A supramolecular hydrogel for manganese immunity therapy and its preparation method and application

Abstract

The present invention provides a supramolecular hydrogel for manganese immunity therapy and its preparation method and application. The supramolecular hydrogel G-TA-I of the present invention can carry drugs and slowly release drugs. The drug-loaded supramolecular hydrogel G-TA-I@aPD-1+Mn 2+ , has a good ability to slowly release manganese ions and PD-1 monoclonal antibodies, and can promote CD45 + CD3 + CD8 + It inhibits the expression of T lymphocytes and DC cells, suppresses the expression of Treg cells, and significantly inhibits the growth of OSCC tumors. It also has excellent biocompatibility, small toxic side effects, and good application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to a supramolecular hydrogel for manganese immunotherapy, a preparation method thereof, and an application thereof. Background Art

[0002] Oral squamous cell carcinoma (OSCC) is the most common malignant tumor of the oral and maxillofacial region. In recent years, the main treatment options for OSCC have been surgery, supplemented by chemotherapy and radiotherapy. However, these options often come with significant drawbacks, such as the potential for severe side effects such as physical deformity and functional disability. These drawbacks not only affect the treatment outcomes but also severely impact patients' quality of life. Therefore, there is an urgent need to develop safer and more effective treatment options that can combat OSCC while minimizing patient suffering.

[0003] At present, immunotherapy is an immune checkpoint inhibitor therapy represented by programmed death protein-1 (PD-1) inhibitors and its ligand (PD-L1) inhibitors, which has become a new strategy for clinical tumor immunotherapy, but its therapeutic effect is not satisfactory. "Manganese immunotherapy" refers to a new therapy that uses manganese ion adjuvants combined with immune checkpoint inhibitor PD-1 monoclonal antibody to enhance local immune response, which has great clinical application prospects. The manganese ions in "manganese immunotherapy" have the ability to activate the cGAS-STING pathway and can be activated by distal dendritic cells (DC), macrophages and lymphocytes (such as CD8 + Manganese ion uptake by T lymphocytes (T lymphocytes) enhances the innate immune response in these immune cells, thereby accelerating the process of adaptive immunity. However, excessive levels of manganese ions in the human body can lead to the side effect of manganese ion poisoning, which is also a bottleneck in the clinical translation of "manganese immunity therapy." There is an urgent need to find new treatment strategies to improve the treatment of oral squamous cell carcinoma.

[0004] The use of hydrogel drug delivery is an effective strategy for the treatment of oral squamous cell carcinoma, but how to effectively deliver drugs to ensure their efficacy and safety still requires further study. Summary of the Invention

[0005] The purpose of the present invention is to provide a supramolecular hydrogel for manganese immunotherapy and a preparation method and application thereof.

[0006] The present invention provides a supramolecular hydrogel, which is prepared by the following steps:

[0007] 1) dissolving guanosine, phenyldiboronic acid or its derivatives and a soluble inorganic base in an aqueous solvent and mixing them to obtain a mixed solution A;

[0008] 2) dissolving and mixing inosine, phenyldiboronic acid or its derivatives and a soluble inorganic base in an aqueous solvent to obtain a mixed solution B;

[0009] 3) adding tannic acid to mixed solution A or mixed solution B, first mixing to obtain mixed solution C, then mixing mixed solution C with mixed solution B or mixed solution A to obtain mixture D, and allowing to stand;

[0010] The molar ratio of guanosine, phenyldiboronic acid or its derivatives and soluble inorganic base in step 1) is 1:(0.5-2):(0.5-2);

[0011] The molar ratio of inosine, phenyldiboronic acid or its derivatives and soluble inorganic base in step 2) is 1:(0.5-2):(0.5-2);

[0012] The molar ratio of the guanosine in step 1) to the inosine in step 2) is 1:(0.5-2);

[0013] The molar ratio of the guanosine in step 1) to the tannic acid in step 3) is 1:(0.1-0.2);

[0014] In step 3), the volume ratio of mixed solution C to mixed solution B or mixed solution A is 1:(0.5-2).

[0015] Furthermore, the phenylenediboronic acid or its derivative is 1,4-phenylenediboronic acid, and the soluble inorganic base is KOH or NaOH.

[0016] Furthermore, the aqueous solvent is water or a buffer solution;

[0017] And / or, the mass concentration of step 1) and the guanosine is 0.0002%-2%, preferably 1.4%;

[0018] and / or, in step 2), the mass concentration of inosine is 0.0002%-2%, preferably 1.4%;

[0019] and / or, in step 1), the molar ratio of guanosine, phenyldiboronic acid or its derivatives and the soluble inorganic base is 1:1:1;

[0020] and / or, in step 2), the molar ratio of inosine, phenylenediboronic acid or its derivatives and the soluble inorganic base is 1:1:1;

[0021] and / or, the molar ratio of the guanosine in step 1) to the inosine in step 2) is 1:1;

[0022] And / or, the molar ratio of the guanosine in step 1) to the tannic acid in step 2) is 1:0.1.

[0023] And / or, in step 3), the volume ratio of mixed solution C to mixed solution B or mixed solution A is 1:1.

[0024] The present invention also provides a drug-loaded supramolecular hydrogel, which is formed by adding drugs into the supramolecular hydrogel as a matrix.

[0025] Furthermore, the drug is Mn 2+ and PD-1 monoclonal antibody; Mn is preferred 2+ It is MnCl2.

[0026] Further, the following method is used to prepare the mixture D prepared by the above method: Mn 2+ and PD-1 monoclonal antibody, mix well and let stand.

[0027] Furthermore, (1-4) mg aPD-1 and 0.5 mg Mn were added to each 1 ml of mixed solution D. 2+ Preferably, (2-2.5) mg aPD-1 and 0.5 mg Mn are added to each 1 ml of mixed solution D. 2+ .

[0028] The present invention also provides a method for preparing the supramolecular hydrogel or drug-loaded supramolecular hydrogel, comprising the following steps:

[0029] 1) dissolving guanosine, phenyldiboronic acid or its derivatives and a soluble inorganic base in an aqueous solvent and mixing them to obtain a mixed solution A;

[0030] 2) dissolving and mixing inosine, phenyldiboronic acid or its derivatives and a soluble inorganic base in an aqueous solvent to obtain a mixed solution B;

[0031] 3) Tannic acid is then added to mixed solution A or mixed solution B, and mixed to obtain mixed solution C. Mixed solution C is then mixed with mixed solution B or mixed solution A to obtain mixture D. The mixture is allowed to stand to obtain a supramolecular hydrogel. Drugs are added to solution D, mixed, and allowed to stand to obtain a drug-loaded supramolecular hydrogel.

[0032] The present invention also provides a use of the supramolecular hydrogel as a drug carrier.

[0033] The present invention also provides a use of the drug-loaded supramolecular hydrogel in preparing anti-tumor drugs, preferably in preparing drugs for treating oral squamous cell carcinoma.

[0034] The present invention uses guanosine, tannic acid, inosine, and 1,4-phenylenediboronic acid to construct a supramolecular hydrogel G-TA-I. When the supramolecular hydrogel is capable of carrying drugs and slowly releasing the drugs, the G-TA-I hydrogel of the present invention can carry manganese ions and PD-1 monoclonal antibodies, which can not only exert the dual-targeting effect of "manganese immunotherapy" to activate cGAS-STING and relieve immune checkpoint inhibition, but also effectively avoid the risk of manganese poisoning. The supramolecular hydrogel provided by the present invention has broad application prospects in the preparation of manganese immunotherapy drugs and biomaterials.

[0035] The supramolecular hydrogel G-TA-I of the present invention is loaded with manganese ions and PD-1 monoclonal antibody to form a drug-loaded supramolecular hydrogel G-TA-I@aPD-1+Mn 2+ , has a good ability to slowly release manganese ions and PD-1 monoclonal antibodies, and can promote the CD45 + CD3 + CD8 + The supramolecular hydrogel can inhibit the expression of T lymphocytes and DC cells, suppress the expression of Treg cells, and significantly inhibit OSCC tumor growth. In addition, the supramolecular hydrogel has excellent biocompatibility, low toxicity and side effects, and has excellent application prospects.

[0036] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

[0037] The following further describes the above content of the present invention in detail through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1Preparation and characterization of G-TA-I hydrogels. A) Preparation process of G-TA-I hydrogels (concentration: 1.4% w / v). B) Investigation of the optimal gelation ratio for G-TA-I hydrogels (gels were constructed using G:TA = 1:0.05, 0.1, 0.2, 0.5, and 1, and observed and photographed after 1 day and 1 month at room temperature); (concentration: 1.4% w / v). C) Rheological analysis of the relationship between viscosity and shear rate of G-TA-I hydrogels (concentration: 1.4% w / v). D) Rheological determination of the storage modulus G′ and loss modulus G″ of G-TA-I hydrogels (concentration: 1.4% w / v). E) Rheological determination of the storage modulus G′ and loss modulus G″ of G-TA-I hydrogels under varying shear stress (concentration: 1.4% w / v). F) Injection experiments verifying the injectability of G-TA-I hydrogels (concentration: 1.4% w / v). G) SEM image of a freeze-dried G-TA-I hydrogel sample (scale bars: 100 μm and 50 μm). H, I) IR spectra of a freeze-dried G-TA-I hydrogel sample, PBA, TA, I, and G powders. J) PXRD pattern of a freeze-dried G-TA-I hydrogel sample. K, L) CD spectra of G-TA-I hydrogel (concentrations: 0.14 mg / mL and 0.028 mg / mL). M) Fluorescence detection spectrum of G-TA-I + ThT (concentration: 1.4% w / v).

[0039] Figure 2 In vitro sustained-release function and in vivo biocompatibility of G-TA-I hydrogel. A) Effects of G-TA-I hydrogel loaded with different drugs (concentration: 1.4% w / v). B) G-TA-I@αPD-1+Mn 2+ Schematic diagram of hydrogel in vitro sustained release system, G-TA-I@αPD-1+Mn 2+ αPD-1 and Mn in hydrogel 2+ Release curve of α-glucose as a function of time (concentration: 1.4% w / v).

[0040] Figure 3 A) After subcutaneous injection of 100 μL of G-TA-I hydrogel and H2O into mice, the experiment was terminated at specific time points and samples were collected to observe gel degradation (hydrogel concentration: 28 mg / mL); B) H&E staining was used to observe the histopathological effects of G-TA-I hydrogel on mouse skin and vital organs (heart, liver, spleen, lungs, and kidneys) at different time points.

[0041] Figure 4G-TA-I hydrogel loaded with manganese ions and PD-1 monoclonal antibody activated local immunity to suppress the growth of OSCC in situ carcinoma. A) OSCC in situ carcinoma model constructed in mice using 4MOSC1 cells and local drug delivery treatment mode. B) Weight ratio of mice in each treatment group at the end of treatment compared to the initial weight. C) Growth curve of tumor volume of mice in each treatment group. D) Comparison of tumor volume at the end of treatment. EG) Flow cytometric analysis of CD45 in the tumor. + CD3 + CD8 + T lymphocytes, CD45 + CD11C + Dendritic cells, FoxP3 + CD4 + Changes in the number of regulatory T cells. DETAILED DESCRIPTION

[0042] The raw materials and equipment used in the present invention are all known products and are obtained by purchasing commercially available products.

[0043] The present invention uses guanosine (G), tannic acid (TA) and inosine (I) to construct a supramolecular hydrogel, which is loaded with manganese ions and PD-1 monoclonal antibody and used in an OSCC carcinoma in situ model.

[0044] Guanosine (G) CAS No. 118-00-3.

[0045] Inosine (I) CAS number: 58-63-9.

[0046] 1,4-phenylenediboronic acid (PBA) CSA No.: 4612-26-4.

[0047] PD-1 monoclonal antibody, also known as αPD-1, is an immune checkpoint inhibitor. αPD-1 was purchased from BioXCell under the catalog number Clone 29F.1A12.

[0048] MnCl2 was purchased from Sigma–Aldrich, product number 244589.

[0049] The full name of 4MOSC1 cells is 4NQO-induced murine oral squamous cells. It is a primary model extracted from a mouse oral cancer model induced by 4NQO (carcinogen). It was provided by J. Silvio Gutkind and recorded in Zhiyong Wang et al., Syngeneic animal models of tobacco-associated oral cancer reveal the activity of in situ anti-CTLA-4, Nature Communications volume 10, Article number: 5546 (2019).

[0050] Example 1: Preparation of the supramolecular hydrogel G-TA-I of the present invention

[0051] 1. Raw materials

[0052] Guanosine (G), 1,4-phenylenediboronic acid (PBA), KOH, inosine (I), tannic acid (TA), and deionized water.

[0053] 2. Preparation method

[0054] ① Dissolve G, PBA, and KOH in equal molar ratios in deionized water (wherein the mass concentration of G is 1.4%), and heat to 90°C to fully dissolve to obtain a mixed solution A;

[0055] ② Dissolve I, PBA, and KOH in equal molar ratios in deionized water (wherein the mass concentration of I is 1.4%) and heat to 95°C to fully dissolve to obtain a mixed solution B; the molar amount of I in solution B is the same as the molar amount of G in solution A;

[0056] ③ Add TA to solution A at a molar ratio of G:TA of 1:0.1 and stir until uniformly mixed to obtain solution C;

[0057] ④ After mixing equal volumes of solution B and solution C, the mixture was allowed to stand at room temperature for several minutes to obtain the supramolecular hydrogel of the present invention.

[0058] from Figure 1 As shown in Figure A, when equal volumes of solution B and solution C were mixed and allowed to stand at room temperature for several minutes, the interface formed a gel, and the resulting supramolecular hydrogel was named G-TA-I.

[0059] Example 2: Preparation of 0.5 mg Mn 2 and 2 mg aPD-1 hydrogel G-TA-I@

[0060] aPD-1+Mn 2+

[0061] 1. Raw materials

[0062] Guanosine (G), 1,4-phenylenediboronic acid (PBA), KOH, inosine (I), tannic acid (TA), deionized water, PD-1 monoclonal antibody, and MnCl2.

[0063] 2. Preparation method

[0064] ① Dissolve G, PBA, and KOH in deionized water at an equal molar ratio (wherein the mass concentration of G is 1.4%), and heat to 90°C to fully dissolve to obtain a mixed solution A;

[0065] ② Dissolve I, PBA, and KOH in deionized water at an equal molar ratio (wherein the mass concentration of I is 1.4%) and heat to 95°C to fully dissolve to obtain a mixed solution B; the molar amount of I in solution B is the same as the molar amount of G in solution A;

[0066] ③ Add TA to solution A at a molar ratio of G:TA of 1:0.1 and stir until uniformly mixed to obtain solution C;

[0067] ④ After mixing equal volumes of solution B and solution C to obtain mixed solution D, MnCl2 and the original mother solution of αPD-1 were quickly added to make the solution contain 0.5 mg Mn per 1 mL. 2 and 2 mg of αPD-1, mixed well and allowed to stand at room temperature for several minutes to obtain the drug-loaded supramolecular hydrogel G-TA-I@aPD-1+Mn 2+ , specifically G-TA-I@2mg aPD-1+0.5mg Mn 2+ .

[0068] Example 3: Preparation of 0.5 mg Mn 2+ and 2.5mg aPD-1 hydrogel G-TA-I@

[0069] aPD-1+Mn 2+

[0070] 1. Raw materials

[0071] Guanosine (G), 1,4-phenylenediboronic acid (PBA), KOH, inosine (I), tannic acid (TA), deionized water, PD-1 monoclonal antibody, and MnCl2.

[0072] 2. Preparation method

[0073] ① Dissolve G, PBA, and KOH in deionized water at an equal molar ratio (wherein the mass concentration of G is 1.4%), and heat to 90°C to fully dissolve to obtain a mixed solution A;

[0074] ② Dissolve I, PBA, and KOH in deionized water at an equal molar ratio (wherein the mass concentration of I is 1.4%) and heat to 95°C to fully dissolve to obtain a mixed solution B; the molar amount of I in solution B is the same as the molar amount of G in solution A;

[0075] ③ Add TA to solution A at a molar ratio of G:TA of 1:0.1 and stir until uniformly mixed to obtain solution C;

[0076] ④ After mixing equal volumes of solution B and solution C to obtain mixed solution D, MnCl2 and the original mother solution of αPD-1 were quickly added to make the solution contain 0.5 mg Mn per 1 mL. 2 and 2.5 mg αPD-1, mixed well and allowed to stand at room temperature for several minutes to obtain the drug-loaded supramolecular hydrogel G-TA-I@aPD-1+Mn 2+ , specifically G-TA-I@2.5mg aPD-1+0.5mg Mn 2+ .

[0077] Example 4: Preparation of aPD-1-carrying hydrogel G-TA-I@aPD-1

[0078] 1. Raw materials

[0079] Guanosine (G), 1,4-phenylenediboronic acid (PBA), KOH, inosine (I), tannic acid (TA), deionized water, and PD-1 monoclonal antibody.

[0080] 2. Preparation method

[0081] ① Dissolve G, PBA, and KOH in deionized water at an equal molar ratio (wherein the mass concentration of G is 1.4%), and heat to 90°C to fully dissolve to obtain a mixed solution A;

[0082] ② Dissolve I, PBA, and KOH in deionized water at an equal molar ratio (wherein the mass concentration of I is 1.4%) and heat to 95°C to fully dissolve to obtain a mixed solution B; the molar amount of I in solution B is the same as the molar amount of G in solution A;

[0083] ③ Add TA to solution A at a molar ratio of G:TA of 1:0.1 and stir until uniformly mixed to obtain solution C;

[0084] ④ Mix equal volumes of solution B and solution C to obtain mixed solution D, quickly add the original αPD-1 mother solution so that the solution contains 2 mg of αPD-1 per 1 mL. After mixing, let it stand at room temperature for several minutes to obtain the drug-loaded supramolecular hydrogel G-TA-I@aPD-1.

[0085] Example 5: Preparation of Mn-loaded 2+ hydrogel G-TA-I@Mn 2+

[0086] 1. Raw materials

[0087] Guanosine (G), 1,4-phenylenediboronic acid (PBA), KOH, inosine (I), tannic acid (TA), deionized water, and MnCl2.

[0088] 2. Preparation method

[0089] ① Dissolve G, PBA, and KOH in deionized water at an equal molar ratio (wherein the mass concentration of G is 1.4%), and heat to 90°C to fully dissolve to obtain a mixed solution A;

[0090] ② Dissolve I, PBA, and KOH in deionized water at an equal molar ratio (wherein the mass concentration of I is 1.4%) and heat to 95°C to fully dissolve to obtain a mixed solution B; the molar amount of I in solution B is the same as the molar amount of G in solution A;

[0091] ③ Add TA to solution A at a molar ratio of G:TA of 1:0.1 and stir until uniformly mixed to obtain solution C;

[0092] ④ After mixing equal volumes of solution B and solution C to obtain mixed solution D, MnCl2 was added quickly so that the solution contained 0.5 mg Mn per 1 mL. 2 After mixing and standing at room temperature for a few minutes, the drug-loaded supramolecular hydrogel G-TA-I@Mn 2+ .

[0093] Example 6: IgG-loaded hydrogel G-TA-I@IgG

[0094] 1. Raw materials

[0095] Guanosine (G), 1,4-phenylenediboronic acid (PBA), KOH, inosine (I), tannic acid (TA), deionized water, IgG, and MnCl2.

[0096] 2. Preparation method

[0097] ① Dissolve G, PBA, and KOH in deionized water at an equal molar ratio (wherein the mass concentration of G is 1.4%), and heat to 90°C to fully dissolve to obtain a mixed solution A;

[0098] ② Dissolve I, PBA, and KOH in deionized water at an equal molar ratio (wherein the mass concentration of I is 1.4%) and heat to 95°C to fully dissolve to obtain a mixed solution B; the molar amount of I in solution B is the same as the molar amount of G in solution A;

[0099] ③ Add TA to solution A at a molar ratio of G:TA of 1:0.1 and stir until uniformly mixed to obtain solution C;

[0100] ④ Mix equal volumes of solution B and solution C to obtain mixed solution D. IgG was quickly added so that the solution contained 2 mg of IgG per 1 mL. After mixing, the mixture was allowed to stand at room temperature for several minutes to obtain the drug-loaded supramolecular hydrogel G-TA-I@IgG.

[0101] Example 7: Preparation of the supramolecular hydrogel G-TA-I of the present invention

[0102] 1. Raw materials

[0103] Guanosine (G), 1,4-phenylenediboronic acid (PBA), KOH, inosine (I), tannic acid (TA), and deionized water.

[0104] 2. Preparation method

[0105] ① Dissolve G, PBA, and KOH in equal molar ratios in deionized water (wherein the mass concentration of G is 1.4%), and heat to 90°C to fully dissolve to obtain a mixed solution A;

[0106] ② Dissolve I, PBA, and KOH in equal molar ratios in deionized water (wherein the mass concentration of I is 1.4%) and heat to 95°C to fully dissolve to obtain a mixed solution B; the molar amount of I in solution B is the same as the molar amount of G in solution A;

[0107] ③ Add TA to solution B at a molar ratio of G:TA of 1:0.1 and stir until uniformly mixed to obtain solution C;

[0108] ④ After mixing equal volumes of solution A and solution C, the mixture was allowed to stand at room temperature for several minutes to obtain the supramolecular hydrogel of the present invention.

[0109] Example 8: Preparation of the supramolecular hydrogel G-TA-I of the present invention

[0110] 1. Raw materials

[0111] Guanosine (G), 1,4-phenylenediboronic acid (PBA), KOH, inosine (I), tannic acid (TA), and deionized water.

[0112] 2. Preparation method

[0113] ① Dissolve G, PBA, and KOH in equal molar ratios in deionized water (wherein the mass concentration of G is 0.002%), and heat to 90°C to fully dissolve to obtain a mixed solution A;

[0114] ② Dissolve I, PBA, and KOH in equal molar ratios in deionized water (wherein the mass concentration of I is 0.002%) and heat to 95°C to fully dissolve to obtain a mixed solution B; the molar amount of I in solution B is the same as the molar amount of G in solution A;

[0115] ③ Add TA to solution B at a molar ratio of G:TA of 1:0.1 and stir until uniformly mixed to obtain solution C;

[0116] ④ After mixing equal volumes of solution A and solution C and letting them stand at room temperature for several minutes, the supramolecular hydrogel of the present invention can be obtained.

[0117] Example 9: Preparation of the supramolecular hydrogel G-TA-I of the present invention

[0118] 1. Raw materials

[0119] Guanosine (G), 1,4-phenylenediboronic acid (PBA), KOH, inosine (I), tannic acid (TA), and deionized water.

[0120] 2. Preparation method

[0121] ① Dissolve G, PBA and KOH in equal molar ratios in deionized water (wherein the mass concentration of G is 2%), and heat to 90°C to fully dissolve to obtain a mixed solution A;

[0122] ② Dissolve I, PBA, and KOH in equal molar ratios in deionized water (wherein the mass concentration of I is 2%) and heat to 95°C to fully dissolve to obtain a mixed solution B; the molar amount of I in solution B is the same as the molar amount of G in solution A;

[0123] ③ Add TA to solution A at a molar ratio of G:TA of 1:0.1 and stir until uniformly mixed to obtain solution C;

[0124] ④ After mixing equal volumes of solution B and solution C and letting them stand at room temperature for several minutes, the supramolecular hydrogel of the present invention can be obtained.

[0125] The beneficial effects of the present invention are demonstrated by the following test examples.

[0126] Experimental Example 1: Characterization of the supramolecular hydrogel G-TA-I of the present invention

[0127] 1. Experimental Methods

[0128] The G-TA-I prepared in Example 1 was subjected to rheological experiments, scanning electron microscopy (SEM) characterization, infrared spectroscopy characterization, and X-ray powder diffraction characterization.

[0129] 2. Experimental Results

[0130] 1. Rheological experimental results

[0131] Rheological experiments evaluated the viscoelasticity of the G-TA-I hydrogel prepared in Example 1. The results showed that the storage modulus G′ was much larger than the loss modulus G″, indicating that G-TA-I was in a gel state and had good solid-like mechanical properties ( Figure 1 D). Figure 1 As shown in Figure E, when the strain is less than 4%, G′ is greater than G″, and G-TA-I is in a gel state; otherwise, G-TA-I is in a sol state, confirming that G-TA-I hydrogel exhibits good gel-sol transition behavior.

[0132] Figure 1 C shows that the viscosity of G-TA-I gradually decreases with increasing shear rate, indicating that G-TA-I hydrogel has shear thinning properties.

[0133] The G-TA-I solution was drawn up with a syringe and after gelation, it was injected into a vial and still showed a gel state. After the vial was inverted, the gel was found to be at the bottom of the vial and did not fall off, further confirming that the G-TA-I hydrogel had good shear thinning properties and injectability ( Figure 1 F).

[0134] The experimental results show that G-TA-I hydrogel has good mechanical properties and injectability.

[0135] 2. SEM Results

[0136] The results of scanning electron microscopy (SEM) observation of freeze-dried G-TA-I powder are as follows: Figure 1 As shown in G, the G-TA-I hydrogel exhibits a loose and porous structure.

[0137] 3. Infrared spectrum results

[0138] Infrared spectroscopy (IR) showed that the vicinal diol on the G ribose was at 3230 cm -1 It showed an obvious peak at the G-TA-I gel, but this peak weakened in the G-TA-I gel, proving that the vicinal diol groups on the G sugar group participated in the gel formation ( Figure 1The characteristic peaks of the vicinal diol groups of TA and I at 3330 cm-1 and the characteristic peaks of the vicinal diol groups of PBA at 3300 cm-1 were significantly weakened in the G-TA-I gel, indicating that the vicinal diol groups of TA, I and PBA were involved in the formation of the G-TA-I gel ( Figure 1 H). At the same time, the presence of νB-OC=1103cm-1 and the disappearance of νB-OH=1350cm-1(PBA) and νB-OH=1087cm-1(G) in G-TA-I further suggest the formation of phenylboronic acid ester bond ( Figure 1 I).

[0139] 4. X-ray powder diffraction results

[0140] Powder X-ray diffraction (PXRD) showed that the freeze-dried sample of G-TA-I hydrogel had a 2θ≈27.0° A peak is shown at , indicating that the G-TA-I hydrogel is formed by G tetramers through π-π stacking ( Figure 1 J). Circular dichroism (CD) spectrum showed two positive absorption peaks at 214 nm and 303 nm, and one negative absorption peak at 250 nm and 333 nm, indicating that G tetramers may exist in G-TA-I hydrogel, and they are stacked in head-to-head and head-to-tail manners ( Figure 1 K, L). Free ThT has very weak fluorescence, but shows strong fluorescence when combined with G tetramer. When ThT is mixed with G-TA-I hydrogel, the hydrogel has strong green fluorescence with an emission peak at around 492nm, further confirming the presence of G tetramer in G-TA-I hydrogel ( Figure 1 M).

[0141] Experimental Example 2: Concentration screening of supramolecular hydrogel G-TA-I of the present invention

[0142] 1. Raw materials

[0143] Guanosine (G), 1,4-phenylenediboronic acid (PBA), KOH, inosine (I), tannic acid (TA), and deionized water.

[0144] 2. Preparation method

[0145] ① Dissolve G, PBA, and KOH in equal molar ratios in deionized water (wherein the mass concentration of G is 1.4%), and heat to 90°C to fully dissolve to obtain a mixed solution A;

[0146] ② Dissolve I, PBA, and KOH in equal molar ratios in deionized water (wherein the mass concentration of I is 1.4%) and heat to 95°C to fully dissolve to obtain a mixed solution B;

[0147] ③ Add TA to solution A according to the molar ratio of G:TA of 1:0.05, 0.1, 0.2, 0.5, and 1, respectively, and stir until the mixture is uniform to obtain solution C;

[0148] ④ After mixing equal volumes of solution B and solution C and letting them stand at room temperature for several minutes, the supramolecular hydrogel of the present invention can be obtained.

[0149] 3. Results

[0150] The results are as follows Figure 1 As shown in Figure B, when G:TA is 1:0.1, gelation can be achieved and the solution remains stable after being placed for 1 day and 1 month. When G:TA is 1:0.0.2, gelation can be achieved, but a small amount of substance precipitates after being placed for 1 day, and the precipitate increases significantly after being placed for 1 month, indicating that the gelation is unstable. When G:TA is 1:0.05, 1:0.5 or 1:1, gelation cannot be achieved.

[0151] Experimental results show that supramolecular hydrogels can be effectively prepared when G:TA is 1:(0.1~0.2), and the gelation properties are optimal when G:TA is 1:0.1.

[0152] Experimental Example 3: Drug loading capacity and in vitro sustained release function of G-TA-I hydrogel

[0153] 1. Experimental Methods

[0154] (1) Drug loading capacity of G-TA-I hydrogel

[0155] The hydrogels prepared in Example 1, Example 4, Example 5, and Example 6 were inverted and observed to investigate the stability of each hydrogel and the drug loading capacity of G-TA-I.

[0156] (II) In vitro sustained release behavior of G-TA-I hydrogel

[0157] 1ml G-TA-I@2mgαPD-1+0.5mg Mn prepared in Example 2 2+ Add 2ml H2O to the hydrogel and construct Figure 2 In the in vitro hydrogel sustained-release system shown in B, at different time points (0h, 2h, 4h, 8h, 12h, 24h, 48h, 72h, 96h and 120h), the content of aPD-1 was detected by BCA, and the content of Mn was detected by ICP. 2+ The content of G-TA-I@2mgαPD-1+0.5mgMn 2+ In vitro sustained release behavior of hydrogels.

[0158] 2. Experimental Results

[0159] (1) Drug loading capacity of G-TA-I hydrogel

[0160] Stability of each hydrogel and drug loading capacity of G-TA-I hydrogel Figure 2 As shown in A, it can be seen that in G-TA-I, 2mg IgG, 2mg αPD-1, 0.5mg Mn 2+ The hydrogel prepared after 5 days still showed a good gel state and remained stable after 5 days. This shows that G-TA-I hydrogel has good drug loading capacity and can stably carry IgG, αPD-1, and Mn 2+ .

[0161] (II) In vitro sustained release behavior of G-TA-I hydrogel

[0162] The experimental results are as follows Figure 2 As shown in B, G-TA-I@2mgαPD-1+0.5mg Mn 2+ aPD-1 is released first and then Mn in the hydrogel sustained-release system 2+ ;aPD-1, Mn 2+ The release ratios of were approximately 73% and 17%, respectively, which indicated that G-TA-I hydrogel had an excellent sustained-release effect.

[0163] Test Example 4: In vivo biocompatibility

[0164] 1. Experimental Methods

[0165] 100 μL of the G-TA-I hydrogel prepared in Example 1 was subcutaneously injected into the subcutaneous tissue of the back of mice, with H2O as the control group, to evaluate the degradation of the G-TA-I hydrogel in vivo.

[0166] 2. Experimental Results

[0167] The degradation degree was observed at different time points. The experimental results are as follows: Figure 3 As shown in Figure A, in the control group, after subcutaneous injection, HO was in a liquid state at the site, diffused to the surrounding area within a few minutes, and then disappeared. However, the G-TA-I hydrogel remained in a hydrogel state at the injection site, its morphology remained largely stable, and the amount of hydrogel gradually decreased until it was completely degraded after 120 hours.

[0168] Similar to the H2O control group, no mouse deaths or obvious pathological abnormalities occurred in the G-TA-I hydrogel group, and no obvious inflammatory response was found at the injection site at different time points.

[0169] The experimental results showed that compared with the control group H2O, the in vivo degradation rate of G-TA-I hydrogel was slower and it had good in vivo stability.

[0170] The in vivo biocompatibility of G-TA-I hydrogel was evaluated from the perspective of histopathology by H&E staining. Figure 3 As shown in Figure B, it can be seen that, like the control group H2O, no obvious organ damage or tissue degeneration was observed in the skin and major organs (including heart, liver, spleen, lung and kidney) of the G-TA-I hydrogel-treated group at different time points.

[0171] The experimental results show that G-TA-I hydrogel has good in vivo biocompatibility and can be used as a good carrier for in vivo biomedical applications.

[0172] Test Example 5: Animal Model Experiment

[0173] 1. Experimental Methods

[0174] In order to further confirm the synergistic effect of the two drugs in co-treatment, reduce drug side effects, and verify the in vivo drug loading and sustained release ability of G-TA-I hydrogel, an OSCC carcinoma in situ model was constructed using 4MOSC1 cells ( Figure 4 A).

[0175] The specific test methods are as follows:

[0176] (I) Construction of OSCC in situ mouse model and grouping

[0177] Female C57BL / 6 mice, aged 4-6 weeks and weighing 16-18 g, were used and 8 × 10 5 4MOSC1 cells were orthotopically transplanted. Tumor development was closely monitored until the tumor reached 20 mm. 3 The OSCC in situ carcinoma mouse model was successfully established.

[0178] The mice with successful modeling were randomly divided into 5 groups, with 6 mice in each group. The treatment methods for each group of mice were as follows:

[0179] Control group: local injection of 20 μL of H2O.

[0180] G-TA-I group: local injection of 20 μL of G-TA-I hydrogel prepared in Example 1;

[0181] G-TA-I@Mn 2+ Group: Local injection of 20 μL volume, G-TA-I@Mn prepared in Example 5 2+ Hydrogel, the hydrogel carries 10ug Mn 2+ .

[0182] G-TA-I@αPD-1 group: Local injection of 20 μL volume of G-TA-I@αPD-1 hydrogel prepared in Example 4, with 50 μg of αPD-1 loaded in the hydrogel.

[0183] G-TA-I@αPD-1+Mn 2+ Group: Local injection of 20 μL of G-TA-I@2.5 mg aPD-1+0.5 mg Mn prepared in Example 3 2+ Hydrogel, the hydrogel carries 10ug Mn 2+ and 50ugαPD-1.

[0184] All drugs were administered by local injection, once every 3 days for a total of 4 times, and the treatment was performed on the day (day 0) when the OSCC in situ carcinoma mouse model was successfully established, the 3rd day, the 6th day, and the 9th day, and the tissues were collected on the 12th day.

[0185] (2) Detection method

[0186] 1. Measure mouse body weight and tumor volume

[0187] The weight and tumor volume of the mice were monitored on days 0, 3, 6, 9, and 12 after the start of medication. The length and width of the tumor on the tongue were measured using a vernier caliper after the mice were locally anesthetized. The tumor volume was calculated as follows: volume = 0.5 * length * width 2 .

[0188] 2. Flow cytometry analysis

[0189] At the end of the experiment, the tumor tissues of each group of mice were collected and washed with PBS to remove blood and impurities. The tumor tissues were minced into 1-2 mm pieces using tissue scissors and forceps. 3 Size. The minced tissue was transferred to a centrifuge tube containing enzymatic solution and incubated at 37°C for 1 hour with gentle stirring or shaking to promote cell separation. Homogenize in cold cell staining buffer and filter to obtain a single-cell suspension. The cells were stained with different fluorescent-labeled antibodies (BioLegend) according to the manufacturer's instructions. The stained cells were evaluated on a cell fluorescence cytometer (Beckman) and analyzed with Kaluza software to measure the number of CD45+CD3+CD8+T lymphocytes (i.e., T cells with tumor-killing function), CD45+CD11c+ dendritic cells (i.e., DC cells with antigen-presenting function), and CD45+CD3+CD4+Foxp3+ immunoregulatory T cells (i.e., Treg cells that suppress immunity).

[0190] 2. Experimental Results

[0191] 1. Mouse body weight and tumor volume results

[0192] At the end of treatment, the body weight of mice was Figure 4 As shown in B, G-TA-I@aPD-1+Mn 2+The body weight of mice in the treatment group was not significantly different from that before treatment, while the body weight of the control group decreased by nearly 20% ( Figure 4 B), indicating G-TA-I@aPD-1+Mn 2+ The treatment combination had little effect on the overall health of mice and showed good tolerance and biocompatibility, indicating that G-TA-I@aPD-1+Mn 2+ It has good safety and can overcome the safety problems of traditional manganese immunity therapy.

[0193] During the 12-day treatment process and at the end of the treatment, the statistical results of the mouse tumor volume were as follows: Figure 4 C and Table 1, the comparison of tumor volume at the end of treatment is shown in Figure Figure 4 As shown in D:

[0194] Compared with the control group, the tumor volume of the G-TA-I group did not change significantly, and the G-TA-I@Mn 2+ group, G-TA-I@αPD-1 group, and G-TA-I@αPD-1+Mn 2+ The tumor volume of mice in the G-TA-I@Mn group was significantly reduced. 2+ , G-TA-I@αPD-1 and G-TA-I@αPD-1+Mn 2+ Both have tumor-suppressing effects;

[0195] Control group, G-TA-I group, G-TA-I@Mn 2+ The volume of mice in the G-TA-I@αPD-1 group and the G-TA-I@αPD-1 group was significantly larger than that before treatment, while the volume of mice in the G-TA-I@αPD-1+Mn 2+ The volume of mice in the G-TA-I@αPD-1+Mn group was reduced to a certain extent compared with that before treatment, indicating that 2+ The treatment effect of the group was particularly excellent, significantly better than that of other groups (p<0.05);

[0196] At 12 days of treatment, G-TA-I@Mn 2+ The volume of the G-TA-I@αPD-1 group and the control group was reduced by 19.39 mm. 3 , 4.84mm 3 , the percentage of decrease was 27.3% and 6.8%, while G-TA-I@αPD-1+Mn 2+ The volume of the group is reduced by 51.42mm 3 , the percentage of decrease is 82.5%. 2+ The effect of the G-TA-I@Mn group was significant. 2+ , G-TA-I@αPD-1 group and their sum, indicating that the present invention G-TA-I@αPD-1+Mn2+ The components of the group have a synergistic effect on inhibiting tumors (especially inhibiting oral squamous cell carcinoma).

[0197] Table 1 Statistical results of mouse tumor volume (mean; mm 3 )

[0198] Day 0 Day 3 Day 6 Day 9 Day 12 Control 20.92 34.34 42.95 57.25 70.94 G-TA-I 20.33 35.25 49.07 52.18 74.26 <![CDATA[G-TA-I@Mn 2+ ]]> 20.58 31.15 40.17 46.83 51.55 G-TA-I@αPD-1 20.75 30.9 35.9 51.95 66.1 <![CDATA[G-TA-I@αPD-1+Mn 2+ ]]> 20.33 31.3 29 21.2 19.52

[0199] 2. Flow cytometry analysis results

[0200] To further verify the activity of G-TA-I hydrogel-loaded drugs and their activation effect on the immune microenvironment, three mice were randomly selected from each group for flow cytometry analysis. The experimental results are as follows: Figure 4 E- Figure 4 As shown in G, it can be seen that G-TA-I@αPD-1+Mn 2+ In all groups, the characteristics of local immune activation in the tumor were very obvious.

[0201] Figure 4 E can be seen that G-TA-I@αPD-1+Mn 2+ The number of CD45+CD3+CD8+T lymphocytes in the mice of the G-TA-I group was significantly increased compared with that in the Control group and the G-TA-I group.

[0202] Figure 4 F It can be seen that the G-TA-I@αPD-1+Mn2+ group also activated the expression of CD11C+ dendritic cells (DC cells) in the immune microenvironment, and the number of DC cells was relatively higher than that of the Control group, G-TA-I group, and G-TA-I@Mn 2+ group increased significantly.

[0203] Figure 4 G can be seen that G-TA-I@αPD-1+Mn 2+ The number of immune suppressive regulatory T cells (Treg cells) in the group was significantly reduced compared with the control group.

[0204] The experimental results show that the G-TA-I@αPD-1+Mn 2+ Hydrogels that promote CD45 + CD3 + CD8 + It downregulates the expression of T lymphocytes and DC cells, inhibits the expression of Treg cells, and significantly inhibits OSCC tumor growth.

[0205] In summary, the present invention provides a supramolecular hydrogel for manganese immunotherapy and its preparation method and application. The present invention uses guanine nucleoside, tannic acid, inosine and 1,4-phenylenediboronic acid to construct a supramolecular hydrogel. When the supramolecular hydrogel is loaded with manganese ions and PD-1 monoclonal antibody, the hydrogel loaded with manganese ions and PD-1 monoclonal antibody has a good ability to slowly release manganese ions and PD-1 monoclonal antibody, which can promote the expression of CD45 in OSCC tumor patients. + CD3 + CD8 + The supramolecular hydrogel can inhibit the expression of T lymphocytes and DC cells, suppress the expression of Treg cells, and significantly inhibit OSCC tumor growth. Furthermore, the supramolecular hydrogel has excellent biocompatibility and minimal toxic side effects. The supramolecular hydrogel provided by the present invention has broad application prospects in the preparation of manganese immunotherapy drugs and biomaterials.

Claims

1. A drug-loaded supramolecular hydrogel, characterized by: It is a drug-loaded hydrogel formed by adding drugs into the supramolecular hydrogel as a matrix; the drug is Mn 2+ and PD-1 monoclonal antibodies; The supramolecular hydrogel is prepared by the following steps: 1) Dissolve guanosine, phenyldiboronic acid, and a soluble inorganic base in an aqueous solvent and mix them to obtain a mixed solution A; 2) Dissolve inosine, phenyldiboronic acid, and a soluble inorganic base in an aqueous solvent and mix them to obtain a mixed solution B; 3) Add tannic acid to mixed solution A or mixed solution B, mix thoroughly to obtain mixed solution C, then mix mixed solution C with mixed solution B or mixed solution A to obtain mixed solution D, and let it stand. The molar ratio of guanosine, phenyldiboronic acid and soluble inorganic base in step 1) is 1:(0.5-2):(0.5-2); The molar ratio of inosine, phenyldiboronic acid and soluble inorganic base in step 2) is 1:(0.5-2):(0.5-2); The molar ratio of guanosine in step 1) to inosine in step 2) is 1:(0.5-2); The molar ratio of the guanosine in step 1) to the tannic acid in step 3) is 1:(0.1-0.2); In step 3), the volume ratio of mixed solution C to mixed solution B or mixed solution A is 1:(0.5-2).

2. The drug-loaded supramolecular hydrogel according to claim 1, characterized in that: The Mn 2+ It is MnCl2.

3. The drug-loaded supramolecular hydrogel according to claim 1, characterized in that: The phenylenediboric acid is 1,4-phenylenediboric acid, and the soluble inorganic base is KOH or NaOH.

4. The drug-loaded supramolecular hydrogel according to claim 1, characterized in that: The aqueous solvent is water or a buffer solution; And / or, the mass concentration of guanosine in step 1) is 0.0002%-2%; and / or, in step 2) the mass concentration of inosine is 0.0002%-2%; and / or, in step 1), the molar ratio of guanosine, phenyldiboronic acid, and soluble inorganic base is 1:1:1; and / or, in step 2), the molar ratio of inosine, phenyldiboronic acid, and soluble inorganic base is 1:1:1; and / or, the molar ratio of the guanosine in step 1) to the inosine in step 2) is 1:1; and / or, the molar ratio of the guanosine in step 1) to the tannic acid in step 2) is 1:0.1; And / or, in step 3), the volume ratio of mixed solution C to mixed solution B or mixed solution A is 1:

1.

5. The drug-loaded supramolecular hydrogel according to claim 4, characterized in that: The mass concentration of the guanosine is 1.4%; And / or, the mass concentration of inosine is 1.4%.

6. The drug-loaded supramolecular hydrogel according to any one of claims 1 to 5, characterized in that: Prepared according to the following method: Add Mn into the mixed solution D prepared according to any one of claims 1 to 5 2+ and PD-1 monoclonal antibody, mix well and let stand.

7. The drug-loaded supramolecular hydrogel according to claim 6, characterized in that: Add (1-4) mg aPD-1 and 0.5 mg Mn per 1 ml of mixed solution D. 2+ .

8. The drug-loaded supramolecular hydrogel according to claim 7, characterized in that: Add (2-2.5) mg aPD-1 and 0.5 mg Mn per 1 ml of mixed solution D. 2+ .

9. The method for preparing the drug-loaded supramolecular hydrogel according to any one of claims 1 to 8, comprising the following steps: 1) Dissolve guanosine, phenyldiboronic acid, and a soluble inorganic base in an aqueous solvent and mix them to obtain a mixed solution A; 2) Dissolve inosine, phenyldiboronic acid, and a soluble inorganic base in an aqueous solvent and mix them to obtain a mixed solution B; 3) Tannic acid is then added to mixed solution A or mixed solution B, and mixed to obtain mixed solution C. Mixed solution C is then mixed with mixed solution B or mixed solution A to obtain mixture D. The mixture is allowed to stand to obtain a supramolecular hydrogel. Drugs are added to solution D, mixed, and allowed to stand to obtain a drug-loaded supramolecular hydrogel.

10. Use of the drug-loaded supramolecular hydrogel according to any one of claims 1 to 8 in the preparation of anti-tumor drugs.

11. The use according to claim 10, characterized in that The tumor is oral squamous cell carcinoma.

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