An aqueous solution of silk fibroin-hydrophobic active aggregates and its preparation method

Abstract

This invention discloses an aqueous solution of silk fibroin-hydrophobic active material aggregates and its preparation method, comprising the following steps: adding a hydrophobic active material with a planar conjugated structure to an aqueous dispersion system of high-crystalline silk fibroin aggregates; and inducing a conjugation reaction between the hydrophobic active material and the silk fibroin aggregates by stirring or homogenization to form silk fibroin-hydrophobic active material aggregates, thereby obtaining an aqueous solution of silk fibroin-hydrophobic active material aggregates. This invention utilizes the mutual conjugation reaction between high-crystalline silk fibroin at the nanoscale and a hydrophobic active material with a planar conjugated structure. Under high-speed stirring or homogenization, the two are induced to directly form nano-aggregates, and the hydrophobic active material is dissolved by utilizing the water dispersibility of silk fibroin. No other solvents, co-solvents, or carriers are required, and the method is simple and controllable.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to an aqueous solution of silk fibroin-hydrophobic active aggregates and its preparation method. Background Technology

[0002] Many active ingredients with anti-inflammatory, antioxidant, and antitumor properties, such as paclitaxel, pterostilbene, asiaticoside, insulin, and minoxidil, possess planar conjugated structures and generally lack water solubility, greatly limiting their application in the medical, pharmaceutical, and cosmetic fields. To address this water solubility issue, researchers have developed various loading methods, including liposomes, nanocarriers, nanoemulsions, and supramolecular structures, to improve their solubility or dispersibility in aqueous solutions. However, all of these methods require first dissolving the active ingredient in a specific solvent and then using complex processes to immobilize / encapsulate it. This not only presents the problem of solvent removal but also significantly limits the concentration and stability in aqueous solutions.

[0003] For example, some researchers used an alkaline solution of sericin after silk degumming to dissolve water-insoluble flavonoids, and then neutralized it to obtain an aqueous dispersion. However, this still requires that the insoluble substances can be dissolved in the alkaline solution. Similarly, the substances need to be dissolved to a specific solution first, and then react with the sericin before being neutralized.

[0004] How to achieve direct high-concentration dissolution / dispersion of the above-mentioned active ingredients in aqueous systems without using relevant solvents or specific solutions has become a bottleneck problem restricting the application of active ingredients. Summary of the Invention

[0005] This invention addresses the shortcomings of existing technologies by providing an aqueous solution of silk fibroin-hydrophobic active material aggregates and its preparation method. The hydrophobic active material is directly added to an aqueous dispersion system of high-crystalline silk fibroin aggregates. Through high-speed stirring or homogenization, the active material and the silk fibroin aggregates come into full contact, inducing a conjugation effect to form nano-aggregates. Simultaneously, the hydrophobic active material dissolves into the aqueous solution along with the aggregates, resulting in a high-concentration aqueous solution of the active material. This method eliminates the need to pre-dissolve the hydrophobic active material in a solvent or specific solution, and avoids solvent removal and post-treatment steps, making the operation simple.

[0006] To address the aforementioned technical problems, the first aspect of this invention provides a method for preparing an aqueous solution of silk fibroin-hydrophobic active aggregates, comprising the following steps:

[0007] A hydrophobic active material with a planar conjugated structure is added to an aqueous dispersion system of high-crystal silk fibroin aggregates. After stirring or homogenization, the hydrophobic active material is induced to undergo conjugation with the silk fibroin aggregates to form silk fibroin-hydrophobic active material aggregates, thus obtaining an aqueous solution of silk fibroin-hydrophobic active material aggregates.

[0008] This invention utilizes the mutual conjugation of high-crystalline silk fibroin at the nanoscale with hydrophobic active substances having planar conjugated structures. Under high-speed stirring or homogenization, the two are induced to directly form nano-aggregates. Then, the water dispersibility of silk fibroin is used to dissolve the hydrophobic active substances. No other solvents, co-solvents or carriers are required. The method is simple and controllable.

[0009] Furthermore, the size of the silk fibroin-hydrophobic active material aggregate is 10-600 nm.

[0010] Furthermore, the silk fibroin aggregates have a β-sheet content >40% and a size of 1-2000 nm.

[0011] Furthermore, the aqueous dispersion system of the silk fibroin aggregate is a silk fibroin aggregate gel or an aqueous solution of silk fibroin aggregate.

[0012] Furthermore, the mass ratio of the hydrophobic active material to the aqueous dispersion system of silk fibroin aggregates is (1-5):100.

[0013] Furthermore, the hydrophobic active ingredient is selected from natural and / or synthetic active ingredients.

[0014] Furthermore, the natural active ingredient is selected from one or more of the following natural active ingredients: alkaloids, flavonoids, terpenes, glycosides, phenols, quinones, phenylpropanoids, coumarins, and lignins.

[0015] Furthermore, the synthetic active ingredient is selected from one or more synthetic compounds of furans, pyrroles, thiophenes, imidazoles, thiazoles, pyrazoles, pyridines, pyrans, and quinolines.

[0016] Furthermore, the stirring speed is 200-3500 rpm, and the time is 2-240 min; the homogenization rate is 500-20000 rpm, and the time is 2-240 min.

[0017] The second aspect of the present invention provides an aqueous solution of silk fibroin-hydrophobic active aggregates prepared by the preparation method described in the first aspect.

[0018] The third aspect of this invention provides the application of the aqueous solution of the silk fibroin-hydrophobic active aggregate described in the second aspect in pharmaceuticals and cosmetics.

[0019] The beneficial effects of this invention are:

[0020] This invention directly adds hydrophobic active materials to an aqueous dispersion system of high-crystalline silk fibroin aggregates. Through high-speed stirring or homogenization, the active materials and silk fibroin aggregates come into full contact, thereby inducing a conjugation effect between the two to form nano-aggregates. At the same time as the nano-aggregates are formed, the hydrophobic active materials dissolve into the aqueous solution along with the aggregates, resulting in a high-concentration aqueous solution of the active materials. No other solvents, co-solvents or carriers are required. The process is simple, the conditions are mild and controllable, and it is conducive to large-scale production.

[0021] The method of this invention is universal and can achieve high-concentration aqueous phase dissolution for hydrophobic substances containing planar conjugated structures, effectively solving the application problems of different active substances.

[0022] The high-crystalline silk fibroin aggregates and hydrophobic active ingredients of this invention both have good bioactivity, and the aggregates of the two can exert a synergistic effect of the two components to achieve better results. Attached Figure Description

[0023] To more clearly illustrate the technical solution of the present invention, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 The images are of the solutions obtained in Example 1 and Comparative Example 1, where a is a macroscopic image of the solution obtained in Comparative Example 1, b is a macroscopic image of the solution obtained in Example 1, and c is an SEM image of the solution obtained in Example 1.

[0025] Figure 2 The ultraviolet and infrared spectra of the solution obtained in Example 1 are analyzed.

[0026] Figure 3 The images are of the solution obtained in Example 2, where a is a macroscopic photograph of the solution obtained in Example 2 and b is an SEM image of the solution obtained in Example 2.

[0027] Figure 4 Ultraviolet and infrared spectra of the solution obtained in Example 2;

[0028] Figure 5 Macroscopic photographs of the solution obtained in Example 3 after dilution with water and storage for different times;

[0029] Figure 6 The image shows the SEM image and transdermal structure diagram of the solution obtained in Example 4.

[0030] Figure 7 Here is a SEM image of the solution obtained in Example 5;

[0031] Figure 8 This is a comparison diagram of inflammatory factors in silk fibroin-methotrexate aggregate cell culture, as shown in Example 5. Figure 9 The fluorescence signal of the silk fibroin-methotrexate aggregate cell culture fluorescent marker staining in Example 5; Figure 10 The results show the average fluorescence intensity of silk fibroin-methotrexate aggregate cell culture in Example 5. Detailed Implementation

[0032] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] This embodiment relates to a method for preparing an aqueous solution of silk fibroin-hydrophobic active material aggregates, comprising the following steps: adding a hydrophobic active material with a planar conjugated structure to an aqueous dispersion system of high-crystalline silk fibroin aggregates; and inducing a conjugation reaction between the hydrophobic active material and the silk fibroin aggregates by stirring or homogenization, thereby forming silk fibroin-hydrophobic active material aggregates to obtain an aqueous solution of silk fibroin-hydrophobic active material aggregates, wherein the size of the silk fibroin-hydrophobic active material aggregates is 10-600 nm. This embodiment utilizes the mutual conjugation reaction between high-crystalline silk fibroin at the nanoscale and a hydrophobic active material with a planar conjugated structure. Under high-speed stirring or homogenization, the two are induced to directly form nano-aggregates, and the hydrophobic active material is dissolved by utilizing the water dispersibility of silk fibroin. No other solvents, co-solvents, or carriers are required, and the method is simple and controllable.

[0034] In a preferred embodiment, the β-sheet content of the silk fibroin aggregate is >40%, and the size is 1-1000 nm; the aqueous dispersion system of the silk fibroin aggregate is a silk fibroin aggregate gel or an aqueous solution of silk fibroin aggregate.

[0035] In a preferred embodiment, the mass ratio of the hydrophobic active material to the aqueous dispersion system of silk fibroin aggregates is (1-5):100.

[0036] In a preferred embodiment, the hydrophobic active ingredient is selected from natural active ingredients and / or synthetic active ingredients. The natural active ingredients are selected from one or more of the following natural active ingredients: alkaloids, flavonoids, terpenes, glycosides, phenols, quinones, phenylpropanoids, coumarins, and lignins. The synthetic active ingredients are selected from one or more of the following synthetic compounds: furans, pyrroles, thiophenes, imidazoles, thiazoles, pyrazoles, pyridines, pyrans, and quinolines.

[0037] In a preferred embodiment, the stirring speed is 200-3500 rpm and the time is 2-240 min; the homogenization rate is 500-20000 rpm and the time is 2-240 min.

[0038] Another embodiment provides an aqueous solution of silk fibroin-hydrophobic active aggregates prepared by the preparation method described in the above embodiments.

[0039] Another embodiment provides the application of the aqueous solution of silk fibroin-hydrophobic active aggregates described in the above embodiments in pharmaceuticals and cosmetics.

[0040] Example 1

[0041] This embodiment relates to a method for preparing an aqueous solution of silk fibroin-hydrophobic active aggregates, comprising the following steps:

[0042] (1) Add 3 grams of Pterostilbene powder directly to 100 grams of 2% (w / w) high-crystalline silk fibroin nanoaggregates (β-sheet content 45%, size 1000 nm) gel;

[0043] (2) Stir the mixture from step (1) at 1500 rpm for 30 min to induce conjugation between silk fibroin aggregates and pterostilbene to form nano-aggregates and obtain an aqueous solution of pterostilbene.

[0044] Example 2

[0045] This embodiment relates to a method for preparing an aqueous solution of silk fibroin-hydrophobic active aggregates, comprising the following steps:

[0046] (1) Add 1 gram of paclitaxel powder directly to 100 grams of 3% (w / w) high-crystalline silk fibroin nanoaggregates (β-sheet content 41%, size 600 nm) gel;

[0047] (2) Stir the mixture from step (1) at 2500 rpm for 30 min to induce the conjugation of silk fibroin aggregates and paclitaxel to form nano-aggregates and obtain an aqueous solution of paclitaxel.

[0048] Example 3

[0049] This embodiment relates to a method for preparing an aqueous solution of silk fibroin-hydrophobic active aggregates, comprising the following steps:

[0050] (1) Add 5 grams of minoxidil powder directly to 100 grams of aqueous solution of 2% high-crystalline silk fibroin nanoaggregates (β-sheet content 50%, size about 100 nm);

[0051] (2) Homogenize the mixture from step (1) at 4500 rpm for 30 min to induce conjugation between silk fibroin aggregates and minoxidil to form nano-aggregates and obtain an aqueous solution of minoxidil.

[0052] Example 4

[0053] This embodiment relates to a method for preparing an aqueous solution of silk fibroin-hydrophobic active aggregates, comprising the following steps:

[0054] (1) Add 1 gram of insulin powder directly to 100 grams of aqueous solution of 1% high-crystalline silk fibroin nanoaggregates (β-sheet content 42%, size approximately 100 nm);

[0055] (2) Stir the mixture from step (1) at 800 rpm for 100 min to induce conjugation between silk fibroin aggregates and insulin, forming nano-aggregates, and obtain an aqueous solution of insulin.

[0056] Example 5

[0057] This embodiment relates to a method for preparing an aqueous solution of silk fibroin-hydrophobic active aggregates, comprising the following steps:

[0058] (1) Add 1 gram of methotrexate powder directly to 100 grams of 2% (by mass) high-crystalline silk fibroin nanofiber (β-sheet content 51%, size approximately 800 nm) gel;

[0059] (2) Stir the mixture from step (1) at 3500 rpm for 60 min to induce the conjugation of silk fibroin aggregates and methotrexate to form nano-aggregates and obtain an aqueous solution of methotrexate.

[0060] Example 6

[0061] This embodiment relates to a method for preparing an aqueous solution of silk fibroin-hydrophobic active aggregates, comprising the following steps:

[0062] (1) Add 5 grams of tea polyphenol powder directly to 100 grams of a solution of 1% mass concentration of high-crystalline silk fibroin nanoparticles (β-sheet content 44%, size 20-50nm);

[0063] (2) Homogenize the mixture from step (1) at 5000 rpm for 30 min to induce conjugation between silk fibroin aggregates and tea polyphenols, forming nano-aggregates, and obtain an aqueous solution of tea polyphenols.

[0064] Example 7

[0065] This embodiment relates to a method for preparing an aqueous solution of silk fibroin-hydrophobic active aggregates, comprising the following steps:

[0066] (1) Add 1 gram of finasteride powder directly to 100 grams of a 2% (w / w) solution of high-crystalline silk fibroin nanofibers (β-sheet content 50%, fiber length 500-800 nm);

[0067] (2) Stir the mixture from step (1) at 2000 rpm for 180 min to induce the silk fibroin aggregates and finasteride to conjugate and form nano-aggregates, thereby obtaining an aqueous solution of finasteride.

[0068] Example 8

[0069] This embodiment relates to a method for preparing an aqueous solution of silk fibroin-hydrophobic active aggregates, comprising the following steps:

[0070] (1) Add 3 grams of ketoconazole powder directly to 100 grams of a 4% (w / w) solution of high-crystalline silk fibroin nanofibers (β-sheet content 44%, nanofiber length 1500-2000 nm);

[0071] (2) Homogenize the mixture from step (1) at 6000 rpm for 15 min to induce conjugation between silk fibroin aggregates and ketoconazole to form nano-aggregates and obtain an aqueous solution of ketoconazole.

[0072] Example 9

[0073] This embodiment relates to a method for preparing an aqueous solution of silk fibroin-hydrophobic active aggregates, comprising the following steps:

[0074] (1) Add 1 gram of methyl jasmonate liquid directly to 100 grams of a 2% (w / w) solution of high-crystalline silk fibroin nanofibers (β-sheet content 52%, nanofiber length 1000-1500 nm);

[0075] (2) Stir the mixture from step (1) at 1500 rpm for 150 min to induce the conjugation of silk fibroin aggregates and methyl jasmonate to form nano-aggregates and obtain an aqueous solution of methyl jasmonate.

[0076] Comparative Example 1

[0077] The mixture was obtained by adding 3 grams of pterostilbene directly to 100 grams of water and stirring at 1500 rpm for 30 minutes.

[0078] Test case

[0079] Figure 1Images a and b are macroscopic photographs of the solutions obtained in Comparative Example 1 and Example 1, respectively. It can be seen that Example 1 is a uniform white paste, which transforms into a stable, translucent aqueous solution upon dilution with water; while in Comparative Example 1, no dissolution of pterostilbene was observed, and it remained a powdery precipitate. (SEM images) Figure 1 In c), it can be observed that the silk fibroin and pterostilbene aggregates are in the form of particles with a size of less than 100 nm.

[0080] Figure 2 The ultraviolet and infrared spectra of the silk fibroin-pterostilbene (SNF-pterostilbene) solution obtained in Example 1 revealed the conjugation effect between silk fibroin and pterostilbene.

[0081] Figure 3 In Figures a and b, respectively, are macroscopic photographs and SEM images of the solution obtained in Example 2. It can be seen that the paclitaxel-silk fibroin aggregates also have good water dispersibility, and can form a semi-transparent stable solution when diluted with water; the aggregate size is less than 100 nm.

[0082] Figure 4 The UV and IR spectra of the silk fibroin-paclitaxel (SNF-paclitaxel) solution obtained in Example 2 also revealed the conjugation effect between silk fibroin and paclitaxel.

[0083] Figure 5 Macroscopic photographs of the solution obtained in Example 3 after dilution with water and storage for different times show that the aqueous solution remained stable after 10 days without precipitation, proving that the silk fibroin-minoxidil aggregate has good water solubility.

[0084] Figure 6 The images show the SEM image and transdermal structure diagram of the solution obtained in Example 4. The SEM image shows that the aggregates are uniformly dispersed. The transdermal structure diagram shows that the silk fibroin-insulin (SNF-insulin) aggregates can be effectively absorbed through the skin, while free insulin is difficult to absorb through the skin. No fluorescent signal was observed in the dermis.

[0085] Figure 7 The image shows the SEM image of the solution obtained in Example 5, which also has a nanoparticle structure. Figure 8This is a comparison of inflammatory factors in cell culture of silk fibroin-methotrexate (SNF-methotrexate) aggregates, as shown in Example 5. Tumor necrosis factor and interleukin-6 are both inflammatory factors, and their high levels indicate that the cells are in a pro-inflammatory state (** indicates significant difference, p<0.01, **** indicates significant difference, p<0.0001). Macrophages undergo lipopolysaccharide-induced transformation into the pro-inflammatory M1 type, exhibiting a significant increase in the secretion of pro-inflammatory factors. When lipopolysaccharide-induced macrophages are cultured with different active ingredients, carbomer-dispersed methotrexate significantly reduces the secretion of pro-inflammatory factors, demonstrating the bioactivity of methotrexate. Furthermore, when silk fibroin-methotrexate aggregates are cultured with lipopolysaccharide-induced macrophages, the secretion of pro-inflammatory factors further decreases, proving that under the same methotrexate concentration, its anti-inflammatory performance is superior to that of free methotrexate. Figure 9 The fluorescent signals are represented by staining with fluorescent markers, where DAPI, DID, and C6 represent the fluorescence signals of cells after staining with these markers, indicating the cytoplasm, nucleus, and methotrexate drug, respectively. Merge is an integrated fluorescence map combining these different signals. The amount of drug entering the cell can be characterized by calculating the fluorescence signal intensity of C6. Figure 10 The mean fluorescence intensity results (* indicates a significant difference, p<0.05, *** indicates a significant difference, p<0.001) show that silk fibroin-methotrexate aggregates are more easily actively phagocytosed by cells compared with free methotrexate and carbomer-dispersed methotrexate.

[0086] The present invention has been described in detail above with reference to specific embodiments and exemplary examples; however, these descriptions should not be construed as limiting the present invention. Those skilled in the art will understand that various equivalent substitutions, modifications, or improvements can be made to the technical solutions and embodiments of the present invention without departing from the spirit and scope of the invention, and all such modifications and improvements fall within the scope of the present invention. The scope of protection of the present invention is defined by the appended claims.

Claims

1. A method for preparing a water-soluble solution of a silk fibroin-hydrophobic active aggregate, characterized by, Includes the following steps: A hydrophobic active material with a planar conjugated structure is added to an aqueous dispersion system of high-crystal silk fibroin aggregates. After stirring or homogenization, the hydrophobic active material is induced to undergo a conjugation effect with the silk fibroin aggregates to form silk fibroin-hydrophobic active material aggregates, thus obtaining an aqueous solution of silk fibroin-hydrophobic active material aggregates. The hydrophobic active ingredient is pterostilbene; The mass ratio of the hydrophobic active material to the aqueous dispersion system of silk fibroin aggregates is (1-5):100; The stirring speed is 200-3500 rpm, and the time is 2-240 min; the homogenization rate is 500-20000 rpm, and the time is 2-240 min.

2. The method for preparing the aqueous solution of silk fibroin-hydrophobic active aggregates as described in claim 1, characterized in that, The silk fibroin aggregates have a β-sheet content >40% and a size of 1-2000 nm.

3. The method for preparing the aqueous solution of silk fibroin-hydrophobic active aggregates as described in claim 1, characterized in that, The aqueous dispersion system of the silk fibroin aggregate is a silk fibroin aggregate gel or an aqueous solution of silk fibroin aggregate.

4. An aqueous solution of silk fibroin-hydrophobic active aggregates prepared by the preparation method according to any one of claims 1-3.

5. The application of the aqueous solution of silk fibroin-hydrophobic active aggregate as described in claim 4 in the preparation of pharmaceuticals and cosmetics.

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