Triptolide hydrogel, preparation method thereof and application of triptolide hydrogel in preparation of medicine for treating hepatocellular carcinoma

Abstract

The invention discloses triptolide hydrogel, a preparation method thereof and application of the triptolide hydrogel in preparation of a medicine for treating hepatocellular carcinoma, and belongs to the field of biological medicine. The stable injectable hydrogel drug is prepared by utilizing the self-assembly capability of glycyrrhizic acid saponins and loading triptolide, and while the anti-tumor effect of triptolide is played, the toxic and side effects are reduced by virtue of the targeting and anti-inflammatory effects of glycyrrhizic acid, and accurate treatment of liver cancer is realized.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a self-assembled hydrogel based on triptolide (TP) and glycyrrhizic acid (GA), its preparation method, and its application. Background Technology

[0002] Hepatocellular carcinoma (HCC) is the major histological subtype of liver cancer, accounting for 90% of primary liver cancers and is one of the six leading causes of cancer-related mortality worldwide. Starting in 2025, more than one million people will be affected by liver cancer each year. Although molecularly targeted drugs, such as sorafenib, and immune checkpoint inhibitors have made some progress in clinical application in recent years, most patients still face the challenges of high relapse rates, rapid development of drug resistance, and severe systemic toxicity after treatment.

[0003] Triptolide (TP) is an epoxy diterpenoid lactone compound extracted from Tripterygium wilfordii, possessing a variety of pharmacological activities including antirheumatic, antitumor, anti-inflammatory, and immunosuppressive effects. Its antitumor activity exhibits broad-spectrum and high efficacy. TP's anti-hepatocellular carcinoma activity in cell and animal models even surpasses that of first-line clinical drugs such as doxorubicin and sorafenib, making it a highly promising candidate drug. However, triptolide suffers from poor water solubility, lack of targeting specific hepatocellular carcinoma cells, and a tendency to cause toxic side effects on other tissues and systems, thus hindering its clinical translation.

[0004] Nanoparticle drug delivery systems have been extensively explored to address the pharmacokinetic challenges of natural products. However, most synthetic polymer or inorganic nanocarriers suffer from drawbacks such as complex preparation, poor biocompatibility, and questionable long-term safety. For example, Chinese invention patent CN109985023A discloses a kidney-targeting nanocarrier capable of reducing the toxicity of triptolide, but its preparation process is relatively complex, and quality control is difficult during preparation. Chinese invention patent CN108635338B discloses a topical triptolide lipid nanoparticle, which does reduce the toxicity of triptolide but fails to increase efficacy. In contrast, self-assembly systems of natural small molecules offer a greener, safer solution that aligns with drug development principles. For instance, Chinese invention patent CN111494319A discloses a triptolide compound composition, its preparation method, and its application. The glycyrrhizic acid ammonium salt in the formulation reduces toxicity and enhances stability when prepared as an emulsion, and it must undergo a phospholipid complex to exert its lipophilic effect and promote TP absorption. It can be seen that, due to the complex formulation of this compound composition, the preparation process is cumbersome and the reaction conditions are relatively harsh, which does not conform to the goal of green development. Summary of the Invention

[0005] In order to overcome the shortcomings of the prior art, the present invention aims to provide a triptolide hydrogel, its preparation method, and its application in the preparation of drugs for treating hepatocellular carcinoma.

[0006] To achieve the above objectives, the present invention employs the following technical solution: This invention discloses a triptolide self-assembled hydrogel, which is composed of triptolide and glycyrrhizic acid in a mass ratio of 1:1-20.

[0007] Preferably, the self-assembled hydrogel is pH responsive and can slowly release drugs in an environment with a pH of 6.5 ± 0.3.

[0008] Preferably, the self-assembled hydrogel is composed of nanoparticles with an average particle size of 10-30 nm.

[0009] More preferably, the zeta potential of the nanoparticles of the self-assembled hydrogel is -30 to -45 mV.

[0010] Preferably, the self-assembled hydrogel has a porous network structure with uniformly distributed pores.

[0011] Preferably, the mass ratio of triptolide to glycyrrhizic acid in the self-assembled hydrogel is 1:5-15.

[0012] More preferably, the mass ratio of triptolide to glycyrrhizic acid in the self-assembled hydrogel is 1:10.

[0013] This invention also discloses a method for preparing the above-mentioned triptolide self-assembled hydrogel, comprising the following steps: 1) Dissolve glycyrrhizic acid in water at 80±10℃ and stir magnetically until completely dissolved to obtain a glycyrrhizic acid solution; 2) Dissolve triptolide in DMSO and sonicate until completely dissolved to obtain triptolide solution; 3) Add triptolide solution to glycyrrhizic acid solution, stir until completely dissolved, and then heat at 80±10℃ to obtain a mixed solution; 4) Let the mixed solution stand at room temperature or 4°C for 30±5 minutes to obtain triptolide self-assembled hydrogel.

[0014] Preferably, in step 1), the magnetic stirring speed is 200-500 r / min, and the stirring time is 10-30 min; In step 2), the ultrasonic power is 100-300W and the ultrasonic time is 5-15min; In step 3), heat for 30-60 minutes; use magnetic stirring with a speed of 300-600 r / min for 5-20 minutes.

[0015] The present invention also discloses the application of the above-mentioned triptolide self-assembled hydrogel in the preparation of drugs for treating liver cancer.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The triptolide self-assembled hydrogel disclosed in this invention possesses significant material advantages and application potential. This is due, in part, to the scientifically sound composition ratio. By precisely controlling the mass ratio of triptolide (TP) to glycyrrhizic acid (GA) from 1:1 to 20, stable self-assembly of the hydrogel is achieved. Furthermore, glycyrrhizic acid simultaneously fulfills three functions: carrier (self-assembly), attenuator, and synergistic pharmacological effect. First, the carrier function of glycyrrhizic acid lays the foundation for the stability of hydrogels. Glycyrrhizic acid achieves stable self-assembly through its own molecular structure, which significantly improves the stability of hydrogels and greatly reduces their fluidity. This effectively solves the problems of easy collapse and drug leakage in traditional hydrogels, and provides a reliable structural carrier for drug loading. Secondly, glycyrrhizic acid's toxicity-reducing function significantly enhances product safety. Tripterygium wilfordii itself is highly toxic, especially posing a potential threat to the liver. Glycyrrhizic acid can reduce its toxicity through two pathways: its molecular structure allows for a mild interaction with triptolide, lowering the concentration of free triptolide and reducing its direct stimulation of normal tissue cells; simultaneously, glycyrrhizic acid possesses clear hepatoprotective activity, alleviating potential liver inflammation caused by triptolide and reducing the risk of liver damage. Furthermore, the pH-responsive sustained-release properties of the hydrogel further prevent the additive local toxicity caused by sudden drug release, allowing the toxicity-reducing effect and targeted drug delivery to work synergistically, significantly improving the clinical safety of the formulation.

[0017] Third, the pharmacological activity of glycyrrhizic acid itself can synergize with triptolide, further enhancing the application value of the formulation.

[0018] Furthermore, glycyrrhizic acid exhibits pH responsiveness, allowing the hydrogel to slowly release the drug in an environment of pH 6.5 ± 0.3. This pH range highly matches the microenvironment of liver cancer tissue (weakly acidic), enabling targeted accumulation and controlled release of the drug at the lesion site. Simultaneously, the viscoelastic properties assessed by G' and G'' are analyzed to ensure the hydrogel possesses both good deformation capacity and structural stability in the in vivo physiological environment, adapting to the needs of different drug delivery scenarios. Preferably, the hydrogel of the present invention has excellent and controllable physicochemical properties. In the nanostructure system formed by the self-assembly of glycyrrhizic acid, the zeta potential of the nanoparticles is in a reasonable range of -10 to -50 mV, especially in the preferred range of -30 to -45 mV, which ensures that the particles are highly stable in the dispersion system and are not prone to agglomeration. The nanoscale particle size of 10-30 nm and the uniform porous network structure not only improve the specific surface area and water retention capacity of the hydrogel, but also provide sufficient space for drug loading and sustained release by relying on the spatial structure advantages of the glycyrrhizic acid carrier.

[0019] Preferably, when TP:GA = 1:10, the hydrogel exhibits optimal stability and significantly reduced fluidity, thus solving the problems of easy collapse and drug leakage inherent in traditional hydrogels.

[0020] Preferably, it has precise pH responsiveness, slowly releasing the drug in an environment of pH 6.5±0.3. This pH range is highly matched with the microenvironment of liver cancer tissue (weakly acidic), which can achieve targeted enrichment and controlled release of the drug at the lesion site, reduce the distribution of the drug in normal tissue, reduce toxic side effects, and simultaneously analyze the viscoelastic properties of G' and G'' to ensure that the hydrogel has good deformation capacity and structural stability in the in vivo physiological environment, adapting to the needs of different drug delivery scenarios.

[0021] The preparation method disclosed in this invention has a simple process, consisting of only three core steps: raw material dissolution, mixing, and static molding. It requires no complex equipment or harsh reaction conditions. Compared to the cross-linking agent-assisted method or high-pressure reaction method commonly used in traditional hydrogel preparation, it can preserve the molecular structural integrity of glycyrrhizic acid to the greatest extent, avoiding the destruction of its self-assembly ability and pharmacological activity by cross-linking agents, while significantly reducing production difficulty and cost. Furthermore, the preparation process is green and environmentally friendly, using water as the main solvent and only a small amount of dimethyl sulfoxide (DMSO) to dissolve triptolide. Subsequent complex solvent residue removal steps are unnecessary, reducing environmental pollution and potential harm to human health. It also avoids interference from residual solvents on the detoxification effect and synergistic pharmacological effects of glycyrrhizic acid, aligning with the development trend of green chemical production.

[0022] In summary, this invention provides a more environmentally friendly and efficient carrier for triptolide, reducing the potential risks of synthetic materials while aligning with current green development principles. Glycyrrhizic acid, possessing the triple functions of carrier, targeting, and toxicity reduction, can help triptolide precisely target its intended purpose, reduce its toxicity, and exert a synergistic effect. Furthermore, it aligns with traditional Chinese medicine principles: based on TCM compatibility theory, TP attacks cancer and eliminates pathogenic factors, while GA supports the body's resistance and protects the liver, achieving a dynamic balance of "attack and supplementation." This not only fully leverages the cancer-fighting advantages of triptolide but also mitigates its potential adverse effects on the body through the compatibility with glycyrrhizic acid, organically combining TCM theory with modern drug therapy, providing a unique and more promising strategy for the application of triptolide in the treatment of cancer and other diseases. Attached Figure Description

[0023] Figure 1 The hydrogel tilt diagram shows the optimal ratio of triptolide and glycyrrhizic acid.

[0024] Figure 2 Zeta potential diagram of triptolide-glycyrrhizic acid hydrogel.

[0025] Figure 3 The histogram of particle size of triptolide-glycyrrhizic acid hydrogel.

[0026] Figure 4 Electron micrograph of triptolide-glycyrrhizic acid hydrogel.

[0027] Figure 5 To evaluate the in vivo retention performance of triptolide-glycyrrhizic acid hydrogel at in situ liver tumor sites.

[0028] Figure 6 Images of mouse hepatocellular carcinoma tumors, including the model group, free TP, free TP and GA, and TP-GA hydrogel.

[0029] Figure 7 The results show the spleen weight of mice in the normal group, model group, and drug-treated group.

[0030] Figure 8 The liver weight results are for mice in the model group and the drug-treated group.

[0031] Figure 9 The results show the changes in body weight of mice in the normal group, model group, and drug-treated group 14 days after drug administration.

[0032] Figure 10 The changes in tumor volume in mice in the model group and the drug administration group 14 days after drug administration were observed.

[0033] Figure 11 A comparison of hemolysis rates of free TP and TP-GA hydrogels at different concentrations. Detailed Implementation

[0034] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0035] It should be noted that the terms "first," "second," etc., in the specification, claims, and accompanying drawings of this invention are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that the embodiments of the invention described herein can be implemented in orders other than those illustrated or described herein. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover a non-exclusive inclusion; for example, a process, method, system, product, or apparatus that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or apparatus.

[0036] The present invention will now be described in further detail with reference to the accompanying drawings: Example 1: Preparation of Tripterygium wilfordii hydrogel 1. Preparation of glycyrrhizic acid hydrogel A GA solution was prepared by dissolving GA in 10 mL of ultrapure water at 80 °C with stirring until completely dissolved, while maintaining the temperature. Different pH values ​​were set. Subsequently, the solution was placed in a refrigerator at room temperature or 4 °C, and the differences in GA gelation were observed to confirm the optimal molar ratio of GA to NaOH.

[0037] 2. Preparation of TP-GA self-assembled hydrogels Based on the above method for preparing glycyrrhizic acid hydrogels, under the same conditions, TP was dissolved in a small amount of DMSO and sonicated until completely dissolved. The solution was maintained at 80±10℃, and triptolide was added to the glycyrrhizic acid solution and stirred until completely dissolved. After mixing, the solution was heated for 30–60 min, and then allowed to stand at room temperature or 4℃ for 30±5 min to form a hydrogel. TP-GA hydrogel samples with different proportions were prepared using the above method. The prepared hydrogel samples were placed into transparent containers, ensuring the containers were well sealed.

[0038] 3. Characterization 3.1 Observation by tilting and tabletop inversion experiment The container containing the hydrogel was slowly tilted to the same angle, and the morphological changes of the hydrogel within the container were observed. Any phenomena such as flow, deformation, or stratification were recorded. Next, the container was inverted and observed; it was quickly inverted and held for 1 minute to observe whether the hydrogel flowed within the container and whether its morphology remained intact. By comparing the morphological changes of TP-GA hydrogel samples with different ratios within the container, the amount of TP added was fixed at 1 mg / mL, while the amount of GA added was varied. It was observed that when the TP:GA ratio was 1:10, the hydrogel began to stabilize, and its fluidity decreased.

[0039] See results Figure 1 As shown in the figure, the hydrogel remains stable and does not flow or deform after the container is tilted. Based on the experimental results, it can be seen that when the amount of TP added is 1 mg / mL and the TP∶GA=1∶10, the hydrogel has good stability and reduced fluidity. It can maintain its shape and form after tilting and inverting without flowing or layering.

[0040] 3.2 Nanoparticle size and Zeta potential Nanoparticle size and zeta potential were verified using nanoparticle tracking analysis. Freshly prepared TP-GA hydrogels were diluted 50-fold with ultrapure water, and the nanoparticle size, concentration, and zeta potential were measured using a ZetaView nanoparticle tracking analyzer. The tests were repeated three times at room temperature.

[0041] See results Figure 2 and Figure 3 ,from Figure 2 The Zeta potential detection plot shows that after dilution, the Zeta potential of the TP-GA hydrogel nanoparticles is -38.6 mV, which is relatively high in absolute value, indicating that the nanoparticles have good stability in the dispersion system and are not prone to aggregation. Combined with repeated testing, the results have a certain degree of reliability. Figure 3 The particle size histogram shows that the average size of the nanoparticles in this TP-GA hydrogel is 17.18±0.17 nm, and the particle size distribution is relatively concentrated.

[0042] 3.3 Transmission Electron Microscopy Freshly prepared TP-GA hydrogel was dehydrated in a freeze dryer for 72 hours. The freeze-dried sample was then attached tightly to a conductive carbon film double-sided adhesive and placed on an ion sputtering stage for approximately 30 seconds to sputter gold. The sample was then observed on the instrument.

[0043] See results Figure 4 This hydrogel has a porous network structure with relatively uniform pore distribution and fine granular protrusions on its surface. The porous structure is a typical characteristic of hydrogels, which helps them achieve functions such as water retention and substance loading.

[0044] 4. In vitro release test Six mL of TP-GA hydrogel was placed in a dialysis bag (molecular weight cutoff 3500), and the bag was immersed in 120 mL of PBS at pH 6.5 and pH 7.4, respectively. The bags were then placed on a shaker at 37 °C and 100 r / min. One mL of release medium was collected at 1, 2, 3, 4, 6, 12, and 24 h, and the same volume of fresh release medium was added. The TP content was determined by HPLC, and the cumulative release rate was calculated. The results showed that the cumulative release rates of TP-GA hydrogel at different pH values ​​were 66.25% and 52.58%, respectively. The release was relatively slow within 24 h, indicating a certain sustained-release effect.

[0045] 5. Small animal imaging The retention of the gel at tumor sites in vivo was qualitatively observed using fluorescence imaging technology. CY7.5 was selected as the fluorescent dye and encapsulated in TGH according to the preparation method of TP-GA hydrogel. The free group was mixed with an equal volume of TP-GA hydrogel. A stock solution of fluorescein was prepared by dissolving a certain amount of CY7.5 in DMSO, and then diluted with deionized water to an equal volume as the free fluorescent solution. Female BABL / C mice (weighing 20 ± 2 g) inoculated with H22 cells were selected and injected intraperitoneally with free PBS containing fluorescent dye and TP-GA hydrogel (0.45 mg / kg). Imaging was performed using a small animal imaging system at 0 h, 1 h, 2 h, 4 h, 6 h, 12 h, 24 h, 36 h, and 48 h post-injection. Figure 5 The results showed that the signal in the TP-GA group was strongly maintained at the tumor site for 24 hours, while the signal in the control group (free Cy7.5) dissipated rapidly within 12 hours. These results indicate that TP-GA hydrogel can effectively anchor and release active ingredients for a long time in the in-situ tumor microenvironment with high perfusion and high interstitial pressure, providing strong support for prolonging the duration of therapeutic action.

[0046] Example 2 Cytotoxicity Experiment HepG2 cells were seeded and cultured in 96-well plates. After 24 hours of cell attachment, different concentrations of free TP, free GA, free TP+GA, and TGH gel were added to the 96-well plates. Cell viability was assessed after 24 hours using CCK-8 staining, and the final drug concentrations were determined to be 80 nm / mL for TP and 400 μm / mL for GA. Subsequently, Annexin V-FITC / PI double staining was used to detect drug-induced apoptosis.

[0047] Example 3 Antitumor activity To establish a HEPG2 tumor-bearing mouse model, 100 μL of hepg2 cell suspension (5 × 10⁻⁶ cells) was subcutaneously injected into the right axillary region of male BALB / c Nude nude mice. 7 (Number of tumor cells / mL). When the tumor volume reaches 100 mm²... 3 Mice were randomly divided into 5 groups and drug administration was initiated. The experiment consisted of six groups: (1) normal group; (2) model group; (3) free TP group; (4) free TP+GA group; and (5) TGH group. TGH was administered via intratumoral injection. Each mouse received 0.45 mg / ml of body weight of TGH on days 1, 3, 5, 7, 9, 11, and 13, with each injection volume being 25 μl. Mouse body weight and tumor volume were recorded every two days during the experiment. Tumor volume was measured using calipers and calculated using the formula: Volume = (Length × Width²) / 2. At the end of the experiment, mice were euthanized using ocular blood collection, and the tumor and major organs (heart, liver, spleen, lungs, and kidneys) were immediately removed. The results are as follows: See Figure 6 The sample volume of the model group is relatively larger, the sample size of the TGH group is more uniform and smaller overall, and the sample size of the other groups is between the two.

[0048] See Figure 7 This is a statistical chart of spleen index in different groups (normal group, model group, TGH group, TP group, and TG group). The spleen index of the normal group is significantly higher than that of the other groups; the spleen index of the model group is lower; the spleen indices of the TGH, TP, and TG groups are slightly higher than those of the model group but lower than those of the normal group. However, the spleen index of the TGH group is closer to that of the model group.

[0049] See Figure 8 The graph shows the liver weight statistics of different groups (model group, TGH group, TP group, and TG group). The liver weight of the model group is significantly higher than that of the TGH, TP, and TG groups. The liver weight of the TGH, TP, and TG groups is similar and much lower than that of the model group. The changes in liver weight are often related to the pathological state of the liver. It can be seen that the TGH group has a significant effect on improving the liver condition of the model group and can effectively reduce liver weight.

[0050] See Figure 9 The graph shows the weight changes over 14 days in different groups (normal group, model group, TGH group, TP group, and TG group). The weight of the normal group is stable; the weight of the model group shows a downward trend; the weight fluctuations of the TGH, TP, and TG groups are smaller and are closer to those of the normal group; the TGH group is very close to the normal group. The weight change reflects the body's condition, indicating that the TGH treatment can improve the weight loss in the model group and help maintain the body's stable condition.

[0051] See Figure 10The bar chart shows the volume changes of different groups over 1-13 days. It can be seen that as time goes on, the volume of some groups shows a significant upward trend, reaching a relatively high level between 11-13 days, and the volume difference between different groups gradually widens.

[0052] Example 4 Safety Evaluation Based on the excellent antitumor effects exhibited by TGH gel in vitro and in vivo, fresh blood was collected using anticoagulant tubes. After centrifugation and removal of the supernatant, 20 μL of red blood cells were added to each tube, followed by different concentrations of free TP and TGH gel. The tubes were incubated at 37°C for 4 hours, centrifuged, and the supernatant was collected. The OD value of the supernatant was measured using a microplate reader, and the hemolysis rate was calculated. The hemolysis rate of both PBS-treated and drug-treated samples was less than 5%, indicating that the drug was non-hemolytic and that the TGH gel had good blood compatibility.

[0053] See results Figure 11 The hemolysis rates of PBS, TP and TGH at different concentrations (45 / 90 / 180 μg / ml) were all far below the safety standard of 5%.

[0054] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A self-assembled hydrogel of triptolide, characterized in that, It is composed of triptolide and glycyrrhizic acid in a self-assembly process, wherein the mass ratio of triptolide to glycyrrhizic acid is 1:1-20.

2. The triptolide self-assembled hydrogel according to claim 1, characterized in that, This self-assembled hydrogel is pH responsive and can slowly release drugs in an environment with a pH of 6.5 ± 0.

3.

3. The triptolide self-assembled hydrogel according to claim 1, characterized in that, This self-assembled hydrogel is composed of nanoparticles with an average particle size of 10-30 nm.

4. The triptolide self-assembled hydrogel according to claim 3, characterized in that, The zeta potential of the nanoparticles in this self-assembled hydrogel is -30 to -45 mV.

5. The triptolide self-assembled hydrogel according to claim 1, characterized in that, The self-assembled hydrogel has a porous network structure with uniformly distributed pores.

6. The triptolide self-assembled hydrogel according to claim 1, characterized in that, The mass ratio of triptolide to glycyrrhizic acid in this self-assembled hydrogel is 1:5-15.

7. The triptolide self-assembled hydrogel according to claim 6, characterized in that, The mass ratio of triptolide to glycyrrhizic acid in this self-assembled hydrogel is 1:

10.

8. The method for preparing the triptolide self-assembled hydrogel according to any one of claims 1-7, characterized in that, Includes the following steps: 1) Dissolve glycyrrhizic acid in water at 80±10℃ and stir magnetically until completely dissolved to obtain a glycyrrhizic acid solution; 2) Dissolve triptolide in DMSO and sonicate until completely dissolved to obtain triptolide solution; 3) Add triptolide solution to glycyrrhizic acid solution, stir until completely dissolved, and then heat at 80±10℃ to obtain a mixed solution; 4) Let the mixed solution stand at room temperature or 4°C for 30±5 minutes to obtain triptolide self-assembled hydrogel.

9. The method for preparing the triptolide self-assembled hydrogel according to claim 8, characterized in that, In step 1), the magnetic stirring speed is 200-500 r / min, and the stirring time is 10-30 min; In step 2), the ultrasonic power is 100-300W and the ultrasonic time is 5-15min; In step 3), heat for 30-60 minutes; use magnetic stirring with a speed of 300-600 r / min for 5-20 minutes.

10. The use of the triptolide self-assembled hydrogel according to any one of claims 1-7 in the preparation of a medicament for treating liver cancer.

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