CN118986860A

CN118986860A - Glycyrrhetinic acid chitosan self-healing hydrogel and preparation method and application thereof

Info

Publication number: CN118986860A
Application number: CN202411143656.9A
Authority: CN
Inventors: 崔明宇; 孙尚辉; 王子函; 阎雪莹; 徐世一
Original assignee: Heilongjiang University of Chinese Medicine
Current assignee: Heilongjiang University of Chinese Medicine
Priority date: 2024-08-20
Filing date: 2024-08-20
Publication date: 2024-11-22

Abstract

The present invention belongs to the technical field of pharmaceutical preparations, and specifically relates to a glycyrrhetinic acid chitosan self-healing hydrogel, and a preparation method and use thereof. The hydrogel is made of glycyrrhetinic acid, chitosan, and polyethylene glycol with benzaldehyde groups at both ends. In the hydrogel, Schiff base bonds are used as bridges, and glycyrrhetinic acid is loaded in a physical mixing manner, and the hydrogel is used for intratumoral injection. The present invention uses glycyrrhetinic acid as the main drug, glycyrrhetinic acid receptors abundant in the liver as the target, self-healing hydrogels as carriers, and intratumoral injection as the administration method to construct a precisely targeted and in vivo sustained-release drug delivery system. At the same time, the accumulation of glycyrrhetinic acid in the tumor lesion site is increased, the effectiveness of the drug in the body is improved, and some toxic side effects caused by the drug are reduced, so as to achieve the purpose of inhibiting hepatocellular carcinoma. The present invention provides ideas and references for the design of hydrogel targeting systems, and provides new methods for the clinical treatment of liver cancer.

Description

Glycyrrhetinic acid chitosan self-healing hydrogel and preparation method and application thereof

Technical Field

The invention belongs to the technical field of pharmaceutical preparations, and particularly relates to glycyrrhetinic acid chitosan self-healing hydrogel, and a preparation method and application thereof.

Background

Liver cancer (HCC) has become the fourth leading cause of tumor-related death worldwide, and world health organization estimates that more than 100 tens of thousands of liver cancer patients will die in 2030. HCC that can be examined is severe, rapidly worsening, prone to migration, lack of early symptoms, and low survival in most patients. Common treatment means comprise surgery, chemotherapy, radiotherapy and the like, but only less than two patients have surgical opportunities, relapse and transfer are easy to occur after surgical excision, and the chemotherapy drugs have larger toxic and side effects on the patients and are easy to cause damage to other tissues and organs. Therefore, the local administration mode is considered, and the targeted drugs are combined, so that the concentration of the drug at the focus part is improved, and the distribution of the tissue in the whole body and the damage to other organs are reduced. The targeted treatment method is simple, easy to prepare and widely applied at present, and is an important method for improving the living standard of patients and prolonging the life expectancy.

Glycyrrhetinic Acid (GA) belongs to triterpene aglycone, is one of natural components in Glycyrrhrizae radix, has multiple pharmacological actions, and has certain therapeutic effects on various diseases, including anti-inflammatory, antiviral, liver protecting and antitumor effects. Glycyrrhetinic acid shows remarkable cytotoxicity to various tumor cells, and therefore has been attracting attention in the study of antitumor activity. The liver is distributed with rich glycyrrhetinic acid receptor, so that the glycyrrhetinic acid can be delivered to the liver in a targeted manner for treating various liver diseases. Although glycyrrhetinic acid can treat various diseases, systemic use can cause serious hypertension and cardiac hypertrophy, so that dosage is limited in clinical application, systemic administration is difficult to obtain enough drug concentration at a targeted tumor site, multiple injections are required to achieve the effect, and the glycyrrhetinic acid has poor usability and compliance. There is a need to develop new drug carriers that address these problems by means of administration by local injection.

The dynamic self-healing hydrogel not only has good water absorbability, can be swelled in water, has high structural consistency with living tissues, and a three-dimensional network structure is beneficial to the loading and release of medicines, but also can repair the self-morphology through the reversibility of the self-dynamic chemical bond after the material is damaged externally, so that the performance is close to or reaches the state before the damage. The controllable injection performance of the hydrogel is the key of treating cancers by taking the hydrogel as a drug carrier, and the hydrogel can be fully filled into tumor tissue gaps after being injected into pathological tissues, so that the transformation of sol-gel is realized, the drug can be concentrated at a required position, the loss of the drug in blood circulation is reduced, and the hydrogel becomes a drug storage library, so that the drug can be effectively controlled and slowly released, the treatment effect of the drug is improved, the toxic and side effects of the drug are reduced, and the dosage of the drug is reduced.

In view of the above, the invention selects glycyrrhetinic acid as a model drug, chitosan and cross-linking agent dialdehyde polyethylene glycol are prepared into hydrogel with dynamic self-repairing property as a drug carrier, and the glycyrrhetinic acid chitosan self-healing hydrogel with anti-liver cancer effect is prepared, so that ideas and methods are provided for developing novel drug carriers for treating liver cancer and local targeting drug delivery systems.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a practical, convenient, stable and reliable glycyrrhetinic acid chitosan self-healing hydrogel for targeted treatment of liver cancer and a preparation method thereof.

In order to achieve the above object, the present invention provides the following technical solutions:

In a first aspect, the present invention provides a glycyrrhetinic acid chitosan self-healing hydrogel made of glycyrrhetinic acid, chitosan and polyethylene glycol (DF-PEG) having benzaldehyde groups at both ends, wherein the glycyrrhetinic acid is loaded in a physical mixing manner with schiff base bond as a bridge in the hydrogel, and the hydrogel is used for intratumoral injection.

Alternatively, in the self-healing hydrogel, the DF-PEG is prepared from PEG2000 and 4-carboxybenzaldehyde through esterification reaction.

Alternatively, in the self-healing hydrogel described above, the hydrogel has good sustained release properties and has acid responsiveness.

In a second aspect, the present invention provides a method for preparing the self-healing hydrogel according to the first aspect, the method comprising the steps of:

Step 1: preparation of chitosan solution: preparing glacial acetic acid solution with volume concentration of 1-5%, preparing chitosan solution with mass volume concentration of 1-5% in g/mL by using the glacial acetic acid solution, and preserving for 24 hours at normal temperature;

Step 2: preparation of DF-PEG solution: DF-PEG is dissolved in ultrapure water to prepare DF-PEG solution with concentration of 2.0-8.0 mg/mL;

Step 3: preparation of self-healing hydrogels: the chitosan solution prepared in the step 1 and the DF-PEG solution prepared in the step 2 are mixed according to the volume ratio of 1-5: 1, mixing in proportion, stirring and standing to prepare hydrogel;

Step 4: preparation of drug-loaded self-healing hydrogel: dissolving glycyrrhetinic acid in methanol to prepare a glycyrrhetinic acid methanol solution, and mixing the glycyrrhetinic acid methanol solution with the blank hydrogel prepared in the step 3 according to a volume ratio of 1: stirring at a ratio of 0.5-5 fully and evenly mixed with the mixture, standing to form gel.

Alternatively, in the above preparation method, the preparation method of the crosslinking agent DF-PEG includes the steps of: obtaining DF-PEG through esterification reaction between PEG2000 and 4-carboxyl benzaldehyde, weighing a proper amount of PEG-2000, 4-aldehyde benzoic acid and 4-Dimethylaminopyridine (DMAP), uniformly dissolving in a proper amount of anhydrous Tetrahydrofuran (THF), cooling the obtained mixed solution to 0 ℃, then dissolving a proper amount of N, N' -Dicyclohexylcarbodiimide (DCC) in a proper amount of anhydrous THF, dropwise adding the DCC solution into the mixed solution under the protection of nitrogen, stirring at 25 ℃ for reaction for 20 hours, filtering the reaction solution after the reaction is finished to obtain white solid, recrystallizing and drying in vacuum to obtain white powder DF-PEG, and storing at-20 ℃ for further use.

Alternatively, in the above preparation method, in step 1, the glacial acetic acid solution has a volume concentration of 1% and the chitosan solution has a mass volume concentration of 2% in g/mL.

Alternatively, in the above preparation method, in step 2, the concentration of DF-PEG solution is 5.0mg/mL.

Alternatively, in the above preparation method, in step 3, the volume ratio of the chitosan solution to the DF-PEG solution is 3:1.

In the preparation method, in step 4, glycyrrhetinic acid is dissolved in methanol to prepare a glycyrrhetinic acid methanol solution with the concentration of 100 mug/mL, and the glycyrrhetinic acid methanol solution and the blank hydrogel prepared in step 3 are mixed according to the volume ratio of 1:1, stirring the materials in proportion, fully and uniformly mixing, and standing the materials to form the adhesive.

In a third aspect, the invention provides the use of the self-healing hydrogel of the first aspect or the self-healing hydrogel prepared by the preparation method of the second aspect in the preparation of a drug for targeting liver cancer.

Alternatively, in the above-mentioned use, the liver cancer is preferably hepatocellular carcinoma.

Compared with the prior art, the invention has the following beneficial effects:

The invention uses glycyrrhetinic acid as a main medicine, uses a glycyrrhetinic acid receptor rich in liver as an action target point, uses self-healing hydrogel as a carrier, and constructs a drug delivery system of a precisely targeted and in-vivo slow-release drug in an intratumoral injection drug delivery mode. Meanwhile, the accumulation of glycyrrhetinic acid in the tumor lesion position is increased, the in vivo effectiveness of the medicine is improved, and some toxic and side effects caused by the medicine are reduced, so that the aim of inhibiting the hepatocellular carcinoma is fulfilled. The invention provides thought and reference for the design of a hydrogel targeting system and provides a new method for clinical treatment of liver cancer.

Drawings

Fig. 1: schematic synthesis of DF-PEG.

Fig. 2: FT-IR analysis of DF-PEG.

Fig. 3: macroscopic self-healing experiments.

Fig. 4: macroscopic injectable experiments.

Fig. 5: SEM microstructure image of hydrogel. Wherein the left image is a panoramic image and the right image is a partial image.

Fig. 6: cumulative release profile (n=3) of GA different formulations in different pH release medium.

Fig. 7: toxicity of different concentrations of blank hydrogels to HepG-2 cells (n=6).

Fig. 8: trend of HepG-2 cell inhibition by different experimental groups at 24h (n=6). Wherein, P <0.05 is represented with significant differences.

Fig. 9: trend of HepG-2 cell inhibition by different experimental groups at 48h (n=6). Wherein, P <0.05 is represented with significant differences.

Fig. 10: trend of HepG-2 cell inhibition by different experimental groups at 72h (n=6). Wherein, P <0.05 is represented with significant differences.

Fig. 11: IC ₅₀ values for Free GA and GA-CS-DF-PEG (n=6).

Fig. 12: in vivo synthesis of hydrogels, subcutaneous hydrogels, in vivo stripping hydrogels.

Fig. 13: subcutaneous injection site tissue sections. Wherein A: control group, B: experimental groups.

Fig. 14: in vivo degradation process diagram of hydrogels.

Fig. 15: mice body weight change profile.

Fig. 16: tumor volume change plot.

Fig. 17: tumor inhibition ratio comparison chart.

Fig. 18: tumor-stripped contrast plots for each group. Wherein A: a physiological saline group; b: CS-DF-PEG group; c: free GA group; d: GA-CS-DF-PEG group.

Detailed Description

The invention will be further illustrated with reference to specific examples. It should be understood that the detailed description and specific examples are intended for purposes of illustration only and are not intended to limit the scope of the invention.

The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or equipment used were conventional products available for purchase through regular channels, with no manufacturer noted.

The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, are all commercially available products.

Example 1: preparation, optimization and characterization of GA-CS-DF-PEG self-healing hydrogel

1. Preparation of self-healing hydrogels

1.1 Synthesis of crosslinker DF-PEG

DF-PEG is obtained through esterification reaction between PEG and 4-carboxybenzaldehyde, and the synthetic steps are shown in figure 1. 1.63g of PEG-2000, 0.49g of 4-aldehyde benzoic acid and 0.025g of 4-Dimethylaminopyridine (DMAP) are weighed and uniformly dissolved in 50mL of anhydrous Tetrahydrofuran (THF), the obtained mixed solution is cooled to 0 ℃, then 0.84g of N, N' -Dicyclohexylcarbodiimide (DCC) is dissolved in 2mL of anhydrous THF, the DCC solution is dropwise added into the mixed solution under the protection of nitrogen, stirring is carried out at 25 ℃ for 20 hours, after the reaction is finished, the reaction solution is filtered to obtain white solid, the crude product is obtained by three times of recrystallization of THF and diethyl ether, and the obtained crude product is dried in vacuum to obtain white powder DF-PEG which is stored at-20 ℃ for further use.

1.2 Preparation of chitosan solution

100ML of glacial acetic acid solution with the volume concentration of 1% is prepared, 2g of chitosan powder is weighed by using a balance, and the solution is poured into the diluted glacial acetic acid solution and stored for 24 hours at normal temperature.

1.3 Preparation of DF-PEG solution

5.0Mg of the dialdehyde group-modified polyethylene glycol is dissolved in ultrapure water to obtain 5.0mg/mL of the dialdehyde group-modified polyethylene glycol solution.

1.4 Preparation of self-healing hydrogels

The chitosan solution and DF-PEG solution are mixed according to the volume ratio of 3: mixing in proportion, stirring and standing to prepare the hydrogel.

1.5 Preparation of drug-loaded self-healing hydrogel

Dissolving glycyrrhetinic acid in methanol to prepare a glycyrrhetinic acid methanol solution with the concentration of 100 mug/mL, and mixing the glycyrrhetinic acid methanol solution with the blank hydrogel prepared in the step 3 according to the volume ratio of 1:1, stirring the materials in proportion, fully and uniformly mixing, and standing the materials to form the adhesive.

Prescription optimization of CS-DF-PEG hydrogels

The invention mainly selects four factors of the concentration of acetic acid solution used for dissolving chitosan, the concentration of cross-linking agent and the volume ratio of chitosan to cross-linking agent as investigation objects to study the prescription and the preparation process of CS-DF-PEG hydrogel. According to the gel time and the result of visual analysis of SEM microscopic appearance, the optimal prescription of CS-DF-PEG hydrogel is glacial acetic acid volume concentration 1%, chitosan mass/volume concentration 2% (g/mL), cross-linking agent concentration 5mg/mL, and the volume ratio of chitosan solution to cross-linking agent solution is 3:1.

The CS-DF-PEG hydrogel is prepared by adopting the optimal prescription, the volume ratio of the glycyrrhetinic acid methanol solution to the blank hydrogel carrier is adjusted, and the optimal ratio is screened by detecting the encapsulation efficiency and the drug loading rate. The results show that when the volume ratio of the glycyrrhetinic acid methanol solution to the blank hydrogel carrier is 1: the encapsulation efficiency and the drug loading rate are the highest in the step 1, so that the volume ratio is 1:1 follow-up study was performed.

3. Characterization of self-healing hydrogels

3.1 Fourier transform Infrared Spectroscopy (FTIR)

The synthesis of DF-PEG was tested by IR spectroscopy. FIG. 2 is an infrared spectrum scan of the product, and compared with PEG-2000 raw powder, characteristic peaks of aldehyde and carbonyl ester (-1715 cm ^-1) can be clearly identified, and the synthesized product is proved to be a double-end benzaldehyde-capped polyethylene glycol (DF-PEG) structure.

3.2 Gel determination

Gel time was determined by tube inversion. The sample bottles were placed in a 37 ℃ incubator for gelation, and at intervals, the sample bottles were tilted 45 ° to see if the solution in the bottles flowed. If the sample in the vial does not flow along the walls of the vial within 30 seconds of tilting, the sample is considered to form a gel and the gel time is recorded. The results show that the hydrogel before the gel is a liquid with good fluidity, is a semisolid gel which does not flow after phase transition at 37 ℃, and is tightly adhered to the bottom when the container is inverted, and does not flow in a short time.

3.3 Self-healing and injectability decisions

Macroscopic self-healing experiments: the hydrogel samples were stained with a dye. And then cutting the dyed hydrogel, splicing and combining the hydrogel blocks dyed into different colors at the cut, placing at room temperature, and monitoring the self-healing condition. The results are shown in FIG. 3. The results show that the scratches become smaller as the hydrogel cuts of different colors meet, and a smooth and complete piece is formed. Macroscopic injectable experiments: the hydrogel samples were aspirated with a 1mL syringe, and then squeezed into a container to examine injectability. The results are shown in FIG. 4. The results show that the preparation has uniform texture and good needle penetrating property, and can not block the needle.

3.4 Microscopic morphology of CS-DF-PEG gel

And (3) putting a proper amount of hydrogel into a freeze drying box for drying treatment, and vacuumizing at the temperature of-51 ℃ to obtain the dried hydrogel under the freezing condition. Cutting the dry gel into blocks with the size of about 2.5 mm-3.5 mm, then adhering the blocks on an aluminum foil with conductive gel, performing metal spraying treatment, and gradually observing the morphology of the CS-DF-PEG hydrogel after the metal spraying treatment by controlling the multiplying power to be 100 times under the condition of accelerating voltage of 3.00 kV. The results are shown in FIG. 5. As shown, the hydrogel showed a clear network structure because the hydrogel contained 95% moisture, and thus showed a larger pore structure when the hydrogel was dehydrated.

Example 2: in vitro release kinetics study of GA-CS-DF-PEG self-healing hydrogels

And respectively taking 10mL of free GA solution with GA content of 1mg/mL and 10mL of GA-CS-DF-PEG hydrogel, assembling the free GA in a pretreated dialysis bag, using a sealing clamp seal number, suspending in a capped centrifuge tube containing 40mL of drug release medium, placing the gel into the capped centrifuge tube containing 40mL of drug release medium after the GA-CS-DF-PEG hydrogel is gelled, screwing the bottle cap, arranging 3 groups of parallel samples in each group, and placing in a constant-temperature water bath with a constant-temperature water bath oscillator at 37 ℃ for shaking at a constant speed (100 rpm). After the drug release is started, 1mL of drug release medium is taken at a preset time point, and simultaneously an equal amount of same-temperature blank medium is added, the measurement is carried out by adopting an HPLC method, the GA concentration is calculated, and the cumulative release percentage Q is calculated according to a formula. The in vitro cumulative release profile is shown in fig. 6, and the cumulative release percentage (Q) is formulated as follows:

In the formula, C _t is the concentration (mg/mL) of the drug in the release medium measured at each time point, W is the total weight (mg) of the drug administered, V ₀ is the total volume of the release medium, and V is the volume of each sample.

As can be seen from the GA release profile of FIG. 6, the GA free drug group was released at a high rate in any medium, 50% at 2 hours, 80% at 8 hours, and almost complete release at 10 hours, without being changed by pH change. The medicine-carrying gel group shows good slow release effect, shows a quick-before-slow release trend, and is influenced by pH. The result shows that under the acidic environment with pH=5.0, the accumulated release rate of the drug at 24 hours can reach 64.32 percent, and the release rate at the final 96 hours can reach 74.72 percent; in a neutral environment with pH=7.4, the accumulated release rate of the drug at 24 hours reaches 61.09%, the release rate at the final 96 hours reaches 67.87%, the preparation group clearly shows the advantage of prolonging the drug release, and the release rate with pH=5.0 is higher than the release rate with pH=7.4, so that the preparation group is sufficiently shown to be more stable at a focus part and can slowly release the drug for a long time after reaching a key target part, thereby exerting the subsequent therapeutic effect. The experimental results show that the GA-CS-DF-PEG has good slow release performance and acid responsiveness.

Meanwhile, the release mechanism of the system is obtained through fitting of different mathematical models, and is caused by double factors that medicine diffusion occurs firstly and then the gel skeleton is eroded.

Example 3: study of GA-CS-DF-PEG hydrogel cytotoxicity on HepG-2 1. Cytotoxicity of blank hydrogel on HepG-2

HepG-2 cell density is regulated to 2.5X10 ⁴/mL, inoculated in a 96-well plate, 100 mu L of cell suspension is added into each well, cultured in a 5% CO ₂ incubator at 37 ℃ for 48 hours, then taken out, old culture solution is sucked, 100 mu L of CS-DF-PEG blank gel series solution is respectively sucked and added into the 96-well plate, meanwhile, a control group which is only added with 100 mu L of cell suspension in equal volume and is not added with medicine is arranged, a blank group which is only added with 100 mu L of culture solution in equal volume is arranged, and 6 compound wells are arranged for each group to be parallel. The cells were further cultured for 24 hours and 48 hours, 10. Mu.L of CCK-8 solution was added to each well of each plate taken out at the same time every day, the culture was continued for 4 hours, the OD value of each well was measured at 450nm with a microplate reader, and the proliferation inhibition ratio of the cells was calculated. The results are shown in FIG. 7.

As can be seen from the figure, although the concentration of the blank gel carrier is increased, the survival rate of cells is not obviously influenced, and the cell viability can still reach more than 85% at the high concentration of 20000 mug/mL, so that the non-loaded gel carrier has smaller toxicity to cells and better biocompatibility, and can be used as a drug carrier.

2. Cytotoxicity of aqueous carrier gels on HepG-2

Preparing HepG-2 cell suspension with the cell density of 2.5X10 ⁴/mL, inoculating 100 mu L of each hole into a 96-well plate, culturing in a 5% CO ₂ incubator at 37 ℃ for 48 hours, taking out and sucking old culture solution, respectively sucking 100 mu L of GA solution with different concentrations and aqueous gel-carrying solution, adding the GA solution into the 96-well plate, simultaneously setting a control group with 100 mu L of cell suspension added with no drug in equal volume, adding a blank group with 100 mu L of culture solution in equal volume, and setting 6 compound holes as parallelism for each group. After the cells were further cultured for 24 hours, 48 hours and 72 hours, 10. Mu.L of CCK-8 solution was added for further culture for 4 hours, the culture solution was aspirated off, the OD value of each well was measured at 450nm with an ELISA reader, and the proliferation inhibition ratio of the cells was calculated. The results are shown in FIGS. 8-10.

The results show that each experimental group has inhibition effect on proliferation of HepG-2 cells, and the inhibition effect of the free GA group is higher than that of the GA-CS-DF-PEG group before 24 hours, which is shown in fig. 8, probably because the gel skeleton cannot be completely eroded and the entrapped drug cannot be completely released, so that the inhibition rate on proliferation of the cells is relatively smaller, and after 48 hours, the inhibition rate of the GA-CS-DF-PEG group is higher than that of the free GA group, so that the gel system has the slow-release long-acting effect while ensuring the treatment effect, slows down the drug release and reduces the drug dosage.

Statistics of IC ₅₀ values

IC ₅₀ values of the two groups of samples, namely Free GA group and GA-CS-DF-PEG, were calculated at 24h, 48h and 72h in combination with data of HepG-2 cell proliferation inhibition results. The analysis results are shown in FIG. 11. As shown in the figure, the Free GA group showed the least IC ₅₀ on the cells and the strongest inhibition (35.19. Mu.g/mL), but the inhibition on the cells by the GA-CS-DF-PEG group was gradually strengthened with the increase of time, and at 48h, the GA-CS-DF-PEG group IC ₅₀ was 64.06. Mu.g/mL smaller than 76.40. Mu.g/mL of the Free GA group, indicating the enhancement of the inhibition rate by the GA-CS-DF-PEG group. Meanwhile, at 72h, the IC ₅₀ value result of the drug on HepG-2 cells shows that the GA-CS-DF-PEG group is obviously lower than the Free GA group, and the inhibition rate is obviously higher than the Free GA group.

Example 4: in vivo feasibility study of GA-CS-DF-PEG hydrogel

Experimental animals: c57BL/6 mice and animal litter and feed used in the experiments were all supplied by Liaoning long Biotechnology Co., ltd. Experimental cells: hepa1-6, mouse hepatoma cells, purchased from Shanghai Saibuten Biotechnology Co., ltd.

1. In vivo loading and injectability of hydrogels

To investigate the hydrogel formation properties in vivo. Taking a healthy mouse, dehairing the pre-injection part, mixing CS-DF-PEG solution, injecting the mixture into the skin of the mouse through a syringe, and gradually forming hydrogel after three minutes of injection, so that the mouse can be touched by hands. Ten minutes later, after the hydrogel had stabilized for a period of time, the skin was cut open to observe the hydrogel formed, and the hydrogel was good at the injection site and could be peeled off from the skin of the mice, as shown in fig. 12. These results demonstrate that hydrogels can be formed by in vivo injection in a short time to ensure well dispersed drug delivery applications.

2. Biocompatibility experiments

The mice in the above section 1 were sacrificed, and the skin on the back of the mice was cut off, so that the skin and the muscle contacted with the gel block were free from redness and swelling and inflammatory reaction. The skin at the contact site was cut off and stored in 4% paraformaldehyde for histopathological analysis, the results are shown in FIG. 13. Blue or purplish blue colored nuclei, red colored cytoplasm and extracellular matrix can be seen. In contrast to the saline group, the gel group showed no apparent pathological changes such as inflammatory cell infiltration or congestion. The gel has good biocompatibility and high safety.

3. In vivo degradation experiments

The GA-CS-DF-PEG is administrated by local injection in situ and directly injected into tumor tissue, so that the in vivo degradation behavior is of great significance to the in vivo release research of the medicine. The gel is injected into mice, the shape and the size of subcutaneous gel blocks are observed at different times, and the skin is dissected to observe the gel degradation condition. The results are shown in fig. 14, which shows that the first day of injection is shown in fig. a, with a significant bulge; b is an anatomic map of the third day of injection, and the gel block volume is reduced, the color is lightened, the texture is thinned, and the gel block is fused with surrounding tissues; c is the seventh day of injection, the gel is basically completely degraded, and the surrounding skin tissues are not red and swollen, thus again proving that the gel carrier has good biocompatibility.

GA-CS-DF-PEG hydrogel in vivo anti-tumor treatment effect

4.1 Establishment of Hepa1-6 liver cancer mouse model

Recovering frozen Hepa1-6 cells, performing conventional in vitro culture, subculturing to digest in logarithmic phase, collecting cells in a centrifuge tube, centrifuging, washing twice with sterile PBS, re-suspending, filtering, counting, subcutaneously injecting the cell suspension into right hindlimb part of mice with 1mL sterile injector, and injecting 0.2mL cell suspension into each mouse. After inoculation, normal water and food feeding are carried out every day, the health condition and the tumor growth condition of each mouse are closely observed, and when a tumor hard mass appears, the model establishment is successful.

4.2 Tumor inhibition experiments

Mice with tumor hard masses are screened out and kept in separate cages for feeding. After the tumor mass volume reached about 150mm ³, mice were randomly divided into 4 groups of 5 mice each and ear tags were made. After the tumor volume had grown to about 200mm ³, the injection of the drug into the mice was started. Each group of mice was injected with physiological saline, free glycyrrhetinic acid physiological saline solution, blank hydrogel and GA-CS-DF-PEG hydrogel, at a concentration of 0.5mg/kg (calculated by GA content) every two days, for a total of 3 times. The first day of administration was recorded as day 0, and the body weight of the mice was weighed every other day and the major (L, mm) and minor (W, mm) diameters of the tumors were measured once, and the physiological state of the mice was observed. After the tumor inhibition test is completed, the mice in each group are killed by cervical removal, the tumor hard blocks under the armpits of each mouse are peeled off, the weight of each tumor is weighed, the tumors are photographed after being divided into groups, and the mice are washed by PBS and then stored in 4% paraformaldehyde solution.

The tumor volume was calculated according to the following formula:

Tumor inhibition was calculated according to the following formula:

Wherein Wc is the average tumor weight (g) of the physiological saline group and Wt is the average tumor weight (g) of the administered treatment group. The results are shown in FIGS. 15-18. The results showed that the tumor growth of the blank hydrogel group was as large as that of the normal saline control group, and that all groups treated with the free drug solution or the drug-loaded hydrogel showed tumor-inhibiting efficacy compared to the control. The experimental results in the graph show that the tumor volume of the normal saline control group mice is continuously increased in the whole administration period, and the blank carrier is the same growing trend, which indicates that the blank carrier has no therapeutic effect; the free GA group and the medicine carrying gel group are slightly increased in the first four days, but the increase is far smaller than that of the normal saline group, the free GA group is in a descending trend in the sixth day, although the increase starts to be increased in the eighth day, the increase is far smaller than that of the normal saline group, the anti-tumor effect of GA is reflected, the GA-CS-DF-PEG group still presents an increasing trend in 5-6 days, the decrease trend is reflected after the seventh day, the effect is equivalent to that of the free GA group in 8-12 days, the tumor volume which is measured to be peeled after the mice are killed in 14 days is smaller than that of the free GA group, so that the early effect is slower probably due to the influence of the coating of the gel carrier on the release of the medicine, the release of the medicine is required to be eroded by the gel carrier, the release rate of the medicine is slowed, the burst release of the medicine is avoided, and the purpose of slow release is achieved while the medicine effect is exerted. It was also found that mice in the free GA group had a low status during the dosing cycle and had poor appetite, probably due to systemic toxicity caused by high concentrations of GA.

It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.

Claims

1. A glycyrrhetinic acid chitosan self-healing hydrogel, which is characterized in that: the hydrogel is prepared from glycyrrhetinic acid, chitosan and polyethylene glycol (DF-PEG) with benzaldehyde groups at two ends, wherein the glycyrrhetinic acid is loaded in a physical mixing mode by taking a Schiff base bond as a connecting bridge, and the hydrogel is used for intratumoral injection.

2. The self-healing hydrogel according to claim 1, wherein: the DF-PEG is prepared from PEG2000 and 4-carboxybenzaldehyde through esterification reaction.

3. The self-healing hydrogel according to claim 1, wherein: the hydrogel has good slow release performance and acid responsiveness.

4. A method for producing a self-healing hydrogel according to any one of claims 1 to 3, characterized in that: the preparation method comprises the following steps:

5. The method of manufacturing according to claim 4, wherein: the preparation method of the cross-linking agent DF-PEG comprises the following steps: obtaining DF-PEG through esterification reaction between PEG2000 and 4-carboxyl benzaldehyde, weighing a proper amount of PEG-2000, 4-aldehyde benzoic acid and 4-Dimethylaminopyridine (DMAP), uniformly dissolving in a proper amount of anhydrous Tetrahydrofuran (THF), cooling the obtained mixed solution to 0 ℃, then dissolving a proper amount of N, N' -Dicyclohexylcarbodiimide (DCC) in a proper amount of anhydrous THF, dropwise adding the DCC solution into the mixed solution under the protection of nitrogen, stirring at 25 ℃ for reaction for 20 hours, filtering the reaction solution after the reaction is finished to obtain white solid, recrystallizing and drying in vacuum to obtain white powder DF-PEG, and storing at-20 ℃ for further use.

6. The method of manufacturing according to claim 4, wherein: in step 1, the volume concentration of the glacial acetic acid solution was 1%, and the mass volume concentration of the chitosan solution in g/mL was 2%.

7. The method of manufacturing according to claim 4, wherein: in step 2, the DF-PEG solution was at a concentration of 5.0mg/mL.

8. The method of manufacturing according to claim 4, wherein: in step 3, the volume ratio of the chitosan solution to the DF-PEG solution is 3:1.

9. The method of manufacturing according to claim 4, wherein: in the step 4, glycyrrhetinic acid is dissolved in methanol to prepare a glycyrrhetinic acid methanol solution with the concentration of 100 mug/mL, and the glycyrrhetinic acid methanol solution and the blank hydrogel prepared in the step 3 are mixed according to the volume ratio of 1:1, stirring the materials in proportion, fully and uniformly mixing, and standing the materials to form the adhesive.

10. Use of the self-healing hydrogel of any one of claims 1 to 3 or the self-healing hydrogel prepared by the preparation method of any one of claims 4 to 9 in the preparation of a drug targeting against liver cancer.