Crosslinked hyaluronic acid hydrogel

Abstract

The invention discloses hyaluronic acid hydrogel prepared on the basis of a cross-linking agent, the cross-linking agent is 1-vinyl-3-ethylidene-2-pyrrolidone (CAS: 373620-97-4, hereinafter referred to as EVP), and the hyaluronic acid hydrogel has the properties of low toxicity and good biocompatibility. Under the action of an initiator potassium persulfate, the cross-linking agent and hyaluronic acid are subjected to a cross-linking reaction, and the generated hyaluronic acid hydrogel is high in softness, good in biocompatibility and wide in application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical chemistry and cosmetics, and particularly relates to a cross-linked hyaluronic acid hydrogel. Technical Background

[0002] Cross-linked hyaluronic acid hydrogel is a polymer gel with a three-dimensional structure obtained by cross-linking and modification reaction of natural hyaluronic acid with a cross-linking agent. Currently known reaction cross-linking agents include 1,4-butanediol diglycidyl ether (BDDE) and divinyl sulfone (DVS).

[0003] The method of cross-linking hyaluronic acid has been reported in the literature. We classify and review the different cross-linking agents used in the literature as follows:

[0004] Mi Pengcheng, Wang Bochu, Feng Wei, et al. [Preparation and biocompatibility of divinyl sulfone cross-linked sodium hyaluronate gel [J]. Chinese Journal of Tissue Engineering Research and Clinical Rehabilitation, 2008, (14): 2675-2678] used divinyl sulfone (DVS) to cross-link and prepare sodium hyaluronate gel and studied its biocompatibility. Although the prepared sodium hyaluronate hydrogel has acceptable biocompatibility, divinyl sulfone itself is a highly toxic and carcinogenic chemical, and the residual amount needs to be strictly controlled during the post-processing process.

[0005] Chen Xueqing [Study on the preparation process of cross-linked hyaluronic acid gel for injection [D]. Northwest Agriculture and Forestry University, 2024] used 1,4-butanediol diglycidyl ether (BDDE) to prepare hyaluronic acid gel. Currently, this method is widely used in the market for the preparation of hyaluronic acid gel. However, the amount of BDDE cross-linking agent is large. According to the provisions of the "People's Republic of China Industry Standard YY / T 0962-2021 Cross-linked Hyaluronic Acid Gel for Plastic Surgery", the residual amount of cross-linking agent BDDE must be strictly controlled.

[0006] The above-mentioned reaction crosslinking agents 1,4-butanediol diglycidyl ether (BDDE) and divinyl sulfone (DVS) have the problems of high toxicity and poor biocompatibility. Therefore, the present invention provides a crosslinking agent 1-vinyl-3-ethylidene-2-pyrrolidone (CAS: 373620-97-4, hereinafter referred to as EVP), which has low toxicity and good biocompatibility. The prepared crosslinked hyaluronic acid hydrogel has high softness and good biocompatibility, and has broad application prospects. Summary of the invention

[0007] The object of the present invention is to provide a hyaluronic acid hydrogel prepared based on a crosslinker, wherein the solid content of the hydrogel is 5-15% by mass, and the rest is water. The present invention provides a hyaluronic acid crosslinker: 1-vinyl-3-ethylidene-2-pyrrolidone (CAS: 373620-97-4, hereinafter referred to as EVP).

[0008] In order to achieve the purpose of the present invention, the technical solution adopted by the present invention is as follows:

[0009] S1: Accurately weigh hyaluronic acid powder, place it in a beaker, and add 0.01 mol / L Na 2 CO 3 The aqueous solution was dissolved and the pH value was adjusted to 9; the mixture was stirred in a water bath at 40° C. for 1 h to obtain a hyaluronic acid aqueous solution;

[0010] S2: After the initiator is dissolved, add 1-vinyl-3-ethylidene-2-pyrrolidone crosslinker, react in a water bath at 25-50°C for 12-36h and then remove;

[0011] S3: Add hydrochloric acid dropwise to the aqueous solution to adjust the pH to 7.4;

[0012] S4: Place in a dialysis bag and purify with ultrapure water for 24 hours, replace the ultrapure water every 6 hours, and then replace with phosphate buffered saline (PBS) for purification for 12 hours. The residual amount of cross-linking agent detected is less than 2ppm;

[0013] S5: After dialysis is completed, collect the gel, sterilize it at high temperature, and seal it for storage.

[0014] The invention provides a cross-linking agent hyaluronic acid hydrogel, wherein the mass ratio of the cross-linking agent 1-vinyl-3-ethylidene-2-pyrrolidone to the hyaluronic acid in the preparation step is 1:50-1:10.

[0015] The cross-linked hyaluronic acid hydrogel proposed by the present invention has a pH value of 7-10 in the hyaluronic acid aqueous solution described in step S1 of its preparation.

[0016] The present invention provides a cross-linked hyaluronic acid hydrogel, wherein the initiator in the preparation step S2 is one or more of potassium persulfate, sodium persulfate, and ammonium persulfate, and the mass ratio of the initiator to the hyaluronic acid is 1:100-1:20.

[0017] The present invention provides a cross-linked hyaluronic acid hydrogel, wherein in the preparation step S2, the cross-linking reaction temperature of 1-vinyl-3-ethylidene-2-pyrrolidone and hyaluronic acid is 25-50° C., and the cross-linking reaction time is 12-36 hours.

[0018] The invention discloses a cross-linked hyaluronic acid hydrogel, wherein the cross-linking agent has low toxicity and good biocompatibility. Under the action of an initiator, potassium persulfate, free radicals initiate polymerization, and a cross-linking agent, EVP, and hyaluronic acid undergo a cross-linking reaction, and the generated hyaluronic acid hydrogel has high softness and good biocompatibility, and has broad application prospects. DETAILED DESCRIPTION

[0019] Example 1

[0020] The steps are as follows:

[0021] S1: Accurately weigh 5 g of hyaluronic acid (molecular weight 100 kDa, Shanghai MacLean Biochemical Technology Co., Ltd.) dry powder, place it in a 1L beaker, add 500 mL of 0.01 mol / L Na 2 CO 3 The aqueous solution was dissolved and the pH value was adjusted to 9; the mixture was stirred in a water bath at 40° C. for 1 h to obtain a hyaluronic acid aqueous solution;

[0022] S2: Add 0.1 g potassium persulfate to dissolve, then add 0.1 g 1-vinyl-3-ethylidene-2-pyrrolidone (EVP) crosslinking agent, react in a water bath at 40° C. and a stirring speed of 500 rpm for 24 hours, and then take out.

[0023] S3: Place the gel in a beaker and soak it in ultrapure water, and add 0.01 mol / L hydrochloric acid to the aqueous solution to adjust the pH to 7.4.

[0024] S4: Place in a 14 kDa dialysis bag and purify with ultrapure water for 24 hours, replace the ultrapure water every 6 hours, and then change to phosphate buffered saline (PBS) for purification for 12 hours. The residual content of the cross-linking agent is detected to be less than 2 ppm.

[0025] S5: After dialysis, the gel was collected and sterilized at 121°C for 30 min to obtain a sterile cross-linked hyaluronic acid gel sample.

[0026] Biocompatibility evaluation:

[0027] The biocompatibility evaluation mainly uses the proportion of inflammatory response in mice and the cell survival rate as evaluation indicators.

[0028] (1) The sample in S5 was prepared into a 0.05 mass fraction aqueous solution. After 20 experimental mice were routinely raised for 1 week, the mice were fixed by hand and anesthetized by intraperitoneal injection of 0.5 mL of tribromoethanol. The hair on the back of the mice was shaved with an electric razor and disinfected with 75% medical alcohol. After the sterile injection needle was inserted into the epidermis, 0.5 mL of the cross-linked hyaluronic acid hydrogel sample was injected. After 24 hours, the mice were observed for inflammatory reactions.

[0029] (2) Cytotoxicity was evaluated in accordance with GB / T 16886.5-2017 “Biological Evaluation of Medical Devices Part 5: In vitro Cytotoxicity Test”. HaCaT cells (human immortalized keratinocytes) were seeded in a 96-well plate and a control group (a hyaluronic acid aqueous solution with a mass fraction of 0.05) and an experimental group (a hyaluronic acid gel aqueous solution with a mass fraction of 0.05) were added. The plates were incubated at 37°C and 5% CO. 2 After 24 hours of culture in the incubator, the original culture medium in the 96-well plate was discarded, and the plate was washed with PBS buffer. Then, 100 μL of incomplete culture medium and 20 μL of 5 mg / mL MTT (thiazolyl blue) solution were added to each well, and the culture was continued for 3 hours. The culture medium was discarded, and dimethyl sulfoxide was added to dissolve the plate. The absorbance value (OD value) at 490 nm was measured with an ELISA reader, and the survival rate of each group of cells was calculated. The cell survival rate was calculated according to the following formula:

[0030] Cell survival rate (%) = [OD (experimental group) / OD (control group)] × 100%

[0031] In Example 2-9, the experimental operation is the same as in Example 1, but the conditions of the cross-linking reaction are changed, as shown in Table 1;

[0032] In Comparative Example 1, the experimental operation is the same as in Example 1, except that the type of cross-linking agent is changed to divinyl sulfone (CVS), as shown in Table 1.

[0033] Comparative Example 2

[0034] The steps are as follows:

[0035] S1: Accurately weigh 5g of hyaluronic acid powder, place it in a 1L beaker, add 500mL of 0.01mol / L Na 2 CO 3 The aqueous solution was dissolved and the pH value was adjusted to 9; the mixture was stirred in a water bath at 40° C. for 1 h to obtain a hyaluronic acid aqueous solution;

[0036] S2: Add 0.5 gg 1,4-butanediol diglycidyl ether (BDDE) crosslinker, react in a water bath at 40°C and 500 rpm stirring speed for 24 hours and then remove;

[0037] S3: Place the gel in a beaker and soak it in ultrapure water, then add 0.01 mol / L hydrochloric acid to the aqueous solution to adjust the pH to 7.4;

[0038] S4: Place in a 14 kDa dialysis bag and purify with ultrapure water for 24 hours, replace the ultrapure water every 6 hours, and then replace with phosphate buffered saline (PBS) for purification for 12 hours. The residual amount of the cross-linking agent is detected to be less than 2 ppm;

[0039] S5: After dialysis, the gel was collected and sterilized at 121° C. for 30 min to obtain a sterile cross-linked hyaluronic acid hydrogel sample.

[0040] Table 1 Examples and Comparative Examples

[0041]

[0042] As can be seen from Table 1, compared with Comparative Examples 1-2, Example 1-9 has a high cell survival rate, low cytotoxicity, and a lower proportion of inflammatory reactions in mice. This shows that the biocompatibility of the hyaluronic acid hydrogel prepared with EVP in Example 1-9 is better than that of the existing hyaluronic acid hydrogel prepared with CVS and BDDE. In Example 1-5, the amount of cross-linking agent EVP is increased, and the cell survival rate is still greater than 70%, far exceeding the 35% and 41% of BDDE using cross-linking agents CVS in Comparative Examples 1-2.

[0043] The above description is only a preferred example of the present invention and is not intended to limit the present invention. Any modification, replacement and improvement made on the basis of the principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A cross-linked hyaluronic acid hydrogel, characterized in that A cross-linking agent is 1-vinyl-3-ethylidene-2-pyrrolidone, the mass ratio of the cross-linking agent to the hyaluronic acid is 1:50-1:10, the solid content of the hydrogel is 5-15% by mass, and the rest is water; The preparation steps are as follows: S1: Weigh hyaluronic acid powder, dissolve it in 0.1 mol / L Na2CO3 aqueous solution, adjust the pH value, and stir in a water bath at 30-60°C for 20-40 min to obtain a hyaluronic acid aqueous solution; S2: After the initiator is dissolved, add 1-vinyl-3-ethylidene-2-pyrrolidone crosslinker, react in a water bath at 20-50°C for 12-36h and then remove; S3: Add 0.01 mol / L hydrochloric acid to the aqueous solution to adjust the pH to 7.4; S4: Place in a dialysis bag and purify with ultrapure water for 24 hours, replace the ultrapure water every 6 hours, and then replace with phosphate buffered saline (PBS) for purification for 12 hours. The residual amount of cross-linking agent detected is less than 2ppm; S5: After dialysis is completed, collect the gel, sterilize it at high temperature, and seal it for storage.

2. A cross-linking agent hyaluronic acid hydrogel as claimed in claim 1, characterized in that: The pH value of the hyaluronic acid aqueous solution described in preparation step S1 is 7-10.

3. A cross-linked hyaluronic acid hydrogel according to claim 1, characterized in that: The initiator described in preparation step S2 is one or more of potassium persulfate, sodium persulfate, and ammonium persulfate, and the mass ratio of the initiator to the hyaluronic acid is 1:100-1:

20.

4. The cross-linked hyaluronic acid hydrogel according to claim 1, characterized in that: The cross-linking reaction temperature of hyaluronic acid and 1-vinyl-3-ethylidene-2-pyrrolidone in preparation step S2 is 20-50°C.

5. The cross-linked hyaluronic acid hydrogel according to claim 1, characterized in that: The cross-linking reaction time of 1-vinyl-3-ethylidene-2-pyrrolidone and hyaluronic acid in preparation step S2 is 12-36 hours.

Images