Metformin injectable hydrogel as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicines, and particularly relates to metformin injectable hydrogel as well as a preparation method and application thereof. The preparation method of the metformin injectable hydrogel comprises the following steps: (1) dissolving metformin in an organic solvent to obtain a metformin solution; the preparation method comprises the following steps: adding glycyrrhetinic acid into a metformin solution, carrying out a stirring reaction under a heating condition, and after the reaction is finished, carrying out organic solvent removal and drying treatment on a reaction solution to obtain glycyrrhetinic acid-metformin salt; (2) dissolving the glycyrrhetinic acid-metformin salt in water, so as to obtain a glycyrrhetinic acid-metformin salt solution; and carrying out heat preservation treatment on the glycyrrhetinic acid-metformin salt solution at 50-70 DEG C, and cooling at room temperature after heat preservation treatment to obtain the metformin injectable hydrogel. The metformin injectable hydrogel prepared by the invention can effectively improve the bioavailability of metformin, improve the enrichment of drugs at tumor sites, and achieve the effect of effectively inhibiting melanoma.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a metformin injectable hydrogel, its preparation method, and its application. Background Technology

[0002] In recent years, the global incidence of melanoma has shown a significant upward trend, becoming one of the key factors in skin cancer deaths. According to data from the International Agency for Research on Cancer, there were approximately 325,000 new cases of melanoma globally in 2020, with about 57,000 deaths. By 2040, it is projected that the number of new melanoma cases worldwide will increase by more than 50% annually, making it a significant public health problem that seriously threatens the health of residents.

[0003] By designing drug delivery systems and regulating their structure and function, targeted, long-acting, and localized chemotherapy can be achieved against tumor tissues, thereby improving cancer treatment efficacy and reducing cancer recurrence rates. This has significant application value and clinical significance. At the 2024 American Society of Clinical Oncology Annual Meeting, a study on a novel fusion protein drug attracted considerable attention during the oral presentation. This drug, administered via intratumoral injection, opens up a new treatment pathway for melanoma patients. Injectable hydrogels, with their unique physical and biological properties, have shown great potential as drug carriers in cancer treatment. Intratumoral injection of hydrogels can deliver therapeutic agents locally, providing a more sustained release and higher dose of therapeutic drugs at the tumor site, reducing toxic side effects on normal tissues. Simultaneously, this method avoids the drawbacks of traditional intravenous administration, such as insufficient drug accumulation at the tumor site, rapid metabolism, the need for repeated administration, and systemic toxicity. By delivering therapeutic agents locally via hydrogels, the following can be achieved: 1) Controllable sustained release of drugs, increasing drug half-life and maximizing drug efficacy; 2) The drugs can be injected into almost any desired location, allowing them to accumulate at the lesion site and effectively reducing systemic toxicity; 3) The sustained-release effect of hydrogels increases the maximum safe dose per administration, reducing the number of administrations while ensuring safety.

[0004] However, traditional hydrogels have many limitations, such as low hydrophobic drug loading capacity, uneven drug distribution, and cytotoxicity induced by cross-linking agents. Driven by the dual goals of drug delivery and therapeutic enhancement, an increasing number of studies are utilizing bioactive materials to construct drug delivery systems. These systems can reduce metabolic toxicity. Therefore, there is an urgent need to develop novel anti-melanoma hydrogels with both high therapeutic efficacy and good safety. Summary of the Invention

[0005] In view of the problems and shortcomings of the existing technology, the purpose of this invention is to provide a metformin injectable hydrogel, its preparation method and application.

[0006] To achieve the objectives of this invention, the technical solution adopted is as follows: The first aspect of this invention provides a method for preparing a metformin injectable hydrogel, comprising the following steps: (1) Metformin was dissolved in an organic solvent to obtain a metformin solution; glycyrrhetinic acid was added to the metformin solution and the mixture was stirred under heating conditions. After the reaction was completed, the reaction solution was removed from the organic solvent and dried to obtain glycyrrhetinic acid-metformin salt. (2) Dissolve the glycyrrhetinic acid-metformin salt in water to obtain a glycyrrhetinic acid-metformin salt solution; keep the glycyrrhetinic acid-metformin salt solution at 50-70℃, and then cool it to room temperature to obtain metformin injectable hydrogel.

[0007] According to the above preparation method, preferably, in step (1), the molar ratio of metformin to glycyrrhetinic acid is 1:(0.5-4); more preferably, the molar ratio of metformin to glycyrrhetinic acid is 1:1.

[0008] According to the above preparation method, preferably, in step (2), the concentration of the glycyrrhetinic acid-metformin salt solution is ≥3% (w / v). When the concentration of the glycyrrhetinic acid-metformin salt solution is less than 3%, a hydrogel cannot be formed; when the concentration is greater than 3%, the gel state can be maintained. More preferably, the concentration of the glycyrrhetinic acid-metformin salt solution is 3% to 10% (w / v). Further preferably, the concentration of the glycyrrhetinic acid-metformin salt solution is 3% to 9% (w / v). Most preferably, the concentration of the glycyrrhetinic acid-metformin salt solution is 3% (w / v).

[0009] According to the above preparation method, preferably, in step (2), the heat preservation treatment time is 5 to 10 minutes.

[0010] According to the above preparation method, preferably, in step (1), the heating temperature is 40℃~70℃ and the stirring reaction time is 12-24h; more preferably, the heating temperature is 50℃ and the stirring reaction time is 24h.

[0011] According to the above preparation method, preferably, in step (1), the preparation method of metformin is as follows: dissolve metformin hydrochloride in an organic solvent to obtain a metformin hydrochloride solution; add potassium hydroxide to the metformin hydrochloride solution, stir to react, filter after the reaction is completed, collect the filtrate, remove the organic solvent from the filtrate and dry it to obtain metformin.

[0012] According to the above preparation method, preferably, the molar ratio of metformin hydrochloride to potassium hydroxide is 1:(0.5-4); more preferably, the molar ratio of metformin hydrochloride to potassium hydroxide is 1:1.

[0013] According to the above preparation method, preferably, the organic solvent is at least one selected from anhydrous methanol, anhydrous ethanol, and dimethylformamide. More preferably, the organic solvent is anhydrous methanol.

[0014] According to the above preparation method, preferably, in step (1), the specific operation of removing organic solvent is to perform rotary evaporation treatment on the reaction solution at 40℃-70℃.

[0015] A second aspect of the present invention provides a metformin injectable hydrogel prepared using the preparation method described in the first aspect above.

[0016] The third aspect of this invention provides the use of the metformin injectable hydrogel described in the second aspect above in the preparation of anti-melanoma drugs.

[0017] A fourth aspect of the present invention provides an anti-melanoma drug comprising the metformin injectable hydrogel described in the second aspect above and pharmaceutically acceptable excipients.

[0018] The reaction principle for preparing metformin injectable hydrogel of the present invention is as follows: during heating and stirring, glycyrrhetinic acid and metformin undergo a neutralization reaction to generate glycyrrhetinic acid-metformin salt. Under the temperature drive of 50-70℃, glycyrrhetinic acid-metformin salt self-assembles in water through non-covalent van der Waals forces, electrostatic interactions and hydrogen bonds to form a three-dimensional network structure hydrogel.

[0019] Compared with the prior art, the technical effects achieved by the present invention are as follows: (1) In this invention, glycyrrhetinic acid and metformin are reacted under heating conditions to generate glycyrrhetinic acid-metformin salt. The aqueous solution of glycyrrhetinic acid-metformin salt is kept at 50-70°C. During the heat preservation process, glycyrrhetinic acid-metformin salt and water self-assemble through non-covalent van der Waals forces, electrostatic interactions and hydrogen bonds to form a three-dimensional network structure hydrogel. This hydrogel can effectively improve the bioavailability of metformin. Compared with metformin alone, the anti-tumor efficacy is increased by 189 times. In addition, the hydrogel can effectively improve the accumulation of drugs at the tumor site. The retention effect after 7 days can reach 75.74%, realizing the effect of single administration and effective inhibition of tumors. Moreover, the metformin injectable hydrogel prepared by this invention has high biosafety and has very important clinical significance.

[0020] (2) The metformin injectable hydrogel prepared by the present invention has shear thinning and self-repairing functions, and can be directly injected for drug administration without additional carrier. Moreover, the hydrogel also has long-term release capability, which can slowly release drug molecules and avoid the rapid elimination of drugs. Attached Figure Description

[0021] Figure 1 The 1H NMR spectrum of metformin-glycyrrhetinic acid obtained in Example 1; Figure 2 The Fourier transform infrared spectrum of metformin-glycyrrhetinic acid obtained in Example 1; Figure 3 The UV-Vis spectrophotometer of metformin-glycyrrhetinic acid obtained in Example 1 is shown below. Figure 4 The X-ray diffraction spectrum of metformin-glycyrrhetinic acid obtained in Example 1 is shown below. Figure 5 Here is a SEM image of the hydrogel obtained in Example 2; Figure 6 This diagram illustrates the formation of hydrogels from metformin-glycyrrhetinic acid solutions of different concentrations under heating conditions. A shows photographs of the gelation effect observed after heat treatment and static cooling of glycyrrhetinic acid-metformin salt solutions of different concentrations; B is a schematic diagram of the gelation process of glycyrrhetinic acid-metformin salt. Figure 7 The mechanical properties of the metformin injectable hydrogel prepared in Example 2 are shown in Figure A. A graph shows the variation of storage modulus (G') / loss modulus (G') of the hydrogel with angular frequency at 1 rad / s. B graph shows the variation of storage modulus (G') / loss modulus (G') of the hydrogel with stress at 1% stress. C graph shows the variation of storage modulus (G') / loss modulus (G') of the hydrogel under different stresses. D graph shows the relationship between the shear rate and viscosity of the hydrogel. Figure 8 The effects of glycyrrhetinic acid, metformin, a physical mixture of glycyrrhetinic acid and metformin, and metformin-glycyrrhetinic acid on the growth inhibition of B16-F10 cells were shown. Among them, A represents the 48-h cytotoxicity of Met on B16-F10 cells, and B represents the 48-h cytotoxicity of Met, GA, and Met-GA on B16-F10 cells. Figure 9This is a preliminary efficacy evaluation of the metformin injectable hydrogel prepared in Example 2 on a mouse model of B16-F10 subcutaneous xenograft tumors. In the figures, A is a schematic diagram of mouse treatment; B shows the statistical results of tumor size recording after tumor removal from euthanized mice; C shows the statistical results of tumor volume changes; D shows the statistical results of tumor inhibition rate; E shows the statistical results of mouse weight changes; F shows the statistical results of the weight of major organs in mice; G1 represents the Ctrl group, G2 represents the Met group, G3 represents the GA group, and G4 represents the Met-GA-H group. Figure 10 The results of the biosafety evaluation of the metformin injectable hydrogel prepared in Example 2 after treatment of a mouse model of B16-F10 subcutaneous xenograft tumors are shown. Among them, A is HE section of important organs; BE is the detection results of biochemical markers ALT, AST, UREA and CREA; G1 represents the Control group and G4 represents the Met-GA-H group. Figure 11 The results show the retention of the metformin injectable hydrogel prepared in Example 2 in a B16-F10 subcutaneous xenograft mouse model; where A is a retention effect diagram of the hydrogel; and B is a quantitative diagram of fluorescence detection. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0023] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. Example 1: A method for preparing glycyrrhetinic acid-metformin salt, the specific steps of which are as follows:

[0024] (1) Add metformin hydrochloride and 20 mL of anhydrous methanol to a 50 mL pear-shaped flask, stir and dissolve to obtain a metformin hydrochloride solution; add potassium hydroxide to the metformin hydrochloride solution, the molar ratio of metformin hydrochloride to potassium hydroxide is 1:1, stir and react, filter after the reaction is complete, and collect the filtrate; evaporate the filtrate at 50 °C by rotary evaporation to remove the anhydrous methanol in the filtrate, and then dry it under vacuum at room temperature for 24-72 hours to obtain metformin.

[0025] (2) Weigh 0.5 g of the obtained metformin and dissolve it in 10 mL of anhydrous methanol to obtain a metformin solution; add glycyrrhetinic acid to the metformin solution and stir magnetically at 50 °C for 24 h. After the reaction is completed, the reaction solution is first evaporated at 50 °C to remove the anhydrous methanol in the reaction solution, and then vacuum dried at room temperature for 24-72 h to obtain glycyrrhetinic acid-metformin salt; wherein the molar ratio of metformin to glycyrrhetinic acid is 1:1. Example 2: A method for preparing a metformin injectable hydrogel, the specific steps of which are as follows:

[0026] S1: Glycyrrhetinic acid-metformin salt is dissolved in water to prepare a 3% (w / v) glycyrrhetinic acid-metformin salt solution, wherein the glycyrrhetinic acid-metformin salt is the glycyrrhetinic acid-metformin salt prepared in Example 1; S2: The glycyrrhetinic acid-metformin salt solution is kept in a water bath at 50°C for 5 min, and then allowed to stand and cool at room temperature for 30 min to obtain metformin injectable hydrogel. Example 3: The content of Example 3 is basically the same as that of Example 2, except that in step S1, the concentration of the glycyrrhetinic acid-metformin salt solution is 5% (w / v). Example 4: The content of Example 4 is basically the same as that of Example 2, except that in step S1, the concentration of the glycyrrhetinic acid-metformin salt solution is 7% (w / v). Example 5: The content of Example 5 is basically the same as that of Example 2, except that in step S1, the concentration of the glycyrrhetinic acid-metformin salt solution is 9% (w / v). Example 6: The content of Example 6 is basically the same as that of Example 1, except that in step (1), the molar ratio of metformin hydrochloride to potassium hydroxide is 1:0.5, and in step (2), the stirring reaction temperature is 40℃. Example 7: The content of Example 7 is basically the same as that of Example 1, except that: in step (1), the molar ratio of metformin hydrochloride to potassium hydroxide is 1:2; in step (2), the molar ratio of metformin to glycyrrhetinic acid is 1:0.5; in step (2), the stirring reaction temperature is 70℃ and the stirring reaction time is 12h. Example 8: The content of Example 8 is basically the same as that of Example 1, except that in step (1), the molar ratio of metformin hydrochloride to potassium hydroxide is 1:3; in step (2), the molar ratio of metformin to glycyrrhetinic acid is 1:2. Example 9: The content of Example 9 is basically the same as that of Example 1, except that in step (1), the molar ratio of metformin hydrochloride to potassium hydroxide is 1:4.

[0027] Example 10: The content of Example 10 is basically the same as that of Example 1, except that in step (2), the molar ratio of metformin to glycyrrhetinic acid is 1:0.5.

[0028] Example 11: The content of Example 11 is basically the same as that of Example 1, except that in step (2), the molar ratio of metformin to glycyrrhetinic acid is 1:2.

[0029] Example 12: The content of Example 12 is basically the same as that of Example 1, except that in step (2), the molar ratio of metformin to glycyrrhetinic acid is 1:4.

[0030] Example 13: The content of Example 13 is basically the same as that of Example 2, except that: in step S1, the glycyrrhetinic acid-metformin salt is the glycyrrhetinic acid-metformin salt prepared in Example 6, and in step S2, the water bath heat treatment temperature is 50°C.

[0031] Example 14: The content of Example 14 is basically the same as that of Example 2, except that: in step S1, the glycyrrhetinic acid-metformin salt is the glycyrrhetinic acid-metformin salt prepared in Example 7, and in step S2, the water bath heat treatment temperature is 70°C.

[0032] Example 15: The content of Example 15 is basically the same as that of Example 2, except that in step S1, the glycyrrhetinic acid-metformin salt is the glycyrrhetinic acid-metformin salt prepared in Example 8.

[0033] Example 16: The content of Example 16 is basically the same as that of Example 2, except that in step S1, the glycyrrhetinic acid-metformin salt is the glycyrrhetinic acid-metformin salt prepared in Example 9.

[0034] Example 17: The content of Example 17 is basically the same as that of Example 2, except that in step S1, the glycyrrhetinic acid-metformin salt is the glycyrrhetinic acid-metformin salt prepared in Example 10.

[0035] Example 18: The content of Example 18 is basically the same as that of Example 2, except that in step S1, the glycyrrhetinic acid-metformin salt is the glycyrrhetinic acid-metformin salt prepared in Example 11.

[0036] Example 19: The content of Example 19 is basically the same as that of Example 2, except that in step S1, the glycyrrhetinic acid-metformin salt is the glycyrrhetinic acid-metformin salt prepared in Example 12.

[0037] (a) Characterization of glycyrrhetinic acid-metformin salt (Met-GA): 1. Nuclear magnetic resonance (NMR) detection The glycyrrhetinic acid-metformin salt (denoted as Met-GA) prepared in Example 1 was subjected to NMR analysis, and its 1H NMR spectrum is shown below. Figure 1 As shown.

[0038] Depend on Figure 1 It can be seen that in Met-GA, the peak of the 30th carboxyl hydrogen proton on GA at around δ 12.2 ppm disappears, and the chemical shift of the 32nd methyl group on Met moves from δ 2.8 ppm to a lower field at δ 3.0 ppm, which preliminarily proves that the synthesis of Met-GA was successful.

[0039] 2. Infrared spectroscopy detection Take 3 mg of Met-GA prepared in Example 1, mix it evenly with KBr in a mortar, compress it into a tablet, and perform infrared spectroscopy testing. The infrared spectrum is as follows: Figure 2 As shown.

[0040] Depend on Figure 2 It can be seen that, compared with glycyrrhetinic acid (GA), Met-GA has a lower concentration at 1710 cm⁻¹. -1 The characteristic peak of -COOH disappears, and is retained at 1650 cm⁻¹. -1 The -C=O- peak is at 1557 cm⁻¹. -1 A new carboxylate (-COO) appears at the site. - Characteristic absorption peak. Additionally, in metformin (Met), 3387 cm⁻¹ is observed. -1 The asymmetric stretching vibration peak of -NH2 is redshifted to 3342 cm⁻¹ in Met-GA. -1 And Met is at 3170 cm -1 The absorption peak of the -NH stretching vibration is blue-shifted to 3207 cm⁻¹ in Met-GA. -1 .

[0041] 3. Ultraviolet-Vis Spectroscopic Detection 1 mg of Met-GA prepared in Example 1 was dissolved in methanol solution and placed in a quartz dish. The peaks were observed in the visible spectrum at 200-350 nm. The results are as follows: Figure 3 As shown.

[0042] Depend on Figure 3 It can be seen that Met-GA exhibits the characteristic absorption peak of GA at 259 nm, while the characteristic absorption peak of Met extends from λ. max =233 nm redshifted to λ max =235 nm, which may be due to the interaction between the amino group in Met and the carboxyl group in GA, such as the formation of hydrogen bonds, which changes the polarity and electron cloud distribution of the molecule, thus leading to a red shift of the absorption peak.

[0043] 4. X-ray diffraction energy dispersive spectroscopy detection Take 30 mg of the glycyrrhetinic acid-metformin salt powder prepared in Example 1 and spread it evenly in the mold, with the sample powder slightly higher than the surface of the sample holder. Gently press a glass slide on the sample surface to remove excess powder sample, ensuring its surface is flush with the mold plane. Place the sample holder in the sample slot and turn on the X-ray machine, scanning within the range of 10-70° at 2θ. The X-ray diffraction energy spectrum is shown below. Figure 4 As shown.

[0044] Depend on Figure 4 It can be seen that the monomer components of GA and Met have obvious crystal peak structures. In contrast, in Met-GA, the characteristic crystal peaks of the monomers disappear, and an amorphous envelope appears at 15.16°. This further indicates that GA and Met form a new two-dimensional structure after being combined by weak bonds. The two-dimensional structure destroys the regularity of the monomer components, further proving the successful preparation of Met-GA.

[0045] (II) Test and characterization of metformin injectable hydrogel: 1. Microstructure testing of metformin injectable hydrogel: 100 μL of the metformin injectable hydrogel prepared in Example 2 was placed on a clean glass slide, freeze-dried for 24 h, and then subjected to SEM testing. Gold sputtering was performed before the testing. The SEM results are as follows: Figure 5 As shown.

[0046] Depend on Figure 5 It can be seen that the microstructure of the metformin injectable hydrogel prepared by the present invention exhibits a three-dimensional network structure.

[0047] 2. Investigating the effect of glycyrrhetinic acid-metformin salt solution concentration on the preparation of metformin injectable hydrogels: The glycyrrhetinic acid-metformin salt prepared in Example 1 was dissolved in water to prepare a series of glycyrrhetinic acid-metformin salt solutions with different mass concentrations (1%, 3%, 5%, 7%, and 9%, respectively). The glycyrrhetinic acid-metformin salt solutions with concentrations of 1%, 3%, 5%, 7%, and 9% were incubated in a water bath at 50°C for 5 min, and then allowed to cool at room temperature for 30 min. After cooling at room temperature, gel formation was observed using an inverted method. The results are as follows... Figure 6 As shown.

[0048] Depend on Figure 6As shown in A, a 1% glycyrrhetinic acid-metformin salt solution cannot form a hydrogel after heat preservation and cooling treatment, while glycyrrhetinic acid-metformin salt solutions with a concentration of 3% or higher can form hydrogels. Furthermore, beyond 3%, the transparency of the hydrogel decreases with increasing glycyrrhetinic acid-metformin salt solution concentration. Therefore, a 3% mass concentration of glycyrrhetinic acid-metformin salt solution is the optimal mass concentration for preparing hydrogels. Figure 6 As shown in B, even with a mass concentration of 3%, the glycyrrhetinic acid-metformin salt solution still failed to gel without heat treatment, demonstrating that the provision of external heat is an important driving force for promoting the self-assembly of hydrogels.

[0049] 3. Rheological property testing The storage modulus (G') and loss modulus (G'') of metformin injectable hydrogel were measured using a rheometer to reflect the state of the product. When G' > G'', the product was in a gel state, and when G' < G'', the product was in a solution state.

[0050] (1) First, the metformin injectable hydrogel prepared in Example 2 was tested at an angular frequency of 1 rad / s, with the stress corresponding to G' and G'' from 0% to 100%. The test results are as follows: Figure 7 As shown in Figure A.

[0051] Depend on Figure 7 As can be seen from A, at an angular frequency of 1 rad / s, when the stress is between 0% and 22.67%, G' is greater than G'', indicating that the gel is in a gel state within this stress range; when the stress exceeds 22.67%, G'' is greater than G', indicating that the gel is in a sol state.

[0052] (2) The metformin injectable hydrogel prepared in Example 2 was tested for angular frequencies ranging from 1 rad / s to 100 rad / s at a stress of 1%. The test results are as follows: Figure 7 As shown in B.

[0053] Depend on Figure 7 As shown in B, when the angular frequency ranges from 1 rad / s to 100 rad / s, G' is greater than G'', indicating that under low stress, the metformin injectable hydrogel remains in a gel state and has good mechanical properties.

[0054] (3) The metformin injectable hydrogel prepared in Example 2 was placed on a rheometer. The experiment was divided into three stages: the first stage was under low stress, with the stress set at 1% and the time held for 100 s; the second stage was under high stress, with the stress set at 50% and the time held for 100 s; the third stage was from high stress back to low stress, with the stress set at 1% and the time held for 100 s. The changes of G' and G'' in each stage were observed. The results are as follows: Figure 7 As shown in C.

[0055] Depend on Figure 7 As shown in C, in the first stage, under low stress of 1%, G' is greater than G'', indicating that the product is in a gel state; in the second stage, when the stress increases to 50%, G' is less than G'', indicating that the product is in a sol state; in the third stage, when returning from high stress to low stress, G' is greater than G'', indicating that when the stress decreases, the product changes from a sol state back to a gel state. The above experimental results demonstrate that the metformin injectable hydrogel prepared in this invention has good self-healing ability.

[0056] (4) The viscosity of the metformin injectable hydrogel prepared in Example 2 was tested as a function of shear rate using a rheometer. The test results are as follows: Figure 7 As shown in D. Figure 7 As shown in D, the viscosity of metformin injectable hydrogel decreases sharply with increasing shear rate, proving that it has good shear-thinning properties.

[0057] (III) Performance testing of glycyrrhetinic acid-metformin salt and metformin injectable hydrogel: 1. Test on the inhibitory effect of glycyrrhetinic acid-metformin salt on the proliferation of B16-F10 cells: The inhibitory effect of glycyrrhetinic acid-metformin salt prepared in Example 1 on the proliferation of B16-F10 cells was detected by the CCK-8 assay.

[0058] The specific experimental method was as follows: Healthy B16-F10 and HaCaT cells in logarithmic growth phase were seeded in 96-well plates at densities of 5000 cells / well and 10000 cells / well per 100 μL, respectively. The plates were incubated for 24 hours. After cell attachment, the culture medium was discarded using a pipette. GA, Met, GA+Met (a simple mixture of GA and Met), and Met-GA prepared in Example 1 were diluted with complete culture medium to concentrations of 10, 20, 30, 40, 50, and 100 μM, respectively, and added to the wells of the 96-well plates. The negative control group was prepared with 100 μL of complete culture medium. After 48 hours of incubation, 10 μL of Cell Counting Kit-8 (CCK-8) solution was added to each well under dark conditions, and the plates were incubated for another hour. Subsequently, the absorbance (Optical Density, OD) of each well was measured using a multi-mode microplate reader. Based on the obtained data, the cell proliferation inhibition rate and the half-maximal inhibitory concentration (IC50) of the drug were calculated. 50 This experiment was repeated three times to ensure the reliability of the results.

[0059] The formula for calculating the cell proliferation inhibition rate is: Cell proliferation inhibition rate (%) = (mean OD value of negative control group - mean OD value of treatment group) / mean OD value of negative control group × 100%.

[0060] The experimental results are as follows Figure 8 As shown. By Figure 8 It can be seen that the Met-GA IC 50 These values ​​were 188.92, 1.25, and 2.10 times higher than those of Met, GA, and GA+Met, respectively, indicating that glycyrrhetinic acid-metformin salt can enhance the tumor-inhibiting effect of Met.

[0061] 2. Validation of the anti-melanoma efficacy of metformin injectable hydrogel. Taking the metformin injectable hydrogel prepared in Example 2 as an example, the anti-melanoma effect of the metformin injectable hydrogel was tested. The specific experimental method is as follows: (1) Experimental animals: Twenty female C57BL / 6 mice, 5-7 weeks old and weighing 16-18g, were purchased from the Experimental Animal Center of Guangxi Medical University, Animal Production License No. SYXK(Gui)2020-0004. The mice were housed in the SPF-grade mouse housing of the Experimental Animal Center of Guangxi Medical University. The experiments were conducted after the mice were acclimatized for one week.

[0062] (2) Experimental method: Construction of the B16-F10 tumor-bearing mouse model: Three days before modeling, the right lower axilla of the mice was shaved and iodine was applied to prevent infection. B16-F10 cells in the logarithmic growth phase were collected, washed with D-PBS, centrifuged at 1000 rpm for 3 min, and the supernatant was discarded. The cells were resuspended in pre-cooled D-PBS and stored in an ice box to maintain a low metabolic state. Before inoculation, the cell suspension was thoroughly dispersed using a 1000 μL pipette, and 100 μL was drawn up using a 1 mL sterile insulin syringe. The mouse was held with its left hand to expose the skin of its right lower axilla. The needle was inserted parallel to the mouse's body with the oblique cut side facing upwards. After injecting the cells, a noticeable bulge was observed. The needle was then slowly rotated out to complete the injection.

[0063] When the tumor volume in the B16-F10 tumor-bearing mouse model reaches 50-100 mm 3 Mice were randomly divided into four groups: Control group, Met group, GA group, and Met-GA-H group, with five mice in each group. Each group was treated with a different drug regimen (e.g., Figure 9 (As shown in Figure A). The dosing regimens for the Control group, Met group, GA group, and Met-GA-H group are as follows: Control group: 0.9% saline was injected into the tumor in situ, 0.1 mL / animal, once. Met group: The equivalent dose of Met was injected into the tumor in situ, with an injection dose of 0.1 mL / animal, for a total of one injection; GA group: The equivalent dose of GA was injected into the tumor in situ, with an injection dose of 0.1 mL / animal, for a total of one injection; Met-GA-H group: Metformin injectable hydrogel (denoted as Met-GA-H) prepared in Example 2 was injected in situ into the tumor, with an injection dose of 0.1 mL / animal, for a total of one injection.

[0064] Each group of B16-F10 tumor-bearing mice was weighed daily, and the maximum diameter (length) and minimum diameter (width) of the transplanted tumor were measured. The tumor volume was calculated using the formula: tumor volume = 0.5 × length × width 2.

[0065] Fifteen days after drug administration, mice in each group were sacrificed. Tumors were dissected, photographed, and their size, volume, and weight were recorded. Changes in tumor volume and weight were statistically analyzed, and the tumor inhibition rate was calculated. Simultaneously, heart, liver, spleen, lung, and kidney tissues from mice in the Control and Met-GA-H groups were fixed in 4% paraformaldehyde and embedded in paraffin. The embedded tissues were then cut into 3-10 μm thick sections and fixed on glass slides. After dewaxing and rehydration, the sections were stained with hematoxylin and eosin sequentially. After staining, the sections were eluted using an ethanol gradient, cleared with xylene, and finally mounted with neutral resin. The mounted sections were observed and images were acquired under an upright fluorescence microscope. In addition, serum samples from mice in the Control and Met-GA-H groups were collected, and the levels of liver function indicators alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and kidney function indicators urea (UREA) and creatinine (CREA) in the mouse serum were detected using kits. All kits were purchased from Redu, including the ALT assay kit (Catalog No. S03030, Batch No. 20240430), the AST assay kit (Catalog No. S03040, Batch No. 20240428), the UREA assay kit (Catalog No. S03036, Batch No. 20240529), and the CREA assay kit (Catalog No. S03076, Batch No. 202400507).

[0066] (3) Experimental results: Among them, the tumor size photos of mice in each group are as follows: Figure 9 As shown in B, the statistical results of tumor volume changes are as follows: Figure 9 As shown in C. Figure 9 As shown in B and 9C, the tumor size and volume in the Met group, GA group, and Met-GA-H group were significantly smaller than those in the Control group. The tumor inhibition rate results for each group of mice are shown in 9D. Figure 9 As shown in Figure D, after treatment, compared with the Control group, Met, GA, and Met-GA-H all exhibited certain inhibitory effects on tumors, with tumor inhibition rates of 21.54%±7.67%, 30.46%±13.89%, and 67.70%±7.13%, respectively. These experimental results demonstrate that the Met-GA-H prepared in this invention possesses excellent anti-tumor effects.

[0067] The statistical results of changes in body weight and the weight of major organs in each group of mice are as follows: Figure 9 As shown in E and 9F. (By...) Figure 9 As shown in E and 9F, no abnormalities were found in the body weight and the weight of major organs of mice in all groups, indicating good biocompatibility.

[0068] HE staining results of heart, liver, spleen, lung, and kidney tissues from mice in the Control group and Met-GA-H group are as follows: Figure 10 As shown in Figure A. Figure 10 As shown in Figure A, the organ tissues of mice in both the Control group and the Met-GA-H group exhibited clear structures with almost no inflammatory cell infiltration, intact cell structures, and no obvious abnormalities. It can be preliminarily concluded that the gel has no systemic toxicity in vivo and has good biosafety.

[0069] The results of serum ALT, AST, UREA, and CREA levels in mice in the Control group and Met-GA-H group are as follows: Figure 10 As shown in B-10E. (By...) Figure 10 According to B-10E, there were no significant differences in liver function indicators (ALT, AST, UREA, CREA) between the Met-GA-H group and the Control group (P>0.05), demonstrating that the gel has good efficacy. 3. Study on the in vivo retention effect of metformin injectable hydrogel Taking the metformin injectable hydrogel prepared in Example 2 as an example, the in vivo retention effect was studied. The specific experimental method is as follows: The B16-F10 tumor-bearing mouse model was constructed according to the method described in step 2 above. The constructed B16-F10 tumor-bearing mouse model was divided into two groups: the AIE group and the AIE@Met-GA-H group. The tumor volume of the B16-F10 tumor-bearing mouse model was approximately 100 mm. 3At approximately 10:00 AM, B16-F10 tumor-bearing mouse models in the AIE group and AIE@Met-GA-H group were administered intratumorally with the drug, once per administration. The administration regimen for the AIE group was: intratumoral injection of 100 μL of AIE solution (AIE solution is prepared by dissolving AIE fluorescent drug in water (see reference: Adv. Funct. Mater. 2024, 34, 2312260.)); the administration regimen for the AIE@Met-GA-H group was: intratumoral injection of 100 μL of AIE@Met-GA-H (AIE@Met-GA-H was prepared by adding glycyrrhetinic acid-metformin salt prepared in Example 1 to water containing AIE to prepare a 3% (w / v) glycyrrhetinic acid-metformin salt solution, which was then incubated in a 50°C water bath for 5 minutes). min, then let stand at room temperature for 30 min to obtain metformin injectable hydrogel loaded with AIE, denoted as AIE@Met-GA-H).

[0070] The AIE group and the AIE@Met-GA-H group underwent in vivo scanning and photography using the AniView animal in vivo imaging system at 0 Day (imaging immediately after injection to observe fluorescence intensity), 0.5 Days, 1 Day, 2 Days, 3 Days, 4 Days, 5 Days, 6 Days, and 7 Days after drug administration to observe the in vivo retention effect of the gel. (Parameter settings: sample stage height 20 cm; exposure time 0.1 s; excitation wavelength 535 nm; emission wavelength 680 nm). Results are as follows: Figure 11 As shown.

[0071] As shown in Figure 11, even on day 7, compared with the AIE group, the retention effect of the AIE@Met-GA-H group was significantly greater (P<0.05), with a retention effect of 75.74% after 7 days. This demonstrates that Met-GA-H has a better intratumoral retention effect, which may be due to the in-situ formation of a drug reservoir by the gel, thus potentially enabling a single dose to effectively inhibit the tumor.

[0072] The above embodiments are specific implementations of the present invention, but the implementation of the present invention is not limited to the above embodiments. Any other combination, change, modification, substitution, or simplification that does not exceed the design concept of the present invention shall fall within the protection scope of the present invention.

Claims

1. A method for preparing a metformin injectable hydrogel, characterized in that, Includes the following steps: (1) Metformin was dissolved in an organic solvent to obtain a metformin solution; glycyrrhetinic acid was added to the metformin solution and the mixture was stirred under heating conditions. After the reaction was completed, the reaction solution was removed from the organic solvent and dried to obtain glycyrrhetinic acid-metformin salt. (2) Dissolve the glycyrrhetinic acid-metformin salt in water to obtain a glycyrrhetinic acid-metformin salt solution; keep the glycyrrhetinic acid-metformin salt solution at 50-70°C, and then cool it to room temperature to obtain metformin injectable hydrogel.

2. The preparation method according to claim 1, characterized in that, In step (1), the molar ratio of metformin to glycyrrhetinic acid is 1:(0.5-4).

3. The preparation method according to claim 1, characterized in that, In step (2), the concentration of the glycyrrhetinic acid-metformin salt solution is ≥3%.

4. The preparation method according to claim 1, characterized in that, In step (2), the heat preservation time is 5-10 min; in step (1), the heating temperature is 40℃~70℃, and the stirring reaction time is 12-24 h.

5. The preparation method according to claim 1, characterized in that, In step (1), the preparation method of metformin is as follows: metformin hydrochloride is dissolved in an organic solvent to obtain a metformin hydrochloride solution; potassium hydroxide is added to the metformin hydrochloride solution, the mixture is stirred and reacted, the mixture is filtered after the reaction is completed, the filtrate is collected, and the filtrate is treated to remove the organic solvent and dry to obtain metformin.

6. The preparation method according to claim 5, characterized in that, The molar ratio of metformin hydrochloride to potassium hydroxide is 1:(0.5-4).

7. The preparation method according to claims 1-6, characterized in that, The organic solvent is at least one of anhydrous methanol, anhydrous ethanol, and dimethylformamide.

8. A metformin injectable hydrogel prepared using any one of the preparation methods described in claims 1-7.

9. The use of the metformin injectable hydrogel according to claim 8 in the preparation of an anti-melanoma drug.

10. An anti-melanoma drug, characterized in that, The drug comprises the metformin injectable hydrogel of claim 8 and pharmaceutically acceptable excipients.

Images