Glycyrrhetinic acid injectable hydrogel, its preparation method and application

Abstract

The application belongs to the technical field of medicine, and specifically discloses a glycyrrhetinic acid injectable hydrogel as well as a preparation method and application thereof. The preparation method of the glycyrrhetinic acid injectable hydrogel comprises the following steps: glycyrrhetinic acid and choline hydroxide are added into pure water, heated and stirred to fully react, and a mixed solution is obtained; the mixed solution is first spin-evaporated and then vacuum-dried to obtain glycyrrhetinic acid-choline; the glycyrrhetinic acid-choline is added into pure water, heated to completely dissolve and cooled at room temperature to form an injectable hydrogel. The hydrogel is used for preparing an antitumor drug in an injectable dosage form. In the application, the glycyrrhetinic acid and the choline hydroxide are self-assembled into a hydrogel with a three-dimensional network microstructure by losing one molecule of water without adding any cross-linking agent and organic solvent. The hydrogel can quickly recover to the original gel state after injection, and the problems of low drug loading, low bioavailability and difficult degradation existing in the prior art are solved.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically referring to an injectable hydrogel of glycyrrhetinic acid, its preparation method, and its application. Background Technology

[0002] Cancer seriously threatens human life and health, and long-acting release and localized treatment have always been challenges in the medical field. In existing technologies, scientists have designed various drug delivery systems to deliver drugs to target sites and release them slowly, with injectable hydrogels being one of them. Hydrogels are flexible materials with open porous structures, possessing good water absorption, water retention, and lubrication properties. After being injected into tumors in situ, they can slowly release drugs, reducing the number of administrations and minimizing adverse reactions from multiple doses. However, the preparation of hydrogels typically requires the addition of various cross-linking agents or organic solvents with strong irritant and toxic side effects, which inevitably cause damage and toxic side effects after injection into the human body. Furthermore, currently developed injectable hydrogels have low drug loading capacity and insignificant sustained-release effects, with insufficient duration of efficacy after a single dose. Therefore, exploring non-toxic and high-loading injectable hydrogels is imperative. Glycyrrhetinic acid (GA), formed by the hydrolysis of glycyrrhizic acid and the removal of its sugar acid chain, has certain anti-cancer and cancer-preventive effects, particularly showing good targeting potential for liver cancer, and is expected to be used as a targeted carrier for liver cancer. Compared to hydrogels formed by chemical and physical cross-linking, self-assembled hydrogels do not require the introduction of bridging agents such as proteins, metal ions, or small molecule surfactants to induce hydrogel formation. Therefore, these hydrogels have higher purity and fewer potential harms to the human body. Currently, there are no reports on the self-assembly of glycyrrhetinic acid to form injectable hydrogels suitable for tumor treatment. Summary of the Invention

[0003] To address the shortcomings of the existing technologies, this invention proposes an injectable hydrogel of glycyrrhetinic acid, its preparation method, and its applications.

[0004] The technical solution of this invention is implemented as follows:

[0005] A method for preparing glycyrrhetinic acid injectable hydrogel includes the following steps:

[0006] (1) Add glycyrrhetinic acid and choline hydroxide to pure water, heat and stir to allow them to react fully, and obtain a mixed solution;

[0007] (2) The mixed solution was first rotary evaporated and then vacuum dried to obtain glycyrrhetinic acid-choline;

[0008] (3) Add the glycyrrhetinic acid-choline to pure water, heat to dissolve completely, and then cool to room temperature.

[0009] Reaction principle: During heating and stirring, the carboxyl group of glycyrrhetinic acid combines with the hydroxide ion in choline hydroxide to remove one molecule of water. The glycyrrhetinic acid that has lost the hydroxide ion combines with the quaternary ammonium structure in choline in the form of an ionic bond. This ionic bond has a dynamic and reversible property. The hydrogel formed can be rapidly sheared and thinned under certain stress, and can quickly return to the original gel state after injection.

[0010] Choline (CC) is an important component of biological membranes and does not have adverse effects on the human body. This invention does not contain any cross-linking agents or organic solvents, and no carrier is needed subsequently. It can be completely absorbed by the human body without concerns about metabolism. This avoids the toxic side effects of existing polymer microspheres, micelles, liposomes, etc., as drug carriers, thus improving medication safety. At the same time, it significantly increases the drug loading capacity, with the amount of glycyrrhetinic acid loaded exceeding 15 wt%, thereby reducing the frequency of administration, improving patient compliance, and reducing adverse reactions. Furthermore, it solves the problem of low degradation rate of existing injectable hydrogels, which has very important clinical significance.

[0011] Preferably, in step (3), the content of glycyrrhetinic acid-choline is at least 20 wt%, and the heating and dissolution temperature is 40-90℃. Creative experimental studies have shown that when the concentration of glycyrrhetinic acid-choline is within the above-mentioned weight percentage range, the gelation is uniform; if the concentration is too low, a hydrogel cannot be formed.

[0012] Specifically, in step (1), the temperature for heating and stirring is 50-70℃.

[0013] Specifically, in step (2), the temperature of rotary evaporation is 50-90℃ and the time is 20-70min.

[0014] Specifically, in step (2), the vacuum drying temperature is 25-50℃ and the time is 12-24h.

[0015] This invention also proposes an injectable hydrogel of glycyrrhetinic acid prepared by the above preparation method. The microstructure of the injectable hydrogel of glycyrrhetinic acid is a three-dimensional network, which can be used to prepare injectable antitumor drugs, especially drugs for treating liver cancer. It has shear-thinning and self-healing properties, and can be directly injected without additional carriers. It has a long-acting release capability, which can slowly release drug molecules and avoid rapid drug clearance. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 SEM images of the hydrogels obtained in Examples 1-3: Labels (1), (2), and (3) correspond to the products obtained in Examples 1, 2, and 3, respectively;

[0018] Figure 2 This is the 1H NMR spectrum of glycyrrhetinic acid-choline;

[0019] Figure 3 The Fourier transform infrared spectrum of glycyrrhetinic acid-choline;

[0020] Figure 4 The graph shows the variation of storage modulus (G') / loss modulus (G”) of the hydrogel with angular frequency at a stress of 1%.

[0021] Figure 5 The graph shows the variation of storage modulus (G') / loss modulus (G”) of the hydrogel with stress at an angular frequency of 1 rad / s.

[0022] Figure 6 The graph shows the variation of storage modulus (G') / loss modulus (G”) of hydrogels under different stresses;

[0023] Figure 7 The graph shows the relationship between the shear rate and viscosity of the hydrogel.

[0024] Figure 8 The variation of hydrogels of different concentrations with temperature;

[0025] Figure 9 The effects of glycyrrhetinic acid, choline, and glycyrrhetinic acid-choline on the growth inhibition of Huh-7 cells were investigated. Detailed Implementation

[0026] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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 are within the scope of protection of the present invention.

[0027] Example 1

[0028] A method for preparing glycyrrhetinic acid injectable hydrogel includes the following steps:

[0029] (1) Place glycyrrhetinic acid in a 50ml pear-shaped flask, add 20ml of pure water, and add choline hydroxide while stirring. The molar ratio of glycyrrhetinic acid to choline hydroxide is 1:1. The entire operation is carried out at 70℃. After the reaction is complete, a mixed solution is obtained.

[0030] (2) The mixed solution was first rotary evaporated and then vacuum dried to remove the solvent water. The rotary evaporation temperature was 70℃ and the time was 40 min. The vacuum drying temperature was 25℃ and the time was 24 h to obtain glycyrrhetinic acid-choline.

[0031] (3) Glycyrrhetinic acid-choline was added to pure water and dissolved completely at 70°C. The content of glycyrrhetinic acid-choline was 20wt%. Then, it was cooled at room temperature to obtain a hydrogel.

[0032] Example 2

[0033] A method for preparing glycyrrhetinic acid injectable hydrogel includes the following steps:

[0034] (1) Place glycyrrhetinic acid in a 50ml pear-shaped flask, add 20ml of pure water, and add choline hydroxide while stirring. The molar ratio of glycyrrhetinic acid to choline hydroxide is 1:1. The entire operation is carried out at 50℃. After the reaction is complete, a mixed solution is obtained.

[0035] (2) The mixed solution was first rotary evaporated and then vacuum dried to remove the solvent water. The rotary evaporation temperature was 50℃ and the time was 70 min. The vacuum drying temperature was 50℃ and the time was 12 h to obtain glycyrrhetinic acid-choline.

[0036] (3) Glycyrrhetinic acid-choline was added to pure water and dissolved completely at 40°C. The content of glycyrrhetinic acid-choline was 23wt%. Then, it was cooled at room temperature to obtain a hydrogel.

[0037] Example 3

[0038] A method for preparing glycyrrhetinic acid injectable hydrogel includes the following steps:

[0039] (1) Place glycyrrhetinic acid in a 50ml pear-shaped flask, add 20ml of pure water, and add choline hydroxide while stirring. The molar ratio of glycyrrhetinic acid to choline hydroxide is 1:1. The entire operation is carried out at 60℃. After the reaction is complete, a mixed solution is obtained.

[0040] (2) The mixed solution was first rotary evaporated and then vacuum dried to remove the solvent water. The rotary evaporation temperature was 90℃ and the time was 20min. The vacuum drying temperature was 35℃ and the time was 18h to obtain glycyrrhetinic acid-choline.

[0041] (3) Glycyrrhetinic acid-choline was added to pure water and dissolved completely at 90°C. The content of glycyrrhetinic acid-choline was 25%. Then, it was cooled at room temperature to obtain a hydrogel.

[0042] Test characterization:

[0043] 1. Microscopic morphology testing

[0044] Take 100 μL of the products obtained in Examples 1-3 respectively on a clean glass slide, freeze-dry for 24 h and then conduct SEM testing. Before testing, sputter coating with gold is carried out. As Figure 1 shown: The microscopic morphologies of the products obtained in Examples 1, 2, and 3 are all three-dimensional network structures.

[0045] 2. NMR testing and infrared spectroscopy testing

[0046] As Figure 2 shown is the 1H NMR spectrum of glycyrrhetinic acid-choline obtained in step (2) of Examples 1-3. As shown in the 1H NMR spectrum of glycyrrhetinic acid-choline, the appearance of the double bond hydrogen at the 12'-position and the appearance of a', b', and c' of choline prove the successful preparation of glycyrrhetinic acid-choline. At the same time, due to the very low solubility of glycyrrhetinic acid in water, the solubility in water is greatly improved after being prepared into glycyrrhetinic acid-choline, indirectly proving the successful preparation of glycyrrhetinic acid-choline.

[0047] Take 3 mg of glycyrrhetinic acid-choline obtained in step (2) of Examples 1-3 respectively in a mortar, mix evenly with KBr and then press into tablets for infrared spectroscopy testing. The obtained infrared spectra are all as Figure 3 shown (Choline, Glycyrrhetinic acid, Glycyrrhetinic acid-Choline): Characteristic absorption bands of glycyrrhetinic acid: The C-O vibration of polysaccharide is at 1000-1100 cm -1 , hydrogen bond association is at 2870-2960 cm -1 , O-H vibration is at 3300 cm -1 , and the -COOH absorption peak is at 1700-1750 cm -1 . After dehydration, the -COOH absorption peak of glycyrrhetinic acid at 1700-1750 cm -1 disappears, and the absorption peak at 1650 cm -1 blue-shifts to 1560-1570 cm -1 , proving that -COOH in glycyrrhetinic acid has changed, indicating the successful preparation of glycyrrhetinic acid-choline.

[0048] 3. Rheological property testing

[0049] The rheometer is used to detect the changes in storage modulus (G') and loss modulus (G") to reflect the state of the product. When G'>G", the product is in a gel state, and when G'<G", the product is in a solution state. As Figure 4 shown: At a stress of 1%, the rheometer is used to test the corresponding G' and G" when the angular frequency ranges from 1 rad / s to 100 rad / s. Within this angular frequency range, G' is greater than G", indicating that at low stress, the product is always in a gel state and has good mechanical properties. As Figure 5 As shown: At an angular frequency of 1 rad / s, the stress values ​​G' and G” were measured using a rheometer from 0% to 100%. When the stress was between 0% and 20%, G' was greater than G”, indicating that the product was in a gel state. When the stress increased to 20%, G” was greater than G', indicating that the product was in a sol state.

[0050] The hydrogels prepared in Examples 1-3 were placed on a rheometer, and the experiment was divided into three stages: the first stage was under low stress, with a stress setting of 1% for 100 s; the second stage was under high stress, with a stress setting of 50% for 100 s; and the third stage was from high stress back to low stress, with a stress setting of 1% for 100 s. The changes in G' and G” were observed in each stage. Figure 6 As shown: 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 experiments show that the hydrogel has good self-healing ability.

[0051] The relationship between gel viscosity and shear rate was detected using a rheometer, such as... Figure 7 As shown, the gel viscosity decreases sharply as the shear rate increases, indicating that the hydrogel has good shear-thinning ability, that is, the gel is injectable.

[0052] 4. Temperature-sensitive change test

[0053] The glycyrrhetinic acid-choline obtained in step (2) of Example 1 was added to pure water and completely dissolved at 50°C. Two samples were prepared with concentrations of 20wt%, 22.5wt%, 23wt%, and 25wt%, respectively. After cooling to room temperature, stable hydrogels were formed in each sample. (Specific details are as follows...) Figure 8 As shown in (A). One sample of each concentration of hydrogel was heated at 37°C for 20 minutes, and the other at 40°C for 20 minutes. Changes in their state were observed, as detailed below. Figure 8As shown in (B): the 20wt% sample formed a sol after heating at 37℃ and 40℃; the 22.5wt% and 23wt% samples formed a gel after heating at 37℃ and a sol after heating at 40℃; and the 25wt% sample formed a gel after heating at 37℃ and 40℃. Clearly, the hydrogel prepared by this invention is a thermosensitive hydrogel. As a local drug delivery formulation, it can utilize the microenvironmental characteristics of the lesion, such as the higher local temperature of the tumor compared to normal tissue, to achieve rapid drug release. Alternatively, photothermal molecules can be added to the gel to achieve controlled drug release, significantly improving drug bioavailability. Furthermore, the concentration can be flexibly adjusted to achieve controllable drug loading.

[0054] 5. Drug efficacy test

[0055] Glycyrrhetinic acid (GA), choline (CC), and a glycyrrhetinic acid-choline mixture (GA-CC) were prepared at concentrations of 0 μM, 1 μM, 5 μM, 10 μM, 20 μM, 40 μM, 80 μM, and 160 μM, respectively. These solutions were then co-incubated with Huh-7 cells for 48 hours before cell viability was measured. Figure 9 As shown, at the same concentration, the IC50 of GA is... 50 IC with a capacity of 115μM and GA-CC 50 At a concentration of 79.66 μM, choline showed a more significant inhibitory effect on Huh-7. The effect of CC on the survival rate of liver cancer cells was almost unrelated to drug concentration, indicating that choline has little toxic side effects on cells.

[0056] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing an injectable hydrogel of glycyrrhetinic acid, characterized in that: The following steps are included: (1) Add glycyrrhetinic acid and choline hydroxide to pure water, heat and stir to allow them to react fully, and obtain a mixed solution. The heating and stirring temperature is 50-70℃. (2) The mixed solution is first rotary evaporated and then vacuum dried to obtain glycyrrhetinic acid-choline; (3) Add the glycyrrhetinic acid-choline to pure water, heat to dissolve completely, and then cool to room temperature. The content of the glycyrrhetinic acid-choline is at least 20 wt%, and the heating temperature for dissolution is 40-90℃.

2. The method for preparing glycyrrhetinic acid injectable hydrogel according to claim 1, characterized in that: In step (2), the temperature of the rotary evaporation is 50-90℃ and the time is 20-70min.

3. The method for preparing glycyrrhetinic acid injectable hydrogel according to claim 1, characterized in that: In step (2), the vacuum drying temperature is 25-50℃ and the time is 12-24h.

4. An injectable hydrogel of glycyrrhetinic acid prepared by the preparation method according to any one of claims 1-3.

5. The application of the glycyrrhetinic acid injectable hydrogel according to claim 4, characterized in that: Used to prepare injectable anti-liver cancer drugs.

Images