Emulsification preparation process of a silk fibroin hyaluronic acid copolymer preparation

Abstract

The application relates to the technical field of biological medicine manufacturing, and discloses an emulsification preparation process of a silk fibroin and hyaluronic acid copolymer preparation, which comprises the following steps: mixing low-molecular-weight hyaluronic acid and silk fibroin, and performing a covalent coupling reaction under heating to construct a copolymer water phase; mixing and stirring an oil phase material with the copolymer water phase to generate a primary emulsion; and performing dispersion treatment on the primary emulsion, so that the copolymer self-assembles into a three-dimensional network interface film at the oil-water interface to obtain a nanoscale emulsion droplet preparation. The application constructs a high viscoelastic network structure at the oil-water interface through covalent combination, blocks droplet coalescence and stratification, and improves the physicochemical stability; the micro-nano scale and the interfacial amphiphilic characteristics are utilized to penetrate the stratum corneum barrier and enter the dermis layer under the condition of no chemical penetration enhancer, and collagen expression and extracellular matrix reconstruction are promoted.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical manufacturing technology, and particularly relates to an emulsification preparation process for a silk fibroin hyaluronic acid copolymer formulation. Background Technology

[0002] In the preparation of biopharmaceutical formulations, natural polymer materials such as silk fibroin and hyaluronic acid are often used to construct micro / nano emulsion delivery systems. Silk fibroin can form a film at the oil-water interface, while hyaluronic acid has strong water absorption and retention capabilities. Mixing and dispersing these two materials in a continuous phase can produce multiphase dispersion formulations. In such systems, the stability of the oil-water interface directly affects the storage stability and shelf life of the formulation. In actual preparation processes, unmodified silk fibroin molecular chains have a strong hydrophobic tendency and easily aggregate at the oil-water interface, but the initial film they form is flexible. Due to insufficient toughness, hyaluronic acid is unable to withstand continuous deformation. It has strong hydrophilicity and usually dissolves and remains in the continuous aqueous phase. It is difficult for it to migrate to the oil-water interface and adhere stably. In conventional physical mixing systems, silk fibroin and hyaluronic acid are prone to disordered competitive adsorption at the interface. When the emulsion is subjected to fluid shear or the droplets collide due to thermal motion, the physical adsorption film formed by van der Waals forces is easy to relax and fall off. After the interfacial film loses mechanical support, the spatial steric hindrance on the surface of the droplets weakens, and the dispersed phase droplets are easy to collide and aggregate with each other, eventually causing irreversible flocculation and oil-water separation.

[0003] To address the issues of emulsion interface instability and the difficulty of large molecules penetrating dense tissues, existing processes typically involve adding chemical penetration enhancers to alter the original arrangement of the lipid bilayer, or adding exogenous chemical cross-linking agents to solidify the interfacial network. These treatments may disrupt normal physiological barriers and cause cytotoxicity due to reagent residues. Another approach avoids chemical reagents by increasing mechanical shear strength or operating temperature to further reduce droplet size. However, when the local energy input exceeds the thermal denaturation threshold of silk fibroin, irreversible thermal aggregation and molecular chain rearrangement occur, disrupting its native spatial conformation. To address the insufficient stability of single-protein emulsion systems, existing technologies... Techniques have attempted to improve emulsification performance through physical pretreatment and exogenous solvent regulation. For example, Chinese invention patent application CN110859776A discloses a silk fibroin-based nanoemulsion, its preparation method, and its application. This method induces the physical aggregation of low molecular weight silk fibroin through high-temperature and high-pressure treatment, and adds high concentrations of polyols such as 1,3-butanediol to the system as stabilizers. However, the interfacial coating structure formed by this process through physical thermal aggregation lacks strong covalent cross-linking support and is still prone to relaxation and detachment under shear or collision. Furthermore, the introduction of a large amount of organic polyols significantly increases the risk of chemical irritation to the skin barrier, making it difficult to balance high interfacial viscoelasticity and deep transdermal safety in a mild aqueous system.

[0004] Therefore, how to form a covalent network with good viscoelasticity and long-term stability at the oil-water interface using a relatively mild process without using chemical penetration enhancers and chemical crosslinking agents, while ensuring that the prepared micro-nano formulations have the ability to penetrate deep tissues, is a technical problem that needs to be solved in this field. Summary of the Invention

[0005] To address the problems in the background art, the technical solution of the present invention is as follows: an emulsification preparation process for a silk fibroin-hyaluronic acid copolymer formulation, comprising the following steps: Step S1: Dissolve hyaluronic acid with a molecular weight of 2kDa to 50kDa in deionized water to prepare a polysaccharide aqueous solution, and then mix the polysaccharide aqueous solution with silk fibroin to obtain a proteoglycan mixed aqueous solution; wherein the mass ratio of silk fibroin to hyaluronic acid is 1:0.2 to 1:4. Step S2: The mixed aqueous solution of proteoglycans is heated for 0.5h to 2h under the conditions of a mass-volume concentration of 8% to 12%, a pH of 4.0 to 8.0, and a temperature of 60℃ to 90℃ to undergo a covalent coupling reaction to generate an aqueous phase of silk fibroin hyaluronic acid copolymer. Step S3, providing at least one oil phase material selected from medium-chain fatty acid triglycerides, triglyceride triglycerides, caprylate-capric triglycerides and 2-octyldodecyl alcohol; Step S4: Mix the oil phase material with the aqueous phase of silk fibroin hyaluronic acid copolymer at a volume ratio of 1:4 to 1:6 and stir to generate a primary emulsion. Step S5: Apply at least one dispersion treatment selected from probe ultrasonic treatment, high pressure homogenization treatment and microfluidic treatment to the primary emulsion, so that the silk fibroin hyaluronic acid copolymer self-assembles at the oil-water interface to form a three-dimensional network interface film, and obtain a silk fibroin hyaluronic acid copolymer formulation with an average particle size of 150nm to 200nm and a polydispersity index of less than 0.3.

[0006] Preferably, in step S1, the molecular weight of hyaluronic acid is 2kDa to 10kDa; the mass ratio of silk fibroin to hyaluronic acid is 1:0.5 to 1:2; and in step S4, the volume ratio of the oil phase material to the aqueous phase of the silk fibroin-hyaluronic acid copolymer is 1:5 to 1:6.

[0007] Preferably, in step S5, the dispersion treatment is ultrasonic treatment with a probe, and the ultrasonic power is controlled to be 200W to 600W and the ultrasonic time is 5min to 15min; in the prepared silk fibroin hyaluronic acid copolymer formulation, the zeta potential on the surface of the droplet is maintained at -10mV to -28mV, so as to construct a coating protective layer with negative charge electrostatic repulsion and three-dimensional network interface film spatial steric hindrance around the droplet.

[0008] Preferably, in step S3, the oil phase material is a medium-chain fatty acid triglyceride; step S4 includes step S41, where the oil phase material is added dropwise to the aqueous phase of the silk fibroin hyaluronic acid copolymer at a temperature of 20°C to 25°C, and mechanically stirred at a speed of 500 rpm to 1500 rpm for 3 min to 10 min, so that the oil phase material is sheared into initially dispersed droplets in the aqueous phase of the silk fibroin hyaluronic acid copolymer to generate a primary emulsion.

[0009] Preferably, after the silk fibroin hyaluronic acid copolymer preparation obtained in step S5 is sealed and stored at an ambient temperature of 40°C to 50°C for 80 to 95 days, the average particle size of the silk fibroin hyaluronic acid copolymer preparation is maintained in the range of 150 nm to 200 nm, and the polydispersity index is maintained at less than 0.3. No oil-water separation boundary or flocculation precipitate is generated in the system.

[0010] Preferably, the silk fibroin-hyaluronic acid copolymer forms an amphiphilic microtopology at the oil-water interface. The amphiphilic microtopology includes hydrophobic amino acid residues of silk fibroin anchored inside the oil phase material and hyaluronic acid polysaccharide chains extending into the continuous aqueous phase. The amphiphilic microtopology enables droplets with an average particle size of 150 nm to 200 nm to have the ability to deform and permeate through epidermal lipid channels.

[0011] Preferably, the surface of the silk fibroin-hyaluronic acid copolymer retains unreacted hyaluronic acid-specific oligosaccharide structures. These hyaluronic acid-specific oligosaccharide structures serve as specific binding targets for CD44 receptors on the surface of fibroblasts. In an in vitro human skin fibroblast photoaging UV radiation model, these structures upregulate the expression of type I collagen to 3.71 pg / mg and the expression of type III collagen to 8.02 pg / mg.

[0012] Preferably, the silk fibroin-hyaluronic acid copolymer formulation is an oil-in-water nanoemulsion with an average droplet size of 150 nm to 200 nm, a polydispersity index of less than 0.3, and a zeta potential of -10 mV to -28 mV.

[0013] Preferably, the silk fibroin-hyaluronic acid copolymer formulation is used to prepare drugs or cosmetics for anti-photoaging of the skin, wherein photoaging of the skin includes skin wrinkles, skin laxity, pigmentation and barrier damage caused by ultraviolet radiation.

[0014] Compared with existing technologies, the emulsification preparation process of the silk fibroin hyaluronic acid copolymer formulation of the present invention has the following advantages: 1. In the emulsification preparation of silk fibroin and hyaluronic acid copolymer formulations, silk fibroin and hyaluronic acid form a stable copolymer through covalent coupling reaction, which avoids disordered competitive adsorption of the two polymers at the oil-water interface and reduces the possibility of the interfacial film falling off under shear or collision. The copolymer can form a three-dimensional network interfacial film at the oil-water interface, and restrict the approach, aggregation and flocculation of emulsion droplets through negative charge repulsion and steric hindrance, thereby reducing oil-water stratification and enabling the formulation to maintain a relatively stable particle size distribution and physicochemical state during storage.

[0015] 2. The combination of the copolymer aqueous phase and dispersion treatment can form droplets with smaller particle size and more uniform distribution, making it easier for the formulation to adhere to the intercellular spaces of the stratum corneum. The hydrophobic regions of silk fibroin on the surface of the droplets have an affinity for stratum corneum lipids, and the hydration layer formed by hyaluronic acid segments helps to reduce the resistance of droplets when passing through lipid channels. Thus, the formulation can improve transdermal ability without the addition of chemical penetration enhancers and reduce the irritation and damage to the skin barrier caused by chemical penetration enhancers.

[0016] 3. The hyaluronic acid receptor binding sites retained on the surface of the copolymer facilitate the binding of the formulation to the receptors on the surface of fibroblasts and its uptake by the cells. After entering the cells, the active fragments of silk fibroin and hyaluronic acid work together to inhibit the accumulation of reactive oxygen species and the overexpression of matrix metalloproteinases caused by ultraviolet radiation, reduce the degradation of existing collagen, and promote the synthesis and secretion of collagen by fibroblasts. Therefore, this formulation helps to restore the expression levels of type I and type III collagen, improve the extracellular matrix structure of the dermis, and reduce tissue damage caused by photoaging of the skin. Attached Figure Description

[0017] Figure 1 This is a flowchart of the copolymer nanoemulsion preparation steps of the present invention; Figure 2 This is a decision diagram of key reaction conditions and dispersion parameters in this invention. Detailed Implementation

[0018] The technical solutions of the embodiments of this application will be clearly described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this application are within the scope of protection of this application.

[0019] An emulsification preparation process for a silk fibroin-hyaluronic acid copolymer formulation includes the following steps: Step S1: Dissolve hyaluronic acid with a molecular weight of 2kDa to 50kDa in deionized water to prepare a polysaccharide aqueous solution, and then mix the polysaccharide aqueous solution with silk fibroin to obtain a proteoglycan mixed aqueous solution; wherein the mass ratio of silk fibroin to hyaluronic acid is 1:0.2 to 1:4. Step S2: The mixed aqueous solution of proteoglycans is heated for 0.5h to 2h under the conditions of a mass-volume concentration of 8% to 12%, a pH of 4.0 to 8.0, and a temperature of 60℃ to 90℃ to undergo a covalent coupling reaction to generate an aqueous phase of silk fibroin hyaluronic acid copolymer. Step S3, providing at least one oil phase material selected from medium-chain fatty acid triglycerides, triglyceride triglycerides, caprylate-capric triglycerides and 2-octyldodecyl alcohol; Step S4: Mix the oil phase material with the aqueous phase of silk fibroin hyaluronic acid copolymer at a volume ratio of 1:4 to 1:6 and stir to generate a primary emulsion. Step S5: Apply at least one dispersion treatment selected from probe ultrasonic treatment, high pressure homogenization treatment and microfluidic treatment to the primary emulsion, so that the silk fibroin hyaluronic acid copolymer self-assembles at the oil-water interface to form a three-dimensional network interface film, and obtain a silk fibroin hyaluronic acid copolymer formulation with an average particle size of 150nm to 200nm and a polydispersity index of less than 0.3.

[0020] Preferably, in step S1, the molecular weight of hyaluronic acid is 2kDa to 10kDa; the mass ratio of silk fibroin to hyaluronic acid is 1:0.5 to 1:2; and in step S4, the volume ratio of the oil phase material to the aqueous phase of the silk fibroin-hyaluronic acid copolymer is 1:5 to 1:6.

[0021] Preferably, in step S5, the dispersion treatment is ultrasonic treatment with a probe, and the ultrasonic power is controlled to be 200W to 600W and the ultrasonic time is 5min to 15min; in the prepared silk fibroin hyaluronic acid copolymer formulation, the zeta potential on the surface of the droplet is maintained at -10mV to -28mV, so as to construct a physical defense layer with negative charge electrostatic repulsion and three-dimensional network interface film spatial steric hindrance on the periphery of the droplet.

[0022] Preferably, in step S3, the oil phase material is a medium-chain fatty acid triglyceride; step S4 includes step S41, where the oil phase material is added dropwise to the aqueous phase of the silk fibroin hyaluronic acid copolymer at a temperature of 20°C to 25°C, and mechanically stirred at a speed of 500 rpm to 1500 rpm for 3 min to 10 min, so that the oil phase material is sheared into initially dispersed droplets in the aqueous phase of the silk fibroin hyaluronic acid copolymer to generate a primary emulsion.

[0023] Preferably, after the silk fibroin hyaluronic acid copolymer preparation obtained in step S5 is sealed and stored at an ambient temperature of 40°C to 50°C for 80 to 95 days, the average particle size of the silk fibroin hyaluronic acid copolymer preparation is maintained in the range of 150 nm to 200 nm, and the polydispersity index is maintained at less than 0.3. No oil-water separation boundary or flocculation precipitate is generated in the system.

[0024] Preferably, the silk fibroin-hyaluronic acid copolymer forms an amphiphilic microtopology at the oil-water interface. The amphiphilic microtopology includes hydrophobic amino acid residues of silk fibroin anchored inside the oil phase material and hyaluronic acid polysaccharide chains extending in the continuous aqueous phase. The amphiphilic microtopology is based on the amphiphilic adaptive deformation capability of the 150nm to 200nm droplet penetrating the epidermal lipid channel.

[0025] Preferably, the surface of the silk fibroin-hyaluronic acid copolymer retains unreacted hyaluronic acid-specific oligosaccharide structures. These hyaluronic acid-specific oligosaccharide structures serve as specific binding targets for CD44 receptors on the surface of fibroblasts. In an in vitro human skin fibroblast photoaging UV radiation model, these structures upregulate the expression of type I collagen to 3.71 pg / mg and the expression of type III collagen to 8.02 pg / mg.

[0026] Preferably, the silk fibroin-hyaluronic acid copolymer formulation is an oil-in-water nanoemulsion with an average droplet size of 150 nm to 200 nm, a polydispersity index of less than 0.3, and a zeta potential of -10 mV to -28 mV.

[0027] Preferably, the silk fibroin-hyaluronic acid copolymer formulation is used to prepare drugs or cosmetics for anti-photoaging of the skin, wherein photoaging of the skin includes skin wrinkles, skin laxity, pigmentation and barrier damage caused by ultraviolet radiation.

[0028] Example 1: In this example, hyaluronic acid with a molecular weight of 2kDa to 50kDa was dissolved in deionized water to prepare a polysaccharide aqueous solution. Then, silk fibroin was added at a mass ratio of 1:0.5 to 1:2 to obtain a proteoglycan mixed aqueous solution. The mass-volume concentration of the mixed aqueous solution was adjusted to 10%, the pH was adjusted to 6.0, and the solution was heated at 60℃ to 90℃ for 1 hour. During the heating process, the reducing sugar groups at the ends of the low molecular weight hyaluronic acid undergo a covalent coupling reaction with the free primary amino groups on the side chains of the silk fibroin molecular chain. After condensation and rearrangement, a silk fibroin-hyaluronic acid copolymer aqueous phase is generated. The low molecular weight hyaluronic acid has a small hydrodynamic volume and low steric hindrance during coupling. The reaction temperature is controlled below the thermal denaturation threshold of silk fibroin to reduce irreversible thermal coagulation and crystal precipitation, so that the obtained silk fibroin-hyaluronic acid copolymer maintains an amphiphilic structure.

[0029] In this embodiment, the pH is controlled at 6.0, which is within the working range of pH 6.0 to 8.0 and is higher than the isoelectric point of silk fibroin, pH 4.0 to 4.5. This causes the carboxyl groups on the side chains of the protein molecules to ionize and carry a negative charge. Before the coupling reaction, hyaluronic acid added in the above mass ratio is wrapped around the silk fibroin molecular chain through hydrogen bonds to form a hydrophilic hydration protective layer, which improves the tolerance of silk fibroin to heating conditions. This ensures that the reaction system remains homogeneous and clear during the heating process at 60°C to 90°C for 1 hour, reducing charge neutralization, thermal coagulation, and precipitation.

[0030] Using medium-chain triglycerides as the oil phase, the oil phase was mixed with the aqueous phase of silk fibroin-hyaluronic acid copolymer at a volume ratio of 1:5 at 20℃ to 25℃, and mechanically stirred at 1000 rpm for 5 min to generate a primary emulsion. The primary emulsion was then ultrasonically treated with a probe at a power of 400 W for 10 min. The shear flow generated by the ultrasound caused the silk fibroin-hyaluronic acid copolymer molecules to migrate to the newly formed oil-water interface and undergo self-assembly. In this process, the hydrophobic amino acid residues of the silk fibroin were anchored within the oil phase. In this process, covalently grafted flexible hyaluronic acid polysaccharide chains extend into a continuous aqueous phase, forming a viscoelastic three-dimensional network interface film at the oil-water interface. The carboxyl groups on the hyaluronic acid polysaccharide chains ionize, maintaining the zeta potential on the surface of the droplets at -10mV to -28mV. The electrostatic repulsion of negative charges and the steric hindrance generated by the three-dimensional network interface film work together to form a double-coated physical defense layer around the droplets, resulting in an oil-in-water silk fibroin hyaluronic acid copolymer formulation with an average particle size of 150nm to 200nm and a polydispersity index of less than 0.3.

[0031] During the ultrasonic treatment process, the cavitation field generated by the 400W ultrasound creates high-frequency pulse shear in local areas, increasing the contact surface area between the oil and water phases. The amphiphilic structure of the silk fibroin hyaluronic acid copolymer lowers the thermodynamic barrier when it adsorbs onto the oil-water interface. In the local shear region formed by the collapse of cavitation bubbles, the hydrophobic amino acid residues of silk fibroin drive the molecular chains to orient towards the oil phase. As the flow gradually decays, the copolymer molecular chains complete conformational relaxation and interweaving, forming a three-dimensional network interface film. Multiple local adsorption, orientation, and relaxation processes occur continuously during the 10-minute ultrasonic treatment, resulting in the three-dimensional network interface film uniformly covering the surface of the emulsion droplets.

[0032] The prepared silk fibroin-hyaluronic acid copolymer formulation was sealed and stored at 45°C for 90 days. The three-dimensional network interface film has high shear modulus and elastic recovery performance, which can resist the interface film rupture and relaxation caused by thermal motion collision, reduce oil droplet aggregation and flocculation stratification. During the storage period, the average particle size of the formulation was maintained at 150nm to 200nm, the polydispersity index was less than 0.3, and no oil-water stratification boundary or flocculation precipitate appeared in the system.

[0033] In the test system without the addition of exogenous chemical penetration enhancers and organic solvents, droplets with a particle size of 150 nm to 200 nm were able to adhere to the intercellular spaces of the stratum corneum. The hydrophobic microdomains of silk fibroin exposed at the droplet interface came into contact with the lipid matrix of the stratum corneum, and the droplets deformed and diffused along the lipid channels between the stratum corneum cells. The hydrating coating layer formed by hyaluronic acid maintained the lubrication state during the barrier crossing process. The test results showed that the formulation crossed the stratum corneum barrier and entered the dermis within 30 min. The fluorescence intensity in the dermis was 2.6 times that of the physical mixed formulation group and 12.8 times that of the free protein formulation group.

[0034] In the aforementioned covalent coupling reaction, hyaluronic acid primarily participates in the reaction via terminal reducing glycosyl groups, while the repeating disaccharide units on its main chain remain unreacted. This allows the surface of the silk fibroin-hyaluronic acid copolymer formulation to retain the hyaluronic acid-specific oligosaccharide structure. This hyaluronic acid-specific oligosaccharide structure serves as a specific binding target for the CD44 receptor on the surface of fibroblasts, mediating the endocytosis and uptake of milk droplets by fibroblasts. After the milk droplets enter the cells, the released active fragments of silk fibroin, together with hyaluronic acid, inhibit the accumulation of reactive oxygen species and the overproduction of matrix metalloproteinases induced by ultraviolet radiation. The drug enhances collagen expression, reduces the degradation of existing collagen networks, and promotes collagen synthesis by fibroblasts. In a human skin fibroblast photoaging UV radiation damage model, the expression level of type I collagen was upregulated to 3.71 pg / mg, and the expression level of type III collagen was upregulated to 8.02 pg / mg. At the same time, the collagen network of the dermal extracellular matrix was reconstructed, and the imbalanced ratio of type I to type III collagen fibers was adjusted. In a mouse photoaging model, the drug also inhibited UV-induced pathological epidermal thickening and reduced skin erythema and wrinkles.

[0035] Collagen expression levels were determined as follows: 24 h after cell drug intervention, fibroblast lysates were collected, and the absolute mass of type I and type III collagen in the lysates was determined using an enzyme-linked immunosorbent assay (ELISA) kit. The total protein content in the cell lysates was determined using the biuret method. Collagen expression levels were expressed as picograms of collagen per milligram of total cell protein. In five independent parallel experimental batches, the expression levels of type I collagen ranged from 3.55 pg / mg to 3.85 pg / mg, and the expression levels of type III collagen ranged from 7.85 pg / mg to 8.20 pg / mg. The results of each batch were within the above range.

[0036] Example 2: This example establishes a platform for evaluating the physicochemical properties and transdermal efficacy of a silk fibroin-hyaluronic acid copolymer formulation. Deionized water is used as the aqueous phase medium, and medium-chain triglycerides are used as the oil phase material. Hyaluronic acid with molecular weights of 2kDa to 10kDa, 10kDa to 50kDa, 50kDa to 100kDa, and 100kDa to 500kDa are selected and covalently coupled with purified and extracted silk fibroin. The grafting rate of the coupling product is determined by the phthalaldehyde method. The average particle size, polydispersity index, and Zeta potential of the silk fibroin-hyaluronic acid copolymer formulation are determined by dynamic light scattering. The centrifugal sedimentation stability parameter Ke value is determined by the centrifugal sedimentation method. The smaller the Ke value, the stronger the formulation's ability to resist centrifugal stratification.

[0037] The centrifugal sedimentation stability parameter Ke value was determined as follows: The preparation sample was centrifuged at 4000 rpm for 30 min; the upper dispersion before and after centrifugation was taken separately and diluted with deionized water to the same factor, and the absorbance was measured at a wavelength of 540 nm; the Ke value was obtained by dividing the difference between the absorbance before and after centrifugation by the absorbance before centrifugation. The conformation of the silk fibroin molecular chain was determined by circular dichroism spectroscopy, with a scanning wavelength range of 190 nm to 250 nm; the ellipticity response values ​​at 208 nm and 222 nm were calculated by baseline subtraction and deconvolution to obtain the relative percentage content of the β-sheet conformation.

[0038] In the material ratio and hyaluronic acid molecular weight screening test, the water phase mass volume concentration was fixed at 10%, the pH was adjusted to 6.0, and the reaction was carried out at 60℃ for 1 hour to generate the water phase of silk fibroin hyaluronic acid copolymer; then the water phase and oil phase were mixed at a volume ratio of 1:5 and ultrasonically treated with a probe with a power of 400W for 10 minutes to obtain the silk fibroin hyaluronic acid copolymer formulations of each group.

[0039] When using hyaluronic acid with a molecular weight of 2kDa to 10kDa, the mass ratio of silk fibroin to hyaluronic acid was set to 1:0.5. The grafting rate of sample group 1 of this invention was 42.5%, the average particle size was 201.38nm, the polydispersity index was 0.21, and the Ke value was 0.102. When the mass ratio was adjusted to 1:1, the grafting rate of sample group 2 of this invention was 41.8%, the average particle size was 202.41nm, the polydispersity index was 0.19, and the Ke value was 0.201. When the mass ratio was adjusted to 1:2, the grafting rate of sample group 3 of this invention was 40.2%, the average particle size was 204.86nm, the polydispersity index was 0.17, and the Ke value was 0.356.

[0040] At the same hyaluronic acid molecular weight, with the mass ratio of silk fibroin to hyaluronic acid set at 1:0.2, the grafting rate of the out-of-range control group 1 was 43.1%, the average particle size was 253.98 nm, the polydispersity index was 0.36, and the Ke value was 0.620. In this group, the amount of hyaluronic acid added was low, and the number of hydrophilic polysaccharide chains covalently grafted onto the periphery of silk fibroin was insufficient. The steric hindrance of the hydration space on the surface of the droplets was weakened, and droplet aggregation and stratification occurred after centrifugation. With the mass ratio set at 1:4, the grafting rate of the out-of-range control group 2 was 38.6%, the average particle size was 200.89 nm, the polydispersity index was 0.22, and the Ke value was 0.505. In this group, the hyaluronic acid that did not participate in the reaction remained in the continuous aqueous phase, which increased the viscosity of the aqueous phase and increased the tendency for phase separation.

[0041] Increasing the molecular weight of hyaluronic acid to 50kDa to 100kDa and setting the mass ratio of silk fibroin to hyaluronic acid to 1:1 reduced the grafting rate of the feature-deficient control group 1 to 18.2%, with an average particle size of 285.60nm, a polydispersity index of 0.42, and a Ke value of 0.715. Further increasing the molecular weight of hyaluronic acid to 100kDa to 500kDa increased the grafting rate of the feature-deficient control group 2 to 9.5%, with an average particle size of 321.15nm, a polydispersity index of 0.51, and a Ke value of 0.832. As the molecular weight of hyaluronic acid increased, the hydrodynamic volume of the polysaccharide chain and the steric hindrance of the coupling reaction increased accordingly. The number of terminal reducing sugar groups that can contact the free primary amino groups of silk fibroin decreased, the grafting rate decreased, and the particle size and polydispersity index of the resulting droplets increased.

[0042] In sample groups 1 to 3 of this invention, when the mass ratio of silk fibroin to hyaluronic acid was adjusted from 1:0.5 to 1:2, the grafting rate remained between 40.2% and 42.5%, the polydispersity index was less than 0.22, and the Ke value was between 0.102 and 0.356. Within this mass ratio range, the covalently grafted hyaluronic acid polysaccharide chains could cover the surface of the droplets and form stable hydration steric hindrance. When the amount of hyaluronic acid added was too low or too high, the dispersion state and centrifugal stability of the droplets decreased.

[0043] To verify the applicability of hyaluronic acid in the molecular weight range of 10kDa to 50kDa, hyaluronic acid with molecular weights of 10kDa to 30kDa and 30kDa to 50kDa were selected and covalently coupled with silk fibroin at a 1:1 mass ratio. When using 10kDa to 30kDa hyaluronic acid, the grafting rate was 37.8%, the average particle size of the prepared formulation was 186.5nm, the polydispersity index was 0.23, and the Ke value was 0.215. When using 30kDa to 50kDa hyaluronic acid, the grafting rate was 35.2%, the average particle size was 195.2nm, the polydispersity index was 0.25, and the Ke value was 0.280. Hyaluronic acid in this molecular weight range can still form silk fibroin-hyaluronic acid copolymers with silk fibroin and form a three-dimensional network interfacial film at the oil-water interface.

[0044] In a comparative experiment on reaction temperature and dispersion treatment, silk fibroin and hyaluronic acid with a molecular weight of 2kDa to 10kDa were mixed at a mass ratio of 1:1. The covalent coupling reaction temperatures were set at 50℃, 60℃, 70℃, 80℃, 90℃ and 100℃, respectively. After the reaction, medium-chain fatty acid triglycerides were added as oil phase material. The primary emulsion was dispersed by probe ultrasonic treatment, microfluidic treatment, high pressure homogenization treatment and high-speed shear treatment, respectively.

[0045] When the reaction temperature is 50℃, the thermal input of the covalent coupling reaction is low, and the average particle size of the prepared formulation is 208.5nm with a polydispersity index exceeding 0.30. When the reaction temperature is between 60℃ and 90℃, the average particle size is between 160nm and 180nm, the polydispersity index is less than 0.22, and the Zeta potential is maintained between -10mV and -28mV. The negative charge electrostatic repulsion formed on the surface of the emulsion droplets by the copolymer and the steric hindrance of the three-dimensional network interface film together restrict the approach and aggregation of the emulsion droplets. When the reaction temperature rises to 100℃, the silk fibroin undergoes thermal coagulation, and the average particle size of the formulation recovers to 198.2nm.

[0046] In the comparison of dispersion treatment methods, probe ultrasonic treatment, microfluidic treatment and high pressure homogenization treatment can all disperse the primary emulsion to the micro-nano scale with an average particle size of no more than 180 nm. When high-speed shear treatment is used, the dispersion energy density is insufficient, the average particle size of the resulting formulation is 360.5 nm, the polydispersity index is 0.82, and oil-water separation occurs within 2 hours of standing.

[0047] When using microfluidic treatment, the microfluidic homogenization pressure was set to 100 MPa, and a Y-type diamond interactive cavity was used for 4 cycles of homogenization. When using high-pressure homogenization, the first-stage homogenization pressure was set to 80 MPa, the second-stage homogenization pressure was set to 10 MPa, and a two-stage high-pressure impact valve was used for 5 cycles of homogenization. Under the above parameters, the shear energy density generated by microfluidic treatment and high-pressure homogenization is comparable to that of ultrasonic treatment with a 400W probe, and both can maintain the average particle size of the formulation in the range of 150 nm to 200 nm.

[0048] In the accelerated stability test, the sample group 1 of the present invention, which was prepared by ultrasonic treatment with a probe, was sealed and stored in a constant temperature oven at 45°C for 90 days. The average particle size, polydispersity index and appearance were measured periodically. During the storage period, the average particle size of the silk fibroin hyaluronic acid copolymer preparation prepared with hyaluronic acid with a molecular weight of 2kDa to 50kDa was maintained at 150nm to 200nm, the polydispersity index was less than 0.3, and no precipitation or oil phase floated out in the system.

[0049] The transdermal properties of each formulation were determined using an in vitro Franz diffusion cell on isolated porcine skin. Fluorescently labeled free fluorescent control samples, free silk fibroin control samples, physical mixture control samples of silk fibroin and hyaluronic acid, and silk fibroin-hyaluronic acid copolymer formulation samples were added to the supply chamber. Samples were collected in the receiving cell at 15 min, 30 min, 60 min, and 120 min, and the cumulative fluorescence intensity and penetration depth in the dermis were measured simultaneously.

[0050] The fluorescence intensities of the free fluorescence control group at 15 min, 30 min, 60 min, and 120 min were 5.2, 8.1, 12.3, and 18.5, respectively, with a dermal penetration depth of 12.0 μm. The fluorescence intensities of the free silk fibroin control group at the same time points were 8.6, 15.4, 28.2, and 45.1, respectively, with a penetration depth of 25.5 μm. The fluorescence intensities of the physical mixing control group were 22.4, 68.5, 115.2, and 182.0, respectively, with a penetration depth of 85.0 μm. The fluorescence intensities of the copolymer formulation group of this invention were 58.6, 178.2, 295.6, and 412.8, respectively, with a penetration depth of 210.0 μm.

[0051] The free fluorescent control sample and the free silk fibroin control sample mainly remained on the outer side of the stratum corneum. In the physically mixed control sample, the adsorption structure of silk fibroin and hyaluronic acid at the oil-water interface was relatively loose, and the droplets were prone to interfacial desorption during shearing and diffusion. The average particle size of the copolymer formulation of the present invention was 150 nm to 200 nm. The hydrophobic microdomains of silk fibroin at the droplet interface contacted the intercellular lipids of the stratum corneum cells, and the hyaluronic acid polysaccharide chains formed a hydrated coating layer, causing the droplets to deform and diffuse along the epidermal lipid channels. Based on the fluorescence intensity at 30 min, the copolymer formulation group of the present invention was 2.6 times that of the physically mixed control group and 11.5 times that of the free silk fibroin control group.

[0052] Further, a human skin fibroblast UVB photoaging damage model and a mouse photoaging model were established. The blank control group was not exposed to UV radiation; the UVB model group was exposed to 20 mJ / cm² UVB radiation for 10 min. After drug intervention, the expression levels of type I collagen in the UVB model group were 1.18 pg / mg and type III collagen were 1.38 pg / mg; the expression levels of type I collagen in the physical mixed control group were 2.84 pg / mg and type III collagen were 6.98 pg / mg; the expression levels of type I collagen in the copolymer formulation group of this invention were 3.71 pg / mg and type III collagen were 8.02 pg / mg. In the mouse photoaging model, the copolymer formulation of this invention restored the epidermal thickness on the back of the mouse from 85.2 μm in the UVB model group to 28.5 μm.

[0053] Example 3: Silk fibroin and hyaluronic acid with a molecular weight of 2kDa to 10kDa were added to deionized water at a mass ratio of 1:1 to obtain a proteoglycan mixed aqueous solution. The mass-volume concentration of the mixed aqueous solution was adjusted to 10%, and the pH was adjusted to 6.0. The solution was heated at 50℃ to 100℃ for 0.5h to 2h. The grafting rate (DG%) of the covalent coupling reaction was determined by the phthalaldehyde method, and the relative content of β-sheet conformation in the silk fibroin molecular chain was determined by circular dichroism spectroscopy.

[0054] When the reaction temperature is 50℃, the grafting rate (DG%) is less than 20%, indicating a low degree of reaction between the terminal reducing glycosyl groups of hyaluronic acid and the free primary amino groups on the side chains of silk fibroin molecules. When the reaction temperature is controlled between 60℃ and 90℃ and heated for 1 hour, the grafting rate (DG%) is 40.2% to 42.5%. Under these conditions, the terminal reducing glycosyl groups of hyaluronic acid undergo nucleophilic addition and Amadori rearrangement with the free primary amino groups on the side chains of silk fibroin molecules, forming a Schiff base covalently bonded product. This process is observed in the silk fibroin molecular chain... The β-sheet conformation content was maintained at 20% to 25%. When the reaction temperature rose to 100°C, the β-sheet conformation content rose to over 48%, and the silk fibroin underwent thermal coagulation and precipitation. The directional arrangement ability of the copolymer at the oil-water interface decreased. Based on the above experimental results, in this embodiment, the temperature of the covalent coupling reaction was controlled at 60°C to 90°C, the pH was set to 6.0, the mass-volume concentration of the proteoglycan mixed aqueous solution was set to 10%, and the heating time was set to 1 hour to generate the aqueous phase of the silk fibroin hyaluronic acid copolymer.

[0055] Using medium-chain fatty acid triglycerides as the oil phase material, the oil phase material and the aqueous phase of silk fibroin hyaluronic acid copolymer were mixed at a volume ratio of 1:4 to 1:6 at 20℃ to 25℃. The mixture was mechanically stirred at 500 rpm to 1500 rpm for 3 min to 10 min to form initial dispersed droplets, thus obtaining a primary emulsion. Subsequently, the primary emulsion was ultrasonically treated with a probe at an ultrasonic power of 200 W to 600 W for 5 min to 15 min.

[0056] When the ultrasonic power is 400W and the ultrasonic time is 10min, the cavitation flow field disperses the initially dispersed droplets to 150nm to 200nm. The silk fibroin hyaluronic acid copolymer molecules migrate with the flow to the newly generated oil-water interface and undergo directional self-assembly. Among them, the hydrophobic amino acid residues of silk fibroin are anchored inside the oil phase material, and the covalently grafted hyaluronic acid flexible polysaccharide chains extend into the continuous aqueous phase, forming a three-dimensional network interface film at the oil-water interface, which restricts the direct contact and aggregation of emulsion droplets.

[0057] In the prepared oil-in-water silk fibroin-hyaluronic acid copolymer formulation, the Zeta potential on the surface of the droplets ranged from -10mV to -28mV. The electrostatic repulsion of the negative charge formed by the ionization of hyaluronic acid polysaccharide chains and the steric hindrance generated by the three-dimensional network interface film worked together to form a double-coated physical defense layer around the droplets. When the formulation was sealed and stored at 45°C for 90 days, the Ke value remained between 0.102 and 0.356, the average droplet size remained between 150nm and 200nm, the polydispersity index was less than 0.3, and no oil-water separation boundary or flocculation precipitate appeared in the system.

[0058] The transdermal performance of this formulation was determined by an in vitro transdermal diffusion assay using isolated porcine skin. Under conditions without the addition of chemical penetration enhancers, droplets with a particle size of 150 nm to 200 nm came into contact with the intercellular spaces of the stratum corneum. The hydrophobic microdomains of silk fibroin at the droplet interface interacted with the intercellular lipids of the stratum corneum. The hyaluronic acid hydration coating maintained the lubrication of the droplet surface, causing the droplets to deform and diffuse along the epidermal lipid channels. The results showed that the formulation penetrated the stratum corneum barrier and entered the dermis within 30 minutes. The fluorescence intensity in the dermis was 2.6 times that of the physical mixture formulation group and 12.8 times that of the free protein formulation group.

[0059] The amphiphilic adaptive deformation capability of emulsion droplets was characterized by the particle size recovery after passing through a confined microporous channel. A silk fibroin-hyaluronic acid copolymer formulation was passed through a polycarbonate microporous membrane with a pore size of 50 nm under a driving pressure of 0.2 MPa. The average particle size and polydispersity index before and after passing through the microporous membrane were measured using a dynamic light scattering instrument. The particle size recovery rate of the emulsion droplets after passing through the microporous membrane reached more than 96.5%, and the polydispersity index did not increase significantly. This indicates that the three-dimensional network interface membrane can maintain the structure of the emulsion droplets under confined extrusion, allowing the emulsion droplets to recover their original particle size distribution after reversible deformation.

[0060] The silk fibroin-hyaluronic acid copolymer formulation retains the hyaluronic acid-specific oligosaccharide structure on its surface. This structure serves as a specific binding target for the CD44 receptor on the surface of fibroblasts, mediating the endocytosis and uptake of emulsion droplets by fibroblasts. After the emulsion droplets enter the cells, the released active fragments of silk fibroin, together with hyaluronic acid, inhibit the accumulation of reactive oxygen species and the expression of matrix metalloproteinases induced by ultraviolet radiation. In an in vitro human skin fibroblast photoaging ultraviolet radiation damage model, the expression level of type I collagen was upregulated to 3.71 pg / mg, and the expression level of type III collagen was upregulated to 8.02 pg / mg. In a mouse photoaging model, this formulation inhibited the pathological epidermal thickening and collagen degradation caused by ultraviolet radiation and reconstructed the three-dimensional mechanical support network of the extracellular matrix in the dermis.

[0061] Example 4: This example combines Figures 1 to 2 The emulsification preparation process of a silk fibroin hyaluronic acid copolymer formulation is described, such as... Figure 1As shown, in step S1, hyaluronic acid with a molecular weight of 2kDa to 50kDa is dissolved in deionized water to prepare a polysaccharide aqueous solution. The polysaccharide aqueous solution is then mixed with silk fibroin to obtain a proteoglycan mixed aqueous solution, wherein the mass ratio of silk fibroin to hyaluronic acid is 1:0.2 to 1:4. In step S2, the proteoglycan mixed aqueous solution is heated for 0.5h to 2h at a mass-volume concentration of 8% to 12%, a pH of 4.0 to 8.0, and a temperature of 60℃ to 90℃ to undergo a covalent coupling reaction, generating a silk fibroin-hyaluronic acid copolymer aqueous phase. In step S3, a medium-chain triglyceride is provided... The product contains at least one oil phase material selected from the following: triglycerides, triglycerides, caprylic / capric triglycerides, and 2-octyldodecyl alcohol; in step S4, the oil phase material is mixed with the aqueous phase of the silk fibroin hyaluronic acid copolymer at a volume ratio of 1:4 to 1:6 and stirred to generate a primary emulsion; in step S5, the primary emulsion is subjected to at least one dispersion treatment selected from probe ultrasonic treatment, high-pressure homogenization treatment, and microfluidic treatment, so that the silk fibroin hyaluronic acid copolymer self-assembles at the oil-water interface to form a three-dimensional network interface film, thereby obtaining a silk fibroin hyaluronic acid copolymer formulation with an average particle size of 150 nm to 200 nm and a polydispersity index of less than 0.3.

[0062] like Figure 2 As shown, in the emulsification preparation process of silk fibroin-hyaluronic acid copolymer formulation, regarding the covalent coupling reaction temperature, when the reaction temperature is 50℃, the grafting rate is less than 20%, and the reaction degree is low; when the reaction temperature is 100℃, silk fibroin undergoes thermal coagulation and precipitation, and its directional distribution ability decreases; when the reaction temperature is 60℃ to 90℃, regarding the mass ratio of silk fibroin to hyaluronic acid, if the amount of hyaluronic acid added is too high, the unreacted hyaluronic acid will remain in the continuous aqueous phase, increasing the tendency for phase separation; when the mass ratio of silk fibroin to hyaluronic acid is 1:0.2 to 1:4, regarding the dispersion treatment of the primary emulsion, if high-speed shearing treatment is used, the dispersion energy density is insufficient, and oil-water stratification occurs; if dispersion treatment selected from probe ultrasonic treatment, microfluidic treatment, or high-pressure homogenization treatment is used, the silk fibroin-hyaluronic acid copolymer self-assembles at the oil-water interface to form a three-dimensional network interface film, resulting in a water-in-oil nanoemulsion with deep tissue penetration capability.

[0063] Example 5: In this example, different batches of silk fibroin raw materials were taken. Before preparing the polysaccharide aqueous solution, the concentration of free primary amino groups on the side chains of the silk fibroin molecular chain was determined by the o-phthalaldehyde method. The amount of feed was calculated based on the stoichiometric relationship between the free primary amino groups and the terminal reducing sugar groups of hyaluronic acid with a molecular weight of 2kDa to 10kDa. The mass ratio of silk fibroin to hyaluronic acid was adjusted to 1:0.5 to 1:2.

[0064] The aqueous phase was adjusted to a mass-volume concentration of 10% and the pH to 6.0. It was then heated at 60℃ to 90℃ for 1 hour to allow the terminal reducing glycosyl groups of hyaluronic acid to undergo a covalent coupling reaction with the free primary amino groups on the side chains of the silk fibroin molecular chain. After condensation and Amadori rearrangement, an aqueous phase of silk fibroin-hyaluronic acid copolymer was generated. The grafting rate (DG%) was monitored by ultraviolet spectrophotometry and controlled between 40% and 43% to reduce the impact of fluctuations in the content of free primary amino groups in different batches of silk fibroin raw materials on the physicochemical properties of the copolymer aqueous phase.

[0065] During the covalent coupling reaction, the reducing glycosyl groups at the ends of the hyaluronic acid molecular chains participate in the reaction, while the repeating disaccharide units on the main chain remain unreacted. By adjusting the feed ratio, the molar amount of hyaluronic acid is made higher than the molar amount of free primary amino groups of silk fibroin. When the grafting rate (DG%) reaches 40% to 43%, the reaction system is cooled in an ice bath to stop the coupling reaction. This reaction process only consumes a portion of the reducing glycosyl groups at the ends of the hyaluronic acid chains, while the remaining unreacted hyaluronic acid chain segments retain the hyaluronic acid-specific oligosaccharide structure and are distributed around the silk fibroin-hyaluronic acid copolymer.

[0066] The aqueous phase of silk fibroin hyaluronic acid copolymer was mixed with medium-chain fatty acid triglycerides at a volume ratio of 1:5 and mechanically stirred at 1000 rpm for 5 min at 20℃ to 25℃ to generate a primary emulsion. The primary emulsion was then treated with ultrasound using a probe at a power of 200W to 600W for 5 min to 15 min. The dispersion energy was controlled according to the acoustic energy density per unit volume to allow the silk fibroin hyaluronic acid copolymer molecules to migrate to the oil-water interface and self-assemble, forming a three-dimensional network interface film on the surface of the emulsion droplets.

[0067] After dispersion treatment, the resulting silk fibroin hyaluronic acid copolymer formulation is an oil-in-water emulsion with an average droplet size of 150 nm to 200 nm, a polydispersity index of less than 0.3, and a zeta potential of -10 mV to -28 mV. When the formulation is sealed and stored at 45 °C for 90 days, the centrifugal sedimentation stability parameter Ke value remains between 0.102 and 0.356. The formulation maintains similar emulsification dispersion and storage stability among different batches of raw materials.

[0068] Based on the reaction kinetics and thermal acceleration equation, it was calculated that when the ambient temperature increased from 25℃ to 45℃, the physical decay and stratification degradation rate of the system increased by 8 to 10 times. After the silk fibroin hyaluronic acid copolymer preparation was sealed and stored at 45℃ for 90 days, no oil-water stratification occurred, and the average droplet size did not increase, indicating that the preparation has a storage stability of more than 24 months under the condition of 25℃.

[0069] Example 6: Hyaluronic acid with a molecular weight of 2kDa to 10kDa was dissolved in deionized water to prepare a polysaccharide aqueous solution. Then, silk fibroin was added at a mass ratio of 1:1 to obtain a protein-polysaccharide mixed aqueous solution. The mass-volume concentration of the mixed aqueous solution was adjusted to 10%, and the pH was adjusted to 6.0. The solution was heated at 60℃ to 90℃ for 1 hour to allow the terminal reducing sugar groups of hyaluronic acid to undergo a covalent coupling reaction with the free primary amino groups of the side chains of the silk fibroin molecular chain, thereby generating an aqueous phase of silk fibroin-hyaluronic acid copolymer.

[0070] Using medium-chain triglycerides as the oil phase material, the oil phase material and the aqueous phase of silk fibroin hyaluronic acid copolymer were mixed at a volume ratio of 1:5 at 20℃ to 25℃ and mechanically stirred at 1000 rpm for 5 min to generate a primary emulsion. Subsequently, the primary emulsion was ultrasonically treated with a probe at a power of 400W for 10 min, causing the silk fibroin hyaluronic acid copolymer to self-assemble at the oil-water interface to form a three-dimensional network interfacial film, thus obtaining an oil-in-water silk fibroin hyaluronic acid copolymer formulation. The average particle size of the resulting droplets was 150 nm to 200 nm, the polydispersity index was less than 0.3, and the zeta potential was -10 mV to -28 mV.

[0071] This silk fibroin-hyaluronic acid copolymer formulation was used as an effective preparation for anti-photoaging drugs or cosmetics. It was applied to treat skin wrinkles, skin laxity, pigmentation, and barrier damage caused by ultraviolet radiation. In a human skin fibroblast photoaging ultraviolet radiation damage model, the ultraviolet B radiation intensity was 20 mJ / cm², and the radiation time was 10 min. After intervention with this preparation, the expression level of type I collagen was upregulated to 3.71 pg / mg, and the expression level of type III collagen was upregulated to 8.02 pg / mg.

[0072] In a mouse photoaging model, after administration of this preparation, the thickness of the dorsal epidermis of mice recovered from 85.2 μm in the UVB model group to 28.5 μm. The pathological epidermal thickening, skin erythema and wrinkles caused by UV radiation were reduced, and the collagen network in the extracellular matrix of the dermis was restored.

[0073] The embodiments of this application have been described above with reference to the accompanying drawings. Unless otherwise specified, the embodiments and features in the embodiments of this application can be combined with each other. This application is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of this application without departing from the spirit of this application and the scope of protection of this invention, and all of these forms are within the protection scope of this application.

Claims

1. An emulsification preparation process for a silk fibroin-hyaluronic acid copolymer formulation, characterized in that, Includes the following steps: Step S1: Dissolve hyaluronic acid with a molecular weight of 2kDa to 50kDa in deionized water to prepare a polysaccharide aqueous solution, and then mix the polysaccharide aqueous solution with silk fibroin to obtain a proteoglycan mixed aqueous solution; wherein the mass ratio of silk fibroin to hyaluronic acid is 1:0.2 to 1:

4. Step S2: The mixed aqueous solution of proteoglycans is heated for 0.5h to 2h under the conditions of a mass-volume concentration of 8% to 12%, a pH of 4.0 to 8.0, and a temperature of 60℃ to 90℃ to undergo a covalent coupling reaction to generate an aqueous phase of silk fibroin hyaluronic acid copolymer. Step S3, providing at least one oil phase material selected from medium-chain fatty acid triglycerides, triglyceride esters, caprylate-capric triglycerides and 2-octyldodecyl alcohol; Step S4: Mix the oil phase material with the aqueous phase of the silk fibroin hyaluronic acid copolymer at a volume ratio of 1:4 to 1:6 and stir to generate a primary emulsion. Step S5: Apply at least one dispersion treatment selected from probe ultrasonic treatment, high pressure homogenization treatment and microfluidic treatment to the primary emulsion, so that the silk fibroin hyaluronic acid copolymer self-assembles at the oil-water interface to form a three-dimensional network interface film, and obtain a silk fibroin hyaluronic acid copolymer formulation with an average particle size of 150nm to 200nm and a polydispersity index of less than 0.

3.

2. The emulsification preparation process of a silk fibroin-hyaluronic acid copolymer formulation according to claim 1, characterized in that, In step S1, the molecular weight of hyaluronic acid is 2kDa to 10kDa; the mass ratio of silk fibroin to hyaluronic acid is 1:0.5 to 1:2; in step S4, the volume ratio of the oil phase material to the aqueous phase of the silk fibroin-hyaluronic acid copolymer is 1:5 to 1:

6.

3. The emulsification preparation process of a silk fibroin-hyaluronic acid copolymer formulation according to claim 1, characterized in that, In step S5, the dispersion treatment is ultrasonic treatment with a probe, and the ultrasonic power is controlled to be 200W to 600W and the ultrasonic time is 5min to 15min. In the prepared silk fibroin hyaluronic acid copolymer formulation, the zeta potential on the surface of the droplet is maintained at -10mV to -28mV, so as to construct a physical defense layer with negative charge electrostatic repulsion and three-dimensional network interface film spatial steric hindrance on the periphery of the droplet.

4. The emulsification preparation process of a silk fibroin-hyaluronic acid copolymer formulation according to claim 1, characterized in that, In step S3, the oil phase material is a medium-chain fatty acid triglyceride; step S4 includes step S41, in which the oil phase material is added dropwise to the aqueous phase of the silk fibroin hyaluronic acid copolymer at a temperature of 20°C to 25°C, and mechanically stirred at a speed of 500 rpm to 1500 rpm for 3 min to 10 min, so that the oil phase material is sheared into initially dispersed droplets in the aqueous phase of the silk fibroin hyaluronic acid copolymer to generate a primary emulsion.

5. The emulsification preparation process of a silk fibroin-hyaluronic acid copolymer formulation according to claim 1, characterized in that, After the silk fibroin hyaluronic acid copolymer preparation obtained in step S5 is sealed and stored at an ambient temperature of 40°C to 50°C for 80 to 95 days, the average particle size of the silk fibroin hyaluronic acid copolymer preparation is maintained in the range of 150 nm to 200 nm, and the polydispersity index is maintained at less than 0.

3. No oil-water separation boundary or flocculation precipitate is generated in the system.

6. The emulsification preparation process of a silk fibroin-hyaluronic acid copolymer formulation according to claim 1, characterized in that, Silk fibroin-hyaluronic acid copolymers form an amphiphilic microtopology at the oil-water interface. The amphiphilic microtopology includes hydrophobic amino acid residues of silk fibroin anchored inside the oil phase material and hyaluronic acid polysaccharide chains extending into the continuous aqueous phase. The amphiphilic microtopology is based on the amphiphilic adaptive deformation capability of droplets penetrating epidermal lipid channels from 150 nm to 200 nm.

7. The emulsification preparation process of a silk fibroin-hyaluronic acid copolymer formulation according to claim 1, characterized in that, The surface of the silk fibroin-hyaluronic acid copolymer retains unreacted hyaluronic acid-specific oligosaccharide structures. These structures serve as specific binding targets for CD44 receptors on the surface of fibroblasts. In an in vitro model of photoaging of human skin fibroblasts under ultraviolet radiation, these structures upregulated the expression of type I collagen to 3.71 pg / mg and type III collagen to 8.02 pg / mg.

8. The emulsification preparation process of a silk fibroin-hyaluronic acid copolymer formulation according to claim 1, characterized in that, The silk fibroin-hyaluronic acid copolymer formulation is an oil-in-water nanoemulsion with an average droplet size of 150 nm to 200 nm, a polydispersity index of less than 0.3, and a zeta potential of -10 mV to -28 mV.

9. The emulsification preparation process of a silk fibroin-hyaluronic acid copolymer formulation according to claim 1, characterized in that, Silk fibroin-hyaluronic acid copolymer formulations are used to prepare drugs or cosmetics for anti-photoaging of the skin, where photoaging of the skin includes skin wrinkles, skin laxity, pigmentation, and barrier damage caused by ultraviolet radiation.

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