CN115246938A - Silk fibroin hydrogel with traditional Chinese medicine polysaccharide activity, and preparation method and application thereof - Google Patents

Abstract

The invention discloses silk fibroin hydrogel with traditional Chinese medicine polysaccharide activity, and a preparation method and application thereof. The preparation method comprises the following steps: adding traditional Chinese medicine polysaccharide into the silk fibroin solution, and adding ethanol to adjust the pH value to acidity, thereby preparing the silk fibroin hydrogel with traditional Chinese medicine polysaccharide activity. The method can shorten the gelling time of the silk fibroin solution to be within 5min, not only improves the working efficiency, but also ensures that the prepared hydrogel has good biocompatibility and is green and safe, and also has the advantages of simple preparation process, lower cost, rapidness and high efficiency.

Description

Silk fibroin hydrogel with traditional Chinese medicine polysaccharide activity, and preparation method and application thereof

technical field

The invention relates to the technical field of hydrogel preparation, in particular to a silk fibroin hydrogel with traditional Chinese medicine polysaccharide activity, a preparation method and application thereof.

Background technique

Silk fibroin is obtained by degumming silk. It is a natural macromolecular fibrin with no physiological activity, accounting for 70% to 80% of silk. It is a natural protein containing essential amino acids for human body and has good biocompatibility. Silk fibroin exists in two different conformations, Silk I and Silk II. The conformation of Silk I includes random coil and a-helix, while the conformation of Silk II is anti-parallel β-sheet. Among them, the SilkI structure is not stable enough, and can be converted into a stable SilkII structure by polar solvents, heat treatment, etc. Silk fibroin can be obtained in a variety of different forms through relatively simple processing, which is conducive to the ingrowth of tissue cells.

Polysaccharides are the main components of plants. Many polysaccharides have important pharmacological activities. Traditional Chinese medicine polysaccharide hydrogels are non-toxic and have good water retention properties, biocompatibility and biodegradability. They have good application prospects in drug delivery systems.

The silk fibroin solution will gradually change to gelation under standing, however, the transformation time is long, and the mass concentration of silk fibroin also affects the gelation time. For example, when the mass concentration of silk fibroin solution is greater than 10% It is easier to form a gel, but it takes a long time to form a gel when the mass concentration is less than 10%. Moreover, the gel formed by the silk fibroin solution with a mass concentration higher than 10% has a lower water content and a lower porosity, and is also limited in application. The long gelation time of silk fibroin solution with a mass concentration of less than 5% also restricts the use of silk fibroin hydrogels.

At present, the main method for shortening the hydrogelation time of silk fibroin is: adding a chemical cross-linking agent to the silk fibroin aqueous solution. Although this method can greatly shorten the gelation time of the silk fibroin aqueous solution, the obtained A certain amount of chemical cross-linking agent will remain in the gel, which brings certain toxicity, and this method also reduces the biocompatibility of silk fibroin hydrogels.

Therefore, how to gel the low-concentration silk fibroin solution in a short time has become the focus of current research.

SUMMARY OF THE INVENTION

Based on this, the purpose of the present invention is to provide a silk fibroin hydrogel with Chinese medicine polysaccharide activity, and a preparation method and application thereof. In the present invention, by adding a certain volume of traditional Chinese medicine polysaccharide and ethanol to adjust the pH value to be acidic, the gelation time of the silk fibroin solution is shortened to less than 5 minutes, and the work efficiency is improved; and the prepared hydrogel has good biocompatibility , does not contain chemical cross-linking agent, green and safe; the invention has the advantages of simple preparation process, low cost, rapidity, high efficiency, etc., and can be widely used in the medical material industry.

Above-mentioned purpose of the present invention is achieved through the following technical solutions:

According to one aspect of the present invention, a preparation method of silk fibroin hydrogel with Chinese medicine polysaccharide activity provided by the present invention comprises:

Mixing the silk fibroin solution with a concentration of 1% to 6% and the traditional Chinese medicine polysaccharide with a concentration of 0.2% to 4% to obtain a mixed solution;

Ethanol is added to adjust the pH value of the mixed solution to be acidic to form a gel to obtain a silk fibroin hydrogel with Chinese medicine polysaccharide activity.

Preferably, the concentration of the silk fibroin solution is 1% to 4%.

Preferably, the concentration of the traditional Chinese medicine polysaccharide is 0.2-2%.

Preferably, the volume ratio of the silk fibroin solution to the traditional Chinese medicine polysaccharide is 1:(0.25-4). More preferably, the volume ratio of the silk fibroin solution to the traditional Chinese medicine polysaccharide may be 1:(0.25-3).

Preferably, the added ethanol concentration is 70% to 100%.

Preferably, the pH value of the mixed solution is adjusted to 4-6.

Preferably, the temperature for gel formation is 25°C to 50°C.

Preferably, the preparation method of the silk fibroin solution includes: placing the degummed silk in a 9.3M lithium bromide solution, heating and dissolving in a water bath; cooling the solution to room temperature, and dialysis; then centrifuging to remove impurities to obtain a silk fibroin solution .

Further, when the water bath is heated and dissolved, the water bath ratio of the degummed silk and the lithium bromide solution is 1:(5-10), and the water bath temperature is 65°C. During dialysis, a dialysis bag with a molecular weight cut-off of 14,000 was used for dialysis at 4°C for 3 d. The centrifugation conditions were: temperature 4°C, rotation speed 9000r/min, and centrifugation time 20min.

Preferably, the traditional Chinese medicine polysaccharide is obtained by extracting the traditional Chinese medicine polysaccharide by water extraction and alcohol precipitation, and using the Sevag method to remove protein, wherein the extract: chloroform: n-butanol=25:5:1.

Further, the preparation method of the traditional Chinese medicine polysaccharide includes: taking the mechanically pulverized Chinese medicinal material, placing it in a water bath to extract multiple times according to the material-to-liquid ratio, combining the filtrates and concentrating, and using the Sevag method to remove protein; The solution is alcohol-precipitated with ethanol, suction filtered, and dried to obtain the traditional Chinese medicine crude polysaccharide; the traditional Chinese medicine crude polysaccharide is dialyzed in deionized water, and freeze-dried to obtain the traditional Chinese medicine polysaccharide. Among them, the volume dosage of ethanol during alcohol precipitation is about 4 times that of the polysaccharide solution after protein removal. Wherein, the Chinese medicinal materials can be astragalus medicinal materials, Huang Jing medicinal materials, Angelica medicinal materials, Rhubarb medicinal materials, Epimedium medicinal materials and the like.

According to another aspect of the present invention, the present invention provides a silk fibroin hydrogel with traditional Chinese medicine polysaccharide activity, which is prepared by the above-mentioned preparation method.

According to yet another aspect of the present invention, the present invention provides the application of a silk fibroin hydrogel with traditional Chinese medicine polysaccharide activity in medical materials, wherein the hydrogel is prepared by the above-mentioned preparation method. Specifically, the medical material can be, for example, an enteric material, a drug release and cell culture material, an injectable hydrogel, and the like.

Compared with the prior art, the present invention obtains the silk fibroin hydrogel by adding traditional Chinese medicine polysaccharide and ethanol to the silk fibroin solution with a concentration of 1% to 6% to adjust the pH value to be acidic, and shortens the time of low-concentration silk fibroin. The gelation time of the protein solution can be shortened to less than 5min, which improves the work efficiency, solves the problem of restricting the use of silk fibroin hydrogel in the prior art because the gelation time of the silk fibroin solution is too long, and also avoids the In order to bring toxicity to the silk fibroin aqueous solution, the chemical cross-linking agent will reduce the biocompatibility of the hydrogel. Moreover, the present invention can make the hydrogel have polysaccharide activity by adding traditional Chinese medicine polysaccharide, so that the hydrogel has the effect of promoting cell proliferation. The hydrogel prepared by the invention has an obvious three-dimensional network structure, a porous internal structure and interconnected pores, which can be used for drug release and is beneficial to the adhesion and migration of cells.

The present invention has the advantages of simple preparation process, low cost, rapidity, high efficiency, etc., and also has the advantages of being sensitive to temperature, sensitive to acidity, and injectable. Moreover, the hydrogel prepared by the invention has good biocompatibility, does not contain chemical cross-linking agents, etc., is green and safe, and can be widely used in the medical material industry.

In addition, the present invention finally obtains the optional and preferred ranges described in the present invention through continuous research on the ranges of parameters such as ratios and concentrations, and the low-concentration polysaccharide, low-concentration silk fibroin and absolute ethanol form a gel faster. In the present invention, by adding the volume of traditional Chinese medicine polysaccharide and ethanol within the concentration range to adjust the pH value to be acidic, the gelation time of the silk fibroin solution can be shortened to less than 5 minutes, and even can be shortened to about 1 minute, which greatly improves the Work efficiency; and the present invention can make the hydrogel have polysaccharide activity by adding traditional Chinese medicine polysaccharide, so that the hydrogel has the effect of promoting cell proliferation.

Description of drawings

Fig. 1 is the silk fibroin hydrogel with Chinese medicine polysaccharide activity that the present invention makes;

Fig. 2 is the release curve diagram of bovine serum albumin coated with silk fibroin hydrogel with Chinese medicine polysaccharide activity prepared in Example 1 of the present invention;

FIG. 3 is a scanning electron microscope image of the silk fibroin hydrogel with Chinese medicine polysaccharide activity prepared in Example 2 of the present invention.

Detailed ways

The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. The following description of at least one exemplary embodiment is merely illustrative in nature and is in no way intended to limit the invention, its application, or uses. Based on the embodiments in the invention, all other embodiments obtained by persons of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

A preparation method of a silk fibroin hydrogel with Chinese medicine polysaccharide activity provided by the present invention may include the following steps:

1. Use water extraction and alcohol precipitation to extract crude polysaccharides from traditional Chinese medicine:

1) Take a certain quality of mechanically pulverized Chinese herbal medicine, place it in a water bath at 80°C for 3 times extraction at a material-to-liquid ratio of 1:10, combine the filtrates and concentrate to a certain volume, and use the Sevag method to remove protein. Wherein, extract: chloroform: n-butanol=25:5:1.

2) Precipitating the polysaccharide solution after protein removal with a certain volume of 95% ethanol, suction filtration, and drying to obtain crude polysaccharide of traditional Chinese medicine. Wherein, the volume dosage of the ethanol can be 4 times that of the polysaccharide solution after protein removal.

2. Preparation of traditional Chinese medicine polysaccharides:

3) Dialyzing the above-mentioned dried Chinese medicine crude polysaccharide in deionized water for 3 d, freeze-drying to obtain the traditional Chinese medicine polysaccharide, for use; during dialysis, the molecular weight cut-off of the dialysis bag is 3500, and the dialysis time is 3 d.

3. Preparation of silk fibroin solution:

4) A certain quality of degummed silk was placed in a 9.3M lithium bromide solution, heated in a 65°C water bath to fully dissolve, cooled to room temperature, transferred to a dialysis bag, and dialyzed in distilled water at low temperature for 3 days. After the dialysis, the solution was centrifuged in a centrifuge to remove insoluble impurities to obtain a clear silk fibroin solution. Wherein, the water bath ratio of degummed silk and lithium bromide solution is 1:(5～10). During dialysis, a dialysis bag with a molecular weight cut-off of 14,000 was used for dialysis at 4°C for 3 d. The centrifugation conditions were: temperature 4°C, rotation speed 9000r/min, and centrifugation time 20min.

4. Preparation of silk fibroin hydrogel with traditional Chinese medicine polysaccharide activity:

5) Mix the silk fibroin solution with a concentration of 1% to 6% and the traditional Chinese medicine polysaccharide with a concentration of 0.2 to 4% in different volume ratios, add a certain volume of ethanol, such as 0.2 to 1 mL, and adjust the pH of the mixture to acidity, For example, adjust the pH to 4-6, place it at different temperatures, such as room temperature, 37°C, and 50°C, and observe the gel formation time. Wherein, the volume ratio of the silk fibroin solution to the traditional Chinese medicine polysaccharide can be 1:(0.25-4), preferably 1:(0.25-3), for example, it can be 1:4, 2:3, 1:1, 3: 2. 4:1 mix. The ethanol concentration is 70% to 100%. Further, the concentration of the silk fibroin solution is preferably 1% to 4%. The concentration of the traditional Chinese medicine polysaccharide is preferably 0.2-2%. Ethanol is preferably absolute ethanol. Low concentrations of polysaccharides, low concentrations of silk fibroin and absolute ethanol will gel faster.

6) Determination of gel formation time: the centrifuge tube is inverted and the solution does not flow, that is, a silk fibroin hydrogel with Chinese medicine polysaccharide activity (ie, silk fibroin Chinese medicine polysaccharide hydrogel) is obtained.

According to statistics, using the preparation method of the present invention to prepare the silk fibroin hydrogel can shorten the gelation time of the silk fibroin solution to within 5 minutes.

The present invention will be described in further detail below with reference to specific embodiments.

Example 1:

1) Take 400 g of mechanically pulverized medicinal materials of Astragalus, and extract crude polysaccharides from Astragalus by water extraction and alcohol precipitation. Wherein, the step of extracting crude polysaccharide from Astragalus membranaceus by water extraction and alcohol precipitation method may specifically include: extracting 3 times in an 80° C. water bath according to a material-to-liquid ratio of 1:10, combining the filtrates and concentrating to a certain volume, using the Sevag method Protein removal (water extract: chloroform: n-butanol = 25:5:1). The polysaccharide solution after protein removal was ethanol-precipitated with 4 times the volume of 95% ethanol, suction filtered, and dried to obtain crude polysaccharide of Astragalus membranaceus.

2) Dialyzing the above-mentioned dried crude polysaccharide of Astragalus membranaceus in deionized water for 3 days, and freeze-drying to obtain Astragalus membranaceus polysaccharide, which is used for later use.

3) A certain mass of degummed silk was placed in a 9.3M lithium bromide solution, heated in a 65°C water bath to fully dissolve, and the solution was cooled to room temperature, transferred to a dialysis bag, and dialyzed with distilled water at 4°C for 3 days. After the dialysis, the solution was centrifuged in a centrifuge to remove insoluble impurities, and the centrifugation conditions were 4° C., 20 min, and 9000 r/min; a clear silk fibroin solution was obtained.

4) After mixing the silk fibroin solution with a concentration of 2% and the astragalus polysaccharide with a concentration of 0.6% in a volume ratio of 1:4, 3:2, 1:1, 2:3, 4:1, add 0.2-1 mL The concentration is 100% ethanol, the pH value of the mixture is adjusted to 4-6, and the gelation time is observed at 37°C.

5) Invert the centrifuge tube and the solution does not flow to obtain a hydrogel of silk fibroin astragalus polysaccharide, as shown in FIG. 1 .

In this embodiment, the gel-forming times under each volume ratio are respectively: no gel-forming within 1 hour, 1min30s, 1min47s, 2min30s, and 1min54s.

FIG. 2 is a release curve diagram of bovine serum albumin coated with silk fibroin hydrogel with traditional Chinese medicine polysaccharide activity prepared by using the example 1. FIG.

Test process: prepare a hydrogel with a polysaccharide solution containing 0.01 g/mL bovine serum albumin, add 10 mL of PBS as the release medium to the EP tube containing the hydrogel, incubate at 37 °C in a shaker, and incubate at intervals time to measure the absorbance of the solution.

It can be seen from Figure 2 that PBS with pH=7.4 was used as the release medium, and every 4h was used as a sampling point for a total of 24h, and the release rate reached more than 80% at 24h.

Example 2:

1) Take 400g of mechanically pulverized Polygonatum chinensis medicinal materials, place them in an 80°C water bath to extract 3 times at a material-to-liquid ratio of 1:10, combine the filtrates and concentrate them to a certain volume and then use the Sevag method to remove protein (water extract: chloroform: n-butanol). = 25:5:1). The polysaccharide solution after protein removal was ethanol precipitated with 4 times the volume of 95% ethanol, suction filtered, and dried to obtain Polygonatum polysaccharide crude polysaccharide.

2) Dialyzing the above-mentioned dried polysaccharide polysaccharide in deionized water for 3 days, freeze-drying to obtain polysaccharide polysaccharide, and standby.

3) A certain quality of degummed silk was placed in a 9.3M lithium bromide solution, heated in a 65°C water bath to fully dissolve, and the solution was cooled to room temperature and transferred to a dialysis bag, and dialyzed with distilled water at 4°C for 3 days. After the dialysis, the solution was centrifuged in a centrifuge to remove insoluble impurities, and the centrifugation conditions were 4°C, 20 min, and 9000 r/min; a clear silk fibroin solution was obtained.

4) After mixing the silk fibroin solution with a concentration of 4% and the polysaccharide polysaccharide with a concentration of 1.6% in a volume ratio of 1:4, 3:2, 1:1, 2:3, 4:1, add 0.2-1 mL The concentration is 85% ethanol, the pH value of the mixture is adjusted to 4-6, and the gelation time is observed at room temperature.

5) Invert the centrifuge tube, and the solution does not flow to obtain a hydrogel of silk fibroin polysaccharide.

In this embodiment, the gel-forming times under each volume ratio are respectively: no gel-forming within 1 hour, 4 min, 4 min 10 s, 3 min 30 s, and 3 min 56 s.

Fig. 3 is a scanning electron microscope image of the silk fibroin hydrogel with Chinese medicine polysaccharide activity prepared in the above-mentioned embodiment 2 of the present invention. It can be seen from Fig. 3 that the prepared hydrogel has an obvious three-dimensional network structure and a porous internal structure. , The interconnection between pores can be applied to drug release, which is beneficial to cell adhesion and migration.

Claims (10)

1. a preparation method of silk fibroin hydrogel with Chinese medicine polysaccharide activity, is characterized in that, comprises: Mixing the silk fibroin solution with a concentration of 1% to 6% and the traditional Chinese medicine polysaccharide with a concentration of 0.2% to 4% to obtain a mixed solution; Ethanol is added to the mixed solution, and the pH value of the mixed solution is adjusted to be acidic to form a gel to obtain a silk fibroin hydrogel with Chinese medicine polysaccharide activity. 2 . The preparation method according to claim 1 , wherein the volume ratio of the silk fibroin solution to the traditional Chinese medicine polysaccharide is 1:(0.25～3). 3 . 3 . The preparation method according to claim 1 , wherein the concentration of the ethanol is 70% to 100%; the pH of the mixed solution is adjusted to be 4 to 6. 4 . 4 . The preparation method according to claim 1 , wherein the temperature of gel formation is 25° C. to 50° C. 5 . 5. The preparation method according to claim 1, wherein the preparation method of the silk fibroin solution comprises: The degummed silk was placed in a 9.3M lithium bromide solution, heated in a water bath to dissolve; The solution was cooled to room temperature and dialyzed; Then centrifuge to remove impurities to obtain a silk fibroin solution. 6. preparation method according to claim 5, is characterized in that, When the water bath is heated and dissolved, the water bath ratio of degummed silk and lithium bromide solution is 1:(5～10), and the water bath temperature is 65°C; During dialysis, a dialysis bag with a molecular weight cut-off of 14000 was used for dialysis at 4°C for 3 days; The centrifugation conditions were: temperature 4°C, rotation speed 9000r/min, and centrifugation time 20min. 7. preparation method according to claim 1, is characterized in that, described Chinese medicine polysaccharide adopts water extraction alcohol precipitation method to extract Chinese medicine polysaccharide, adopts Sevag method to remove protein and obtains, wherein, extracting solution: chloroform: n-butanol=25: 5:1. 8. preparation method according to claim 7, is characterized in that, the preparation method of described Chinese medicine polysaccharide, comprises: Take the mechanically pulverized Chinese medicinal materials, place them in a water bath to extract several times according to the material-to-liquid ratio, combine the filtrates and concentrate, and use the Sevag method to remove proteins; The polysaccharide solution after protein removal is ethanol precipitation, suction filtration, and drying to obtain crude polysaccharide of traditional Chinese medicine; The crude Chinese medicine polysaccharide is dialyzed in deionized water and freeze-dried to obtain the traditional Chinese medicine polysaccharide. 9 . A silk fibroin hydrogel with traditional Chinese medicine polysaccharide activity, characterized in that, it is prepared by the preparation method according to any one of claims 1 to 8 . 10 . The application of a silk fibroin hydrogel having traditional Chinese medicine polysaccharide activity prepared by the preparation method according to any one of claims 1 to 8 in medical materials. 11 .

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