Preparation and application of carrier-free hydrogel co-assembled by effective part extract of traditional Chinese medicine euphorbia kansui, glycyrrhizic acid and manganese chloride

Abstract

The invention discloses preparation and application of a carrier-free hydrogel which is formed by assembling a traditional Chinese medicine euphorbia kansui effective part extract (KE), glycyrrhizic acid (GA) and manganese chloride (MnCl2). The hydrogel can be self-assembled to form the gel under the condition that no auxiliary material is added. The hydrogel has excellent gel properties such as injectability, heat sensitivity and stability, and can be used as an ideal drug delivery system to be applied to treatment of low-immunogenicity tumors such as advanced malignant liver cancer ascites and melanoma. The hydrogel has the effects of inducing apoptosis and ferroptosis of tumor cells and reprogramming an immunosuppressive tumor microenvironment. The tumor inhibition rate reaches 82% when the compound is combined with alpha-PD-L1 immunotherapy checkpoint blocking treatment in a liver cancer malignant ascites mouse model, and the tumor inhibition rate reaches 98% when the compound is combined with adoptive cell therapy in a melanoma mouse model. In addition, the hydrogel has good biocompatibility, and is a novel anti-tumor hydrogel which is simple to prepare, green and has clinical transformation potential.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical chemistry and pharmaceutical preparations, and in particular relates to a novel pharmaceutical preparation for anti-tumor and immunotherapy. Background Art

[0002] Hepatocellular carcinoma (HCC) is the leading cause of cancer-related death worldwide, causing more than 7 million deaths each year. Existing treatment strategies, including resection, transplantation, ablation, or new combination regimens such as immunotherapy and targeted therapy, are not suitable or effective for some patients with malignant ascites caused by advanced HCC. The treatment of low-immunogenic malignant ascites has long been a challenge. In advanced malignant ascites, tumor cells invade the peritoneal blood vessels, causing hemorrhagic ascites, marking an uncontrollable disease state with a median survival of only 1.1 years. Advanced HCC is widely resistant to systemic treatment, with low drug accumulation and limited drug residence time in the peritoneal cavity, resulting in unsatisfactory treatment effects. Malignant ascites is present in 23% of patients diagnosed with HCC, which is considered a sign of tumor progression and poor prognosis. Emerging immunotherapy strategies such as immune checkpoint blockade (ICB) and adoptive cell therapy can treat cancer by reactivating exhausted cytotoxic T lymphocytes (CTL) to avoid strong immune responses of cancer cells. However, only about 20% of patients can benefit from these therapies. A series of inhibitory factors, such as low immunogenicity, defective antigen presentation, and insufficient immune cell infiltration in tumors, limit the clinical application of immunotherapy in cancer patients.

[0003] At present, most of the methods for treating liver cancer ascites that have been applied for approval are traditional Chinese medicine compound drugs, such as Chinese patent application CN108210839A discloses a traditional Chinese medicine for treating ascites of middle and late liver cancer. And traditional Chinese medicine compound drugs for adjuvant treatment of liver cancer ascites, such as Chinese patent application CN108210839B a traditional Chinese medicine for treating ascites of middle and late liver cancer. All failed to directly target the abdominal ascites tissue. At present, there are no targeted drug delivery, sustained-release drug delivery and high bioavailability drugs for the treatment of liver cancer ascites. Therefore, drugs related to the treatment of liver cancer ascites have failed to meet the current treatment needs. Due to its variable physicochemical and biological properties, hydrogels have been widely explored and applied in drug delivery. Although many anti-tumor hydrogels have been reported, the number of them converted into clinical applications is limited. This limitation may be due to the need for carrier excipients or chemical modification in the preparation of anti-tumor hydrogels. Synthetic polymer hydrogel materials may produce toxic metabolites during human metabolism, which brings unpredictable risks to clinical use and hinders the progress of clinical trials. In addition, some hydrogels are not only complex to prepare and expensive, but also face the challenge of low drug loading, which limits their wide clinical application. Therefore, the development of simple, environmentally friendly, efficient, and high-drug-loading carrier-free hydrogels is of great significance for clinical tumor treatment.

[0004] Based on the above challenges, the present invention prepares a manganese metal-mediated glycyrrhizic acid and ethyl acetate site ternary carrier-free co-assembled injectable hydrogel (KEEM carrier-free hydrogel), which induces tumor cell apoptosis, ferroptosis and reprograms the tumor microenvironment. Combined immunotherapy can more effectively prevent tumor progression. Good therapeutic effects were achieved in low-immunogenic refractory liver cancer malignant ascites mouse models and melanoma mouse models. The hydrogel has good mechanical properties, a long-acting sustained release effect, and no obvious side effects on normal tissues and organs. The advantages of targeted tumors, minimally invasive drug delivery and sustained release make it have great application prospects in the biomedical field. In addition, there is currently no report on fat-soluble multi-component co-assembled carrier-free hydrogel. This is the first study on the self-assembly of natural fat-soluble multi-components into hydrogels, which provides a new research approach for the delivery of fat-soluble natural products. Although it has been reported that glycyrrhizic acid has self-assembly function, whether the self-assembly can be successful is greatly affected by the solvent, temperature, concentration, pH value, and the structure and properties of other components / impurities of the system. At present, there is no research report on the co-assembly of glycyrrhizic acid with two or more components into a gel, and there has never been a report on the co-assembly of multiple terpenoid components and glycyrrhizic acid, which is of great significance for the delivery of poorly soluble terpenoid drugs. Therefore, the present invention is highly innovative and can enrich the current research field. Summary of the invention

[0005] The present invention aims to solve various problems existing in the prior art by preparing a ternary carrier-free co-assembled injectable hydrogel of ethyl acetate part of gansui, glycyrrhizic acid and manganese. The carrier-free hydrogel does not require chemical modification and auxiliary additives, does not require additional carrier materials, has significant anti-tumor effects, can more effectively prevent tumor development and invasion after combined immunotherapy, has high drug loading and good biocompatibility, and has great potential to be developed into a new drug.

[0006] One of the purposes of the present invention is to provide a carrier-free hydrogel of ethyl acetate part of gansuiji-glycyrrhizic acid-manganese.

[0007] The second object of the present invention is to provide a method for preparing the ethyl acetate fraction of gansui-glycyrrhizic acid-manganese carrier-free hydrogel.

[0008] The third object of the present invention is to provide excellent gel rheological material properties of ethyl acetate part of gansui-glycyrrhizic acid-manganese carrier-free hydrogel.

[0009] The fourth purpose of the present invention is to provide an application of the ethyl acetate fraction of gansuiji-glycyrrhizic acid-manganese carrier-free hydrogel in inducing apoptosis and ferroptosis of H22 cells in liver cancer ascites, thereby inhibiting tumor cells.

[0010] The fifth purpose of the present invention is to provide the application of ethyl acetate fraction of gansui-glycyrrhizic acid-manganese carrier-free hydrogel in preventing malignant ascites of liver cancer.

[0011] The sixth purpose of the present invention is to provide an application of the ethyl acetate fraction of gansui-glycyrrhizic acid-manganese carrier-free hydrogel combined with immune checkpoint blockade therapy in the field of anti-malignant ascites of liver cancer.

[0012] The seventh object of the present invention is to provide an application of the ethyl acetate fraction of gansuiji-glycyrrhizic acid-manganese carrier-free hydrogel in the field of anti-melanoma.

[0013] The eighth object of the present invention is to provide an application of the ethyl acetate fraction of gansui-glycyrrhizic acid-manganese carrier-free hydrogel combined with adoptive cell therapy in the field of anti-melanoma.

[0014] The ninth objective of the present invention is to provide a carrier-free hydrogel of ethyl acetate fraction of gansuiji - glycyrrhizic acid - manganese and its safety evaluation on mice after combined immune checkpoint blockade therapy and adoptive cell therapy.

[0015] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0016] 1. The preparation process of the carrier-free hydrogel of glycyrrhizic acid manganese in the ethyl acetate part of gansui was determined by screening different experimental factors such as mass ratio, heating temperature, reaction time, etc. The hydrogel was assembled from the ethyl acetate part of gansui, glycyrrhizic acid, and manganese chloride in a mass ratio of 3-5:4-7:0.3-0.5. UHPLC-Q-Orbitrap HRMS, rheometer, Malvern particle size analyzer and other technical means were further used to determine the chemical composition structure information, injectability, recoverability and stability of the hydrogel.

[0017] The preparation method of the ethyl acetate fraction of Kansui comprises the following steps:

[0018] (1) Powder the gansui slices in a grinder, and then sieve through a 100-mesh stainless steel sieve;

[0019] (2) Place the sieved kansui powder into a round-bottom flask, then add 10 times the amount of 95% ethanol and reflux for 1 h. Repeat twice, filter the extract while hot, and combine the filtrates.

[0020] (3) The filtrate is concentrated to a certain volume under reduced pressure, and an equal amount of ethyl acetate is taken and extracted in a separatory funnel. This is repeated several times until the ethyl acetate layer becomes colorless after extraction. After freeze-drying, the ethyl acetate fraction of Gansui is obtained.

[0021] The preparation method of the carrier-free hydrogel of manganese glycyrrhizinate in the ethyl acetate part of Gan Sui comprises the following steps:

[0022] (1) dissolving the ethyl acetate fraction of gansui, glycyrrhizic acid, and manganese chloride in a mass ratio of 3-5:4-7:0.3-0.5 in deionized water, wherein the mass ratio of glycyrrhizic acid to manganese chloride does not exceed 1:0.15;

[0023] (2) Glycyrrhizic acid was dissolved by ultrasound at room temperature and then added to the ethyl acetate part of Gan Sui and heated for co-assembly;

[0024] (3) after stabilization, the ethyl acetate fraction of the gansui and glycyrrhizic acid binary hot solution is mixed with manganese chloride using a reflux extraction device and reflux extracted;

[0025] (4) The extract is transferred to a centrifuge tube and allowed to stand to return to room temperature, thereby obtaining the ethyl acetate fraction of the gansui extract-manganese glycyrrhizinate-carrier-free hydrogel.

[0026] Preferably, it includes:

[0027] (1) dissolving glycyrrhizic acid in deionized water by ultrasonication for 10-40 min;

[0028] (2) the heating temperature is 70°C-100°C and the ultrasonic time is 10 min-40 min to uniformly disperse the ethyl acetate part of the gansui;

[0029] (3) adding the dissolved glycyrrhizic acid solution to the ethyl acetate fraction of the gansui extract (ultrasound / heating environment) and rapidly stirring (pipette blowing) to dissolve it;

[0030] (4) The ethyl acetate fraction of gansui and glycyrrhizic acid solutions prepared in steps (1), (2) and (3) are mixed, and an aqueous solution of manganese chloride is added, and the mixture is refluxed for 0.5-2.0 hours, and then allowed to stand and cool to obtain a hydrogel.

[0031] 2. Rheological study of the ethyl acetate part of Gan Sui-lilycyrrhizic acid-manganese carrier-free hydrogel was carried out. The main research steps are as follows:

[0032] The material properties of hydrogels are characterized by frequency change scanning and amplitude change scanning.

[0033] 3. The present invention provides the application of the hydrogel in inhibiting liver cancer ascites H22 cells, and the main research steps are as follows:

[0034] (1) The CCK-8 live cell assay was used to detect the survival rate of H22 cells in ascites of hepatocellular carcinoma after 24 h of drug culture. The Calcein / PI / Hoechst live and dead cell staining detection kit (Biyuntian C2015S) was used to evaluate its inhibitory effect on H22 cells in vitro.

[0035] (2) The tumor volume and body weight of H22 tumor-bearing mice were monitored after administration of the drug to evaluate its anti-malignant ascites effect in vivo.

[0036] (3) The drug was administered to the H22 tumor-bearing mouse model with hepatocellular carcinoma ascites and combined with α-PD-L1 immune checkpoint blockade therapy to monitor the tumor volume and body weight, and to evaluate its combined effect with emerging immunotherapies against malignant ascites of hepatocellular carcinoma in vivo.

[0037] 4. The present invention provides the use of the hydrogel in inhibiting melanoma, and the main research steps are as follows:

[0038] (1) Luciferase-labeled B16F10 melanoma cells were used to detect the survival rate of B16F10 cells after 24 hours of drug culture. CD8 + T cells were co-cultured with B16F10 to evaluate the ability of hydrogels to synergistically kill tumor cells with immune cells in vitro.

[0039] (2) The anti-melanoma effect in vivo was evaluated by monitoring the tumor volume and body weight of the B16F10 melanoma model after administration.

[0040] (3) The tumor volume and body weight of the B16F10 melanoma model were monitored after administration and combination with adoptive cell therapy to evaluate its anti-melanoma effect in vivo after combination with emerging immunotherapies.

[0041] 5. Comprehensive safety evaluation is carried out through in vitro hemolysis experiments and mouse organ toxicity experiments.

[0042] The safety evaluation of the ethyl acetate fraction of gansui-glycyrrhizic acid-manganese carrier-free hydrogel and combined immunotherapy includes the following steps:

[0043] (1) The ethyl acetate fraction of gansui-glycyrrhizic acid-manganese carrier-free hydrogel was incubated with rat erythrocytes, and the hemolysis rate of the hydrogel at different concentrations at 570 nm was measured using an ELISA instrument to evaluate the hemolytic activity of the drug.

[0044] (2) The toxicity of the drug on organs was evaluated by H&E staining of the heart, liver, spleen, lung, and kidney of mice treated with the ethyl acetate fraction of glycyrrhizic acid-manganese carrier-free hydrogel.

[0045] The present invention has at least the following beneficial effects:

[0046] (1) The ethyl acetate part of the gansuiji-glycyrrhizic acid-manganese carrier-free hydrogel of the present invention is formed by co-assembly of the ethyl acetate part of the gansuiji-glycyrrhizic acid and manganese metal through non-covalent forces, does not require chemical modification, does not contain drug excipients such as carriers, and does not use organic solvents. The hydrogel is both the drug itself and the carrier, and can be self-delivered and self-released, avoiding the side effects of carrier excipients on the body. It is green to prepare and has high safety.

[0047] (2) The carrier-free hydrogel of the ethyl acetate fraction of gansuiji-glycyrrhizic acid-manganese of the present invention has excellent shear thinning and stability. The hydrogel can be sucked up through a syringe and injected into the patient's body by shear thinning, actively targeting the drug effect site, and improving the drug effect and action time.

[0048] (3) The present invention is derived from a classic Chinese medicine compound drug pair. The anti-hepatocellular carcinoma ascites and anti-melanoma activities of the hydrogel were evaluated using an in vitro hepatocellular carcinoma malignant ascites H22 cell model, an in vivo hepatocellular carcinoma malignant ascites tumor-bearing mouse model, and an in vivo melanoma model. It was demonstrated that the hydrogel has the effects of inducing tumor cell apoptosis and ferroptosis, while reprogramming the tumor microenvironment, with significant efficacy, and can improve the patient's immunity, reduce the recurrence rate of ascites, and significantly inhibit hemorrhagic ascites.

[0049] (4) The present invention evaluated the anti-tumor activity of the ethyl acetate fraction of Gan Sui-glycyrrhizic acid-manganese hydrogel combined with immune checkpoint blockade and adoptive cell therapy through an in vivo liver cancer malignant ascites tumor-bearing mouse model and a melanoma mouse model. It was found that after combined use, the anti-liver cancer ascites activity was significantly stronger than that of other control groups (tumor inhibition rate 82%) and hemorrhagic ascites was significantly inhibited; the melanoma model also showed excellent anti-tumor efficacy (tumor inhibition rate 98%). BRIEF DESCRIPTION OF THE DRAWINGS

[0050] In the attached figure, MN is glycyrrhizic acid-manganese; KEE is ethyl acetate part of gansui acid-glycyrrhizic acid; KEEM is ethyl acetate part of gansui acid-glycyrrhizic acid-manganese (the same below).

[0051] Figure 1 This is a macroscopic image of the KEEM carrier-free hydrogel of the present invention.

[0052] Figure 2 This is a scanning electron micrograph of the KEEM carrier-free hydrogel of the present invention.

[0053] Figure 3 The particle size (Figure A), zeta potential (Figure B) and Tyndall effect (Figure C) of the KEEM carrier-free hydrogel of the present invention.

[0054] Figure 4 The rheological properties of the KEEM carrier-free hydrogel of the present invention are characterized. Figure A shows the frequency scanning result of the hydrogel formed by KEEM, Figure B shows the healing test result of the hydrogel formed by KEEM, Figure C shows the amplitude scanning result of the hydrogel formed by KEEM, and Figure D shows the viscosity test result of the hydrogel formed by KEEM.

[0055] Figure 5 This is a graph showing the inhibition rate of the KEEM carrier-free hydrogel of the present invention on in vitro malignant ascites H22 cells of liver cancer (CCK8 method).

[0056] Figure 6This is an image of live and dead cell staining (Calcein-green / PI-red / Hoechst-blue) of in vitro malignant ascites H22 cells of liver cancer using the KEEM carrier-free hydrogel of the present invention.

[0057] Figure 7 This is a photograph of ascites tissue of mice bearing malignant ascites of liver cancer in vivo after 7 days of treatment with the KEEM carrier-free hydrogel combined with α-PD-L1 immune checkpoint blockade therapy.

[0058] Figure 8 The images of the organs of mice bearing liver cancer malignant ascites treated with KEEM carrier-free hydrogel for 7 days ( Figure 8 A) and H&E staining ( Figure 8 B).

[0059] Fig. 9 The changes in tumor volume of melanoma mice treated with KEEM carrier-free hydrogel combined with adoptive cell therapy for 7 days in vivo ( Fig. 9 A) Tumor inhibition rate ( Fig. 9 B) Macroscopic images of tumors in each group ( Fig. 9 C).

[0060] Fig.10 This is a graph showing the results of the hemolysis experiment of the KEEM carrier-free hydrogel of the present invention. DETAILED DESCRIPTION

[0061] The following examples are intended to further illustrate the present invention. It should be understood by those skilled in the art that the examples are only provided to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0062] It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0063] A novel carrier-free hydrogel with anti-malignant ascites effect of liver cancer is assembled from the ethyl acetate part of gansui, glycyrrhizic acid and manganese chloride in a mass ratio of 3-5:4-7:0.3-0.5, wherein the mass ratio of glycyrrhizic acid to manganese chloride does not exceed 1:0.15.

[0064] Preferably, the mass ratio of the ethyl acetate part of Gansui, glycyrrhizic acid, and manganese chloride is 3-4:4-7:0.3-0.5, or 3-4:5-6:0.3-0.4, and the mass ratio of glycyrrhizic acid to manganese chloride does not exceed 1:0.15. Further preferably, the mass ratio of the ethyl acetate part of Gansui, glycyrrhizic acid, and manganese chloride is 3:4:0.3, or 4:5:0.3, or 4:7:0.5.

[0065] At the same time, the solvent selected is water or PBS (pH 7.0-8.0), and the glycyrrhizic acid must be completely dissolved and mixed with the ethyl acetate part of the glycyrrhizic acid. Experiments have shown that stable hydrogel drugs can only be prepared within the above-mentioned specific component ratio range. When this range is exceeded, the hydrogel will disintegrate into a solution state or produce precipitation. For example: if the molar ratio of glycyrrhizic acid to manganese ions exceeds 1:0.15, a white precipitate will be formed, and a stable hydrogel drug system cannot be formed.

[0066] Whether the ethyl acetate part of gansui, glycyrrhizic acid and manganese metal can be co-assembled to form a carrier-free hydrogel is greatly affected by the solvent, temperature and pH value of the system. The preparation method of the carrier-free hydrogel comprises:

[0067] (1) Dissolve glycyrrhizic acid in deionized water (PBS) by ultrasonication for 10-40 min;

[0068] (2) heating the temperature to 70°C-100°C and ultrasonicating for 10 min-40 min to dissolve the ethyl acetate fraction of the gansui in water (PBS) and disperse it evenly;

[0069] (3) adding the dissolved glycyrrhizic acid solution to the ethyl acetate fraction of the gansui extract (ultrasound / heating environment) and rapidly stirring (pipette blowing) to dissolve it;

[0070] (4) The glycyrrhizic acid solutions of the ethyl acetate fraction of Gan Sui prepared in steps (1), (2) and (3) are mixed, and an aqueous solution of manganese chloride is added, and the mixture is refluxed for extraction for 0.5-2.0 hours, and allowed to stand and cool to obtain a hydrogel.

[0071] The application of the KEEM carrier-free hydrogel in anti-liver cancer malignant ascites and anti-melanoma drugs. The drug is prepared into oral drugs, gastrointestinal release drugs, sustained-release drugs, external or transdermal drugs with the carrier-free hydrogel as the main drug. The drug is prepared into pharmaceutically acceptable dosage forms with the carrier-free injectable hydrogel as the main drug, including oral gels, tablets, capsules, soft capsules, external gels, and injectable hydrogels. It plays a dual role of both exerting anti-ascites activity and acting as a matrix to carry active ingredients.

[0072] The application of the carrier-free injectable hydrogel in combination with immune checkpoint blockade therapy can synergistically resist the proliferation of liver cancer ascites cells by activating the immune system.

[0073] The application of the carrier-free injectable hydrogel in combination with adoptive cell therapy synergistically fights melanoma proliferation by activating the immune system.

[0074] The use of the carrier-free injectable hydrogel in the preparation of a drug, wherein the drug includes a main drug and a drug delivery carrier, and the carrier-free injectable hydrogel is used as a drug delivery carrier to carry and deliver the main drug. Preferably, the main drug is an anti-tumor active drug. It plays a dual role of exerting synergistic pharmacological activity and carrying active ingredients.

[0075] Embodiment 1:

[0076] Take 200g of Gan Sui decoction pieces and make them into powder in a grinder, then pass through a 100-mesh sieve, put the sieved Gan Sui powder into a round-bottom flask, then add 2000mL of 95% ethanol and reflux for 2.0h, repeat twice, filter the extract while hot, combine the filtrate, concentrate the filtrate under reduced pressure to 50mL, measure an equal amount of ethyl acetate and extract in a separatory funnel, repeat 3 times until the ethyl acetate layer is colorless after extraction. After freeze drying, the ethyl acetate part of Gan Sui is obtained.

[0077] Embodiment 2:

[0078] Weigh the ethyl acetate part of Gansui, glycyrrhizic acid, and manganese chloride in a mass ratio of 3:4:0.3, and dissolve them in deionized water respectively. The concentration of glycyrrhizic acid should not be less than 4 mg / mL. Sonicate the glycyrrhizic acid for 20 min, and sonicate the ethyl acetate part of Gansui at 70°C for 20 min. Add the glycyrrhizic acid solution to the ethyl acetate part of Gansui solution and mix quickly. Add the manganese chloride aqueous solution to the mixed solution, reflux and stir for 1.0 h, and let it stand to room temperature to obtain KEEM carrier-free hydrogel.

[0079] Figure 1 This is the appearance of KEEM carrier-free injectable hydrogel. Figure 1 As shown, the carrier-free injectable hydrogel has the appearance of a white, uniform, transparent jelly-like gel.

[0080] Embodiment 3:

[0081] Weigh the ethyl acetate part of Gansui, glycyrrhizic acid, and manganese chloride in a mass ratio of 4:4:0.3, and dissolve them in deionized water respectively. The concentration of glycyrrhizic acid should not be less than 4 mg / mL. Sonicate the glycyrrhizic acid for 25 min, and sonicate the ethyl acetate part of Gansui at 70°C for 25 min. Add the glycyrrhizic acid solution to the ethyl acetate part of Gansui solution and mix quickly. Add the manganese chloride aqueous solution to the mixed solution, reflux and stir for 1.0 h, and let it stand to room temperature to obtain KEEM carrier-free hydrogel.

[0082] Example 4

[0083] Weigh the ethyl acetate part of Gansui, glycyrrhizic acid, and manganese chloride in a mass ratio of 4:4.5:0.4, and dissolve them in deionized water respectively. The concentration of glycyrrhizic acid should not be less than 4 mg / mL. Sonicate the glycyrrhizic acid for 25 min, and sonicate the ethyl acetate part of Gansui at 80°C for 25 min. Add the glycyrrhizic acid solution to the ethyl acetate part of Gansui solution and mix quickly. Add the manganese chloride aqueous solution to the mixed solution, reflux with stirring for 1.0 h, and let it stand to room temperature to obtain KEEM carrier-free hydrogel.

[0084] Example 5

[0085] Weigh the ethyl acetate part of Gansui, glycyrrhizic acid, and manganese chloride in a mass ratio of 4:5:0.4, and dissolve them in deionized water respectively. The concentration of glycyrrhizic acid should not be less than 4 mg / mL. Sonicate the glycyrrhizic acid for 25 min, and sonicate the solution of the ethyl acetate part of Gansui at 85°C for 25 min. Add the glycyrrhizic acid solution to the solution of the ethyl acetate part of Gansui and mix quickly. Add the manganese chloride aqueous solution to the mixed solution, reflux and stir for 1.5 h, and let it stand to room temperature to obtain KEEM carrier-free hydrogel.

[0086] Example 6

[0087] Weigh the ethyl acetate part of Gansui, glycyrrhizic acid, and manganese chloride in a mass ratio of 5:6:0.4, and dissolve them in deionized water respectively. The concentration should not be less than 4 mg / mL based on glycyrrhizic acid. Ultrasonicate the glycyrrhizic acid for 25 min, and ultrasonicate the solution of the ethyl acetate part of Gansui for 25 min at 85°C. Add the glycyrrhizic acid solution to the solution of the ethyl acetate part of Gansui and mix quickly. Add the manganese chloride aqueous solution to the mixed solution, reflux and stir for 1.5 h, and let it stand to room temperature to obtain KEEM carrier-free hydrogel.

[0088] Example 7

[0089] Weigh the ethyl acetate part of Gansui, glycyrrhizic acid, and manganese chloride in a mass ratio of 4:6:0.4, and dissolve them in deionized water respectively. The concentration of glycyrrhizic acid should not be less than 4 mg / mL. Sonicate the glycyrrhizic acid for 25 min, and sonicate the solution of the ethyl acetate part of Gansui at 80°C for 25 min. Add the glycyrrhizic acid solution to the solution of the ethyl acetate part of Gansui and mix quickly. Add the manganese chloride aqueous solution to the mixed solution, reflux and stir for 1.5 h, and let it stand to room temperature to obtain KEEM carrier-free hydrogel.

[0090] Comparative Example 1

[0091] 4 mg / mL glycyrrhizic acid was weighed and dissolved in deionized water and ultrasonicated for 20 minutes; 4 mg / mL glycyrrhizic acid and 0.3 mg / mL manganese chloride were weighed and dissolved in deionized water and ultrasonicated for 20 minutes. After standing for 1 hour, it was found that the glycyrrhizic acid monomer solution could not form a hydrogel, and the glycyrrhizic acid-manganese solution could form a stable hydrogel with mechanical strength, indicating that manganese ions can be co-assembled with glycyrrhizic acid to form a more stable hydrogel.

[0092] Comparative Example 2

[0093] 3 mg / mL glycyrrhizic acid and 0.3 mg / mL manganese chloride were weighed and dissolved in deionized water and ultrasonicated for 20 minutes; 3 mg / mL glycyrrhizic acid, 0.3 mg / mL manganese chloride and 3 mg / mL ethyl acetate of Gansui were weighed and prepared according to the method described in the instruction manual. It was found that the glycyrrhizic acid-manganese solution could not form a hydrogel, while the ethyl acetate of Gansui-glycyrrhizic acid-manganese solution could form a stable hydrogel with mechanical strength, indicating that the ternary system can be co-assembled into a stable hydrogel system and the material properties are better than those of the binary and glycyrrhizic acid monomers.

[0094] Comparative Example 3

[0095] The ethyl acetate part of Gansui, glycyrrhizic acid and manganese chloride were weighed in a mass ratio of 3:4:0.3 to prepare aqueous solutions respectively. The glycyrrhizic acid solution and the ethyl acetate part of Gansui solution were mixed without using a pipette or ultrasound. The obtained ethyl acetate part of Gansui-glycyrrhizic acid solution and manganese chloride solution were refluxed and extracted for 1.0 h, left to cool, and it was found that the precipitate was still oily after reflux extraction, and a stable hydrogel system could not be formed.

[0096] Comparative Example 4

[0097] The ethyl acetate part of Gansui, glycyrrhizic acid and manganese chloride were weighed in a mass ratio of 3:4:0.65, wherein the mass ratio of glycyrrhizic acid to manganese chloride exceeded 1:0.15, and aqueous solutions were prepared respectively. Among them, the ethyl acetate part of Gansui-glycyrrhizic acid solution was prepared and the manganese chloride solution was added and refluxed for extraction for 1.5 hours. After standing and cooling, it was found that only flocculent precipitates could be formed after the solutions were mixed, and a stable hydrogel system could not be formed.

[0098] Example 8 Morphological Characterization of Carrier-free Hydrogel

[0099] (1) The structure of the hydrogel prepared in Example 2 was observed by scanning electron microscopy. Figure 2 shown.

[0100] (2) The hydrogel prepared in Example 2 was subjected to UHPLC-Q-Orbitrap HRMS analysis to analyze its chemical structure composition.

[0101]

[0102]

[0103] (4) The hydrogel prepared in Example 2 was analyzed for particle size, potential, and Tyndall effect. The hydrogel prepared in Example 2 was placed in a small glass dish, and the glass dish was illuminated with a laser pen, and a digital camera was used to take pictures ( Figure 3 C), the hydrogel prepared in Example 2 was diluted 1000 times and placed in a particle size test dish and a ZETA test dish, and the particle size of the hydrogel was measured respectively ( Figure 3 A) ZETA size ( Figure 3 B), indicating good stability of the hydrogel after co-assembly.

[0104] Example 10 Characterization of rheological properties of hydrogel

[0105] The KEEM carrier-free hydrogel prepared in Example 2 was subjected to rheological testing as follows:

[0106] According to Example 2, a KEEM injectable hydrogel with a final concentration of 10 mg / mL was prepared, placed on the rheometer measuring plate, the gap was set to 5 mm, and the temperature was 25°C or 37°C. Frequency sweep: The strain was constant at 0.1%, and the measurement frequency range was 0.1Hz-10Hz. Amplitude sweep: The frequency was set to a constant value of 1Hz, and the strain variation range was 0.001%-10%. Viscosity test: The strain was constant at 0.1%, and the measurement ω range was 0.1-10rad / s. Healability test: The frequency was constant at 1Hz, and low strain (0.1%) and high strain (300%) were cycled to obtain the storage modulus (G′) and loss modulus (G″) that varied with strain. The frequency sweep of the KEEM carrier-free injectable hydrogel is as follows. Figure 4 As shown in A, the healing test is as follows Figure 4 As shown in B, the amplitude sweep is Figure 4 C, the viscosity test is as follows Figure 4 D. The rheological properties of the prepared hydrogels were further analyzed. The G′ reaction gel has solid-like properties, and the G″ reaction gel has liquid-like properties. Figure 4As shown, G′ of KEEM carrier-free injectable hydrogel is much larger than G″, and both G′ and G″ are independent of frequency (0.1-10Hz), proving that the sample exists as a stable hydrogel with an elastic network inside. Such characteristics are common to all hydrogel materials. The oscillatory stress scanning process of KEEM carrier-free hydrogel shows that when the oscillation strain is large enough, G′ and G″ intersect, indicating that KEEM carrier-free hydrogel has good shear thinning properties. The viscoelastic property test of KEEM carrier-free injectable hydrogel shows that it has a certain viscosity, and the viscosity decreases with increasing frequency, proving that it is injectable. The healing test of KEEM carrier-free hydrogel shows that under low strain conditions of 0.1%, the hydrogel can maintain a gel state, while under high strain conditions of 300%, G′ drops rapidly, the hydrogel structure is destroyed, and turns into a sol state. After repeated cycles, G′ can recover to the same height as the first cycle, proving that the hydrogel has rapid healing ability.

[0107] Embodiment 11:

[0108] The in vitro anti-ascites H22 cells activity of the KEEM carrier-free hydrogel prepared in Example 2 was verified by the following method:

[0109] Cell plating: H22 mouse hepatocellular carcinoma malignant ascites cells in the logarithmic growth phase were inoculated into 96-well plates at a plating density of 10 4 The cells were cultured in a cell culture incubator for 24 h (culture conditions: 5% CO2, temperature 37°C).

[0110] Cell administration: negative blank group, control group and drug administration group were set up. Only H22 cells were added to the negative control group, only culture medium was added to the blank group, and the drug administration groups included the following groups: KEEM group, MN group, KEE group. Drug-containing culture medium was added to each drug administration group, and the concentration in each well (calculated by the weight of the ethyl acetate part) was 120, 60, 30, 15, 7.5, and 0 mg / mL, respectively. The cells were placed in the cell culture incubator and cultured for 24 hours.

[0111] CCK-8 experiment: add 10 μL of CCK-8 solution to each experimental well, continue to place in the incubator for 2 hours, take out the 96-well plate, place it on a shaker in the dark, and measure the absorbance (OD value) at a wavelength of 450 nm. Calculate the survival rate according to the calculation formula:

[0112] Cell survival rate (%) = (OD 给药组 -OD 空白组 ) / (OD 阴性组 -OD 空白组 )×100%

[0113] The specific experimental results are shown in Table 1.

[0114] Table 1: Survival rate of H22 cells after treatment with KEE carrier-free hydrogel prepared by the present invention and control group (%)

[0115]

[0116] The results showed that the anti-malignant ascites activity of the KEEM group was significantly enhanced compared with other groups, and it had excellent in vitro anti-malignant ascites activity of the liver cancer.

[0117] Example 12: In vivo validation of KEEM carrier-free hydrogel's anti-malignant ascites activity against liver cancer

[0118] The in vivo anti-malignant ascites activity of the KEEM carrier-free hydrogel prepared in Example 2 was evaluated as follows:

[0119] The mouse model of malignant ascites of liver cancer was established by intraperitoneal injection of H22 cells into SPF male Balb / c mice (6-8 weeks old) to observe the anti-malignant ascites effect of hydrogel drugs after intraperitoneal injection. The mice were provided by Beijing Weitong Lihua, animal certificate number (SCXK (Beijing) 2020-0006).

[0120] Adaptive feeding of mice: The experimental animals were adaptively fed for 7 days at a temperature of (23±2)℃, humidity of (60±5)%, and a 12-h day-night cycle. They were allowed to drink water and eat freely during this period, and were given standard feed throughout the entire experimental period.

[0121] Modeling, screening and grouping: H22 cells in the logarithmic growth phase were collected and prepared into 1.0×10 7 / mL of cell suspension. The cells were inoculated intraperitoneally, and 200μL of cell suspension was injected intraperitoneally into each mouse. Three days after inoculation, the mice with swollen abdomen were randomly divided into groups. They were divided into 7 groups, including normal group (untreated mice), model group, KEEM carrier-free hydrogel group, KEE group, MN group, α-PD-L1 group, KEEM carrier-free hydrogel + α-PD-L1 group, with 5 mice in each group.

[0122] Drug administration: Each group of mice was administered every other day by intraperitoneal injection, with a volume of 200 μL and a dose of 3 mg / kg based on the ethyl acetate of gansui. The drug was administered 5 times in total. The weight change, abdominal circumference change, and ascites volume of each group of mice were measured, and the ascites inhibition rate was calculated according to the formula:

[0123] Ascites inhibition rate = (ascites volume in model group - ascites volume in drug administration group) / ascites volume in drug administration group × 100%

[0124] See Table 2-3 for specific results.

[0125] Table 2: Changes in the body weight of mice over time after treatment with KEEM carrier-free hydrogel prepared by the present invention (g)

[0126]

[0127]

[0128] Table 3: Ascites volume (mL) and ascites inhibition rate (%) of mice after treatment with KEEM carrier-free hydrogel prepared by the present invention

[0129]

[0130] All data are mean ± standard deviation (n = 5). There is no significant difference between each group and the model group (ns), *P < 0.05, **P < 0.01, ***P < 0.001.

[0131] The results showed that after treatment, the weight of mice in each group was not significantly different from that in the normal group, indicating that the drug was safe. All drug groups were detected with different degrees of inhibition of malignant ascites of liver cancer. Among them, the KEEM+α-PD-L1 group had the best tumor growth inhibition effect (tumor inhibition rate reached 82.83%), which was significantly better than the control drug groups. The ascites photos of each group are shown in the figure below. Figure 7 shown.

[0132] Example 13: Verification of the anti-melanoma activity of KEEM carrier-free hydrogel in vivo

[0133] The in vivo anti-melanoma activity of the ethyl acetate fraction of gansui-glycyrrhizic acid-manganese carrier-free hydrogel prepared in Example 2 was evaluated as follows:

[0134] The melanoma mouse model was established by subcutaneously injecting B16F10 cells into SPF-grade male C57 mice (6-8 weeks old) to observe the anti-melanoma effect of hydrogel drugs injected into the tumor. The mice were provided by Beijing Weitong Lihua, with the animal qualification certificate number (SCXK (Beijing) 2020-0006).

[0135] Adaptive feeding of mice: The experimental animals were adaptively fed for 7 days at a temperature of (23±2)℃, humidity of (60±5)%, and a 12-h day-night cycle. They were allowed to drink water and eat freely during this period, and were given standard feed throughout the entire experimental period.

[0136] Modeling, screening and grouping: B16F10 cells in the logarithmic growth phase were collected and prepared into 5.0×10 6 Cells were injected subcutaneously, with 100 μL of cell suspension injected subcutaneously into each mouse. Three days after inoculation, the mice with successful modeling were randomly divided into four groups, including the model group, KEEM group, OT-1T cell group (pure CD8 isolated and cultured from the spleen of OT-1 mice), and +T cells, tail vein injection for adoptive cell therapy treatment), KEEM+OT-1T cell group, 5 mice in each group.

[0137] Drug administration: Each group of mice was administered every other day, with a subcutaneous injection, a volume of 25 μL, and a dose of 3 mg / kg based on the ethyl acetate of gansui. A total of 5 doses were administered. The weight change, tumor volume, and tumor size of each group of mice were measured, and the ascites inhibition rate was calculated according to the formula:

[0138] Tumor inhibition rate = (tumor weight of model group - tumor weight of drug administration group) / tumor weight of drug administration group

[0139] ×100%

[0140] See Table 4 for specific results.

[0141] Table 4: Tumor weight (g) and tumor inhibition rate (%) of mice after treatment with KEEM carrier-free hydrogel prepared by the present invention

[0142]

[0143]

[0144] All data are mean ± standard deviation (n = 5). There is no significant difference between each group and the model group (ns), *P < 0.05, **P < 0.01, ***P < 0.001.

[0145] The results showed that after treatment, all drug-administered groups had different degrees of melanoma inhibition. Among them, the KEEM+OT-1T cell group had the best tumor growth inhibition effect (tumor inhibition rate reached 98.08%), which was significantly better than the control drug-administered groups. The tumor photos of each group are shown in the figure below. Fig. 9 As shown in C.

[0146] Example 14: Biosafety Verification of KEEM Carrier-free Hydrogel

[0147] In this example, the safety of the hydrogel prepared in Example 2 was evaluated as follows:

[0148] Hemolysis experiment: Fresh SD rat blood was used to determine the in vitro hemolysis effect of the sample. First, blood was taken from the abdominal aorta of SD rats and centrifuged at 3000rpm / min for 15 minutes. The red blood cells were collected and washed with normal saline (PBS) 3 times. Then 3mL of the red blood cells obtained by centrifugation were mixed into 11mL of normal saline for storage and dispersion. The hydrogel was diluted to the corresponding concentration with normal saline. Then the test solution (1mL) was mixed with 100μL of red blood cell stock solution to obtain a 4% red blood cell solution, which was incubated at 37°C for 4h. Then centrifuged at 3000rpm / min for 15min, and the absorbance value of the supernatant was measured at 570nm by an enzyme reader. The positive control was deionized water and the negative control was normal saline. The hemolysis rate was calculated according to the following formula:

[0149] Hemolysis rate (%) = (A 给药组 -A PBS组 ) / (A 去离子水组 -A PBS组 )×100%

[0150] The results showed that the hydrogel had no obvious hemolytic properties. Even at a concentration of up to 240 μg / mL, the hemolysis rate was still lower than the internationally recognized standard of 5%.

[0151] H&E staining experiment: After the animal experiment, mice from each group were taken out for dissection to obtain the heart, liver, spleen, lung tissue, and kidney organs. Their morphology was observed and fixed with 4% paraformaldehyde solution for H&E staining.

[0152] The results are as follows Figure 6 As shown, compared with the normal group, no obvious pathological changes were observed in the heart, liver, spleen, lung, and kidney tissues of the KEEM hydrogel group and other drug-treated groups.

[0153] The results of blood routine and blood biochemistry showed that there were no significant differences in red blood cells, white blood cells, platelets, liver function, kidney function and other indicators between the KEEM gel group and the combined drug group and the normal group.

[0154] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes, and they can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the illustrations shown and described herein.

Claims

1. A carrier-free hydrogel of ethyl acetate fraction of gansui, glycyrrhizic acid and manganese, characterized in that: The ethyl acetate part of gansui-glycyrrhizic acid-manganese carrier-free hydrogel is prepared from the following components: ethyl acetate part of gansui-glycyrrhizic acid, and manganese chloride in a mass ratio of 3-5:4-7:0.3-0.5, wherein the mass ratio of glycyrrhizic acid to manganese chloride does not exceed 1:0.

15.

2. The ethyl acetate fraction of gansuiji-glycyrrhizic acid-manganese carrier-free hydrogel according to claim 1 is prepared by the following method: (1) Add appropriate amount of deionized water to the ethyl acetate fraction of Glehnia littoralis, glycyrrhizic acid, and manganese chloride; (2) After dissolving glycyrrhizic acid by ultrasonic at room temperature, the ethyl acetate fraction of glycyrrhizic acid was added, and the two were heated to dissolve; (3) The hot solution of (2) is mixed with manganese chloride and heated to reflux; and the mixture is allowed to stand at room temperature to obtain the ethyl acetate fraction of glycyrrhizic acid-manganese glycyrrhizinate-carrier-free hydrogel.

3. The ethyl acetate fraction of gansuiji-glycyrrhizic acid-manganese carrier-free hydrogel according to claim 1, characterized in that: The ethyl acetate part of the gansui is prepared by the following method: (1) Take kansui and make it into powder; (2) adding 95% ethanol by weight to reflux the kansui powder, filtering the extract, and combining the filtrate; (3) The filtrate was concentrated under reduced pressure, and an equal volume of ethyl acetate was taken for extraction. The extraction was repeated several times until the ethyl acetate layer was colorless. The ethyl acetate layers were combined and freeze-dried to obtain the ethyl acetate fraction of Gansui.

4. The ethyl acetate fraction of gansuiji-glycyrrhizic acid-manganese carrier-free hydrogel according to any one of claims 1 to 3, characterized in that: The heating temperature is 70°C-100°C; the ultrasonic time is 10min- 40min; the reflux extraction time is 0.5-2.0h.

5. A composition comprising the ethyl acetate fraction of gansuiji-glycyrrhizic acid-manganese carrier-free hydrogel as described in any one of claims 1 to 4.

6. The composition according to claim 5, characterized in that Also contains α-PD-L1.

7. The composition according to claim 5, characterized in that The composition is an oral preparation, an injection, a sustained-release preparation, and an external preparation; preferably, the oral preparation is an oral gel, a tablet, a capsule, or a soft capsule, the injection is an injection gel, the sustained-release preparation is a gastrointestinal release preparation, and the external preparation is a transdermal preparation.

8. Use of the ethyl acetate fraction of gansuiji-glycyrrhizic acid-manganese carrier-free hydrogel as described in any one of claims 1 to 2 or the composition as described in any one of claims 6 to 7 in the preparation of anti-tumor drugs.

9. Use of the ethyl acetate fraction of gansui-glycyrrhizic acid-manganese carrier-free hydrogel as described in any one of claims 1 to 2 or the composition as described in any one of claims 6 to 7 in the preparation of a drug for enhancing the effect of anti-tumor immunotherapy.

10. Use of the ethyl acetate fraction of gansui-glycyrrhizic acid-manganese carrier-free hydrogel as described in any one of claims 1-2 or the composition as described in any one of claims 6-7 in the preparation of drugs for preventing liver cancer ascites and melanoma and enhancing the effect of immunotherapy.

11. The use according to claims 8-10, characterized in that The ethyl acetate part of gansuiji-glycyrrhizic acid-manganese carrier-free hydrogel is used as the only active ingredient of the medicine, or as one of the active ingredients, or as a carrier.

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