Preparation of copper ion mediated glycyrrhizic acid and norcantharidin ternary carrier-free co-assembled injectable hydrogel and anti-tumor application of copper ion mediated glycyrrhizic acid and norcantharidin ternary carrier-free co-assembled injectable hydrogel

Abstract

The invention discloses preparation and application of a ternary carrier-free injectable hydrogel formed by co-assembling glycyrrhizic acid, norcantharidin and metal copper ions. The hydrogel can form gel under the condition of not adding auxiliary materials, has excellent gel material properties such as injectability, heat sensitivity and stability, and can be used as an ideal anti-tumor drug to be clinically applied. The hydrogel has the effects of inducing tumor cell apoptosis and copper death and resisting inflammation, and cooperatively regulates and controls a tumor microenvironment; and laser irradiation is combined to assist in enhancing the Fenton-like effect, so that chemical kinetic treatment of tumors is realized. In a Hepa1-6 tumor-bearing liver cancer mouse model, the hydrogel has an excellent anti-tumor effect, and the drug effect is obviously superior to that of each monomer drug group. 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 discloses a preparation method of an injectable glycyrrhizic acid-copper-norcantharidin hydrogel and its anti-tumor application. The invention can coordinately regulate the tumor microenvironment by promoting tumor cell apoptosis, copper cell death and anti-inflammation, and realize laser-assisted Fenton-like anti-tumor effect. Glycyrrhizic acid-copper-norcantharidin has co-assembly performance and can be prepared into a carrier-free hydrogel drug, belonging to the field of pharmaceutical technology. Background Art

[0002] Cancer is the leading cause of death in the world, with 19.3 million new cases and nearly 10 million deaths in 2020. Currently, the main clinical treatments for tumors are surgical resection, radiotherapy, and chemotherapy. Although chemotherapy drugs can effectively kill tumor cells, their clinical application is limited by their shortcomings such as easy development of drug resistance, poor targeting, and severe adverse reactions. Some emerging treatment strategies such as Cart T cell therapy, antibodies, and targeted drugs have made great progress. However, due to the complex preparation and high cost of treatment, their clinical application worldwide has been hindered and has not yet been widely used. In addition to the above-mentioned difficulties in tumor treatment, the tumor microenvironment plays a vital role in the occurrence, invasion, metastasis, and recurrence of tumors, and is the main challenge in tumor treatment.

[0003] In recent years, more and more studies have begun to develop treatment strategies targeting the weak acidity, overexpression of hydrogen peroxide, hypoxia, and severe inflammatory response of the tumor microenvironment. Chemodynamic therapy mediated by metal ions with Fenton / Fenton-like effects and coordinated inflammation regulation has become a promising new strategy for tumor treatment due to its high specificity, low side effects, and non-invasiveness. Many bio-nanomaterials and hydrogels for chemodynamic therapy have been developed. Although bio-nanomaterials have active or passive tumor targeting effects when administered systemically, recent studies have shown that only a small portion of nanoparticles (median 0.7%) can reach tumor tissues after intravenous injection, which may cause unnecessary toxicity to normal tissues and organs [S. Wilhelm, AJ Tavares, Q. Dai, et al., Analysis of nanoparticle delivery to tumours, Nature Reviews Materials 1 (2016) 16014. https: / / doi.org / 10.1038 / natrevmats.2016.14.]. At the same time, the hydrogels that have been developed so far are mostly used as carriers of various drugs, which require the participation of polymer materials such as hyaluronic acid, alginate, and chitosan. However, in the tumor microenvironment, the inflammation-regulating efficacy of these polymer carriers is usually weaker than the anti-inflammatory ability of active molecules such as glycyrrhizic acid. In addition, the intervention of carriers may lead to low drug loading, poor biodegradability and biocompatibility, and potential side effects, which are the main reasons hindering industrial production and clinical transformation. These inevitable obstacles have prompted an urgent need to develop synergistic anticancer strategies with different mechanisms of action to provide alternative pathways to chemodynamic therapy.

[0004] Copper ions have a Fenton-like effect. They can react with hydrogen peroxide overexpressed in the tumor microenvironment to generate reactive oxygen and oxygen, and react with glutathione to protect the generated reactive oxygen, thereby inducing cell apoptosis and fighting hypoxia. In addition, the Fenton-like effect of copper ions is more efficient under 808nm laser irradiation and can be used for chemokinetic therapy of tumors. Recent studies have shown that copper ions can also kill tumor cells through the copper death pathway. Compound Cantharidae Capsules achieve efficacy and toxicity reduction by combining with licorice. Currently, they are widely used in the treatment of various cancers. Among them, glycyrrhizic acid is the main active ingredient of traditional Chinese medicine licorice and has good anti-inflammatory effects; norcantharidae is derived from traditional Chinese medicine Cantharidae, which can induce tumor cell apoptosis and is an effective anticancer drug for the clinical treatment of liver cancer, gastric cancer, etc. However, norcantharidae has poor water solubility, a short half-life in the blood, and low tumor targeting efficiency, which seriously hinders its clinical application.

[0005] Based on the above challenges, the present invention prepares a copper ion-mediated glycyrrhizic acid and norcantharidin ternary carrier-free co-assembly injectable hydrogel, which coordinates apoptosis, copper death and anti-inflammatory regulation of tumor microenvironment, and realizes the chemokinetic treatment of tumors. The hydrogel has good mechanical properties, just like a drug storage warehouse, which can continuously release drugs to the target site, with little side effects on normal tissues and organs. The advantages of local targeting and minimally invasive drug delivery make it have great application prospects in the biomedical field, and open up new ways for the development of cancer treatment. Although some scholars have found that glycyrrhizic acid has a self-assembly effect, whether the self-assembly can be successful is greatly affected by the solvent, temperature, concentration, pH value, structure and properties of other components / impurities of the system. At present, there is no research report on the co-assembly of glycyrrhizic acid and two or more components into a gel, and there is no report on the application of the obtained gel as a carrier-free anti-tumor drug. Therefore, the present invention is very innovative and can enrich the current research field. Summary of the invention

[0006] Based on the above background, the present invention aims at various problems existing in the aforementioned prior art and innovatively prepares a glycyrrhizic acid-copper-norcantharidin ternary carrier-free co-assembled injectable hydrogel. The hydrogel not only avoids many shortcomings of the current application of polymer materials, but also has good safety itself, significant and long-lasting anti-tumor and anti-inflammatory effects, and has great potential to be developed into a new drug.

[0007] One of the objects of the present invention is to provide a glycyrrhizic acid-copper-norcantharidin injectable hydrogel.

[0008] The second object of the present invention is to provide a method for preparing the glycyrrhizic acid-copper-norcantharidin injectable hydrogel.

[0009] The third object of the present invention is to provide glycyrrhizic acid-copper-norcantharidin injectable hydrogel with excellent gel material properties.

[0010] The fourth object of the present invention is to provide the application of glycyrrhizic acid-copper-norcantharidin injectable hydrogel in the anti-tumor field.

[0011] The fifth object of the present invention is to provide the application of glycyrrhizic acid-copper-norcantharidin injectable hydrogel in the anti-inflammatory field.

[0012] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0013] 1. The preparation process of the ternary hydrogel was determined by exploring different inorganic copper salts, heating temperatures, molar ratios, etc. The hydrogel was co-assembled by glycyrrhizic acid, inorganic copper salts, and norcantharidin in a molar ratio of 1-10:0.1-1:1-10, wherein the molar ratio of glycyrrhizic acid to copper ions did not exceed 1:0.7. The structural information of the ternary hydrogel was further determined by infrared spectroscopy, nuclear magnetic resonance hydrogen spectroscopy, and other technical means.

[0014] Preferably, the inorganic copper salt is copper chloride or copper sulfate.

[0015] The self-assembly preparation method of the ternary hydrogel comprises the following steps:

[0016] (1) heating to dissolve glycyrrhizic acid in water;

[0017] (2) heating to dissolve norcantharidin in water;

[0018] (3) heating to dissolve the inorganic copper salt in water;

[0019] (4) The glycyrrhizic acid, norcantharidin and copper ion aqueous solutions prepared in steps (1), (2) and (3) are mixed, with the concentration of glycyrrhizic acid being not less than 5 mmol / L, heated, and allowed to stand and cool to obtain a hydrogel.

[0020] Preferably, it includes:

[0021] (1) heating to 60-100° C. to dissolve glycyrrhizic acid in water;

[0022] (2) heating to 60-100° C. to dissolve norcantharidin in water;

[0023] (3) heating to 25-100°C to dissolve the inorganic copper salt in water;

[0024] (4) The glycyrrhizic acid, norcantharidin and copper ion aqueous solutions prepared in steps (1), (2) and (3) are mixed, with the concentration of glycyrrhizic acid being not less than 5 mmol / L, heated to 60-100° C., and allowed to stand and cool to obtain a hydrogel.

[0025] 2. Rheological study of ternary hydrogel, the main research steps are as follows:

[0026] The materials characterization of the ternary hydrogel was carried out through frequency scanning and amplitude scanning.

[0027] 3. The present invention provides the use of the hydrogel in the preparation of anti-tumor drugs, and the main research steps are as follows:

[0028] (1) The MTT method was used to detect the survival rate of human hepatoma cell (HepG2) cells after 72 h of culture with the drug to evaluate its in vitro antitumor effect.

[0029] (2) The antitumor effect in vivo was evaluated by monitoring the tumor volume and body weight after administration to Hepa1-6 tumor-bearing mice.

[0030] (3) The Fenton-like effect of copper ions was enhanced by 808 nm laser irradiation to enhance the anti-tumor effects in vitro and in vivo.

[0031] (4) The anti-tumor mechanism of the ternary hydrogel was analyzed by qPCR (real-time quantitative polymerase chain reaction) and IF (immunofluorescence).

[0032] 4. Safety evaluation is carried out through cell safety experiments, in vitro hemolysis experiments, and whole mouse toxicity experiments.

[0033] The safety evaluation of ternary hydrogel includes the following steps:

[0034] (1) The MTT method was used to evaluate the survival rate of canine kidney cells (MDCK) after 72 h of drug addition and to assess the cytotoxicity of the drugs.

[0035] (2) The ternary hydrogel was co-incubated with rat erythrocytes, and the hemolysis rate of each drug group was measured at 570 nm using an ELISA reader to evaluate the hemolytic activity of the drug.

[0036] (3) HE staining of the organs of treated mice was performed to evaluate the toxicity of the drugs on the organs.

[0037] (4) The safety of the drug was evaluated by observing the animal condition, measuring blood routine and blood biochemical indicators, observing the main organs, and performing HE staining after subcutaneous injection of the hydrogel into normal mice.

[0038] The present invention has at least the following beneficial effects:

[0039] (1) The ternary hydrogel of the present invention is formed by co-assembly of glycyrrhizic acid, copper ions and norcantharidin, does not contain drug excipients such as carriers, is not chemically modified, and does not use organic solvents, and is a ternary carrier-free hydrogel;

[0040] (2) The self-assembly method of the present invention can be used to prepare ternary hydrogels, and the assembly mechanism and structure of the hydrogels formed after assembly are confirmed by infrared and nuclear magnetic resonance methods;

[0041] (3) The present invention evaluated the anti-tumor and anti-inflammatory activity of the ternary hydrogel using a Hepa1-6 tumor-bearing liver cancer mouse model and found that the hydrogel has the effects of inducing tumor cell apoptosis, copper cell death, and anti-inflammatory effects, synergistically regulating the tumor microenvironment, and achieving chemokinetic treatment of tumors. The efficacy is significantly better than that of the control drug groups.

[0042] (4) The ternary hydrogel of the present invention can also be used as a drug delivery carrier to carry other drugs, such as anti-tumor drugs, playing a dual role of both exerting pharmacological activity and carrying active ingredients.

[0043] Other advantages, objectives and features of the present invention will be embodied in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 It is a macroscopic picture of the ternary carrier-free hydrogel of the present invention.

[0045] Figure 2 The scanning electron microscope images of the binary and ternary carrier-free hydrogels of the present invention are shown.

[0046] Figure 3 The present invention characterizes the rheological properties of the binary carrier-free hydrogel.

[0047] Figure 4 The present invention characterizes the rheological properties of the ternary carrier-free hydrogel.

[0048] Figure 5 This is a photograph of tumor tissue in Hepa1-6 liver cancer-bearing mice treated with the ternary carrier-free hydrogel of the present invention for 13 days.

[0049] Figure 6 These are pictures of organs and HE staining of Hepa1-6 tumor-bearing liver cancer mice treated with the ternary carrier-free hydrogel of the present invention for 13 days.

[0050] Figure 7 The photos of liver and kidney tissues and HE staining of the whole toxicity experiment of the ternary carrier-free hydrogel in mice of the present invention are shown. DETAILED DESCRIPTION

[0051] The present invention is further described in detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

[0052] It should be understood that the terms such as “having”, “including” and “comprising” used herein do not exclude the existence or addition of one or more other elements or combinations thereof.

[0053] 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.

[0054] A novel ternary carrier-free injectable hydrogel with anti-tumor effect is formed by co-assembly of glycyrrhizic acid, inorganic copper salt and norcantharidin in a molar ratio of 1-10:0.1-1:1-10, wherein the molar ratio of glycyrrhizic acid to copper ions does not exceed 1:0.7.

[0055] Preferably, it is made of glycyrrhizic acid, inorganic copper salt, and norcantharidin in a molar ratio of 1-5:0.1-1:1-5, wherein the molar ratio of glycyrrhizic acid to copper ions does not exceed 1:0.7. For example, 1:0.2:1, or 1:0.5:1, or 1:0.7:1, or 1:0.5:2. Experiments have shown that a stable hydrogel drug can be prepared within the above-mentioned specific range, and when this range is exceeded, the hydrogel will disintegrate into a solution state. For example: if the molar ratio of glycyrrhizic acid to copper ions exceeds 1:0.7, a flocculent precipitate is formed, and a stable hydrogel drug system cannot be formed.

[0056] The inorganic copper salt is copper chloride or copper sulfate.

[0057] Whether the self-assembly is successful is greatly affected by the solvent, temperature, pH value, and the structure and properties of other components / impurities of the system. The self-assembly preparation method of the ternary hydrogel comprises:

[0058] (1) heating to 60-100° C. to dissolve glycyrrhizic acid in water;

[0059] (2) heating to 60-100° C. to dissolve norcantharidin in water;

[0060] (3) heating to 25-100°C to dissolve the inorganic copper salt in water;

[0061] (4) The glycyrrhizic acid, norcantharidin and copper ion aqueous solutions prepared in steps (1), (2) and (3) are mixed, with the concentration of glycyrrhizic acid being not less than 5 mmol / L, heated to 60-100° C., and allowed to stand and cool to obtain a hydrogel.

[0062] The application of the ternary hydrogel in the preparation of anti-tumor drugs. The drug is prepared into oral drugs, gastrointestinal release drugs, sustained-release drugs, external or transdermal drugs with the ternary hydrogel as the main drug. The drug is prepared into pharmaceutically acceptable dosage forms with the ternary hydrogel as the main drug, including oral gels, tablets, capsules, soft capsules, external gels, and hydrogels for injection. It plays a dual role of both exerting pharmacological activity and carrying active ingredients.

[0063] The ternary hydrogel is used in the preparation of a drug, wherein the drug includes a main drug and a drug delivery carrier, and the ternary hydrogel is used as a drug delivery carrier to carry and deliver the main drug. Preferably, the main drug is an anti-tumor drug. It plays a dual role of exerting synergistic pharmacological activity and carrying active ingredients.

[0064] Example 1

[0065] Weigh glycyrrhizic acid and different metal ions (copper chloride, calcium chloride, magnesium chloride, ferrous chloride, ferric chloride, aluminum chloride, zinc chloride) in a molar ratio of 10:1 to 10:10, dissolve them in water, heat and mix, and let stand to cool. Glycyrrhizic acid and copper can form hydrogels in the range of 10:1 to 10:7, while other metal ions (calcium, magnesium, ferrous chloride, iron, aluminum, zinc) react with glycyrrhizic acid mostly to form flocculent precipitation, and only a few ratios can form gels, for example: glycyrrhizic acid-calcium forms hydrogels in the range of 10:1 to 10:4, glycyrrhizic acid-magnesium forms hydrogels in the range of 10:1 to 10:5, glycyrrhizic acid-ferrous chloride forms hydrogels in the range of 10:1 to 10:4, glycyrrhizic acid-iron forms hydrogels in the range of 10:1 to 10:2, glycyrrhizic acid-aluminum forms hydrogels in the range of 10:1 to 10:5, and glycyrrhizic acid-zinc forms hydrogels in the range of 10:1 to 10:3. Moreover, each metal ion and glycyrrhizic acid are easier to gel when they are in a small proportion, and the color is clearer and more transparent; when the metal ion concentration increases, the hydrogel is easy to disintegrate, and the color is darker and more turbid. Compared with other metal ions, the glycyrrhizic acid-copper binary hydrogel has the best material properties. It can be seen that whether different raw materials and proportions can form hydrogels is unpredictable and accidental. The technical solution of the present invention requires creative labor to obtain and is innovative.

[0066] Example 2

[0067] The ternary hydrogel was prepared by the following method.

[0068] Weigh glycyrrhizic acid (GA) and inorganic copper salt (Cu) in a molar ratio of 1:0.5:1. 2+ ), norcantharidin (NCTD), are dissolved in water, mixed, and heated to 60-100°C to a concentration of not less than 5 mmol / L based on glycyrrhizic acid to obtain a clear and transparent hydrogel (NCTD Gel) with a macroscopic morphology as shown in FIG. Figure 1 shown.

[0069] Example 3

[0070] Weigh glycyrrhizic acid, inorganic copper salt and norcantharidin in a molar ratio of 1:0.2:1, dissolve them in water respectively, mix them, and heat them to a concentration of not less than 5 mmol / L based on glycyrrhizic acid, and prepare a clear and transparent hydrogel.

[0071] Example 4

[0072] Weigh glycyrrhizic acid, inorganic copper salt and norcantharidin in a molar ratio of 1:0.7:1, dissolve them in water respectively, mix them, and heat them to a concentration of not less than 5 mmol / L based on glycyrrhizic acid to 60-100° C. to prepare a clear and transparent hydrogel.

[0073] Example 5

[0074] Weigh glycyrrhizic acid, inorganic copper salt and norcantharidin in a molar ratio of 1:0.5:2, dissolve them in water respectively, mix them, and heat them to a concentration of not less than 5 mmol / L based on glycyrrhizic acid to 60-100° C. to prepare a clear and transparent hydrogel.

[0075] Comparative Example 1

[0076] Glycyrrhizic acid, inorganic copper salt, and norcantharidin were weighed in a molar ratio of 1:0.1-1:1, dissolved in water respectively, heated and mixed, and allowed to stand and cool. It was found that if the ratio of glycyrrhizic acid to copper ions exceeded 1:0.7, a flocculent precipitate was formed and a stable hydrogel system could not be formed.

[0077] Comparative Example 2

[0078] Glycyrrhizic acid, inorganic copper salt, and cantharidin (a structural analogue of norcantharidin) were weighed in a molar ratio of 1:0.5:1, dissolved in water respectively, heated and mixed, and allowed to stand and cool. It was found that no stable hydrogel system could be formed, indicating that the chemical structure of norcantharidin plays a unique role in the formation of the ternary hydrogel. Although cantharidin has a similar structure to it, they cannot be replaced by each other. This is consistent with the current research status that the discovery of small molecule carrier-free hydrogels is accidental.

[0079] Structural characterization of hydrogels

[0080] (1) The structures of the glycyrrhizic acid-copper binary hydrogel and the glycyrrhizic acid-copper-norcantharidin ternary hydrogel prepared in Example 1-2 were observed by scanning electron microscopy. Figure 2 A is an electron micrograph of the binary hydrogel, which is in the form of arranged nanofibers; Figure 2 B is the electron micrograph of the ternary hydrogel, which is a uniformly distributed regular spherical nanoparticle. The results show that after the addition of norcantharidin, the microscopic morphology of the binary hydrogel and the ternary hydrogel changed significantly, proving that norcantharidin and glycyrrhizic acid-copper did co-assemble to form a ternary hydrogel.

[0081] (2) The hydrogel prepared in Example 2 was subjected to infrared analysis. The infrared analysis conditions were: the scanning range was 4000 cm -1 -400cm -1 , resolution 4cm -1 , the scanning speed is 7.5KHz, and the infrared peak positions of the hydrogel obtained by infrared analysis are as follows:

[0082] The infrared attribution of the hydrogel is: 3202.74 (-OH stretching vibration), 1720.07 (-C=O stretching vibration), 1593.81, 1416.96 (-COO symmetric and asymmetric stretching vibration), 1007.55 (-CO vibration peak). After adding copper ions, the -OH group of glycyrrhizic acid blue-shifted to 3202.74 cm -1 , and the shape and strength of the -COO- group changed significantly, indicating that the copper ion is likely to form a coordination bond with the carboxyl group of glycyrrhizic acid; after adding norcantharidin, the -CO group changed from 1032.37 cm -1 Shift to lower wavenumbers to 1007.55 cm -1 , and the absorption peak of -C=O was significantly enhanced, indicating that norcantharidin may form a hydrogen bond with the hydroxyl group on glycyrrhizic acid glucuronic acid.

[0083] (3) The hydrogel prepared in Example 2 was characterized by nuclear magnetic resonance, and the results were as follows:

[0084] Ternary hydrogel NMR assignment: 5.39 (s, 1H, 12-H, GA), 4.67 (m, 2H, 2-H, 5-H, NCTD), 4.47 (d, J = 7.6 Hz, 1H, 1'-H, GA), 4.35 (d, J = 7.6 Hz, 1H, 1"-H, GA), 3.50-3.00 (m, 14H, 3-H, Glu-H, GA), 2.86 (s, 2H, 1-H, 6-H, NCTD), 2.56 (d, J = 13.2 Hz, 1H, 18-H, GA), 2.33 (s, 1H, 9-H, GA), 2.08-0.71 (m, 40H, parent nucleus-H,GA),1.56-1.48(m,4H,3-H,4-H,NCTD).After binding with norcantharidin, the shape of the proton peak on glucuronic acid of glycyrrhizic acid changed significantly, and the proton peak of the carboxyl group of norcantharidin disappeared, indicating that the carboxyl group of norcantharidin was combined with the hydroxyl group of glucuronic acid of glycyrrhizic acid in the form of hydrogen bond.

[0085] Rheological characterization of hydrogels

[0086] The binary and ternary hydrogels prepared in Example 1-2 were subjected to rheological tests as follows:

[0087] According to Example 1-2, binary and ternary hydrogels with a final concentration of 10 mmol / L were 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 0.1 Hz-10 Hz. Amplitude sweep: the frequency was set to a constant value of 1 Hz, and the strain variation range was 0.01%-10%. Viscosity test: the strain was constant at 0.1%, and the measurement ω range was 0.6-85 rad / s. Healability test: the frequency was constant at 1 Hz, 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 binary hydrogel formed by glycyrrhizic acid and different metal ions (calcium, magnesium, ferrous iron, iron, aluminum, zinc) is as follows Figure 3 As shown in A, the amplitude sweep is Figure 3 As shown in B; the frequency scanning of the ternary hydrogel formed by glycyrrhizic acid-copper-norcantharidin is as follows Figure 4 As shown in A, the amplitude sweep is Figure 4 As shown in B, the viscosity test is Figure 4 C, the healing test is as follows Figure 4 As shown in D.

[0088] The rheological properties of the prepared hydrogels were further analyzed. The G′ reaction gel had solid-like properties, while the G″ reaction gel had liquid-like properties. Figure 4As shown, G′ of the ternary hydrogel is much larger than G″, and both G′ and G″ are independent of the 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 the ternary hydrogel shows that when the oscillatory strain is large enough, G′ and G″ intersect, indicating that the ternary hydrogel has good shear thinning properties. The viscoelastic properties test of the ternary hydrogel shows that the ternary hydrogel has a certain viscosity, and the viscosity decreases with increasing frequency, proving that it is injectable. The healing test of the ternary hydrogel shows that under low strain conditions of 0.1%, the ternary hydrogel can maintain a gel state, while under high strain conditions of 300%, G′ drops rapidly, the structure of the ternary hydrogel is destroyed, and it is transformed into a sol state. After repeating several cycles, G′ can recover to the same height as the first cycle, proving that the ternary has rapid healing ability. Figure 3 In the experiment, compared with other metal ions (calcium, magnesium, ferrous iron, iron, aluminum, and zinc), the G′ and G″ of the binary hydrogel formed by glycyrrhizic acid-copper were the highest, showing better mechanical strength. At the same concentration, compared with the glycyrrhizic acid-copper binary hydrogel, the G′ and G″ of the glycyrrhizic acid-copper-norcantharidin ternary hydrogel were higher and the mechanical strength was better, indicating that the co-assembly synergistic effect of glycyrrhizic acid, copper ions, and norcantharidin can obtain a hydrogel with better mechanical strength.

[0089] Verification of the antitumor activity of hydrogel in vitro

[0090] The in vitro antitumor activity of the ternary hydrogel prepared in Example 2 was evaluated as follows:

[0091] Cell plating: Logarithmically growing human liver cancer cell HepG2 cells were inoculated into a 96-well plate with a cell density of 3000 cells per well and cultured in a cell culture incubator for 24 hours.

[0092] Cell administration: set up blank cell group, blank group and drug administration group. Only culture medium was added to the blank cell group, and no liquid was added to the blank group. Drug administration group was added with drug-containing culture medium. The concentration of each well was 32, 16, 8, 4, 2, and 1 μg / mL, respectively. The cells were cultured in the cell culture incubator for 72 hours.

[0093] MTT experiment: Except for the blank group, 20 μL of MTT solution was added to each group, and the culture was continued in the incubator for 4 h. The culture medium in the well was replaced with 150 μL of dimethyl sulfoxide, and the well was placed on a shaker in the dark and shaken well. The absorbance (OD value) was measured at a wavelength of 490 nm, and the survival rate was calculated according to the formula:

[0094] Cell survival rate (%) = (OD 给药组 -OD 空白组 ) / (OD 正常组 -OD空白组 )×100%

[0095] The specific results are shown in Table 1.

[0096] Table 1: Survival rate of HepG2 cells after treatment with the ternary hydrogel prepared in the present invention (%)

[0097]

[0098]

[0099] The results show that the antibacterial activity of most ternary hydrogels is significantly enhanced compared with that of monomer raw materials, and the activity is further enhanced after laser irradiation. It has excellent in vitro anti-tumor activity and is worthy of in-depth research and further clinical development.

[0100] Verification of hydrogel antitumor activity in vivo

[0101] The in vivo antitumor activity of the ternary hydrogel prepared in Example 2 was evaluated as follows:

[0102] The Hepa1-6 tumor-bearing liver cancer mouse model was established by subcutaneous injection of Hepa1-6 cells, and the anti-tumor effect of intratumoral injection of hydrogel drugs was observed. The mice used were SPF-grade female C57BL / 6J mice, weighing 20±1g, provided by Beijing Weitong Lihua, with animal certificate number (SCXK (Beijing) 2020-0006).

[0103] 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. During this period, they were allowed to drink water and eat freely, and were given standard feed throughout the whole process.

[0104] Modeling, screening and grouping: Hepa1-6 cells in the logarithmic growth phase were collected and prepared into 7.5×10 7 The cells were inoculated in the right armpit of the mice, and 100 μL of cell suspension was injected subcutaneously into each mouse. After 11 days of inoculation, the mice without tumors were eliminated, and the mice with tumors of 100 mm 3 The mice were randomly divided into 6 groups, including the normal group (not inoculated with Hepa1-6 cells), the model group, the glycyrrhizic acid-copper group, the norcantharidin group, the ternary hydrogel group, and the ternary hydrogel+808nm laser irradiation group, with 5 mice in each group.

[0105] Drug administration: Each group of mice was given the drug once every 2 days, with the drug administration method being intratumoral injection (4 mg / mL in terms of norcantharidin), the dosage being 5 mg / kg, and a total of 6 administrations. The normal group was injected with an equal volume of saline, and the ternary hydrogel + 808nm laser irradiation group was irradiated with an 808nm laser at the administration site for 8 minutes after administration. The weight of the mice and the length and width of the tumor were recorded at the same time as the drug administration, and the tumor volume was calculated according to the formula:

[0106] Tumor volume = (length × width 2 ) / 2

[0107] See Table 2-3 for specific results.

[0108] Table 2: Changes in the body weight of mice after treatment with the ternary hydrogel prepared by the present invention over time (g)

[0109]

[0110] Table 3: Changes in tumor volume over time in mice after treatment with the ternary hydrogel prepared by the present invention (mm 3 )

[0111]

[0112]

[0113] The results showed that after the ternary hydrogel treatment, the weight of the mice was not significantly different from that of the normal group, indicating that the drug was safe. All drug groups were detected to have different degrees of tumor growth inhibition effects. Among them, the ternary hydrogel plus laser group had the best tumor growth inhibition effect, followed by the ternary hydrogel group, which was better than the control drug groups. The tumor photos of each group are shown in the figure below. Figure 5 shown.

[0114] Analysis of the anti-tumor efficacy mechanism of hydrogel

[0115] The anti-tumor mechanism of the ternary hydrogel prepared in Example 2 was evaluated as follows:

[0116] qPCR (real-time quantitative polymerase chain reaction) analysis: Take the tumor tissue of the treated mice and use it according to the instructions Total RNA was extracted from tumor tissues using the Reagent, and then reverse transcribed into cDNA using the first-strand reverse transcription kit. qPCR was performed using a fluorescent quantitative detection kit. Gene expression was normalized and calculated using the 2-ΔΔCт method.

[0117] See Table 4-5 for specific results.

[0118] Table 4: Gene expression levels in mouse tumor tissues after treatment with the ternary hydrogel prepared by the present invention

[0119]

[0120]

[0121] Table 5: Gene expression levels in mouse tumor tissues after treatment with the ternary hydrogel prepared in the present invention

[0122]

[0123] The results showed that compared with the model group, the expression of apoptosis and metastasis-related genes CD44, ICAM-1, and RAPGEF3 in the ternary hydrogel plus laser irradiation group was downregulated, and the expression of pro-inflammatory gene IL-1β was downregulated; the expression of anti-inflammatory genes TNFAIP8L2, IL-1RN and copper death-related genes DLD, DLAT, and PDHA1 were all upregulated. This shows that the ternary hydrogel can induce tumor cell apoptosis, copper death, and anti-inflammation, and synergistically regulate the tumor microenvironment to exert an anti-tumor effect.

[0124] IF (immunofluorescence) analysis: The tumor tissues of the glycyrrhizic acid-copper group, demethylcantharidin group, ternary hydrogel group, and ternary hydrogel + 808nm laser irradiation group were paraffin sectioned, dewaxed and hydrated, and then immersed in immune blocking solution, and the antigen was extracted in antigen buffer (pH6.0). According to the target protein, the primary and secondary antibodies were incubated, washed and colored, and then the nucleus was counterstained with DAPI. After dehydration and transparency with ethanol and xylene, the sections were sealed with sealing solution containing anti-fluorescence quencher. Observation under a fluorescence microscope showed that the loss of FDX1 and the aggregation of DLAT were observed in the ternary hydrogel plus laser irradiation group, which are typical characteristics of copper death; the expression of TNF-α, IL-1β and IL-6 pro-inflammatory factors in the ternary hydrogel plus laser irradiation group was significantly downregulated. In addition, the observation results of TUNEL staining of the above-mentioned tumor tissues showed that more cell apoptosis occurred in the ternary hydrogel plus laser irradiation group than in the control drug groups (glycyrrhizic acid-copper group, norcantharidin group, and ternary hydrogel group); the results of immunofluorescence analysis and TUNEL staining showed that the ternary hydrogel had good pro-apoptotic, copper-death and anti-inflammatory effects, which were consistent with the qPCR results.

[0125] Biosafety Verification of Hydrogels

[0126] The safety of the ternary hydrogel prepared in Example 2 was evaluated as follows:

[0127] Cytotoxicity experiment: After treating MDCK cells with drugs ranging from 3.13 to 25 μg / mL for 72 hours, add 20 μL of MTT mixed solution to the cells in the 96-well plate and continue incubation for 4 hours. Then add 150 μL of DMSO to dissolve, and measure its absorbance at 490 nm. The cell survival rate is calculated according to the following formula:

[0128] Cell survival rate (%) = (OD 给药组 -OD 空白组 ) / (OD 正常组 -OD 空白组 )×100%

[0129] The results showed that the inhibition rate of glycyrrhizic acid-copper and ternary hydrogel on MDCK cells was always less than 20%, with good biosafety. The inhibition rate of norcantharidin was 40% at a concentration of 25 μg / mL, indicating that the biosafety of the monomer can be improved after glycyrrhizic acid-copper and norcantharidin are co-assembled into a ternary carrier-free hydrogel. This is because the weak acidity of the tumor microenvironment, overexpression of hydrogen peroxide and hypoxia are more conducive to the biosafety of Cu 2+ Produce a Fenton-like effect; however, normal tissues and cells such as MDCK cells do not have these characteristics, therefore, the ternary hydrogel is safe for normal tissues and cells.

[0130] Hemolysis experiment: Fresh rat blood was used to determine the in vitro hemolysis effect of the sample. First, the red blood cells were collected by centrifugation at a speed of 3000rmp / min for 15 minutes, and the red blood cells were washed 3 times with normal saline. Then 3mL of the red blood cells obtained by centrifugation were mixed into 11mL of normal saline for storage and dispersion. The ternary 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, and incubated at 37°C for 4h. Then centrifuged at 3000rmp / min for 15min, and the absorbance value of the supernatant was measured at 570nm by the microplate reader. The positive control was deionized water and the negative control was normal saline. The hemolysis rate was calculated according to the following formula:

[0131] Hemolysis rate (%) = (A 给药组 -A PBS组 ) / (A 去离子水组 -A PBS组 )×100%

[0132] The results showed that the ternary hydrogel had no obvious hemolytic properties. Even at a concentration of up to 128 μg / mL, the hemolysis rate was still lower than the internationally recognized standard of 5%.

[0133] HE staining experiment: The ternary hydrogel drug was injected intratumorally into Hepa1-6 liver cancer-bearing mice. After 6 treatments, the mouse heart, liver, spleen, lung tissue, and kidney organs were dissected and their morphology was observed. They were fixed and preserved with formalin fixative and HE staining was performed.

[0134] The results are as follows Figure 6 As shown in the figure, compared with the normal group, no obvious pathological changes were observed in the heart, liver, spleen, lung, and kidney tissues of the ternary hydrogel and other drug-administered groups. Compared with the model group, the red and white pulp boundaries of the spleen tissue of the mice in the model group were blurred, the white pulp was dissociated, and the cells were arranged in a disordered manner, while the spleen tissue of the ternary hydrogel and other drug-administered groups was normal. Since the spleen is the largest peripheral immune organ, the changes in the model group suggest that the immune dysfunction caused by the tumor may be accompanied by an inflammatory response in the spleen, while the spleen tissue of the mice in the drug-administered group remained intact, indicating that the ternary hydrogel has an effective anti-tumor effect and can inhibit the splenomegaly caused by the tumor.

[0135] Mouse overall toxicity experiment: Hepa1-6 normal mice were subcutaneously injected with ternary hydrogel (4 mg / mL in terms of norcantharidin) at a dose of 5 mg / kg, and the normal group was injected with an equal volume of saline. The state of the animals was observed 24 hours and 48 hours after administration, blood was collected for routine blood tests and blood biochemical tests, and the liver and kidney of the mice were dissected for morphological observation and HE staining.

[0136] See Tables 6-9 for specific results.

[0137] Table 6: Routine blood test of the overall toxicity test of the ternary hydrogel prepared by the present invention in mice

[0138]

[0139]

[0140] Table 7: Routine blood test of the ternary hydrogel prepared by the present invention in mice for overall toxicity test

[0141]

[0142] Table 8: Routine blood test of the overall toxicity test of the ternary hydrogel prepared by the present invention in mice

[0143]

[0144] Table 9: Blood biochemistry of the ternary hydrogel prepared by the present invention in the overall toxicity experiment of mice

[0145]

[0146]

[0147] 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, and kidney function between the ternary hydrogel group and the normal group. Figure 7 As shown in A, the morphology and HE staining results of the kidney are as follows Figure 7 As shown in B, the morphology and HE staining of the liver and kidney in the ternary hydrogel group were consistent with those in the normal group, and no pathological damage was found in the liver and kidney, further demonstrating the safety of the ternary hydrogel.

[0148] The number of devices and processing scales described here are used to simplify the description of the present invention. Applications, modifications and variations of the present invention will be obvious to those skilled in the art.

[0149] 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 novel ternary carrier-free injectable hydrogel, characterized in that: From the molar ratio of 1-10: The glycyrrhizic acid, inorganic copper salt and norcantharidin are co-assembled in a ratio of 0.1-1:1-10, wherein the molar ratio of glycyrrhizic acid to copper ions does not exceed 1:0.

7.

2. The ternary hydrogel according to claim 1, characterized in that The inorganic copper salt is copper chloride or copper sulfate.

3. The ternary hydrogel according to claim 1, characterized in that No excipients need to be added, as the drug molecules can co-assemble themselves into a hydrogel.

4. The method for preparing the ternary hydrogel by self-assembly according to any one of claims 1 to 2, characterized in that: The method is: The steps include: (1) heating to dissolve glycyrrhizic acid in water; (2) heating to dissolve norcantharidin in water; (3) heating to dissolve the inorganic copper salt in water; (4) The glycyrrhizic acid, norcantharidin and copper ion aqueous solutions prepared in steps (1), (2) and (3) are mixed, with the concentration of glycyrrhizic acid being not less than 5 mmol / L, heated, and allowed to stand and cool to obtain a hydrogel.

5. The method for preparing the ternary hydrogel by self-assembly as claimed in claim 4, characterized in that: include: (1) heating to 60-100° C. to dissolve glycyrrhizic acid in water; (2) heating to 60-100° C. to dissolve norcantharidin in water; (3) heating to 25-100°C to dissolve the inorganic copper salt in water; (4) The glycyrrhizic acid, norcantharidin and copper ion aqueous solutions prepared in steps (1), (2) and (3) are mixed, with the concentration of glycyrrhizic acid being not less than 5 mmol / L, heated to 60-100° C., and allowed to stand and cool to obtain a hydrogel.

6. Use of the ternary hydrogel according to any one of claims 1 to 2 in the preparation of anti-tumor drugs.

7. The use according to claim 6, characterized in that The drug prepares the ternary hydrogel into an oral drug, a gastrointestinal release drug, a sustained-release drug, or an external or transdermal drug.

8. The use according to claim 7, characterized in that The medicine prepares the ternary hydrogel into pharmaceutically acceptable dosage forms, including oral gel, tablet, capsule, soft capsule, external gel, and injectable hydrogel.

9. Use of the ternary hydrogel according to any one of claims 1 to 2 in the preparation of medicines, characterized in that: The drug comprises a main drug and a drug delivery carrier, and the ternary hydrogel serves as a drug delivery carrier to carry and deliver the main drug.

10. The use according to claim 9, characterized in that The main drug is an anti-tumor drug.

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