Preparation of Copper Ion-Mediated Ternary Carrier-Free Injectable Hydrogels Conjugated from Glycyrrhizic Acid and Norcantharidin and Their Antitumor Application

Abstract

This invention discloses the preparation and application of a ternary carrier-free injectable hydrogel co-assembled from glycyrrhizic acid, norcantharidin, and copper ions. The hydrogel forms a gel without the addition of excipients and possesses excellent gel material properties such as injectability, thermosensitivity, and stability, making it an ideal anti-tumor drug for clinical application. This hydrogel induces tumor cell apoptosis, copper death, and anti-inflammation, synergistically regulating the tumor microenvironment; combined with laser irradiation to enhance the Fenton-like effect, it achieves chemokinetic therapy of tumors. In the Hepa1-6 hepatocellular carcinoma mouse model, this hydrogel exhibits excellent anti-tumor activity, with significantly better efficacy than individual drug groups. Furthermore, this hydrogel has good biocompatibility and is a novel anti-tumor hydrogel that is simple to prepare, green, and has potential for clinical translation.

Description

Technical Field

[0001] This invention relates to a method for preparing an injectable hydrogel of glycyrrhizic acid-copper-norcantharidin and its anti-tumor application. It can synergistically regulate the tumor microenvironment by promoting tumor cell apoptosis, copper death and anti-inflammation, thereby achieving a laser-assisted Fenton-like effect in anti-tumor treatment. The glycyrrhizic acid, copper and norcantharidin have co-assembly properties, which can be used to prepare carrier-free hydrogel drugs, belonging to the field of pharmaceutical technology. Background Technology

[0002] Cancer is a leading cause of death worldwide, with 19.3 million new cases and nearly 10 million deaths in 2020. Currently, the main clinical treatments for cancer are surgical resection, radiotherapy, and chemotherapy. While chemotherapy drugs can effectively kill tumor cells, their clinical application is limited by drawbacks such as drug resistance, poor targeting, and severe adverse reactions. Some emerging treatment strategies, such as CAR-T cell therapy, antibodies, and targeted drugs, have made significant progress; however, their complex preparation and high treatment costs hinder their widespread clinical application globally. In addition to the aforementioned challenges in cancer treatment, the tumor microenvironment plays a crucial role in the occurrence, invasion, metastasis, and recurrence of tumors, representing a major challenge in cancer treatment.

[0003] In recent years, an increasing number of studies have begun to develop treatment strategies targeting the characteristics of the tumor microenvironment, such as its weak acidity, hydrogen peroxide overexpression, hypoxia, and severe inflammatory response. Chemokinetic therapy mediated by metal ions with Fenton / Fenton-like effects, in conjunction with inflammation modulation, has become a promising new strategy for tumor treatment due to its high specificity, low side effects, and non-invasiveness. Many bionanomaterials and hydrogels for chemokinetic therapy have been developed. Although bionanomaterials have active or passive tumor-targeting effects when administered systemically, recent studies have shown that only a small fraction of nanoparticles (median 0.7%) reach tumor tissue after intravenous injection, potentially causing unnecessary toxicity to normal tissues and organs [S. Wilhelm, AJ Tavares, Q. Dai, et al., Analysis of nanoparticle delivery to tumors, Nature Reviews Materials 1 (2016) 16014]. https: / / doi.org / 10.1038 / natrevmats.2016.14Meanwhile, currently developed hydrogels are mostly used as carriers for various drugs, requiring the participation of polymeric materials such as hyaluronic acid, alginate, and chitosan. However, in the tumor microenvironment, the inflammatory modulatory effects of these polymeric carriers are generally weaker than the anti-inflammatory capabilities of active molecules such as glycyrrhizic acid. Furthermore, the involvement of carriers may lead to low drug loading, poor biodegradability and biocompatibility, and potential side effects, all of which are major obstacles to industrial production and clinical translation. These unavoidable obstacles necessitate the urgent need to develop synergistic anticancer strategies with different mechanisms of action to provide alternatives to chemokinetic therapy.

[0004] Copper ions exhibit a Fenton-like effect, reacting with overexpressed hydrogen peroxide in the tumor microenvironment to generate reactive oxygen species (ROS) and oxygen. They also react with glutathione to protect these ROS, thereby inducing apoptosis and combating hypoxia. Furthermore, the Fenton-like effect of copper ions is even more efficient under 808nm laser irradiation, making it suitable for chemokinetic therapy of tumors. Recent studies have shown that copper ions can also kill tumor cells via the copper death pathway. Compound cantharidin capsules, combined with licorice to achieve synergistic effects and reduce toxicity, are widely used clinically for the treatment of various cancers. Glycyrrhizic acid, the main active ingredient in licorice, has excellent anti-inflammatory properties. Norcantharidin, derived from the Chinese herb cantharidin, can induce tumor cell apoptosis and is an effective anticancer drug for treating liver cancer, gastric cancer, and other cancers. However, norcantharidin has poor water solubility, a short half-life in the blood, and low tumor targeting efficiency, severely hindering its clinical application.

[0005] To address the aforementioned challenges, this invention prepares a copper-mediated, carrier-free, injectable hydrogel of glycyrrhizic acid and norcantharidin, which synergistically regulates the tumor microenvironment through apoptosis, copper death, and anti-inflammation, achieving chemokinetic therapy for tumors. This hydrogel possesses excellent mechanical properties, acting as a drug reservoir that can continuously release drugs to the target site with minimal side effects on normal tissues and organs. Its advantages of local targeting and minimally invasive drug delivery make it highly promising for applications in the biomedical field, opening new avenues for cancer treatment. Although some researchers have discovered that glycyrrhizic acid exhibits a self-assembly effect, the success of this self-assembly is significantly influenced by the solvent, temperature, concentration, pH value, and the structure and properties of other components / impurities. Currently, there are no reports of glycyrrhizic acid being co-assembled with two or more components into a gel, nor are there any reports of using the resulting gel as a carrier-free antitumor drug. Therefore, this invention is highly innovative and can enrich the current research field. Summary of the Invention

[0006] Based on the above background, this invention addresses the various problems existing in the prior art by innovatively preparing a ternary carrier-free injectable hydrogel of glycyrrhizic acid-copper-norcantharidin. This hydrogel not only avoids many of the shortcomings of current applications of polymer materials, but also has good safety, 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 objectives of this invention is to provide an injectable hydrogel of glycyrrhizic acid-copper-norcantharidin.

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

[0009] The third objective of this invention is to provide excellent gel material properties for glycyrrhizic acid-copper-norcantharidin injectable hydrogels.

[0010] The fourth objective of this invention is to provide the application of glycyrrhizic acid-copper-norcantharidin injectable hydrogel in the field of antitumor therapy.

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

[0012] To achieve this objective, the present invention adopts the following technical solution:

[0013] 1. The preparation process of the ternary hydrogel was determined by exploring different inorganic copper salts, heating temperatures, and molar ratios of feed ingredients. This hydrogel was formed by the co-assembly of 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. Further, infrared spectroscopy and proton nuclear magnetic resonance spectroscopy were used to determine the structural information of the ternary hydrogel.

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

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

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

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

[0018] (3) Heating dissolves the inorganic copper salt in water;

[0019] (4) Mix the glycyrrhizic acid, norepinephrine, and copper ion aqueous solution prepared in steps (1), (2), and (3). The concentration of glycyrrhizic acid should not be less than 5 mmol / L. Heat and let stand to cool to obtain hydrogel.

[0020] Preferably, it includes:

[0021] (1) Heat to 60-100℃ to dissolve glycyrrhizic acid in water;

[0022] (2) Heat to 60-100℃ to dissolve norcantharidin in water;

[0023] (3) Heat to 25-100℃ to dissolve the inorganic copper salt in water;

[0024] (4) Mix the glycyrrhizic acid, norepinephrine, and copper ion aqueous solution prepared in steps (1), (2), and (3). The concentration of glycyrrhizic acid should not be less than 5 mmol / L. Heat to 60-100℃ and let stand to cool to obtain hydrogel.

[0025] 2. Rheological studies of ternary hydrogels were conducted, and the main research steps are as follows:

[0026] The ternary hydrogel was characterized by frequency scanning and amplitude scanning.

[0027] 3. This invention provides the application of the hydrogel in the preparation of antitumor drugs, and the main research steps are as follows:

[0028] (1) The survival rate of human liver cancer cells (HepG2) after 72 h of drug-treated culture was detected by the MTT method to evaluate its in vitro anti-tumor effect.

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

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

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

[0032] 4. Safety was evaluated through cell safety experiments, in vitro hemolysis experiments, and whole mouse toxicity experiments.

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

[0034] (1) The MTT assay was used to assess the cell viability of canine kidney cells (MDCK) after 72 h of drug culture and to evaluate the cytotoxicity of the drug.

[0035] (2) The ternary hydrogel was incubated with rat red blood cells, and the hemolysis rate of each drug group at 570 nm was measured by an enzyme-linked immunosorbent assay (ELISA) reader to evaluate the hemolytic activity of the drug.

[0036] (3) The toxicity of the drug to the organs was evaluated by HE staining of the organs of mice after treatment.

[0037] (4) The safety of the drug was evaluated by observing the animal condition, measuring blood routine and blood biochemical indicators, observing major organs, and performing HE staining after subcutaneous injection of 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 the co-assembly of glycyrrhizic acid, copper ions and norepinephrine, without carriers or other pharmaceutical excipients, without chemical modification, and without the use of 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 formed after assembly of the hydrogels were confirmed by infrared and nuclear magnetic resonance methods.

[0041] (3) The present invention evaluated the antitumor and anti-inflammatory activity of the ternary hydrogel using the Hepa1-6 tumor-bearing liver cancer mouse model. It was found that the hydrogel has the effects of inducing tumor cell apoptosis, copper death and anti-inflammation, and synergistically regulating the tumor microenvironment to achieve chemokinetic therapy of tumors. Moreover, the efficacy is significantly better than that of each control drug group.

[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, and play a dual role of both exerting pharmacological activity and carrying active ingredients.

[0043] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

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

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

[0046] Figure 3 Rheological properties characterization of the binary carrier-free hydrogel of the present invention.

[0047] Figure 4 Rheological properties characterization of the ternary carrier-free hydrogel of the present invention.

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

[0049] Figure 6 Images of organs and HE staining of Hepa1-6 hepatocellular carcinoma-bearing mice treated with the ternary carrier-free hydrogel of this invention 13 days later.

[0050] Figure 7 The images show liver and kidney tissues and HE staining results from the overall toxicity test of the ternary carrier-free hydrogel in mice according to the present invention. Detailed Implementation

[0051] The present invention will now be described in further detail with reference to the accompanying drawings, so that those skilled in the art can implement it based on the description.

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

[0053] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0054] A novel ternary carrier-free injectable hydrogel with antitumor activity is assembled from 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, the hydrogel is prepared from 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, 1:0.5:1, 1:0.7:1, or 1:0.5:2. Experiments have shown that stable hydrogel drugs can only be prepared within the above specific range; 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, flocculent precipitate will form, and a stable hydrogel drug system cannot be formed.

[0056] The inorganic copper salts are copper chloride and copper sulfate.

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

[0058] (1) Heat to 60-100℃ to dissolve glycyrrhizic acid in water;

[0059] (2) Heat to 60-100℃ to dissolve norcantharidin in water;

[0060] (3) Heat to 25-100℃ to dissolve the inorganic copper salt in water;

[0061] (4) Mix the glycyrrhizic acid, norepinephrine, and copper ion aqueous solution prepared in steps (1), (2), and (3). The concentration of glycyrrhizic acid should not be less than 5 mmol / L. Heat to 60-100℃ and let stand to cool to obtain hydrogel.

[0062] The application of the ternary hydrogel in the preparation of antitumor drugs. The drugs are prepared using the ternary hydrogel as the main ingredient in oral, gastrointestinal release, sustained-release, controlled-release, topical, or transdermal formulations. The drugs are also prepared using the ternary hydrogel as the main ingredient in pharmaceutically acceptable dosage forms, including oral gels, tablets, capsules, soft capsules, topical gels, and hydrogels for injection. This provides a dual function: both exerting pharmacological activity and carrying the active ingredient.

[0063] The ternary hydrogel is used in the preparation of drugs, wherein the drug comprises an active pharmaceutical ingredient (API) and a drug delivery carrier, and the ternary hydrogel serves as the drug delivery carrier, carrying and delivering the API. Preferably, the API is an antitumor drug. It serves a dual purpose: exerting synergistic pharmacological activity and carrying the active ingredient.

[0064] Example 1

[0065] Glycyrrhizic acid in molar ratios of 10:1 to 10:10 was dissolved separately in water with different metal ions (copper chloride, calcium chloride, magnesium chloride, ferrous chloride, ferric chloride, aluminum chloride, and zinc chloride), heated and mixed, and then allowed to stand and cool. Glycyrrhizic acid and copper formed a hydrogel in the range of 10:1 to 10:7, while other metal ions (calcium, magnesium, ferrous, iron, aluminum, and zinc) mostly formed flocculent precipitates with glycyrrhizic acid, with only a few ratios forming a gel. For example, glycyrrhizic acid-calcium formed a hydrogel in the range of 10:1 to 10:4, glycyrrhizic acid-magnesium in the range of 10:1 to 10:5, glycyrrhizic acid-ferrous in the range of 10:1 to 10:4, glycyrrhizic acid-iron in the range of 10:1 to 10:2, glycyrrhizic acid-aluminum in the range of 10:1 to 10:5, and glycyrrhizic acid-zinc in the range of 10:1 to 10:3. Furthermore, the various metal ions readily form gels with glycyrrhizic acid in small proportions, resulting in a clearer and more transparent color. As the concentration of metal ions increases, the hydrogel easily disintegrates, becoming darker and more turbid. Moreover, compared to other metal ions, the glycyrrhizic acid-copper binary hydrogel exhibits the best material properties. Therefore, whether different raw materials and proportions can form a hydrogel is unpredictable and involves chance. The technical solution of this invention requires creative effort and is therefore innovative.

[0066] Example 2

[0067] Ternary hydrogels were prepared using the following steps.

[0068] Weigh out glycyrrhizic acid (GA) and inorganic copper salt (Cu) in a molar ratio of 1:0.5:1. 2+ The following ingredients were dissolved separately in water: norcantharidin (NCTD), mixed, and heated to 60-100℃ to prepare a clear and transparent hydrogel (NCTD Gel). The macroscopic morphology is as follows: Figure 1 As shown.

[0069] Example 3

[0070] Weigh out glycyrrhizic acid, inorganic copper salt, and norcantharidin in a molar ratio of 1:0.2:1, dissolve them separately in water, mix them, and the concentration, calculated as glycyrrhizic acid, shall not be less than 5 mmol / L. Heat to 60-100℃ to prepare a clear and transparent hydrogel.

[0071] Example 4

[0072] Weigh out glycyrrhizic acid, inorganic copper salt, and norcantharidin in a molar ratio of 1:0.7:1, dissolve them separately in water, mix them, and the concentration, calculated as glycyrrhizic acid, shall not be less than 5 mmol / L. Heat to 60-100℃ to prepare a clear and transparent hydrogel.

[0073] Example 5

[0074] Weigh out glycyrrhizic acid, inorganic copper salt, and norcantharidin in a molar ratio of 1:0.5:2, dissolve them separately in water, mix them, and the concentration, calculated as glycyrrhizic acid, shall not be less than 5 mmol / L. Heat to 60-100℃ 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 to 1:1, dissolved in water, 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, 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 analog of norcantharidin) in a molar ratio of 1:0.5:1 were weighed, dissolved in water, heated and mixed, and allowed to stand and cool. It was found that a stable hydrogel system could not be formed, indicating that the chemical structure of norcantharidin plays a unique role in the formation of this ternary hydrogel. Although cantharidin is structurally similar to it, they cannot be substituted for each other. This is consistent with the current research status that the discovery of small molecule carrier-free hydrogels is somewhat accidental.

[0079] Structural characterization of hydrogels

[0080] (1) The structures of glycyrrhizic acid-copper binary hydrogel and glycyrrhizic acid-copper-norcantharidin ternary hydrogel prepared in Examples 1-2 were observed by scanning electron microscopy. Figure 2 A is an electron micrograph of the binary hydrogel, which appears as neatly arranged nanofibers. Figure 2 Image B is an electron micrograph of the ternary hydrogel, showing uniformly distributed, regularly shaped spherical nanoparticles. The results indicate that the addition of norcantharidin significantly altered the microstructure of both the binary and ternary hydrogels, proving that norcantharidin and glycyrrhizic acid-copper did indeed co-assemble to form a ternary hydrogel.

[0081] (2) Infrared analysis was performed on the hydrogel prepared in Example 2. The infrared analysis conditions were: scanning range of 4000 cm⁻¹. -1 -400cm -1 The resolution is 4cm. -1 The scanning speed was 7.5 kHz, and the infrared peak positions of the hydrogel were obtained from infrared analysis as shown below:

[0082] Infrared signatures of the hydrogel: 3202.74 (-OH stretching vibration), 1720.07 (-C=O stretching vibration), 1593.81, 1416.96 (-COO symmetric and asymmetric stretching vibrations), 1007.55 (-CO vibration peak). Upon addition of copper ions, the -OH group of glycyrrhizic acid blue-shifted to 3202.74 cm⁻¹. -1 Furthermore, the shape and strength of the -COO- group changed significantly, indicating that copper ions likely formed a coordination bond with the carboxyl group of glycyrrhizic acid; after the addition of norcantharidin, the -CO group changed from 1032.37 cm⁻¹ -1 Shift to a lower wavenumber of 1007.55 cm -1 Furthermore, the absorption peak of -C=O is significantly enhanced, indicating that norcantharidin may form hydrogen bonds with the hydroxyl groups on glycyrrhizic acid glucuronic acid.

[0083] (3) The hydrogel prepared in Example 2 was characterized by NMR, and the results are as follows:

[0084] NMR assignments of ternary hydrogels: 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 proton peak shape on glycyrrhizic acid glucuronic acid changed significantly, and the proton peak of the carboxyl group of norcantharidin disappeared, indicating that the carboxyl group of norcantharidin is bound to the hydroxyl group of glycyrrhizic acid glucuronic acid in the form of hydrogen bonds.

[0085] Rheological property characterization of hydrogels

[0086] The rheological tests of the binary and ternary hydrogels prepared in Examples 1-2 were performed as follows:

[0087] Binary and ternary hydrogels with a final concentration of 10 mmol / L were prepared according to Examples 1-2 and placed on a rheometer measuring plate with a gap of 5 mm and a temperature of 25℃ or 37℃. Frequency scanning: strain was constant at 0.1%, and the measured frequency range was 0.1 Hz-10 Hz. Amplitude scanning: the frequency was constant at 1 Hz, and the strain variation range was 0.01%-10%. Viscosity testing: strain was constant at 0.1%, and the measured ω range was 0.6-85 rad / s. Healing ability testing: the frequency was constant at 1 Hz, and low strain (0.1%) and high strain (300%) cycles were performed to obtain the storage modulus (G′) and loss modulus (G″) as a function of strain. The frequency scanning of binary hydrogels formed by glycyrrhizic acid with different metal ions (calcium, magnesium, ferrous, iron, aluminum, zinc) is as follows. Figure 3 As shown in A, the amplitude scan is as follows: Figure 3 As shown in B; the frequency scan of the ternary hydrogel formed by glycyrrhizic acid-copper-norcantharidin is as follows. Figure 4 As shown in A, the amplitude scan is as follows: Figure 4 As shown in B, the viscosity test is as follows: Figure 4 As shown in C, the healingability test is as follows: Figure 4 As shown in D.

[0088] Further rheological property analysis was performed on the prepared hydrogels. The solid-like properties of the G′-reactive gel and the liquid-like properties of the G″-reactive gel were analyzed. Figure 4As shown, the G′ of the ternary 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 internal elastic network, a characteristic common to all hydrogel materials. The oscillatory stress scanning process of the ternary hydrogel shows that G′ and G″ intersect when the oscillatory strain is sufficiently large, indicating that the ternary hydrogel has good shear thinning properties. Viscoelastic property tests of the ternary hydrogel show that it has a certain viscosity, and the viscosity decreases with increasing frequency, proving its injectability. The healability 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′ decreases rapidly, the ternary hydrogel structure is destroyed, and it transforms into a sol state. After several cycles, G′ can recover to the same height as in the first cycle, proving that the ternary hydrogel has rapid healing capabilities. Figure 3 Among the various metal ions (calcium, magnesium, ferrous, iron, aluminum, and zinc), the glycyrrhizic acid-copper binary hydrogel exhibits the highest G′ and G″ values, demonstrating superior mechanical strength. At the same concentration, the glycyrrhizic acid-copper-norcantharidin ternary hydrogel shows even higher G′ and G″ values ​​and better mechanical strength compared to the glycyrrhizic acid-copper binary hydrogel, indicating that the synergistic effect of the co-assembly of glycyrrhizic acid, copper ions, and norcantharidin can yield hydrogels with superior mechanical strength.

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

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

[0091] Cell seeding: Logarithmically growing human liver cancer cells (HepG2) were seeded into 96-well plates at a density of 3000 cells per well and cultured in a cell culture incubator for 24 hours.

[0092] Cell drug delivery: The cell groups were set up as blank cell group, blank group and drug delivery group. The blank cell group was only added with culture medium, the blank group was not added with any liquid, and the drug delivery group was added with drug-containing culture medium. The concentrations per well were 32, 16, 8, 4, 2 and 1 μg / mL respectively. The cells were then cultured in a cell culture incubator for 72 h.

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

[0094] Cell viability (%) = (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 this invention

[0097]

[0098]

[0099] The results show that the majority of the antibacterial activities of the ternary hydrogel are significantly enhanced compared with the monomer raw materials, and the activity is further enhanced after laser irradiation, possessing excellent in vitro anti-tumor activity, and having the value of in-depth research and further clinical development.

[0100] Verification of in vivo anti-tumor activity of the hydrogel

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

[0102] A Hepa1-6 tumor-bearing liver cancer mouse model was established by subcutaneous injection of Hepa1-6 cells, and the anti-tumor effect after intratumoral injection of the hydrogel drug was observed. The mouse strain used was SPF-grade female C57BL / 6J mice, with a body weight of 20±1 g, provided by Beijing Vital River Laboratory Animal Technology Co., Ltd., and the animal certificate number was (SCXK(Beijing)2020-0006).

[0103] Mouse adaptation feeding: The experimental animals were adaptively fed for 7 d in an environment with a temperature of (23±2)°C, a humidity of (60±5)%, and a 12-h day-night cycle. During this period, they had free access to water and food, and were fed with standard feed throughout the process.

[0104] Model establishment, screening and grouping: Hepa1-6 cells in the logarithmic growth phase were collected and prepared into a cell suspension of 7.5×10 7 cells / mL. They were inoculated under the right axilla of the mice, and 100 μL of the cell suspension was subcutaneously injected into each mouse. Eleven days after inoculation, the mice that did not form tumors were excluded, and the mice with a tumor volume reaching 100 mm 3 were randomly grouped. A total of 6 groups were divided, including a normal group (without inoculation of Hepa1-6 cells), a model group, a glycyrrhizic acid-copper group, a norcantharidin group, a ternary hydrogel group, and a ternary hydrogel + 808 nm laser irradiation group, with 5 mice in each group.

[0105] Administration: Mice in each group were administered the drug every two days via intratumoral injection (4 mg / mL of norcantharidin), at a dose of 5 mg / kg, for a total of 6 administrations. The normal control group received an equal volume of physiological saline, while the ternary hydrogel + 808nm laser irradiation group received 808nm laser irradiation at the administration site for 8 min after drug administration. Mouse weight and tumor length and width were recorded simultaneously with drug administration, and tumor volume was calculated using the formula:

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

[0107] The specific results are shown in Table 2-3.

[0108] Table 2: Changes in mouse body weight (g) over time after treatment with the ternary hydrogel prepared in this invention.

[0109]

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

[0111]

[0112]

[0113] The results showed that after treatment with the ternary hydrogel, the body weight of mice was not significantly different from that of the normal group, indicating good drug safety. All treatment groups showed varying degrees of tumor growth inhibition. The ternary hydrogel plus laser group showed the best tumor growth inhibition, followed by the ternary hydrogel group, both superior to the control groups. Tumor images of each group are shown below. Figure 5 As shown.

[0114] Analysis of the antitumor pharmacodynamic mechanism of hydrogels

[0115] The antitumor mechanism of the ternary hydrogel prepared in Example 2 was evaluated using the following methods:

[0116] qPCR (quantitative real-time polymerase chain reaction) analysis: Tumor tissue was collected from treated mice and analyzed according to the instructions. Reagent extracted total RNA from tumor tissue and then reverse transcribed it into cDNA using a first-strand reverse transcription kit. qPCR was performed using a quantitative real-time PCR kit. Gene expression was normalized and calculated using the 2-ΔΔCt method.

[0117] The specific results are shown in Table 4-5.

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

[0119]

[0120]

[0121] Table 5: Gene expression levels in mouse tumor tissues after treatment with the ternary hydrogel prepared in this 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 was downregulated in the ternary hydrogel plus laser irradiation group, as was the expression of the pro-inflammation-related gene IL-1β; while the expression of anti-inflammatory genes TNFAIP8L2 and IL-1RN and copper death-related genes DLD, DLAT, and PDHA1 were all upregulated. This indicates that the ternary hydrogel can exert its anti-tumor effect by synergistically regulating the tumor microenvironment through inducing tumor cell apoptosis, copper death, and anti-inflammation.

[0124] Immunofluorescence (IF) analysis: Tumor tissues from the glycyrrhizic acid-copper group, norcantharidin group, ternary hydrogel group, and ternary hydrogel + 808nm laser irradiation group were sectioned in paraffin. After dewaxing and hydration, the paraffin sections were immersed in immunoblocking solution, and antigens were extracted in antigen buffer (pH 6.0). Primary and secondary antibodies were incubated according to the target proteins, and after washing, staining was performed. The nuclei were counterstained with DAPI, and the sections were dehydrated and cleared with ethanol and xylene, respectively, before mounting with mounting solution containing an anti-fluorescence quencher. Fluorescence microscopy revealed the loss of FDX1 and the aggregation of DLAT in the ternary hydrogel + laser irradiation group, typical characteristics of copper death. The expression of pro-inflammatory factors TNF-α, IL-1β, and IL-6 was significantly downregulated in the ternary hydrogel + laser irradiation group. Furthermore, observations of tumor tissues in the above groups using TUNEL staining showed that the ternary hydrogel plus laser irradiation group had more apoptosis than the control drug groups (glycyrrhizic acid-copper group, norcantharidin group, and ternary hydrogel group); immunofluorescence analysis and TUNEL staining results indicated that the ternary hydrogel had good pro-apoptotic, copper death and anti-inflammatory effects, consistent with the qPCR results.

[0125] Biosafety verification of hydrogels

[0126] The safety of the ternary hydrogel prepared in Example 2 was evaluated using the following method:

[0127] Cytotoxicity assay: MDCK cells were treated with a drug concentration ranging from 3.13 to 25 μg / mL for 72 h. Then, 20 μL of MTT solution was added to each well of a 96-well plate, and the cells were incubated for another 4 h. Subsequently, 150 μL of DMSO was added to dissolve the cells, and the absorbance was measured at 490 nm. Cell viability was calculated using the following formula:

[0128] Cell viability (%) = (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 the ternary hydrogel on MDCK cells remained below 20%, indicating good biocompatibility. Meanwhile, the inhibition rate of norcantharidin at a concentration of 25 μg / mL was 40%, suggesting that the co-assembly of glycyrrhizic acid-copper and norcantharidin into a ternary carrier-free hydrogel can improve the biocompatibility of the monomers. This is because the weakly acidic environment of the tumor microenvironment, the overexpression of hydrogen peroxide, and the hypoxic environment are more conducive to the growth of Cu. 2+ It produces a Fenton-like effect; however, normal tissues and cells such as MDCK cells do not possess these characteristics, therefore, ternary hydrogels are safe for normal tissues and cells.

[0130] Hemolysis Assay: Fresh rat blood was used to determine the in vitro hemolysis effect of the samples. First, red blood cells were collected by centrifugation at 3000 rpm for 15 minutes, and washed three times with physiological saline. Then, 3 mL of the centrifuged red blood cells were mixed with 11 mL of physiological saline for storage and dispersion. The ternary hydrogel was diluted with physiological saline to the appropriate concentration. Then, 1 mL of the test solution was mixed with 100 μL of red blood cell stock solution to obtain a 4% red blood cell solution, and incubated at 37°C for 4 hours. Subsequently, the solution was centrifuged at 3000 rpm for 15 minutes, and the absorbance of the supernatant was measured at 570 nm using a microplate reader. Deionized water was used as the positive control, and physiological saline was used as the negative control. The hemolysis rate was calculated using 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, and even at a concentration as high as 128 μg / mL, the hemolysis rate was still lower than the internationally recognized standard of 5%.

[0133] HE staining experiment: Hepa1-6 hepatocellular carcinoma mice were injected intratumorally with a ternary hydrogel drug. After 6 treatments, the mice were dissected and their heart, liver, spleen, lung tissue and kidney organs were collected to observe their morphology. They were fixed and preserved with formalin fixative and then stained with HE.

[0134] The results are as follows Figure 6 As shown, compared with the normal group, no significant pathological changes were observed in the heart, liver, spleen, lung, and kidney tissues of the ternary hydrogel and other drug-treated groups. Compared with the model group, the spleen tissue of the model group mice showed blurred red and white pulp boundaries, white pulp dissociation, and disordered cell arrangement, while the spleen tissue of the ternary hydrogel and other drug-treated groups was normal. Since the spleen is the largest peripheral immune organ, the changes in the model group suggest tumor-induced immune dysfunction, possibly accompanied by an inflammatory response within the spleen. The spleen tissue of the drug-treated mice remained intact, indicating that the ternary hydrogel has an effective anti-tumor effect and can inhibit tumor-induced splenomegaly.

[0135] Overall toxicity test in mice: Hepa1-6 normal mice were subcutaneously injected with a ternary hydrogel (4 mg / mL based on norcantharidin) at a dose of 5 mg / kg, while the normal control group was injected with an equal volume of physiological saline. The animals were observed at 24 h and 48 h after administration, and blood was collected for complete blood count and blood biochemistry tests. The liver and kidneys of the mice were dissected for morphological observation and HE staining.

[0136] The specific results are shown in Table 6-9.

[0137] Table 6: Blood routine tests of the ternary hydrogel prepared in this invention in mice for overall toxicity.

[0138]

[0139]

[0140] Table 7: Blood routine tests of the ternary hydrogel prepared in this invention in mice for overall toxicity.

[0141]

[0142] Table 8: Blood routine tests of the ternary hydrogel prepared in this invention in mice for overall toxicity.

[0143]

[0144] Table 9: Blood biochemistry of the ternary hydrogel prepared in this invention in mouse whole toxicity test

[0145]

[0146]

[0147] Complete blood count and blood biochemistry results showed no significant differences between the ternary hydrogel group and the normal group in terms of red blood cells, white blood cells, platelets, liver function, and kidney function. Liver morphology and HE staining results are as follows: Figure 7 As shown in Figure A, the morphological and HE staining results of the kidney are as follows: Figure 7 As shown in Figure 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 observed in the liver and kidney, further demonstrating the safety of the ternary hydrogel.

[0148] The number of devices and processing scale described herein are for the purpose of simplifying the description of the invention. Applications, modifications, and variations of the invention will be readily apparent to those skilled in the art.

[0149] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A method for the self-assembly preparation of a ternary carrier-free injectable hydrogel, characterized in that, Ternary carrier-free injectable hydrogels are available in molar ratios of 1-10: A hydrogel is formed by the self-assembly of glycyrrhizic acid, inorganic copper salt, and norcantharidin in a ratio of 0.1-1:1-10. No excipients are required; the drug molecules themselves can self-assemble to form a hydrogel. The molar ratio of glycyrrhizic acid to copper ions does not exceed 1:0.

7. The self-assembly preparation method includes the following steps: Step 1: Heat to 60-100℃ to dissolve glycyrrhizic acid in water; Step 2: Heat to 60-100℃ to dissolve norcantharidin in water; Step 3: Heat to 25-100℃ to dissolve the inorganic copper salt in water; Step 4: Mix the glycyrrhizic acid, norcantharidin, and copper ion aqueous solution prepared in steps 1, 2, and 3. The concentration of glycyrrhizic acid should not be less than 5 mmol / L. Heat to 60-100℃ and let stand to cool to obtain hydrogel. The inorganic copper salts are copper chloride and copper sulfate.

2. The application of the ternary carrier-free injectable hydrogel obtained by the self-assembly preparation method of the ternary carrier-free injectable hydrogel as described in claim 1 in the preparation of anti-liver cancer drugs.

3. The application as described in claim 2, characterized in that... The ternary carrier-free injectable hydrogel is prepared into an oral or transdermal drug.

4. The application as described in claim 2, characterized in that... The ternary carrier-free injectable hydrogel is prepared into a sustained-release drug.

5. The application as described in claim 2, characterized in that... The ternary carrier-free injectable hydrogel is prepared into pharmaceutically acceptable dosage forms, including oral gels, tablets, capsules, topical gels, and injectable hydrogels.

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