A method and application for encapsulating active ingredients in silk fibroin nanofibers with high β-sheet content.

Abstract

This invention relates to the technical field of active ingredient loading, and more specifically, to a method and application of encapsulating active ingredients with high β-sheet content silk fibroin nanofibers. The key technical solution includes the following steps: S1, mixing a water-insoluble active ingredient and an emulsifier, heating and stirring until completely dissolved to obtain a first mixture; S2, adding high β-sheet content silk fibroin nanofibers to water and mixing evenly to obtain a high β-sheet content silk fibroin nanofiber gel; S3, adding the high β-sheet content silk fibroin nanofiber gel to the first mixture and stirring evenly to obtain a second mixture; S4, cooling the second mixture to obtain the target product. This invention, by encapsulating water-insoluble active ingredients with high β-sheet content silk fibroin nanofibers, has the advantages of improving the loading capacity and loading stability of water-insoluble active ingredients.

Description

Technical Field

[0001] This invention relates to the technical field of active ingredient loading, and more specifically, to a method and application of encapsulating active ingredients in silk fibroin nanofibers with high β-sheet content. Background Technology

[0002] In the food, cosmetic, and pharmaceutical industries, there are various active ingredients, which are classified into water-soluble and water-insoluble active ingredients according to their solubility in water. Water-insoluble active ingredients are hydrophobic and soluble in oils or alcohols. Most of them have characteristics such as high melting points. The solubility and melting point characteristics of these active ingredients increase the difficulty of adding them to the formulation, thus limiting their application.

[0003] To address the above issues, researchers have developed various liposome carriers and encapsulated active ingredients to create compositions that achieve encapsulation of different active ingredients and better water dispersibility. However, liposomes are generally complex systems with multiple components and poor stability, which limits their application in various fields. The oil-phase characteristic of liposomes further affects their application in medical and functional cosmetic fields, and there is an urgent need for new carrier systems that are more biocompatible and skin-compatible to replace them.

[0004] Silk fibroin possesses excellent biocompatibility, superior mechanical properties, and adjustable degradation rates. It also exhibits the ability to stabilize drugs and promote drug dispersion / loading / controlled release, making it a versatile carrier for various drug types. Through encapsulation, adsorption, and encapsulation, silk fibroin can stabilize a variety of active ingredients, enabling their controlled release. Researchers have developed stable silk fibroin nanofibers (SFNs) with high β-sheet content by modifying the structure of silk fibroin. By controlling the concentration, these nanofibers can form gels, allowing for the loading of active ingredients via physical adsorption. However, using silk fibroin as a carrier to transfer or encapsulate water-insoluble active ingredients is limited by the inherent properties of the silk fibroin carrier, making it difficult to achieve high concentrations of active ingredients. Furthermore, the inherent hydrophobicity and large size of the silk fibroin carriers used to load active ingredients cause the drug-loaded silk fibroin to easily separate in water, affecting practical applications. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a method and application for encapsulating active substances with high β-sheet content silk fibroin nanofibers, which has the advantages of improving the loading capacity and loading stability of water-insoluble active substances.

[0006] The above-mentioned technical objective of the present invention is achieved through the following technical solution: a method for encapsulating active substances in silk fibroin nanofibers with high β-sheet content, comprising the following steps:

[0007] S1. Mix the water-insoluble active ingredient and emulsifier, heat and stir until completely dissolved to obtain the first mixture;

[0008] S2. Add silk fibroin nanofibers with high β-sheet content to water and mix evenly to obtain silk fibroin nanofiber gel with high β-sheet content;

[0009] S3. Add the high β-sheet content silk protein nanofiber gel to the first mixture and stir evenly to obtain the second mixture;

[0010] S4. Cool the second mixture to obtain the target product.

[0011] In one embodiment, the water-insoluble active ingredient is a hydrophobic active ingredient.

[0012] In one embodiment, the concentration of the high β-sheet content silk fibroin nanofibers in the high β-sheet content silk fibroin nanofiber gel is 0.3-10%.

[0013] In one embodiment, the heating temperature of S1 is 40-100°C, and the cooling temperature of S4 is 2-50°C.

[0014] In one embodiment, S3 further includes the following steps:

[0015] S31. The high β-sheet content silk protein nanofiber gel and volatile co-solvent are added to the first mixture;

[0016] S32. Stir the first mixture evenly to obtain the second mixture;

[0017] S33. Stir the second mixture for 1 min to 2 h under a heat preservation condition of 40-90℃.

[0018] In one embodiment, the stirring speed in S32 is greater than or equal to 200 rpm and the stirring time is 1-30 min, and the stirring speed in S33 is greater than or equal to 10 rpm.

[0019] In one embodiment, the volatile co-solvent includes one or more of ethanol, tetrahydrofuran, acetone, volatile plant essential oils, and methyl salicylate.

[0020] In one embodiment, the water-insoluble active ingredient in the target product is 0.5-20% by mass.

[0021] A composition containing a water-insoluble active ingredient is prepared by the above-described method of encapsulating the active ingredient in silk fibroin nanofibers with high β-sheet content.

[0022] The above-mentioned compositions containing water-insoluble active ingredients are used in the preparation of skin care products or pharmaceuticals.

[0023] The above-mentioned method and application of encapsulating active substances with high β-sheet content silk fibroin nanofibers have the following beneficial effects:

[0024] Firstly, this invention utilizes high β-sheet content silk fibroin nanofibers and emulsifiers to form liposome-like nanostructures, achieving uniform distribution and encapsulation of water-insoluble active ingredients. This not only improves the water dispersibility of the active ingredients but also prevents their precipitation, thereby increasing the amount of water-insoluble active ingredients added to the emulsion system, reducing the difficulty of addition, and significantly improving its applicability in different directions.

[0025] Secondly, the composition obtained by the present invention is encapsulated by silk fibroin nanofibers with high β-sheet content. Compared with liposome encapsulation, the present invention has better stability, and the silk fibroin encapsulating the active ingredients has better hydration, thus improving the skin feel.

[0026] Thirdly, all components in the composition obtained by the present invention have good biocompatibility and skin affinity, and can reduce irritation, and have a significant beneficial effect on the application of sensitive skin. Attached Figure Description

[0027] Figure 1 This is a flowchart of the steps of the present invention;

[0028] Figure 2 This is a fluorescent labeling image of the high β-sheet content silk fibroin nanofiber gel and composition in Example 1;

[0029] Figure 3 This is a microscope image of the composition in Example 1;

[0030] Figure 4 This is a ZETA potential diagram of the high β-sheet content silk fibroin nanofiber gel and composition in Example 1;

[0031] Figure 5 The infrared spectrum of the high β-sheet content silk fibroin nanofiber gel, ceramide, and composition in Example 1 is shown.

[0032] Figure 6 This is the diffraction spectrum of the ceramide in Example 1;

[0033] Figure 7 This is the diffraction spectrum of the silk fibroin nanofiber gel with high β-sheet content in Example 1;

[0034] Figure 8 This is the diffraction spectrum of the composition in Example 1. Detailed Implementation

[0035] The present invention will be further described in detail below with reference to embodiments. It should be noted that the embodiments described below are intended to facilitate understanding of the present invention and are not intended to limit it in any way. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In case of any conflict, the definitions in this specification shall prevail.

[0036] As used herein, the terms “prepared from” and “comprising” are synonymous. The terms “comprising,” “including,” “having,” “containing,” or any other variations thereof, as used herein, are intended to cover non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that includes the listed elements is not necessarily limited to those elements, but may include other elements not expressly listed or elements inherent to such composition, step, method, article, or apparatus.

[0037] The conjunction "composed of..." excludes any unspecified elements, steps, or components. If used in a claim, this phrase makes the claim closed, excluding materials other than those described, except for associated conventional impurities. When the phrase "composed of..." appears in a clause of the body of a claim rather than immediately following it, it limits only the elements described in that clause; other elements are not excluded from the claim as a whole.

[0038] When a quantity, concentration, or other value or parameter is expressed as a range, a preferred range, or a range defined by a series of upper and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pair of any upper or preferred value with any lower or preferred value, regardless of whether the range is disclosed individually. For example, when the range “1 to 5” is disclosed, the described range should be interpreted as including the ranges “1 to 4”, “1 to 3”, “1 to 2”, “1 to 2 and 4 to 5”, “1 to 3 and 5”, etc. When numerical ranges are described herein, unless otherwise stated, the range is intended to include its endpoints and all integers and fractions within that range.

[0039] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0040] See Figure 1 A method for preparing a composition, comprising the following steps:

[0041] S1. Mix the water-insoluble active ingredient and emulsifier, heat and stir until completely dissolved to obtain the first mixture;

[0042] S2. Add silk fibroin nanofibers with high β-sheet content to water and mix evenly to obtain silk fibroin nanofiber gel with high β-sheet content;

[0043] In this invention, the β-sheet content of the high β-sheet silk fibroin nanofibers used is greater than or equal to 30%;

[0044] S3. Add the high β-sheet content silk protein nanofiber gel to the first mixture and stir evenly to obtain the second mixture;

[0045] In practice, when stirred until homogeneous, the resulting second mixture is milky white.

[0046] S4. Cool the second mixture to obtain the target product.

[0047] This invention utilizes high β-sheet content silk fibroin nanofibers and emulsifiers to form liposome-like nanostructures, achieving uniform distribution and encapsulation of water-insoluble active ingredients. This not only improves the water dispersibility of the active ingredients but also prevents crystallization, allowing for increased addition amounts of water-insoluble active ingredients in emulsion systems, reducing addition difficulty, and significantly enhancing their applicability in different directions.

[0048] Specifically, water-insoluble active ingredients are hydrophobic active ingredients;

[0049] Preferably, the hydrophobic active ingredient is sphingolipid;

[0050] In practical applications, sphingolipids are selected from one or more of ceramides, phytosphingosine, phytosterols, and phytosterol esters.

[0051] Specifically, the concentration of high β-sheet content silk fibroin nanofibers in the high β-sheet content silk fibroin nanofiber gel is 0.3-10%.

[0052] Specifically, the heating temperature of S1 is 40-100℃, and the cooling temperature of S4 is 2-50℃;

[0053] In actual operation, the heating temperature is controlled within the above range in S1 to avoid the active material losing its activity due to excessively high temperature, and to prevent the active material and emulsifier from dissolving and mixing due to excessively low temperature.

[0054] Specifically, S3 also includes the following steps:

[0055] S31. Add the high β-sheet content silk protein nanofiber gel and volatile co-solvent to the first mixture;

[0056] S32. Stir the first mixture evenly to obtain the second mixture;

[0057] S33. Stir the second mixture for 1 min to 2 h under a heat preservation condition of 40-90℃;

[0058] For water-insoluble active ingredients that are difficult to disperse, adding volatile co-solvents can improve the encapsulation effect of silk fibroin nanofibers with high β-sheet content. The volatile co-solvents are then removed by incubation and stirring.

[0059] Specifically, in S32, the stirring speed is greater than or equal to 200 rpm and the stirring time is 1-30 min, while in S33, the stirring speed is greater than or equal to 10 rpm.

[0060] Specifically, volatile cosolvents include one or more of ethanol, tetrahydrofuran, acetone, volatile plant essential oils, and methyl salicylate.

[0061] Specifically, the mass percentage of water-insoluble active ingredients in the target product is 0.5-20%.

[0062] A composition containing a water-insoluble active ingredient is prepared by the above-described method of encapsulating the active ingredient in silk fibroin nanofibers with high β-sheet content;

[0063] Compositions containing water-insoluble active ingredients are encapsulated by silk fibroin nanofibers with high β-sheet content, avoiding the use of oil-phase liposome carriers. The silk fibroin encapsulating the active ingredients is easy to add to the formulation, improving the skin feel.

[0064] A composition comprising a water-insoluble active ingredient is used in the preparation of skin care products or pharmaceuticals;

[0065] All components of the composition have good biocompatibility and skin affinity, which can significantly reduce skin irritation and have a significant beneficial effect on sensitive skin.

[0066] The following is a specific example:

[0067] Example 1

[0068] A method for encapsulating ceramides with silk fibroin nanofibers containing high β-sheet content, the specific steps of which are as follows:

[0069] S1. Mix 0.2g of ceramide and 0.4g of emulsifier (polyglycerol-6 distearate, jojoba esters, polyglycerol-3 beeswax ester and cetyl alcohol), heat to 90°C, and stir until completely dissolved to obtain the first mixture;

[0070] S2. Add high β-sheet content silk protein nanofibers to water and mix evenly to obtain a 1% high β-sheet content silk protein nanofiber gel.

[0071] S3. Add 20 ml of 1% high β-sheet content silk protein nanofiber gel and 10 ml of ethanol to the first mixture, stir evenly at 500 rpm for 5 min until the above solution is milky white, and obtain the second mixture. Stir the second mixture at 75℃ and 200 rpm for 10 min-2 h.

[0072] S4. Cool the second mixture to 25°C to obtain a composition containing ceramides.

[0073] In this embodiment, the emulsifier used is Emulium Mellifera MB emulsifier manufactured by GAFRA (France).

[0074] Experimental analysis was conducted on the high β-sheet content silk fibroin nanofiber gel, ceramide, and composition in this embodiment.

[0075] like Figure 2 As shown, the high β-sheet content silk fibroin nanofiber gel and composition in this embodiment were observed using a fluorescence co-concentration microscope. Figure 2 (a) shows the fluorescence labeling pattern of silk fibroin nanofiber gel with high β-sheet content. Figure 2 Image (b) shows the fluorescence labeling of the composition after ceramide was encapsulated by high β-sheet content silk fibroin nanofibers. Before encapsulation, the high β-sheet content silk fibroin nanofibers were aggregated particles. After encapsulation, the encapsulated particles were uniformly dispersed in the composition and their size was less than 1 μm. The composition obtained in this example was observed using an electron microscope. Figure 3 As shown, the particles are uniformly distributed after the silk fibroin nanofibers with high β-sheet content encapsulate ceramide, with a particle size within 500 nm.

[0076] like Figure 4 As shown, the zeta potential of the high β-sheet content silk fibroin nanofiber gel and the composition coated with ceramide in this embodiment was measured. After loading ceramide, the particle size distribution of the high β-sheet content silk fibroin nanofibers increased slightly, especially the particles around 100 nm. This particle size increase indicates that the high β-sheet content silk fibroin nanofibers, emulsifier, and ceramide combine to achieve particle aggregation, rather than simple mixing. The particles in the composition are mostly below 1000 nm because the silk fibroin coating maintains a high charge density for the ceramide particles, allowing for uniform dispersion.

[0077] like Figure 5 As shown, the high β-sheet content silk fibroin nanofiber gel, ceramide, and the composition after encapsulating ceramide in this embodiment were freeze-dried and subjected to infrared testing. Figure 5In (a), the dark line represents the infrared spectrum of the high β-sheet content silk fibroin nanofiber gel, and the light line represents the infrared spectrum of the composition of the high β-sheet content silk fibroin nanofiber gel after encapsulating ceramides. Figure 5 (b) shows the infrared spectrum of ceramide. By comparing the changes in the infrared spectrum before and after loading, an increase in the typical ceramide peak can be observed in the composition, proving that the ceramide has been successfully dispersed in the composition.

[0078] like Figure 6-8 As shown, X-ray diffraction spectroscopy analysis was performed on the high β-sheet content silk fibroin nanofiber gel, ceramide, and the composition after ceramide encapsulation in this embodiment. Ceramide crystals exhibited strong diffraction peaks near 5-8° and 20°, while the high β-sheet content silk fibroin nanofibers showed weaker diffraction peaks near 20°, 30°, and 40°. After encapsulating ceramide with high β-sheet content silk fibroin nanofibers to form liposomes, it was observed that the majority of the composition was high β-sheet content silk fibroin nanofibers, and no typical strong ceramide crystal peaks were observed, indicating that the ceramide did not crystallize after encapsulation. Simultaneously, the crystallization peak near 20° in the mixture showed shape changes and enhancement, which should be attributed to the influence of ceramide, further demonstrating the successful encapsulation of ceramide.

[0079] Example 2

[0080] A method for encapsulating plant sphingosine with high β-sheet content silk fibroin nanofibers, the specific steps of which are as follows:

[0081] S1. Mix 0.2g of phytosphingosine and 0.4g of emulsifier (polyglycerol-6 distearate, jojoba esters, polyglycerol-3 beeswax ester and cetyl alcohol), heat to 90°C, and stir until completely dissolved to obtain the first mixture;

[0082] S2. Add high β-sheet content silk protein nanofibers to water and mix evenly to obtain a 1% high β-sheet content silk protein nanofiber gel.

[0083] S3. Add 20 ml of 1% high β-sheet content silk protein nanofiber gel to the first mixture and stir at 500 rpm for 5 min until the above solution is milky white to obtain the second mixture.

[0084] S4. Cool the second mixture to 20°C to obtain a composition containing phytosphingosine.

[0085] In this embodiment, the emulsifier used is Emulium Mellifera MB emulsifier manufactured by GAFMA (France). The composition obtained in this embodiment can be diluted and miscible with water or added as an intermediate to skin care product formulations.

[0086] Example 3

[0087] A method for encapsulating plant sphingosine and ceramides with high β-sheet content silk fibroin nanofibers, the specific steps of which are as follows:

[0088] S1. Mix 0.1g phytosphohydrin, 0.1g ceramide and 0.4g emulsifier (polyglycerol-6 distearate, jojoba esters, polyglycerol-3 beeswax ester and cetyl alcohol), heat to 90°C and stir until completely dissolved to obtain the first mixture;

[0089] S2. Add high β-sheet content silk protein nanofibers to water and mix evenly to obtain a high β-sheet content silk protein nanofiber gel with a concentration of 2%.

[0090] S3. Add 20 ml of 2% high β-sheet content silk protein nanofiber gel to the first mixture and stir at 500 rpm for 5 min until the above solution is milky white to obtain the second mixture.

[0091] S4. Cool the second mixture to 20°C to obtain a composition containing phytosphingosine and ceramide.

[0092] In this embodiment, the emulsifier used is Emulium Mellifera MB emulsifier manufactured by GAFMA (France). The composition obtained in this embodiment is a white cream and is miscible with water or can be added as an intermediate to skin care product formulations.

[0093] Example 4

[0094] The application of a composition includes the following specific steps:

[0095] (1) Mix 84g water, 10g sunflower seed oil and 3g emulsifier (hydroxyethyl acrylate / sodium acryloyl dimethyl taurate copolymer, squalane and polysorbate-60) and emulsify at 2000rpm for 3min to obtain a uniform emulsion;

[0096] (2) Add 3g of the composition in Example 1 to the emulsion and stir at 500rpm for 1min to obtain a skin care product.

[0097] In this embodiment, the emulsifier used is SIMULGEL NS emulsifier manufactured by Seppic AG, France. The skincare product obtained in this embodiment is an oil-water gel system. The composition of Example 1 is uniformly mixed with the emulsion, resulting in a skincare product with high stability and a refreshing feel. Under normal conditions, ceramides do not precipitate out of the skincare product.

[0098] The amounts of raw materials used in Examples 1-4 of this invention are shown in the table below:

[0099]

[0100] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A method for encapsulating active ingredients in silk fibroin nanofibers with high β-sheet content, characterized in that, Includes the following steps: S1. Mix the water-insoluble active ingredient and emulsifier, heat and stir until completely dissolved to obtain the first mixture; S2. Add high β-sheet content silk fibroin nanofibers to water and mix evenly to obtain high β-sheet content silk fibroin nanofiber gel. The β-sheet content of the high β-sheet content silk fibroin nanofibers is greater than or equal to 30%, and the concentration of the high β-sheet content silk fibroin nanofibers in the high β-sheet content silk fibroin nanofiber gel is 0.3-10%. S3. Add the high β-sheet content silk protein nanofiber gel to the first mixture and stir evenly to obtain the second mixture; S4. Cool the second mixture to obtain the target product; The target product contains 0.5-20% by mass of the water-insoluble active ingredient, and the water-insoluble active ingredient is a hydrophobic active ingredient, which is one or more of ceramide, phytosphoamine, phytosterol and phytosterol ester.

2. The method for encapsulating active ingredients in silk fibroin nanofibers with high β-sheet content according to claim 1, characterized in that, The heating temperature of S1 is 40-100℃, and the cooling temperature of S4 is 2-50℃.

3. The method for encapsulating active ingredients in silk fibroin nanofibers with high β-sheet content according to claim 1, characterized in that, S3 further includes the following steps: S31. The high β-sheet content silk protein nanofiber gel and volatile co-solvent are added to the first mixture; S32. Stir the first mixture evenly to obtain the second mixture; S33. Stir the second mixture for 1 min to 2 h under a heat preservation condition of 40-90℃.

4. The method for encapsulating active ingredients with high β-sheet content silk fibroin nanofibers according to claim 3, characterized in that: The stirring speed in S32 is greater than or equal to 200 rpm and the stirring time is 1-30 min; the stirring speed in S33 is greater than or equal to 10 rpm.

5. The method for encapsulating active ingredients with high β-sheet content silk fibroin nanofibers according to claim 3, characterized in that: The volatile cosolvents include one or more of ethanol, tetrahydrofuran, acetone, volatile plant essential oils, and methyl salicylate.

6. A composition comprising a water-insoluble active ingredient, characterized in that: It is prepared by a method for encapsulating active ingredients in silk fibroin nanofibers with high β-sheet content according to any one of claims 1-5.

7. The use of the composition comprising a water-insoluble active ingredient as described in claim 6 in the preparation of skin care products or pharmaceuticals.

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