阅读说明：本技术 一种壳聚糖/海藻酸钠双网络水凝胶的制备方法 (Preparation method of chitosan/sodium alginate double-network hydrogel ) 是由 毛宏理 顾忠伟 何雨芯 于 2020-07-23 设计创作，主要内容包括：本发明公开了一种壳聚糖/海藻酸钠双网络水凝胶的制备方法,将由改性壳聚糖、改性海藻酸钠、光引发剂和水组成的预聚液通过席夫碱反应,得到单网络水凝胶；再将单网络水凝胶于紫外光照射下反应,得到二次光交联的壳聚糖/海藻酸钠双网络水凝胶；其中,所述的改性壳聚糖为接枝胶原蛋白肽的羧甲基壳聚糖；所述的改性海藻酸钠为甲基丙烯酸改性的醛基化海藻酸钠。本发明所述的胶原蛋白肽具有非免疫原性,在不使用有毒交联剂的情况下,可通过酶促反应接枝在壳聚糖上,提高材料的细胞相容性和促细胞增殖能力。(The invention discloses a preparation method of chitosan/sodium alginate double-network hydrogel, which comprises the steps of carrying out Schiff base reaction on prepolymerization liquid consisting of modified chitosan, modified sodium alginate, a photoinitiator and water to obtain single-network hydrogel; then reacting the single-network hydrogel under ultraviolet irradiation to obtain secondary photo-crosslinked chitosan/sodium alginate double-network hydrogel; wherein, the modified chitosan is carboxymethyl chitosan grafted with collagen peptide; the modified sodium alginate is methacrylic acid modified aldehyde sodium alginate. The collagen peptide has non-immunogenicity, and can be grafted on chitosan through enzymatic reaction under the condition of not using toxic cross-linking agents, so that the cell compatibility and the cell proliferation promoting capacity of the material are improved.)

1. A preparation method of chitosan/sodium alginate double-network hydrogel is characterized in that a prepolymerization solution consisting of modified chitosan, modified sodium alginate, a photoinitiator and water is reacted to obtain single-network hydrogel; then reacting the single-network hydrogel under ultraviolet irradiation to obtain secondary photo-crosslinked chitosan/sodium alginate double-network hydrogel; wherein, the modified chitosan is carboxymethyl chitosan grafted with collagen peptide; the modified sodium alginate is methacrylic acid modified aldehyde sodium alginate.

2. The preparation method of claim 1, wherein the modified chitosan is prepared by reacting chitosan with chloroacetic acid under alkaline conditions to obtain carboxymethyl chitosan; and then catalyzing the collagen peptide to graft on the carboxymethyl chitosan through transglutaminase so as to obtain the carboxymethyl chitosan grafted with the collagen peptide.

3. The method according to claim 2, wherein the molar ratio of chitosan to chloroacetic acid is 1: 2-4; the mass ratio of the carboxymethyl chitosan to the transglutaminase to the collagen peptide is 1: 0.1: 0.5-1.5.

4. The preparation method of claim 2, wherein the modified chitosan is prepared by dissolving chitosan in 50 wt% aqueous NaOH solution, the concentration of chitosan is 0.1g/mL, freezing for 24h, thawing in isopropanol and dispersing, then adding chloroacetic acid to the dispersed material, stirring for 5-12h at room temperature, dialyzing the stirred material for three days, and freeze-drying to obtain carboxymethyl chitosan; respectively dissolving carboxymethyl chitosan, transglutaminase and collagen peptide in PBS buffer solution, reacting at 30-60 deg.C for 4-12h, keeping in boiling water for 10min, cooling, filtering, dialyzing the filtrate for four days, and freeze-drying to obtain carboxymethyl chitosan grafted with collagen peptide; wherein the concentration of the collagen peptide is 1g/30mLPBS buffer solution.

5. The preparation method of claim 1, wherein the modified sodium alginate is prepared by reacting sodium alginate with sodium periodate to obtain aldehyde sodium alginate, and then methylacrylating the aldehyde sodium alginate with 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide.

6. The method according to claim 5, wherein the molar ratio of sodium alginate to sodium periodate is 1: 0.4 to 1; the mol ratio of sodium alginate, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is 2: 1: 2.

7. the preparation method of claim 5, wherein the modified sodium alginate is prepared by adding sodium periodate into 0.02g/mL sodium alginate aqueous solution, keeping away from light, reacting at room temperature for 3-12h, adding ethylene glycol to terminate the reaction, stirring for 15-30min to obtain a first reaction solution, wherein the molar ratio of sodium periodate to ethylene glycol is 1: 1; dialyzing the first reaction solution for three days, and freeze-drying to obtain aldehyde sodium alginate; dissolving aldehyde sodium alginate in MES buffer solution, wherein the concentration of the aldehyde sodium alginate is 0.02g/mL, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, and reacting at room temperature for 30-45min to obtain a second reaction solution; adding diethylaminoethyl methacrylate into the second reaction solution, wherein the molar ratio of sodium alginate to diethylaminoethyl methacrylate is 1:0.3-0.7, and reacting at room temperature in the dark for 24-48h to obtain a third reaction solution; dialyzing the third reaction solution for three days, and freeze-drying to obtain the final product.

8. The preparation method of claim 1, wherein in the pre-polymerization solution, the mass percentage concentration of the modified chitosan is 5-10%, the mass percentage concentration of the modified sodium alginate is 15-25%, and the mass percentage concentration of the photoinitiator is 0.05-0.2%.

9. The method according to claim 1, wherein the pre-polymerization solution is reacted at 25 ℃ for 1 to 5 min.

10. The production method according to claim 1, wherein the single-network hydrogel is produced at a wavelength of 365nm and a power of 1mW/cm²The reaction is carried out for 15-20min under the irradiation of ultraviolet light.

Technical Field

The invention belongs to the technical field of medical biomaterials, and particularly relates to a preparation method of chitosan/sodium alginate double-network hydrogel.

Background

The skin is on the surface of the body and is directly contacted with the external environment, has the functions of protecting, excreting, regulating body temperature, sensing external stimulation and the like, and is the largest organ of the human body. However, the skin is exposed to a large area, and is very susceptible to various injuries in daily life, such as mechanical wounds, burns, chronic ulcers and the like. Skin wound healing is a major clinical problem, and wound infection caused by bacteria and the like during wound recovery is the most common complication, and death can be caused in serious cases. The hydrogel can absorb a large amount of exudates due to the natural porous structure and high moisture content, can serve as a barrier for blocking bacteria, can maintain a moist environment at a skin defect part, keeps good oxygen and water permeability, and realizes the slow release effect of the drug, thereby drawing the attention of Chinese and foreign students.

Chitosan, also known as chitosan, is obtained by deacetylating chitin, which is widely present in nature. Since 1859, after the first obtained chitosan by rubet, this natural polymer has attracted much attention from various industries due to its excellent biocompatibility, hemocompatibility, antibacterial properties, biodegradability, etc. But the application of the chitosan is limited due to poor water solubility and mechanical property, so that the modification and modification of the chitosan are very important. Typical modification methods include chemical modification of the primary structure of chitosan, which may alter its initial properties, particularly the reactions in which its amino groups participate. In addition, most chitosan modifications require the use of cross-linking agents, such as glutaraldehyde, which are generally toxic and have a significant negative impact on subsequent applications.

Disclosure of Invention

The purpose of the invention is as follows: the invention aims to solve the technical problem of providing a preparation method of chitosan/sodium alginate double-network hydrogel aiming at the defects of the prior art.

In order to solve the technical problems, the invention discloses a preparation method of chitosan/sodium alginate double-network hydrogel, which is prepared by performing Schiff base reaction and secondary photocrosslinking reaction on carboxymethyl chitosan grafted with collagen peptide and methacrylated sodium alginate modified by methacrylic acid.

Specifically, a prepolymerization solution consisting of modified chitosan, modified sodium alginate, a photoinitiator and water is subjected to Schiff base reaction to obtain single-network hydrogel; then reacting the single-network hydrogel under ultraviolet irradiation to obtain secondary photo-crosslinked chitosan/sodium alginate double-network hydrogel; wherein, the modified chitosan is carboxymethyl chitosan grafted with collagen peptide; the modified sodium alginate is methacrylic acid modified aldehyde sodium alginate.

The preparation method of the modified chitosan comprises the following steps of reacting chitosan with chloroacetic acid under an alkaline condition to obtain carboxymethyl chitosan; and then catalyzing the collagen peptide to graft on the carboxymethyl chitosan through transglutaminase so as to obtain the carboxymethyl chitosan grafted with the collagen peptide.

Wherein the molar ratio of the chitosan to the chloroacetic acid is 1: 2-4, preferably 1: 3; the mass ratio of the carboxymethyl chitosan to the transglutaminase to the collagen peptide is 1: 0.1: 0.5-1.5, preferably 1: 0.1: 1.

further preferably, the preparation method of the modified chitosan comprises the steps of dissolving chitosan in 50 wt% of NaOH aqueous solution, freezing for 24 hours, unfreezing and dispersing in isopropanol, adding chloroacetic acid into the dispersed material, stirring at room temperature for 5-12 hours, preferably 5 hours, dialyzing the stirred material for three days, and freeze-drying to obtain carboxymethyl chitosan; respectively dissolving carboxymethyl chitosan, transglutaminase and collagen peptide in PBS buffer solution, reacting at 30-60 deg.C for 4-12h, preferably at 40 deg.C for 4h, keeping in boiling water for 10min, cooling, filtering, dialyzing the filtrate for four days, and freeze-drying to obtain collagen peptide grafted carboxymethyl chitosan.

Wherein the concentration of the chitosan in 50 wt% NaOH aqueous solution is 0.1 g/mL; the isopropanol is used as a dispersing solvent, does not need a certain dosage, and is preferably 150-200mL/5g chitosan; wherein the PBS buffer solution is 0.2mol/L PBS buffer solution with the pH value of 6; wherein the concentration of the collagen peptide is 1g/30mL of PBS buffer solution.

The preparation method of the modified sodium alginate comprises the steps of reacting sodium alginate with sodium periodate to obtain aldehyde sodium alginate, and then acidifying the aldehyde sodium alginate by 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to obtain the modified sodium alginate.

Wherein the molar ratio of sodium alginate to sodium periodate is 1: 0.4 to 1, preferably 1: 0.6; the molar ratio of sodium alginate to EDC to NHS is 2: 1: 2.

further preferably, the preparation method of the modified sodium alginate comprises the steps of adding sodium periodate into 0.02g/mL sodium alginate aqueous solution, keeping out of the sun, reacting at room temperature for 3-12h, preferably 4h, adding ethylene glycol to terminate the reaction, stirring for 15-30min to obtain a first reaction solution, preferably 30min, wherein the molar ratio of the sodium periodate to the ethylene glycol is 1: 1; dialyzing the first reaction solution for three days, and then freeze-drying to obtain aldehyde sodium alginate (OSA); dissolving aldehydized sodium alginate in MES buffer solution with the concentration of 0.02g/mL, adding EDC and NHS, and reacting at room temperature for 30-45min to obtain a second reaction solution; adding diethylaminoethyl methacrylate (AEMA) into the second reaction solution, wherein the molar ratio of sodium alginate to AEMA is 1:0.3-0.7, preferably 1:0.5, and reacting at room temperature in a dark place for 24-48h, preferably 24h to obtain a third reaction solution; dialyzing the third reaction solution for three days, and freeze-drying to obtain the final product.

Wherein the MES buffer solution is pH 5.5-6, 50mM/L MES, 0.5M NaCl.

In the preparation method of the chitosan/sodium alginate double-network hydrogel, in the pre-polymerization solution, the mass percentage concentration of the modified chitosan is 5-10%, the mass percentage concentration of the modified sodium alginate is 15-25%, and the mass percentage concentration of the photoinitiator is 0.05-0.2%; preferably, the mass percentage concentration of the modified chitosan is 7.5%, the mass percentage concentration of the modified sodium alginate is 20%, and the mass percentage concentration of the photoinitiator is 0.05%.

Preferably, the photoinitiator is I2959.

Wherein the pre-polymerization solution is reacted at 25 ℃ for 1-5min, preferably 3 min.

Wherein, the single-network hydrogel is prepared at the wavelength of 365nm and the power of 1mW/cm²Under the irradiation of ultraviolet light, the reaction is carried out for 15-20min, preferably for 15 min.

Has the advantages that: compared with the prior art, the invention has the following advantages:

1. the invention selects chitosan and sodium alginate as hydrogel raw materials, the two raw materials can generate crosslinking effect through simple modification, the preparation is simple, and the raw materials are commercialized. Therefore, the two raw materials are selected to have very important values for establishing and popularizing the hydrogel method and promoting the application of the hydrogel method in tissue engineering and regenerative medicine.

2. The traditional chitosan modification needs cross-linking agents such as glutaraldehyde and the like, and has high cytotoxicity, while the collagen peptide has nonimmunogenicity and is beneficial to fibroblast proliferation, and is grafted on the chitosan through enzymatic reaction, so that the cell compatibility is improved, the cell proliferation is facilitated, and meanwhile, toxic cross-linking agents cannot be introduced. Therefore, the double-network hydrogel prepared by the invention has low cytotoxicity.

3. Compared with other crosslinking modes, the Schiff base reaction is simple to operate, green and non-toxic, and the method has the advantages that the Schiff base reaction is carried out on the aldehyde sodium alginate and the chitosan, so that no by-product is generated, and no crosslinking agent is required to be added; and after methacrylic acid is modified, double-network hydrogel can be prepared by secondary photocrosslinking, so that the mechanical property of the double-network hydrogel is improved.

Drawings

The foregoing and/or other advantages of the invention will become further apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings.

FIG. 1 shows modified chitosan and modified sodium alginate¹H NMR spectrum.

Figure 2 is a compressive stress-strain curve for a single network hydrogel (lower) and a double network hydrogel (upper).

FIG. 3 is SEM photographs of a single network hydrogel (left) and a double network hydrogel (right).

FIG. 4 shows the relative cell viability (4A) and live-dead staining (4B) of L929 cells cultured with hydrogel extracts; in 4A, each group was, from left to right, control (control), OSA/CMC, OMSA/CMC and OMSA/CMC-COP.

Detailed Description

The chitosan used in the examples below was S11064 chitosan from a leafy organism of shanghai origin, having a number average molecular weight of about 20w and a monomer molecular weight of about 161.2; the sodium alginate is S11053 sodium alginate of Shanghai leaf organisms, the number average molecular weight of the sodium alginate is 24w, and the molecular weight of a monomer is about 216.1.