Composite microsphere containing silk material as well as preparation method and application of composite microsphere

Abstract

The invention discloses a composite microsphere containing a silk material and a preparation method and application of the composite microsphere, and relates to the technical field of biomedical materials. The shell raw materials comprise a chitosan solution, a cross-linking agent and a catalyst; the encapsulated microsphere raw materials comprise microspheres and vesicles; the raw materials of the microspheres comprise a silk material, an extracellular matrix, poly-desoxyribonucleic acid and a cationic substance. Raw materials of the vesicles comprise an oleate solution and a glutamic acid solution.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to a composite microsphere containing silk material, its preparation method, and its application. Background Technology

[0002] Medical gel dressings, as core carriers for skin damage repair, inflammation intervention, and local treatment, rely heavily on two key performance characteristics for their clinical value: first, to achieve efficient transdermal absorption of large-molecule therapeutic ingredients such as fibrin and polydeoxyribonucleic acid (PDRN); and second, to leverage technology to enable the extracellular matrix (ECM), which is inherently difficult to penetrate the skin, to play an auxiliary role in transdermal absorption, and to synergistically enhance overall transdermal absorption and skin repair performance through self-assembly and encapsulation of therapeutic ingredients. Current bottlenecks in the development of medical gel dressings lie in how to overcome the skin's stratum corneum barrier to achieve precise delivery of active ingredients, while simultaneously mitigating the safety risks associated with complex excipients and processes.

[0003] The "lipid sandwich" structure of the stratum corneum of the skin has an extremely high barrier rate for substances with a molecular weight exceeding 500 Da. In particular, charged macromolecules (such as PDRN and filacrin) are easily repelled by the negative charge of the lipid layer. As a result, the active ingredients in medical gel dressings tend to remain on the skin surface after application, making it difficult for them to penetrate into the dermis to exert their repair, anti-inflammatory, or antibacterial effects. This problem is particularly prominent in cases requiring deep local treatment, such as acne and skin ulcers. To overcome this barrier, traditional solutions often rely on formulation technologies such as nanoemulsions and liposomes. While these can improve permeability to some extent, they require the introduction of excipients such as solubilizers and emulsifiers. These ingredients not only increase production costs but may also cause skin irritation or allergic reactions, which contradicts the clinical requirements of "low allergenicity and high safety" for medical dressings. Furthermore, some formulation carriers are too large, making it difficult to achieve long-term controlled release of active ingredients and failing to meet the continuous treatment needs of chronic skin damage.

[0004] While macromolecular self-assembly microsphere technology can reduce reliance on excipients, it faces the dual challenges of single-component compression difficulties and the complexity of multi-component sphere formation. Existing technologies often produce microspheres with rigid structures and poor biocompatibility, with particle sizes exceeding 10 μm, far exceeding the transdermal threshold of the stratum corneum (approximately 50 nm). These are only suitable for injection administration and cannot meet the application requirements of medical gel dressings. Other technologies achieve multi-component composite microsphere formation through electrostatic adsorption, but the surface coating structure hinders the release and penetration of active ingredients, further reducing transdermal efficiency. More importantly, the synthesis of some microspheres requires alcohol precipitation using organic solvents such as ethanol. Residual solvents can disrupt the skin microenvironment, contradicting the clinical contraindications for medical dressings, and is particularly unsuitable for repairing damaged skin.

[0005] Therefore, inventing a gel formulation containing composite microspheres is of great significance. Summary of the Invention

[0006] The purpose of this invention is to provide a composite microsphere containing silk material, its preparation method, and its application, so as to solve the problems raised in the prior art.

[0007] To achieve the above objectives, the present invention provides the following technical solution: A composite microsphere containing silk material, wherein the raw materials of the composite microsphere, by mass parts, include 0.001-2 parts of silk material, 0.001-2 parts of polydeoxyribonucleic acid and 0.008-0.16 parts of cationic substance; wherein the mass ratio of silk material to polydeoxyribonucleic acid is 1:1.

[0008] Furthermore, the raw materials for the composite microspheres, by mass fraction, include 0.001-2 parts of silk material, 0.001-3 parts of extracellular matrix, 0.002-5 parts of polydeoxyribonucleic acid and 0.008-0.16 parts of cationic substances; wherein the total mass ratio of silk material and extracellular matrix to polydeoxyribonucleic acid is 1:1.

[0009] Furthermore, the composite microspheres have a core-shell structure, with the core made of materials including silk, extracellular matrix, polydeoxyribonucleic acid, and cationic substances; the shell is a vesicle covering the surface of the core. Furthermore, the raw materials for the vesicles include an oleate solution and a glutamic acid solution, wherein the concentration of the oleate solution is 8.5-9 mM and the concentration of the glutamic acid solution is 4-5 mM; the oleate solution includes any one of glyceryl oleate citrate solution and choline oleate solution.

[0010] Furthermore, the composite microsphere has a double-shell structure, with the first shell being a vesicle covering the surface of the core and the second shell being an outer shell covering the surface of the vesicle. Furthermore, the raw materials of the shell include chitosan solution, crosslinking agent, and catalyst; in the shell, the mass concentration of chitosan solution is 1-10%, the crosslinking agent is a 90-98% mass concentration 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloric acid solution, the catalyst is a 95-100% mass concentration N-hydroxysuccinimide solution, and the mass ratio of chitosan solution: crosslinking agent: catalyst is (4-10):1:1.

[0011] A method for preparing composite microspheres containing silk material includes the following steps: S1: Silk material, extracellular matrix, polydeoxyribonucleic acid (PDA), and cationic substances are added to deionized water and stirred evenly to obtain silk material solution, extracellular matrix solution, PDA solution, and cationic substance solution, respectively; the silk material solution, extracellular matrix solution, and PDA solution are added to a reaction vessel, heated to 60-65℃ for 1-1.5 h, the pH is adjusted to 6, the cationic substance solution is added, and the mixture is homogenized under high pressure 5-6 times to obtain microspheres; Furthermore, the silk material includes any one or more of the following: silk protein, hydrolyzed silk, hydrolyzed silk protein, and silk amino acids; Furthermore, the cationic substance includes either polyethyleneimine or copper sulfate; S2: Oleic acid ester and glutamic acid are added to deionized water and stirred until homogeneous to obtain oleic acid ester solution and glutamic acid solution respectively; the glutamic acid aqueous solution is added to the oleic acid ester solution and allowed to stand to obtain vesicles; S3: Add the microspheres to dimethyl sulfone and stir until homogeneous to obtain a microsphere solution; add the microsphere solution to vesicles, let stand, centrifuge, and freeze-dry to obtain encapsulated microspheres; S4: Add chitosan solution, crosslinking agent, and catalyst to a reaction vessel, stir and react for 12-24 hours, then freeze-dry to obtain the outer shell; S5: Disperse the shell and encapsulated microspheres in deionized water to form a suspension, mix them evenly by ultrasonication, centrifuge, remove the lower precipitate, freeze-dry, and obtain composite microspheres.

[0012] Furthermore, the method for preparing the choline oleate includes the following steps: A methanol solution of oleic acid was added to a methanol solution of choline hydroxide at a temperature of 10-15°C. After the addition was complete, the mixture was stirred at room temperature for 20-24 hours. The mixture was then rotary evaporated, washed with diethyl ether, and dried under vacuum at room temperature to obtain choline oleate. In the preparation of choline oleate, the molar ratio of oleic acid to choline hydroxide is 1:1.

[0013] Furthermore, the chitosan solution is prepared by dissolving modified chitosan and carboxylated chitosan in acetic acid solution at a mass ratio of (0.5-1.5):(0.5-1.5). Furthermore, the preparation method of the modified chitosan includes the following steps: Succinic anhydride was heated to 135-140℃ to melt, and 6-deoxy-6-aminochitosan was added. The reaction was maintained at this temperature for 10-15 min to obtain a succinic anhydride derivative. The succinic anhydride derivative was added to N,N-dimethylformamide, and COMU peptide coupling agent was added for activation at room temperature for 1-1.5 h. Phytosphingosine was added, and the reaction was carried out at room temperature for 23-24 h. After purification, a phytosphingosine conjugate was obtained. Fatty acid ethyl esters were added to the phytosphingosine conjugate, and a lipase catalyst was added. The mixture was reacted at 50-55℃ and 90 mbar vacuum for 8-9 h. After purification, modified chitosan was obtained.

[0014] Furthermore, in the preparation process of the succinic anhydride derivative, the molar ratio of succinic anhydride to 6-deoxy-6-aminochitosan is 1:3; Furthermore, in the preparation process of the phytosphingosine conjugate, the molar ratio of succinic anhydride derivative: COMU peptide conjugate: phytosphingosine is 1:2:10; Furthermore, in the preparation process of the modified chitosan, the ratio of plant sphingosine coupling compound: fatty acid ethyl ester: lipase catalyst is 100mg:2mL:100mg.

[0015] Furthermore, the preparation method of the gel formulation includes the following steps: adding poly(N-vinylcaprolactam) to deionized water at 3-4℃, stirring evenly, and allowing it to stand to obtain a poly(N-vinylcaprolactam) dispersion; adding hydroxypropyl methylcellulose to the poly(N-vinylcaprolactam) dispersion, stirring at 3-4℃ until completely dissolved to obtain a mixture; Polyvinyl alcohol was added to deionized water, heated to 95-100℃ and stirred until homogeneous, then cooled to obtain polyvinyl alcohol hydrogel; the polyvinyl alcohol hydrogel was added to starch aqueous solution, heated to 95-100℃ and stirred until homogeneous, then cooled to obtain composite gel phase; The composite microspheres and hyaluronic acid were added to the acetic acid solution and stirred until homogeneous to obtain the aqueous phase. Add the aqueous phase to the mixture, stir at room temperature, add the composite gel phase, stir until homogeneous, homogenize at high speed, let stand at room temperature, and store at 3-4℃ in the dark to obtain the gel formulation.

[0016] Furthermore, in the composite microspheres, the mass ratio of the outer shell to the encapsulated microspheres is (10-80):(1-10).

[0017] Furthermore, in the encapsulated microspheres, the concentration of the microsphere solution is 0.5-5 mM.

[0018] Furthermore, the concentration of the acetic acid solution is 2% v / v.

[0019] Furthermore, the proportions of each component in the gel formulation, by mass percentage, include: 25-30% poly(N-vinylcaprolactam), 1.5-2% hydroxypropyl methylcellulose, 0.25-0.5% polyvinyl alcohol, 0.5-0.75% starch, 0.5-3% composite microspheres, 0.05-0.3% hyaluronic acid, 3-5% acetic acid solution, and the remainder being deionized water.

[0020] Furthermore, during the preparation of the gel formulation, the rate at which the aqueous phase solution is added to the mixture is 1 mL / min.

[0021] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention achieves enhanced transdermal absorption and long-lasting antibacterial properties of composite microspheres and their gel formulations through multi-component synergistic design. At the same time, the silk material, polydeoxyribonucleic acid and extracellular matrix in the composite microspheres work synergistically, and with the help of the transdermal absorption performance of the double-layer coating of the composite microspheres, the gel formulation is further endowed with better skin tissue repair ability. It can also optimize the biocompatibility of the composite microspheres and reduce the foreign body sensation on the skin. The original functions of the silk material and polydeoxyribonucleic acid remain unchanged, still bringing anti-wrinkle and basic repair functions to the gel formulation. In particular, the extracellular matrix, as a key component of the composite microsphere core, forms a precise 1:1 ratio of total mass and polydeoxyribonucleic acid when combined with silk material. It not only serves as a core auxiliary transdermal absorption substance, but also works synergistically with the cationic properties of the microspheres, the pH-responsive permeability of the vesicles, and the temporary transdermal channels of the shell, facilitating the penetration of active ingredients through the stratum corneum barrier to the dermis. Furthermore, through its rich content of collagen, elastin, laminin, and other active ingredients, it provides a natural regenerative and repair scaffold for skin cells, significantly promoting the proliferation and migration of fibroblasts. It acts directly on damaged skin to accelerate tissue remodeling and regeneration, significantly improving wound healing rate and shortening the time for complete skin repair. At the same time, the extracellular matrix, silk material, and polydeoxyribonucleic acid produce significant synergistic effects, effectively inhibiting MMP-1 expression and promoting type I collagen synthesis, thereby filling skin wrinkles, improving skin elasticity and firmness, optimizing the biocompatibility of composite microspheres, reducing the foreign body sensation on the skin, and giving the gel formulation excellent anti-wrinkle effects, making it show outstanding application potential in skin rejuvenation repair and various skin damage repair scenarios.

[0022] 2. Regarding transdermal absorption, this invention firstly utilizes the dual effects of pH responsiveness and enhanced permeability in the vesicles formed by choline oleate and glutamate. Choline oleate, as a surface-active ionic liquid, allows its oleic acid chains to fuse with lipids in the stratum corneum and adipose layer, disrupting the lipid barrier. Furthermore, the cationic choline head group can electrostatically attract negatively charged groups on the skin surface, prolonging the vesicle's residence time. Glutamate, on the other hand, possesses pH-responsive properties. In the neutral environment of normal skin, it keeps the vesicles stable. When exposed to the acidic environment of inflamed areas, the protonation state of glutamate changes, disrupting the vesicle bilayer structure and releasing the encapsulated microspheres, achieving targeted transdermal absorption. Simultaneously, the vesicle structure protects the microspheres from enzymatic degradation, maintaining long-lasting release. Next, microspheres composed of silk, polydeoxyribonucleic acid, extracellular matrix, and cationic substances are homogenized under high pressure to form uniform nanoscale particles, which can penetrate through the lipid gaps of the stratum corneum or hair follicles and sweat gland channels. The cationic substances give the microspheres a positive charge, which can electrostatically attract the negatively charged surface of the stratum corneum, prolonging the contact time between the microspheres and the skin and reducing the loss of components. The silk material itself has good moisturizing properties, which can maintain the skin's moist environment to soften the stratum corneum, and its excellent biocompatibility can reduce the irritation of the microspheres to the skin and improve the tolerance of the transdermal process. Finally, the gel matrix enhances the transdermal effect through the synergistic effect of temperature-sensitive reservoirs and penetration enhancers. The low critical dissolution temperature of poly(N-vinylcaprolactam) is close to skin temperature, fixing the composite microspheres on the skin surface to form a localized high-concentration reservoir for continuous skin penetration. The modified chitosan structure in the composite microsphere shell, containing plant sphingolipid arms, fatty acid chains, and hyaluronic acid, works synergistically. The plant sphingolipid arms, containing a sphingosine backbone consistent with the sphingosine structure of ceramides in the stratum corneum, can insert into the lipid bilayer through hydrophobic interactions, disrupting the original ordered arrangement and promoting transdermal absorption. The fatty acid chains match the length of free fatty acid chains in the stratum corneum, further filling lipid gaps and increasing membrane fluidity, ultimately forming "temporary permeation channels" in the intercellular lipids, reducing transdermal resistance.

[0023] 3. In terms of antibacterial properties, the hydroxyl groups of the core chitosan backbone can form hydrogen bonds with the phosphate groups of bacterial DNA, interfering with DNA replication and transcription, thus providing basic antibacterial protection for the gel formulation; the choline oleate vesicles themselves can also enhance the antibacterial effect by disrupting the bacterial cell membrane, achieving a long-lasting and precise antibacterial effect. Detailed Implementation

[0024] The technical solutions in the embodiments of the present invention will be clearly and completely described below. 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.

[0025] It should be noted that there are no special restrictions on the manufacturers of the raw materials involved in this invention. Exemplary examples include: silk core protein (Suzhou Suhao Biomaterials Technology Co., Ltd.); polydeoxyribonucleic acid (PDRN, Ruijiming Biotechnology Co., Ltd.; Shanghai Huiwen Biotechnology Co., Ltd.); polyvinyl alcohol (PVA2000); and other raw materials are commercially available.

[0026] The following examples illustrate a method for preparing choline oleate, comprising the following steps: A methanol solution containing 1 mM oleic acid was added to a methanol solution containing 1 mM choline hydroxide at a temperature of 10°C. After the addition was complete, the mixture was stirred at room temperature for 20 h, rotary evaporated, washed with ether, and dried under vacuum at room temperature to obtain choline oleate.

[0027] The preparation method of modified chitosan includes the following steps: 1 mM succinic anhydride was heated to 135 °C to melt, and 3 mM 6-deoxy-6-aminochitosan was added. The reaction was maintained at this temperature for 10 min to obtain a succinic anhydride derivative. 1 mM of the succinic anhydride derivative was added to N,N-dimethylformamide, and 2 mM COMU peptide coupling agent was added to activate it at room temperature for 1 h. 10 mM phytosphingosine was added, and the reaction was carried out at room temperature for 23 h. After purification, a phytosphingosine conjugate was obtained. 2 mL of ethyl decanoate was added to 100 mg of the phytosphingosine conjugate, and 100 mg of lipase catalyst was added. The mixture was reacted at 50 °C and 90 mbar vacuum for 8 h. After purification, modified chitosan was obtained.

[0028] The preparation method of 6-deoxy-6-aminochitosan includes the following steps: 7.37 mmol chitosan and 18.6 mmol phthalic anhydride were added to 50 mL of AcOH / H2O mixed solution, heated to 120 °C and refluxed for 24 h. After centrifugation, filtration, washing of the precipitate, purification, and drying under reduced pressure, N-phthalylated protected chitosan was obtained. 7.89 g of N-phthalylated protected chitosan was suspended in 190 mL of pyridine, cooled to 0 °C, and 20.4 g of p-toluenesulfonyl chloride was added. The mixture was reacted at room temperature for 48 h. The reaction solution was then introduced into ice water, filtered, the precipitate was washed, and dried under reduced pressure to obtain intermediate A. 0.188 g of intermediate A and 0.0595 g of potassium phthalimide were suspended in DMAc, heated to 120 °C and reacted for 4.5 h. After cooling to room temperature, 5 mL of deionized water was added, filtered, the precipitate was washed, and dried under reduced pressure to obtain intermediate B. 5.13 g of intermediate B and hydrazine hydrate were added to anhydrous ethanol, heated to 95 °C and reacted for 4 h. After cooling to room temperature, 300 mL of ethanol was added and filtered to obtain intermediate C. 1.76 g of intermediate C was added to 40 mL of 45% sodium hydroxide solution, heated to 100 °C and refluxed for 1 h, cooled to room temperature, filtered, the pH of the filtrate was adjusted to 7, dialyzed, and freeze-dried to obtain 6-deoxy-6-aminochitosan.

[0029] Example 1: A method for preparing a gel formulation of composite microspheres containing silk material, comprising the following steps: S1: Silk protein, polydeoxyribonucleic acid (PDA), and copper sulfate are added to deionized water and stirred evenly to obtain a silk material solution, a PDA solution, and a cationic substance solution, respectively; the silk material solution and the PDA solution are added to a reaction vessel, heated to 60°C and reacted for 1 hour, the pH is adjusted to 6, the cationic substance solution is added, and the mixture is homogenized under high pressure 5 times to obtain microspheres; wherein, the mass ratio of silk protein: PDA: copper sulfate is 1:1:0.096; S2: Choline oleate and glutamic acid were added to deionized water and stirred until homogeneous to obtain 8.5 mM choline oleate solution and 4 mM glutamic acid solution, respectively; the glutamic acid aqueous solution was added to the choline oleate solution and allowed to stand to obtain vesicles; S3: Add the microspheres to dimethyl sulfone and stir until homogeneous to obtain a 2.5 mM microsphere solution; add the microsphere solution to the vesicles, let stand, centrifuge, and take the supernatant to obtain the encapsulated microspheres; S4: Add 4g of chitosan solution, 1g of crosslinking agent, and 1g of catalyst to a reaction vessel, stir and react for 12h, and freeze-dry to obtain the outer shell; the chitosan solution is prepared by dissolving modified chitosan and carboxylated chitosan in acetic acid solution at a mass ratio of 0.5:1.5. S5: Disperse 10g of shell and 1g of encapsulated microspheres in deionized water to form a suspension, mix evenly by ultrasonication, centrifuge, take the lower precipitate, freeze dry, and obtain composite microspheres; S6: Add 25wt% poly(N-vinylcaprolactam) to deionized water at 3℃, stir until homogeneous, and let stand to obtain a poly(N-vinylcaprolactam) dispersion; add 1.5wt% hydroxypropyl methylcellulose to the poly(N-vinylcaprolactam) dispersion, stir at 3℃ until completely dissolved to obtain a mixture; 0.5 wt% polyvinyl alcohol was added to deionized water, heated to 95°C and stirred until homogeneous, then cooled to obtain polyvinyl alcohol hydrogel; the polyvinyl alcohol hydrogel was added to an aqueous solution containing 0.5 wt% starch, heated to 95°C and stirred until homogeneous, then cooled to obtain a composite gel phase; 0.5 wt% composite microspheres and 0.05 wt% hyaluronic acid were added to a 3 wt% acetic acid solution and stirred until homogeneous to obtain an aqueous phase. The composite gel phase was added to the mixture, stirred at room temperature, and then the aqueous phase was added. The mixture was stirred until homogeneous, homogenized at high speed, allowed to stand at room temperature, and stored at 3°C ​​in the dark to obtain the gel formulation.

[0030] Example 2: A method for preparing a gel formulation of composite microspheres containing silk material, comprising the following steps: S2: Choline oleate and glutamic acid are added to deionized water and stirred evenly to obtain 9mM choline oleate solution and 5mM glutamic acid solution respectively; the glutamic acid aqueous solution is added to the choline oleate solution and allowed to stand to obtain vesicles; The remaining steps are the same as in Example 1.

[0031] Example 3: A method for preparing a gel formulation of composite microspheres containing silk material, comprising the following steps: S4: 4g chitosan solution, 1g crosslinking agent, and 1g catalyst are added to a reaction vessel, stirred and reacted for 12h, and then freeze-dried to obtain the outer shell; the chitosan solution is prepared by dissolving modified chitosan and carboxylated chitosan in acetic acid solution at a mass ratio of 1.5:0.5. The remaining steps are the same as in Example 2.

[0032] Example 4: A method for preparing a gel formulation of composite microspheres containing silk material, comprising the following steps: S6: 25 wt% poly(N-vinylcaprolactam) is added to deionized water at 3°C, stirred evenly, and allowed to stand to obtain a poly(N-vinylcaprolactam) dispersion; 1.5 wt% hydroxypropyl methylcellulose is added to the poly(N-vinylcaprolactam) dispersion, stirred at 3°C ​​until completely dissolved to obtain a mixture; 0.5 wt% polyvinyl alcohol was added to deionized water, heated to 95°C and stirred until homogeneous, then cooled to obtain polyvinyl alcohol hydrogel; the polyvinyl alcohol hydrogel was added to an aqueous solution containing 0.5 wt% starch, heated to 95°C and stirred until homogeneous, then cooled to obtain a composite gel phase; 3 wt% composite microspheres and 0.3 wt% hyaluronic acid were added to a 3 wt% acetic acid solution and stirred until homogeneous to obtain an aqueous phase. The composite gel phase was added to the mixture, stirred at room temperature, the aqueous phase was added, stirred until homogeneous, homogenized at high speed, allowed to stand at room temperature, and stored at 3°C ​​in the dark to obtain the gel formulation. The remaining steps are the same as in Example 3.

[0033] Example 5: A method for preparing a gel formulation of composite microspheres containing silk material, comprising the following steps: S1: Silk core protein, extracellular matrix, polydeoxyribonucleic acid (PDA), and copper sulfate are added to deionized water and stirred evenly to obtain silk material solution, extracellular matrix solution, PDA solution, and cationic substance solution, respectively; the silk material solution, extracellular matrix solution, and PDA solution are added to a reaction vessel, heated to 60°C and reacted for 1 hour, the pH is adjusted to 6, the cationic substance solution is added, and the mixture is homogenized under high pressure 5 times to obtain microspheres; wherein, the mass ratio of silk core protein: extracellular matrix: PDA: copper sulfate is 0.5:0.5:1:0.096; The remaining steps are the same as in Example 1.

[0034] Example 6: A method for preparing a gel formulation of composite microspheres containing silk material, comprising the following steps: S1: Silk core protein, extracellular matrix, polydeoxyribonucleic acid (PDA), and copper sulfate are added to deionized water and stirred evenly to obtain silk material solution, extracellular matrix solution, PDA solution, and cationic substance solution, respectively; the silk material solution, extracellular matrix solution, and PDA solution are added to a reaction vessel, heated to 60°C and reacted for 1 hour, the pH is adjusted to 6, the cationic substance solution is added, and the mixture is homogenized under high pressure 5 times to obtain microspheres; wherein, the mass ratio of silk core protein: extracellular matrix: PDA: copper sulfate is 1:1:3:0.096; The remaining steps are the same as in Example 1.

[0035] Example 7: A method for preparing a gel formulation of composite microspheres containing silk material, comprising the following steps: S1: Silk protein, polydeoxyribonucleic acid (PDA), and copper sulfate are added to deionized water and stirred evenly to obtain a silk material solution, a PDA solution, and a cationic substance solution, respectively; the silk material solution and the PDA solution are added to a reaction vessel, heated to 60°C and reacted for 1 hour, the pH is adjusted to 6, the cationic substance solution is added, and the mixture is homogenized under high pressure 5 times to obtain microspheres; wherein, the mass ratio of silk protein: PDA: copper sulfate is 1:1:0.096; S2: Glyceryl oleate citrate and glutamic acid were added to deionized water and stirred until homogeneous to obtain 8.5 mM glyceryl oleate citrate solution and 4 mM glutamic acid solution, respectively; the glutamic acid aqueous solution was added to the glyceryl oleate citrate solution and allowed to stand to obtain vesicles; S3: Add the microspheres to dimethyl sulfone and stir until homogeneous to obtain a 2.5 mM microsphere solution; add the microsphere solution to the vesicles, let stand, centrifuge, and take the supernatant to obtain the encapsulated microspheres; S4: Add 4g of chitosan solution, 1g of crosslinking agent, and 1g of catalyst to a reaction vessel, stir and react for 12h, and freeze-dry to obtain the outer shell; the chitosan solution is prepared by dissolving modified chitosan and carboxylated chitosan in acetic acid solution at a mass ratio of 0.5:1.5. S5: Disperse 10g of shell and 1g of encapsulated microspheres in deionized water to form a suspension, mix evenly by ultrasonication, centrifuge, take the lower precipitate, freeze dry, and obtain composite microspheres; S6: Add 25wt% poly(N-vinylcaprolactam) to deionized water at 3℃, stir until homogeneous, and let stand to obtain a poly(N-vinylcaprolactam) dispersion; add 1.5wt% hydroxypropyl methylcellulose to the poly(N-vinylcaprolactam) dispersion, stir at 3℃ until completely dissolved to obtain a mixture; 0.5 wt% polyvinyl alcohol was added to deionized water, heated to 95°C and stirred until homogeneous, then cooled to obtain polyvinyl alcohol hydrogel; the polyvinyl alcohol hydrogel was added to an aqueous solution containing 0.5 wt% starch, heated to 95°C and stirred until homogeneous, then cooled to obtain a composite gel phase; 0.5 wt% composite microspheres and 0.05 wt% hyaluronic acid were added to a 3 wt% acetic acid solution and stirred until homogeneous to obtain an aqueous phase. The composite gel phase was added to the mixture, stirred at room temperature, and then the aqueous phase was added. The mixture was stirred until homogeneous, homogenized at high speed, allowed to stand at room temperature, and stored at 3°C ​​in the dark to obtain the gel formulation.

[0036] Comparative Example 1: A method for preparing a gel formulation of composite microspheres containing silk material, comprising the following steps: S2: Choline oleate and glutamic acid are added to deionized water and stirred evenly to obtain 8.5 mM choline oleate solution and 3 mM glutamic acid solution respectively; the glutamic acid aqueous solution is added to the choline oleate solution and allowed to stand to obtain vesicles; The remaining steps are the same as in Example 1.

[0037] Comparative Example 2: A method for preparing a gel formulation of composite microspheres containing silk material, comprising the following steps: S2: Choline oleate and glutamic acid are added to deionized water and stirred evenly to obtain 8.5 mM choline oleate solution and 7 mM glutamic acid solution respectively; the glutamic acid aqueous solution is added to the choline oleate solution and allowed to stand to obtain vesicles; The remaining steps are the same as in Example 1.

[0038] Comparative Example 3: A method for preparing a gel formulation of composite microspheres containing silk material, comprising the following steps: S4: 4g of carboxylated chitosan solution, 1g of crosslinking agent and 1g of catalyst are added to a reaction vessel, stirred and reacted for 12h, and then freeze-dried to obtain the outer shell; The remaining steps are the same as in Example 1.

[0039] Comparative Example 4: A method for preparing a gel formulation containing composite microspheres of silk material, comprising the following steps: The preparation method of modified chitosan includes the following steps: 1 mM succinic anhydride was heated to 135 °C to melt, and 3 mM 6-deoxy-6-amino chitosan was added. The reaction was maintained at this temperature for 10 min to obtain a succinic anhydride derivative. 1 mM succinic anhydride derivative was added to N,N-dimethylformamide, and 2 mM COMU peptide coupling agent was added to activate it at room temperature for 1 h. 10 mM phytosphingosine was added, and the reaction was carried out at room temperature for 23 h. After purification, modified chitosan was obtained. The remaining steps are the same as in Example 1.

[0040] Test: Transdermal absorption performance: Using porcine skin (simulating the stratum corneum barrier of human skin) as a transdermal model, the gel formulation was continuously stirred at 32°C, pH 7.4, and PBS buffer (simulating dermal fluid). The skin was laid flat between the donor and receiver chambers of the diffusion cell, with the stratum corneum facing the donor chamber and the dermis facing the receiver chamber, and sealed with clamps to prevent leakage. Samples were taken periodically to test the concentration of the active ingredient, and the cumulative transdermal absorption rate (Q, μg / cm³) over 24 hours was calculated. 2 ) and transdermal flux (J, μg / cm) 2 •h), reflecting the total amount and rate at which active ingredients penetrate the skin.

[0041] Antibacterial properties: The antibacterial properties of the gel formulation were tested using agarocanthiate.

[0042] The test results are shown in Table 1 below.

[0043] Table 1 Performance test data of gel formulation

[0044] Conclusion: The gel formulation containing composite microspheres prepared in this invention has excellent transdermal absorption and antibacterial properties.

[0045] In Comparative Example 1, the vesicles formed rod-shaped micelles. These rod-shaped micelles could not penetrate the stratum corneum, and only a small amount of small molecules could pass through, resulting in reduced transdermal absorption. The antibacterial components in the rod-shaped micelles were difficult to release, and the antibacterial zone was reduced.

[0046] In Comparative Example 2, the vesicles formed an insoluble precipitate, making it difficult for the active ingredients in the precipitate to be released, resulting in extremely low transdermal penetration; the antibacterial components in the precipitate could not diffuse, and the inhibition zone shrank.

[0047] In Comparative Example 3, the plant sphingolipid arms and fatty acid chains lacking modified chitosan could not form "temporary permeation channels," resulting in a significant reduction in transdermal permeability.

[0048] In Comparative Example 4, the modified chitosan lacked fatty acid chains, which prevented it from filling the lipid gaps in the stratum corneum and increasing membrane fluidity, thus reducing its transdermal promotion effect.

[0049] Anti-wrinkle properties: Human fibroblasts were seeded in 12-well plates, starved in serum-free medium for 24 h, and then injected with 40 mJ / cm². 2 UVB irradiation induced oxidative damage. Samples from Examples 1, 5, and 6 (50 μg / mL deionized water solution of composite microspheres) were added and co-cultured for 48 h, after which the supernatant was collected. The inhibition rate of MMP-1 expression and the promotion rate of type I collagen synthesis in Samples 1, 5, and 6 were detected to evaluate their anti-wrinkle performance.

[0050] Set up a blank control group: basal culture medium; and a model control group: UVB irradiation, without adding samples.

[0051] The test results are shown in Table 2 below.

[0052] Table 2 Anti-wrinkle efficacy performance test data table

[0053] Conclusion: The composite microsphere gel of the present invention (Examples 1, 5, and 6) has a significantly better inhibitory effect on MMP-1 and a better effect on promoting type III collagen synthesis than single components and commercially available products. Among them, Example 6 (containing ECM) showed the best performance, and the effects of each example far exceeded the sum of the single components, indicating that there is a significant synergistic anti-wrinkle effect between silk materials, PDRN and ECM.

[0054] Skin repair performance: Using a deep second-degree burn model in SD rats, the wound healing time and healing rate at different time points were recorded, and histological observation was conducted to investigate the promoting effect of the gel preparations in Examples 1, 4, and 5 on wound healing.

[0055] SD rats weighing approximately 200g each, with half males and half females, were placed in contact with an 80℃ hot plate for 5 seconds. After shaving the hair on their backs, deep second-degree burn wounds with a diameter of 2cm were prepared. The animals were randomly divided into 6 groups of 10 each, and covered with the gel preparations of Examples 1, 5, and 6 respectively. The dressings were changed once a day. The wound healing was observed on the 3rd, 7th, 14th, and 21st days after surgery, and the wound healing rate was calculated.

[0056] Set up a blank control group: no wound treatment; commercially available dressing group: covered with sterile petroleum jelly gauze.

[0057] The test results are shown in Table 3 below.

[0058] Table 3. Skin Repair Performance Test Data

[0059] Conclusion: The composite microsphere gel of the present invention can significantly accelerate the healing of scald wounds in rats. Among them, Example 6 (containing ECM) showed the fastest healing speed and the best tissue repair quality, confirming its excellent skin repair efficacy.

[0060] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

Claims

1. A composite microsphere containing silk material, characterized in that: The raw materials for the composite microspheres, by mass parts, include 0.001-2 parts of silk material, 0.001-2 parts of polydeoxyribonucleic acid and 0.008-0.16 parts of cationic substances; wherein the mass ratio of silk material to polydeoxyribonucleic acid is 1:

1.

2. The composite microsphere containing silk material according to claim 1, characterized in that... The raw materials for the composite microspheres, by mass, also include 0.001-2 parts of silk material, 0.001-3 parts of extracellular matrix, 0.002-5 parts of polydeoxyribonucleic acid and 0.008-0.16 parts of cationic substances; wherein, the total mass ratio of silk material and extracellular matrix to polydeoxyribonucleic acid is 1:

1.

3. The composite microsphere containing silk material according to claim 2, characterized in that: The composite microspheres have a core-shell structure, and the raw materials of the core include silk materials, extracellular matrix, polydeoxyribonucleic acid and cationic substances; The shell consists of vesicles covering the surface of the nucleus; The raw materials for the vesicles include an oleate solution and a glutamic acid solution, wherein the concentration of the oleate solution is 8.5-9 mM and the concentration of the glutamic acid solution is 4-5 mM; the oleate solution includes any one of glyceryl oleate citrate solution and choline oleate solution.

4. The composite microsphere containing silk material according to claim 3, characterized in that: The composite microspheres have a double-shell structure, with the first shell consisting of vesicles covering the core surface and the second shell consisting of an outer shell covering the vesicle surface. The outer shell is made of chitosan solution, crosslinking agent, and catalyst. In the outer shell, the chitosan solution has a mass concentration of 1-10%, the crosslinking agent is a 90-98% mass concentration 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloric acid solution, and the catalyst is a 95-100% mass concentration N-hydroxysuccinimide solution. The mass ratio of chitosan solution: crosslinking agent: catalyst is (4-10):1:

1.

5. The composite microsphere containing silk material according to claim 3, characterized in that: The method for preparing the choline oleate includes the following steps: A methanol solution of oleic acid was added to a methanol solution of choline hydroxide at a temperature of 10-15°C. After the addition was complete, the mixture was stirred at room temperature for 20-24 hours. The mixture was then rotary evaporated, washed with diethyl ether, and dried under vacuum at room temperature to obtain choline oleate. In the preparation of choline oleate, the molar ratio of oleic acid to choline hydroxide is 1:

1.

6. A method for preparing composite microspheres containing silk material, characterized in that: The composite microspheres as described in claim 4 are prepared as follows: Includes the following steps: S1: Silk material, extracellular matrix, polydeoxyribonucleic acid (PDA), and cationic substances are added to deionized water and stirred until homogeneous to obtain silk material solution, extracellular matrix solution, PDA solution, and cationic substance solution, respectively. The silk material solution, extracellular matrix solution, and PDA solution are added to a reaction vessel and heated to 60-65℃ for 1-1.5 hours. The pH is adjusted to 6, and the cationic substance solution is added. The mixture is homogenized under high pressure 5-6 times to obtain microspheres. S2: Oleic acid ester and glutamic acid are added to deionized water and stirred until homogeneous to obtain oleic acid ester solution and glutamic acid solution respectively; the glutamic acid aqueous solution is added to the oleic acid ester solution and allowed to stand to obtain vesicles; S3: Add the microspheres to dimethyl sulfone and stir until homogeneous to obtain a microsphere solution; add the microsphere solution to vesicles, let stand, centrifuge, and freeze-dry to obtain encapsulated microspheres; S4: Add chitosan solution, crosslinking agent, and catalyst to a reaction vessel, stir to react, and freeze-dry to obtain the outer shell; S5: Disperse the shell and encapsulated microspheres in deionized water to form a suspension, mix them evenly by ultrasonication, centrifuge, remove the lower precipitate, freeze-dry, and obtain composite microspheres.

7. The method for preparing composite microspheres containing silk material according to claim 6, characterized in that: The chitosan solution was prepared by dissolving modified chitosan and carboxylated chitosan in acetic acid solution at a mass ratio of (0.5-1.5):(0.5-1.5). The method for preparing the modified chitosan includes the following steps: Succinic anhydride was heated to 135-140℃ to melt, and 6-deoxy-6-aminochitosan was added. The reaction was maintained at this temperature for 10-15 min to obtain a succinic anhydride derivative. The succinic anhydride derivative was added to N,N-dimethylformamide, and COMU peptide coupling agent was added for activation at room temperature for 1-1.5 h. Phytosphingosine was added, and the reaction was carried out at room temperature for 23-24 h. After purification, a phytosphingosine conjugate was obtained. Fatty acid ethyl esters were added to the phytosphingosine conjugate, and a lipase catalyst was added. The mixture was reacted at 50-55℃ and 90 mbar vacuum for 8-9 h. After purification, modified chitosan was obtained.

8. The method for preparing composite microspheres containing silk material according to claim 7, characterized in that: In the preparation of succinic anhydride derivatives, the molar ratio of succinic anhydride to 6-deoxy-6-aminochitosan is 1:3; In the preparation of the phytosphingosine conjugate, the molar ratio of succinic anhydride derivative: COMU peptide conjugate: phytosphingosine is 1:2:10; In the preparation of modified chitosan, the ratio of plant sphingosine conjugate: fatty acid ethyl ester: lipase catalyst is 100mg:2mL:100mg.

9. An application of a composite microsphere containing silk material, characterized in that: Using the composite microspheres according to any one of claims 1-8 to prepare a gel formulation includes the following steps: adding poly(N-vinylcaprolactam) to deionized water at 3-4°C, stirring until homogeneous, and allowing to stand to obtain a poly(N-vinylcaprolactam) dispersion; adding hydroxypropyl methylcellulose to the poly(N-vinylcaprolactam) dispersion, stirring at 3-4°C until completely dissolved to obtain a mixture; Polyvinyl alcohol was added to deionized water, heated to 95-100℃ and stirred until homogeneous, then cooled to obtain polyvinyl alcohol hydrogel; the polyvinyl alcohol hydrogel was added to starch aqueous solution, heated to 95-100℃ and stirred until homogeneous, then cooled to obtain composite gel phase; The composite microspheres and hyaluronic acid were added to the acetic acid solution and stirred until homogeneous to obtain the aqueous phase. Add the aqueous phase to the mixture, stir at room temperature, add the composite gel phase, stir until homogeneous, homogenize at high speed, let stand at room temperature, and store at 3-4℃ in the dark to obtain the gel formulation.

10. The application of a composite microsphere containing silk material according to claim 9, characterized in that: The components of the gel formulation, by mass percentage, include: 25-30% poly(N-vinylcaprolactam), 1.5-2% hydroxypropyl methylcellulose, 0.25-0.5% polyvinyl alcohol, 0.5-0.75% starch, 0.5-3% composite microspheres, 0.05-0.3% hyaluronic acid, 3-5% acetic acid solution, and the remainder is deionized water.

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