Injectable beta-glucan hydrogel with immunologic adjuvant function as well as preparation method and application thereof

Abstract

The invention provides injectable beta-glucan hydrogel with an immunologic adjuvant function as well as a preparation method and application of the beta-glucan hydrogel, and relates to the field of biological medicines. The hydrogel is a macromolecular network which is formed by crosslinking an aldehyde group-containing beta-glucan derivative and an amino-containing polymer through a Schiff base bond and has a remarkable immunologic adjuvant function. The hydrogel and the drug-loading sustained-release system thereof can be directly implanted into a focus position through a conventional medical injector, immune cells are remarkably activated, and the immunogenicity of tumor cells is enhanced, so that the immune microenvironment of solid tumors is effectively adjusted. Particularly, when the beta-glucan derivative hydrogel and the drug sustained-release system thereof provided by the invention are applied in combination with radiotherapy, the beta-glucan derivative hydrogel and the drug sustained-release system thereof show excellent tumor treatment efficiency. The preparation process is simple and mild, the obtained hydrogel has good biocompatibility and controllable drug release characteristics, and a novel and efficient adjuvant therapy strategy is provided for tumor immunotherapy.

Description

Technical Field

[0001] This invention belongs to the field of biomedical products technology, and in particular relates to a method for preparing an injectable β-glucan hydrogel with immune adjuvant function and its application in drug sustained-release systems. Background Technology

[0002] Hydrogels are highly hydrated three-dimensional (3D) macromolecular networks with broad application prospects in the medical and biomedical fields due to their excellent biocompatibility, chemical modifiability, physical tunability, and relatively simple processing procedures. Currently, hydrogels have demonstrated significant advantages in constructing tissue engineering scaffolds to repair damaged tissues and organs, and as drug carriers for controlled release. Especially in anti-tumor therapy, the sustained release of drugs at the tumor site via hydrogels not only improves therapeutic targeting but also reduces systemic toxicity, making it an ideal drug delivery platform (see Adv. Mater. 2024, 36, 2313188).

[0003] In recent years, tumor immunotherapy, as an emerging strategy to activate the body's own immune system to recognize and eliminate tumor cells, has become an important direction in cancer treatment (see Chem. Soc. Rev. 2023, 52, 47-96). In this process, immune adjuvants, as key components for enhancing antigen-specific immune responses, can effectively activate innate and adaptive immune responses, thereby significantly improving the efficacy of immunotherapy.

[0004] Existing hydrogel adjuvant systems often rely on inert polymeric backbones to load adjuvants. In these systems, although the hydrogel backbone can achieve local sustained release of adjuvants, it faces the following core problems: (1) The inert backbone itself has no immune function, so a high dose of adjuvant is required to achieve sufficient immune stimulation, and a large amount of inert material occupies the administration volume, reducing the effective activity per unit volume; (2) The inert backbone cannot participate in antigen presentation or immune cell activation, and exists only as an "empty carrier", and cannot provide multiple and continuous stimulation signals for the immune microenvironment; (3) When the large amount of inert polymer introduced is degraded or cleared in vivo, it may increase the biological metabolic stress. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing β-glucan derivative hydrogels and their application in drug sustained-release systems. The β-glucan derivative hydrogels and their drug sustained-release systems provided by this invention not only possess excellent biocompatibility and immune-activating functions, but also enable controlled sustained release of drugs, effectively improving drug release kinetics and demonstrating broad clinical application prospects.

[0006] To address the limitations of existing hydrogel adjuvant systems that often rely on inert polymeric frameworks to load adjuvants, this invention directly constructs a hydrogel framework using immunologically active molecules. On the one hand, this combines the adjuvant carrier and the active source into one, significantly increasing the intensity of local immune stimulation. On the other hand, it significantly reduces the amount of inert material used, alleviating degradation burden and potential toxicity, and achieving synergistic amplification of immune signals between the carrier and the adjuvant.

[0007] Naturally derived immune adjuvants are gaining increasing attention in vaccine development and immunotherapy due to their low toxicity, high stability, and cost advantages. Among them, β-glucan, a polysaccharide formed by glucose molecules linked by glycosidic bonds, is widely found in various organisms such as oats, yeast, mushrooms, and bacteria. Glucans from different sources differ in structure, physiological activity, and immunomodulatory capacity. Yeast-β-glucan was the first to be discovered to possess significant immunomodulatory activity, significantly promoting the maturation of dendritic cells and macrophages and antigen presentation, and activating T cell-mediated adaptive immune responses, thus showing broad prospects for immunotherapy applications.

[0008] The present invention adopts the following technical solution.

[0009] An injectable hydrogel with immune adjuvant function is prepared from amino-containing polymer A and aldehyde-containing β-glucan derivative B.

[0010] In this invention, an amino-containing polymer A is crosslinked with an aldehyde-containing β-glucan derivative B to form the injectable hydrogel with immunoadjuvant function. The hydrogel network of this invention is obtained by crosslinking amino-rich polymer A with aldehyde-modified β-glucan derivative B through the formation of Schiff base bonds.

[0011] In this invention, the amino-containing polymer A is selected from one or more of the following reagents: chitosan, trimethyl chitosan, carboxymethyl chitosan, hydroxyethyl chitosan, hydroxypropyl chitosan, and chitosan grafted with benzothiazole-5-carboxylic acid; the aldehyde-containing β-glucan derivative B is prepared by modifying β-glucan with small molecules containing both aldehyde and carboxyl functional groups; the small molecules containing both aldehyde and carboxyl functional groups are selected from one or more of the following reagents: p-aldehyde benzoic acid, o-carboxybenzaldehyde, m-carboxybenzaldehyde, 3-(4-aldehydephenyl)propionic acid, 3-aldehyde indole-5-carboxylic acid, 3-oxopropionic acid, 5-oxovalerate, and succinic acid.

[0012] Preferably, the molecular weight of chitosan grafted with trimethyl chitosan, carboxymethyl chitosan, hydroxyethyl chitosan, hydroxypropyl chitosan, or benzothiazole-5-carboxylic acid is 5 kDa to 1000 kDa.

[0013] In this invention, the aldehyde-containing β-glucan derivative B is prepared by modifying β-glucan with a small molecule containing both carboxyl and aldehyde functional groups; specifically, the chemical structural formula of the glucan derivative of this invention is as follows: Wherein, R represents a hydrogen atom or a small molecule residue containing an aldehyde group, and n is the number of repeating units in the sugar ring of the dextran, which is a conventional representation. The small molecule containing both aldehyde and carboxyl functional groups is selected from one or more of the following reagents: p-aldehyde benzoic acid, o-carboxybenzaldehyde, m-carboxybenzaldehyde, 3-(4-aldehydephenyl)propionic acid, 3-aldehyde indole-5-carboxylic acid, 3-oxopropionic acid, 5-oxovalerate, and succinic half-aldehyde.

[0014] In this invention, the grafting rate of small molecules containing both carboxyl and aldehyde functional groups to the hydroxyl groups on β-glucan is 0.1% to 100%; the small molecules containing both carboxyl and aldehyde functional groups are selected from p-aldehyde benzoic acid (CAS No. 619-66-9), o-carboxybenzaldehyde (CAS No. 119-67-5), m-carboxybenzaldehyde (CAS No. 619-21-6), 3-(4-aldehydephenyl)propionic acid (CAS No. 34961-64-3), 3-aldehyde indole-5-carboxylic acid (CAS No. 148563-41-1), 3-oxopropionic acid (CAS No. 926-61-4), 5-oxovalerate (CAS No. 51306-02-1), and succinic acid (CAS No. 61452-29-5).

[0015] This invention constructs a β-glucan immunoadjuvant hydrogel by modifying β-glucan and chemically crosslinking it with an amino-rich polymer. In the β-glucan modification process, the carboxyl groups in the small molecule compound structure are first activated using a coupling agent (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC×HCl)) and a co-coupling agent (4-dimethylaminopyridine (DMAP)). This allows the carboxyl groups to react efficiently with the hydroxyl groups in the β-glucan structure, forming stable ester bonds. This successfully grafts the small molecule onto the β-glucan molecular backbone, achieving the modification of β-glucan and ultimately yielding an aldehyde-modified β-glucan derivative.

[0016] In this invention, β-glucan is derived from black yeast and has a molecular weight between 50 kDa and 2000 kDa. This invention discloses a method for preparing the above-mentioned β-glucan derivative, comprising the following steps: (1) React small molecules containing both carboxyl and aldehyde functional groups with coupling agents and co-coupling agents to form a carboxyl-activated small molecule solution; (2) The carboxyl-activated small molecule solution is mixed with the β-glucan solution to obtain the β-glucan derivative.

[0017] Specifically, the preparation method of the above-mentioned β-glucan derivative includes the following steps: (1) Dissolve the powder of a small molecule compound containing both carboxyl and aldehyde functional groups in DMSO, and react it with a coupling agent and a co-coupling agent to form solution A; (2) Dissolve β-glucan in DMSO to form solution B; (3) Add solution A to solution B, react for a certain time, and then obtain the target product by dialysis and freeze drying.

[0018] In this invention, in step (1), the molar ratio of the carboxyl group of the coupling agent to the small molecule compound is 0.05:1 to 20:1, preferably 0.5:1 to 5:1; the molar ratio of the carboxyl group of the co-coupling agent to the small molecule compound is 0.05:1 to 20:1, preferably 0.1:1 to 2:1; the reaction temperature is 10 to 90 °C, preferably 25 to 37 °C, and the reaction time is 0 to 120 hours, preferably 24 to 96 hours.

[0019] In this invention, in step (1), the solvent in the small molecule solution containing both carboxyl and aldehyde functional groups is DMSO, and the concentration of the small molecule is 0.01 mg / mL to 1000 mg / mL, preferably 10 mg / mL to 500 mg / mL; in step (3), the molar ratio of the small molecule to the hydroxyl group on β-glucan is 0.001:1 to 100:1, preferably 0.01:1 to 20:1, most preferably 0.1:1 to 6:1, the reaction temperature is 10 to 90 °C, preferably 25 to 37 °C, and the reaction time is 0 to 120 hours, preferably 24 to 96 hours.

[0020] In this invention, the grafting rate (molar ratio of aldehyde group to hydroxyl group in the dextran structure) of the small molecule containing both carboxyl and aldehyde functional groups is preferably 1% to 33%, more preferably 1% to 16%, and even more preferably 1% to 8%. In solution A, the small molecule containing both carboxyl and aldehyde functional groups is p-aldehyde benzoic acid, o-carboxybenzaldehyde, m-carboxybenzaldehyde, 3-(4-aldehydephenyl)propionic acid, 3-aldehyde indole-5-carboxylic acid, 3-oxopropionic acid, 5-oxovalerate, or succinic acid, and its concentration is preferably 0.01 mg / mL to 1000 mg / mL, more preferably 10 mg / mL to 500 mg / mL, and even more preferably 20 mg / mL to 100 mg / mL.

[0021] In this invention, the coupling agent is preferably 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC×HCl), dicyclohexylcarbodiimide (DCC), or carbonyldiimidazole (CDI), with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC×HCl) being the most preferred. The co-coupling agent is preferably hydroxybenzotriazole (HOBt), N-hydroxysuccinimide (NHS), pyridine, 4-dimethylaminopyridine (DMAP), or triethylamine, with 4-dimethylaminopyridine (DMAP) being the most preferred. Preferably, the molar ratio of 4-dimethylaminopyridine to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:5 to 1:1.

[0022] This invention discloses a method for preparing the above-mentioned injectable β-glucan hydrogel with immune adjuvant function, comprising the following steps: (1) Adjust the pH of the amino-containing polymer A solution to 5-8, and use it as solution a; (2) Adjust the pH of β-glucan derivative B containing aldehyde group to 5-8, and use it as solution b; (3) Mix solution a and solution b, let stand, and obtain injectable β-glucan hydrogel with immune adjuvant function; the ratio of the number of amino moles of polymer A to the number of aldehyde moles of aldehyde-containing β-glucan derivative B is 60:1 to 1:60, preferably, the molar ratio is 20:1 to 5:1; the standing time is 1 to 720 minutes.

[0023] Furthermore, the preparation method of the above-mentioned injectable β-glucan hydrogel with immune adjuvant function includes the following steps: (1) Dissolve the amino-containing polymer A in water or a buffer solution (such as PBS buffer), and adjust the pH of the solution to 5-8 using dilute hydrochloric acid and dilute sodium hydroxide solution, as solution a; in solution a, the mass concentration of the amino-containing polymer A is 0.1 wt%-40 wt%; (2) Dissolve the aldehyde-containing β-glucan derivative B in water, a buffer solution (such as PBS buffer), or an organic solvent (such as ethanol), and adjust the pH of the solution to 5-8 using dilute hydrochloric acid and dilute sodium hydroxide solution to obtain solution b; in solution b, the mass concentration of the aldehyde-containing β-glucan derivative B is 0.1 wt%-40 wt%; (3) Mix solution a and solution b evenly and let stand at room temperature to obtain injectable hydrogel. The ratio of the number of amino moles of polymer A in solution a to the number of aldehyde moles of aldehyde-modified β-glucan derivative B in solution b is 60:1~1:60. The gelation time at room temperature is 1~720 minutes.

[0024] Preferably, in solution a, the mass concentration of amino-rich polymer A is 0.1 wt% to 40 wt%, more preferably 1 wt% to 10 wt%. In solution b, the mass concentration of aldehyde-containing β-glucan derivative B is 0.1 wt% to 40 wt%, more preferably 1 wt% to 10 wt%.

[0025] This invention discloses an injectable hydrogel drug delivery system with immune adjuvant function, comprising the aforementioned injectable hydrogel with immune adjuvant function and drug molecules.

[0026] In the process of mixing the above-mentioned amino-containing polymer A and aldehyde-containing β-glucan derivative B into a gel, the present invention encapsulates the drug molecule C in the hydrogel matrix to prepare an injectable hydrogel drug-loaded sustained-release system with immune adjuvant function. The drug molecule C is selected from one or more of small molecule inhibitors or anti-tumor drugs.

[0027] Furthermore, this invention provides an injectable, drug-encapsulated hydrogel system with immune adjuvant function and its preparation method, comprising: (1) Dissolve the amino-containing polymer A in water or a buffer solution (such as PBS buffer), and adjust the pH of the solution to 5-8 using dilute hydrochloric acid and dilute sodium hydroxide solution, as solution aa; in solution aa, the mass concentration of the amino-containing polymer A is 0.1 wt%-40 wt%; (2) Dissolve β-glucan derivative B containing aldehyde group in water, buffer solution (such as PBS buffer) or organic solvent (such as ethanol), add drug molecule C solution that is pre-dissolved in water, buffer solution or organic solvent, and adjust the pH of the mixed solution to 5 ~ 8 by dilute hydrochloric acid solution and dilute sodium hydroxide solution, as solution bb; in solution bb, the mass concentration of β-glucan derivative B containing aldehyde group is 0.1 wt% ~ 40 wt%, and the concentration of drug molecule C is 0.01 mg / mL ~ 100 mg / mL; (3) Mix solution aa and solution bb evenly and let stand at room temperature to obtain a hydrogel sustained-release system containing the drug.

[0028] Preferably, in solution bb, the mass concentration of aldehyde-containing β-glucan derivative B is 0.1 wt% to 40 wt%, more preferably 1 wt% to 10 wt%, and the concentration of drug molecule C is 0.01 mg / mL to 100 mg / mL, more preferably 0.1 mg / mL to 10 mg / mL.

[0029] In this invention, the drug molecule C includes small molecule inhibitors and antitumor drugs. Further, the small molecule inhibitors include, but are not limited to, one or more of the CD39 inhibitor POM-1 and the IDO1 inhibitor 1-methyl-D-tryptophan (1-MT); the antitumor drugs include, but are not limited to, one or more of doxorubicin hydrochloride (DOX), gemcitabine (GEM), and decitabine (DAC).

[0030] This invention discloses the application of the above-mentioned injectable hydrogel with immune adjuvant function or injectable hydrogel drug-loaded sustained-release system with immune adjuvant function in drug delivery, immunotherapy and combination therapy; or the application of the above-mentioned injectable hydrogel with immune adjuvant function or injectable hydrogel drug-loaded sustained-release system with immune adjuvant function in the preparation of drug delivery drugs, immunotherapy drugs and combination therapy drugs.

[0031] This invention discloses a hydrogel system comprising the aforementioned β-glucan derivative hydrogel and its drug encapsulation system. This invention discloses the application of the aforementioned β-glucan derivative in the field of drug delivery. Compared to traditional hydrogels, β-glucan derivative hydrogels not only have widely available and inexpensive raw materials, but also possess good biocompatibility, ensuring their stability and safety in vivo. Furthermore, they inherently possess certain immunomodulatory functions, stimulating the immune system and promoting antigen presentation, thereby improving the efficacy of immunotherapy and showing great potential in tumor immunotherapy. This invention discloses the application of the aforementioned β-glucan derivative hydrogel and its encapsulation system in drug delivery. The preparation method of this hydrogel is simple, easy to scale up, and has good economic benefits and scalability. Attached Figure Description

[0032] Figure 1 The grafting rate is the β-glucan derivative prepared under the conditions of Examples 3 and 4.

[0033] Figure 2 The in vitro activation of DC cells by the β-glucan derivative prepared under the conditions of Example 3 is shown.

[0034] Figure 3 Photograph of the injectable hydrogel with immune adjuvant function prepared under the conditions of Example 43.

[0035] Figure 4 Storage modulus (G') and loss modulus (G'') of the injectable hydrogels with immune adjuvant function prepared under the conditions of Examples 43 and 55.

[0036] Figure 5The in vitro activation of DC cells by the injectable hydrogel with immune adjuvant function prepared under the conditions of Example 43.

[0037] Figure 6 The injectable hydrogel with immune adjuvant function prepared under the conditions of Example 43 is effective for CD11c. + MHC II of cells + The function of expression.

[0038] Figure 7 The effect of an injectable hydrogel with immune adjuvant function prepared under the conditions of Example 43 on macrophage polarization.

[0039] Figure 8 The MC38 tumor volume on day 18 following combined radiotherapy with the injectable hydrogel or its drug-release system with immunoadjuvant function provided in Examples 43 and 58 (with an average mouse tumor volume of approximately 100 mm²) was measured. 3 Treatment should begin at that time. Detailed Implementation

[0040] This invention provides an injectable hydrogel with immunoadjuvant function and a sustained-release system for encapsulating drugs. The hydrogel framework is prepared by chemical cross-linking of an amino-rich polymer A with an aldehyde-modified β-glucan derivative B. The amino groups in polymer A and the aldehyde-modified β-glucan derivative B can form Schiff base bonds; this process is a dynamically reversible reaction, thus the prepared hydrogel exhibits self-healing properties.

[0041] The present invention does not limit the specific modification method described herein; any method known to those skilled in the art is acceptable.

[0042] In this invention, the injectable β-glucan hydrogel with immune adjuvant function is preferably prepared by the following method: The amino-rich polymer A is dissolved in water or a buffer solution, and the pH of the solution is adjusted to 5-8 using dilute hydrochloric acid solution and dilute sodium hydroxide solution to obtain solution a; Aldehyde-modified β-glucan derivative B is dissolved in water, a buffer solution (such as PBS buffer), or an organic solvent (such as ethanol), and the pH of the solution is adjusted to 5-8 using dilute hydrochloric acid solution and dilute sodium hydroxide solution to obtain solution b; Mix solution a and solution b evenly and let stand at room temperature to obtain an injectable hydrogel with immune adjuvant function.

[0043] In this invention, the amino-rich polymer A is preferably chitosan and its derivatives, and more preferably carboxymethyl chitosan.

[0044] In this invention, the degree of carboxymethyl substitution of carboxymethyl chitosan is preferably ≥1%, more preferably ≥50%, and even more preferably ≥90%, with an isoelectric point of 3-4.

[0045] In this invention, β-glucan is preferably yeast-derived β-glucan, and more preferably yeast-derived water-soluble β-glucan.

[0046] In this invention, the small molecule used for β-glucan aldehyde modification that contains both aldehyde and carboxyl functional groups is preferably an aromatic compound, more preferably a benzene ring derivative, and even more preferably p-aldehyde benzoic acid.

[0047] In this invention, the coupling agent is preferably 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl), dicyclohexylcarbodiimide (DCC), or carbonyldiimidazole (CDI), with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) being the most preferred. The co-coupling agent is preferably hydroxybenzotriazole (HOBt), N-hydroxysuccinimide (NHS), pyridine, 4-dimethylaminopyridine (DMAP), or triethylamine, with 4-dimethylaminopyridine (DMAP) being the most preferred.

[0048] In this invention, the grafting ratio of p-aldehyde benzoic acid to the hydroxyl groups of β-glucan is preferably 1% to 33%, more preferably 1% to 16%, and even more preferably 1% to 8%.

[0049] In this invention, the mass concentration of amino-rich polymer A in solution a is 0.1 wt% to 40 wt%, more preferably 1 wt% to 10 wt%, and most preferably 4 wt%.

[0050] In this invention, the mass concentration of aldehyde-modified β-glucan derivative B in solution b is 0.1 wt% to 40 wt%, more preferably 0.1 wt% to 10 wt%, and most preferably 4 wt%.

[0051] In this invention, the temperature for preparing injectable hydrogels with immune adjuvant function is preferably 10 to 50°C, more preferably 25 to 37°C, and most preferably 25°C.

[0052] In a specific embodiment of the present invention, the preparation process of the injectable hydrogel with immune adjuvant function includes the following steps: First, β-glucan grafted with p-aldehyde benzoic acid was prepared: 1 g of β-glucan was dissolved in 50 mL of DMSO to prepare a β-glucan solution. 926 mg of p-aldehyde benzoic acid was dissolved in 10 mL of DMSO to prepare a p-aldehyde benzoic acid solution. The carboxyl group of p-aldehyde benzoic acid was activated for 1 h using EDC·HCl and DMAP. The concentration of EDC·HCl was 925 mmol / L, and the concentration of DMAP was 310 mmol / L. Subsequently, the DMSO solution of p-aldehyde benzoic acid was slowly added to the β-glucan solution, and the reaction was stirred for 72 h. After dialysis and freeze-drying, p-aldehyde benzoic acid grafted β-glucan was obtained.

[0053] Carboxymethyl chitosan was dissolved in deionized water to form solution a, with a pH of 5-8 and a concentration of 4 wt%. β-glucan grafted with p-aldehyde benzoic acid was dissolved in water as solution b, with a pH of 5-8 and a concentration of 4 wt%. Mix solution a and solution b at a volume ratio of 1:1 and incubate at 25°C for 3 minutes to obtain an injectable hydrogel with immune adjuvant function.

[0054] In a specific embodiment of the present invention, the preparation process of the injectable, immunoadjuvant-functional β-glucan hydrogel-encapsulated drug sustained-release system includes the following steps: First, β-glucan grafted with p-aldehyde benzoic acid was prepared: 1 g of β-glucan was dissolved in 50 mL of DMSO to prepare a β-glucan solution. 926 mg of p-aldehyde benzoic acid was dissolved in 10 mL of DMSO to prepare a p-aldehyde benzoic acid solution. The carboxyl group of p-aldehyde benzoic acid was activated for 1 h using EDC·HCl and DMAP. The concentration of EDC·HCl was 925 mmol / L, and the concentration of DMAP was 310 mmol / L. Subsequently, the DMSO solution of p-aldehyde benzoic acid was added to the β-glucan solution, and the reaction was stirred for 72 h. After dialysis and freeze-drying, p-aldehyde benzoic acid grafted β-glucan was obtained.

[0055] Carboxymethyl chitosan was dissolved in deionized water to prepare solution aa, with a pH of 5-8 and a concentration of 4 wt%. β-glucan grafted with p-aldehyde benzoic acid was dissolved in deionized water, and CD39 inhibitor POM-1 pre-dissolved in deionized water was added. The mixture was mixed evenly to form solution bb with a pH of 5-8. The concentration of β-glucan grafted with p-aldehyde benzoic acid was 4 wt%, and the concentration of POM-1 was 1 mg / mL. Mix solution aa and solution bb at a volume ratio of 1:1 and incubate at 25°C for 3 min to obtain an injectable, sustained-release system with β-glucan hydrogel encapsulating the drug and possessing immune adjuvant function.

[0056] This invention provides an application of the injectable β-glucan hydrogel with immune adjuvant function described in the above-described technical solution, or the injectable β-glucan hydrogel with immune adjuvant function prepared by the preparation method described in the above-described solution, in immunotherapy.

[0057] According to the present invention, the injectable β-glucan hydrogel with immune adjuvant function has a strong immune activation function and can be used for immunotherapy of tumors, combination therapy, or immunotherapy of other diseases.

[0058] The raw materials involved in this invention are existing products, and the specific preparation operations and performance testing are conventional techniques, briefly described below: Cell culture: MC38, B16F10, 4T1, RAW264.7 cell lines and BMDC and BMDM extracted from C57BL / 6 mice were used. The cell culture method can be any method well known to those skilled in the art and is not particularly limited. Preferably, the cells were cultured in DMEM medium containing 10% inactivated fetal bovine serum under the following conditions: 37°C in an incubator containing 5% carbon dioxide (v / v).

[0059] In vitro immune activation assay: This invention applies the resulting injectable hydrogel with immune adjuvant function to cells. Flow cytometry was used to verify changes in the expression of relevant markers on the surface of BMDCs and BMDMs after cell treatment. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was used to verify the mRNA expression levels of relevant cytokines (e.g., IFN-β, IL-6, IL-1β, etc.) in the treated cells. mRNA expression levels were measured using 2^... (-ΔΔCt) The method is used to calculate.

[0060] In vivo tumor treatment efficiency: This invention combines the resulting injectable β-glucan hydrogel with immunoadjuvant function with radiation to treat MC38 mouse subcutaneous colon cancer tumor models, B16F10 mouse subcutaneous melanoma tumor models, and 4T1 mouse subcutaneous breast cancer tumor models. Tumor volume changes and mouse survival were monitored.

[0061] The results show that the injectable β-glucan hydrogel with immune adjuvant function provided by the present invention can exert its immune adjuvant effect, has excellent immune activation effect, good tumor treatment function, and significantly improves the efficacy of radiation-induced tumor treatment.

[0062] To further illustrate the present invention, the preparation method and application of a β-glucan hydrogel and its drug-loaded sustained-release system provided by the present invention are described in detail below with reference to embodiments. However, these descriptions should not be construed as limiting the scope of protection of the present invention. The raw materials used in the present invention are all existing products, and the specific preparation operations, performance tests, and data statistical analyses are all conventional techniques. Animal experiments meet the relevant requirements of Soochow University.

[0063] Examples 1-32: Preparation of aldehyde-modified β-glucan derivatives A β-glucan solution was prepared by dissolving 1 g of β-glucan (CAS: 9012-72-0, Beijing Puxitang Biotechnology Co., Ltd.) in 50 mL of DMSO and stirring until dissolved. A small molecule containing both aldehyde and carboxyl functional groups was prepared by dissolving 6.18 mmol of the small molecule in 10 mL of DMSO and stirring until dissolved. The carboxyl group in the small molecule containing both aldehyde and carboxyl functional groups was activated for 1 h using EDC·HCl and DMAP, with 9.25 mmol of EDC·HCl and 3.08 mmol of DMAP used. The reaction was carried out at 37 °C with stirring for 1 h.

[0064] A carboxyl-activated small molecule solution was added to a β-glucan solution, and the reaction was allowed to proceed for 72 h. After the reaction, the reaction solution was placed in a dialysis bag with a molecular weight cutoff of 7000 Da for dialysis: dialysis with deionized water was performed for 72 h to completely remove residual organic solvents and free small molecules. After dialysis, the solution in the dialysis bag was collected and freeze-dried to obtain the final product.

[0065] Table 1 shows the types of small molecules containing both aldehyde and carboxyl functional groups, as well as the molar ratio of hydroxyl groups on β-glucan to small molecules containing both aldehyde and carboxyl functional groups.

[0066] Table 1 Examples 1-32 ;

[0067] This invention performs grafting assays on the aldehyde-modified β-glucans prepared in Examples 1-32. Among them, Figure 1 The grafting rate of the aldehyde-modified β-glucan derivatives prepared under the conditions of Examples 3 and 4 was measured; and their adjuvant properties were verified. Figure 2 The effect of DC activation on the aldehyde-modified β-glucan derivative prepared under the conditions of Example 3.

[0068] Examples 33-56: Preparation of injectable hydrogels with immunoadjuvant function Adjuvant hydrogels were prepared using the β-glucan grafted with aldehyde benzoic acid prepared in Example 3.

[0069] Amino-rich polymer A (component A) was dissolved in deionized water to form solution a, with a mass concentration of 3-4 wt%. The pH of the solution was adjusted to 5.5 using dilute hydrochloric acid and dilute sodium hydroxide solutions. Aldehyde-modified β-glucan B (component B) was dissolved in deionized water to form solution b, with a mass concentration of 3-6 wt%. The pH of the solution was adjusted to 7 by dilute hydrochloric acid solution and dilute sodium hydroxide solution. Mix solution a and solution b thoroughly at a volume ratio of 1:1 and let stand at room temperature for 3 minutes to obtain an injectable β-glucan hydrogel with immunoadjuvant properties. Table 2 shows the types of amino-rich polymers and the mass ratio of amino-rich polymers to the β-glucan grafted with aldehyde benzoic acid prepared in Example 2.

[0070] Table 2 Examples 33-56 ;

[0071] The polymers used in the above embodiments, including chitosan (CAS: 9012-76-4), carboxymethyl chitosan (CAS: 83512-85-0), trimethyl chitosan (CAS: 52349-26-5), hydroxyethyl chitosan (CAS: 123938-86-3), and hydroxypropyl chitosan (CAS: 84069-44-3), were all commercially available. The chitosan grafted with benzothiazole-5-carboxylic acid (CAS No.: 68867-17-4) was prepared by modifying the chitosan structure after activating the carboxyl group in the benzothiazole-5-carboxylic acid structure with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and N-hydroxysuccinimide (NHS), using conventional techniques. The grafting rate of benzothiazol-5-carboxylic acid onto the amino groups of chitosan backbone was 9.35% (mole fraction), as determined by UV-Vis spectrophotometry.

[0072] Performance testing is a conventional technique, and the experimental method involved in this invention is preferably performed as follows.

[0073] The injectability of the prepared β-glucan hydrogel was verified. Images and injectability of the hydrogel prepared in Example 43 (a hydrogel formed from carboxymethyl chitosan and p-aldehyde benzoic acid-modified β-glucan) are shown below. Figure 3As shown. The storage modulus and loss modulus results of the hydrogels prepared under the conditions of Examples 43 and 55 (hydrogels formed by chitosan grafted with benzothiazole-5-carboxylic acid and β-glucan modified with p-aldehyde benzoic acid) are as follows. Figure 4 As shown.

[0074] Cell culture: Common cell lines such as MC38, B16F10, 4T1, and RAW264.7 were used. Bone marrow extracted from C57BL / 6 cells was used to culture BMDC and BMDM. The cell culture method can be any method well known to those skilled in the art and there are no special limitations. In this invention, cells are preferably cultured in 1640 medium containing 10% inactivated fetal bovine serum under the following conditions: 37°C in an incubator containing 5% carbon dioxide (v / v).

[0075] In vitro immune activation assay: To verify the immunoadjuvant function of the β-glucan hydrogel, the in vitro immune activation of dendritic cells (DCs) and macrophages by the hydrogel formed from β-glucan grafted with aldehyde benzoic acid and carboxymethyl chitosan provided in Example 43 was tested. BMDCs and BMDM cells were incubated at 1.0 × 10⁻⁶ cells / cells. 6 The dendritic cells (DCs) were seeded at a density of 1 cells / well in 12-well plates, and pre-prepared hydrogel degradation solution was added to achieve a final concentration of 1.0 mg / mL for each well. The cells were then incubated at 37°C and 5% CO2 for 8 hours. Flow cytometry was used to verify changes in relevant biomarkers on the surface of the DCs after treatment. Figure 5 , 6 Section 7 shows a comparison of the in vitro activation ability of the hydrogel formed by the β-glucan derivative and carboxymethyl chitosan provided in Example 43 on DC cells. The results show that the hydrogel can also effectively activate DC cells and enhance MHC II activity. + The expression rate of [something]. Simultaneously, the hydrogel can also promote M1 polarization of macrophages.

[0076] Examples 57-71: Preparation of an injectable, sustained-release system for drugs encapsulated in β-glucan hydrogel with immunoadjuvant function. Referring to Example 43, an injectable β-glucan hydrogel with immune adjuvant function was prepared to load drug molecules.

[0077] Carboxymethyl chitosan was dissolved in deionized water to form solution aa. The pH of this solution was adjusted to 7 using dilute hydrochloric acid and dilute sodium hydroxide solutions, and the concentration was selected as 4 wt%. Aldehyde-modified β-glucan derivative B was dissolved in water or an organic solvent (such as ethanol), and a solution of drug molecule C pre-dissolved in water or an organic solvent was added. The mixture was then thoroughly mixed to form solution b. In solution b, the mass concentration of aldehyde-modified β-glucan B was 4 wt%, and the concentration of drug molecule C was 0.1 mg / mL to 10 mg / mL. The pH of the solution was adjusted to 7 using dilute hydrochloric acid and dilute sodium hydroxide solutions.

[0078] Mix solutions aa and bb thoroughly at a volume ratio of 1:1, and let stand at room temperature for 3 minutes to obtain a hydrogel sustained-release system containing the drug.

[0079] Injectable hydrogels with immune adjuvant function are preferred among small molecule inhibitors and antitumor drugs such as CD39 inhibitor POM-1, IDO1 inhibitor 1-MT, decitabine (DAC), doxorubicin hydrochloride (DOX), and gemcitabine (GEM). The types of small molecule inhibitors and antitumor drugs selected, as well as their mass concentrations in solution bb, are shown in Table 3.

[0080] Table 3 Examples 57-71

[0081] Performance testing is a conventional technique, and the experimental method involved in this invention is preferably performed as follows.

[0082] In vivo tumor treatment efficiency: This invention combines the obtained hydrogel (e.g., the hydrogel prepared in Example 43) and the hydrogel drug sustained-release system (e.g., the POM-1-loaded hydrogel in Example 58) with radiotherapy in a subcutaneous MC38 colon cancer tumor model. MC38 colon cancer cells were subcutaneously inoculated into mice to establish a subcutaneous tumor model. Each mouse was inoculated with approximately 1 × 10⁻⁶ cells. 6 One MC38 tumor cell, until the tumor volume reaches 80-100 mm. 3 Day 0 was designated as day 0, and treatment began. The treatment protocol was as follows: On day 0, 50 μL of hydrogel or hydrogel drug sustained-release system was injected into the tumor area of ​​the mice. On day 2, the mouse tumors were locally irradiated with a dose of 12 Gy of X-rays. Throughout the treatment, changes in tumor volume were monitored regularly, and mouse survival was recorded to assess the impact of the combination therapy on tumor growth inhibition and mouse survival. Figure 8 The MC38 tumor volume in mice on day 18 after treatment with radiotherapy combined with the hydrogel provided in Example 43 or radiotherapy combined with the hydrogel drug sustained-release system provided in Example 58 (when the tumor volume in mice is approximately 80-100 mm). 3(Treatment begins at the specified time). Results show that the β-glucan hydrogel and drug sustained-release system provided by this invention, combined with radiotherapy, exhibit excellent tumor treatment performance.

[0083] In this invention, chitosan or its derivatives are assembled with β-glucan derivatives into a hydrogel, which is of great significance for constructing an immune adjuvant capable of achieving long-lasting immune activation. As the hydrogel slowly degrades, β-glucan is continuously released, thereby fully leveraging its excellent role as a natural immune adjuvant and enhancing local immune activation. Through the meticulous design of multiple strategies including local immune activation, systemic regulation, drug synergy, and intelligent response, the β-glucan derivative-based hydrogel and its drug delivery system provide a novel design approach and application prospect for precision tumor immunotherapy.

[0084] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An injectable hydrogel with immune adjuvant function, characterized in that, The injectable hydrogel with immune adjuvant function is prepared from amino-containing polymer A and aldehyde-containing β-glucan derivative B.

2. The injectable hydrogel with immune adjuvant function according to claim 1, characterized in that, The amino-containing polymer A is selected from one or more of the following reagents: chitosan, trimethyl chitosan, carboxymethyl chitosan, hydroxyethyl chitosan, hydroxypropyl chitosan, and chitosan grafted with benzothiazole-5-carboxylic acid; the aldehyde-containing β-glucan derivative B is prepared by modifying β-glucan with small molecules containing both aldehyde and carboxyl functional groups; the small molecules containing both aldehyde and carboxyl functional groups are selected from one or more of the following reagents: p-aldehyde benzoic acid, o-carboxybenzaldehyde, m-carboxybenzaldehyde, 3-(4-aldehydephenyl)propionic acid, 3-aldehyde indole-5-carboxylic acid, 3-oxopropionic acid, 5-oxovalerate, and succinic acid.

3. The injectable hydrogel with immune adjuvant function according to claim 2, characterized in that, The molecular weights of chitosan, trimethyl chitosan, carboxymethyl chitosan, hydroxyethyl chitosan, hydroxypropyl chitosan, and benzothiazole-5-carboxylic acid-grafted chitosan range from 5 kDa to 1000 kDa. In benzothiazole-5-carboxylic acid-grafted chitosan, the grafting rate of benzothiazole-5-carboxylic acid to the amino groups of the polymer backbone is 0.1% to 100%.

4. The method for preparing the injectable hydrogel with immune adjuvant function as described in claim 1, characterized in that, The amino-containing polymer A is crosslinked with the aldehyde-containing β-glucan derivative B to form the injectable hydrogel with immune adjuvant function.

5. The method for preparing the injectable β-glucan hydrogel with immunoadjuvant function according to claim 4, characterized in that, Includes the following steps: (1) Adjust the pH of the amino-containing polymer A solution to 5-8, and use it as solution a; (2) Adjust the pH of β-glucan derivative B containing aldehyde group to 5-8, and use it as solution b; (3) Mix solution a and solution b, let stand, and obtain injectable β-glucan hydrogel with immune adjuvant function; the ratio of the number of amino moles of polymer A to the number of aldehyde moles of aldehyde-containing β-glucan derivative B is 60:1~1:60, and the standing time is 1~720 minutes.

6. An injectable hydrogel drug delivery system with immune adjuvant function, characterized in that, It includes the injectable hydrogel with immune adjuvant function as described in claim 1, and drug molecules.

7. The injectable hydrogel drug-loaded sustained-release system with immune adjuvant function according to claim 6, characterized in that, The drug molecules include one or more of small molecule inhibitors and anti-tumor drugs.

8. The method for preparing the injectable hydrogel drug-loaded sustained-release system with immune adjuvant function as described in claim 6, characterized in that, Includes the following steps: (1) Adjust the pH of the amino-containing polymer A solution to 5-8, and use it as solution aa; (2) Adjust the pH of the β-glucan derivative B containing aldehyde group and the drug molecule solution to 5-8, and use it as solution bb; (3) Mix solution aa and solution bb, let stand, and obtain injectable β-glucan hydrogel with immune adjuvant function; the ratio of the number of amino moles of polymer A to the number of aldehyde moles of aldehyde-containing β-glucan derivative B is 60:1 to 1:60, and the standing time is 1 to 720 minutes.

9. A drug whose active ingredient comprises the injectable hydrogel with immune adjuvant function as described in claim 1 or the injectable hydrogel drug-loaded sustained-release system with immune adjuvant function as described in claim 7.

10. The use of the injectable hydrogel with immune adjuvant function as described in claim 1 or the injectable hydrogel drug-loaded sustained-release system with immune adjuvant function as described in claim 7 in drug delivery, immunotherapy, and combination therapy; or the use of the injectable hydrogel with immune adjuvant function as described in claim 1 or the injectable hydrogel drug-loaded sustained-release system with immune adjuvant function as described in claim 7 in the preparation of drug delivery drugs, immunotherapy drugs, and combination therapy drugs.

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