A glucomannan hydrogel, its preparation method and application

Abstract

The present invention discloses a glucomannan hydrogel, a preparation method thereof and an application thereof. The glucomannan hydrogel is obtained by adding glycidyl methacrylate to a glucomannan solution for grafting reaction under the action of a catalyst to obtain methacryloylated glucomannan; the methacryloylated glucomannan solution is subjected to a crosslinking reaction under the action of a photoinitiator and ultraviolet light irradiation to obtain the hydrogel. The method is simple and has high efficiency. The hydrogel has good biocompatibility. The glucomannan therein belongs to polysaccharide, can mimic the extracellular matrix, promote cell growth, accelerate blood vessel formation at the wound site and wound healing. The hydrogel can be used as a wound healing promoter, directly used for in-situ injection at the wound site, filling the skin defect and bone defect sites, which is beneficial to wound closure and reduces the risk of infection.

Description

Technical Field

[0001] The present invention relates to a preparation method of a hydrogel, specifically a glucomannan hydrogel, and also relates to its preparation method and application, belonging to the fields of biomaterials and biomedical engineering. Background Art

[0002] Delayed wound healing and non-healing are major challenges in clinical practice. In clinical work, a large number of trauma patients face slow healing of the trauma site, which increases the risk of infection and poses a severe challenge to medical staff. According to statistics, the annual treatment demand for wound repair and regeneration in China is about 100 million person-times, and among them, the number of patients with difficult wound treatment reaches 30 million person-times. Now China is gradually entering an aging society, and the incidence of chronic wounds has increased sharply. In particular, the difficult-to-heal wounds caused by burns, traumas, neurotrophic ulcers, severe postoperative infections, diabetic foot, pressure sores, fistulas, etc. are increasing year by year. The proportion of such patients among inpatients is 1.7‰; the high-incidence age groups are 40 - 60 years old and 60 - 80 years old (31% and 38% respectively), caused by diabetes (32.6%); caused by trauma (23.8%); pressure sores (10.5%); the repair rate at the time of patient discharge is only 53%, and the recurrence rate of ulcers is as high as 70%. Currently, the wound dressings used clinically have a single function and a high cost, and cannot achieve a multi-functional treatment effect well. Most of the currently studied wound dressings need to additionally add drugs such as growth factors, with good effects, but high costs and uncontrollable release processes. Therefore, wound dressings with a single function can no longer meet the current needs of wound repair, and there is an urgent need to develop a multi-functional material to cope with the complex current situation of wound repair. Summary of the Invention

[0003] Aiming at the deficiencies of the existing technology, such as single function, complex synthesis method, and the need to provide various drugs from the outside, the first object of the present invention is to provide a glucomannan hydrogel. This hydrogel has good biocompatibility. The polysaccharide hydrogel can mimic the components of the extracellular matrix, promote cell growth and proliferation, and promote wound healing, effectively solving the problems of delayed wound healing, slow blood vessel formation, and easy infection.

[0004] The second object of the present invention is to provide a preparation method of a glucomannan hydrogel. This method is simple and easy to operate, has a low cost, and a high production efficiency, and is suitable for large-scale production.

[0005] The third object of the present invention is to provide an application of a glucomannan hydrogel, which can be used as a wound healing promoter. This promoter can be directly used for in-situ injection at the trauma site, filling the skin defect and bone defect sites, facilitating wound closure, and reducing the risk of infection.

[0006] To achieve the above technical objectives, the present invention provides a method for preparing a glucomannan hydrogel, which involves adding glycidyl methacrylate to a glucomannan solution and conducting a grafting reaction under the action of a catalyst to obtain methacrylated glucomannan; and then conducting a crosslinking reaction on the methacrylated glucomannan solution under the irradiation of a photoinitiator and ultraviolet light, thus obtaining the product.

[0007] The present invention uses glucomannan as a raw material, which has good biocompatibility and is easy to carry out graft modification of chemical groups. The hydrogel is prepared by the method of ultraviolet light crosslinking, with a fast gelation speed and simple operation.

[0008] As a preferred embodiment, the mass-volume ratio of glucomannan to glycidyl methacrylate in the glucomannan solution is 5 g: 3 mL - 5 mL.

[0009] During this grafting reaction process, if the dosage of glycidyl methacrylate (GMA) is too small, it will lead to insufficient grafting rate and poor photocuring effect, and the gel cannot be formed smoothly; on the contrary, if its dosage is too large, it will lead to a long subsequent dialysis time and waste of raw materials. Therefore, the dosage of glycidyl methacrylate needs to be controlled within a reasonable range.

[0010] As a preferred embodiment, the addition method of glycidyl methacrylate is: dropping it into the glucomannan solution at a rate of 0.4 - 0.6 mL / min. The process needs to control the slow addition of glycidyl methacrylate. Otherwise, too fast addition rate will lead to a low grafting rate of the final product.

[0011] As a preferred embodiment, the concentration of the glucomannan solution is 0.05 - 0.15 g / mL. The solvent in the glucomannan solution is dimethyl sulfoxide. Controlling the concentration of the glucomannan solution within a suitable range is beneficial to the grafting reaction. If its concentration is too high, glucomannan cannot be completely dissolved in dimethyl sulfoxide, and if the concentration is too low, it will lead to waste of the solvent and increase the preparation cost.

[0012] As a preferred embodiment, the catalyst is 4-dimethylaminopyridine.

[0013] As a preferred embodiment, the mass ratio of the catalyst to glucomannan in the glucomannan solution is 1 - 2: 5, and further preferably 1: 5.

[0014] As a preferred embodiment, the grafting reaction conditions are: reacting in the dark for 48 - 72 h. During this grafting reaction process, the reaction time needs to be controlled within a suitable range. If the reaction time is too short, it will lead to insufficient grafting rate and the gel cannot be formed smoothly, while if the time is too long, the preparation efficiency will be reduced.

[0015] As a preferred embodiment, the concentration of the methacrylated glucomannan solution in the crosslinking reaction is 0.01 - 0.03 g / mL. Controlling the concentration of the methacrylated glucomannan solution within a reasonable range is beneficial to the synthesis of the gel. If the concentration is too high, it will increase the dissolution difficulty and affect the use after gel formation, while if the concentration is too low, the gel cannot be formed.

[0016] As a preferred embodiment, the concentration of the photoinitiator in the methacrylated glucomannan solution is 0.002 - 0.01 g / mL. The photoinitiator is preferably lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP). Controlling the amount of the photoinitiator within a suitable range is beneficial to improving the crosslinking reaction efficiency. If the amount is too low, the gel cannot be formed, while if the amount is too large, it will cause waste of raw materials and increase the preparation cost.

[0017] As a preferred embodiment, the crosslinking reaction conditions are: ultraviolet light irradiation for 10 - 20 s.

[0018] The present invention also provides a glucomannan hydrogel, which is prepared by the above method. This hydrogel has good biocompatibility. The polysaccharide hydrogel can mimic the components of the extracellular matrix, promote cell growth and proliferation, play a role in promoting wound healing, accelerate blood vessel formation, and shorten the healing time.

[0019] The present invention also provides an application of the glucomannan hydrogel as a wound healing promoter. This hydrogel can be directly used for in-situ injection at the wound site to fill the skin defect and bone defect sites, which is beneficial to wound closure, reduces the risk of infection, and can be widely applied in the fields of wound repair, bone defect and other wound repair fields.

[0020] Compared with the prior art, the advantages of the present invention are as follows:

[0021] (1) Using glucomannan as the raw material improves the biocompatibility of the gel, is easy to carry out graft modification of chemical groups, and the hydrogel formed after crosslinking is beneficial to cell adhesion and proliferation.

[0022] (2) Using the ultraviolet light crosslinking method to prepare the hydrogel has a fast gelation speed, simple operation, high preparation efficiency, low cost, and is suitable for large-scale production.

[0023] (3) The present invention can be directly used for in-situ injection at the wound site to fill the skin defect and bone defect sites, which is beneficial to wound closure, reduces the risk of infection. The polysaccharide hydrogel can mimic the components of the extracellular matrix, promote cell growth and proliferation, play a role in promoting wound healing, accelerate blood vessel formation, and shorten the healing time. Description of the Drawings

[0024] Figure 1Appearance diagram of the glucomannan hydrogel prepared in Example 1.

[0025] Figure 2 1H NMR comparison diagram of methacrylated glucomannan (KGMMA) and glucomannan (KGM).

[0026] Figure 3 Effect diagram of the glucomannan hydrogel prepared in Example 1 promoting the proliferation of human umbilical vein endothelial cells (HUVEC).

[0027] Figure 4 Effect diagram of the glucomannan hydrogel prepared in Example 1 promoting the expression of CD31 in human umbilical vein endothelial cells. Among them, (a) is the immunofluorescence staining diagram of CD31 expression in HUVEC cultured in the KGMMA group and the blank group, and (b) is the relative quantification diagram of CD31 protein expression in HUVEC. Detailed implementation manners

[0028] To facilitate the understanding of the present invention, the present invention will be described more comprehensively and meticulously below in conjunction with the specification drawings and preferred embodiments. However, the protection scope of the present invention is not limited to the following specific embodiments.

[0029] Unless otherwise defined, all professional terms used hereinafter have the same meaning as commonly understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the protection scope of the present invention.

[0030] Unless otherwise specifically stated, various raw materials, reagents, instruments, and equipment used in the present invention can be obtained through market purchase or can be prepared by existing methods.

[0031] Example 1

[0032] The following provides a preparation method of a glucomannan hydrogel, including the following preparation steps:

[0033] (1) Weigh 5 g of glucomannan and add it to 50 mL of dimethyl sulfoxide. Stir it under nitrogen protection until it is fully dissolved to prepare a 10% w / v glucomannan solution. Add 1 g of 4-dimethylaminopyridine (DMAP) to it and stir for 1 h to dissolve. Drop 3 mL of glycidyl methacrylate (GMA) into the solution at a rate of 0.5 mL / min and continue the reaction for 48 h. The whole reaction is carried out at room temperature in the dark. Drop the reacted solution into an ethanol solution and centrifuge it using a centrifuge at a speed of 2000 r / min for 5 min. Discard the supernatant, collect the solid, dissolve the solid with 15 mL of deionized water, place the obtained solution in a dialysis bag with a molecular weight cut-off of 3500 Da, and dialyze it in deionized water for 7 days, then carry out freeze-drying to obtain a white solid, namely methacrylated glucomannan (KGMMA).

[0034] (2) Weigh 0.2 g of KGMMA and dissolve it in 10 mL of deionized water to prepare a 2% w / v KGMMA solution. Add 0.1 g of photoinitiator LAP to the above solution. After KGMMA is completely dissolved, pour the solution into a mold and irradiate it with ultraviolet light for 10 s for crosslinking to obtain a glucomannan hydrogel.

[0035] Example 2

[0036] The following is another preparation method of glucomannan hydrogel, including the following preparation steps:

[0037] (1) Weigh 5 g of glucomannan and add it to 50 mL of dimethyl sulfoxide. Stir it under nitrogen protection until it is fully dissolved to prepare a 10% w / v glucomannan solution. Add 1 g of 4-dimethylaminopyridine (DMAP) to it and stir for 1 h to dissolve. Drop 4 mL of glycidyl methacrylate (GMA) into the solution at a rate of 0.5 mL / min and continue the reaction for 48 h. The whole reaction is carried out at room temperature in the dark. Drop the reacted solution into an ethanol solution and centrifuge it using a centrifuge at a speed of 2000 r / min for 5 min. Discard the supernatant, collect the solid, dissolve the solid with 15 mL of deionized water, place the obtained solution in a dialysis bag with a molecular weight cut-off of 3500 Da, and dialyze it in deionized water for 7 days, then carry out freeze-drying to obtain a white solid, namely methacrylated glucomannan (KGMMA).

[0038] (2) Weigh 0.2 g of KGMMA and dissolve it in 10 mL of deionized water to prepare a 2% w / v KGMMA solution. Add 0.1 g of photoinitiator LAP to the above solution. After KGMMA is completely dissolved, pour the solution into a mold and irradiate it with ultraviolet light for 10 s for crosslinking to obtain a glucomannan hydrogel.

[0039] Example 3

[0040] The following is the third preparation method of glucomannan hydrogel, including the following preparation steps:

[0041] (1) Weigh 5 g of glucomannan and add it to 50 mL of dimethyl sulfoxide. Stir it under nitrogen protection until it is fully dissolved to prepare a 10% w / v glucomannan solution. Add 1 g of 4-dimethylaminopyridine (DMAP) to it and stir to dissolve for 1 h. Drop 5 mL of glycidyl methacrylate (GMA) into the solution at a rate of 0.5 mL / min, and continue to react for 48 h. The whole reaction is carried out at room temperature in the dark. Drop the reacted solution into an ethanol solution, and centrifuge it using a centrifuge at a speed of 2000 r / min for 5 min. Discard the supernatant, collect the solid, dissolve the solid with 15 mL of deionized water, place the obtained solution in a dialysis bag with a molecular weight cut-off of 3500 Da, dialyze it in deionized water for 7 days, and then perform freeze-drying to obtain a white solid, namely methacrylated glucomannan (KGMMA).

[0042] (2) Weigh 0.2 g of KGMMA and dissolve it in 10 mL of deionized water to prepare a 2% w / v KGMMA solution. Add 0.1 g of photoinitiator LAP to the above solution. After KGMMA is completely dissolved, pour the solution into a mold and irradiate it with ultraviolet light for 10 s for crosslinking to obtain glucomannan hydrogel.

[0043] As Figure 1 shown, the prepared glucomannan hydrogel has a yellow gel-like appearance, and the nuclear magnetic resonance hydrogen spectrum of KGMMA is as Figure 2 shown. The proliferation of human umbilical vein endothelial cells (HUVEC) and the expression level of CD31 in human umbilical vein endothelial cells of the glucomannan hydrogel were tested using a CCK-8 kit and cell immunofluorescence staining. Each condition was performed with at least three independent experiments and repeated three times. Statistical tests were performed using the SPSS software package (version 12.0; using SPSS Inc.). A p value < 0.05 was considered to indicate a significant difference.

[0044] As Figure 3 shown, the absorbance of HUVEC detected by the CCK-8 kit after culturing for 3 days and 7 days in the KGMMA group was significantly better than that in the blank group. This result indicates that the glucomannan hydrogel can promote the proliferation of HUVEC in vitro.

[0045] As Figure 4 shown, the expression of CD31 (green fluorescence) protein in HUVEC in the KGMMA group was better than that in HUVEC in the blank group, indicating that the glucomannan hydrogel can promote the expression of CD31 in HUVEC and can promote its differentiation into blood vessels in vitro.

Claims

1. A method for preparing glucomannan hydrogel, characterized in that: Glycidyl methacrylate was added to the glucomannan solution and grafting reaction was carried out under the action of a catalyst to obtain methacryloylated glucomannan; the methacryloylated glucomannan solution was subjected to a crosslinking reaction under the action of a photoinitiator and ultraviolet light irradiation, thus obtaining the product; In the glucomannan solution, the mass-volume ratio of glucomannan to glycidyl methacrylate is 5 g: 3 mL - 5 mL; The concentration of the glucomannan solution is 0.05 - 0.15 g / mL; The catalyst is 4-dimethylaminopyridine; In the crosslinking reaction, the concentration of the methacryloylated glucomannan solution is 0.01 - 0.03 g / mL; The concentration of the photoinitiator in the methacryloylated glucomannan solution is 0.002 - 0.01 g / mL; The crosslinking reaction conditions are: ultraviolet light irradiation reaction for 10 - 20 s.

2. The method for preparing glucomannan hydrogel according to claim 1, characterized in that: The addition method of glycidyl methacrylate is: dropping it into the glucomannan solution at a rate of 0.4 - 0.6 mL / min.

3. The method for preparing glucomannan hydrogel according to claim 1 or 2, characterized in that: The mass ratio of the catalyst to glucomannan in the glucomannan solution is 1 - 2:

5.

4. The method for preparing glucomannan hydrogel according to claim 1, characterized in that: The grafting reaction conditions are: light-shielded reaction for 48 - 72 h.

5. A glucomannan hydrogel, characterized in that: Prepared by the method according to any one of claims 1 - 4.

6. The application of the glucomannan hydrogel according to claim 5, characterized in that: As a wound healing promoter.

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