CN104861214A - Pullulan containing injectable hydrogel and preparation method thereof - Google Patents

Abstract

The invention relates to a preparation method of pullulan injectable hydrogel. Pullulan is weighed and dissolved in deionized water, then a cross-linking agent solution is added to stir for reaction, and the reaction solution is heated in a water bath to obtain a hydrogel. The hydrogel was sequentially soaked with pyrogen-free water and phosphate buffer solution, and made into injectable granules with a granulator. The present invention adopts a chemical cross-linking method, and two adjacent hydroxyl groups in pullulan polysaccharides of different molecular weights are docked with epoxy groups at both ends of BDDE to form a docked polymer, which reacts at 50°C to form a jelly-like The hydrogel can be made into millimeter-sized particles by a granulator, and is used for injectable hydrogels. It has the advantages of high stability, good biological properties, and can avoid surgical trauma.

Description

Pullulan injectable hydrogel and preparation method thereof

technical field

The invention relates to a hydrogel, in particular to a pullulan injectable hydrogel and a preparation method thereof.

Background technique

The design and development of hydrogels has become a hotspot in the research of multifunctional polymers for tissue repair. Pullulan is a non-reducing polymer substance, a water-soluble extracellular neutral polysaccharide secreted by Aureobasidium pullulans, which has non-toxicity, safety, solubility, stability and lubricity , Adhesiveness, coagulation, film covering, decomposing, improving physical properties and other properties, widely used in food, medicine, light industry, chemical industry and petroleum and other fields. Because pullulan has good non-toxic effect, water solubility, dispersibility and hygroscopicity, it can be used in cosmetics as a viscous additive; its price is far lower than that of hyaluronic acid, but its effect is different from that of hyaluronic acid Not much.

Amphiphilic graft or block polymers can self-assemble into nanoparticles or nanobundles in aqueous solution, and have good biocompatibility, so they can be used as soft tissue fillers in the field of tissue engineering.

Contents of the invention

The purpose of the present invention is to provide a pullulan injectable hydrogel and its preparation method, which uses a chemical cross-linking method to prepare more degradable, biocompatible and safer skin filling materials and implants Scaffolds.

The realization process of the present invention is as follows:

A preparation method of pullulan injectable hydrogel, comprising the following steps:

(1) dissolving pullulan in deionized water to obtain a pullulan solution with a mass volume fraction of 50-300 mg/mL, and controlling the temperature at 0-27°C;

(2) Add the cross-linking agent 1,4-butanediol diglycidyl ether solution with a mass fraction of 1-10%, stir evenly, and control the temperature at 20-30°C; among them, the mass fraction of pullulan and cross-linking agent The ratio is 300~1800:1~40;

(3) React the reaction solution at 45-55°C for 3-6 hours to obtain a hydrogel;

(4) The prepared hydrogel is sequentially soaked with pyrogen-free water and phosphate buffer solution for 2 to 6 days, and made into injectable granules by a granulator.

The aforementioned pullulan is a water-soluble extracellular neutral polysaccharide secreted by Aureobasidium pullulans, with a molecular weight of 100,000 g/mol-530,000 g/mol; the pH of the phosphate buffer solution is 7.4.

Specifically, the preparation method of the above-mentioned pullulan injectable hydrogel comprises the following steps:

(1) Dissolve pullulan in deionized water to obtain 6 mL of pullulan solution with a mass volume fraction of 50-300 mg/mL, control the reaction temperature at 0-27°C, and react under stirring conditions for 0.5-1 h;

(2) Add 0.1-0.35mL cross-linking agent 1,4-butanediol diglycidyl ether solution with a mass fraction of 1-10%, control the reaction temperature at 20-30°C, and react under stirring conditions for 0.2-0.5h ;

(3) Put the reaction solution in a constant temperature water bath at 50°C for 3-6 hours to prepare a hydrogel;

(4) The prepared hydrogel is sequentially soaked with pyrogen-free water and phosphate buffer solution for 2 to 6 days, and made into injectable granules by a granulator.

The hydrogel obtained by the above method for preparing the pullulan injectable hydrogel.

The present invention has the following advantages:

The pullulan hydrogel involved in the present invention is a skin filling material made by combining pullulan with different molecular weights and BDDE. The pullulan is secreted by Aureobasidium pullulans The water-soluble extracellular neutral pullulan with different molecular weights is used as raw material, and it is made by chemical cross-linking method. The two adjacent hydroxyl groups in the different molecular weight pullulan are docked with the epoxy groups at both ends of BDDE to form A docking polymer. The polymer reacts at 50°C to form a jelly-like hydrogel, which can be made into millimeter-sized particles by a granulator for use in injectable hydrogels, with high stability, good biological properties, and avoidable Trauma advantages of surgery.

Description of drawings

Fig. 1 is the infrared absorption spectrum of hydrogel; Among the figure, in A, No. 1 is the infrared spectrum of the pullulan polysaccharide that molecular weight is 100,000g/moL, and No. 2 is the molecular weight of the pullulan polysaccharide that No. 2 is 530,000g/moL Infrared image; in B, No. 1 is the infrared image of the hydrogel prepared by pullulan with a molecular weight of 100,000 g/moL, and No. 2 is the infrared image of the hydrogel prepared by pullulan with a molecular weight of 530,000 g/moL Figure; C is the infrared image of 1,4-butanediol diglycidyl ether (BDDE);

Fig. 2 is the residual amount of BDDE in the hydrogel; Among the figure, A is the residual amount of BDDE in the hydrogel prepared by the pullulan polysaccharide that the molecular weight is 100,000g/moL, and B is the pullulan that the molecular weight is 530,000g/moL The residual amount of BDDE in the hydrogel prepared by blue polysaccharide;

Figure 3 is a sample diagram of the hydrogel; among the figures, No. 1 and No. 2 are photographs of the hydrogel; No. 1 is a sample diagram of the hydrogel prepared by pullulan with a molecular weight of 100,000 g/moL; No. 2 A sample image of a hydrogel prepared for pullulan with a molecular weight of 530,000 g/moL;

Figure 4 is the SEM image of the hydrogel; in the figure, No. 1 is the SEM image of the hydrogel (magnified 100 times), No. 2 is the SEM image of the hydrogel (magnified 250 times), and No. 3 is the water The scanning electron micrograph of the gel (magnification 100 times), No. 4 is the scanning electron micrograph of the hydrogel (magnification 250 times); No. 1 and No. 2 are the hydrogel prepared by pullulan with a molecular weight of 100,000 g/moL Internal structure; Nos. 3 and 4 are the internal structure of the hydrogel prepared by pullulan with a molecular weight of 530,000g/moL;

Figure 5 is a picture of hydrogel injected subcutaneously in mice; in the figure, No. 1 is the picture of hydrogel prepared by pullulan with a molecular weight of 100,000 g/moL injected subcutaneously in mice for two weeks, and No. 2 is the molecular weight The hydrogel prepared for 530,000g/moL pullulan is the picture after two weeks of subcutaneous injection in mice, No. 3 is pullulan with molecular weight of 100,000g/moL (left) and 530,000g/moL (right) The hydrogel prepared by lantosaccharide is the figure after two weeks of subcutaneous injection in the mouse, and 4 is the figure of the hydrogel taken out after the hydrogel is injected into the mouse subcutaneous for two weeks;

Figure 6 is the H&E staining diagram of the hydrogel injected subcutaneously in the mouse; in the figure, Nos. 1 and 2 are the H&E tissues of the hydrogel prepared by the pullulan with a molecular weight of 100,000 g/moL injected subcutaneously in the mouse Staining; Nos. 3 and 4 are H&E tissue staining after the hydrogel prepared by pullulan with a molecular weight of 530,000 g/moL was injected subcutaneously in mice.

Detailed ways

The present invention will be described in detail below in combination with specific embodiments.

The pullulan hydrogel involved in the present invention uses water-soluble extracellular neutral pullulan of different molecular weights secreted by Aureobasidium pullulans as raw materials, and uses BDDE cross-linking agent for chemical cross-linking. The reaction solution was placed at 50° C. for 3-6 hours to prepare a hydrogel. The prepared crude product is purified, that is, pyrogen-free water and phosphate buffer solution are used to replace residual BDDE, unreacted pullulan molecules and impurities in the hydrogel.

The pullulan in the raw material is a water-soluble extracellular neutral polysaccharide of different molecular weight secreted by Aureobasidium pullulans. Compared with hyaluronic acid, it has good water solubility, mild reaction conditions and stability Good performance and other significant advantages. In addition, pullulan itself has good biocompatibility and degradability, and can be applied in the field of tissue engineering.

The preparation method of the pullulan injectable hydrogel related to the present invention is realized by the following steps:

Step 1: Weigh pullulan powders with different molecular weights and dissolve them in deionized water to obtain 6 mL of pullulan solution with a mass volume fraction of 50-300 mg/mL. The reaction temperature is controlled at 0-27 ° C. Lower reaction time 0.5～1h;

Step 2: Add 0.1-0.35mL cross-linking agent solution with a mass fraction of 1-10%, control the reaction temperature at 20-30°C, and react under stirring conditions for 0.2-0.5h;

Step 3: Put the reaction solution in a constant temperature water bath at 50°C for 3h to 6h to prepare a hydrogel;

Step 4: The prepared hydrogel is sequentially soaked with pyrogen-free water and phosphate buffer solution for 2-6 days, and is made into injectable granules with different particle sizes by a granulator.

In step 1, the pullulan is a water-soluble extracellular neutral polysaccharide with different molecular weights secreted by Aureobasidium pullulans, and the molecular weight is 100000g/mol-530000g/mol.

In the second step, the crosslinking agent is 1,4 butanediol diglycidyl ether.

In step 4, the pH of the phosphate buffer solution is 7.4.

Among them, the cross-linking agent 1,4-butanediol diglycidyl ether is docked with pullulan molecular chains of different molecular weights to form a polymer, and the docked polymer forms a jelly-like hydrogel at a temperature of 50 °C. Pyrogen-free water and phosphate buffer solution were used to wash the remaining impurities and some unreacted 1,4-butanediol diglycidyl ether in the hydrogel. The cleaning time is 2 to 6 days. The purified hydrogel was made into millimeter-sized particles of different particle sizes by a granulator, and then loaded into a syringe for injection.

Example 1

Step 1: Weigh pullulan powders with different molecular weights and dissolve them in deionized water to obtain 6 mL of pullulan solution with a mass volume fraction of 50 mg/mL. The reaction temperature is controlled at 27 °C and reacted for 0.5 h under stirring conditions ;

Step 2: Add 0.35 mL of a cross-linking agent solution with a mass fraction of 1%, control the reaction temperature at 30°C, and react for 0.2 h under stirring conditions;

Step 3: Put the reaction solution in a constant temperature water bath at 50°C for 6 hours to prepare a hydrogel;

Step 4: The prepared hydrogel was successively soaked with pyrogen-free water and phosphate buffer solution for 2 days, and the injectable granules of different particle sizes were made by a granulator.

In step 1, the pullulan is a water-soluble extracellular neutral polysaccharide with different molecular weights secreted by Aureobasidium pullulans, and the molecular weight is 380000g/mol-530000g/mol.

In the second step, the crosslinking agent is 1,4 butanediol diglycidyl ether.

In step 4, the pH of the phosphate buffer solution is 7.4.

Example 2

Step 1: Weigh pullulan powders with different molecular weights and dissolve them in deionized water to obtain 6 mL of pullulan solution with a mass volume fraction of 180 mg/mL. The reaction temperature is controlled at 14°C and reacted for 0.7 h under stirring conditions ;

Step 2: Add 0.14 mL of a cross-linking agent solution with a mass fraction of 5%, control the reaction temperature at 25°C, and react for 0.3 h under stirring conditions;

Step 3: Put the reaction solution in a constant temperature water bath at 50°C for 4.5 hours to prepare a hydrogel;

Step 4: The prepared hydrogel was successively soaked with pyrogen-free water and phosphate buffer solution for 4 days, and the injectable granules of different particle sizes were made by a granulator.

In step 1, the pullulan is a water-soluble extracellular neutral polysaccharide of different molecular weights secreted by Aureobasidium pullulans, and the molecular weight is 240000g/mol-380000g/mol.

In the second step, the crosslinking agent is 1,4 butanediol diglycidyl ether.

In step 4, the pH of the phosphate buffer solution is 7.4.

Example 3

Step 1: Weigh pullulan powders with different molecular weights and dissolve them in deionized water to obtain 6 mL of pullulan solution with a mass volume fraction of 300 mg/mL. The reaction temperature is controlled at 0°C, and the reaction is carried out for 1 h under stirring;

Step 2: Add 0.1 mL of a cross-linking agent solution with a mass fraction of 10%, control the reaction temperature at 20°C, and react for 0.5 h under stirring conditions;

Step 3: Put the reaction solution in a constant temperature water bath at 50°C for 3 hours to prepare a hydrogel;

Step 4: The prepared hydrogel was sequentially soaked with pyrogen-free water and phosphate buffer solution for 6 days, and the injectable granules with different particle sizes were made by a granulator.

In step 1, the pullulan is a water-soluble extracellular neutral polysaccharide with different molecular weights secreted by Aureobasidium pullulans, and the molecular weight is 100000g/mol-240000g/mol.

In the second step, the crosslinking agent is 1,4 butanediol diglycidyl ether.

In step 4, the pH of the phosphate buffer solution is 7.4.

Scanning electron microscope analysis, sample observation, and H&E staining analysis are carried out to the product of above embodiment, and the results are as follows:

1. The infrared absorption spectrum of the hydrogel is shown in Figure 1. There are new absorption peaks at 1645 cm ^-1 and 1159 cm ^-1 , which are the absorption peaks of carbonyl and secondary hydroxyl. It shows that the docking polymerization forms a new polymer during the crosslinking process of pullulan.

2. The residual BDDE content of the hydrogel after different purification treatments is shown in Figure 2. The longer the purification time, the lower the residual BDDE content, and it can still meet the standard requirements after 2 days of treatment.

3. A sample of the hydrogel filling material is shown in Figure 3, and the hydrogel is similar to a jelly-like milky white gel. No. 1 is a sample picture of a hydrogel prepared by pullulan with a molecular weight of 100,000 g/moL, which is transparent; No. 2 is a sample picture of a hydrogel prepared by a pullulan with a molecular weight of 530,000 g/moL, which is milky.

4. Scanning electron microscope analysis of the hydrogel filling material As shown in Figure 4, the hydrogel is a three-dimensional network-like porous structure, and the arrangement is relatively regular.

5. The hydrogel filling material was injected subcutaneously in the mouse and analyzed as shown in Figure 5. The hydrogel existed in the subcutaneous area of the mouse in the second week and was slightly degraded; after the hydrogel filling material was injected subcutaneously in the mouse for 2 weeks, the water The gel gradually adapts to the surrounding tissue.

6. The H&E staining analysis of the hydrogel filling material injected into the mouse skin is shown in Figure 6. No. 1 and No. 2 are the hydrogel injection mouse skin prepared by pullulan with a molecular weight of 530,000 g/moL 1 1 week and 2 weeks; 3, 4 hydrogels prepared from pullulan with a molecular weight of 100,000 g/moL were injected into the skin of mice for 1 week and 2 weeks. Nos. 1 and 2 degrade the slowest.

The animal experiment that the hydrogel filling material prepared by the present invention is used in adult female ICR mice shows that: the hydrogel prepared by the chemical cross-linking method has good biocompatibility and tissue compatibility, and does not degrade Slow, good for tissue filling.

The content of the present invention is not limited to the examples listed, and any equivalent transformation of the technical solution of the present invention adopted by those of ordinary skill in the art by reading the description of the present invention is covered by the claims of the present invention.

Claims (5)

1. a preparation method for pulullan polysaccharide injection aquagel, is characterized in that comprising the following steps:

(1) be dissolved in deionized water by pulullan polysaccharide and obtain the pulullan polysaccharide solution that quality volume fraction is 50 ~ 300mg/mL, it is 0 ~ 27 DEG C that temperature controls;

(2) add the linking agent Isosorbide-5-Nitrae butanediol diglycidyl ether solution that massfraction is 1 ~ 10%, stir, it is 20 ~ 30 DEG C that temperature controls; Wherein, the mass ratio of pulullan polysaccharide and linking agent is 300 ~ 1800:1 ~ 40;

(3) reaction solution is obtained hydrogel at 45 ~ 55 DEG C of reaction 3 ~ 6h;

(4) obtained hydrogel embathes 2 ~ 6 days with apirogen water and phosphate buffer soln in succession, makes injectable particle with nodulizer.

2. the preparation method of pulullan polysaccharide injection aquagel according to claim 1, is characterized in that: pulullan polysaccharide is the outer neutral polysaccharide of water-soluble born of the same parents of Aureobasidium pullulans secretion, and molecular weight is 100000g/mol ~ 530000g/mol.

3. the preparation method of pulullan polysaccharide injection aquagel according to claim 3, is characterized in that: the pH of phosphate buffer soln is 7.4.

4. the preparation method of pulullan polysaccharide injection aquagel according to claim 1, is characterized in that:

(1) be dissolved in deionized water by pulullan polysaccharide, obtain the pulullan polysaccharide solution 6mL that quality volume fraction is 50 ~ 300mg/mL, it is 0 ~ 27 DEG C that temperature of reaction controls, and reacts 0.5 ~ 1h under agitation;

(2) add 0.1 ~ 0.35mL, massfraction be 1 ~ 10% linking agent Isosorbide-5-Nitrae butanediol diglycidyl ether solution, temperature of reaction control be 20 ~ 30 DEG C, react 0.2 ~ 0.5h under agitation;

(3) reaction solution is put in 3 ~ 6h in 50 DEG C of thermostat water baths, obtained hydrogel;

(4) obtained hydrogel embathes 2 ~ 6 days with apirogen water and phosphate buffer soln in succession, makes injectable particle with nodulizer.

5. the pulullan polysaccharide injection aquagel that obtains of preparation method according to claim 1.