CN102070786B - Hyaluronic acid-sodium alginate composite hydrogel and preparation method thereof - Google Patents

Abstract

The invention discloses a composite hydrogel of hyaluronic acid and sodium alginate and a preparation method thereof. The composite hydrogel is prepared according to a method comprising the following steps: 1) dissolving hyaluronic acid and sodium alginate in water to obtain solution 1, 2) adding a cross-linking agent to said solution 1, and using a buffer adjusting the pH value of the solution to 4.0-5.5 to obtain solution 2; 3) adding a carboxyl activator to the solution 2 and stirring, using a buffer solution to control the pH value of the solution 2 at 2.0-5.5 until a gel is formed during the stirring process , to obtain hyaluronic acid and sodium alginate composite hydrogel. The present invention provides a new approach for the modification of hyaluronic acid, and the composite modified product prepared by the method of the present invention has the advantages of high mechanical properties, hyaluronidase resistance, biocompatibility, etc. , Tissue engineering has potential applications.

Description

A kind of hyaluronic acid-sodium alginate composite hydrogel and preparation method thereof

technical field

The invention relates to a hyaluronic acid-sodium alginate composite hydrogel and a preparation method thereof.

Background technique

Hyaluronic acid (HA) is a linear, unbranched acidic mucopolysaccharide composed of N-acetylglucosamine and D-glucuronic acid alternately. It is a valuable raw material for medicine and cosmetics. HA has good hydrophilicity, biocompatibility and moisturizing function. It is widely used in cosmetics and is known as a natural moisturizing factor. HA also has good biocompatibility and does not produce rejection in the body. Due to HA's unique rheological properties, lubricity and biocompatibility and other physical and chemical properties, HA can be used as an elastic material in eye surgery, treatment of bone and joint diseases, and has the effect of promoting wound healing and certain anticancer effects. HA also plays an important role in drug sustained release, postoperative anti-adhesion and tissue engineering.

However, defects such as poor mechanical properties and rapid degradation by hyaluronidase in vivo limit the clinical application of hyaluronic acid. In order to improve the physical and chemical stability of HA while maintaining its natural biocompatibility, biodegradability and non-rejection characteristics in vivo, people have used various methods to modify its hydroxyl and carboxyl groups. Chemical modification methods include cross-linking, grafting, esterification and composite modification. At present, because compound modification can often maintain the original characteristics of the two raw materials, it has attracted people's attention. For composite modification, many macromolecular substances form cross-links with carboxyl groups of HA through carboxyl or hydroxyl groups. Substances successfully compounded and cross-linked include sodium carboxymethyl cellulose, collagen, gelatin, polylactic acid, chitosan, etc. Sodium alginate (SAL for short) is an acidic mucopolysaccharide composed of α-L-guluronic acid and β-D-mannuronic acid connected by 1,4-glycosidic bonds, and is widely used in Food processing and pharmaceutical fields, including drug slow-release carriers, cell culture, etc. SAL has good biocompatibility and, once gelled, has good mechanical properties. Compounds of SAL and other polysaccharides, such as chitosan, chondroitin sulfate, etc., have been reported, and some people have conducted rheological investigations on the compound of SAL-HA, but this compound is only two kinds of polymers. mixture of substances.

Contents of the invention

The object of the present invention is to provide a hyaluronic acid-sodium alginate composite hydrogel and a preparation method thereof.

Hyaluronic acid (HA) and sodium alginate (SAL) composite hydrogel provided by the present invention are prepared according to the method comprising the following steps:

1) Dissolving hyaluronic acid and sodium alginate in water to obtain solution 1,

2) adding a cross-linking agent into the solution 1, and adjusting the pH value to 4.0-5.5 with a buffer solution to obtain a solution 2;

3) Add a carboxyl activator to the solution 2 and stir. During the stirring process, adjust the pH value of the solution 2 to 2.0-5.5 with a buffer solution until a gel is formed, that is, to obtain a composite hydrogel of hyaluronic acid and sodium alginate glue.

In the above solution 1, the mass ratio of hyaluronic acid and sodium alginate can be 1:4-4:1, preferably 1:2. In solution 1, the sum of the concentrations of hyaluronic acid and sodium alginate may be 0.5-1.5g/100ml, preferably 1.0g/100ml.

The cross-linking agent described in the above step 2) can be selected from at least one of the following: adipate dihydrazide (ADH), polyethylene glycol (PEG), glutaraldehyde and divinyl sulfone (GTA), preferably It is adipic acid dihydrazide (ADH); the ratio of the added amount of the crosslinking agent to the solution 1 can be 0.1g-1.0g:100mL, preferably 0.52g:100mL.

The carboxyl activator in step 3) can be selected from at least one of the following: carbodiimide (EDC), N-hydroxysuccinimide (NHS) and N, N, N ", N "-tetramethyl Ethylenediamine (TEMED), preferably carbodiimide; the ratio of the added amount of the carboimide to the solution 2 may be 10mmol-100mmol:1L, preferably 50mmol:1L.

The stirring in step 3) can be carried out at 15°C-40°C, preferably 20°C-30°C; during the stirring process, a buffer solution is required to control the pH value of solution 2 to 2.0-5.5, preferably the pH value is 4.75. When stirring, the stirring speed should not be too fast, so as not to cause the gel to become thinner.

Above-mentioned step 2) and the buffer solution used in step 3) are identical, and described buffer solution can be selected from following any one: phosphate buffer solution, citrate buffer solution and acetate buffer solution; The concentration can be 0.01-0.5 mol/L, the pH value is 2.0-4.5, preferably acetate buffer solution, the most preferred concentration is 0.1 mol/L, the pH value is 3.6 acetate buffer solution.

The method of the present invention also includes the step of washing the hyaluronic acid and sodium alginate composite hydrogel obtained in step 3). The number of washings should not be less than 5 times, and the gel should be turned over slowly during washing, so that the residual reaction impurities can be dissolved in water and removed.

The molecular weight of the hyaluronic acid used in the present invention can be 5.0×10 ⁵ Da-2.1×10 ⁶ Da, preferably 1.5×10 ⁶ Da; the particle size of the sodium alginate powder used is 50 mesh-300 mesh, preferably 200 mesh head.

In the present invention, in the presence of a carboxyl activator, the polymer or oligomer (cross-linking agent) with two or more amino groups in the molecular structure undergoes amidation reaction cross-linking with hyaluronic acid and sodium alginate. A hyaluronic acid-sodium alginate composite cross-linked product with good hardness and viscoelasticity is obtained. The obtained composite cross-linked hydrogel has the dual advantages of HA and SAL. On the one hand, the ability to resist enzymatic degradation is improved, and on the other hand, the mechanical properties and textural characteristics of the hydrogel are improved. After the physical and chemical properties of the composite cross-linked hydrogel are improved, it has the potential use as a dermal filler and tissue scaffold material in the fields of surgery and tissue engineering.

Description of drawings

Fig. 1 is a process flow chart of the present invention for preparing hyaluronic acid-sodium alginate composite hydrogel.

Fig. 2 is the infrared spectrogram of the hyaluronic acid-sodium alginate composite hydrogel prepared in Example 1.

Detailed ways

The method of the present invention will be described below through specific examples, but the present invention is not limited thereto.

The experimental methods described in the following examples, unless otherwise specified, are conventional methods; the reagents and materials, unless otherwise specified, can be obtained from commercial sources.

Example 1. Preparation of hyaluronic acid-sodium alginate hydrogel and verification of its physical and chemical properties

Prepare hyaluronic acid-sodium alginate hydrogel according to the process flow diagram shown in Figure 1, and the specific steps are as follows:

①Polysaccharide dissolution: hyaluronic acid (Shandong Freda Biochemical Co., Ltd.) with a molecular weight of 1.5×10 ⁶ Da and sodium alginate (a particle size of 200 mesh sieve, 10 mg/ml aqueous solution, the viscosity at 25°C is 205mPa·s, Qingdao Jingyan Biotechnology Co., Ltd.) was dissolved in water at a ratio (mass ratio) of 1:2, so that its total concentration was 1.0% (g/100ml), and solution 1 was obtained.

②Add a cross-linking agent and adjust the pH of the solution: After the polysaccharide in step ① is completely dissolved, add the cross-linking agent ADH (Beijing Jinyuan Chemical Group Co., Ltd.) into the solution 1 at a ratio of 0.52g:100ml, mix uniform. And carry out pH adjustment to above-mentioned mixed solution, adjust pH to 4.75 with the HAc-NaAc buffer solution (pH 3.6) of 0.1mol/L, obtain solution 2.

③Add carboxyl activator: add EDC (Shanghai Covalent Chemical Co., Ltd.) to Solution 2 to make the EDC concentration reach 50mmol/L.

④Stir to react and control pH: Stir the reaction solution in step ③ and control pH, and start stirring at the same time as EDC is added. The stirring speed should not be too fast to avoid shear thinning of the formed gel. Stirring at room temperature for 4 hours, during the reaction, the pH value of the reaction solution was kept at 4.75 by dropwise adding 0.1 mol/L HAc-NaAc buffer solution to obtain a composite hydrogel of hyaluronic acid and sodium alginate.

⑤ Gel washing: For the composite hydrogel formed in step ④, wash repeatedly with deionized water. The number of washings should not be less than 5 times, and each time the washing time should be more than 20 minutes. Slowly turn the gel when washing to make the residual reaction impurities Dissolved in water and removed.

⑥ Gel storage: Store the gel after washing in step ⑤ at 4°C.

The hyaluronic acid-sodium alginate composite hydrogel prepared according to the above method is colorless and has certain hardness and viscoelasticity.

The characteristics of the hyaluronic acid-sodium alginate composite hydrogel were measured: 1, using FT-IR spectrometer (Nicolet Nexus, Thermo Electron, USA) to determine whether a covalent bond was formed; 2, using a scanning electron microscope (Hitachi SEM-2500 , Japan) to observe the microstructure of the gel; 3. Use a texture analyzer to measure the texture characteristics of the gel (pre test speed 2.0mm/s, test speed 0.8mm/s, post test speed 2.0mm/s, rupture test distance 4.0%, distance 50%, temperature 25°C.).

The infrared spectrogram is shown in Figure 2. The measurement results show that the gel has formed a new covalent bond, and hyaluronic acid and sodium alginate are cross-linked by adipate dihydrazide (ADH). Scanning electron microscopy showed that a relatively uniform network structure with suitable pore size was formed in the gel. The gel hardness measured by a texture analyzer (Cns-Farnell, Britain) was 357.06g, the fragmentation degree was 253.56g, and the elasticity was 0.961.

Swelling test:

The gel after vacuum freeze-drying was weighed and placed in deionized water for 12 hours. After removing the water on the surface with filter paper, the gel was weighed. The results showed that the gel could absorb water up to 40-50 times its own weight.

Anti-enzyme degradation test:

The gel sample was placed in 10ml of hyaluronidase solution with an enzyme activity of 10U/ml, and 1ml of the supernatant was taken every 3 hours for measurement, and then 1ml of freshly prepared hyaluronidase solution was added to the reaction system. The determination method is as follows: 5mL of concentrated _H2SO4 is placed in a test tube and cooled to 4°C, 1mL of sample solution _or standard solution _is carefully placed on top of the concentrated _H2SO4 layer, shaken gently at first, then vigorously, And make it cool continuously in ice bath, then heat the test tube in boiling distilled water for 10min, then cool to room temperature, add 0.2mL carbazole test solution, shake well again, and heat in boiling water bath for 15min, cool to room temperature , Pour the reaction solution into a 1cm cuvette, and measure the absorbance (A530) at 530nm. The content of hyaluronic acid in the solution was determined by a standard curve. Calculate the degree of degradation of the gel by hyaluronidase according to the following formula:

Wi＝C _i V+∑C _i-1 V _s

Among them, C _i and C _i-1 are the HA concentration (mg/ml) that the gel is decomposed at time i and i-1, V is the total volume of enzyme solution (10ml), and Vs is the sample volume taken out each time ( 1ml).

The results showed that with the prolongation of treatment time, the gel was gradually decomposed by hyaluronidase, and it was almost completely decomposed at 9 hours, while the hyaluronic acid gel in the control group was completely decomposed after 3 hours.

Example 2. Preparation of hyaluronic acid-sodium alginate hydrogel and verification of its physical and chemical properties

Prepare hyaluronic acid-sodium alginate hydrogel according to the process flow diagram shown in Figure 1, and the specific steps are as follows:

①Polysaccharide dissolution: hyaluronic acid (Shandong Freda Biochemical Co., Ltd.) with a molecular weight of 5.0×10 ⁵ Da and sodium alginate (a particle size of 50 mesh sieve, 10 mg/ml aqueous solution, the viscosity at 25 °C is 100mPa·s, Qingdao Jingyan Biotechnology Co., Ltd.) was dissolved in water at a ratio (mass ratio) of 1:4 so that its total concentration was 0.5% (g/100ml) to obtain solution 1.

②Add a cross-linking agent and adjust the pH of the solution: After the polysaccharide in step ① is completely dissolved, add the cross-linking agent ADH (Beijing Jinyuan Chemical Group Co., Ltd.) into the solution 1 at a ratio of 0.1g:100ml, mix uniform. And carry out pH adjustment to above-mentioned mixed solution, adjust pH to 4.75 with the HAc-NaAc buffer solution (pH 3.6) of 0.1mol/L, obtain solution 2.

③Add carboxyl activator: add EDC (Shanghai Covalent Chemical Co., Ltd.) to solution 2 to make the EDC concentration reach 10mmol/L.

④Stir to react and control pH: Stir the reaction solution in step ③ and control pH, and start stirring at the same time as EDC is added. The stirring speed should not be too fast to avoid shear thinning of the formed gel. Stirring at room temperature for 4 hours, during the reaction, the pH value of the reaction solution was kept at 4.75 by dropwise adding 0.1 mol/L HAc-NaAc buffer solution to obtain a composite hydrogel of hyaluronic acid and sodium alginate.

⑤ Gel washing: For the composite hydrogel formed in step ④, wash repeatedly with deionized water. The number of washings should not be less than 5 times, and each time the washing time should be more than 20 minutes. Slowly turn the gel when washing to make the residual reaction impurities Dissolved in water and removed.

⑥ Gel storage: Store the gel after washing in step ⑤ at 4°C.

The hyaluronic acid-sodium alginate composite hydrogel prepared according to the above method is colorless and has certain hardness and viscoelasticity.

Infrared spectroscopy showed that the gel formed new covalent bonds, and hyaluronic acid and sodium alginate were cross-linked by adipate dihydrazide (ADH). Scanning electron microscopy showed that a relatively uniform network structure with suitable pore size was formed in the gel. The gel hardness measured by a texture analyzer (Cns-Farnell, Britain) was 71.02g, the fragmentation degree was 66.89g, and the elasticity was 0.97.

Swelling test:

The gel after vacuum freeze-drying was weighed and placed in deionized water for 12 hours. After removing the water on the surface with filter paper, the gel was weighed. The results showed that the gel could absorb water up to 55 times its own weight.

Anti-enzyme degradation test:

The gel sample was placed in 10 ml of hyaluronidase solution with an enzyme activity of 10 U/ml, and 1 ml of supernatant was taken from it every 3 hours for determination.

The results showed that with the prolongation of treatment time, the gel was gradually decomposed by hyaluronidase, and it was almost completely decomposed at 6 hours, while the hyaluronic acid gel in the control group was completely decomposed after 3 hours.

Example 3. Preparation of hyaluronic acid-sodium alginate hydrogel and verification of its physical and chemical properties

Prepare hyaluronic acid-sodium alginate hydrogel according to the process flow diagram shown in Figure 1, and the specific steps are as follows:

①Polysaccharide dissolution: hyaluronic acid (Shandong Freda Biochemical Co., Ltd.) with a molecular weight of 2.1×10 ⁶ Da and sodium alginate (a particle size of 300 mesh sieve, 10 mg/ml aqueous solution, the viscosity at 25°C is 300mPa·s, Qingdao Jingyan Biotechnology Co., Ltd.) was dissolved in water at a ratio (mass ratio) of 4:1 so that its total concentration was 1.5% (g/100ml) to obtain solution 1.

②Add a cross-linking agent and adjust the pH of the solution: After the polysaccharide in step ① is completely dissolved, add the cross-linking agent ADH (Beijing Jinyuan Chemical Group Co., Ltd.) into the solution 1 at a ratio of 1.0g:100ml, mix uniform. And carry out pH adjustment to above-mentioned mixed solution, adjust pH to 4.75 with the HAc-NaAc buffer solution (pH 3.6) of 0.1mol/L, obtain solution 2.

③Add carboxyl activator: add EDC (Shanghai Covalent Chemical Co., Ltd.) to Solution 2 to make the EDC concentration reach 100mmol/L.

④Stir to react and control pH: Stir the reaction solution in step ③ and control pH, and start stirring at the same time as EDC is added. The stirring speed should not be too fast to avoid shear thinning of the formed gel. Stirring at room temperature for 4 hours, during the reaction, the pH value of the reaction solution was kept at 4.75 by dropwise adding 0.1 mol/L HAc-NaAc buffer solution to obtain a composite hydrogel of hyaluronic acid and sodium alginate.

⑤ Gel washing: For the composite hydrogel formed in step ④, wash repeatedly with deionized water. The number of washings should not be less than 5 times, and each time the washing time should be more than 20 minutes. Slowly turn the gel when washing to make the residual reaction impurities Dissolved in water and removed.

⑥ Gel storage: Store the gel after washing in step ⑤ at 4°C.

The hyaluronic acid-sodium alginate composite hydrogel prepared according to the above method is colorless and has certain hardness and viscoelasticity.

Infrared spectroscopy showed that the gel formed new covalent bonds, and hyaluronic acid and sodium alginate were cross-linked by adipate dihydrazide (ADH). Scanning electron microscopy showed that a relatively uniform network structure with suitable pore size was formed in the gel. The gel hardness measured by a texture analyzer (Cns-Farnell, Britain) was 209.50g, the fragmentation degree was 190.28g, and the elasticity was 1.14.

Swelling test:

The gel after vacuum freeze-drying was weighed and placed in deionized water for 12 hours. After removing the water on the surface with filter paper, the gel was weighed. The results showed that the gel could absorb water up to 20 times its own weight.

Anti-enzyme degradation test:

The gel sample was placed in 10 ml of hyaluronidase solution with an enzyme activity of 10 U/ml, and 1 ml of supernatant was taken from it every 3 hours for determination.

The results showed that with the prolongation of treatment time, the gel was gradually decomposed by hyaluronidase, and it was almost completely decomposed at 8 hours, while the hyaluronic acid gel in the control group was completely decomposed after 3 hours.

Claims (10)

1. the preparation method of mucinase and sodium-alginate composite aquogel comprises the steps:

1) mucinase and sodium-alginate is soluble in water, obtain solution 1,

2) linking agent is added in the said solution 1, and use buffered soln adjusting pH value to be 4.0-5.5, obtain solution 2;

3) in said solution 2, add carboxyl activator and stirring, the pH value of regulating and control said solution 2 with buffered soln in the whipping process until forming gel, promptly obtains mucinase and sodium-alginate composite aquogel at 2.0-5.5.

2. method according to claim 1 is characterized in that: the mass ratio of mucinase and sodium-alginate is 1 in the said solution 1: 4-4: 1; The concentration sum of mucinase and sodium-alginate is 0.5-1.5g/100ml in the said solution 1.

3. method according to claim 1 and 2 is characterized in that: step 2) described in linking agent be selected from following at least a: adipic dihydrazide, polyoxyethylene glycol, LUTARALDEHYDE and divinylsulfone; The proportioning of the add-on of said linking agent and said solution 1 is 0.1g-1.0g: 100mL.

4. method according to claim 1 is characterized in that: the activator of carboxyl described in the step 3) is selected from following at least a: carbodiimide, N-hydroxy-succinamide and N, N, N ", a N " Tetramethyl Ethylene Diamine; The proportioning of the addition of said carbodiimide and said solution 2 is 10mmol-100mmol: 1L.

5. method according to claim 1 is characterized in that: be stirred in described in the step 3) under 15 ℃ of-40 ℃ of conditions and carry out; The pH value of regulation and control solution 2 is 4.75 in the said whipping process.

6. method according to claim 1; It is characterized in that: step 2) identical with buffered soln described in the step 3); Be selected from following any one: phosphate buffer soln; Citrate buffer solution and acetate buffer solution, the concentration of said buffered soln are 0.01-0.5mol/L, and the pH value is 2.0-4.5.

7. method according to claim 6 is characterized in that: step 2) and step 3) described in buffered soln be that concentration is that 0.1mol/L, pH value are 3.6 acetate buffer.

8. method according to claim 1 is characterized in that: said hyaluronic molecular weight is 5.0 * 10 ⁵-2.1 * 10 ⁶Da; The granularity of said sodium-alginate is 50 orders-300 orders.

9. method according to claim 1 is characterized in that: said method also comprises the step that mucinase that step 3) is obtained and sodium-alginate composite aquogel water wash.

10. arbitrary said method prepares among the claim 1-9 mucinase and sodium-alginate composite aquogel.

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