Injectable, self-healing hydrogel and preparation method and application thereof

Abstract

The application relates to an injectable self-healing hydrogel and a preparation method and application thereof, and belongs to the technical field of hydrogels. The preparation method of the injectable self-healing hydrogel comprises the following steps: adding a ferrous salt solution, a carbonic anhydrase inhibitor solution and a glucose oxidase solution into an aldehyde group sodium hyaluronate solution to obtain a mixed solution containing the aldehyde group sodium hyaluronate; and mixing the obtained mixed solution containing the aldehyde group sodium hyaluronate and a spermidine modified dextran solution to perform a cross-linking reaction, so as to obtain the injectable self-healing hydrogel. The aldehyde group sodium hyaluronate, the ferrous salt, the carbonic anhydrase inhibitor, the glucose oxidase and the spermidine modified dextran are cross-linked to obtain the injectable self-healing hydrogel which has high biocompatibility, high tissue affinity, is biodegradable and has tumor pH homeostasis regulation function, and has good anti-tumor effect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of hydrogels, in particular to an injectable and self-healing hydrogel as well as a preparation method and application thereof. BACKGROUND

[0002] In recent years, tumor immunotherapy represented by programmed death 1 / programmed death ligand 1 (PD-1 / PD-L1) has been widely used in various malignant tumors. At present, a variety of PD-L1 antibodies have been approved for single-agent therapy of metastatic malignant melanoma and non-small cell lung cancer. However, more and more evidence shows that due to the influence of tumor heterogeneity and immunosuppressive tumor microenvironment (TME) and other factors, the effect of immunotherapy for clinical tumors is not ideal, and the response rate of patients is only 5-30%. The immune tolerance mechanism mediated by regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) is highly related to poor prognosis effect. Metabolomics-related studies show that tumor dysbiosis characterized by tumor acidosis will directly lead to gene re-editing, metabolic reorganization and T cell anergy in tumor tissues, and further promote the occurrence, development and metastasis of tumors.

[0003] Injectable hydrogels, as a class of injectable three-dimensional networks formed by physical or chemical crosslinking of polymers, have been widely used in biomedical fields including drug delivery, tissue regeneration, etc. due to their excellent biochemical properties. At present, the market all over the world shows great interest in the transformation application of hydrogels. According to relevant statistical data, the global market of hydrogels was estimated to be about 10 billion US dollars in 2017, and it is expected to grow to 15 billion US dollars by 2020. After a period of clinical application practice, injectable hydrogels have been proved to be an effective anti-tumor drug delivery platform, which can control the release of the loaded drugs in time and space, thereby improving the therapeutic index of clinical conventional chemotherapy drugs. So far, including CS / organic phosphate PLGA-PEG-PLGA Poloxamer 407 Poly(vinyl methyl ether co-maleic anhydride) A series of injectable hydrogel preparations including hydrogels have been approved for listing and applied in the fields of tumors, vaccine adjuvants, etc. Some preparations are currently in the clinical trial stage, such as radiopaque PEG hydrogel (PEG-PEG-PEG) and biodegradable PEG hydrogel (PEG-PLGA-PEG). and ), which can be used for precise radiotherapy positioning to reduce radiation dose. However, sterilization process, scale-up production process, shelf life and user compliance (professionals and / or patients) are the main limiting problems in the clinical transformation of current injectable hydrogel preparations. It is certain that injectable hydrogels based on the delivery of antitumor drugs have great transformation potential and application value in the clinical treatment of cancer, and even have the potential to replace traditional systemic injection administration mode, providing new treatment strategies and treatment modes for the clinical treatment of tumors. In order to better accelerate the clinical efficiency, multi-modal therapeutic hydrogels based on the loading of related drugs to achieve chemotherapy, hyperthermia, immunotherapy and radiotherapy are being accelerated in research in recent years. However, at present, the research on hydrogel matrix with physiological function is relatively scarce, and how to effectively use bioactive ingredients to construct multifunctional hydrogels with biological activity is the focus of current research. SUMMARY

[0004] The present application aims to overcome the deficiencies of the prior art and provide a hydrogel with high biocompatibility, high tissue affinity, biodegradability, tumor pH homeostasis regulation function, and injectability and self-healing performance.

[0005] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0006] In a first aspect, the present application provides a preparation method of an injectable and self-healing hydrogel, comprising the following steps:

[0007] (1) adding a ferrous salt solution, a carbonic anhydrase inhibitor solution and a glucose oxidase solution into an aldehyde-modified sodium hyaluronate solution to obtain a mixed solution containing aldehyde-modified sodium hyaluronate;

[0008] (2) mixing the mixed solution containing aldehyde-modified sodium hyaluronate obtained in step (1) and a spermidine-modified dextran solution to perform cross-linking reaction, to obtain an injectable and self-healing hydrogel; the volume ratio of the mixed solution containing aldehyde-modified sodium hyaluronate and the spermidine-modified dextran solution is 1:(1-2);

[0009] In step (2), the mass ratio of aldehyde-modified sodium hyaluronate, ferrous salt, carbonic anhydrase inhibitor, glucose oxidase and spermidine-modified dextran in the spermidine-modified dextran solution in the mixed solution containing aldehyde-modified sodium hyaluronate is aldehyde-modified sodium hyaluronate: ferrous salt: carbonic anhydrase inhibitor: glucose oxidase: spermidine-modified dextran = 50:(0.1-0.2):(1-1.6):(0.1-0.2):50.

[0010] The application takes agmatine as a skeleton, and a biocompatible, high-tissue-affinity, biodegradable, injectable, self-healing hydrogel with tumor pH homeostasis regulation function and good anti-tumor effect is obtained by cross-linking reaction of aldehyde-modified sodium hyaluronate, ferrous salt, carbonic anhydrase inhibitor, glucose oxidase (GOx) and agmatine-modified dextran. The dynamic chemical cross-linking between aldehyde-modified sodium hyaluronate and agmatine-modified dextran, i.e. Schiff base reaction, enables the hydrogel to be injectable and self-healing.

[0011] Agmatine, as a kind of polyamine compound, has been found in recent years to effectively mediate and regulate tumor inhibition and immunoregulation by promoting autophagy, and has great potential in developing tumor diagnosis and treatment platform. In terms of tumor inhibition, by promoting tumor cell autophagy, agmatine can promote tumor cells to release damage-associated molecular patterns (DAMPs) such as ATP, activate innate immune cells, cause innate immune response, and directly or indirectly initiate adaptive immune response, thereby improving the microenvironment of immune suppression in tumor tissue and effectively improving the treatment effect of tumor. At the same time, by reducing the stimulation of adenosine receptors that play an immunosuppressive function, agmatine can reduce the infiltration of regulatory T cells in tumor tissue, thereby improving the microenvironment of immune escape in tumor tissue and improving the treatment effect of immunotherapy such as PD-L1. Studies have shown that fatty acid oxidation (FAO) can be enhanced by directly binding and activating mitochondrial trifunctional protein (MTP), and exogenous supplementation of agmatine can enhance the inhibition of FAO activity in cells, improve the mitochondrial activity and cytotoxic function of T lymphocytes, and effectively improve the efficiency of immunotherapy of tumors.

[0012] Based on the physiological characteristics of tumor immunotherapy tolerance dominated by tumor microenvironment pH homeostasis, the application fully utilizes the natural expression of carbonic anhydrase between normal tissues and tumor tissues, and for the first time adds carbonic anhydrase inhibitors and glucose oxidase to the hydrogel with agmatine as a skeleton to prepare an injectable, self-healing hydrogel loaded with carbonic anhydrase inhibitors and glucose oxidase. The addition of carbonic anhydrase inhibitors and glucose oxidase can regulate the H + metabolism and transport in tumor tissue cells, and synergistically break the pH homeostasis of tumor cells to improve the immune tolerance microenvironment of tumor tissue.

[0013] As a preferred embodiment of the preparation method described in the application, in step (2), the mass ratio of aldehyde-modified sodium hyaluronate, ferrous salt, carbonic anhydrase inhibitor, glucose oxidase and spermidine-modified dextran in the mixed solution of aldehyde-modified sodium hyaluronate is aldehyde-modified sodium hyaluronate: ferrous salt: carbonic anhydrase inhibitor: glucose oxidase: spermidine-modified dextran = 50:0.2:1.6:0.1:50.

[0014] Under the preferred ratio conditions, the injectable, self-healing hydrogel of the application has good anti-tumor effect, and the obtained hydrogel has uniform shape and regular three-dimensional porous structure inside, which is beneficial to load carbonic anhydrase and glucose oxidase.

[0015] As a preferred embodiment of the preparation method described in the application, in step (1), the aldehyde-modified sodium hyaluronate in the aldehyde-modified sodium hyaluronate solution is prepared by the following method: adding potassium periodate aqueous solution dropwise into sodium hyaluronate aqueous solution, reacting at room temperature in the dark, adding ethylene glycol to terminate the reaction, dialyzing, and freeze-drying to obtain aldehyde-modified sodium hyaluronate.

[0016] As a preferred embodiment of the preparation method described in the application, in step (2), the spermidine-modified dextran in the spermidine-modified dextran solution is prepared by the following method:

[0017] S1, reacting the dextran aqueous solution and the potassium periodate aqueous solution at room temperature in the dark until a clear yellow solution appears, dialyzing, and freeze-drying to obtain oxidized dextran;

[0018] S2, preparing the oxidized dextran obtained in step S1 into an oxidized dextran aqueous solution, adding spermidine borate buffer, and obtaining product a after reaction at room temperature;

[0019] S3, mixing the product a obtained in step S2 with sodium borohydride for reduction reaction, dialyzing, and freeze-drying to obtain spermidine-modified dextran.

[0020] As a preferred embodiment of the preparation method described in the application, in step S1, the molar ratio of dextran in the dextran aqueous solution to potassium periodate in the potassium periodate aqueous solution is 1:(1-5);

[0021] In step S2, the molar ratio of oxidized dextran in the oxidized dextran aqueous solution to spermidine in the spermidine borate buffer is oxidized dextran:spermidine = 1:(1-3);

[0022] In step S2, the concentration of boric acid in the spermidine borate buffer is 0.1M, and the pH value of the spermidine borate buffer is 11;

[0023] In step S3, the molar ratio of the product a to sodium borohydride is product a:sodium borohydride = 1:(2-5).

[0024] The present application can further improve the anti-tumor effect of the injectable and self-healing hydrogel by oxidizing dextran and then reacting with spermidine to obtain spermidine-modified dextran, so that the dextran can cross-link with aldehyde-modified sodium hyaluronate while having the physiological activity of spermidine. Under the preferred ratio range, when the molar ratio of dextran to potassium periodate in step S1 is 1:1, the yield of the obtained oxidized dextran is higher; when the molar ratio of the oxidized dextran to spermidine in step S2 is 1:(2-3), the yield of the obtained product a is higher, and the excess of spermidine can significantly reduce the generation of by-products, further improving the yield of product a; when the molar ratio of product a to sodium borohydride in step S3 is 1:(2-5), the excess of sodium borohydride can ensure the occurrence of reduction reaction, and the yield of the obtained spermidine-modified dextran is higher.

[0025] As a preferred embodiment of the preparation method described in the present application, the preparation method satisfies at least one of the following (I)-(III):

[0026] (I) In step (1), the ferrous salt in the ferrous salt solution is at least one of ferrous sulfate, ferrous chloride and ferrous nitrate;

[0027] (II) In step (1), the carbonic anhydrase inhibitor in the carbonic anhydrase inhibitor solution is at least one of acetazolamide, methazolamide, ethoxzolamide and dichlorphenamide;

[0028] (III) In step (2), the volume ratio of the mixed solution containing aldehyde-modified sodium hyaluronate to the spermidine-modified dextran solution is 1:1.

[0029] Under the preferred ratio conditions, the anti-tumor effect obtained by using acetazolamide (ACZ) as the carbonic anhydrase inhibitor is better.

[0030] In a second aspect, the present application provides an injectable and self-healing hydrogel prepared according to the above preparation method.

[0031] In a third aspect, the present application provides the use of the above injectable and self-healing hydrogel in the preparation of an anti-tumor material.

[0032] The applicant injects the injectable and self-healing hydrogel as an anti-tumor material into the tumor of a tumor-bearing mouse, which can significantly reduce the tumor volume and weight of the tumor-bearing mouse. Further research shows that the injectable and self-healing hydrogel of the present application can amplify the iron apoptosis signal in the tumor tissue through a cascade, effectively activate the immunogenic death in the tumor, thereby improving the tumor immune microenvironment and achieving the effect of anti-tumor.

[0033] In a fourth aspect, the application provides a use of the injectable, self-healing hydrogel and the immune checkpoint inhibitor in the preparation of an anti-tumor material.

[0034] As a preferred embodiment of the use of the application, the immune checkpoint inhibitor comprises at least one of a PD-1 / PD-L1 inhibitor and a CTLA-4 inhibitor.

[0035] The applicant found that the injectable, self-healing hydrogel combined with the PD-L1 inhibitor can amplify the iron apoptosis signal in the tumor tissue through a cascade, effectively activate the immunogenic death in the tumor, promote the release of DAMPs in the tumor tissue, improve the tumor immune microenvironment, and improve the infiltration of lymphocytes, thereby improving the treatment response rate and treatment effect of the PD-L1 inhibitor, and achieving the effects of anti-tumor and inhibiting tumor metastasis.

[0036] In a fifth aspect, the application provides an anti-tumor material comprising the injectable, self-healing hydrogel and / or the immune checkpoint inhibitor.

[0037] Compared with the prior art, the application has the following beneficial effects:

[0038] (1) The application uses spermidine as a skeleton, and an injectable, self-healing hydrogel with high biocompatibility, high tissue affinity, biodegradability, and tumor pH homeostasis regulation function is obtained by cross-linking reaction of aldehyde-modified hyaluronic acid, ferrous salt, carbonic anhydrase inhibitor, glucose oxidase, and spermidine-modified dextran, which has good anti-tumor effect.

[0039] (2) Based on the physiological characteristics of tumor immunotherapy tolerance dominated by tumor microenvironment pH homeostasis, and fully utilizing the natural expression of carbonic anhydrase between normal tissues and tumor tissues, the carbonic anhydrase inhibitor and glucose oxidase are first added to the hydrogel with spermidine as the skeleton to prepare the injectable, self-healing hydrogel loaded with carbonic anhydrase inhibitor and glucose oxidase. The addition of carbonic anhydrase inhibitor and glucose oxidase can regulate the intracellular H + metabolism and transport of tumor cells, and cooperatively break the pH homeostasis of tumor cells to improve the immune tolerance microenvironment of tumor tissues.

[0040] (3) The injectable, self-healing hydrogel based on spermidine as the skeleton can be combined with tumor immunotherapy to achieve the effects of anti-tumor and inhibiting tumor metastasis, and can be used as a drug loading platform for personalized multi-modal tumor therapy in anti-tumor materials and drugs. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 Preparation schematic of injectable self-healing hydrogel of the present application;

[0042] Figure 2 Scanning electron micrographs of injectable self-healing hydrogel of Example 1 (ACZ / GOx@SPM-HA), Example 2 (ACZ / GOx@SPM-HA2), Example 3 (ACZ / GOx@SPM-HA), Example 4 (ACZ / GOx@SPM-HA4) and Comparative Example 2 (SPM-HA) in Experimental Example 1 of the present application (scale bar 100 pm, scale bar in lower left corner of inset in Example 1 and Comparative Example 2 is 20 pm);

[0043] Figure 3 Fourier transform infrared spectra of aldehyde-modified sodium hyaluronate (ALD-HA) and spermidine-modified dextran (SPM-DEX) obtained in Example 1, sodium hyaluronate and SPM-HA of Comparative Example 2 in Experimental Example 1 of the present application;

[0044] Figure 4 Swelling coefficient determination results of injectable self-healing hydrogel of Example 1 and Comparative Example 2 in Experimental Example 1 of the present application;

[0045] Figure 5 Hemolysis rate determination results of injectable self-healing hydrogel of Example 1 and Comparative Example 2 in Experimental Example 1 of the present application;

[0046] Figure 6 Thermogravimetric analysis results of injectable self-healing hydrogel of Example 1, Comparative Examples 1-4 in Experimental Example 1 of the present application;

[0047] Figure 7 Biodegradation rate determination results of injectable self-healing hydrogel of Example 1 and Comparative Example 2 in Experimental Example 1 of the present application;

[0048] Figure 8 Self-healing and injectability characterization results of injectable self-healing hydrogel of Example 1 in Experimental Example 1 of the present application;

[0049] Figure 9 Flow chart of mouse treatment in Experimental Example 2 of the present application;

[0050] Figure 10 Representative photographs of tumor excision of mice in each treatment group after 12 days in Experimental Example 2 of the present application;

[0051] Figure 11 Tumor volume curve of mice in each treatment group in Experimental Example 2 of the present application;

[0052] Figure 12 Relative tumor growth rate of mice in each treatment group in Experimental Example 2 of the present application;

[0053] Figure 13 Tumor weight and body weight of mice in each treatment group on day 12 in Experimental Example 2 of the present application;

[0054] Figure 14 HE, TUNEL, Ki67 and CRT staining results of tumor tissues of mice in each treatment group on day 12 in Experimental Example 2 of the present application, scale bar is 100 μm;

[0055] Figure 15 Semi-quantitative analysis of TUNEL, Ki67 and CRT staining results of tumor tissues of mice in each treatment group on day 12 in Experimental Example 2 of the present application;

[0056] Figure 16 Determination results of cytokines TNF-α, IL-6 and IFN-γ in tumor tissues of mice in each treatment group on day 12 in Experimental Example 2 of the present application;

[0057] Figure 17 Flow chart of mouse treatment in Experimental Example 3 of the present application;

[0058] Figure 18 Volume curves of primary tumors (upper row) and distant tumors (lower row) of mice in each treatment group in Experimental Example 3 of the present application;

[0059] Figure 19 Relative growth rates of primary tumors and distant tumors of mice in each treatment group in Experimental Example 3 of the present application;

[0060] Figure 20 Weights of primary tumors and distant tumors of mice in each treatment group on day 12 in Experimental Example 3 of the present application;

[0061] Figure 21 HE, TUNEL, CD31, Ki67 and CD3 / CD8 staining results of distant tumors of mice in each treatment group on day 12 in Experimental Example 3 of the present application, scale bar is 100 μm;

[0062] Figure 22 Semi-quantitative analysis of TUNEL, CD31 and Ki67 staining results of distant tumors of mice in each treatment group on day 12 in Experimental Example 3 of the present application;

[0063] Figure 23 Semi-quantitative analysis of CD3+ lymphocytes and CD8+ lymphocytes of distant tumors of mice in each treatment group on day 12 in Experimental Example 3 of the present application;

[0064] Figure 24 Determination results of cytokines TNF-α, IL-6 and IFN-γ in serum of mice in each treatment group on day 12 in Experimental Example 3 of the present application.

[0065] In the above figures, * is P<0.05, ** is P<0.01, *** is P<0.001, and **** is P<0.0001. DETAILED DESCRIPTION

[0066] For better illustrating the purpose, technical scheme and advantages of the present application, the present application will be further described in combination with specific examples.

[0067] The materials, reagents and the like used in the examples, comparative examples and experimental examples can be obtained from commercial channels unless otherwise specified.

[0068] Example 1

[0069] An embodiment of the injectable, self-healing hydrogel and the preparation method thereof according to the present application, the injectable, self-healing hydrogel described in the present embodiment is prepared by the following method:

[0070] A1, 648 mg / mL potassium periodate aqueous solution is added to 0.4 mg / mL sodium hyaluronate aqueous solution, and the reaction is carried out at room temperature in the dark for 24 h, 500 μL of ethylene glycol is added and stirred for 1 h, dialysis is performed using a dialysis bag with a molecular weight cut-off of 3500 Da, and freeze-drying is performed to obtain aldehyde-modified sodium hyaluronate;

[0071] A2, the dextran aqueous solution and the potassium periodate aqueous solution are reacted at room temperature in the dark until the solution is clear yellow, dialysis is performed using a dialysis bag with a molecular weight cut-off of 12000 Da, and freeze-drying is performed to obtain oxidized dextran; the molar ratio of dextran to potassium periodate is dextran: potassium periodate = 1:1;

[0072] A3, the oxidized dextran obtained in step A2 is prepared into an oxidized dextran aqueous solution, and a pH = 11, spermidine borate buffer (boric acid concentration is 0.1 M) is added, and the reaction is carried out at room temperature for 24 h to obtain product a; the molar ratio of oxidized dextran to spermidine is oxidized dextran: spermidine = 1: (1-3);

[0073] A4, the product a obtained in step A3 is mixed with sodium borohydride to carry out a reduction reaction to obtain a reduction product, and then sodium borohydride is added, dialysis is performed using a dialysis bag with a molecular weight cut-off of 3500 Da, and freeze-drying is performed to obtain spermidine-modified dextran; the molar ratio of product a to sodium borohydride is product a: sodium borohydride = 1: (2-5);

[0074] A5, ferrous sulfate solution, acetazolamide solution and glucose enzyme solution are added to the aldehyde-modified sodium hyaluronate obtained in step A1 to obtain a mixed solution containing aldehyde-modified sodium hyaluronate;

[0075] A6, the mixture solution of aldehyde group-containing sodium hyaluronate and spermidine-modified dextran solution obtained in step A5 is mixed in a volume ratio of 1:1 at room temperature to carry out crosslinking reaction, to obtain an injectable, self-healing hydrogel; the mass ratio of aldehyde group-containing sodium hyaluronate in the mixture solution of aldehyde group-containing sodium hyaluronate, ferrous sulfate, acetazolamide, glucose oxidase and spermidine-modified dextran in the spermidine-modified dextran solution is aldehyde group-containing sodium hyaluronate: ferrous sulfate: acetazolamide: glucose oxidase: spermidine-modified dextran = 50:0.2:1.6:0.1:50.

[0076] The obtained injectable, self-healing hydrogel is named as ACZ / GOx@SPM-HA hydrogel, and the preparation schematic diagram is shown in Figure 1 .

[0077] Example 2

[0078] An embodiment of the injectable, self-healing hydrogel and the preparation method thereof, the preparation method of the injectable, self-healing hydrogel in this embodiment is similar to that in example 1, the difference is that the volume ratio of the mixture solution of aldehyde group-containing sodium hyaluronate and spermidine-modified dextran solution in step A6 is mixture solution of aldehyde group-containing sodium hyaluronate: spermidine-modified dextran solution = 1:2, and the rest of the parameter conditions are unchanged, and the obtained injectable, self-healing hydrogel is named as ACZ / GOx@SPM-HA2 hydrogel.

[0079] Example 3

[0080] An embodiment of the injectable, self-healing hydrogel and the preparation method thereof, the preparation method of the injectable, self-healing hydrogel in this embodiment is similar to that in example 1, the difference is that in step A6, the mass ratio of aldehyde group-containing sodium hyaluronate, ferrous sulfate, acetazolamide, glucose oxidase and spermidine-modified dextran in the mixture solution of aldehyde group-containing sodium hyaluronate is aldehyde group-containing sodium hyaluronate: ferrous sulfate: acetazolamide: glucose oxidase: spermidine-modified dextran = 50:0.1:1:0.2:50, and the rest of the parameter conditions are unchanged, and the obtained injectable, self-healing hydrogel is named as ACZ / GOx@SPM-HA3 hydrogel.

[0081] Example 4

[0082] An embodiment of the injectable, self-healing hydrogel and the preparation method thereof, the preparation method of the injectable, self-healing hydrogel in this embodiment is similar to that in example 1, the difference is that the ferrous sulfate solution in step A5 is replaced by ferrous chloride solution, and the acetazolamide solution is replaced by dichlorobenzenesulfonamide solution, and the rest of the parameter conditions are unchanged, and the obtained injectable, self-healing hydrogel is named as ACZ / GOx@SPM-HA4 hydrogel.

[0083] Comparative Example 1

[0084] A comparative example of the injectable, self-healing hydrogel and the preparation method thereof, the preparation method of the injectable, self-healing hydrogel of the comparative example is similar to that of Example 1, the difference is that: no ferrous salt solution, carbonic anhydrase inhibitor solution and glucose oxidase solution are added in the aldehyde group sodium hyaluronate solution in step A5, and the rest of the parameter conditions remain unchanged, the obtained injectable, self-healing hydrogel is named as SPM-HA hydrogel without Fe 2+ .

[0085] Comparative Example 2

[0086] A comparative example of the injectable, self-healing hydrogel and the preparation method thereof, the preparation method of the injectable, self-healing hydrogel of the comparative example is similar to that of Example 1, the difference is that: no carbonic anhydrase inhibitor solution and glucose oxidase solution are added in the aldehyde group sodium hyaluronate solution in step A5, and the rest of the parameter conditions remain unchanged, the obtained injectable, self-healing hydrogel is named as SPM-HA hydrogel.

[0087] Comparative Example 3

[0088] A comparative example of the injectable, self-healing hydrogel and the preparation method thereof, the preparation method of the injectable, self-healing hydrogel of the comparative example is similar to that of Example 1, the difference is that: no glucose oxidase solution is added in the aldehyde group sodium hyaluronate solution in step A5, and the rest of the parameter conditions remain unchanged, the obtained injectable, self-healing hydrogel is named as ACZ@SPM-HA hydrogel.

[0089] Comparative Example 4

[0090] A comparative example of the injectable, self-healing hydrogel and the preparation method thereof, the preparation method of the injectable, self-healing hydrogel of the comparative example is similar to that of Example 1, the difference is that: no carbonic anhydrase inhibitor solution is added in the aldehyde group sodium hyaluronate solution in step A5, and the rest of the parameter conditions remain unchanged, the obtained injectable, self-healing hydrogel is named as GOx@SPM-HA hydrogel.

[0091] Experimental Example 1

[0092] I. The physicochemical properties of the prepared injectable, self-healing hydrogel are characterized, including the following indexes:

[0093] 1. The injectable, self-healing hydrogel of Example 1 and the injectable, self-healing hydrogel of Comparative Example 2 are observed under a scanning electron microscope, and the observation results are shown in Figure 2 .

[0094] As Figure 2As shown, the injectable, self-healing hydrogel of the present application presents a regular three-dimensional porous structure, and the loading of acetazolamide and glucose oxidase does not significantly affect its spatial structure.

[0095] 2、The spermidine-modified dextran (SPM-DEX) obtained in Example 1, the aldehyde- modified sodium hyaluronate (HA-ALD), the sodium hyaluronate (HA), and the injectable, self- healing hydrogel of Comparative Example 2 were detected by a Fourier infrared spectrometer, and the results are shown in Figure 3 .

[0096] As shown in Figure 3 , the carbonyl peak of SPM-DEX is red-shifted from 1660 cm -1 to 1648 cm -1 after the formation of the hydrogel, proving the occurrence of the Schiff base reaction and the successful preparation of the injectable, self-healing hydrogel.

[0097] 3、The swelling coefficients of the injectable, self-healing hydrogels of Example 1 and Comparative Example 2 were determined, and the results are shown in Figure 4 .

[0098] As shown in Figure 4 , the hydrogels of Example 1 and Comparative Example 2 both have good swelling coefficients, although the loading of drugs has a certain influence on the swelling performance, the swelling coefficient of Example 1 can still ensure that it effectively absorbs water and is anchored around the tumor tissue after injection.

[0099] 4、The hemolysis rates of the injectable, self-healing hydrogels of Example 1 and Comparative Example 2 were determined, and the results are shown in Figure 5 .

[0100] As shown in Figure 5 , the hemolysis rates of the hydrogels of Example 1 and Comparative Example 2 are both about 0%, showing excellent blood compatibility, which also reflects that the loading of drugs does not significantly leak, thereby causing the increase of the hemolysis rate.

[0101] 5、The thermogravimetric analysis of the injectable, self-healing hydrogels of Example 1 and Comparative Examples 1-4 was performed, and the results are shown in Figure 6 .

[0102] As shown in Figure 6 , near 100℃, the sharp decrease of the mass of each hydrogel formulation corresponds to the removal of water in the system, the sharp loss of mass at 200-300℃ is caused by the combustion and decomposition of dextran and hyaluronic acid and the contained compounds, and above 300℃, the mass loss corresponds to the combustion of the remaining carbonized components, and the degree of mass loss of each formulation can well correspond to the loaded components.

[0103] 6、The in vitro degradation rates of the injectable, self-healing hydrogels of Example 1 and Comparative Example 2 were determined, and the results are shown in Figure 7.

[0104] As shown in Figure 7 , the hydrogel of Example 1 and Comparative Example 2 has a degradation rate of 60% at 21 days, has good biodegradable potential, and the loading of carbonic anhydrase inhibitors and glucose oxidase does not significantly affect the degradation rate of the hydrogel.

[0105] II. Characterization of the self-healing and injectable properties of the prepared injectable, self-healing hydrogel.

[0106] As shown in Figure 8 , the injectable hydrogel of Example 1 can achieve self-healing within 5 minutes after physical cutting, which is due to the dynamic chemical cross-linking (Schiff base reaction) that achieves self-healing of the hydrogel, and still has injectable properties after gelation, indicating that the injectable, self-healing hydrogel of the present application has the properties of short-time self-healing and long-time injectability.

[0107] From the above characterization results, the injectable, self-healing hydrogel of the present application has a regular three-dimensional porous structure, good swelling coefficient, excellent blood compatibility, biodegradability, and excellent self-healing and injectability.

[0108] Experimental Example 2

[0109] The injectable, self-healing hydrogel of the present application was evaluated for its anti-tumor effect as an anti-tumor material, and the specific scheme is as follows:

[0110] 1. Establishment of tumor-bearing mouse model

[0111] C57BL / 6 mice were subcutaneously injected with 1 x 10 6 After feeding the mice with oral squamous cell carcinoma cells (MOC1) for one week, a tumor-bearing mouse model was obtained.

[0112] 2. Experimental setup and treatment

[0113] Tumor-bearing mice with a tumor volume of about 150mm 3 were selected as experimental objects, and randomly divided into 6 groups, with 5 mice in each group. The treatment of each group is shown in Table 1.

[0114] Table 1 Treatment of mice in different treatment groups

[0115] Group Mouse Treatment Control Group Intratumoral injection of 100 μL of saline Example 1 Intratumoral injection of 100 μL of the injectable, self-healing hydrogel of Example 1 Comparative Example 1 Intratumoral injection of 100 μL of the injectable, self-healing hydrogel of Comparative Example 1 Comparative Example 2 Intratumoral injection of 100 μL of the injectable, self-healing hydrogel of Comparative Example 2 Comparative Example 3 Intratumoral injection of 100 μL of the injectable, self-healing hydrogel of Comparative Example 3 Comparative Example 4 Intratumoral injection of 100 μL of the injectable, self-healing hydrogel of Comparative Example 4

[0116] According to Figure 9 , the body weight and tumor volume of the mice were recorded every 1 day after injection, and the main organs and tumors were removed for histological analysis after euthanasia of the mice on the 12th day, including HE, Ki67, TUNE L and CRT staining, and detection of cytokines TNF-α, IL-6 and IFN-γ. The results of the statistical analysis of the body weight and tumor volume of the mice are shown inFigures 10-13 , the staining results are shown in Figures 14-15 , the cytokine detection results are shown in Figure 16 .

[0117] 3. Experimental results

[0118] As shown in Figures 10-11 , intratumoral injection of the hydrogel of Example 1 can effectively inhibit the growth of tumor volume, compared with the control group (about 1200mm 3 ), the tumor volume is reduced by about 900mm 3 on average, compared with the groups of Comparative Example 3 and Comparative Example 4 (single drug groups), the tumor volume is effectively reduced by about 300mm 3 . The tumor volume relative growth rate graph (as shown in Figure 12 ) can further show that, compared with the control group, the relative growth rate of the group of Example 1 is reduced by 75%, and compared with the single drug groups, the relative growth rate is reduced by 52% (Comparative Example 3) and 55% (Comparative Example 4) respectively, which proves that the combined use of ACZ and GOx can more effectively inhibit the growth of tumor volume.

[0119] As shown in Figure 13 , the tumor weight of each treatment group removed on the 12th day of evaluation can further prove the above conclusion, specifically, compared with the control group, the tumor weight of the group of Example 1 is reduced by 78%, and compared with the single drug groups, the tumor weight is also reduced by about 30% (Comparative Example 3) and 50% (Comparative Example 4) respectively. And from the weight change of the mice during the evaluation period, it can be preliminarily inferred that the injectable, self-healing hydrogel of the application has good biocompatibility and biosafety.

[0120] As shown in Figures 14-15 , further analysis of tumor tissue sections can find that the hydrogel of Example 1 can effectively inhibit tumor cell proliferation and promote apoptosis, combined with its semi-quantitative analysis Figure 15 , it can be further found that, compared with the control group, the expression amount of Ki67 in the tumor tissue of the group of Example 1 is down-regulated by about 97.5%, and compared with the single drug groups, the expression amount is also reduced by about 85% (Comparative Example 3) and 93% (Comparative Example 4) respectively. Correspondingly, the group of Example 1 effectively increases the TUNEL signal representing cell apoptosis, compared with the control group, the TUNEL signal of the group of Example 1 is increased by 30 times, and compared with the single drug groups, the signal is increased by about 2.6 times (Comparative Example 3) and 4 times (Comparative Example 4) respectively. Overall, the above data shows that, on the one hand, the Fe 2+, the ACZ / GOx@SPM-HA hydrogel of embodiment 1 effectively triggered the ferroptosis of tumor tissues in situ through Fenton reaction; on the other hand, in the combined drug system of ACZ / GOx, GOx catalyzes the degradation of glucose to produce gluconic acid and hydrogen peroxide, and ACZ efficiently antagonizes carbonic anhydrase to inhibit the efflux of H + , thereby continuously providing oxygen free radicals for Fenton reaction, thereby cascading amplifying the ferroptosis signal in tumor tissues.

[0121] Based on this, the levels of damage molecule related patterns (DAMPs) and related inflammatory factor signals in tumor tissues were further detected. From Figures 14-15 It can be seen that compared with the ACZ@SPM-HA hydrogel of comparative example 3 (**p<0.01) and the GOx@SPM-HA hydrogel of comparative example 4 (****p<0.0001), the combined drug system of ACZ / GOx can significantly increase the expression of calreticulin (CRT) in tumor tissues (about 0.6, 1 times respectively), thereby triggering immunogenic death in tumor tissues. Correspondingly, the levels of related inflammatory factors in tumor tissues (such as Figure 16 ) are also significantly improved. Compared with the single drug group, the levels of TNF-α, IL-6 and IFN-γ are increased by 34%, 60%, 18.8% (comparative example 3) and 100%, 80%, 53% (comparative example 4) respectively.

[0122] In summary, in situ injection of the injectable, self-healing hydrogel ACZ / GOx@SPM-HA of the present application can cascade amplify the ferroptosis signal in tumor tissues, effectively activate the immunogenic death in tumor, which has important significance for improving the tumor immune microenvironment and enhancing the response rate of tumor immunotherapy.

[0123] Experimental Example 3

[0124] The effect of the injectable, self-healing hydrogel of the present application combined with immune checkpoint inhibitors on anti-tumor metastasis was evaluated, and the specific scheme was as follows:

[0125] 1. Experimental setup and treatment

[0126] MOC1 tumor-bearing mouse models were prepared according to the method of experimental example 1, and tumor-bearing mice with right limb subcutaneous tumor volume reaching about 150mm 3 were selected as experimental objects, which were randomly divided into 4 groups, 5 mice in each group, and each group was treated according to table 2, and the treatment schedule is shown in Figure 17 In this experiment, the immune checkpoint inhibitor used was a PD-L1 inhibitor.

[0127] Table 2 Treatment of mice in different treatment groups

[0128]

[0129]

[0130] according to Figure 17 As shown, mouse body weight and tumor volume were recorded every day after injection. Blood samples were collected on day 14 for the detection of cytokines TNF-α, IL-6, and IFN-γ. Histological analysis was performed on the major organs and tumors excised after euthanasia, including Ki67, TUNEL, and CD31 staining, and detection of lymphocyte infiltration (CD3 / CD8). The statistical results and analysis of mouse body weight and tumor volume are shown below. Figures 18-20 The staining results are shown in Figures 21-22 The results of the cytokine detection are shown in [the image]. Figure 23 .

[0131] 2. Experimental Results

[0132] like Figure 18 As shown, ACZ / GOx@SPM-HA hydrogel + PD-L1 inhibitor can effectively inhibit the growth of primary and distant tumor volume, compared with the control group (distal tumor size approximately 500 mm). 3 In comparison, its distal tumor volume was controlled at approximately 200 mm. 3 The following. Furthermore, due to tumor heterogeneity and the presence of a tumor immunosuppressive microenvironment, the single-drug group (ACZ / GOx@SPM-HA hydrogel and PD-L1 inhibitor) did control the growth of distant tumors to some extent (approximately 300 mm). 3 However, individual effects varied considerably, and the overall response rate to immunotherapy was lower than that of the combination therapy group. Figure 19 The tumor volume relative growth rate plot further shows that, compared with the control group, the relative growth rate of distal tumors in the ACZ / GOx@SPM-HA hydrogel + PD-L1 inhibitor group was reduced by 66.7%. Compared with the single-drug group, this indicator was significantly reduced by 55.6% (ACZ / GOx@SPM-HA hydrogel, *p<0.05) and 40% (PD-L1 inhibitor, *p<0.05), respectively. This demonstrates that the injectable, self-healing hydrogel ACZ / GOx@SPM-HA combined with the immune checkpoint inhibitor PD-L1 inhibitor can more effectively inhibit the growth of distal tumors.

[0133] like Figure 20As shown, the tumor quality resected on day 14 in each treatment group further confirms the above conclusions. Specifically, compared with the control group, the primary tumor quality and distant tumor quality of the ACZ / GOx@SPM-HA hydrogel + PD-L1 inhibitor group were reduced by 71% and 66.5%, respectively. Compared with the monotherapy group, the tumor quality was also reduced by approximately 37.3% and 59.4% (ACZ / GOx@SPM-HA hydrogel) and 44% and 41.5% (PD-L1 inhibitor), respectively.

[0134] like Figure 21 As shown, further analysis of distal tumor tissue sections revealed that ACZ / GOx@SPM-HA hydrogel combined with PD-L1 inhibitors effectively inhibited distal tumor proliferation and promoted apoptosis. This was further supported by semi-quantitative analysis (e.g., Figure 22 As shown in the figure, further investigation revealed that, compared to the control group, the expression level of Ki67 in tumor tissues of the ACZ / GOx@SPM-HA hydrogel + PD-L1 inhibitor group was downregulated by approximately 99%. Compared to the single-drug groups, this expression level was also reduced by approximately 55% (ACZ / GOx@SPM-HA hydrogel) and 77% (PD-L1 inhibitor), respectively. Correspondingly, the combination of ACZ / GOx@SPM-HA hydrogel and PD-L1 inhibitor effectively enhanced the TUNEL signal, a characterization of apoptosis. Furthermore, CD31, which characterizes angiogenesis, was significantly downregulated. Specifically, compared to the control group, the expression level of CD31 in tumor tissue was downregulated by approximately 96% in the ACZ / GOx@SPM-HA hydrogel + PD-L1 inhibitor group. Compared to the single-drug group, the expression level was also reduced by approximately 82.8% (ACZ / GOx@SPM-HA hydrogel) and 88% (PD-L1 inhibitor), respectively. This result indicates to some extent that the combination of ACZ / GOx@SPM-HA hydrogel and PD-L1 inhibitor can inhibit the occurrence and development of metastatic tumors by downregulating CD31 expression.

[0135] Figure 21 Lymphocyte infiltration within mid-to-distal tumor tissue further demonstrates that the ACZ / GOx@SPM-HA hydrogel combined with a PD-L1 inhibitor can effectively improve the tumor immune microenvironment and enhance the response rate to immunotherapy. Figure 23As shown, according to the semi-quantitative analysis results, compared with the control group, each treatment group can significantly improve the immunogenicity in the tumor tissue, and the proportion of CD3+ lymphocytes is increased to 5.9% (ACZ / GOx@SPM-HA hydrogel group), 8.6% (PD-L1 inhibitor group), and 31.8% (ACZ / GOx@SPM-HA hydrogel + PD-L1 inhibitor group). Further CD8+ immune marker analysis shows that although the ACZ / GOx@SPM-HA hydrogel can induce the immunogenic death of the tumor tissue to a certain extent and improve the lymphocyte infiltration, the proportion of specific CD3+CD8+ lymphocytes is relatively low, only 1.6%, indicating that the induced immune response is more focused on the innate immunity. Correspondingly, the specific CD3+CD8+ lymphocytes in the PD-L1 inhibitor group can reach about 7.7%, but because of the factors of the tumor immune microenvironment, the anti-tumor metastasis effect is not very ideal. Compared with the single drug group, the proportion of CD3+ lymphocytes and specific CD3+CD8+ lymphocytes (14.2%) in the combination therapy group is increased by about 3, 6 times (****p<0.0001) and 1.5, 0.8 times (****p<0.0001), respectively, indicating that the systemic tumor-specific immune response has been generated. The expression level of related cytokines in the blood further proves the above speculation (such as Figure 24

[0136] In summary, the injectable, self-healing hydrogel ACZ / GOx@SPM-HA of the present application combined with the immune checkpoint inhibitor PD-L1 inhibitor can amplify the ferroptosis signal in the tumor tissue through a cascade, effectively activate the immunogenic death in the tumor, promote the release of DAMPs in the tumor tissue, improve the immunosuppressive microenvironment, and improve the lymphocyte infiltration, thereby effectively improving the treatment response rate and treatment effect of the PD-L1 inhibitor, and significantly inhibiting the metastasis of the tumor.

[0137] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application and not to limit the protection scope of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present application.​

Claims

1. A method for preparing an injectable, self-healing hydrogel, characterized in that, The method comprises the following steps: (1) adding a ferrous salt solution, a carbonic anhydrase inhibitor solution and a glucose oxidase solution into an aldehyde group sodium hyaluronate solution to obtain a mixed solution containing aldehyde group sodium hyaluronate; (2) mixing the mixed solution containing aldehyde group sodium hyaluronate obtained in step (1) and a spermidine modified dextran solution to perform a cross-linking reaction to obtain an injectable, self-healing hydrogel; the volume ratio of the mixed solution containing aldehyde group sodium hyaluronate and the spermidine modified dextran solution is 1:(1-2); In step (2), the mass ratio of aldehyde group sodium hyaluronate, ferrous salt, carbonic anhydrase inhibitor, glucose oxidase in the mixed solution containing aldehyde group sodium hyaluronate and spermidine modified dextran in the spermidine modified dextran solution is: aldehyde group sodium hyaluronate: ferrous salt: carbonic anhydrase inhibitor: glucose oxidase: spermidine modified dextran = 50:(0.1-0.2):(1-1.6):(0.1-0.2):50; In step (1), the carbonic anhydrase inhibitor in the carbonic anhydrase inhibitor solution is acetazolamide; In step (2), the spermidine modified dextran in the spermidine modified dextran solution is prepared by the following method: S1, the dextran aqueous solution and the potassium periodate aqueous solution are reacted at room temperature in the dark until the solution becomes clear yellow, then dialysis, freeze-drying to obtain oxidized dextran; S2, the oxidized dextran obtained in step S1 is prepared into an oxidized dextran aqueous solution, and a spermidine borate buffer solution is added, and after reaction at room temperature, the product a is obtained; S3, the product a obtained in step S2 is mixed with sodium borohydride to perform a reduction reaction, dialysis, freeze-drying to obtain spermidine modified dextran.

2. The production method according to claim 1, wherein In step (2), the mass ratio of aldehyde group sodium hyaluronate, ferrous salt, carbonic anhydrase inhibitor, glucose oxidase in the mixed solution containing aldehyde group sodium hyaluronate and spermidine modified dextran in the spermidine modified dextran solution is: aldehyde group sodium hyaluronate: ferrous salt: carbonic anhydrase inhibitor: glucose oxidase: spermidine modified dextran = 50:0.2:1.6:0.1:

50.

3. The production method according to claim 1, wherein In step (1), the aldehyde group sodium hyaluronate in the aldehyde group sodium hyaluronate solution is prepared by the following method: drop the potassium periodate aqueous solution into the sodium hyaluronate aqueous solution, react at room temperature in the dark, add ethylene glycol to terminate the reaction, then dialysis, freeze-drying to obtain aldehyde group sodium hyaluronate.

4. The production method according to claim 1, wherein In step S1, the molar ratio of dextran in the dextran aqueous solution and potassium periodate in the potassium periodate aqueous solution is dextran: potassium periodate = 1:(1-5); In step S2, the molar ratio of oxidized dextran in the oxidized dextran aqueous solution and spermidine in the spermidine borate buffer solution is oxidized dextran: spermidine = 1:(1-3); In step S2, the concentration of boric acid in the spermidine borate buffer solution is 0.1M, and the pH value of the spermidine borate buffer solution is 11; In step S3, the molar ratio of product a and sodium borohydride is product a: sodium borohydride = 1:(2-5).

5. The production method according to claim 1, wherein The preparation method satisfies at least one of the following (I)~(II): (I) in step (1), the ferrous salt in the ferrous salt solution is at least one of ferrous sulfate, ferrous chloride and ferrous nitrate; (II) in step (2), the volume ratio of the mixed solution of sodium hyaluronate containing aldehyde group and the solution of spermidine modified dextran is 1:

1.

6. An injectable, self-healing hydrogel, characterized in that, Prepared according to the preparation method of any one of claims 1-5.

7. The use of the injectable, self-healing hydrogel of claim 6 in the preparation of materials for resisting oral squamous cell carcinoma.

8. The use of the injectable, self-healing hydrogel of claim 6 in combination with an immune checkpoint inhibitor in the preparation of materials for resisting oral squamous cell carcinoma.

9. An antitumor material, characterized by, The injectable, self-healing hydrogel of claim 6 and an immune checkpoint inhibitor.

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