CN106334192A - Sericin hydrogel, preparation method and application thereof - Google Patents

Abstract

The present invention provides a sericin hydrogel and its preparation method and application. The preparation method comprises 1) extracting sericin, making it into powder, 2) preparing hydrazide-modified sericin, and making it 3) prepare oxidized dextran, and make it into powder, 4) prepare hydrazide-modified sericin solution and oxidized dextran solution, 5) mix hydrazide-modified sericin solution and oxidized dextran sugar solution to prepare sericin hydrogels. The sericin hydrogel prepared by the scheme of the present invention has good biodegradability and biocompatibility, and can be used as a drug carrier to carry a variety of macromolecular and small molecular drugs, and can be injected into the body by subcutaneous injection or intramuscular injection to release drugs . The hydrogel has fluorescence properties, and the degradation of the gel and the release of drugs can be monitored through fluorescence. And the sericin hydrogel can also be applied to skin repair. The sericin hydrogel scaffold is obtained through freeze-drying, which can also be used as a biomedical material.

Description

A kind of sericin hydrogel and its preparation method and application

technical field

The invention relates to the field of biomedical composite materials, in particular to a sericin hydrogel and its preparation method and application.

Background technique

Silk Sericin is a natural macromolecular protein wrapped on the surface of silk fibre. For a long time, due to the lack of understanding of sericin and the limitations of research, about 50,000 tons of sericin are treated as waste in the silk reeling industry every year, wasting a lot of valuable natural resources and causing serious damage to the environment. pollution. If the recycling of waste sericin is used as a green material in the field of tissue engineering and regenerative medicine, it may bring huge social, economic and environmental benefits.

In recent years, in the field of tissue engineering, sericin has attracted more and more attention due to its good biological activities, such as biodegradability, low immunogenicity, antioxidant activity, and cell adhesion. At present, some research teams have simply mixed sericin protein extracted by high-temperature alkaline extraction with other polymer materials such as gelatin, polyvinyl alcohol, hydroxymethyl cellulose, polyacrylamide, etc. to prepare thin films, biological scaffolds, and hybrid gels. and so on in the field of tissue engineering. However, currently the most important method for preparing sericin into hydrogel form is to simply incorporate it into other polymer networks instead of forming cross-links autonomously. This is mainly because the complete separation of sericin and silk fibroin in silkworm cocoons requires treatment methods such as high temperature or lye, which will inevitably lead to serious degradation of sericin protein. This may be the reason why sericin cannot form a gel by cross-linking.

So far, there is no report that sericin extracted by high temperature or alkaline extraction can be prepared into injectable hydrogel by self-crosslinking.

Contents of the invention

The invention provides a method for preparing sericin hydrogel, which can prepare sericin with good stability and mechanical properties by mixing a hydrazide-modified sericin solution with an oxidized dextran solution Hydrogel, the operation method is simple and easy.

The present invention also provides a sericin hydrogel, which has good biodegradability and biocompatibility, can be used as a drug carrier to carry various macromolecular and small molecular drugs, and can be injected by subcutaneous injection or intramuscular injection. The body releases the drug.

The invention also provides the application of the sericin hydrogel in fluorescent probes, drug carriers, and preparation of tissue engineering materials.

The present invention also provides a sericin hydrogel freeze-dried scaffold, which is obtained by freezing the sericin hydrogel below zero and drying in vacuum.

The invention also provides the application of the sericin hydrogel freeze-dried scaffold in tissue engineering.

The preparation method of a kind of sericin hydrogel provided by the invention comprises the following steps:

1) Extract sericin and make it into powder:

Take silkworm cocoon, and cut it into pieces, wash with water, remove moisture;

Add 10-50mL of deionized water or 10-50mL of 0.01-0.2mol/L Na ₂ CO ₃ aqueous solution per gram of cocoon fragments, and then stir at 70-100°C for 0.5-3 hours to dissolve sericin and obtain silk Colloid solution;

centrifuging to remove insoluble substances in the sericin solution, and dialyzing the sericin solution for 12-72 hours to obtain a clear sericin solution;

freeze-drying the clear sericin solution to obtain sericin powder;

2) Preparation of hydrazide-modified sericin:

Dissolve the sericin powder obtained in step 1) in a ratio of 0.2-10g/mL in 30-200mL of dimethyl sulfoxide, then add 0.1-2g per gram of sericin powder, add N,N - carbonyldiimidazole reacted for 1 to 3 hours to obtain a dimethyl sulfoxide solution of sericin;

At the same time, adipic acid dihydrazide was dissolved in 50-400 mL of dimethyl sulfoxide at a ratio of 0.5-50 g/mL to obtain a dimethyl sulfoxide solution of adipate dihydrazide;

And add 6-10g of adipate dihydrazide per gram of sericin, mix the dimethyl sulfoxide solution of the adipate dihydrazide with the dimethyl sulfoxide solution of the sericin, and set React at 20-45°C for 0.5-36 hours, dialyze the reaction solution for 12-72 hours to obtain a hydrazide-modified sericin solution, and then freeze-dry to obtain a hydrazide-modified sericin powder; or

Dissolve the sericin powder obtained in step 1) in a ratio of 0.001-0.1 g/mL in 100-1000 mL of 2-morpholineethanesulfonic acid solution with a mass percentage of 2%, and then add 8 -10g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-40g of N-hydroxysuccinimide, stirred at room temperature for 12-24 hours to obtain sericin Reaction solution: Add 2.5-25mL per liter of the sericin reaction solution, add 85% hydrazine hydrate solution dropwise into the sericin reaction solution, and react at room temperature for 12-24 hours , dialyzing the reaction solution for 12 to 72 hours to obtain a hydrazide-modified sericin solution, and then freeze-drying to obtain a hydrazide-modified sericin powder;

3) Preparation of oxidized dextran:

Weigh dextran and sodium periodate and dissolve them in 10-50mL water at a ratio of 0.1-0.8g/mL and 0.01-0.2g/mL respectively to obtain an aqueous solution of dextran and an aqueous solution of sodium periodate;

Slowly add 10-50mL of sodium periodate solution dropwise to 10-50mL of dextran aqueous solution in an ice bath, and react in the dark at 4°C for 1-24 hours;

Dialyzing the reaction solution for 12-72 hours, and then freeze-drying to obtain oxidized dextran powder with an oxidation degree of 10-40%;

4) Preparation of hydrazide-modified sericin solution and oxidized dextran solution

Dissolving the hydrazide-modified sericin powder obtained in step 2) in distilled water to prepare a hydrazide-modified sericin solution with a concentration of 50-400 g/L; dissolving the oxidized dextran powder obtained in step 3) in In distilled water, make a concentration of 5-500g/L oxidized dextran solution;

5) Mix the hydrazide-modified sericin solution obtained in 4) with the oxidized dextran solution at a volume ratio of 1:1-5 to obtain the sericin hydrogel.

Further, in the scheme of the present application, the concentration of the hydrazide-modified sericin solution is 0.2 g/L.

Furthermore, the concentration of the oxidized dextran solution is 100-350 g/L.

Furthermore, the oxidation degree of the oxidized dextran is 10%-35%.

In the solution of this application, the concentration of the hydrazide-modified sericin solution and the oxidized dextran solution is easy to control, and various properties of the gel can be controlled by adjusting the concentration, such as water swelling, degradation, drug release, etc.

Further, in the solution of the present invention, the molecular weight of the dextran is 40-120kDa.

Furthermore, the dialysis in step 1) is performed using a dialysis bag with a molecular weight cut-off of 8-14 kDa.

Furthermore, the dialysis in steps 2) and 3) is performed using a dialysis bag with a molecular weight cut-off of 3.5 kDa.

The present invention also provides a preparation method of sericin hydrogel, comprising the following steps:

1) Extract sericin and make it into powder:

Take silkworm cocoon, and cut it into pieces, wash with water, remove moisture;

Add 10-50mL of deionized water or 10-50mL of 0.01-0.2mol/L Na ₂ CO ₃ aqueous solution per gram of cocoon fragments, and then stir at 70-100°C for 0.5-3 hours to dissolve sericin and obtain silk Colloid solution;

centrifuging to remove insoluble substances in the sericin solution, and dialyzing the sericin solution for 12-72 hours to obtain a clear sericin solution;

freeze-drying the clear sericin solution to obtain sericin powder;

2) Preparation of hydrazide-modified sericin:

Dissolve the sericin powder obtained in step 1) in a ratio of 0.2-10g/mL in 30-200mL of dimethyl sulfoxide, then add 0.1-2g per gram of sericin powder, add N,N - carbonyldiimidazole reacted for 1 to 3 hours to obtain a dimethyl sulfoxide solution of sericin;

At the same time, adipic acid dihydrazide was dissolved in 50-400 mL of dimethyl sulfoxide at a ratio of 0.5-50 g/mL to obtain a dimethyl sulfoxide solution of adipate dihydrazide;

And add 6-10g of adipate dihydrazide per gram of sericin, mix the dimethyl sulfoxide solution of the adipate dihydrazide with the dimethyl sulfoxide solution of the sericin, and set React at 20-45°C for 0.5-36 hours, dialyze the reaction solution for 12-72 hours to obtain a hydrazide-modified sericin solution, and then freeze-dry to obtain a hydrazide-modified sericin powder; or

Dissolve the sericin powder obtained in step 1) in a ratio of 0.001-0.1 g/mL in 100-1000 mL of 2-morpholineethanesulfonic acid solution with a mass percentage of 2%, and then add 8 -10g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-40g of N-hydroxysuccinimide, stirred at room temperature for 12-24 hours to obtain sericin Reaction solution: Add 2.5-25mL per liter of the sericin reaction solution, add 85% hydrazine hydrate solution dropwise into the sericin reaction solution, and react at room temperature for 12-24 hours , dialyzing the reaction solution for 12 to 72 hours to obtain a hydrazide-modified sericin solution, and then freeze-drying to obtain a hydrazide-modified sericin powder;

3) Preparation of hydrazide-modified sericin solution

Dissolving the hydrazide-modified sericin powder obtained in step 2) in distilled water to prepare a hydrazide-modified sericin solution with a concentration of 50-400 g/L;

4) Mix the hydrazide-modified sericin solution obtained in 3) with the genipin aqueous solution at a concentration of 10 g/L at a volume of 6:1-3:1 to prepare the sericin hydrogel.

The present invention also provides a preparation method of sericin hydrogel, comprising the following steps:

1) Extract sericin and make it into powder:

Take silkworm cocoon, and cut it into pieces, wash with water, remove moisture;

Add 10-50mL of deionized water or 10-50mL of 0.01-0.2mol/L Na ₂ CO ₃ aqueous solution per gram of cocoon fragments, and then stir at 70-100°C for 0.5-3 hours to dissolve sericin and obtain silk Colloid solution;

centrifuging to remove insoluble substances in the sericin solution, and dialyzing the sericin solution for 12-72 hours to obtain a clear sericin solution;

freeze-drying the clear sericin solution to obtain sericin powder;

2) Preparation of hydrazide-modified sericin:

Dissolve the sericin powder obtained in step 1) in a ratio of 0.2-10g/mL in 30-200mL of dimethyl sulfoxide, then add 0.1-2g per gram of sericin powder, add N,N - carbonyldiimidazole reacted for 1 to 3 hours to obtain a dimethyl sulfoxide solution of sericin;

At the same time, adipic acid dihydrazide was dissolved in 50-400 mL of dimethyl sulfoxide at a ratio of 0.5-50 g/mL to obtain a dimethyl sulfoxide solution of adipate dihydrazide;

And add 6-10g of adipate dihydrazide per gram of sericin, mix the dimethyl sulfoxide solution of the adipate dihydrazide with the dimethyl sulfoxide solution of the sericin, and set React at 20-45°C for 0.5-36 hours, dialyze the reaction solution for 12-72 hours to obtain a hydrazide-modified sericin solution, and then freeze-dry to obtain a hydrazide-modified sericin powder; or

Dissolve the sericin powder obtained in step 1) in a ratio of 0.001-0.1 g/mL in 100-1000 mL of 2-morpholineethanesulfonic acid solution with a mass percentage of 2%, and then add 8 -10g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-40g of N-hydroxysuccinimide, stirred at room temperature for 12-24 hours to obtain sericin Reaction solution: Add 2.5-25mL per liter of the sericin reaction solution, add 85% hydrazine hydrate solution dropwise into the sericin reaction solution, and react at room temperature for 12-24 hours , dialyzing the reaction solution for 12 to 72 hours to obtain a hydrazide-modified sericin solution, and then freeze-drying to obtain a hydrazide-modified sericin powder;

3) Preparation of hydrazide-modified sericin solution

Dissolving the hydrazide-modified sericin powder obtained in step 2) in distilled water to prepare a hydrazide-modified sericin solution with a concentration of 50-400 g/L;

7) Mix the hydrazide-modified sericin solution obtained in 3) with 250 g/L glutaraldehyde aqueous solution at a volume of 100:1-5 to prepare the sericin hydrogel.

The invention provides a sericin hydrogel prepared by the preparation method.

Further, the present invention also provides the application of the sericin hydrogel in the preparation of fluorescent probes.

Furthermore, the present invention also provides the application of the sericin hydrogel in the preparation of drug carriers.

Furthermore, the present invention also provides the application of the sericin hydrogel in preparing tissue engineering materials. The tissue engineering material can be, for example, gel and biological glue for skin repair.

The invention also provides a preparation method of the sericin hydrogel freeze-dried scaffold, which is obtained by freezing the sericin hydrogel below zero and drying in vacuum. The sericin hydrogel freeze-dried scaffold made by the method can be used as a biological scaffold for tissue engineering.

In the solution of the present invention, the slave silkworm is a wild-type silkworm.

The present invention also provides a sericin hydrogel pharmaceutical preparation, including the sericin hydrogel, and chemotherapeutic drugs for killing tumors.

Further, the chemotherapeutic drug is doxorubicin. Furthermore, the sericin hydrogel pharmaceutical preparation is a unit preparation. The amount of sericin hydrogel in the unit preparation is 200 μL, and the amount of doxorubicin is 100 μg. In the scheme of the present application, the unit preparation is a preparation that satisfies the active ingredient required for one administration to one mouse, such as a unit (needle) injection or the like.

The present invention also provides a pharmaceutical preparation for wound healing, including the sericin hydrogel, and a dressing for wound healing. Further, the wound healing dressing is Duoaifu.

The solution of the present invention has the following advantages:

1) The present invention uses wild-type silkworm cocoons as raw materials, extracts them with high-temperature lye, and modifies them with hydrazide to obtain a sericin solution in which sericin retains good natural characteristics. The solution can be processed by simple filtration. Sterilized; and prepared into powder form by freeze-drying for long-term storage.

2) For the first time in the present invention, hydrazide-modified sericin is cross-linked with natural macromolecular compounds such as oxidized dextran to make a hydrogel, and the operation method is simple and easy. A hydrogel with good biodegradability and biocompatibility, which can be used as a drug carrier to carry a variety of macromolecular and small molecular drugs, has been obtained.

3) The sericin hydrogel provided by the present invention can regulate the gelation speed by adjusting the concentration and temperature of the hydrazide-modified sericin solution and the oxidized dextran solution (in-situ gelation can be realized as required).

4) As a drug carrier, the sericin hydrogel of the present invention can be injected into the body by subcutaneous injection or intramuscular injection to release drugs, and can be controlled by adjusting the concentration of hydrazide-modified sericin solution and oxidized dextran solution. Effectively control the release rate of macromolecular and small molecule drugs, effectively solve the problem of drug burst release, and can carry anti-tumor drugs to effectively inhibit the growth of tumors.

5) The sericin hydrogel of the present invention has fluorescence properties, and the degradation of the gel and the release of drugs can be monitored through fluorescence.

6) The sericin hydrogel of the present invention has good skin repair function.

7) The sericin hydrogel of the present invention can be freeze-dried to obtain sericin hydrogel scaffolds with different pore diameters and sizes, which can be used to prepare various tissue damage repair materials.

Description of drawings

Figure 1A is the SDS-PAGE electrophoresis patterns of silk fibroin-deficient sericin extracted from lithium bromide (2), sericin extracted from sodium carbonate (3), and hydrazide-modified sericin (4).

Fig. 1B is the infrared spectrum of sericin, hydrazide-modified sericin and sericin hydrogel SDH-2.

Figure 1C is the infrared spectrum of dextran and oxidized dextran DEX-AL-3.

Figure 1D is the NMR spectra of dextran and oxidized dextran DEX-AL-3.

Fig. 2A is a histogram of gelation time of sericin hydrogels (SDH-1, SDH-2, SDH-3) under different temperature conditions.

Fig. 2B is a histogram of the maximum water swelling rate (37° C.) of sericin hydrogels (SDH-1, SDH-2, SDH-3) under different pH environments.

Fig. 2C is a graph showing the rheological properties of sericin hydrogels (SDH-1, SDH-2, SDH-3).

Fig. 3 is a view of the internal structure of sericin hydrogel freeze-dried products with different concentrations observed under a scanning electron microscope.

Figure 4 is a graph of the degradation rate (37°C) of sericin hydrogels (SDH-1, SDH-2, SDH-3) in PBS with different pH values

Fig. 5 is a statistical histogram of sericin hydrogel cytotoxicity detection in normal human liver cells (HL7702) and mouse myocytes (C2C12).

Fig. 6A is a graph showing HRP release curves from sericin hydrogels (SDH-1, SDH-2, SDH-3) in vitro.

Fig. 6B is a graph showing release curves of doxorubicin DOX from sericin hydrogels (SDH-1, SDH-2, SDH-3) in vitro.

Fig. 7A is the fluorescence spectrum of sericin extracted with sodium carbonate at high temperature. Fig. 7B is the fluorescence spectrum of hydrazide-modified sericin.

7C, 7D, and 7E are fluorescence spectra of sericin hydrogels.

Figure 7F and 7G are images of sericin hydrogel (SDH-1, SDH-2, SDH-3) lyophilized products observed under laser confocal microscope.

Fig. 8A is a small animal imaging image of sericin hydrogel injected into the back subcutaneous of C57BL/6J mice.

Figure 8B is a small animal imaging image of sericin hydrogel loaded with doxorubicin injected into the back subcutaneous of C57BL/6J mice at different time points (green-sericin hydrogel (SDH-2), red-doxorubicin ( DOX)).

Fig. 8C is a graph showing the changes in fluorescence intensity of doxorubicin (DOX) at different time points after injection of sericin hydrogel with doxorubicin into the back of C57BL/6J mice.

Fig. 8D is a graph showing the degradation and fluorescence changes of sericin hydrogel at different time points in vivo.

Fig. 8E is a graph showing the correlation analysis between the degradation rate of sericin hydrogel and the fluorescence intensity in vivo.

Fig. 9A is a graph showing the tumor size of melanoma C57BL/6J mice inhibited by doxorubicin loaded on sericin hydrogel.

Fig. 9B is a graph showing body weight changes of melanoma C57BL/6J mice loaded with doxorubicin on sericin hydrogel.

Fig. 9C is a graph showing the survival period of melanoma C57BL/6J mice loaded with doxorubicin on sericin hydrogel.

Fig. 9D is a physical picture of the tumor size of melanoma C57BL/6J mice loaded with doxorubicin in the sericin hydrogel (14th day after treatment).

Fig. 9E is H&E staining diagram of paraffin-embedded H&E staining of melanoma C57BL/6J mouse tumors loaded with doxorubicin inhibited by sericin hydrogel (14th day after treatment).

Fig. 9F is a picture of paraffin-embedded TUNEL fluorescence staining of melanoma C57BL/6J mouse tumors loaded with doxorubicin inhibited by sericin hydrogel (14th day after treatment).

Figure 10 is a statistical chart of the wound size of sericin hydrogel repairing C57BL/6J mice scalded at different time points. Duoaifu is a commercial wound dressing as a control.

detailed description

Embodiment 1 Preparation of sericin hydrogel provided by the present invention

Choice of cocoons:

Wild-type silkworm cocoons (provided by the Institute of Sericulture, Chinese Academy of Agricultural Sciences) are selected, and the main chemical components are: sericin and silk fibroin.

Step 1), extract sericin and make it into powder

1. Weigh wild-type silkworm cocoons (100g and cut into 1cm fragments, put them in a clean beaker, wash ³ times with ultrapure water, and remove moisture by centrifugation at 3500rpm for 5 minutes;

2. Add 3-4L of Na ₂ CO ₃ solution with a molar concentration of 0.02mol/L or 3-4L of deionized water to the silkworm cocoon fragments obtained in step 1, and put the beaker into a constant temperature water bath at 100°C 1h, dissolving sericin to obtain a sericin solution;

3. Transfer the solution obtained in step 2 into a centrifuge tube and centrifuge at 3500rpm for 5 minutes to remove insoluble substances and obtain the centrifuged solution;

4. Transfer the centrifuged solution in step 3 into the pretreated dialysis bag (molecular weight cut-off: 8-14kDa), and clamp the two ends of the dialysis bag containing the centrifuged sericin solution with dialysis bag clips , placed in a beaker containing ddH ₂ O; place the beaker on a stirrer and slowly stir for dialysis, change the water every 3 hours, and dialyze for a total of 72 hours to obtain a clear sericin solution;

5. Optionally, transfer the clarified sericin solution to a centrifuge tube and centrifuge at 3500rpm for 5 minutes to remove the precipitate;

6. Place the sericin solution in a freeze dryer to freeze-dry it into sericin powder;

7. Store the sericin powder in a -20°C refrigerator for later use.

Step 2), preparation of hydrazide-modified sericin

1. Weigh 6g of sericin powder and dissolve it in 30mL of dimethyl sulfoxide at 35°C;

2. Add 4.3g of N,N-carbonyldiimidazole to 1) and react for 1 to 3 hours to obtain a dimethyl sulfoxide solution of sericin;

3. Weigh 54 g of adipic acid dihydrazide and dissolve it in 100 mL of dimethyl sulfoxide at 45°C to obtain a dimethyl sulfoxide solution of adipic acid dihydrazide;

4. Add 6-10g adipate dihydrazide per gram of sericin, mix the dimethyl sulfoxide solution of adipate dihydrazide with the dimethyl sulfoxide solution of sericin , placed at 20-45°C to fully react for 0.5-36 hours;

5. Transfer the reaction solution in step 4 into the pretreated dialysis bag (molecular weight cut-off: 3500Da), clamp both ends of the dialysis bag containing sericin solution with dialysis bag clips, place the ddH ₂ O in a beaker; place the beaker on a stirrer and slowly stir for dialysis, changing the water every 3 hours for a total of 48 hours of dialysis;

6. The hydrazide-modified sericin solution obtained in step 5 was freeze-dried in a freeze dryer to obtain hydrazide-modified sericin powder, and stored in a -20°C refrigerator for later use.

Step 3), preparation of oxidized dextran

1. Weigh 10g of dextran with a relative molecular weight of 40kDa and dissolve it in 50mLddH ₂ O;

2. Weigh 2.64g of NaIO ₄ and dissolve in 20mLddH ₂ O;

3. Slowly add the NaIO ₄ aqueous solution to the oxidized dextran aqueous solution dropwise under ice bath conditions, and then place it in a -4°C refrigerator for a full reaction for 24 hours;

4. Transfer the solution in step 3 into a dialysis bag (3500Da), place the dialysis bag in a beaker containing ddH ₂ O; One time of water, a total of 48 hours of dialysis;

5. Freeze-dry the oxidized dextran aqueous solution in a freeze dryer to form oxidized dextran powder, and store in a -20°C refrigerator;

6. The degree of oxidation of the prepared oxidized dextran measured by redox titration is 35%.

Step 4) Prepare hydrazide-modified sericin solution and oxidized dextran solution

1. Dissolve the hydrazide-modified silk protein powder in ddH ₂ O and adjust the concentration to about 200g/L to obtain a hydrazide-modified sericin solution;

2. Dissolving the oxidized dextran powder in ddH ₂ O and adjusting the concentration to about 100 g/L to obtain an oxidized dextran solution.

Step 5) Preparation of Sericin Hydrogel

Add 1mL of oxidized dextran solution to every 1mL of the hydrazide-modified sericin solution in 4); stir and mix well, and place them at 4°C, 25°C, and 37°C respectively to obtain sericin Hydrogel SDH-1.

Example 2 Preparation of sericin hydrogel provided by the present invention

In the present embodiment, the preparation method of sericin solution is the same as that of Example 1, except that step 4) and step 5) are carried out as follows:

1. Dissolve the hydrazide-modified silk protein powder in ddH ₂ O, adjust the concentration to about 200g/L, and obtain the hydrazide-modified sericin solution;

2. Dissolve the oxidized dextran powder in ddH ₂ O and adjust the concentration to about 200g/L to obtain the oxidized dextran solution;

3. Add 5mL oxidized dextran solution to every 1mL sericin solution;

4. After fully stirring and mixing, place at 4°C, 25°C, and 37°C respectively to obtain sericin hydrogel SDH-2.

Example 3 Preparation of sericin hydrogel provided by the present invention

The difference between the preparation method of sericin hydrogel in this example and the preparation method of Example 1 is that step 4) and step 5) are carried out as follows:

1. Dissolve the hydrazide-modified silk protein powder in ddH ₂ O, adjust the concentration to about 200g/L, and obtain the hydrazide-modified sericin solution;

2. Dissolve the oxidized dextran powder in ddH ₂ O, and adjust the concentration to about 350g/L to obtain the oxidized dextran solution;

3. Add 1mL oxidized dextran solution to every 1mL sericin solution;

4. After fully stirring and mixing, place at 4°C, 25°C, and 37°C respectively to obtain sericin hydrogel SDH-3.

Example 3-1

Preparation of sericin hydrogel freeze-dried scaffold

The sericin hydrogels SDH-1, SDH-2, and SDH-3 obtained at 25°C were quickly placed at -80°C, frozen for 24 hours, and then taken out; the samples were dried in a freezer vacuum dryer (determined according to the size of the sample) Suitable drying time), three kinds of sericin freeze-dried scaffolds with different pore diameters were obtained, the pore diameters were respectively 128nm, 47.1nm and 10.4nm.

Example 4 Preparation of sericin hydrogel provided by the present invention

The difference between the preparation method of sericin hydrogel in this example and the preparation method of Example 1 is that step 3) is carried out as follows:

1. Weigh 10g of dextran with a relative molecular weight of 40kDa and dissolve it in 50mLddH2O;

2. Weigh 0.66g of NaIO ₄ and dissolve it in 10mLddH ₂ O;

3. Slowly add the NaIO ₄ aqueous solution to the oxidized dextran aqueous solution dropwise under ice bath conditions, and then place it in a -40°C refrigerator for a full reaction for 24 hours;

4. Transfer the solution in step 3 into a dialysis bag (3500Da), place the dialysis bag in a beaker containing ddH ₂ O; One time of water, a total of 48 hours of dialysis;

5. Place the oxidized dextran aqueous solution in a freeze dryer to freeze-dry the oxidized dextran powder, and store it in a refrigerator at -200°C; the oxidation degree of the prepared oxidized dextran is 10% as measured by redox titration .

Then the hydrogel was prepared according to the method of Example 1, and its performance parameters were tested to be similar to those of SDH-1, SDH-2, and SDH-3.

The applicant also utilizes different ratios of dextran and sodium periodate to make oxidized dextran with an oxidation degree of 10-40% and its parameters:

Table 1

^a The molecular weight (Mw) of commercial dextran is 40kDa.

Example 5 Preparation of sericin hydrogel provided by the present invention

The difference between the preparation method of sericin hydrogel in this example and the preparation method of Example 1 is that step 2) is carried out as follows:

1. Weigh 1 g of sericin powder and dissolve it in 1 L of 2-morpholineethanesulfonic acid solution with a mass percentage of 2% at 35°C;

2. Add 8g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-40g of N-hydroxysuccinimide to the solution in step 1, and stir at room temperature for 24 hours to obtain Sericin reaction solution;

3. Add 2.5 ml of 85% by mass hydrazine hydrate solution dropwise to the sericin reaction liquid in step 2, and fully react for 24 hours at room temperature.

4. Transfer the solution in step 3 into the pretreated dialysis bag (3500Da), clamp both ends of the dialysis bag containing the sericin solution with dialysis bag clips, and place it in a beaker containing ddH ₂ O; The beaker was placed on a stirrer and slowly stirred for dialysis, and the water was changed every 3 hours for a total of 48 hours of dialysis;

6. The hydrazide-modified sericin solution obtained in step 4 was freeze-dried in a freeze dryer to obtain hydrazide-modified sericin powder, and stored in a -20°C refrigerator for later use.

Then the hydrogel was prepared according to the method of Example 1, and its performance parameters were tested to be similar to those of SDH-1, SDH-2, and SDH-3.

Example 6 Preparation of sericin hydrogel provided by the present invention

The difference between the preparation method of sericin hydrogel in this example and the preparation method of Example 1 is that step 4) and step 5) are carried out as follows:

1. Dissolve the hydrazide-modified silk protein powder in ddH ₂ O, adjust the concentration to about 200g/L, and obtain the hydrazide-modified sericin solution;

2. Add 10-50μL glutaraldehyde aqueous solution (250g/L) to every 1mL sericin solution;

3. After fully stirring and mixing, place them respectively at 25° C. to obtain light yellow sericin hydrogel.

Example 7 Preparation of sericin hydrogel provided by the present invention

The difference between the preparation method of sericin hydrogel in this example and the preparation method of Example 1 is that step 4) and step 5) are carried out as follows:

1. Dissolve the hydrazide-modified silk protein powder in ddH ₂ O, adjust the concentration to about 200g/L, and obtain the hydrazide-modified sericin solution;

2. Dissolve genipin powder in ddH ₂ O and adjust the concentration to about 10g/L to obtain genipin solution;

2. Add 300μL genipin aqueous solution to every 1mL～1.8mL sericin solution;

3. After fully stirring and mixing, place at 4°C respectively to obtain dark blue sericin hydrogel.

The sericin hydrogels provided in Examples 6 and 7 have good biocompatibility and can be used in the fields of tissue engineering and regenerative medicine, while unmodified sericin cannot form hydrogels.

Example 8 Characterization test of sericin, hydrazide-modified sericin, sericin hydrogel, and freeze-dried scaffold of said sericin hydrogel

Figure 1A shows sericin extracted from silk fibroin-deficient silkworm cocoons using lithium bromide in the prior art, sericin extracted from sodium carbonate made in step 1) of Example 1 of the present application, and hydrazide made in step 2) of Example 1 SDS-PAGE electrophoresis pattern of modified sericin.

The sodium carbonate-extracted sericin and the hydrazide-modified sericin produced in steps 1) and 2) in Example 1 were respectively passed through SDS-PAGE electrophoresis analysis, the results are shown in Figure 1A. Among them, 1 is a standard band (marker), 2 is sericin extracted from silk fibroin-deficient silkworm cocoons extracted with lithium bromide, 3 is sericin extracted from sodium carbonate, and 4 is hydrazide-modified sericin. Compared with the fibroin-deficient sericin extracted from lithium bromide, the sericin extracted from sodium carbonate and hydrazide-modified sericin are mainly in the diffuse type, but the diffuse-type sericin can also be prepared after hydrazide modification into a hydrogel.

Figure 1B shows the hydrazide-modified sericin (Sericin-ADH) made in step 2) of Example 1, the sericin (Sericin) made in step 1), and the sericin made in step 5) of Example 2 Infrared spectrum of protein hydrogel (SDH-2).

The characteristic peaks of sericin, hydrazide-modified sericin and sericin hydrogel at 4000-650cm ^-1 were determined by Fourier transform infrared spectrometer (Nexus, Thermal Nicolet, USA).

As shown in Figure 1B: from the characteristic peaks amide I, amide II, and amide III, it can be seen that sericin (Sericin), hydrazide-modified sericin (Sericin-ADH), sericin hydrogel (SDH- The infrared spectra of 2) are basically the same, indicating that the secondary structure of the polypeptide in sericin hydrogel (SDH-2) is similar to that of pure sericin, and sericin hydrogel (SDH-2) can well maintain Native conformation of sericin.

Figure 1C is the nuclear magnetic resonance spectra of dextran (Dextran) and oxidized dextran (DEX-Al-3) prepared in step 3) in Example 1.

It can be seen from FIG. 1C that the peaks appearing at shifts between 5.0-6.0 are the carbonyl peaks attached to the oxidized dextran (DEX-Al-3), which proves that the oxidized dextran has been successfully prepared.

Figure 1D is the infrared spectrum of dextran and oxidized dextran.

The characteristic peaks of sericin hydrogel at 4000–650 cm ^-1 were determined by Fourier transform infrared spectrometer (Nexus, Thermal Nicolet, USA).

As shown in Figure 1D: there is a peak at 1729 in the infrared spectrum of oxidized dextran (DEX-AL-3), which is the -C=O- stretching vibration peak on the aldehyde group, while in the infrared spectrum of dextran There is no such peak on the graph, indicating that the hydroxyl group on the dextran molecule has become an aldehyde group after oxidation, and the dextran has become an oxidized dextran; in addition, it can be seen from the graph that the oxidized dextran Except for the -C=O-stretching vibration peak, the other peaks are basically consistent with the infrared absorption peaks of dextran, indicating that only a part of the hydroxyl groups on the dextran molecule have changed into aldehyde groups.

Fig. 2A is a histogram of the gelation time of sericin hydrogels SDH-1, SDH-2 and SDH-3 prepared in Examples 1-3 under different temperature conditions.

The sericin hydrogel was composed of hydrazide-modified sericin and oxidized dextran (the degree of oxidation was 35%, the concentrations were 100g/L, 200g/L, 350g/L) at 4°C, 25°C and 37°C. Prepared according to the volume ratio (1:1) at ℃, observe and record the crosslinking time.

As shown in Figure 2A, as the concentration of oxidized dextran increases, the required cross-linking time becomes shorter; as the temperature increases, the required cross-linking time becomes shorter. It shows that by controlling the concentration of oxidized dextran and adjusting the gelation time by temperature, this property can be used to prepare the hydrogel into injectable hydrogel. Moreover, the injectable hydrogel as a drug carrier can overcome the determination that most injectable hydrogels are easy to diffuse when injected into the body, and can reduce the initial burst release of drugs.

Fig. 2B is a histogram of the maximum water swelling rate (37° C.) of the sericin hydrogels SDH-1, SDH-2 and SDH-3 prepared in Examples 1-3 under different pH environments.

The above-mentioned water swelling rate is determined by freeze-drying, weighing, and soaking the sericin hydrogel in three kinds of PBS solutions with different pH values (pH 6.0, pH 7.4, pH 11.0), and taking them out at different time points according to the following formula . (where Ws is the weight in the expanded state, Wd is the dry weight).

$\mathrm{<span\; class="notranslate">\; the\; s</span>}\mathrm{<span\; class="notranslate">\; w</span>}\mathrm{<span\; class="notranslate">\; e</span>}\mathrm{<span\; class="notranslate">\; l</span>}\mathrm{<span\; class="notranslate">\; l</span>}\mathrm{<span\; class="notranslate">\; i</span>}\mathrm{<span\; class="notranslate">\; no</span>}\mathrm{<span\; class="notranslate">\; g</span>}<span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; \%</span>}<span\; class="notranslate">\; )</span><span\; class="notranslate">\; =</span>\frac{\mathrm{<span\; class="notranslate">\; W</span>}\mathrm{<span\; class="notranslate">\; the\; s</span>}<span\; class="notranslate">\; -</span>\mathrm{<span\; class="notranslate">\; W</span>}\mathrm{<span\; class="notranslate">\; d</span>}}{\mathrm{<span\; class="notranslate">\; W</span>}\mathrm{<span\; class="notranslate">\; d</span>}}<span\; class="notranslate">\; \&times;</span>\mathrm{<span\; class="notranslate">\; 100</span>}$

As shown in Figure 2B: SDH-1 sericin hydrogel had a maximum expansion rate of 5.3 times, 9.1 times and 9.3 times at pH 6.0, pH 7.4 and pH 11.0 respectively; SDH-2 sericin hydrogel Under the environment of pH 6.0, pH 7.4 and pH 11.0, the maximum swelling rate of the hydrogel can reach 4.2 times, 7.3 times and 7.7 times respectively; Under the environment, the maximum expansion rate can reach 3.7 times, 6.2 times, 6.5 times respectively. It shows that the sericin hydrogel has good water absorption performance, and the water absorption performance of the hydrogel can be adjusted by controlling the concentration of oxidized dextran.

Fig. 2C is a graph showing the rheological properties of sericin hydrogels SDH-1, SDH-2 and SDH-3 prepared in Examples 1-3.

As shown in Figure 2C, when G'>G", it means that sericin has become a gel. The gelation of sericin hydrogel with oxidized dextran concentration of 350g/L, 200g/L and 100g/L The glue time is about 5 seconds, 145 seconds and 618 seconds respectively. The maximum elastic modulus are: 2218Pa, 1355Pa, 369Pa respectively. The maximum viscous modulus are respectively: 487.5Pa, 351.2Pa, 62.29Pa. It shows that the sericin protein water The gel has good elastic-viscosity properties, and the elastic-viscosity properties of the hydrogel can be adjusted by controlling the concentration of oxidized dextran.

Fig. 3 is a view of the internal structure of sericin hydrogel freeze-dried products with different concentrations observed under a scanning electron microscope.

As shown in Figure 3: Sericin hydrogels SDH-1(A), SDH-2(B), SDH-3 with different oxidized dextran concentrations (100g/L, 200g/L, 350g/L) The freeze-dried product of (C) has a large number of hole structures inside under the observation of scanning electron microscope.

Table 3 is a statistical table of pore size and porosity of freeze-dried products of sericin hydrogels with different concentrations

As shown in Table 3: the pore diameters of sericin hydrogel freeze-dried products with different oxidized dextran concentrations (100g/L, 200g/L, 350g/L were about 128.83±34.88μm, 47.13±27.77μm, and 10.41± 4.83μm.In addition, the porosity of three sericin hydrogel freeze-dried products with different oxidized dextran concentrations (100g/L, 200g/L, 350g/L) were statistically calculated to be 81.06±0.46, 76.58 ± 4.00, 73.82 ± 3.28. It shows that the pore size and porosity of sericin hydrogel freeze-dried products can be regulated by adjusting the concentration of oxidized dextran. And the pore structure inside the sericin hydrogel is a powerful condition as a drug carrier .

Fig. 4 is a graph showing degradation (37° C.) curves of sericin hydrogels (SDH-1, SDH-2, SDH-3) in PBS with different pH values.

To test the effect of pH environment on degradation, the sericin hydrogel was soaked in PBS solutions with different pH values (pH 6.0, pH 7.4, pH 11.0), and at different time points, the PBS was taken out and dried and weighed. The results are shown in Figure 4.

As shown in Figure 4, the sericin hydrogel degraded rapidly within the first day, and the sericin hydrogel gradually began to degrade slowly after 7 days, and the hydrogel degradation was pH-responsive. Among them, under the condition of pH 11.0, the degradation rate of sericin hydrogel (SDH-1, SDH-2, SDH-3) is the fastest, and can be basically completely degraded in 65 days; while the degradation rate is the slowest at pH 6.0, The degradation rates of sericin hydrogels (SDH-1, SDH-2, SDH-3) in 65 days were 40.58%, 43.45%, 38.14%, respectively.

Example 9 In Vitro Biocompatibility Test of Sericin Hydrogel

1. Experimental process

1. First use DMEM high-glucose medium to culture human liver cells HL7702 and mouse myoblasts C2C12 for 24 hours at 37°C under 5% CO ₂ and 100% humidity. Liver cells HL7702 and mouse myoblasts C2C12 were resuspended in culture medium, blown out, and planted in 96-well plates, with the number of cells per well being about 8000;

2. Then spread the sericin hydrogel (SDH-1, SDH-2, SDH-3) in the 96-well plate containing the cells, and set the sericin hydrogel addition amount compared to the cell culture medium to be respectively 4g/L, 8g/L, 16g/L, 32g/L, 64g/L, each concentration and each mass of sericin hydrogel in various three-well parallel samples;

3. Next, incubate for 48 hours;

4. After 48 hours, add 10 μL CCK-8 to each well in 3 and incubate for 1 hour;

5. Use a microplate reader to measure its absorbance at 490nm and 630nm.

2. Experimental analysis

1. Test of the cytotoxicity of sericin hydrogel (SDH-1, SDH-2, SDH-3) to HL7702 and C2C12 cells: as shown in Figure 5, sericin hydrogel (SDH-1, SDH-2 , SDH-3) has basically no toxicity to HL7702 and C2C12 cells, and the cell survival rate (ie, cell viability) is basically maintained at about 90%;

2. Experimental results prove that the sericin hydrogel has better biocompatibility.

Example 10 Sericin hydrogel as a macromolecular drug HRP controlled release carrier

1. Experimental process

1. Mix 4 μL of horseradish peroxidase (HRP) solution (2 μg/μL) with 150 μL of hydrazide-modified sericin solution (200 g/L);

2. Add oxidized dextran with a volume ratio of 150 μL (concentrations are 100 g/L, 200 g/L, and 350 g/L) to 1, leave it at room temperature for 0.5 hours, and then put it in a refrigerator at 4°C for 24 hours;

3. Add 2 mL of PBS (pH 7.4) to the sample and place at 37°C;

4. On days 0.5, 1, 2, 3, 4, 6, 8, 12, 19, 24, 30, 40, and 50, carefully remove the supernatant and re-add 1 mL of PBS (pH 7.4);

5. The HRP content in the supernatant was measured by enzyme-linked immunosorbent assay (ELISA), and the cumulative release rate was obtained by calculating the ratio of the accumulated HRP content in the supernatant to the actual drug loading at each time point.

2. Experimental analysis

HRP Release Analysis from Sericin Hydrogel

Mix the horseradish peroxidase (HRP) solution and the hydrazide-modified sericin solution at a volume ratio of 2/75, and add oxidized dextran with an oxidation degree of 35% equal to the volume of the hydrazide-modified sericin solution. Sugar solution (concentration 100g/L, 200g/L, 350g/L), placed at room temperature for 0.5 hours, then placed in a refrigerator at 4°C for 24 hours, added PBS (pH 7.4) to the sample and placed at 37°C. At the corresponding time points, carefully collect the supernatant, and re-add PBS (pH 7.4), measure the HRP content in the collected supernatant by enzyme-linked immunoassay (ELISA), and calculate the HRP content in the supernatant at each time point. The ratio of the cumulative HRP content to the actual drug loading is the cumulative release rate.

As shown in Figure 6A: Sericin hydrogel has a good controlled release effect on macromolecular protein drugs (HRP), and the release rate can be adjusted by adjusting the concentration of oxidized dextran, which is a good carrier material for macromolecular protein drugs .

Example 11 Sericin Hydrogel as a Controlled Release Carrier of Small Molecule Antitumor Drug Doxorubicin

1. Experimental process

1. Mix 6 μL doxorubicin (DOX) solution (20 μg/μL) with 150 μL hydrazide-modified sericin solution (20%, g/L);

2. Add oxidized dextran with a volume ratio of 150 μL (concentrations are 100 g/L, 200 g/L, and 350 g/L) to 1, leave it at room temperature for 0.5 hours, and then put it in a refrigerator at 4°C for 24 hours;

3. Add 1 mL of PBS (pH 7.4) to the sample and place at 37°C;

4. On days 0.5, 1, 2, 3, 4, 7, 10, 15, 22, 32, and 42, carefully remove the supernatant and re-add 1 mL of PBS (pH 7.4);

5. Use the Nanodrop to measure the absorbance value of the supernatant, calculate the accumulated doxorubicin content in the supernatant at each time point through the standard curve, and calculate the cumulative release rate by the ratio of the obtained value to the actual drug loading.

2. Experimental analysis

Analysis of doxorubicin release from sericin hydrogel

Mix the doxorubicin (DOX) solution with the hydrazide-modified sericin solution at a volume ratio of 1/50, and add an oxidized dextran solution ( Concentration 100g/L, 200g/L, 350g/L), placed at room temperature for 0.5 hours, then placed in a refrigerator at 4°C for 24 hours, added PBS (pH 7.4) to the sample and placed at 37°C. At the corresponding time points, carefully collect the supernatant, and add PBS (pH 7.4) again, use the Nanodrop to measure the absorbance value of the supernatant, and calculate the accumulated doxorubicin content in the supernatant at each time point through the standard curve, and the obtained The ratio of the value to the actual drug loading is the cumulative release rate

As shown in Figure 6B: sericin hydrogel has a good controlled release effect on small molecule chemical drugs, and can regulate the release rate of small molecule chemical drugs by adjusting the concentration of oxidized dextran, which is a good small molecule chemical drug carrier material .

Example 12 In Vitro Fluorescent Properties of Sericin Hydrogel

1. Fluorescent property test of sericin

1. Take 100 μg each of freeze-dried sodium carbonate-extracted sericin powder and hydrazide-modified sericin powder respectively to prepare a sericin solution with a concentration of 100 μg/mL;

2. Test the fluorescence spectrum of the sericin solution in 1 from 320nm to 600nm with a fluorescence spectrum tester.

2. Testing of fluorescence properties of sericin hydrogels (SDH-1, SDH-2, SDH-3)

1. Take 100 μL each of sericin hydrogels (SDH-1, SDH-2, SDH-3) to prepare sericin hydrogel membranes;

2. Test the fluorescence spectrum of the sericin hydrogels (SDH-1, SDH-2, SDH-3) in 1 from 320nm to 600nm with a fluorescence spectrum tester.

3. Laser confocal test of sericin hydrogel (SDH-1, SDH-2, SDH-3) freeze-dried products

1. Prepare sericin hydrogels (SDH-1, SDH-2, SDH-3) and obtain lyophilized products after lyophilization;

2. Observing the freeze-dried products of sericin hydrogels (SDH-1, SDH-2, SDH-3) by laser confocal microscope.

5. Experimental analysis

1. As shown in Figures 7A and 7B, compared with sericin extracted from sodium carbonate, the fluorescence spectrum of hydrazide-modified sericin does not change significantly, indicating that hydrazide modification has no effect on the fluorescence characteristics of sericin. no significant impact;

2. As shown in Figure 7C, 7D, and 7E, the fluorescence spectrum of sericin hydrogel did not change significantly compared with sericin and hydrazide-modified sericin, indicating that sericin hydrogel still Maintain the fluorescent properties of sericin;

3. As shown in Figures 7F and 7G, laser confocal results show that the sericin hydrogel freeze-dried product still maintains the fluorescence properties of sericin.

Example 13 In vivo fluorescence properties of sericin hydrogel

1. In vivo fluorescence test of sericin hydrogel (SDH-1, SDH-2, SDH-3) mice

1. Inject 200 μL each of sericin hydrogels (SDH-1, SDH-2, SDH-3) into the back of C57BL/6J mice by injection;

2. Use the small animal imager to observe its fluorescence when the excitation light is 420nm and the emission light is 520nm.

2. Fluorescence tracer test of doxorubicin released from sericin hydrogel in mice

1. Mix 2 μL doxorubicin (DOX) solution (20 μg/μL) with 100 μL hydrazide-modified sericin solution (200 g/L);

2. Add oxidized dextran with a volume ratio of 100 μL (concentration: 200 g/L) to 1, and inject it into C57BL/6J mice subcutaneously by injection;

3. The C57BL/6J mice were tested on Day 1, 0, 2, 4, 8, 12, and 18 using a small animal imager.

3. Experimental analysis

1. As shown in Figure 8A, the small animal imaging results show that sericin hydrogels (SDH-1, SDH-2, SDH-3) still maintain their fluorescence properties in C57BL/6J mice, which can be used to Fluorescent properties for in vivo tracer drug release.

2. As shown in Figures 8B and 8C, the specific position of the drug carrier in the body can be located through the fluorescence properties of the sericin hydrogel, and the release of the drug in the body can be monitored by detecting the fluorescence intensity of doxorubicin.

Example 14

1. Fluorescence properties of sericin hydrogel to monitor degradation in vivo

1. Prepare 200 μL each of sericin hydrogel (SDH-2) and inject it into the back of C57BL/6J mice by injection;

2. On days 4, 8, 12, 16, 22, 40, and 70, carefully remove the gel from the back of the mouse, freeze-dry the gel and weigh it.

2. Experimental analysis

As shown in Figures 8D and 8E, the sericin hydrogel has a strong correlation with its degradation in vivo, and its degradation in vivo can be known by monitoring the fluorescence intensity of the sericin hydrogel.

Example 15

1. Sericin hydrogel loaded with doxorubicin (DOX) inhibits melanoma

1. Inject 0.2 mL of PBS solution containing 2×10 ⁵ B16F10 mouse melanoma cells into the back of C57BL/6J mice by injection;

^2. When the tumor grows to 30-40mm2, the mice (each weighing 25g) are randomly divided into four groups, namely: SDH-2+DOX group; SDH-2 group; DOX group; PBS group;

3. SDH-2 (200 μL/mouse body weight)+DOX (4 mg/kg mouse body weight); SDH-2 (200 μL); DOX (4 mg/kg); PBS (200 μL) was injected into the tumor by injection nearby;

4. Detect the tumor size of the mice every other day, and record the survival period and body weight;

5. On the 15th day, one mouse was randomly selected from the four groups, the tumor was cut out to observe the size, the tumor tissue was embedded in paraffin, and the sections were stained with H&E and TUNEL.

2. Experimental analysis

1. As shown in Figure 9A, the tumor size of the SDH-2+DOX group was significantly smaller than that of the other three groups, indicating that the sericin hydrogel loaded with doxorubicin can significantly inhibit melanoma;

2. As shown in Figure 9B, the body weight of the mice in the four groups did not change significantly, indicating that the sericin hydrogel loaded with doxorubicin has no obvious side effects;

3. As shown in Figure 9C, the survival period of the SDH-2+DOX group was significantly longer than that of the other three groups, indicating that the sericin hydrogel loaded with doxorubicin can significantly inhibit melanoma;

4. As shown in Figure 9D, on the 15th day, the tumor size of the SDH-2+DOX group was significantly smaller than that of the other three groups, indicating that the sericin hydrogel loaded with doxorubicin can significantly inhibit melanoma;

5. As shown in Figure 9E, the degree of apoptosis in the SDH-2+DOX group was significantly greater than that in the other three groups, indicating that the sericin hydrogel loaded with doxorubicin can significantly inhibit melanoma;

6. As shown in Figure 9F, the degree of apoptosis in the SDH-2+DOX group was significantly greater than that in the other three groups, indicating that the sericin hydrogel loaded with doxorubicin can significantly inhibit melanoma.

In summary, compared with the DOX group alone or the SDH-2 group alone, the sericin hydrogel carrying doxorubicin (SDH-2+DOX) group of the present application has a significantly enhanced ability to inhibit melanoma

Example 16

1. Application of Sericin Hydrogel in Tissue Repair

^1. Immerse a cylindrical stainless steel column with a bottom diameter of 1cm2 in water at 100°C for 10 minutes;

2. Take out the cylinder 1 and press it on the back of the C57BL/6J mouse for 5 seconds;

3. The C57BL/6J mice in 2 were randomly divided into two groups: Duoderm (Duoderm) group, Duoderm+SDH-2 group;

4. On the third day, cut off the scalded skin on the back of the C57BL/6J mice in 2, and apply Duoaifu+SDH-2;

5. Take step 4 as the starting point of treatment, and observe the wound recovery on the 7th, 14th, and 21st days after treatment.

2. Experimental analysis

1. As shown in Figure 10, the wound size of C57BL/6J mice in the Duoderm+SDH-2 group was smaller than that in the Duoderm group on the 7th and 14th day. It shows that the use of the sericin hydrogel of the present application and Duoaifu at the same time has a significantly improved wound repair effect compared with the separate commercial Duoaifu dressing.

Claims (10)

1. a kind of preparation method of sericin hydrogel is it is characterised in that comprise the following steps:

1) extract sericin, and be made into powder:

Weigh silkworm Bombyx bombycis, and be cut into fragment, cleaned with water, remove moisture removal；

Add the 0.01-0.2mol/l of 10-50ml deionized water or 10-50ml by every gram of Bombyx bombycis fragment na₂co₃Aqueous solution, then stirs 0.5-3 hour under the conditions of 70-100 DEG C, so that sericin is dissolved, Obtain sericin solution；

Centrifugation removes the insoluble substance in described sericin solution, and sericin solution 12～72 of dialysing Hour, obtain clarification sericin solution；

This clarification sericin solution of lyophilizing, obtains sericin powder；

2) prepare hydrazides modification sericin:

By step 1) in the sericin powder that obtains 30-200ml is dissolved in 0.2-10g/ml ratio Dimethyl sulfoxide in, add the ratio of 0.1-2g according still further to every gram of sericin powder, add n, n- carbonyl Base diimidazole reacts 1～3 hour, obtains the dimethyl sulphoxide solution of sericin；

Adipic dihydrazide is dissolved in 50-400ml dimethyl sulfoxide with 0.5-50g/ml ratio simultaneously, Obtain the dimethyl sulphoxide solution of adipic dihydrazide；

And add 6-10g adipic dihydrazide by every gram of sericin, by the two of described adipic dihydrazide Methyl sulfoxide solution is mixed with the dimethyl sulphoxide solution of described sericin, is placed in reaction at 20～45 DEG C 0.5～36 hour, reactant liquor is dialysed 12～72 hours, obtains the sericin solution that hydrazides is modified, The freeze-dried sericin powder obtaining hydrazides modification again；Or

By step 1) in the sericin powder that obtains be dissolved in 0.001-0.1g/ml ratio The mass percent of 100-1000ml is 2% MES solution, according still further to every gram of sericin Powder adds 1- (3- the dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride of 8-10g and the n- of 4-40g N-Hydroxysuccinimide, is stirred at room temperature 12-24 hour, obtains sericin reactant liquor；Will be according to every liter Described sericin reactant liquor adds the ratio of 2.5-25ml, and the hydrazine hydrate that mass percent is 85% is molten Drop is added in described sericin reactant liquor, reacts 12-24 hour, reactant liquor is dialysed under room temperature 12～72 hours, obtain the sericin solution that hydrazides is modified, the more freeze-dried sericin obtaining hydrazides modification Protein powder；

3) prepare oxidized dextran:

Weigh glucosan, sodium metaperiodate respectively with 0.1-0.8g/ml, 0.01-0.2g/ml ratio is dissolved in Glucan aqueous solution and sodium metaperiodate aqueous solution is obtained in 10-50ml water；

The sodium periodate solution of 10-50ml is slowly dropped to the glucosan water of 10-50ml under ice bath In solution, under the conditions of 4 DEG C, lucifuge is reacted 1～24 hour；

By reactant liquor dialyse 12～72 hours, more freeze-dried obtain oxidizability be 10-40% oxidized dextran Powder；

4) sericin solution and the oxidized dextran solution of hydrazides modification are prepared

By step 2) the sericin powder modified of the hydrazides that obtains is dissolved in distilled water, and making concentration is The sericin solution that 50-400g/l hydrazides is modified；By step 3) the oxidized dextran powder that obtains is dissolved in In distilled water, making concentration is 5-500g/l oxidized dextran solution；

5) by 1:1-5 volume ratio by 4) in the sericin solution modified of the hydrazides that obtains and oxidation Portugal Polysaccharide solution is mixed to get described sericin hydrogel.

2. preparation method according to claim 1 is it is characterised in that the molecular weight of described glucosan For 40-120kda.

3. a kind of preparation method of sericin hydrogel is it is characterised in that comprise the following steps:

1) extract sericin, and be made into powder:

Weigh silkworm Bombyx bombycis, and be cut into fragment, cleaned with water, remove moisture removal；

Add the 0.01-0.2mol/l of 10-50ml deionized water or 10-50ml by every gram of Bombyx bombycis fragment na₂co₃Aqueous solution, then stirs 0.5-3 hour under the conditions of 70-100 DEG C, so that sericin is dissolved, Obtain sericin solution；

Centrifugation removes the insoluble substance in described sericin solution, and sericin solution 12～72 of dialysing Hour, obtain clarification sericin solution；

This clarification sericin solution of lyophilizing, obtains sericin powder；

2) prepare hydrazides modification sericin:

By step 1) in the sericin powder that obtains 30-200ml is dissolved in 0.2-10g/ml ratio Dimethyl sulfoxide in, add the ratio of 0.1-2g according still further to every gram of sericin powder, add n, n- carbonyl Base diimidazole reacts 1～3 hour, obtains the dimethyl sulphoxide solution of sericin；

Adipic dihydrazide is dissolved in 50-400ml dimethyl sulfoxide with 0.5-50g/ml ratio simultaneously, Obtain the dimethyl sulphoxide solution of adipic dihydrazide；

And add 6-10g adipic dihydrazide by every gram of sericin, by the two of described adipic dihydrazide Methyl sulfoxide solution is mixed with the dimethyl sulphoxide solution of described sericin, is placed in reaction at 20～45 DEG C 0.5～36 hour, reactant liquor is dialysed 12～72 hours, obtains the sericin solution that hydrazides is modified, The freeze-dried sericin powder obtaining hydrazides modification again；Or

By step 1) in the sericin powder that obtains be dissolved in 0.001-0.1g/ml ratio The mass percent of 100-1000ml is 2% MES solution, according still further to every gram of sericin Powder adds 1- (3- the dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride of 8-10g and the n- of 4-40g N-Hydroxysuccinimide, is stirred at room temperature 12-24 hour, obtains sericin reactant liquor；Will be according to every liter Described sericin reactant liquor adds the ratio of 2.5-25ml, and the hydrazine hydrate that mass percent is 85% is molten Drop is added in described sericin reactant liquor, reacts 12-24 hour, reactant liquor is dialysed under room temperature 12～72 hours, obtain the sericin solution that hydrazides is modified, the more freeze-dried sericin obtaining hydrazides modification Protein powder；

3) prepare the sericin solution of hydrazides modification

By step 2) the sericin powder modified of the hydrazides that obtains is dissolved in distilled water, and making concentration is The sericin solution that 50-400g/l hydrazides is modified；

4) by 6:1-3:1 volume by 3) in the sericin solution modified of the hydrazides that obtains with concentration be The genipin aqueous solution of 10g/l is obtained described sericin hydrogel.

4. a kind of preparation method of sericin hydrogel is it is characterised in that comprise the following steps:

1) extract sericin, and be made into powder:

Weigh silkworm Bombyx bombycis, and be cut into fragment, cleaned with water, remove moisture removal；

Add the 0.01-0.2mol/l of 10-50ml deionized water or 10-50ml by every gram of Bombyx bombycis fragment na₂co₃Aqueous solution, then stirs 0.5-3 hour under the conditions of 70-100 DEG C, so that sericin is dissolved, Obtain sericin solution；

Centrifugation removes the insoluble substance in described sericin solution, and sericin solution 12～72 of dialysing Hour, obtain clarification sericin solution；

This clarification sericin solution of lyophilizing, obtains sericin powder；

2) prepare hydrazides modification sericin:

By step 1) in the sericin powder that obtains 30-200ml is dissolved in 0.2-10g/ml ratio Dimethyl sulfoxide in, add the ratio of 0.1-2g according still further to every gram of sericin powder, add n, n- carbonyl Base diimidazole reacts 1～3 hour, obtains the dimethyl sulphoxide solution of sericin；

Adipic dihydrazide is dissolved in 50-400ml dimethyl sulfoxide with 0.5-50g/ml ratio simultaneously, Obtain the dimethyl sulphoxide solution of adipic dihydrazide；

And add 6-10g adipic dihydrazide by every gram of sericin, by the two of described adipic dihydrazide Methyl sulfoxide solution is mixed with the dimethyl sulphoxide solution of described sericin, is placed in reaction at 20～45 DEG C 0.5～36 hour, reactant liquor is dialysed 12～72 hours, obtains the sericin solution that hydrazides is modified, The freeze-dried sericin powder obtaining hydrazides modification again；Or

By step 1) in the sericin powder that obtains be dissolved in 0.001-0.1g/ml ratio The mass percent of 100-1000ml is 2% MES solution, according still further to every gram of sericin Powder adds 1- (3- the dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride of 8-10g and the n- of 4-40g N-Hydroxysuccinimide, is stirred at room temperature 12-24 hour, obtains sericin reactant liquor；Will be according to every liter Described sericin reactant liquor adds the ratio of 2.5-25ml, and the hydrazine hydrate that mass percent is 85% is molten Drop is added in described sericin reactant liquor, reacts 12-24 hour, reactant liquor is dialysed under room temperature 12～72 hours, obtain the sericin solution that hydrazides is modified, the more freeze-dried sericin obtaining hydrazides modification Protein powder；

3) prepare the sericin solution of hydrazides modification

By step 2) the sericin powder modified of the hydrazides that obtains is dissolved in distilled water, and making concentration is The sericin solution that 50-400g/l hydrazides is modified；

4) by 100:1-5 volume by 3) in the sericin solution modified of the hydrazides that obtains and 250g/l Glutaraldehyde water solution be mixed to prepare described sericin hydrogel.

5. a kind of sericin hydrogel is it is characterised in that prepare described in any one of claim 1-4 Method obtains.

6. application in fluorescent probe for the sericin hydrogel described in claim 5.

7. application in pharmaceutical carrier for the sericin hydrogel described in claim 5.

8. application in preparing tissue engineering material for the sericin hydrogel described in claim 5.

9. a kind of sericin hydrogel lyophilizing support is it is characterised in that by will be according to claim 5 Described sericin hydrogel is placed in subzero and freezes and be vacuum dried and obtains.

10. application in organizational project for the sericin hydrogel lyophilizing support described in claim 9.