhibition rate was calculated. (Refer to...) Figure 8 As shown, the heaviest tumor in the Saline group was 1.73±0.08g. There was no significant difference in tumor weight between the gel, GM+1MT and 1MT@gel groups and the Saline group (p>0.05). This indicates that the single IDO inhibitor treatment is not effective for tumors. Free drugs cannot accumulate at the tumor site for a long time and cannot effectively inhibit tumor growth.

[0092] The tumor inhibition rate is shown in Table 1 below:

[0093] Table 1. Tumor inhibition rate of each treatment group

[0094]

[0095]

[0096] Table 1 shows that both GM@gel and PTX exhibited varying degrees of tumor inhibition effects, with inhibition rates of 58.75% and 44.96%, respectively. GM-1MT@gel showed the strongest anti-tumor effect, with a tumor weight of only 0.07±0.03g and an inhibition rate as high as 96.18%, which was significantly different from the other groups (p<0.05).

[0097] Example 7

[0098] GM-1MT@gel regulates the tumor immune microenvironment.

[0099] After treatment, tumors were collected from the mice, and a portion was used to extract single-cell suspensions of the tumor cells. Flow cytometry was then used to detect T lymphocytes (CD3+) in the tumor microenvironment. + CD4 + / CD8 + ) and regulatory T cells (CD3) + CD4 + CD25 + FoxP3 + Another portion was ground into a tissue homogenate, and the content of cytokines in the tumor was determined using cytometric bead array (CBA) technology.

[0100] (1) Flow cytometry detection of T lymphocytes in the tumor microenvironment.

[0101] CTLs(CD3 + CD8 + It directly kills tumor cells by releasing membrane-soluble substances such as perforin and granzymes. Detection of CD3 in the mouse tumor microenvironment. + CD8 + T lymphocyte count. For example... Figures 9-10 As shown, CD3 of the Saline group + CD8 + The proportion of T lymphocytes was low, only 5.69±1.77%; CD3 in the gel, GM+1MT and PTX groups + CD8 + The proportion of T lymphocytes was not significantly different from that in the Saline group; however, CD3 levels were significantly lower after treatment with 1MT@gel, GM@gel, and GM-1MT@gel. + CD8 +The proportion of T lymphocytes increased to varying degrees in both. 1MT increased CTL infiltration by relieving the immunosuppressive effect of IDO, while GM increased CTL infiltration by inducing tumor ICD and activating T cells. GM-1MT@gel fully leverages the synergistic effect of 1MT and GM to further increase CTL infiltration and CD3+ in tumors. + CD8 + The proportion of T lymphocytes reached 23.08±1.60%, which was 4 times that of the Saline group. Furthermore, GM-1MT@gel showed significant differences compared to both 1MT@gel and GM@gel (p<0.01). These results indicate that GM-1MT@gel primarily increases CD3+. + CD8 + T lymphocytes infiltrate the TME, thereby enhancing anti-tumor immunity and prompting CTLs to kill tumor cells.

[0102] (2) Flow cytometry detection of Tregs in the tumor microenvironment.

[0103] Tregs, or T cell subsets expressing Foxp3, CD25, and CD4, possess significant immunosuppressive effects. They can suppress anti-tumor immune responses, hindering their immune function. Therefore, flow cytometry was used to detect Tregs (CD3+) in the mouse tumor microenvironment. + CD4 + CD25 + FoxP3 + The proportion of ). The results are as follows Figures 11-12 As shown, the Saline group had the highest proportion of Treg cells in the tumor microenvironment, reaching 53.26±2.32%, indicating that mouse breast cancer cells were in a highly immunosuppressed state. Compared with the Saline group, gel and PTX had no significant effect on Treg cells (p>0.05). After treatment with GM+1MT, 1MT@gel, GM@gel, and GM-1MT@gel, Treg cells were reduced to varying degrees, with the lowest reduction in the GM-1MT@gel group, at only 22.13±1.93%, and there was a significant difference between GM-1MT@gel and the other groups (p<0.05). This indicates that GM-1MT@gel has the ability to reverse the immunosuppressive tumor microenvironment, thereby enhancing the tumor-killing effect of CTLs.

[0104] (3) The levels of cytokines (TNF-α, IFN-γ and IL-6) in tumors were determined using cytometric bead array (CBA) technology.

[0105] Activated T cells release cytokines such as TNF-α, IFN-γ, and IL-6, thereby further enhancing the anti-tumor immune response. The inventors used the CBA method to detect the levels of IFN-γ, TNF-α, and IL-6 in mouse tumor tissues. The results are as follows... Figure 13 As shown, compared with other groups, the levels of IFN-γ, TNF-α, and IL-6 in the tumors of mice treated with GM-1MT@gel were significantly higher in the GM-1MT@gel group, being 4, 2.4, and 3.1 times higher, respectively, than in the Saline group (p<0.05). This indicates that GM-1MT@gel can activate T cells, release related inflammatory factors, enhance T cell killing ability, and further strengthen the anti-tumor immune response.

[0106] Example 8

[0107] Conduct stability experiments on the gel.

[0108] Hydrogels with different proportions were prepared according to Example 1. After mixing in different proportions, the mixtures were placed in a 4°C refrigerator and at room temperature, and their state was observed at regular intervals. The stability of hydrogels with different proportions was investigated.

[0109] The stability of hydrogels with different ratios was investigated at 4℃ and room temperature. As shown in Table 2, all hydrogels with different ratios gelled at room temperature. The hydrogels prepared with ratios of 2:1 and 3:1 were the most stable at 4℃, maintaining a clear and transparent solution state. However, the hydrogel prepared with the 2:1 ratio gelled after only 1 hour at room temperature, which was not conducive to subsequent experimental operations. Therefore, the 3:1 ratio was preferred for preparing the hydrogel.

[0110] Table 2. Gel formation time and stability of hydrogels with different proportions

[0111]

[0112] In summary, this invention provides a dual-drug co-loaded hydrogel, GM-1MT@gel, which exists as a solution at room temperature and as a gel at body temperature, exhibiting injectability and good biocompatibility. In vivo and in vitro, it allows the drug to remain at the tumor site for extended periods, acting as a reservoir effect. In antitumor efficacy studies, GM-1MT@gel demonstrated a synergistic inhibitory effect of GM and 1MT on tumor growth, with efficacy greater than single-drug therapy and superior to the positive control drug paclitaxel-albumin. Further analysis of T cells and Treg cells in the mouse tumor microenvironment revealed that GM-1MT@gel significantly increased CTL infiltration, inhibited IDO-induced immunosuppression, and downregulated the proportion of Treg cells, thereby reversing the tumor immunosuppressive microenvironment and transforming the "cold" immune environment of breast cancer into a "hot" environment.

[0113] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A temperature-sensitive dual-drug co-loaded hydrogel, characterized in that, It includes: The esterification reaction of polyethylene glycol and gambogeylic acid, an IDO inhibitor, and a thermosensitive gel are prepared by encapsulating gambogeylic acid and an IDO inhibitor in a thermosensitive gel. The thermosensitive gel contains 0.006-0.01 M gambogeylic acid and 0.01-0.02 M IDO inhibitor. The IDO inhibitor is 1-methyl-D-tryptophan (1MT). The thermosensitive gel is a hydrogel with a three-dimensional network structure formed by a natural cationic polymer and a crosslinking agent. The natural cationic polymer is selected from chitosan. The crosslinking agent is selected from sodium β-glycerophosphate. The volume ratio of chitosan to sodium β-glycerophosphate in the thermosensitive gel is 10:1 to 1:

1.

2. The temperature-sensitive dual-drug co-loaded hydrogel according to claim 1, characterized in that, The volume ratio of chitosan to sodium β-glycerophosphate in the thermosensitive gel is 3:

1.

3. The temperature-sensitive dual-drug co-loaded hydrogel according to claim 1, characterized in that, The relative molecular mass of polyethylene glycol in the esterification reaction product is 1000-4000 Da.

4. The temperature-sensitive dual-drug co-loaded hydrogel according to claim 3, characterized in that, The relative molecular mass of polyethylene glycol in the esterification reaction product is 2000 Da; The temperature-sensitive dual-drug co-loaded hydrogel is in a solution state at 0°C–10°C and in a gel state at temperatures above 36°C.

5. A method for preparing a temperature-sensitive dual-drug co-loaded hydrogel as described in any one of claims 1-4, characterized in that, It includes the following steps: A temperature-sensitive dual-drug co-loaded hydrogel was prepared by mixing a mixed solution of a product synthesized from the esterification reaction of polyethylene glycol and gambogeylic acid and an IDO inhibitor with a solution of raw materials used to prepare a temperature-sensitive gel.

6. The method for preparing the temperature-sensitive dual-drug co-loaded hydrogel according to claim 5, characterized in that, The preparation method includes: blending the mixed solution of the esterification reaction compound and the IDO inhibitor with an unloaded hydrogel solution composed of a natural cationic polymer and a crosslinking agent to obtain a temperature-sensitive dual-drug co-loaded hydrogel.

7. The method for preparing the temperature-sensitive dual-drug co-loaded hydrogel according to claim 5, characterized in that, The preparation method includes: first, mixing the esterification reaction compound and the IDO inhibitor solution with a natural cationic polymer solution to obtain a drug-loaded hydrogel precursor; then, mixing the drug-loaded hydrogel precursor with a crosslinking agent solution to obtain a temperature-sensitive dual-drug co-loaded hydrogel.

8. The method for preparing the temperature-sensitive dual-drug co-loaded hydrogel according to claim 6 or 7, characterized in that, The mass-volume concentration of the natural cationic polymer is 2-3%, and the mass-volume concentration of the crosslinking agent solution is 40%-60%. The volume ratio of chitosan to sodium β-glycerophosphate in the thermosensitive gel is 10:1 to 1:

1.

9. A composition, characterized in that, It includes: The temperature-sensitive dual-drug co-loaded hydrogel according to any one of claims 1-4, or the temperature-sensitive dual-drug co-loaded hydrogel prepared by the preparation method of the temperature-sensitive dual-drug co-loaded hydrogel according to any one of claims 5-8.

10. The composition according to claim 9, characterized in that, The composition also includes pharmaceutically acceptable excipients.

11. The composition according to claim 9, characterized in that, The composition is an injectable preparation.

12. The use of the temperature-sensitive dual-drug co-loaded hydrogel according to any one of claims 1-4, or the temperature-sensitive dual-drug co-loaded hydrogel prepared by the method of preparation of the temperature-sensitive dual-drug co-loaded hydrogel according to any one of claims 5-8, in the preparation of antitumor drugs or reagents, characterized in that, The tumor is selected from any one of oral cancer, lung cancer, liver cancer, colon cancer, breast cancer, osteosarcoma, brain tumor, melanoma, stomach cancer, pancreatic cancer, and prostate cancer.

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