CN117599247B - Silk fibroin microcarrier skin filling preparation, and preparation method and application thereof - Google Patents

Abstract

The invention belongs to the technical field of silk fibroin, and particularly relates to a silk fibroin microcarrier skin filling preparation, a preparation method and application thereof. The method comprises the following steps: degumming, concentrating and drying mulberry silk to obtain dry silk fibroin; preparing a dry silk fibroin into an aqueous solution, mixing the aqueous solution with vitamin C, stirring, and freeze-drying to obtain VC-loaded silk fibroin; preparing VC-loaded silk fibroin into silk fibroin solution, mixing the silk fibroin solution, hyaluronic acid, maltodextrin and a cross-linking agent, standing at 35-40 ℃ to obtain composite gel, and sterilizing to obtain the silk fibroin microcarrier skin filling preparation. The silk fibroin microcarrier skin filling preparation prepared by the invention has better mechanical property, good biocompatibility and high application value.

Description

Silk fibroin microcarrier skin filling preparation and preparation method and application thereof

Technical Field

The invention belongs to the technical field of silk fibroin, and in particular relates to a silk fibroin microcarrier skin filling preparation and a preparation method and application thereof.

Background technique

Silk fibroin is a high molecular weight protein that mainly contains 18 amino acids. Silk fibroin has excellent mechanical properties, biocompatibility and biodegradability, and has good application prospects in the fields of biomaterials and tissue engineering.

At present, silk fibroin is used in various forms (Lin Nan, Zuo Baoqi. Preparation and research progress of medical regenerated silk fibroin materials [J]. Modern Silk Science and Technology, 2020, 35(3):9.), including:

(1) preparing a regenerated silk fibroin solution, specifically including a degumming step and a dissolving step. Since the synergistic effect of silk fibroin and sericin will produce a certain immune response, degumming is a key step in using silk fibroin as a biomaterial. Silk can be dissolved after degumming, and the dissolved silk fibroin can be made into a variety of dosage forms.

(2) Preparation of silk fibroin materials, including preparation of hydrogels, porous scaffolds, films, microspheres and other materials. The preparation methods of hydrogels include cyclonic shearing, ultrasonic oscillation, acid treatment, natural gelation, etc.; porous scaffolds are prepared by freeze drying, particle pore formation and other steps; films are prepared by casting and other methods; microspheres are prepared by spray drying, freeze drying and other methods.

The main function of skin filling preparations is tissue repair. Through local injection of minimally invasive therapy, substances with skin tissue repair function are injected into the local soft tissue with loose structure to improve skin laxity, so that the tissue can restore normal function and appearance. Existing patents have studied the technology of preparing tissue filling materials using silk fibroin, such as the scheme recorded in Chinese patent CN102836465B. The patent combines silk fibroin with hyaluronic acid to prepare a composite gel, which significantly improves the in vitro anti-enzymatic ability of the gel, thereby slowing down the degradation rate of the composite gel, and finally prolonging the action time of the composite gel in tissue repair and filling. In addition, the patent adds hyaluronic acid to the silk fibroin particles, which increases the water absorption and water storage function, so the water storage function and filling effect of the prepared composite gel are also good. However, the skin repair function of hyaluronic acid alone cannot meet the needs of skin fillers. Some patients with poor skin need to add other auxiliary factors to improve skin conditions.

Summary of the invention

In order to solve the technical problem of "adding other auxiliary factors to improve skin conditions", we use silk fibroin and other substances as raw materials to make gel and develop a new silk fibroin microcarrier skin filling preparation. Specifically, the present invention provides a silk fibroin microcarrier skin filling preparation and its preparation method and application. Compared with the composite gel of the prior art, our technical solution can load multiple auxiliary factors such as vitamin B5 and type I collagen, with a high auxiliary factor loading rate, which helps to improve skin conditions.

An object of the present invention is to provide a method for preparing a silk fibroin microcarrier skin filling preparation, comprising:

Degumming, concentrating and drying the mulberry silk to obtain dry silk fibroin;

The dried silk fibroin is prepared into an aqueous solution, the aqueous solution is mixed with vitamin C, stirred, and freeze-dried to obtain VC-loaded silk fibroin;

The VC-loaded silk fibroin is prepared into a silk fibroin aqueous solution, and the silk fibroin aqueous solution, hyaluronic acid, maltodextrin and a cross-linking agent are mixed, and allowed to stand at 35-40° C. to obtain a composite gel, and then sterilized to obtain a silk fibroin microcarrier skin filling preparation.

Preferably, in the preparation method of the above-mentioned silk fibroin microcarrier skin filling preparation, double distilled water is used as a solvent for drying silk fibroin to prepare an aqueous solution with a mass fraction of 5-10%, the aqueous solution is mixed with vitamin C in a mass ratio of 90-110:1, stirred for 15-30 minutes, and freeze-dried to obtain VC-loaded silk fibroin.

Preferably, in the preparation method of the above-mentioned silk fibroin microcarrier skin filling preparation, double distilled water is used as a solvent for drying silk fibroin to prepare a 5% by mass aqueous solution, the aqueous solution is mixed with vitamin C in a mass ratio of 100:1, stirred for 20 minutes, and freeze-dried to obtain VC-loaded silk fibroin.

Preferably, the preparation method of the above-mentioned silk fibroin microcarrier skin filling preparation is to use PEG300-PEG600 as solvent, configure VC loaded silk fibroin into a 30-40g/L silk fibroin solution, mix the silk fibroin solution, hyaluronic acid, maltodextrin and cross-linking agent in a mass ratio of 10:1:0.5-0.8:0.2-0.5, and stand at 35-40° C. to obtain a composite gel. The addition of maltodextrin can improve the auxiliary factor loading performance of the composite gel.

Preferably, the preparation method of the above-mentioned silk fibroin microcarrier skin filler preparation is to use PEG300 as solvent, configure VC-loaded silk fibroin into a 30 g/L silk solution, mix the silk solution, hyaluronic acid, maltodextrin and cross-linking agent in a mass ratio of 10:1:0.5:0.2, and let stand at 40°C to obtain a composite gel.

Preferably, in the preparation method of the above-mentioned silk fibroin microcarrier skin filling preparation, the crosslinking agent is one or more of octyl sodium sulfate solution and sodium dodecyl sulfate. Further preferably, the crosslinking agent is one or more of octyl sodium sulfate solution with a concentration of 15g/L and sodium dodecyl sulfate with a concentration of 15g/L. Preferably, after the crosslinking agent is mixed with the polymer compound, it is mixed with the silk fibroin solution and maltodextrin, and allowed to stand at 35-40°C to obtain a composite gel.

Under conditions suitable for cross-linking, the polymer compound undergoes a cross-linking reaction in the presence of the cross-linking agent, for example, the polymer compound is one or more selected from the group consisting of hyaluronic acid, sodium hyaluronate, chitosan, carboxymethyl chitosan, chitin, carboxymethyl chitin, collagen, gelatin, silk fibroin, carboxymethyl dextran, carboxymethyl cellulose, amino polyethylene glycol, carboxyl polyethylene glycol. The initial mass concentration of the polymer compound in the solution is 0.5%-10%, for example 0.6%-8%.

Preferably, in the preparation method of the above-mentioned silk fibroin microcarrier skin filling preparation, the degumming method comprises:

(1) boiling the silk with an alkaline solution for 30 minutes, filtering, and collecting the precipitate; wherein the alkaline solution is a mixture of a sodium carbonate aqueous solution with a mass fraction of 0.05% and a sodium L-glutamate aqueous solution with a mass fraction of 0.03% in a volume ratio of 20:1;

(2) boiling the precipitate with the alkaline solution for 30 minutes and collecting the solution;

(3) Repeat step (2) until the silk is completely degummed. The completely degummed material is called silk fibroin solution.

The present invention also provides a silk fibroin microcarrier skin filling preparation prepared by the method.

The present invention also provides a silk fibroin microcarrier skin filling preparation loaded with auxiliary factors, and the preparation method is as follows: degumming and drying mulberry silk to obtain dry silk fibroin;

The dried silk fibroin is prepared into an aqueous solution, the aqueous solution is mixed with vitamin C, stirred, and freeze-dried to obtain VC-loaded silk fibroin;

Using PEG300-PEG600 as solvent, VC loaded silk protein is prepared into silk solution, silk solution, hyaluronic acid, maltodextrin, auxiliary factor and cross-linking agent are mixed in a mass ratio of 10:1:0.5-0.8:0.1:0.2-0.5, and allowed to stand at 35-40°C to obtain a composite gel, which is sterilized to obtain a silk protein microcarrier skin filling preparation. Preferably, PEG300, PEG400 or PEG600 is used as solvent.

It should be noted that, under the inventive concept of the present invention, the auxiliary factors can be selected from the following: vitamin B5, skin growth factor, vitamin E, vitamin A, type I collagen and any other substance with skin repair effect.

The present invention also provides an application of a silk fibroin microcarrier skin filling preparation. Specifically, the silk fibroin microcarrier skin filling preparation is used to fill skin wrinkles or skin depressions.

Compared with the prior art, the present invention has the following beneficial effects:

The present invention improves silk fibroin so that it is first loaded with VC and then used to prepare a filling preparation, which can improve the anti-enzymatic ability of the filling preparation and present a good skin filling effect. Principle: Silk fibroin has adsorption properties. Studies have shown that it can adsorb metals. The present invention uses silk fibroin to adsorb VC, and then uses the VC to load silk fibroin to prepare a filling preparation. VC can be wrapped in the filling preparation and used as a nutrient component, and the anti-enzymatic ability of the filling preparation can be improved, thereby improving the effectiveness of the material in filling the skin.

Furthermore, in the present invention, maltodextrin is used in combination with a modified silk fibroin solution, and the prepared filling preparation has better anti-enzymatic effect and auxiliary factor loading performance. Principle: Maltodextrin is a low-conversion product between starch and starch sugar, has a filling effect, good carrier properties, and good stability. Maltodextrin is commonly used as a drug carrier in the medical field. The present invention takes into account the chemical structures of maltodextrin and various auxiliary factors such as vitamin B5 and type I collagen, and provides a specific loading method. Under the loading method of the present invention, the loading rate of the auxiliary factor is high, and the anti-enzymatic performance of the prepared filling preparation is also improved.

Based on the above principles and our experimental results, the silk fibroin microcarrier skin filling preparation prepared by the present invention has good anti-enzymatic effect and auxiliary factor loading performance, good biocompatibility, and high application value.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 Skin filling results of volunteer 1, A, before filling, B, after filling.

Figure 2 Skin filling results of volunteer 2, A, before filling, B, after filling.

Figure 3 Skin filling results of volunteer 3, A, before filling, B, after filling.

In Figures 1, 2 and 3, the direction indicated by the arrow is the filling position.

Detailed ways

In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention is further described below in conjunction with specific embodiments and drawings.

In the description of the present invention, unless otherwise specified, all reagents used are commercially available and all methods used are conventional techniques in the art.

1. Main experimental reagents

Mulberry silk: Available in Zhejiang, it is a natural fiber. It is made by cutting open the silkworm cocoons, removing the pupae and molts inside, and obtaining the white cocoon shell.

Type I collagen: CAS No. 9007-34-5, Shanghai Yuanye Biotechnology Co., Ltd.

Hyaluronic acid: CAS No. 9004-61-9, Shanghai Bangcheng Chemical Co., Ltd.

Maltodextrin: CAS No. 9050-36-6, West Asia Chemical Technology (Shandong) Co., Ltd. It has good chemical stability and biocompatibility.

Vitamin C: has antioxidant properties.

2. Example

Example 1

A method for preparing a silk fibroin microcarrier skin filling preparation comprises the following steps:

1.1 Degumming

1.1.1 Boil the silk with an alkaline solution for 30 minutes, filter and collect the precipitate. The alkaline solution is a mixture of a sodium carbonate aqueous solution with a mass fraction of 0.05% and a sodium L-glutamate aqueous solution with a mass fraction of 0.03% in a volume ratio of 20:1. Sodium L-glutamate has a certain stability to light and heat. Adding it to the silk for treatment can not only improve the stability of silk fibroin, but also accelerate degumming.

1.1.2 Continue to boil the precipitate with the alkaline solution in 1.1.1 for 30 minutes and collect the solution.

1.1.3 Repeat step 1.1.2 until the mulberry silk is completely degummed. The completely degummed material is called silk fibroin solution. Here, the picric acid carmine solution is used to detect whether the degumming is complete.

1.2 Processing

1.2.1 Dialysis, concentration and drying

The silk protein solution of 1.1.3 was filtered with a sand core funnel, and the filtrate was adjusted to pH 4 with acetic acid solution, and then salted out with a saturated sulfate solution for 2 hours. The precipitate was centrifuged to obtain a protein precipitate. The protein precipitate was transferred to a sand core suction filtration funnel with a microporous filter membrane with a pore size of 0.2 μm, and filtered and washed with 75% ethanol and deionized water by volume. The filter cake after elution was ultrasonically dispersed in deionized water and transferred into a dialysis bag with a molecular weight cutoff of 3500Da. The solution in the dialysis bag was concentrated with a 30% aqueous solution of polyethylene glycol 20000 by volume. The concentrated solution was placed in a freeze dryer, pre-frozen at -30°C, and vacuum dried for 10 hours in a frozen state to obtain dry silk.

1.2.2 Improvement of silk fibroin:

Using double distilled water as solvent, the dry silk collected in 1.2.1 was prepared into a 5% aqueous solution by mass, and the aqueous solution was mixed with vitamin C in a mass ratio of 100:1, stirred for 20 minutes, and freeze-dried at -20°C to obtain VC-loaded silk protein.

1.3 Preparation of composite gel

Using PEG300 as solvent, 1.2.2 VC-loaded silk fibroin was configured into a 30 g/L silk fibroin solution; the silk fibroin solution, hyaluronic acid, maltodextrin, auxiliary factors and cross-linking agent were mixed in a mass ratio of 10:1:0.5:0.1:0.2, and allowed to stand at 40°C for 20 min to obtain a composite gel. The composite gel was steam sterilized at 121°C for 15 min and used as a silk fibroin microcarrier skin filler preparation with good biocompatibility and good filling effect.

The cross-linking agent is a sodium octyl sulfate aqueous solution with a concentration of 15 g/L, and the auxiliary factor is vitamin B5.

Example 2

A method for preparing a silk fibroin microcarrier skin filling preparation comprises the following steps:

1.1 Degumming

1.1.1 Boil the silk with an alkaline solution for 30 minutes, filter and collect the precipitate. The alkaline solution is a mixture of a 0.05% sodium carbonate aqueous solution and a 0.03% sodium L-glutamate aqueous solution in a volume ratio of 20:1.

1.1.2 Continue to boil the precipitate with the alkaline solution in 1.1.1 for 30 minutes and collect the solution.

1.1.3 Repeat step 1.1.2 until the mulberry silk is completely degummed. The completely degummed material is called silk fibroin solution. Here, the picric acid carmine solution is used to detect whether the degumming is complete.

1.2 Processing

1.2.1 Dialysis, concentration and drying

The silk protein solution of 1.1.3 was filtered with a sand core funnel, and the filtrate was adjusted to pH 4 with acetic acid solution, and then salted out with a saturated sulfate solution for 2 hours. The precipitate was centrifuged to obtain a protein precipitate. The protein precipitate was transferred to a sand core suction filtration funnel with a microporous filter membrane with a pore size of 0.2 μm, and filtered and washed with 75% ethanol and deionized water by volume. The filter cake after elution was ultrasonically dispersed in deionized water and transferred into a dialysis bag with a molecular weight cutoff of 3500Da. The solution in the dialysis bag was concentrated with a 30% aqueous solution of polyethylene glycol 20000 by volume. The concentrated solution was placed in a freeze dryer, pre-frozen at -30°C, and vacuum dried for 10 hours in a frozen state to obtain dry silk.

1.2.2 Improvement of silk fibroin:

Using double distilled water as solvent, the dry silk collected in 1.2.1 was prepared into a 5% aqueous solution by mass, and the aqueous solution was mixed with vitamin C in a mass ratio of 90:1, stirred for 20 minutes, and freeze-dried at -20°C to obtain VC-loaded silk protein.

1.3 Preparation of composite gel

Using PEG300 as solvent, 1.2.2 VC-loaded silk fibroin was configured into 30 g/L silk fibroin aqueous solution, and the silk fibroin aqueous solution, hyaluronic acid, maltodextrin, auxiliary factors and cross-linking agent were mixed in a mass ratio of 10:1:0.5:0.1:0.2, and allowed to stand at 40°C for 20 min to obtain a composite gel. The composite gel was steam sterilized at 121°C for 15 min and used as a silk fibroin microcarrier skin filling preparation with good biocompatibility and good filling effect.

The cross-linking agent is a 15 g/L sodium octyl sulfate solution, and the auxiliary factor is vitamin B5.

Example 3

A method for preparing a silk fibroin microcarrier skin filling preparation comprises the following steps:

1.1 Degumming

1.1.1 Boil the silk with an alkaline solution for 30 minutes, filter and collect the precipitate. The alkaline solution is a mixture of a sodium carbonate aqueous solution with a mass fraction of 0.05% and a sodium L-glutamate aqueous solution with a mass fraction of 0.03% in a volume ratio of 20:1. Sodium L-glutamate has a certain stability to light and heat. Adding it to the silk for treatment can not only improve the stability of silk fibroin, but also accelerate degumming.

1.1.2 Continue to boil the precipitate with the alkaline solution in 1.1.1 for 30 minutes and collect the precipitate solution.

1.1.3 Repeat step 1.1.2 until the mulberry silk is completely degummed. The completely degummed material is called silk fibroin solution. Here, the picric acid carmine solution is used to detect whether the degumming is complete.

1.2.1 Dialysis, concentration and drying

The silk protein solution of 1.1.3 was filtered with a sand core funnel, and the filtrate was adjusted to pH 4 with acetic acid solution, and then salted out with a saturated sulfate solution for 2 hours. The precipitate was centrifuged to obtain a protein precipitate. The protein precipitate was transferred to a sand core suction filtration funnel with a microporous filter membrane with a pore size of 0.2 μm, and filtered and washed with 75% ethanol and deionized water by volume. The filter cake after elution was ultrasonically dispersed in deionized water and transferred into a dialysis bag with a molecular weight cutoff of 3500Da. The solution in the dialysis bag was concentrated with a 30% aqueous solution of polyethylene glycol 20000 by volume. The concentrated solution was placed in a freeze dryer, pre-frozen at -30°C, and vacuum dried for 10 hours in a frozen state to obtain dry silk.

1.2.2 Improvement of silk fibroin:

Using double distilled water as solvent, the dry silk collected in 1.2.1 was prepared into a 5% aqueous solution by mass, and the aqueous solution was mixed with vitamin C in a mass ratio of 110:1, stirred for 20 minutes, and freeze-dried at -20°C to obtain VC-loaded silk protein.

1.3 Preparation of composite gel

Using PEG300 as solvent, 1.2.2 VC-loaded silk fibroin was configured into a 30 g/L silk fibroin solution. The silk fibroin solution, hyaluronic acid, maltodextrin, auxiliary factors and cross-linking agent were mixed in a mass ratio of 10:1:0.5:0.1:0.2, and the mixture was allowed to stand at 40°C for 20 min to obtain a composite gel. The composite gel was steam sterilized at 121°C for 15 min and used as a silk fibroin microcarrier skin filler preparation with good biocompatibility and filling effect.

The cross-linking agent is a 15 g/L sodium octyl sulfate solution, and the auxiliary factor is vitamin B5.

Example 4

A method for preparing a silk fibroin microcarrier skin filling preparation comprises the following steps:

1.1 Degumming

1.1.1 Boil the silk with an alkaline solution for 30 minutes, filter and collect the precipitate. The alkaline solution is a mixture of a sodium carbonate aqueous solution with a mass fraction of 0.05% and a sodium L-glutamate aqueous solution with a mass fraction of 0.03% in a volume ratio of 20:1. Sodium L-glutamate has a certain stability to light and heat. Adding it to the silk for treatment can not only improve the stability of silk fibroin, but also accelerate degumming.

1.1.2 Continue to boil the precipitate with the alkaline solution in 1.1.1 for 30 minutes and collect the solution.

1.1.3 Repeat step 1.1.2 until the mulberry silk is completely degummed. The completely degummed material is called silk fibroin solution. Here, the picric acid carmine solution is used to detect whether the degumming is complete.

1.2 Processing

1.2.1 Dialysis, concentration and drying

The silk protein solution of 1.1.3 was filtered with a sand core funnel, and the filtrate was adjusted to pH 4 with acetic acid solution, and then salted out with a saturated sulfate solution for 2 hours. The precipitate was centrifuged to obtain a protein precipitate. The protein precipitate was transferred to a sand core suction filtration funnel with a microporous filter membrane with a pore size of 0.2 μm, and filtered and washed with 75% ethanol and deionized water in turn. The filter cake after elution was ultrasonically dispersed in deionized water and transferred into a dialysis bag with a molecular weight cutoff of 3500Da. The solution in the dialysis bag was concentrated with a 30% volume fraction of polyethylene glycol 20000 aqueous solution. The concentrated solution was placed in a freeze dryer, pre-frozen at -30°C, and vacuum dried for 10 hours in a frozen state to obtain dry silk.

1.2.2 Improvement of silk fibroin:

Using double distilled water as solvent, the dried silk collected in 1.2.1 was prepared into a 5% aqueous solution, the aqueous solution was mixed with vitamin C in a mass ratio of 100:1, stirred for 20 minutes, and freeze-dried at -20°C to obtain VC-loaded silk protein. 1.3 Preparation of composite gel.

Using PEG300 as solvent, 1.2.2 VC-loaded silk fibroin was prepared into 30 g/L silk fibroin solution, and the silk solution, hyaluronic acid, maltodextrin, auxiliary factors and cross-linking agent were mixed in a mass ratio of 10:1:0.6:0.1:0.2, and allowed to stand at 40°C for 20 min to obtain a composite gel. The composite gel was steam sterilized at 121°C for 15 min and used as a silk fibroin microcarrier skin filler preparation with good biocompatibility and good filling effect.

The cross-linking agent is a 15 g/L sodium octyl sulfate solution, and the auxiliary factor is vitamin B5.

Example 5

A method for preparing a silk fibroin microcarrier skin filling preparation comprises the following steps:

1.1 Degumming

1.1.1 Boil the silk with an alkaline solution for 30 minutes, filter and collect the precipitate. The alkaline solution is a mixture of a sodium carbonate aqueous solution with a mass fraction of 0.05% and a sodium L-glutamate aqueous solution with a mass fraction of 0.03% in a volume ratio of 20:1. Sodium L-glutamate has a certain stability to light and heat. Adding it to the silk for treatment can not only improve the stability of silk fibroin, but also accelerate degumming.

1.1.2 Continue to boil the precipitate with the alkaline solution in 1.1.1 for 30 minutes and collect the solution.

1.1.3 Repeat step 1.1.2 until the mulberry silk is completely degummed. The completely degummed material is called silk fibroin solution. Here, the picric acid carmine solution is used to detect whether the degumming is complete.

1.2 Processing

1.2.1 Dialysis, concentration and drying

The silk protein solution of 1.1.3 was filtered with a sand core funnel, and the filtrate was adjusted to pH 4 with acetic acid solution, and then salted out with a saturated sulfate solution for 2 hours. The precipitate was centrifuged to obtain a protein precipitate. The protein precipitate was transferred to a sand core suction filtration funnel with a microporous filter membrane with a pore size of 0.2 μm, and filtered and washed with 75% ethanol and deionized water by volume. The filter cake after elution was ultrasonically dispersed in deionized water and transferred into a dialysis bag with a molecular weight cutoff of 3500Da. The solution in the dialysis bag was concentrated with a 30% aqueous solution of polyethylene glycol 20000 by volume. The concentrated solution was placed in a freeze dryer, pre-frozen at -30°C, and vacuum dried for 10 hours in a frozen state to obtain dry silk.

1.2.2 Improvement of silk fibroin:

Using double distilled water as solvent, the dry silk collected in 1.2.1 was prepared into a 5% aqueous solution by mass, and the aqueous solution was mixed with vitamin C in a mass ratio of 100:1, stirred for 20 minutes, and freeze-dried at -20°C to obtain VC-loaded silk protein.

1.3 Preparation of composite gel

Using PEG300 as solvent, 1.2.2 VC-loaded silk fibroin was configured into a 30 g/L silk fibroin solution. The silk fibroin solution, hyaluronic acid, maltodextrin, auxiliary factors and cross-linking agent were mixed in a mass ratio of 10:1:0.7:0.1:1, and the mixture was allowed to stand at 40°C for 20 min to obtain a composite gel. The composite gel was steam sterilized at 121°C for 15 min and used as a silk fibroin microcarrier skin filler preparation with good biocompatibility and filling effect.

The cross-linking agent is a 15 g/L sodium octyl sulfate solution, and the auxiliary factor is vitamin B5.

Example 6

A method for preparing a silk fibroin microcarrier skin filling preparation comprises the following steps:

1.1 Degumming

1.1.1 Boil the silk with an alkaline solution for 30 minutes, filter and collect the precipitate. The alkaline solution is a mixture of a sodium carbonate aqueous solution with a mass fraction of 0.05% and a sodium L-glutamate aqueous solution with a mass fraction of 0.03% in a volume ratio of 20:1. Sodium L-glutamate has a certain stability to light and heat. Adding it to the silk for treatment can not only improve the stability of silk fibroin, but also accelerate degumming.

1.1.2 Continue to boil the precipitate with the alkaline solution in 1.1.1 for 30 minutes and collect the solution.

1.1.3 Repeat step 1.1.2 until the mulberry silk is completely degummed. The completely degummed material is called silk fibroin solution. Here, the picric acid carmine solution is used to detect whether the degumming is complete.

1.2 Processing

1.2.1 Dialysis, concentration and drying

The silk protein solution of 1.1.3 was filtered with a sand core funnel, and the filtrate was adjusted to pH 4 with acetic acid solution, and then salted out with a saturated sulfate solution for 2 hours. The precipitate was centrifuged to obtain a protein precipitate. The protein precipitate was transferred to a sand core suction filtration funnel with a microporous filter membrane with a pore size of 0.2 μm, and filtered and washed with 75% ethanol and deionized water in turn. The filter cake after elution was ultrasonically dispersed in deionized water and transferred into a dialysis bag with a molecular weight cutoff of 3500Da. The solution in the dialysis bag was concentrated with a 30% volume fraction of polyethylene glycol 20000 aqueous solution. The concentrated solution was placed in a freeze dryer, pre-frozen at -30°C, and vacuum dried for 10 hours in a frozen state to obtain dry silk.

1.2.2 Improvement of silk fibroin:

Using double distilled water as solvent, the dry silk collected in 1.2.1 was prepared into a 5% aqueous solution by mass, and the aqueous solution was mixed with vitamin C in a mass ratio of 100:1, stirred for 20 minutes, and freeze-dried at -20°C to obtain VC-loaded silk protein.

1.3 Preparation of composite gel

Using PEG300 as solvent, 1.2.2 VC-loaded silk fibroin was configured into a 30 g/L silk fibroin solution. The silk fibroin solution, hyaluronic acid, maltodextrin, auxiliary factors and cross-linking agent were mixed in a mass ratio of 10:1:0.8:0.1:1, and the mixture was allowed to stand at 40°C for 20 min to obtain a composite gel. The composite gel was steam sterilized at 121°C for 15 min and used as a silk fibroin microcarrier skin filler preparation with good biocompatibility and filling effect.

The cross-linking agent is a 15 g/L sodium octyl sulfate solution, and the auxiliary factor is vitamin B5.

The silk fibroin microcarrier skin filling preparation prepared in the above examples has good anti-enzyme effect and good biocompatibility.

3. Description of the innovative features of the present invention

(1) Innovation 1: The present invention adopts an improved alkaline solution formula to improve degumming efficiency and shorten degumming time. Different methods are designed as follows:

Test 1-1: Boil the silk with an alkaline solution for 30 minutes, filter and collect the precipitate. The alkaline solution is a mixture of a sodium carbonate aqueous solution with a mass fraction of 0.05% and a sodium L-glutamate aqueous solution with a mass fraction of 0.03% in a volume ratio of 20:1. Boil the precipitate with the alkaline solution for another 30 minutes to check whether the degumming is complete. Repeat the step of "boil the silk with the alkaline solution for 30 minutes to check whether the degumming is complete" until the silk is completely degummed.

Experiment 1-2: The operation is basically the same as that of Experiment 1-1, except that the alkaline solution is replaced by a sodium carbonate aqueous solution with a mass fraction of 0.05%.

In this test, picric acid carmine solution was used to detect whether the degumming was complete. The comparison results of degumming time of different methods are shown in Table 1. Among them, the sodium carbonate aqueous solution with a mass fraction of 0.05% in the prior art was used as the control group.

Table 1 Comparison results of degumming time of different methods

Note: In Table 1, the efficiency improvement rate is calculated based on the time of Test 1-2, so the meaning of “-” is that the efficiency improvement rate of Test 1-2 is not calculated.

The results in Table 1 show that the degumming time of mulberry silk is the shortest when the mixture of sodium carbonate aqueous solution and sodium L-glutamate aqueous solution is boiled. Therefore, we also explored the degumming time of different 0.05% sodium carbonate aqueous solution and 0.03% sodium L-glutamate aqueous solution in order to obtain the best ratio. The results are shown in Table 2.

Table 2 Degumming time of different sodium carbonate aqueous solutions and sodium L-glutamate aqueous solutions

Proportion Degumming time (min) 40:1 240min 30:1 150min 20:1 120min 10:1 150min 5:1 240min

The results in Table 2 show that as the amount of sodium carbonate is gradually reduced, the degumming time shows a trend of first decreasing and then increasing. Among them, when the ratio of the sodium carbonate aqueous solution to the L-sodium glutamate aqueous solution is in the range of 10-30:1, the degumming time is shorter, which is beneficial to saving time cost.

(2) Innovation 2: Improving the ability to resist enzymatic degradation by improving silk fibroin. The experimental setup is as follows.

Experiment 2-1: The method of Example 1 was used to prepare completely degummed dry silk fibroin. Double distilled water was used as solvent to prepare dry silk fibroin into an aqueous solution with a mass fraction of 5%, and the aqueous solution was mixed with vitamin C in a mass ratio of 100:1, stirred for 20 minutes, and freeze-dried at -20°C to obtain VC-loaded silk fibroin protein; PEG300 was used as solvent to prepare VC-loaded silk fibroin protein into a 30g/L silk fibroin solution, and the silk fibroin solution, hyaluronic acid, maltodextrin, auxiliary factor and cross-linking agent were mixed in a mass ratio of 10:1:0.5:0.1:0.2, and allowed to stand at 40°C for 20 minutes to obtain a composite gel. The cross-linking agent was a 15g/L sodium octyl sulfate aqueous solution. The auxiliary factor was vitamin B5.

Experiment 2-2: The method of Example 1 was used to prepare completely degummed dry silk. PEG300 was used as solvent to prepare a 30 g/L silk solution. The silk solution, hyaluronic acid, maltodextrin, auxiliary factor and cross-linking agent were mixed in a mass ratio of 10:1:0.5:0.1:0.2, and allowed to stand at 40°C for 20 min to obtain a composite gel. The cross-linking agent was a 15 g/L sodium octyl sulfate aqueous solution. The auxiliary factor was vitamin B5.

The test method is as follows: after hyaluronic acid is hydrolyzed by hyaluronidase, glucuronic acid can be produced. By testing the glucuronic acid concentration before and after hyaluronidase hydrolysis, the anti-enzymatic ability of the composite gel can be determined.

The results of in vitro enzymatic hydrolysis experiments of composite gels prepared by different silk fibroin treatment methods are shown in Table 3.

Table 3 Results of in vitro enzymatic hydrolysis experiments of composite gels prepared by different silk fibroin treatment methods

The results in Table 3 show that the glucuronic acid concentration after enzymatic hydrolysis in Test 2-1 is lower than that in Test 2-2, indicating that the anti-enzymatic hydrolysis effect is better after adding VC.

We also investigated the effect of different VC dosages in the mixed solution on the in vitro anti-enzymatic performance of the composite gel (the method of experiment 2-1 changed the ratio of aqueous solution to vitamin C). The results are shown in Table 4.

Table 4 Effect of VC dosage on the in vitro anti-enzymatic performance of composite gel

The results in Table 4 show that under the system conditions of the composite gel, with the gradual increase in the amount of VC added, the anti-enzymatic performance shows a trend of first increasing and then tending to be stable, indicating that VC has a strengthening effect on the anti-enzymatic ability of the composite gel system.

(3) Innovation point 3: The composite gel prepared by combining maltodextrin with modified silk fibroin solution has better anti-enzymatic effect and auxiliary factor loading performance.

1) Test on the anti-enzymatic effect of composite gel

The different methods are designed as follows:

Experiment 3-1: The method of Example 1 was used to prepare completely degummed dry silk. Using double distilled water as solvent, the dry silk was prepared into an aqueous solution with a mass fraction of 5%, the aqueous solution was mixed with vitamin C in a mass ratio of 100:1, stirred for 20 minutes, and freeze-dried at -20°C to obtain VC-loaded silk protein; using PEG300 as solvent, the VC-loaded silk protein was prepared into a 30g/L silk solution, the silk solution, hyaluronic acid, maltodextrin, auxiliary factor and cross-linking agent were mixed in a mass ratio of 10:1:0.5:0.1:0.2, and allowed to stand at 40°C for 20 minutes to obtain a composite gel. The cross-linking agent was a 15g/L sodium octyl sulfate aqueous solution. The auxiliary factor was vitamin B5.

Experiment 3-2: The method of Example 1 was used to prepare completely degummed dry silk. Using double distilled water as solvent, the dry silk was prepared into an aqueous solution with a mass fraction of 5%, and the aqueous solution was mixed with vitamin C in a mass ratio of 100:1, stirred for 20 minutes, and freeze-dried at -20°C to obtain VC-loaded silk protein; using PEG300 as solvent, the VC-loaded silk protein was prepared into a 30g/L silk solution, and the silk solution, hyaluronic acid, auxiliary factor and cross-linking agent were mixed in a mass ratio of 10:1:0.1:0.2, and allowed to stand at 40°C for 20 minutes to obtain a composite gel. The cross-linking agent was a 15g/L sodium octyl sulfate aqueous solution. The auxiliary factor was vitamin B5.

The comparison results of the in vitro anti-enzymatic performance of the composite gel of silk fibroin microcarrier skin filling preparations treated by different methods are shown in Table 5.

Table 5 Effects of different methods on the in vitro anti-enzymatic properties of composite gel

The results in Table 5 show that the anti-enzymatic effect of Test 3-1 is better.

We also explored the effects of different ratios of silk fibroin solution, hyaluronic acid, maltodextrin and cross-linking agent on the in vitro anti-enzymatic performance of the composite gel, and the results are shown in Table 6. The results in Table 6 show that within the range of 10:0.5-0.8:0.1:0.2, the anti-enzymatic performance of the material is better.

Table 6 Effect of silk fibroin aqueous solution, maltodextrin and cross-linking agent ratio on the in vitro anti-enzymatic properties of composite gel

2) Auxiliary factor loading rate test

Experiment 3-3: The method of Example 1 was used to prepare completely degummed dry silk. Using double distilled water as solvent, the dry silk was prepared into an aqueous solution with a mass fraction of 5%, the aqueous solution was mixed with vitamin C in a mass ratio of 100:1, stirred for 20 minutes, and freeze-dried at -20°C to obtain VC-loaded silk protein; using PEG300 as solvent, the VC-loaded silk protein was prepared into a 30g/L silk solution, the silk solution, hyaluronic acid, maltodextrin and cross-linking agent were mixed in a mass ratio of 10:1:0.5:1, and allowed to stand at 40°C for 20 minutes to obtain a composite gel. Among them, the cross-linking agent was a 15g/L sodium octyl sulfate aqueous solution. The auxiliary factor was vitamin B5.

Experiment 3-4: The method of Example 1 was used to prepare completely degummed dry silk. Using double distilled water as solvent, the dry silk was prepared into an aqueous solution with a mass fraction of 5%, the aqueous solution was mixed with vitamin C in a mass ratio of 100:1, stirred for 20 minutes, and freeze-dried at -20°C to obtain VC-loaded silk protein; using PEG300 as solvent, the VC-loaded silk protein was prepared into a 30g/L silk solution, the silk solution, hyaluronic acid and cross-linking agent were mixed in a mass ratio of 10:1:1, and allowed to stand at 40°C for 20 minutes to obtain a composite gel. The cross-linking agent was a 15g/L sodium octyl sulfate aqueous solution. The auxiliary factor was vitamin B5.

This experiment explored the loading rate of vitamin B5. The results are shown in Table 7. The results show that the loading rate of vitamin B5 can be increased after adding maltodextrin.

Table 7 Auxiliary factor loading rate test results

test group Cofactor loading rate (%) Test 3-3 5.8 Test 3-4 1.2

4. Application

We also conducted a skin filling and repair test. Figures 1, 2 and 3 show the skin repair results of different volunteers. The arrows in Figures 1, 2 and 3 indicate the filling area. Through the application experiment, it can be seen that the silk fibroin microcarrier skin filling preparation prepared by the present invention is more conducive to skin repair. It should be noted that in order to protect the privacy of the subjects, Figures 1, 2 and 3 only use eye effect pictures.

It should be noted that when the present invention involves a numerical range, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as those in the embodiments, in order to avoid redundancy, the present invention describes a preferred embodiment. Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they know the basic creative concept. Therefore, the attached claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims (9)

1. A method for preparing a silk fibroin microcarrier skin filling preparation, comprising: Degumming, concentrating and drying the mulberry silk to obtain dry silk fibroin; The dried silk fibroin is configured into an aqueous solution, the aqueous solution is mixed with vitamin C, and freeze-dried to obtain VC-loaded silk fibroin; VC-loaded silk fibroin is prepared into a silk fibroin solution, the silk fibroin solution, hyaluronic acid, maltodextrin and a cross-linking agent are mixed, and the mixture is allowed to stand at 35-40° C. to obtain a composite gel, which is then sterilized to obtain a silk fibroin microcarrier skin filling preparation. 2. The method for preparing a silk fibroin microcarrier skin filling preparation according to claim 1 is characterized in that double distilled water is used as a solvent for drying silk fibroin to prepare an aqueous solution with a mass fraction of 5-10%, the aqueous solution is mixed with vitamin C in a mass ratio of 90-120:1, stirred for 15-30 minutes, and freeze-dried to obtain VC-loaded silk fibroin. 3. The method for preparing the silk fibroin microcarrier skin filling preparation according to claim 2, characterized in that it is prepared into a 5% by mass aqueous solution, the aqueous solution is mixed with vitamin C in a mass ratio of 100:1, stirred for 20 minutes, and freeze-dried to obtain VC-loaded silk fibroin. 4. The method for preparing the silk fibroin microcarrier skin filling preparation according to claim 3 is characterized in that VC-loaded silk fibroin is prepared into a 30-40 g/L silk fibroin solution using PEG300-PEG600 as a solvent, the silk fibroin solution, hyaluronic acid, maltodextrin and a cross-linking agent are mixed, and the mixture is allowed to stand at 35-40° C. to obtain a composite gel. 5. The method for preparing the silk fibroin microcarrier skin filling preparation according to claim 4 is characterized in that VC-loaded silk fibroin is prepared into a 30 g/L silk fibroin solution using PEG300 as a solvent, the silk fibroin solution, hyaluronic acid, maltodextrin and a cross-linking agent are mixed in a mass ratio of 10:1:0.5:0.2, and the mixture is allowed to stand at 40° C. to obtain a composite gel. 6 . The method for preparing the silk fibroin microcarrier skin filling preparation according to claim 5 , wherein the cross-linking agent is one or more of octyl sodium sulfate solution and sodium dodecyl sulfate. 7. The method for preparing the silk fibroin microcarrier skin filling preparation according to claim 1, characterized in that the degumming method comprises: (1) boiling mulberry silk with an alkaline solution, filtering, and collecting precipitates; wherein the alkaline solution is a mixture of an aqueous sodium carbonate solution and an aqueous sodium L-glutamate solution; (2) boiling the precipitated silk with the alkaline solution and collecting the solution; (3) Repeat the operation (2) until the mulberry silk is completely degummed. The completely degummed material is called silk fibroin solution. 8. A silk fibroin microcarrier skin filling preparation prepared according to any one of claims 1-7. 9. The silk fibroin microcarrier skin filling preparation according to claim 8, characterized in that the silk fibroin microcarrier skin filling preparation is loaded with auxiliary factors, and the preparation method is as follows: Degumming, concentrating and drying the mulberry silk to obtain dry silk fibroin; The dried silk fibroin is prepared into an aqueous solution, the aqueous solution is mixed with vitamin C, stirred, and freeze-dried to obtain VC-loaded silk fibroin; Using PEG300-PEG600 as solvent, VC-loaded silk protein is prepared into a silk solution, the silk solution, hyaluronic acid, maltodextrin, auxiliary factors and cross-linking agent are mixed, and allowed to stand at 35-40°C to obtain a composite gel, which is sterilized to obtain a silk protein microcarrier skin filling preparation.