Silk protein-active matter nano aggregate and preparation method thereof

Abstract

The invention relates to the technical field of cosmetic raw materials, in particular to a fibroin-active matter nano aggregate and a preparation method thereof.The preparation method of the fibroin-active matter nano aggregate is characterized by comprising the following steps that S1, a fibroin aggregate aqueous solution and large-molecular-weight active matter are obtained, mixing the fibroin aggregate aqueous solution and the high-molecular-weight active matter to obtain a first mixed solution; and S2, continuously stirring the first mixed solution for 2-240 minutes to obtain a target product, wherein the large-molecular-weight active matter comprises at least one of protein, polysaccharide, glycosaminoglycan, sugar alcohol and a compound of the protein, the polysaccharide, the glycosaminoglycan and the sugar alcohol, and the mass ratio of the silk fibroin aggregate in the silk fibroin aggregate water solution to the large-molecular-weight active matter is (100: 1)-(1: 2). The transdermal property of the active matter can be simply and effectively improved while the structure and the performance of the active matter are not changed.

Description

Technical Field

[0001] The invention relates to the technical field of cosmetic raw materials, and more specifically, to a silk protein-active substance nano-aggregate and a preparation method thereof. Background Art

[0002] Active ingredients can accelerate the renewal rate of epidermal cells, reduce the accumulation of aging cells, stimulate the activity of key cells such as fibroblasts, promote the synthesis and rearrangement of collagen and elastic fibers, and effectively stimulate and promote the regeneration and repair ability of skin cells through a series of complex biochemical processes. However, most macromolecular active ingredients have difficulty penetrating the skin barrier, which affects the efficacy of active ingredients and restricts the development and application of active ingredients in the field of functional skin care.

[0003] With the development of technology, some researchers have improved the transdermal permeability of active ingredients by reducing the molecular weight, but this will reduce the activity of the active ingredients themselves; some researchers have used transdermal carrier technology to improve the transdermal permeability of active ingredients, but its preparation process is complicated and the cost is relatively high. Developing a simple and effective method to improve the transdermal permeability of active ingredients without changing the structure and performance of active ingredients is the key to promoting the widespread application of active ingredients in the field of functional skin care. Summary of the invention

[0004] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a silk protein-active substance nano-aggregate and a preparation method thereof, which can simply and effectively improve the skin permeability of the active substance without changing the structure and performance of the active substance.

[0005] The above technical objectives of the present invention are achieved through the following technical solutions: a method for preparing a silk protein-active substance nano-aggregate, comprising the following steps: S1, obtaining a silk fibroin aggregate aqueous solution and a high molecular weight active substance, and mixing the silk fibroin aggregate aqueous solution and the high molecular weight active substance to obtain a first mixed solution; S2, continue stirring the first mixed solution for 2-240 minutes to obtain the target product; The high molecular weight active substance comprises at least one of proteins, polysaccharides, glycosaminoglycans, sugar alcohols and their mixtures, and the mass ratio of the fibroin aggregates to the high molecular weight active substance in the fibroin aggregate aqueous solution is 100:1-1:2.

[0006] In one embodiment, the mass ratio of the fibroin aggregate to the high molecular weight active substance in the fibroin aggregate is 20:1-2:1.

[0007] In one embodiment, the step S1 further comprises: using a pH regulator to adjust the pH of the first mixed solution to 3-10; The pH adjuster includes citric acid, vitamin C, fruit acid, hydrochloric acid and sodium carbonate.

[0008] In one embodiment, the concentration of the fibroin aggregates in the fibroin aggregate aqueous solution is 0.1-4 wt %.

[0009] In one embodiment, the beta-sheet content of the silk fibroin aggregate is greater than 40%, and the size range of the silk fibroin aggregate is 20-2000 nm.

[0010] In one embodiment, the stirring rate of S2 is 200-3500 rpm.

[0011] In one embodiment, the protein includes at least one of animal-derived collagen and recombinant collagen; The recombinant collagen includes at least one of recombinant type I, recombinant type II, recombinant type III, recombinant type IV and recombinant type XVII collagen.

[0012] In one embodiment, the polysaccharide comprises at least one of hyaluronic acid and plant-extracted polysaccharides.

[0013] A silk protein-active substance nano-aggregate is prepared by the above-mentioned silk protein-active substance nano-aggregate preparation method.

[0014] The application of the above-mentioned silk protein-active ingredient nanoaggregates in functional skin care products.

[0015] The above-mentioned silk protein-active substance nano-aggregate and preparation method thereof have the following beneficial effects: First, the silk fibroin aggregates and high molecular weight active substances are mixed and stirred, and the active substances are attached through the non-covalent interaction between the two, and the attached active substances are used to regulate the reassembly of the silk fibroin aggregates. After stirring, the silk fibroin-active substance nano-aggregates are obtained, and the transdermal property of the active substances is improved. The method is simple and highly controllable. Secondly, the transdermal properties of silk fibroin aggregates are used to carry non-transdermal active substances through the stratum corneum, and the nanostructure of the aggregates is combined to optimize the transdermal ability, achieving dual transdermal enhancement of hydrophilicity and nanostructure; Thirdly, the good biological activities of the silk fibroin aggregates and active substances of the present invention are not destroyed, and the aggregates of the two can exert the synergistic effect of the two components; Fourthly, the entire preparation process does not require high temperature, thus avoiding the impact of the preparation process on the biocompatibility of silk fibroin and active ingredients, making it convenient for direct use in cosmetics and topical medicines. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1is a flow chart of the steps of the present invention; Figure 2 It is a schematic diagram of the results of macroscopic observation test of Example 1 and Comparative Example 1; Figure 3 It is a schematic diagram of the results of scanning electron microscope testing of Example 1 and Comparative Example 1; Figure 4 It is a schematic diagram of the results of the fluorescent staining test of Example 1, Comparative Example 2 and Comparative Example 3; Figure 5 It is a schematic diagram of the results of freeze-drying dissolution test of Example 1 and Comparative Example 3; Figure 6 is a schematic diagram of the results of scanning electron microscope testing of Example 6 and Example 7; Figure 7 It is a schematic diagram of the results of the fluorescent staining test in Example 6. DETAILED DESCRIPTION

[0017] The present invention is described in further detail below in conjunction with the embodiments. It should be noted that the embodiments described below are intended to facilitate the understanding of the present invention and do not have any limiting effect on it. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those of ordinary skill in the art to which the present invention belongs. When there is a conflict, the definitions in this specification shall prevail.

[0018] As used herein, the term "prepared from" is synonymous with "comprising." As used herein, the terms "comprising," "including," "having," "containing," or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises the listed elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0019] The conjunction "consisting of excludes any unspecified element, step, or component. If used in a claim, this phrase renders the claim closed-ended so that it does not include materials other than those described, except for conventional impurities associated therewith. When the phrase "consisting of" appears in a clause of the body of a claim rather than immediately following the subject matter, it limits only the elements described in that clause; other elements are not excluded from the claim as a whole.

[0020] When amount, concentration or other value or parameter is expressed as range, preferred range or a series of upper preferred value and lower preferred value limit range, this should be understood as specifically disclosing all ranges formed by any pairing of any range upper limit or preferred value and any range lower limit or preferred value, regardless of whether the range is disclosed separately. For example, when disclosing range "1 to 5", described range should be interpreted as including range "1 to 4", "1 to 3", "1 to 2", "1 to 2 and 4 to 5", "1 to 3 and 5" etc. When numerical range is described in this article, unless otherwise stated, the range is intended to include its end value and all integers and fractions within the range.

[0021] The present invention is described in detail below in conjunction with the accompanying drawings and embodiments.

[0022] A method for preparing a silk protein-active substance nano-aggregate, such as Figure 1 As shown, the following steps are included: S1, obtaining a silk fibroin aggregate aqueous solution and a high molecular weight active substance, and mixing the silk fibroin aggregate aqueous solution and the high molecular weight active substance to obtain a first mixed solution; S2, continue stirring the first mixed solution for 2-240 min to obtain the target product; The high molecular weight active substances include at least one of proteins, polysaccharides, glycosaminoglycans, sugar alcohols and their mixtures, and the mass ratio of the silk fibroin aggregates to the high molecular weight active substances in the silk fibroin aggregate aqueous solution is 100:1-1:2.

[0023] First, a high molecular weight active substance solution is mixed with a silk fibroin nanofiber gel or aqueous solution, wherein the mass ratio of the silk fibroin aggregate to the high molecular weight active substance is 100:1-1:2, the silk fibroin nanofiber and the high molecular weight active substance mixture are stirred at a rate of 200-3500 rpm for 2-240 min, and the high molecular weight active substance attached to the silk fibroin nanofiber is used to induce the reassembly of the silk fibroin nanofiber to form a silk protein-active substance nanoaggregate. Among them, the high molecular weight active substance is one or more of active substances such as proteins, polysaccharides, glycosaminoglycans and sugar alcohols and their composites. Affected by the ratio of the added components of the system, the form of the first mixed solution can be liquid and gel. The type of high molecular weight active substance is not limited here, and other active substances that are difficult to penetrate the skin are also included in the scope of the present invention.

[0024] The present invention mixes and stirs the silk fibroin aggregate and the high molecular weight active substance, realizes the attachment of the active substance through the non-covalent interaction between the two, and uses the attached active substance to regulate the reassembly of the silk fibroin aggregate, obtains the silk fibroin-active substance nano-aggregate after stirring, and improves the transdermal property of the active substance. The method is simple and highly controllable. The non-transdermal active substance is carried through the stratum corneum through the transdermal property of the silk fibroin aggregate, and the transdermal ability is optimized in combination with the nanostructure of the aggregate, so as to realize the dual transdermal enhancement of hydrophilicity and nanostructure.

[0025] In addition, the silk fibroin aggregates and active ingredients of the present invention both have good biological activity, and the aggregates of the two can exert the synergistic effect of the two components. The entire preparation process does not require high temperature, thus avoiding the influence of the preparation process on the biocompatibility of silk fibroin and active ingredients, and facilitating the direct use of functional skin care products.

[0026] Specifically, the mass ratio of the fibroin aggregates to the high molecular weight active substances is 20:1-2:1. In S1, the mixing mass ratio of the fibroin aggregates to the high molecular weight active substances is preferably 20:1-2:1.

[0027] Specifically, S1 also includes: using a pH regulator to adjust the pH of the first mixed solution to 3-10; the pH regulator includes citric acid, vitamin C, fruit acid, hydrochloric acid and sodium carbonate. Before stirring the first mixed solution, the pH value is adjusted to a suitable range so that the fibroin aggregates and the high molecular weight active substances have suitable non-covalent interactions. The pH value mainly affects the reassembly of the fibroin aggregates, and then affects the reassembly of the fibroin aggregates and the high molecular weight active substances. Too high or too low a pH value will affect the non-covalent interaction between the fibroin aggregates and the high molecular weight active substances, resulting in the formed aggregates failing to achieve the expected goal. The optimal pH values ​​for the interaction between different high molecular weight active substances and fibroin aggregates are different.

[0028] Furthermore, the concentration of the fibroin aggregates in the fibroin aggregate aqueous solution is 0.1-4wt%, wherein the common concentration is 0.5-2%. When the concentration is greater than or equal to 2wt%, the fibroin aggregate aqueous solution is in a gel state.

[0029] Specifically, the beta-sheet content of the silk fibroin aggregate is greater than 40%, and the size range is 20-2000nm. The silk fibroin aggregate used in the present invention is a high-crystalline silk fibroin aggregate, containing more than 40% of the beta-folded structure, having good structural stability and mechanical properties, so as to better achieve the attachment of large molecular weight active substances.

[0030] Specifically, the stirring rate of S2 is 200-3500rpm. The goal is to form a homogeneous system of fibroin aggregates and high molecular weight active substances. When the stirring rate is too low, it will be difficult for fibroin aggregates and high molecular weight active substances to form uniform nano-aggregates. When the stirring rate is too high, the aggregation of fibroin aggregates and high molecular weight active substances will be too strong, resulting in phase separation or precipitation.

[0031] Specifically, the protein includes at least one of animal-derived collagen and recombinant collagen; the recombinant collagen includes at least one of recombinant type I, recombinant type II, recombinant type III, recombinant type IV and recombinant type XVII collagen. The type of protein is not limited here, and other proteins that are difficult to penetrate the skin are also included in the scope of the present invention.

[0032] Specifically, the polysaccharides include hyaluronic acid and various plant-extracted polysaccharides. The plant-extracted polysaccharides include Cassia seed extract, Tremella polysaccharide and Bosin, etc. The types of polysaccharides are not limited here, and other polysaccharides that are difficult to penetrate the skin are also included in the scope of the present invention.

[0033] A silk protein-active substance nano-aggregate is prepared by the above-mentioned silk protein-active substance nano-aggregate preparation method.

[0034] The application of the above-mentioned silk protein-active ingredient nanoaggregates in functional skin care products.

[0035] Silk fibroin aggregates and high molecular weight active ingredients form stable aggregates through non-covalent interactions. The aggregates give full play to the synergistic effect of the two, achieving dual transdermal enhancement of hydrophilicity and nanostructure, and have good transdermal properties. Effective skin care products can be developed based on these aggregates, which have high practical value. Embodiment 1

[0036] This embodiment provides a silk protein-active substance nano-aggregate, and the specific preparation steps are as follows: S1, mixing a fibroin aggregate gel with a length of 2000 nm and a concentration of 2 wt% and freeze-dried recombinant collagen, wherein the mass ratio of the fibroin aggregate to the freeze-dried recombinant collagen in the fibroin aggregate gel is 7:1, and after the recombinant collagen is dissolved in the fibroin aggregate gel, adjusting the pH value to 7 to form a uniform first mixed solution; S2. Stir the first mixed solution at 1000 rpm for 20 min to induce the fibroin aggregates and recombinant collagen to form a nano-aggregate solution. Embodiment 2

[0037] This embodiment provides a silk protein-active substance nano-aggregate, and the specific preparation steps are as follows: S1. Adding freeze-dried recombinant collagen to water to prepare a recombinant collagen aqueous solution with a concentration of 3wt%, and then mixing it with a fibroin aggregate aqueous solution with a length of 600-1000 nm and a concentration of 3wt%, wherein the mass ratio of fibroin aggregate to freeze-dried recombinant collagen in the fibroin aggregate aqueous solution is 50:1, and adjusting the pH value to 5 to form a uniform first mixed solution; S2. Stir the first mixed solution at 600 rpm for 60 min to induce the fibroin aggregates and recombinant collagen to form nano-aggregates. Embodiment 3

[0038] This embodiment provides a silk protein-active substance nano-aggregate, and the specific preparation steps are as follows: S1, mixing a fibroin aggregate aqueous solution with a length of 1000-1500 nm and a concentration of 4wt% and freeze-dried recombinant collagen, wherein the mass ratio of the fibroin aggregate in the fibroin aggregate aqueous solution to the freeze-dried recombinant collagen is 2:1, and after the recombinant collagen is dissolved in the fibroin aggregate aqueous solution, adjusting the pH value to 4 to form a uniform first mixed solution; S2. Stir the first mixed solution at 1800 rpm for 30 min to induce the fibroin aggregates and recombinant collagen to form nano-aggregates. Embodiment 4

[0039] This embodiment provides a silk protein-active substance nano-aggregate, and the specific preparation steps are as follows: S1. Adding freeze-dried recombinant collagen to water to prepare a 3% recombinant collagen aqueous solution, and then mixing it with a 1wt% silk fibroin aggregate gel with a length of 500-800 nm, wherein the mass ratio of the silk fibroin aggregate to the freeze-dried recombinant collagen in the silk fibroin aggregate gel is 9:1, and adjusting the pH value to 9 to form a uniform first mixed solution; S2. Stir the first mixed solution at 2200 rpm for 40 min to induce the fibroin aggregates and recombinant collagen to form nano-aggregates. Embodiment 5

[0040] This embodiment provides a silk protein-active substance nano-aggregate, and the specific preparation steps are as follows: S1, mixing a fibroin aggregate gel with a length of 1000-2000 nm and a concentration of 2 wt% and freeze-dried recombinant collagen, wherein the mass ratio of the fibroin aggregate to the freeze-dried recombinant collagen in the fibroin aggregate gel is 10:1. After the recombinant collagen is dissolved in the fibroin aggregate gel, the pH value is adjusted to 7 to form a uniform first mixed solution; S2. Homogenize the first mixed solution at 3500 rpm for 40 min to induce the silk fibroin aggregates and recombinant collagen to form nano-aggregates. Embodiment 6

[0041] This embodiment provides a silk protein-active substance nano-aggregate, and the specific preparation steps are as follows: S1, mixing a 600-1000 nm-long, 3 wt% fibroin aggregate aqueous solution and freeze-dried bosogenin, wherein the mass ratio of fibroin aggregate to freeze-dried bosogenin in the fibroin aggregate aqueous solution is 2:1. After bosogenin is dissolved in the fibroin nanofiber aggregate gel, a uniform first mixed solution is formed; S2. Stir the first mixed solution at 2000 rpm for 20 min to induce the fibroin aggregates and bosera to form nano-aggregates. Embodiment 7

[0042] This embodiment provides a silk protein-active substance nano-aggregate, and the specific preparation steps are as follows: S1, mixing a silk fibroin aggregate gel with a length of 1000-2000 nm and a concentration of 2 wt% and freeze-dried hyaluronic acid, wherein the mass ratio of the silk fibroin aggregate to the freeze-dried hyaluronic acid in the silk fibroin aggregate gel is 3:1. After the hyaluronic acid is dissolved in the silk fibroin nanofiber aggregate gel, a uniform first mixed solution is formed; S2. Homogenize the first mixed solution at 4000 rpm for 10 min to induce the silk fibroin aggregates and hyaluronic acid to form nano-aggregates.

[0043] The present invention provides three comparative examples for performance test comparison of the embodiments.

[0044] Preparation method of comparative example 1: a 2000 nm long, 2 wt% fibroin aggregate gel and freeze-dried recombinant collagen are mixed, wherein the mass ratio of fibroin aggregate to freeze-dried recombinant collagen in the fibroin aggregate gel is 7:1. The mixture is stirred at 100 rpm for 20 min to obtain the target product of comparative example 1.

[0045] Comparative Example 2 is a pure recombinant collagen aqueous solution, and the recombinant collagen concentration is 2.28%.

[0046] Preparation method of comparative example three: mix a silk fibroin aggregate gel with a length of 2000nm and a concentration of 2wt% and freeze-dried recombinant collagen, wherein the mass ratio of silk fibroin aggregate to freeze-dried recombinant collagen in the silk fibroin aggregate gel is 1:3, and stir at a speed of 1000rpm for 20min to obtain the target product of comparative example three.

[0047] Macro observation and analysis: The products obtained in Example 1 and Comparative Example 1 were macroscopically observed and analyzed. Figure 2 As shown, it can be found that the transparency and fluidity of comparative example 1 (Figure a) are poor and still maintain a gel state, while Example 1 (Figure b) has changed from a gel state to a translucent solution state with good fluidity, indicating that Example 1 has a different aggregation structure compared with Comparative Example 1.

[0048] Scanning electron microscopy analysis: The microstructures of the products obtained in Example 1, Comparative Example 1 and Comparative Example 3 were observed using a scanning electron microscope. Figure 3 As shown, it was found that comparative example 1 (Fig. a) still maintained the nanofiber structure, comparative example 3 (Fig. c) was an aggregate with a size of more than 300 nm due to the change in the fibroin-collagen ratio, and embodiment 1 (Fig. b) was nanoparticles with a size between 40-200 nm.

[0049] The products obtained in Example 6 and Example 7 were observed by scanning electron microscope. Figure 6 As shown, it was found that both Example 6 (Figure a) and Example 7 (Figure b) formed nano-aggregate particles, and the size of each particle was less than 200 nm.

[0050] Fluorescent staining test: The products obtained in Example 1, Comparative Example 2 and Comparative Example 3 were subjected to a fluorescence staining test. After the fluorescence staining, the three products were smeared on fresh pig skin and left for 24 hours. The transdermal conditions were observed by fluorescence microscopy. Figure 4 As shown, it can be found that both Comparative Example 2 (Figure a) and Comparative Example 3 (Figure b) have no transdermal fluorescence signal, while Example 1 (Figure c) can observe obvious transdermal aggregation phenomenon.

[0051] The product obtained in Example 6 was subjected to a fluorescence staining test. The fibroin-bosine aggregate of Example 6 was fluorescently stained and then applied to pig skin. The mixture was left for 24 hours and the transdermal state was observed by fluorescence microscopy. Figure 7 As shown, it can be found that the aggregates have penetrated into the dermis.

[0052] Freeze-dried dissolution test: The products obtained in Example 1 and Comparative Example 3 were freeze-dried and then dissolved in water. Figure 5 As shown, it can be observed that Example 1 (Figure a) cannot be dispersed in water, indicating that it is hydrophobic, while Comparative Example 3 (Figure b) is transformed back into a solution, indicating that it is hydrophilic, indicating that changes in the ratio of fibroin aggregates and collagen can affect the hydrophilicity and thus the transdermal performance.

[0053] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for preparing a silk protein-active substance nano-aggregate, characterized in that: The following steps are involved: S1, obtaining a silk fibroin aggregate aqueous solution and a high molecular weight active substance, and mixing the silk fibroin aggregate aqueous solution and the high molecular weight active substance to obtain a first mixed solution; S2, continue stirring the first mixed solution for 2-240 minutes to obtain the target product; The high molecular weight active substance comprises at least one of proteins, polysaccharides, glycosaminoglycans, sugar alcohols and their mixtures, and the mass ratio of the fibroin aggregates to the high molecular weight active substance in the fibroin aggregate aqueous solution is 100:1-1:

2.

2. The method for preparing a silk protein-active substance nano-aggregate according to claim 1, characterized in that: The mass ratio of the silk fibroin aggregate to the high molecular weight active substance is 20:1-2:

1.

3. The method for preparing a silk protein-active substance nano-aggregate according to claim 1, characterized in that: The step S1 further comprises: using a pH regulator to adjust the pH of the first mixed solution to 3-10; The pH adjuster includes citric acid, vitamin C, fruit acid, hydrochloric acid and sodium carbonate.

4. The method for preparing a silk protein-active substance nano-aggregate according to claim 2, characterized in that: The concentration of the fibroin aggregates in the fibroin aggregate aqueous solution is 0.1-4 wt %.

5. The method for preparing a silk protein-active substance nano-aggregate according to claim 1, characterized in that: The beta-sheet content of the silk fibroin aggregate is greater than 40%, and the size range is 20-2000nm.

6. The method for preparing a silk protein-active substance nano-aggregate according to claim 1, characterized in that: The stirring rate of S2 is 200-3500 rpm.

7. The method for preparing a silk protein-active substance nano-aggregate according to claim 1, characterized in that: The protein includes at least one of animal-derived collagen and recombinant collagen; The recombinant collagen includes at least one of recombinant type I, recombinant type II, recombinant type III, recombinant type IV and recombinant type XVII collagen.

8. The method for preparing a silk protein-active substance nano-aggregate according to claim 1, characterized in that: The polysaccharide includes at least one of hyaluronic acid and plant-extracted polysaccharides.

9. A silk protein-active substance nanoaggregate, characterized in that: The silk protein-active substance nanoaggregate is prepared according to the preparation method of any one of claims 1-8.

10. Use of the silk protein-active substance nanoaggregates according to claim 9 in functional skin care products.

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