Injectable composite hydrogel, its preparation method and application in regulating lactic acid metabolism and remodeling tumor immune microenvironment

Abstract

The present application relates to the technical fields of biological medicine and functional biological material, and particularly relates to an injectable composite hydrogel, a preparation method thereof and application thereof in regulating lactic acid metabolism and remodeling tumor immune microenvironment. The injectable composite hydrogel is prepared by synthesizing copper-gossypol complex through coordination reaction of soluble divalent copper salt and ligand gossypol in a solution system, and then dispersing the copper-gossypol complex uniformly in a sodium magnesium lithium silicate hydrogel network to form a composite system. The preparation method is simple, the component content can be precisely controlled, the preparation conditions are mild and controllable, the energy consumption is low, and the prepared injectable composite hydrogel can target and regulate lactic acid metabolism of osteosarcoma, remodel tumor immune microenvironment, promote bone tissue repair, and effectively induce immunogenic death of tumor cells, thereby effectively activating the body's anti-tumor immune response. The application of the injectable composite hydrogel in the preparation of a drug for treating osteosarcoma has a good application prospect.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and functional biomaterials, specifically to an injectable composite hydrogel, its preparation method, and its application in regulating lactate metabolism and remodeling the tumor immune microenvironment. Background Technology

[0002] Osteosarcoma and other malignant bone tumors pose a serious threat to human health, especially that of adolescents. Current standard treatments include surgical resection combined with radiotherapy and chemotherapy, but these still face many significant challenges: First, surgical resection often results in extensive bone defects, while the acidic microenvironment at the tumor site and persistent osteoclast activity severely hinder bone regeneration and repair. Second, tumor cells produce large amounts of lactic acid through aerobic glycolysis (the Warburg effect), which not only exacerbates the acidification of the extracellular environment, providing favorable conditions for tumor growth, invasion, and metastasis, but also inhibits the function of immune cells. Finally, traditional chemotherapy drugs suffer from poor targeting, high systemic toxicity, and a tendency to develop drug resistance, and are difficult to effectively activate the body's own anti-tumor immune response, leading to a high recurrence and metastasis rate. To address these challenges, researchers are attempting to develop multifunctional biomaterials. For example, bioactive materials with pH buffering capacity (such as magnesium- and calcium-containing silicates) are used to neutralize the acidic tumor microenvironment, inhibit osteoclast activity, and promote osteogenic formation, creating conditions for bone repair. However, simply adjusting the pH value is insufficient to effectively kill tumor cells and reverse their metabolic abnormalities.

[0003] In tumor metabolic regulation, lactate dehydrogenase A (LDHA) is a key enzyme in glycolysis, responsible for converting pyruvate to lactate, and is an important target for reversing tumor acidification and inhibiting its progression. Gossypol, a naturally occurring LDHA inhibitor, can effectively inhibit lactate production. However, its poor water solubility, rapid in vivo metabolism, and low targeted delivery efficiency limit its effectiveness when used alone. On the other hand, inducing immunogenic cell death (ICD) is considered a key strategy for activating anti-tumor immunity and generating long-lasting immune memory. In recent years, copper death, a newly discovered cell death mechanism dependent on copper ion accumulation and mitochondrial respiration, has been shown to effectively induce strong ICD effects, thereby promoting dendritic cell maturation and subsequent T-cell immune responses. However, precisely delivering copper ions to the tumor site and controlling their release to maximize ICD effects while minimizing systemic toxicity remains a technical challenge. Currently, there is a lack of a synergistic therapeutic strategy that integrates tumor microenvironment regulation, metabolic intervention, immune activation, and bone repair. Existing materials or drugs often focus on only one aspect, making it difficult to simultaneously address multiple bottlenecks in bone tumor treatment, such as acidic environment disruption, metabolic reprogramming, immunosuppression, and bone defect repair. Summary of the Invention

[0004] To overcome the shortcomings of the prior art, the first objective of this invention is to provide a method for preparing an injectable composite hydrogel. This method is simple, the component content can be precisely controlled, the preparation conditions are mild and controllable, the energy consumption is low, and the resulting injectable composite hydrogel can target and regulate lactic acid metabolism in osteosarcoma, reshape the tumor immune microenvironment, promote bone tissue repair, and effectively induce immunogenic death of tumor cells.

[0005] To overcome the shortcomings of the prior art, the second objective of this invention is to provide an injectable composite hydrogel that can target and regulate lactate metabolism in osteosarcoma, reshape the tumor immune microenvironment, promote bone tissue repair, and effectively induce immunogenic death of tumor cells.

[0006] A third objective of this invention is to provide the use of an injectable composite hydrogel in the preparation of a medicament for the treatment of osteosarcoma.

[0007] The fourth objective of this invention is to provide an injectable composite hydrogel for use in the preparation of a drug for targeted regulation of lactic acid metabolism.

[0008] The fifth objective of this invention is to provide an injectable composite hydrogel for use in the preparation of a drug for remodeling the tumor immune microenvironment.

[0009] To achieve the first objective of the invention, the technical solution adopted by the present invention is as follows:

[0010] This invention provides a method for preparing an injectable composite hydrogel, comprising the following steps:

[0011] S1. A soluble divalent copper salt solution is mixed with a gossypol solution to obtain a mixed solution, and the soluble divalent copper salt and gossypol undergo a coordination reaction in the mixed solution. After centrifugation, washing and drying, a copper-gossypol complex is obtained.

[0012] S2. The copper-gossypol complex and sodium magnesium lithium silicate are mixed to obtain a mixed powder, then water is added, and the mixture is stirred and allowed to stand to solidify, thus obtaining the injectable composite hydrogel.

[0013] This invention discloses a method for preparing an injectable composite hydrogel, which involves synthesizing a copper-gossypol complex through a coordination reaction between a soluble divalent copper salt and the ligand gossypol in a solution system. The copper-gossypol complex is then uniformly dispersed in a sodium magnesium lithium silicate hydrogel network to form a composite system. This preparation method is simple, allows for precise control of component content, provides mild and controllable preparation conditions, and has low energy consumption.

[0014] In this method, the copper-gossypol complex and sodium magnesium lithium silicate are first mixed to obtain a mixed powder, and then water is added to form an injectable composite hydrogel, which can make the copper-gossypol complex well dispersed in the hydrogel system.

[0015] The copper-gossypol complex obtained in step S1 is composed of Cu 2+ It coordinates with the phenolic oxygen atom in the gossypol molecule to form a Cu-O coordination bond, thereby forming a stable coordination structure.

[0016] Furthermore, in step S1, the molar ratio of soluble divalent copper salt to gossypol in the mixed solution is (1~3):(1~3).

[0017] Furthermore, in step S1, the soluble divalent copper salt solution is a copper chloride solution; and / or

[0018] The molar concentration of the soluble divalent copper salt in the mixed solution is 1 mmol / L to 20 mmol / L; and / or

[0019] The molar concentration of gossypol in the mixed solution is 1 mmol / L to 20 mmol / L.

[0020] Furthermore, in step S1, the coordination reaction temperature is 30℃~50℃, and the coordination reaction time is 6h~24h. This coordination reaction operates under mild conditions with low energy consumption, and these mild conditions contribute to the formation of a structurally stable copper-gossypol complex with excellent biological activity.

[0021] Furthermore, in step S1, the centrifugation is performed at a speed of 7000 rpm to 9000 rpm for 5 min to 15 min; and / or

[0022] The washing process involves alternating between deionized water and anhydrous ethanol 2-3 times to remove unreacted raw materials and byproducts; and / or

[0023] The drying process involves drying at 55℃~65℃ for 10h~14h.

[0024] In addition, the coordination reaction product of step S1 is prone to agglomeration after centrifugation and drying. In order to obtain fine particles with uniform particle size, the product can be ground.

[0025] Further, in step S2, the mass ratio of the copper-gossypol complex to sodium magnesium lithium silicate is (1~5):(20~100); and / or

[0026] The mass-to-volume ratio of the mixed powder to the water is (1~5) g: 1 mL; and / or

[0027] The water is deionized water; and / or

[0028] The stirring time is 20s~40s; and / or

[0029] The static curing time is 5 min to 10 min.

[0030] To achieve the second objective of the invention, the technical solution adopted by the present invention is as follows:

[0031] This invention provides an injectable composite hydrogel, which is prepared by the above-described method for preparing an injectable composite hydrogel.

[0032] To achieve the third objective of the invention, the technical solution adopted by the present invention is as follows:

[0033] This invention provides the application of an injectable composite hydrogel in the preparation of a drug for treating osteosarcoma.

[0034] To achieve the fourth objective of the invention, the technical solution adopted by the present invention is as follows:

[0035] This invention provides the application of an injectable composite hydrogel in the preparation of a drug for targeted regulation of lactate metabolism.

[0036] The present invention relates to the application of an injectable composite hydrogel in the preparation of a drug for targeted regulation of lactate metabolism. The injectable composite hydrogel can specifically regulate the lactate metabolism process of osteosarcoma cells. Its mechanism of action is manifested as follows: the active ingredient gossypol released by the injectable composite hydrogel can specifically inhibit the activity of lactate dehydrogenase (LDH) in tumor cells, thereby reducing lactate synthesis from the source.

[0037] To achieve the fifth objective of the invention, the technical solution adopted by the present invention is as follows:

[0038] This invention provides the application of an injectable composite hydrogel in the preparation of a drug for remodeling the tumor immune microenvironment.

[0039] This invention relates to the application of an injectable composite hydrogel in the preparation of drugs for reshaping the tumor immune microenvironment. This injectable composite hydrogel can provide favorable conditions for tumor immunotherapy by inhibiting lactate production and reversing the immunosuppressive state in the tumor microenvironment. Its mechanism of action lies in: by inhibiting lactate metabolism, reducing the local lactate concentration in the tumor, thereby alleviating lactate-mediated functional suppression of immune cells such as T cells and NK cells, promoting the infiltration and activation of immune cells in tumor tissue, and achieving a reversal of the tumor microenvironment from a "cold tumor" to a "hot tumor."

[0040] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0041] (1) A method for preparing an injectable composite hydrogel according to the present invention involves synthesizing a copper-gossypol complex by coordinating a soluble divalent copper salt with a gossypol ligand in a solution system, and then uniformly dispersing the copper-gossypol complex in a sodium magnesium lithium silicate hydrogel network to form a composite system. This preparation method has the advantages of simple process, precise control of component content, effective control of the composition and release performance of the complex by precisely controlling the proportion, concentration and reaction conditions of reactants; good product stability, mild and controllable preparation conditions, low energy consumption, and suitability for large-scale production.

[0042] (2) The injectable composite hydrogel of the present invention can actively neutralize the acidic tumor microenvironment, optimize the bone repair microenvironment, precisely inhibit the lactic acid metabolism pathway of tumor cells, target and regulate lactic acid metabolism in osteosarcoma, reshape the tumor immune microenvironment, promote bone tissue repair, and effectively induce immunogenic death of tumor cells, thereby effectively activating the body's anti-tumor immune response. Through the synergistic effect of the above multiple functions, it is expected to achieve efficient synergistic treatment and functional bone reconstruction of bone tumors, providing a new strategy for clinical treatment of bone tumors.

[0043] (3) An injectable composite hydrogel of the present invention achieves stable loading of gossypol, a natural compound with definite lactate dehydrogenase (LDH) inhibitory activity, into a copper-gossypol complex through a coordination reaction with copper. This injectable composite hydrogel system can achieve continuous and controllable release of active ingredients at the tumor site, effectively inhibit the glycolysis pathway of osteosarcoma cells, and then target and regulate lactate metabolism to directly address the core pathological link, precisely regulate abnormal lactate metabolism, and fundamentally improve the acidification state of the tumor microenvironment.

[0044] (4) Application of an injectable composite hydrogel of the present invention in the preparation of a drug for treating osteosarcoma. The present invention constructs a benign regulatory cycle through the dual-function synergistic effect of the injectable composite hydrogel, synergistically exerting the dual effects of lactate metabolism regulation and tumor microenvironment regulation. Among them, the active ingredient gossypol can effectively inhibit lactate production, while the sodium magnesium lithium silicate hydrogel itself has acid-base buffering capacity, which can actively neutralize the acidic microenvironment already formed in the tumor area; the synergistic effect of the two can rapidly reverse the acidic immunosuppressive microenvironment of the tumor, laying the foundation for the initiation and implementation of subsequent immune responses.

[0045] (5) The application of an injectable composite hydrogel of the present invention in the preparation of a drug for treating osteosarcoma. This injectable composite hydrogel can not only directly inhibit tumor growth by regulating tumor cell metabolism, but also improve the tumor immune microenvironment and form a synergistic effect with existing immunotherapeutic drugs such as PD-1 / PD-L1 inhibitors, significantly improving the overall therapeutic effect of tumors. At the same time, the sodium magnesium lithium silicate component in this injectable composite hydrogel has good biocompatibility and osteoproliferative activity. On the basis of osteosarcoma treatment, it can simultaneously provide structural and bioactive support for bone defect repair, realizing the integrated function of anti-tumor and bone repair, and therefore has a very good application prospect.

[0046] (6) The application of an injectable composite hydrogel of the present invention in the preparation of a drug for treating osteosarcoma. This composite hydrogel is an in-situ moldable or injectable hydrogel that can fully fill the tumor defect cavity formed after surgical resection, achieving local sustained-release delivery of the drug, effectively increasing the drug concentration at the lesion site, and minimizing the toxic side effects caused by systemic drug exposure. This injectable composite hydrogel provides a novel material solution for metabolic targeted therapy and immune microenvironment remodeling of osteosarcoma, possessing significant theoretical research value and potential clinical application value.

[0047] (7) The application of an injectable composite hydrogel of the present invention in the preparation of a drug for targeted regulation of lactate metabolism, wherein the injectable composite hydrogel can specifically regulate the lactate metabolism process of osteosarcoma cells, improve the tumor immunosuppressive microenvironment, and provide favorable conditions for tumor treatment.

[0048] (8) The application of an injectable composite hydrogel of the present invention in the preparation of a drug for remodeling the tumor immune microenvironment, wherein the injectable composite hydrogel can provide favorable conditions for tumor immunotherapy by inhibiting lactic acid production and reversing the immunosuppressive state in the tumor microenvironment. Attached Figure Description

[0049] To more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0050] Figure 1 This is the FT-IR image of gossypol and the copper-gossypol complex (Cu-GP) of Example 1 of the present invention.

[0051] Figure 2 This is a SEM image of the injectable composite hydrogel of Example 1 of the present invention.

[0052] Figure 3 This is a graph showing the detection results of lactate levels in the cell supernatant of human osteosarcoma MG-63 cells after different culturing times in various experimental groups during the in vitro lactate metabolism regulation performance test of this invention.

[0053] Figure 4 This is a graph showing the results of the in vitro immune microenvironment regulation ability detection for each tumor conditioned medium (TCM) treatment group in this invention. Figure a shows the CD8+ regulation ability. + The results of T cell proportion detection are shown in Figure b, which shows the relative content of interferon-γ (IFN-γ), Figure c shows the relative content of tumor necrosis factor-α (TNF-α), and Figure d shows the killing rate of T cells against MG-63 tumor cells.

[0054] Figure 5 This is a graph showing the evaluation results of the anti-osteosarcoma and immunomodulatory effects of each experimental group in vivo in the animal body. Figure a shows the tumor volume detection results for each group of mice, figure b shows the survival cycle change curves of mice, and figure c shows the serum lactate content detection results for mice.

[0055] Figure 6 These are figures showing the results of the preliminary evaluation of the biocompatibility and in vivo safety of the present invention for each experimental group. Figure a shows the results of the hemolysis experiment, and Figure b shows the results of the live / dead staining of mouse fibroblasts (L929). Detailed Implementation

[0056] To make the technical problem to be solved, the technical solution, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.

[0057] The terminology used in the embodiments of this invention is for the purpose of describing particular embodiments only and is not intended to limit the invention. In this invention, the singular forms “a,” “the,” and “the” as used in the embodiments and appended claims are also intended to include the plural forms unless the context clearly indicates otherwise.

[0058] In this embodiment of the invention, a method for preparing an injectable composite hydrogel includes the following steps:

[0059] S1. A soluble divalent copper salt solution is mixed with a gossypol solution to obtain a mixed solution, and the soluble divalent copper salt and gossypol undergo a coordination reaction in the mixed solution. After centrifugation, washing and drying, a copper-gossypol complex is obtained.

[0060] S2. The copper-gossypol complex and sodium magnesium lithium silicate are mixed to obtain a mixed powder, then water is added, and the mixture is stirred and allowed to stand to solidify, thus obtaining the injectable composite hydrogel.

[0061] In some embodiments, in step S1, the molar ratio of soluble divalent copper salt to gossypol in the mixed solution is (1~3):(1~3).

[0062] In some embodiments, in step S1, the soluble divalent copper salt solution is a copper chloride solution; and / or

[0063] The molar concentration of the soluble divalent copper salt in the mixed solution is 1 mmol / L to 20 mmol / L; and / or

[0064] The molar concentration of gossypol in the mixed solution is 1 mmol / L to 20 mmol / L.

[0065] In some embodiments, in step S1, the temperature of the coordination reaction is 30°C to 50°C, and the time of the coordination reaction is 6h to 24h.

[0066] In some embodiments, in step S1, the centrifugation is performed at a speed of 7000 rpm to 9000 rpm for 5 min to 15 min; and / or

[0067] The washing process involves alternating between deionized water and anhydrous ethanol 2-3 times; and / or

[0068] The drying process involves drying at 55℃~65℃ for 10h~14h.

[0069] In some embodiments, in step S2, the mass ratio of the copper-gossypol complex to sodium magnesium lithium silicate is (1~5):(20~100); and / or

[0070] The mass-to-volume ratio of the mixed powder to the water is (1~5) g: 1 mL; and / or

[0071] The water is deionized water; and / or

[0072] The stirring time is 20s~40s; and / or

[0073] The static curing time is 5 min to 10 min.

[0074] In this embodiment of the invention, an injectable composite hydrogel is prepared by the above-described method for preparing an injectable composite hydrogel.

[0075] In this embodiment of the invention, the above-described injectable composite hydrogel is used in the preparation of a drug for treating osteosarcoma.

[0076] In this embodiment of the invention, the above-described injectable composite hydrogel is used in the preparation of a drug for targeted regulation of lactic acid metabolism.

[0077] In this embodiment of the invention, the application of the injectable composite hydrogel described above in the preparation of a drug for remodeling the tumor immune microenvironment is illustrated.

[0078] The following description is based on specific embodiments. Example 1

[0079] A method for preparing an injectable composite hydrogel includes the following steps:

[0080] S1. Under magnetic stirring, a 10 mmol / L copper chloride solution was slowly added dropwise to a 10 mmol / L gossypol ethanol solution. Stirring continued at room temperature for 10 min to obtain a dark brown, homogeneous mixed solution. The mixed solution was transferred to a reaction vessel, sealed, and placed in an oven at 40°C for 12 h to allow the copper chloride and gossypol to undergo a coordination reaction. After the reaction, the mixture was allowed to cool naturally to room temperature. The product in the reaction vessel was transferred to a centrifuge tube and centrifuged at 8000 rpm for 10 min. It was then washed three times alternately with deionized water and anhydrous ethanol, and dried in a vacuum drying oven at 60°C for 12 h. After grinding, a brownish-black copper-gossypol complex (denoted as Cu-GP) was obtained. In this embodiment, the molar ratio of copper chloride to gossypol in the mixed solution was 1:1. In this embodiment, the molar concentration of copper chloride in the mixed solution was 5 mmol / L; the molar concentration of gossypol in the mixed solution was 5 mmol / L.

[0081] S2. The copper-gossypol complex and sodium magnesium lithium silicate (Na2MgLiSiO4) are placed in a mortar and ground to achieve preliminary physical mixing, resulting in a mixed powder. Deionized water is then added, and the mixture is stirred with a glass rod for 30 seconds until a uniform paste is formed. The paste is then placed in a 37°C constant temperature oven and allowed to solidify for 8 minutes to obtain the injectable composite hydrogel (denoted as Gel-CuGP-1). This injectable composite hydrogel is an elastic, brownish-red hydrogel block. In this embodiment, the mass ratio of the copper-gossypol complex to sodium magnesium lithium silicate is 1:20; the mass-to-volume ratio of the mixed powder to deionized water is 2.1 g:1 mL. Example 2

[0082] A method for preparing an injectable composite hydrogel. The difference between this embodiment and Example 1 is that in this embodiment, the mass ratio of copper-gossypol complex to sodium magnesium lithium silicate is 1:40; the rest of the preparation method is the same as in Example 1, and an injectable composite hydrogel (denoted as Gel-CuGP-2) is obtained. Example 3

[0083] A method for preparing an injectable composite hydrogel. The difference between this embodiment and Example 1 is that in this embodiment, the mass ratio of copper-gossypol complex and sodium magnesium lithium silicate is 1:10; the rest of the preparation methods are the same as in Example 1, and an injectable composite hydrogel (denoted as Gel-CuGP-3) is obtained. Example 4

[0084] A method for preparing an injectable composite hydrogel. The difference between this embodiment and Example 1 is that in this embodiment, the mass ratio of copper-gossypol complex and sodium magnesium lithium silicate is 1:6.7; the rest of the preparation method is the same as in Example 1, and an injectable composite hydrogel (denoted as Gel-CuGP-4) is obtained. Example 5

[0085] A method for preparing an injectable composite hydrogel includes the following steps:

[0086] S1. Under magnetic stirring, a 10 mmol / L copper chloride solution was slowly added dropwise to a 10 mmol / L gossypol ethanol solution. Stirring continued at room temperature for 10 min to obtain a dark brown, homogeneous mixed solution. The mixed solution was transferred to a reaction vessel, sealed, and placed in an oven at 30°C for 24 h to allow the copper chloride and gossypol to undergo a coordination reaction. After the reaction, the mixture was allowed to cool naturally to room temperature. The product in the reaction vessel was transferred to a centrifuge tube and centrifuged at 7000 rpm for 15 min. The product was then washed twice alternately with deionized water and anhydrous ethanol, and dried in a vacuum drying oven at 55°C for 14 h. After grinding, a brownish-black copper-gossypol complex (denoted as Cu-GP) was obtained. In this embodiment, the molar ratio of copper chloride to gossypol in the mixed solution was 1:2; the molar concentration of copper chloride in the mixed solution was 10 mmol / L; and the molar concentration of gossypol in the mixed solution was 20 mmol / L.

[0087] S2. The copper-gossypol complex and sodium magnesium lithium silicate (Na2MgLiSiO4) are placed in a mortar and ground to achieve preliminary physical mixing, resulting in a mixed powder. Deionized water is then added, and the mixture is stirred with a glass rod for 20 seconds until a uniform paste is formed. The paste is then placed in a 37°C constant temperature oven and allowed to solidify for 10 minutes to obtain the injectable composite hydrogel (denoted as Gel-CuGP-1). This injectable composite hydrogel is an elastic, brownish-red hydrogel block. In this embodiment, the mass ratio of the copper-gossypol complex to sodium magnesium lithium silicate is 3:50; the mass-to-volume ratio of the mixed powder to deionized water is 1 g:1 mL. Example 6

[0088] A method for preparing an injectable composite hydrogel includes the following steps:

[0089] S1. Under magnetic stirring, a 10 mmol / L copper chloride solution was slowly added dropwise to a 10 mmol / L gossypol ethanol solution. Stirring was continued at room temperature for 10 min to obtain a dark brown, homogeneous mixed solution. The mixed solution was transferred to a reaction vessel, sealed, and placed in an oven at 50°C for 6 h to allow the copper chloride and gossypol to undergo a coordination reaction. After the reaction, the mixture was allowed to cool naturally to room temperature. The product in the reaction vessel was transferred to a centrifuge tube and centrifuged at 9000 rpm for 5 min. The product was then washed three times alternately with deionized water and anhydrous ethanol, and dried in a vacuum drying oven at 65°C for 10 h. After grinding, a brownish-black copper-gossypol complex (denoted as Cu-GP) was obtained. In this embodiment, the molar ratio of copper chloride to gossypol in the mixed solution was 3:1; the molar concentration of copper chloride in the mixed solution was 15 mmol / L; and the molar concentration of gossypol in the mixed solution was 5 mmol / L.

[0090] S2. The copper-gossypol complex and sodium magnesium lithium silicate (Na2MgLiSiO4) are placed in a mortar and ground to achieve preliminary physical mixing, resulting in a mixed powder. Deionized water is then added, and the mixture is stirred with a glass rod for 40 seconds until a uniform paste is formed. The paste is then placed in a 37°C constant temperature oven and allowed to solidify for 5 minutes to obtain the injectable composite hydrogel (denoted as Gel-CuGP-1). This injectable composite hydrogel is an elastic, brownish-red hydrogel block. In this embodiment, the mass ratio of the copper-gossypol complex to sodium magnesium lithium silicate is 5:90; the mass-to-volume ratio of the mixed powder to deionized water is 5 g:1 mL. Example 7

[0091] A method for preparing an injectable composite hydrogel includes the following steps:

[0092] S1. Under magnetic stirring, a 10 mmol / L copper chloride solution was slowly added dropwise to a 10 mmol / L gossypol ethanol solution. Stirring continued at room temperature for 10 min to obtain a dark brown, homogeneous mixed solution. The mixed solution was transferred to a reaction vessel, sealed, and placed in an oven at 35°C for 18 h to allow the copper chloride and gossypol to undergo a coordination reaction. After the reaction, the mixture was allowed to cool naturally to room temperature. The product in the reaction vessel was transferred to a centrifuge tube and centrifuged at 7500 rpm for 12 min. It was then washed three times alternately with deionized water and anhydrous ethanol, and dried in a vacuum drying oven at 58°C for 13 h. After grinding, a brownish-black copper-gossypol complex (denoted as Cu-GP) was obtained. In this embodiment, the molar ratio of copper chloride to gossypol in the mixed solution was 2:1. In this embodiment, the molar concentration of copper chloride in the mixed solution was 14 mmol / L, and the molar concentration of gossypol in the mixed solution was 7 mmol / L.

[0093] S2. The copper-gossypol complex and sodium magnesium lithium silicate (Na2MgLiSiO4) are placed in a mortar and ground to achieve preliminary physical mixing, resulting in a mixed powder. Deionized water is then added, and the mixture is stirred with a glass rod for 25 seconds until a uniform paste is formed. The paste is then placed in a 37°C constant temperature oven and allowed to solidify for 9 minutes to obtain the injectable composite hydrogel (denoted as Gel-CuGP-1). This injectable composite hydrogel is an elastic, brownish-red hydrogel block. In this embodiment, the mass ratio of the copper-gossypol complex to sodium magnesium lithium silicate is 2:70; the mass-to-volume ratio of the mixed powder to deionized water is 3 g:1 mL. Example 8

[0094] A method for preparing an injectable composite hydrogel includes the following steps:

[0095] S1. Under magnetic stirring, a 10 mmol / L copper chloride solution was slowly added dropwise to a 10 mmol / L gossypol ethanol solution. Stirring continued for 10 min at room temperature to obtain a dark brown, homogeneous mixed solution. The mixed solution was transferred to a reaction vessel, sealed, and placed in an oven at 45°C for 8 h to allow the copper chloride and gossypol to undergo a coordination reaction. After the reaction, the mixture was allowed to cool naturally to room temperature. The product in the reaction vessel was transferred to a centrifuge tube and centrifuged at 8500 rpm for 7 min. The product was then washed twice alternately with deionized water and anhydrous ethanol, and then dried in a vacuum drying oven at 62°C for 11 h. After grinding, a brownish-black copper-gossypol complex (denoted as Cu-GP) was obtained. In this embodiment, the molar ratio of copper chloride to gossypol in the mixed solution was 1:3. In this embodiment, the molar concentration of copper chloride in the mixed solution was 6 mmol / L, and the molar concentration of gossypol in the mixed solution was 18 mmol / L.

[0096] S2. The copper-gossypol complex and sodium magnesium lithium silicate (Na2MgLiSiO4) are placed in a mortar and ground to achieve preliminary physical mixing, resulting in a mixed powder. Deionized water is then added, and the mixture is stirred with a glass rod for 35 seconds until a uniform paste is formed. The paste is then placed in a 37°C constant temperature oven and allowed to solidify for 7 minutes to obtain the injectable composite hydrogel (denoted as Gel-CuGP-1). This injectable composite hydrogel is an elastic, brownish-red hydrogel block. In this embodiment, the mass ratio of the copper-gossypol complex to sodium magnesium lithium silicate is 4:65; the mass-to-volume ratio of the mixed powder to deionized water is 4 g:1 mL. Example 9

[0097] Application of any one of the injectable composite hydrogels in Examples 1 to 8 in the preparation of a drug for treating osteosarcoma. Example 10

[0098] Application of any one of the injectable composite hydrogels in Examples 1 to 8 in the preparation of drugs for targeted regulation of lactic acid metabolism. Example 11

[0099] Application of any one of the injectable composite hydrogels in Examples 1 to 8 in the preparation of drugs for remodeling the tumor immune microenvironment.

[0100] Structural morphology characterization

[0101] (I) Infrared spectral characterization

[0102] The gossypol used in Example 1 and the prepared copper-gossypol complex (Cu-GP) were characterized by Fourier transform infrared spectroscopy (FT-IR), as follows: Figure 1 As shown.

[0103] Depend on Figure 1 As can be seen, compared with the infrared spectrum of the raw material gossypol, the characteristic absorption peaks related to the phenolic hydroxyl group (-OH) in the FT-IR spectrum of Cu-GP show a significant shift and a marked decrease in intensity, indicating that Cu... 2+ It coordinates with the phenolic oxygen atom in gossypol to form Cu-O coordination bonds, thereby forming a stable coordination structure.

[0104] (II) Morphological characterization by scanning electron microscopy

[0105] The injectable composite hydrogels (Gel-CuGP-1, Gel-CuGP-2, Gel-CuGP-3, and Gel-CuGP-4) prepared in Examples 1 to 4 were freeze-dried at -60°C for 72 hours, and their cross-sectional morphology was characterized by scanning electron microscopy (SEM). The SEM image of the injectable composite hydrogel (Gel-CuGP-1) in Example 1 is shown below. Figure 2 As shown.

[0106] The injectable composite hydrogels of Examples 1 to 4, as observed by SEM, all exhibited a typical three-dimensional interconnected porous network structure with interconnected pores. Figure 2 It is evident that Cu-GP particles are uniformly loaded and embedded in the pore walls and framework structure of the sodium magnesium lithium silicate hydrogel, without obvious aggregation.

[0107] Performance testing and effect evaluation

[0108] (I) In vitro lactate metabolism regulation performance test

[0109] The in vitro lactate metabolism regulation performance test was used to verify that the injectable composite hydrogel of the present invention has a significant inhibitory effect on lactate production and lactate dehydrogenase (LDH) activity in osteosarcoma cells.

[0110] The testing method is as follows:

[0111] Cell Culture and Experimental Groups: In vitro cell culture experiments were conducted using the human osteosarcoma cell line MG-63. Five groups were set up: a blank control group (MG-63 cells only), a sodium magnesium lithium silicate gel group (Gel-Base, without Cu-GP), a gossypol solution group (GP-Sol, with a concentration consistent with the gossypol release in Gel-CuGP-3), and injectable composite hydrogels (Gel-CuGP-1 and Gel-CuGP-3 groups).

[0112] Sample preparation: Equal amounts (20 mg) of Gel-Base, Gel-CuGP-1 and Gel-CuGP-3 were placed in the upper chamber of a Transwell chamber and then placed into a 24-well plate pre-inoculated with MG-63 cells for indirect co-culture; the GP-Sol group was cultured directly in medium containing gossypol.

[0113] The testing indicators are as follows:

[0114] Lactic acid content determination: Cell culture supernatant was collected after 24h, 48h and 72h of co-culture, and the lactic acid concentration in the supernatant was quantitatively detected using a lactic acid detection kit.

[0115] Test results as follows Figure 3As shown, the results indicate that, compared to the blank control group, the Gel-Base group, due to its buffering capacity, can reduce lactate concentration to a certain extent. The GP-Sol group can significantly reduce lactate levels in the early stage of culture (24 h), but its effect diminishes significantly after 48 h with prolonged culture time. Both Gel-CuGP-1 and Gel-CuGP-3 groups can continuously and significantly inhibit lactate production throughout the entire detection period, with the Gel-CuGP-3 group, which has a higher drug loading ratio, showing the best inhibitory effect. The above results fully demonstrate that the injectable composite hydrogel system of the present invention can effectively regulate the acidic tumor microenvironment through the dual effects of inhibiting lactate production and acid-base buffering, providing reliable experimental evidence for intervention in the osteosarcoma microenvironment and possessing good application potential.

[0116] (II) Detection of in vitro immune microenvironment regulation capacity

[0117] The ability of the injectable composite hydrogel of the present invention to regulate the immune microenvironment was tested in vitro to verify its effect on improving and activating immune cell function.

[0118] The method is as follows:

[0119] Preparation of conditioned medium: Using the above-mentioned method for testing the in vitro lactate metabolism regulation performance, MG-63 cells were co-cultured with blank medium, Gel-Base and Gel-CuGP-3 for 48 h, respectively. The culture supernatant was collected to prepare the corresponding tumor conditioned medium (TCM).

[0120] T cell co-culture: CD8 cells were isolated and purified from peripheral blood mononuclear cells of healthy individuals. + T cells were cultured in the three groups of tumor conditioned media mentioned above.

[0121] The testing indicators are as follows:

[0122] T cell proliferation level: CD8+ levels were measured by flow cytometry after 72 hours of culture. + The proportion of T cells.

[0123] Cytokine secretion levels: The levels of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) in the culture supernatant were detected using an ELISA kit.

[0124] Cytotoxicity assay: T cells pretreated with different tumor conditioning media (TCM) were used as effector cells, and fresh MG-63 cells were used as target cells. They were co-cultured at an effector-to-target ratio (E:T) of 10:1. The killing efficiency of T cells against tumor cells was quantitatively detected by lactate dehydrogenase (LDH) release assay.

[0125] Test results as follows Figure 4As shown, by Figure 4 As can be seen, compared with the blank TCM treatment group, the T cell proliferation activity and cytokine secretion level of the Gel-Base TCM treatment group were slightly improved, indicating that pH adjustment alone can improve T cell function to a certain extent. The T cells in the Gel-CuGP-3 TCM treatment group exhibited the strongest proliferation capacity, the highest IFN-γ and TNF-α secretion levels, and the most significant killing activity against MG-63 cells. These results confirm that the injectable composite hydrogel of the present invention can effectively reverse lactate-mediated T cell immunosuppression by regulating tumor cell metabolism and reshaping the tumor microenvironment, thereby restoring and significantly enhancing its anti-tumor immune function.

[0126] (III) Evaluation of anti-osteosarcoma effects and immunomodulatory effects in animals

[0127] The evaluation aims to systematically verify the anti-osteosarcoma efficacy and immunomodulatory effect of the injectable composite hydrogel of the present invention in nude mice or humanized immune system mouse osteosarcoma models, clarify its in vivo therapeutic effect, and provide reliable experimental evidence for subsequent clinical applications.

[0128] The method is as follows:

[0129] Model establishment: Balb / c nude mice were used, and MG-63-Luc cells that stably express luciferase were injected into their tibias to construct an orthotopic osteosarcoma animal model, ensuring that the model was successfully constructed and had good stability.

[0130] Grouping and Treatment: After the tumors in the tibia of nude mice had grown for one week and the tumor size met the experimental requirements, the nude mice were randomly divided into 4 groups of 5 mice each (n=5). The specific grouping and treatment methods for each group are as follows: (1) PBS control group: PBS solution was injected intratumorally; (2) Gel-Base group: Sodium magnesium lithium silicate gel was injected intratumorally; (3) Free gossypol group: Free gossypol preparation was injected intratumorally; (4) Gel-CuGP-3 group: Gel-CuGP-3 was injected intratumorally. All groups were treated with a single intratumoral injection to ensure that the injection dosage and method were standardized.

[0131] The therapeutic effect is evaluated as follows:

[0132] Tumor growth monitoring: The tumor bioluminescence signal was detected twice a week using a small animal in vivo imaging system, and the changes in tumor volume were observed and calculated simultaneously.

[0133] Survival period analysis: The survival time of mice in each group was continuously observed and recorded, and statistical analysis of survival period was carried out.

[0134] Serological marker detection: Peripheral serum of mice was collected and the level of lactate in the serum was quantitatively detected.

[0135] Test results as follows Figure 5 As shown, by Figure 5 As can be seen, compared with the PBS control group, both the Gel-Base group and the free gossypol group showed some inhibitory effect on osteosarcoma growth, but the inhibitory effect was limited and could not effectively stop tumor progression. The Gel-CuGP-3 group showed the most significant tumor inhibitory effect, with tumor bioluminescence signal growth almost completely halted, and the survival time of mice in this group was significantly prolonged, showing a statistically significant difference compared with other groups. Serological tests showed that the serum lactate level of mice in the Gel-CuGP-3 group was significantly lower than that of other groups, reaching the lowest level. The above experimental results confirm that the injectable composite hydrogel of the present invention can effectively inhibit the growth and progression of osteosarcoma in vivo. Its anti-tumor mechanism is closely related to regulating tumor cell lactate metabolism, inducing tumor cell apoptosis, and activating the body's local anti-tumor immune response, further verifying the good in vivo therapeutic effect of the injectable composite hydrogel of the present invention.

[0136] (iv) Preliminary evaluation of biocompatibility and in vivo safety performance

[0137] Hemolysis experiments were conducted using the injectable composite hydrogel (Gel-CuGP-3) prepared in Example 3 of this invention, a PBS control group, and a Triton-X-100 control group. Cytotoxicity assays were also conducted using the injectable composite hydrogel (Gel-CuGP-3) prepared in Example 3 of this invention and a blank control group (human osteosarcoma MG-63 cells only). The results are as follows: Figure 6 As shown.

[0138] Depend on Figure 6 As can be seen, the hemolysis rate of the injectable composite hydrogel (Gel-CuGP-3) of this invention is far below the 5% safety limit specified for biomaterials; after co-culturing it with L929 cells in direct contact for 72 hours, the cell viability remained above 85%. These results demonstrate that the injectable composite hydrogel of this invention possesses excellent blood and cell compatibility and exhibits good biosafety performance.

[0139] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A method for preparing an injectable composite hydrogel, characterized in that, Includes the following steps: S1. A soluble divalent copper salt solution is mixed with a gossypol solution to obtain a mixed solution, and the soluble divalent copper salt and gossypol undergo a coordination reaction in the mixed solution. After centrifugation, washing and drying, a copper-gossypol complex is obtained. S2. The copper-gossypol complex and sodium magnesium lithium silicate are mixed to obtain a mixed powder, then water is added, stirred and allowed to stand to solidify, thus obtaining the injectable composite hydrogel. In step S1, the molar ratio of soluble divalent copper salt to gossypol in the mixed solution is (1~3):(1~3); the soluble divalent copper salt solution is a copper chloride solution. In step S1, the temperature of the coordination reaction is 30℃~50℃, and the time of the coordination reaction is 6h~24h. In step S2, the mass ratio of the copper-gossypol complex to sodium magnesium lithium silicate is (1~5):(20~100).

2. The method for preparing an injectable composite hydrogel as described in claim 1, characterized in that, In step S1 The molar concentration of the soluble divalent copper salt in the mixed solution is 1 mmol / L to 20 mmol / L; and / or The molar concentration of gossypol in the mixed solution is 1 mmol / L to 20 mmol / L.

3. The method for preparing an injectable composite hydrogel as described in claim 1, characterized in that, In step S1, the centrifugation is performed at a speed of 7000 rpm to 9000 rpm for 5 min to 15 min; and / or The washing process involves alternating between deionized water and anhydrous ethanol 2-3 times; and / or The drying process involves drying at 55℃~65℃ for 10h~14h.

4. The method for preparing an injectable composite hydrogel as described in claim 1, characterized in that, In step S2, the mass-to-volume ratio of the mixed powder to the water is (1~5) g:1 mL; and / or The water is deionized water; and / or The stirring time is 20s~40s; and / or The static curing time is 5 min to 10 min.

5. An injectable composite hydrogel, characterized in that, It is prepared by the method of any one of claims 1 to 4 for the preparation of an injectable composite hydrogel.

6. The use of the injectable composite hydrogel of claim 5 in the preparation of a medicament for treating osteosarcoma.

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