Artificial human skin and method for manufacturing the same

Abstract

The present invention provides an artificial human skin comprising a modified silk fibroin (SF) film, the artificial human skin having a first surface and a second surface, wherein the SF film is modified with one or both of: a crosslinking agent that forms crosslinks between SF fibers, wherein when the crosslinking agent is used alone, the artificial human skin has a thickness of 105 μm to 130 μm and a surface free energy of the first surface of 10 mN / m to 65 mN / m; and a lipid mimic selected from one or more of: a fatty alcohol, more particularly, a fatty acid and a fatty amine, wherein: the fatty alcohol forms an ester linkage with a carboxylic acid functionality in the SF, the fatty acid forms an ester linkage with a hydroxyl functionality in the SF, and the fatty amine forms an amide linkage with a carboxylic acid functionality in the SF. The present invention also provides a method of manufacturing the artificial human skin.

Description

Technical Field

[0001] This invention provides an artificial human skin, and more specifically, an artificial human skin comprising a modified silk fibroin (SF) membrane. That is, this invention relates to the protein reconstruction of human skin. Background Technology

[0002] The listing or discussion of previously published documents in this specification should not necessarily be construed as an admission that such documents are part of the prior art or common general knowledge.

[0003] A growing number of countries have enacted policies banning animal testing, and many others are planning to join in the future. As a result, the market for non-animal testing alternatives is becoming increasingly attractive. In fact, it is estimated that the market size for non-animal testing will reach approximately $29.4 billion by 2030.

[0004] Current alternatives for animal testing, such as human skin cell-based models and synthetic polymer-based skin, face significant limitations. Human skin cell models are highly accurate but extremely expensive and difficult to mass-produce, while polymer-based models, although cheaper, cannot replicate the biological complexity of real skin. This creates a significant gap in the market for a solution that combines the economics of polymer models with the accuracy of human skin cell models.

[0005] The average R&D intensity for cosmetic companies is approximately 2.2%, which refers to the percentage of R&D expenditure to operating revenue. This is significantly lower than the average level (approximately 20.9%) in the healthcare industry (including pharmaceuticals). Therefore, cosmetic companies are more price-sensitive than pharmaceutical companies. Consequently, the most suitable artificial skin for the market is a low-priced product with a moderate level of similarity to human skin.

[0006] Therefore, there is an urgent need for alternative and / or improved artificial human skin and its manufacturing methods applicable to various applications. Summary of the Invention

[0007] Various aspects and embodiments of the invention will now be described with reference to the following numbered clauses.

[0008] 1. An artificial human skin comprising a modified silk fibroin (SF) membrane, the artificial human skin having a first surface and a second surface, wherein the SF membrane is modified by one or both of the following: A crosslinking agent that forms crosslinks between SF fibers, wherein when the crosslinking agent is used alone, the artificial human skin has the following characteristics: The thickness ranges from 105 μm to 130 μm; and The surface free energy of the first surface is 10 mN / m to 65 mN / m; and Lipid mimics, selected from one or more of the following: fatty alcohols; more particularly, fatty acids and fatty amines, wherein: The fatty alcohol forms an ester bond with the carboxylic acid functional group in the SF; The fatty acid forms an ester bond with the hydroxyl functional group in the SF; and The fatty amine forms an amide bond with the carboxylic acid functional group in the SF.

[0009] 2. The artificial human skin according to Clause 1, wherein the modified SF membrane has a first surface shaped to mimic the structure of human skin.

[0010] 3. The artificial human skin according to Clause 1 or 2, wherein the crosslinking agent is a crosslinking agent derived from polyethylene glycol (PEG).

[0011] 4. The artificial human skin according to Clause 3, wherein the crosslinking agent is selected from one or more of the group consisting of: O'O-bis[2-(N-succiniminosuccinamido)ethyl]polyethylene glycol (NHSP), polyethylene glycol dithiol, 4-arm PEG-thiol, 4-arm PEG-epoxide, 4-arm PEG-isocyanate, isocyanate PEG isocyanate and polyethylene glycol diglycidyl ether (e.g., polyethylene glycol dithiol, 4-arm PEG-thiol, 4-arm PEG-epoxide, 4-arm PEG-isocyanate, isocyanate PEG isocyanate and polyethylene glycol diglycidyl ether).

[0012] 5. The artificial human skin as described in Clause 4, wherein the crosslinking agent is polyethylene glycol diglycidyl ether.

[0013] 6. The artificial human skin according to Clause 4 or Clause 5, wherein the number average molecular weight of said crosslinking agent is from 150 Daltons to 4,000 Daltons, for example from 150 Daltons to 1,000 Daltons, for example from 200 Daltons to 700 Daltons, for example from 250 Daltons to 500 Daltons.

[0014] 7. The artificial human skin according to any one of clauses 3 to 6, wherein when the SF membrane is modified by crosslinking, it mimics the sensory behavior of human skin (sensory skin).

[0015] 8. The artificial human skin as described in Clause 7, wherein the thickness of the sensory skin is approximately 120 μm.

[0016] 9. The artificial human skin according to any one of clauses 3 to 8, wherein the surface free energy of the first surface of the artificial human skin is: 10mN / m to 20mN / m; 25mN / m to 35mN / m; 30mN / m to 40mN / m; 39mN / m to 45mN / m; 42mN / m to 58mN / m; or 59mN / m to 65mN / m.

[0017] 10. The artificial human skin according to any one of Clause 9, wherein the surface free energy of the first surface of the artificial human skin is about 30 mN / m.

[0018] 11. The artificial human skin according to any one of clauses 7 to 10, wherein 2 to 20 layers of sensory skin are formed as a single stack to mimic the absorption behavior of human skin (absorbent skin).

[0019] 12. The artificial human skin as described in Clause 11, wherein the absorbent skin comprises 2 to 10 layers, such as 3 to 5 layers, of the sensory skin.

[0020] 13. The artificial human skin according to clause 11 or 12, wherein said absorbent skin has one or more of the following characteristics: (aa) In the absorbent skin, more than 50% of the silk fibroin is in a β-sheet / β-turn conformation, less than 30% of the silk fibroin is in a random coil, and if the remaining silk fibroin is present, it is in an α-helix form (e.g., in the absorbent skin, 51% to 75% of the silk fibroin is in a β-sheet / β-turn conformation, 3% to 25% of the silk fibroin is in a random coil, and the remaining silk fibroin is in an α-helix form). (ab) Crystallinity value greater than 25%, for example, 27% to 50%; (ac) has a caffeine permeation rate of 1 μg / cm 2 / h to 3μg / cm 2 / h, for example, about 2.5 μg / cm 2 / h; (ad) The permeation rate of nicotinamide was 7 μg / cm. 2 / h to 15μg / cm 2 / h, for example, about 10 μg / cm 2 / h; and (ae) has a permeation rate of 2 μg / cm for salicylic acid. 2 / h to 7μg / cm 2 / h, for example, about 5 μg / cm 2 / h.

[0021] 14. The artificial human skin according to any one of the preceding clauses, wherein the lipid mimic has 14 to 20 carbon atoms arranged in a branched chain, or more particularly in a straight chain.

[0022] 15. The artificial human skin according to any one of the preceding clauses, wherein when the SF membrane is modified with the lipid mimicry, the lipid mimicry is hexadecylamine.

[0023] 16. The artificial human skin according to any one of the preceding clauses, wherein when the SF membrane is modified with the lipid mimicry, the artificial human skin mimics the deposition behavior of human skin (deposited skin).

[0024] 17. The artificial human skin as described in Clause 16, wherein the deposited skin has a brick-wall structure that mimics the stratum corneum structure in the human epidermis.

[0025] 18. The artificial human skin as described in Clause 16 or Clause 17, wherein the thickness of the deposited skin is from 20 μm to 400 μm.

[0026] 19. The artificial human skin according to any one of the preceding clauses, wherein when the SF membrane is modified with the lipid mimicry, the SF membrane further comprises one or both of the following: a surfactant and a suspended solid material containing unreacted lipid mimicry.

[0027] 20. The artificial human skin according to any one of the preceding clauses, wherein the artificial human skin is coated with sebum, optionally wherein the sebum is artificial sebum, and further optionally wherein one or more of the following are applicable: (a) The artificial sebum comprises triglycerides, paraffin, free fatty acids, squalene, and cholesterol, optionally wherein the weight ratio of triglycerides:paraffin:free fatty acids:squalene:cholesterol is 8:5:5:3:1; and (b) The sebum content ratio is 0.5% to 10% by weight, for example, 0.6% to 8% by weight, relative to the weight of the modified SF film.

[0028] 21. The artificial human skin according to any one of the preceding clauses, wherein the SF membrane is modified only by one or more lipid mimics selected from: fatty acids; and fatty amines.

[0029] 22. The artificial skin according to any one of the preceding clauses, wherein the artificial skin is configured to have a water contact angle of 60° to 135° before the test material is applied to the artificial skin to simulate one of oily skin, normal skin or dry skin.

[0030] 23. The artificial skin according to Clause 22, wherein the artificial skin has: (i) A water contact angle of 60° to 85° to simulate oily skin; (ii) A water contact angle of 80° to 90° to simulate neutral skin; or (iii) A water contact angle of 90° to 135° to simulate dry skin.

[0031] 24. The artificial skin described under Clause 22 or Clause 23, when subject to Clause 7, wherein said artificial skin is sensory skin.

[0032] 25. A method for manufacturing artificial skin, the method comprising the following steps: (a) Providing a concentrated solution comprising cross-linked silk fibroin (SF) fibers, wherein the concentration of the cross-linked SF fibers is from 11% to 21% by weight of the total weight of the solution; and (b) The concentrated solution is poured onto a substrate, and the poured material is spread out using a doctor blade coating technique to achieve a solution height of approximately 350 μm to 450 μm. The resulting material is then dried to provide the artificial skin, wherein the artificial skin has a second surface and a first surface in contact with the substrate, and has the following characteristics: The thickness ranges from 105 μm to 130 μm; and The surface free energy of the first surface is between 10 mN / m and 65 mN / m.

[0033] 26. The method according to Clause 25, wherein the crosslinking agent is a crosslinking agent derived from polyethylene glycol (PEG).

[0034] 27. The method according to Clause 25, wherein the crosslinking agent is selected from one or more of the group consisting of: O'O-bis[2-(N-succiniminosuccinamido)ethyl]polyethylene glycol (NHSP), polyethylene glycol dithiol, 4-arm PEG-thiol, 4-arm PEG-epoxide, 4-arm PEG-isocyanate, isocyanate PEG isocyanate and polyethylene glycol diglycidyl ether (e.g., polyethylene glycol dithiol, 4-arm PEG-thiol, 4-arm PEG-epoxide, 4-arm PEG-isocyanate, isocyanate PEG isocyanate and polyethylene glycol diglycidyl ether).

[0035] 28. The method according to Clause 27, wherein the crosslinking agent is polyethylene glycol diglycidyl ether.

[0036] 29. The method according to Clause 27 or Clause 28, wherein the number average molecular weight of said crosslinking agent is from 150 Daltons to 4,000 Daltons, for example from 150 Daltons to 1,000 Daltons, for example from 200 Daltons to 700 Daltons, for example from 250 Daltons to 500 Daltons.

[0037] 30. The method according to any one of clauses 25 to 29, wherein the thickness of the artificial skin is about 120 μm.

[0038] 31. The method according to any one of clauses 25 to 29, wherein the surface free energy of the first surface of the artificial human skin is: 10mN / m to 20mN / m; 25mN / m to 35mN / m; 30mN / m to 40mN / m; 39mN / m to 45mN / m; 42mN / m to 58mN / m; or The surface free energy of the first surface of the artificial human skin is 59 mN / m to 65 mN / m, optionally with a surface free energy of about 30 mN / m.

[0039] 32. The method according to any one of clauses 25 to 31, wherein the concentrated solution in step (a) of clause 25 is provided by the following steps: (ai) Provides a solution comprising cross-linked silk fibroin (SF) fibers, said solution having been filtered to remove suspended solids, and the concentration of cross-linked SF fibers being less than 7% by weight of the total weight of said solution; and (aii) The solution is concentrated to provide the concentrated solution by keeping the solution at a high temperature for a period of time, optionally wherein the high temperature is 55°C to 70°C, for example about 60°C, and the period of time is 1 hour to 5 hours, for example about 3 hours.

[0040] 33. The method according to Clause 32, wherein the solution in step (ai) of Clause 32 is provided by the following steps: (bi) A cross-linking agent is added to a solution of silk fibroin fibers, the concentration of which is less than 7% by weight of the total weight of the solution, and the mixture is allowed to react for a period of time to provide a cross-linked silk fibroin solution; and (bii) The solution is filtered to remove suspended solids (e.g., one or both of unreacted crosslinking agent and unreacted silk fibroin) to provide a solution containing silk fibroin (SF) fibers crosslinked by the crosslinking agent.

[0041] 34. The method according to Clause 33, wherein the weight ratio is 20:1 to 5:1, for example about 10:1.

[0042] 35. A method for forming artificial skin, comprising the following steps: (ci) provides multiple sensory skins as described in Clause 7 and Clauses 8 to 24 which are subordinate to Clause 7; and (cii) The plurality of sensory skins are attached together to provide a single stack to mimic the absorption behavior of human skin (absorbent skin).

[0043] 36. The method according to Clause 35, wherein the absorbent skin comprises 2 to 10 layers, such as 3 to 5 layers of the sensory skin.

[0044] 37. The method described in Clause 35 or Clause 36, wherein the attachment is performed using hot pressing. Attached Figure Description

[0045] Figure 1 The WSS values ​​between different materials (i.e., silk fibroin (SF), wool, feathers, and hair) and keratinized capsule (CE) are shown.

[0046] Figure 2 Three types of artificial skin (i.e., D-skin, S-skin, and A-skin) are shown, along with their functions and application examples.

[0047] Figure 3 The water contact angles of human forearm skin and S-shaped artificial skin after treatment with different skin care products are shown.

[0048] Figure 4 The coefficients of friction for human cheek skin and S-shaped artificial skin are shown after treatment with different skincare products. A) Human cheek skin. B) S-shaped artificial skin.

[0049] Figure 5 The preparation of concentrated polyethylene glycol-modified silk fibroin (CPSF) films is shown. a, Schematic diagram of the CPSF film preparation process. b, Photograph of the dried CPSF film. c, Photograph of the hydrated CPSF film.

[0050] Figure 6 shows the characteristics of CPSF solutions at different concentrations. a, Turbidity of CPSF solutions at different concentrations. Data are expressed as the mean ± standard deviation of three independent measurements. b, Zeta potential and gelation time of CPSF solutions at different concentrations. Gelation conditions: 25°C. Data are expressed as the mean ± standard deviation of three independent measurements. c, Photographs of CPSF solutions at different concentrations and two standard turbidity solutions. The leftmost solution is the 0 NTU standard solution, and the rightmost solution is the 100 NTU standard solution. d, Photograph showing the gelation of CPSF solutions after 13 days of storage at room temperature.

[0051] Figure 7 The thickness uniformity of CPSF films prepared by blade coating and PSF films prepared by solvent casting is shown. Data are expressed as the mean ± standard deviation of thirteen independent measurements.

[0052] Figure 8The diagram illustrates a mold-based SFE modification technique. a) Schematic diagram of different materials (including silicone, aluminum foil, PS, PVC, and glass) used for doctor blade coating. b) Schematic diagram showing the mold-facing side of the CPSF film in contact with the mold material.

[0053] Figure 9 The following figures show the water contact angle measurements of CPSF films made using different molds, with water on the left and diiodomethane on the right. a) CPSF film on silicone. b) CPSF film on aluminum foil. c) CPSF film on polystyrene (PS). d) CPSF film on polyvinyl chloride (PVC). e) CPSF film on glass.

[0054] Figure 10 Various mold materials and the surface contact angle (SFE) of CPSF films formed using these molds are shown. a) Measurements of the water contact angle (water and diiodomethane) and SFE data for CPSF films formed on different mold materials (including silicone, aluminum foil, PS, PVC, and glass). Data are expressed as the mean ± standard deviation of six independent measurements. b) Comparison of SFE data for different mold materials and CPSF films formed on these molds.

[0055] Figure 11 XPS characterization of CPSF films prepared using different molds is shown. a, XPS spectra of CPSF films prepared using different molds. b, Quantitative analysis of the chemical environment of carbon atoms in CPSF films prepared using different molds.

[0056] Figure 12 This is a schematic diagram of hot pressing a CPSF film into PraE.

[0057] Figure 13 These are photographs of CPSF films hot-pressed at different temperatures.

[0058] Figure 14 The images include cross-sectional SEM images of PraE prepared by hot pressing at different temperatures. a) SEM cross-sectional images of PraE prepared at different temperatures at 70x magnification. b) SEM cross-sectional images of PraE prepared at different temperatures at 10,000x magnification. c) SEM cross-sectional images of PraE prepared at different temperatures at 20,000x magnification.

[0059] Figure 15FTIR characterization of PraE prepared by hot pressing at different temperatures is shown. a, FTIR spectra of PraE prepared by hot pressing at 4 MPa and different temperatures for 3 minutes. b, Deconvolution of the amide I band peak in the FTIR spectra to quantitatively calculate the percentage of different secondary structures (including α-helices, β-sheets / β-turns, and random coils). c, Quantitative analysis of the secondary structures of PraE prepared at different temperatures. Data are expressed as mean ± standard deviation of five / six independent measurements.

[0060] Figure 16 A control experiment is shown involving heating or pressurizing CPSF films.

[0061] Figure 17 XRD characterization of PraE prepared by hot pressing at different temperatures is shown. a, XRD spectra of PraE prepared by hot pressing at 4 MPa and different temperatures for 3 minutes. b, Deconvolution of the XRD spectra, and quantitative calculation of the crystallinity percentage by calculating the crystalline peak (blue dashed line) and amorphous peak (red dashed line). c, Quantitative analysis of the crystallinity of PraE prepared at different temperatures.

[0062] Figure 18 The density of PraE prepared by hot pressing at different temperatures is shown.

[0063] Figure 19 XPS characterization of PraE prepared by hot pressing at different temperatures is shown. a) XPS spectra of PraE prepared by hot pressing at 4 MPa and different temperatures for 3 minutes. b) Deconvolution of C1s spectra, and quantitative analysis of the chemical environment of carbon atoms by calculating C–C peak (blue dashed line), C–O peak (green dashed line), and C=O peak (red dashed line). c) Quantitative analysis of the chemical environment of carbon atoms in PraE prepared at different temperatures.

[0064] Figure 20 It consists of CPSF thin films (original sample) and PraE prepared at different temperatures. 1 ¹H NMR spectroscopy. These films were dissolved in 9.3 M LiBr at 60 °C for 4 hours, and then dialyzed in tubes (molecular weight cutoff: 3.5 kJ) for 2 days to remove LiBr. The solution was then lyophilized and redissolved in D₂O. 1 H NMR analysis.

[0065] Figure 21Different methods for preparing films of varying thicknesses (60 μm to 600 μm) are illustrated, including solvent casting, blade coating, and hot pressing. a) Thickness uniformity distribution of films of different thicknesses prepared by solvent casting, blade coating, and hot pressing. Data are expressed as the mean ± standard deviation of fifteen independent measurements. b) Schematic diagrams of solvent casting, blade coating, and hot pressing.

[0066] Figure 22 The process of coating PraE with artificial lipids is shown. a, Schematic diagram of the process of coating PraE with artificial lipids at 72°C to obtain PLraE. b, Photographs of PraE and PLraE.

[0067] Figure 23 The following diagram illustrates the relationship between thickness and permeation rate: a) Graph and calculation formula of steady-state permeation rate, influenced by concentration gradient, path length, and diffusion coefficient. b) Secondary structures of PraE-180 with varying thicknesses (200 μm to 650 μm) prepared by changing the number of CPSF film layers and the thickness of a single CPSF film. c) Fitting curve analysis to verify the relationship between permeation rate and path length.

[0068] Figure 24 The caffeine permeation rate test of PraE is shown. a) Cumulative permeation of caffeine in PraE prepared by hot pressing at 120°C, 140°C, 160°C, and 180°C. Data are expressed as the mean ± standard deviation of three independent measurements. b) Diffusion coefficient of PraE prepared by hot pressing at 120°C, 140°C, 160°C, and 180°C. Data are expressed as the mean ± standard deviation of three independent measurements. c) Photographs of PraE prepared by hot pressing at 120°C, 140°C, 160°C, and 180°C. d) Photograph of PraE fixed in a Franz diffusion cell for transdermal permeation testing.

[0069] Figure 25 The caffeine partition coefficients of different secondary structures in PraE are shown. a) Quantitative analysis of the secondary structures of PraE prepared at different temperatures. Data are expressed as mean ± standard deviation of five / six independent measurements. b) Diffusion coefficients of different secondary structures (including β-sheets / β-turns, α-helices, and random coils). Data are expressed as mean ± standard deviation of three independent measurements.

[0070] Figure 26 The mechanism by which small molecule compounds (caffeine) permeate through PraE is shown.

[0071] Figure 27 This paper compares the caffeine penetration rate of PraE prepared at different temperatures with that of human skin. Data are expressed as the mean ± standard deviation of three independent measurements.

[0072] Figure 28 The permeation rates of four compounds in human skin, PraE, PLraE, Strat-M, and Episkin are compared. a, Caffeine permeation rate in different skin types. b, Niacinamide permeation rate in different skin types. c, Salicylic acid permeation rate in different skin types. d, Rhodamine B permeation rate in different skin types. Data are expressed as mean ± standard deviation of three independent measurements. The human skin permeation rate for each molecule was calculated by averaging published study data.

[0073] Figure 29 These are photographs of different skin types in Franz diffusion pools 8 hours after a Rhodamine B penetration test. a, PLraE. b, PraE. c, Strat-M. d, Episkin.

[0074] Figure 30 The permeation rates of PLraE and PraE in aqueous solution are shown as MW and log S. aq The correlation between them.

[0075] Figure 31 This diagram illustrates an R2R system utilizing slot coating for thin film production. a) Schematic diagram of an R2R system with slot coating, showing the film-forming process, including coating, drying, and web travel. b) Structural details of the slot coating section. c) Photograph of a CPSF film produced by the R2R system. d) Thickness of CPSF films produced by the R2R system at different flow rates.

[0076] Figure 32 The structures of protein-lipid linkages in the stratum corneum (SC) and D-skin are shown. A) Structure of the SC, B) Schematic diagram of the synthesis and structure of D-skin.

[0077] Figure 33 Includes a schematic diagram of the fragrance deposition test, and a comparison of the fragrance molecule ratios of D skin (SH artificial skin) relative to the palms of three different testers (i.e., tester 1, tester 2, and tester 3).

[0078] Figure 34 This includes a schematic diagram of the preparation of SHSSs artificial skin, and a comparison of the fragrance molecule ratio of SHSSs artificial skin to human forearm skin.

[0079] Figure 35Includes: a) A schematic diagram of the stepwise preparation of Skin A, demonstrating precise control over protein structure at the molecular level, starting from the silkworm cocoon. First, sericin is removed by degumming to extract silk fibroin. Then, the silk fibroin is regenerated into a protein solution by breaking internal hydrogen bonds. Subsequently, a protein film is formed through self-assembly, and then multiple thinner films are pressed together by thermo-pressing to form a single film (Skin A), thus significantly altering its secondary structure. b) Selective transdermal penetration is characterized by three key principles: (i) different compounds penetrate into the protein matrix through random coil regions; (ii) smaller molecules penetrate faster than larger molecules; and (iii) compounds with higher water solubility exhibit a faster penetration rate. c) A schematic diagram of the transdermal penetration process, showing how therapeutic agents penetrate through the epidermis and dermis. This illustration emphasizes that some compounds can penetrate the skin, while others cannot. Detailed Implementation

[0080] Surprisingly, silk fibroin has been found to be processed in a variety of ways to create skin testing platforms that can more realistically simulate the properties of real human skin types. Therefore, in a first aspect of the invention, an artificial human skin is provided comprising a modified silk fibroin (SF) membrane, the artificial human skin having a first surface and a second surface, wherein the SF membrane is modified by one or both of the following: A crosslinking agent that forms crosslinks between SF fibers, wherein when the crosslinking agent is used alone, the artificial human skin has the following characteristics: The thickness ranges from 105 μm to 130 μm; and The surface free energy of the first surface is 10 mN / m to 65 mN / m; and Lipid mimics, selected from one or more of the following: fatty alcohols; more particularly, fatty acids and fatty amines, wherein: The fatty alcohol forms an ester bond with the carboxylic acid functional group in the SF; The fatty acid forms an ester bond with the hydroxyl functional group in the SF; and The fatty amine forms an amide bond with the carboxylic acid functional group in the SF.

[0081] To avoid ambiguity, three possible types of artificial skin are listed in this regard: (a) Skin formed from SF and a crosslinking agent; (b) Skin formed of SF and a lipid mimic, wherein the lipid mimic forms a covalent bond with SF; and (c) Skin formed from SF, cross-linking agent and lipid mimicry.

[0082] In the embodiments herein, the word "comprising" can be interpreted as the features that need to be mentioned, but does not limit the presence of other features. Alternatively, the word "comprising" can also refer to situations where only the listed components / features are intended to be present (e.g., the word "comprising" can be replaced by the phrases "consisting of" or "substantially consisting of"). It is clearly anticipated that both broad and narrow interpretations can apply to all aspects and embodiments of the invention. In other words, the word "comprising" and its synonyms can be replaced by the phrases "consisting of" or "substantially consisting of" or their synonyms, and vice versa.

[0083] The phrase "consistently made up of..." and its synonyms can be interpreted in this text as meaning that a small amount of impurities may be present in the material. For example, the purity of the material may be greater than or equal to 90%, such as greater than 95%, greater than 97%, greater than 99%, greater than 99.9%, greater than 99.99%, greater than 99.999%, or 100%.

[0084] It should be understood that the modified SF film may have a first surface shaped to mimic the structure of human skin. This human skin structure may be provided through the interaction described above (a) to (c) with the mold surface, which may impart different properties to the resulting first surface, or it may be provided through the chemical structure of SF with lipid mimics and / or crosslinking agents.

[0085] Any suitable crosslinking agent may be used herein. For example, the crosslinking agent may be one that provides ionic or covalent crosslinking between SF fibers. More specifically, the crosslinking may be covalent. For example, in some embodiments that may be mentioned herein, the crosslinking agent may be a crosslinking agent derived from polyethylene glycol (PEG). Therefore, any suitable PEG-derived crosslinking agent may be used herein, provided that it contains functional groups capable of achieving crosslinking. The crosslinking agent may be bidentate, tripentate, or tetradentate in terms of the functional groups that can be used to form crosslinks with SF. In more specific embodiments mentioned herein, the crosslinking agent may be selected from one or more of the group consisting of: O'O-bis[2-(N-succinimidylsuccinamido)ethyl]polyethylene glycol (NHSP), polyethylene glycol dithiol, 4-arm PEG-thiol, 4-arm PEG-epoxide, 4-arm PEG-isocyanate, isocyanate PEG isocyanate, and polyethylene glycol diglycidyl ether (e.g., polyethylene glycol dithiol, 4-arm PEG-thiol, 4-arm PEG-epoxide, 4-arm PEG-isocyanate, isocyanate PEG isocyanate, and polyethylene glycol diglycidyl ether). In specific embodiments of the invention, when the crosslinking agent is present in the artificial skin, it may be polyethylene glycol diglycidyl ether. For the avoidance of doubt, although the crosslinking agents mentioned above relate to unreacted materials, it should be understood that the crosslinkable functional groups therein will be covalently bonded to the SF fibers in the artificial skin described herein.

[0086] Any suitable weight ratio of SF fiber to crosslinking agent may be used in this document. For example, the weight ratio of SF fiber to crosslinking agent may be from 20:1 to 5:1, such as about 10:1. It should be understood that these values ​​refer to the relative amounts of chemical components derived from silk fibroin and crosslinking agent in the final product (where the SF fiber is crosslinked by the crosslinking agent).

[0087] The crosslinking agent can have any suitable molecular weight. For example, the number average molecular weight of the crosslinking agent can be from 150 Daltons to 4,000 Daltons, such as 150 Daltons to 1,000 Daltons, such as 200 Daltons to 700 Daltons, such as 250 Daltons to 500 Daltons.

[0088] As mentioned above, the crosslinking agent can be used alone without combining with lipid mimics. In such cases, the artificial skin can have a thickness of 105 μm to 130 μm, and the surface free energy of the first surface can be 10 mN / m to 65 mN / m. This makes the artificial skin suitable for use as sensory skin (or S-skin). S-skin is designed to mimic the feel of different types of skin after treatment with cosmetic products. In this type of skin, the water contact angle can be used to define different types of skin (dry skin, oily skin, and normal skin) prepared by varying different methods. For cosmetic products, the feel of the product on the skin is quite important, as many consumers choose products based on how they feel on their skin. Therefore, S-skin is manufactured to mimic the feel of human skin when applying the product. Here, two factors can be used to evaluate the effectiveness of the artificial skin: one is the water contact angle, which describes the spreadability of the product on human skin; the other is the coefficient of friction, which describes the smoothness of the skin after treatment with the product.

[0089] In an embodiment where the artificial skin is S-skin, its thickness can be from 105 μm to 130 μm, for example from 110 μm to 125 μm, for example about 120 μm.

[0090] In some embodiments, the water contact angle of the artificial skin can be from 60° to 135° to mimic one of oily, normal, or dry skin. While this may be a general characteristic of the artificial skin prepared herein, it is particularly suitable for S-skin materials. More specifically, the artificial skin disclosed herein may have: (i) A water contact angle of 60° to 85° to simulate oily skin; (ii) A water contact angle of 80° to 90° to simulate neutral skin; or (iii) A water contact angle of 90° to 135° to simulate dry skin.

[0091] It is worth noting that the S-skin with the above-mentioned properties not only realistically simulated the properties of real human skin in terms of water contact angle for various skin types in sensory tests, but also realistically simulated the properties of real human skin in terms of the coefficient of friction generated after applying cosmetic products, as shown in the examples below.

[0092] In embodiments of the invention utilizing a crosslinking agent derived from polyethylene glycol (PEG), the first surface of the artificial human skin can be configured to have any suitable surface free energy in the range of 10 mN / m to 65 mN / m. For example, the surface free energy of the first surface of the artificial human skin can be: 10mN / m to 20mN / m; 25mN / m to 35mN / m; 30mN / m to 40mN / m; 39mN / m to 45mN / m; 42mN / m to 58mN / m; or 59mN / m to 65mN / m.

[0093] In a specific embodiment of the invention that may be mentioned herein, the surface free energy of the first surface of the artificial human skin may be approximately 30 mN / m.

[0094] This can be achieved by using different materials in the molds used to create the artificial skin, more details of which are described below.

[0095] While S-skin itself is useful, it can also be used to form absorbent skin. Absorption is another important research topic for cosmetic and skincare products. For skincare products such as whitening, moisturizing, and anti-aging products, researchers and consumers are concerned about how much of the active molecules (including niacinamide, hyaluronic acid, and ascorbic acid) can be absorbed by the skin. Surprisingly, it has been found that forming 2 to 20 layers of sensory skin into a single stack can yield products that mimic the absorption behavior of human skin (absorbent skin (or A-skin)). For example, absorbent skin can contain 2 to 10 layers, such as 3 to 5 layers of sensory skin. This integration can be achieved by heat-pressing the layers of S-skin together to form a unified whole.

[0096] Such absorbent skin may have one or more of the following characteristics: (aa) In the absorbent skin, more than 50% of the silk fibroin is in a β-sheet / β-turn conformation, less than 30% of the silk fibroin is in a random coil, and if the remaining silk fibroin is present, it is in an α-helix form (e.g. in the absorbent skin, 51% to 75% of the silk fibroin is in a β-sheet / β-turn conformation, 3% to 25% of the silk fibroin is in a random coil, and the remaining silk fibroin is in an α-helix form). (ab) Crystallinity value greater than 25%, for example, 27% to 50%; (ac) has a caffeine permeation rate of 1 μg / cm 2 / h to 3μg / cm 2 / h, for example, about 2.5 μg / cm 2 / h; (ad) The permeation rate of nicotinamide was 7 μg / cm. 2 / h to 15μg / cm 2 / h, for example, about 10 μg / cm 2 / h; and (ae) has a permeation rate of 2 μg / cm for salicylic acid. 2 / h to 7μg / cm 2 / h, for example, about 5 μg / cm 2 / h.

[0097] It is worth noting that the aforementioned penetration rate is close to that of real human skin. Furthermore, it is noteworthy that skin A is non-permeable to Rhodamine B, thus making this test platform more closely resemble the performance of real human skin than other testing platforms.

[0098] In embodiments of the present invention providing lipid mimics, the artificial human skin can mimic the deposition behavior of human skin (deposited skin (or D-skin)). For example, when the lipid mimic is used in combination with a crosslinking agent or without (e.g., without) a crosslinking agent. As described above, the lipid mimic can be one or more of the following: fatty alcohols; more particularly, fatty acids and fatty amines, wherein: Fatty alcohols form ester bonds with the carboxylic acid functional groups in SF; Fatty acids form ester bonds with the hydroxyl functional groups in SF; and Aliphatic amines form amide bonds with the carboxylic acid functional groups in SF.

[0099] The amount of lipids in SF can be from 5% to 20% of the total weight of SF.

[0100] D-skin is used for deposition tests, such as depositing substances to be applied to the skin (e.g., perfumes, cosmetics, sunscreens, antibacterial agents, and cleansers) onto D-skin to simulate the effect of perfume deposition on human skin.

[0101] Lipid mimics can be selected to provide properties that more realistically simulate the deposition characteristics of human skin. For example, lipid mimics may have 14 to 20 carbon atoms, such as 15 to 18 carbon atoms, or even 16 carbon atoms, arranged in a branched chain, or more particularly, in a straight chain. In specific embodiments mentioned herein, the lipid mimic may be hexadecylamine.

[0102] Not wanting to be bound by theory, it is believed that the use of lipid mimics makes the resulting artificial skin closer to real skin. For example, deposited skin can have a brick-wall structure that mimics the stratum corneum structure in the human epidermis. Not wanting to be bound by theory, it is believed that this structure is obtained because the artificial skin contains both a protein portion (SF) and a lipid portion, which mimic the bonding patterns found in the stratum corneum of real human skin, containing ester bonds between skin proteins and ceramides (lipids).

[0103] D-skin can have any suitable thickness. For example, the thickness of deposited skin can range from 20 μm to 400 μm.

[0104] In embodiments of the present invention where SF membranes are modified with lipid mimics, the SF membrane may further comprise one or both of a surfactant and / or a suspended solids material. It should be noted that the suspended solids material may simply be an unreacted (i.e., free) lipid mimic (e.g., unreacted hexadecylamine). Any suitable surfactant may be used herein, such as those mentioned in the examples below.

[0105] In some embodiments, the artificial human skin may be coated with sebum. While any suitable sebum (e.g., human, animal, or artificial sebum) may be used herein, in some embodiments the sebum may be artificial sebum. In embodiments where sebum is present, one or more of the following may apply: (a) When the sebum is synthetic, it may contain triglycerides, paraffin, free fatty acids, squalene, and cholesterol, optionally wherein the weight ratio of triglycerides:paraffin:free fatty acids:squalene:cholesterol is 8:5:5:3:1; and (b) The sebum content ratio relative to the weight of the modified SF film may be from 0.5% to 10% by weight, for example from 0.6% to 8% by weight.

[0106] In some embodiments that may be mentioned herein, the SF membrane is modified only by one or more lipid mimics selected from: fatty acids; and fatty amines. In this case, the resulting artificial skin may be deposited skin.

[0107] As described in the examples, D-skin is a good substitute for human palm skin in the deposition test.

[0108] In another aspect of the present invention, a method for manufacturing artificial skin is provided, the method comprising the following steps: (a) Provides a concentrated solution comprising cross-linked silk fibroin (SF) fibers, wherein the concentration of the cross-linked SF fibers is from 11% to 21% by weight of the total weight of the solution; and (b) A concentrated solution is poured onto a substrate, and the poured material is spread using a doctor blade coating technique to achieve a solution height of approximately 350 μm to 450 μm. The resulting material is then dried to provide an artificial skin, wherein the artificial skin has a second surface and a first surface in contact with the substrate, and has the following characteristics: The thickness ranges from 105 μm to 130 μm; and The surface free energy of the first surface ranges from 10 mN / m to 65 mN / m.

[0109] The crosslinking agent may be as described above. The resulting artificial human skin may have the properties described above.

[0110] As shown in the embodiments disclosed herein, the zeta potential of a concentrated solution containing cross-linked silk fibroin fibers decreases with increasing concentration, indicating reduced electrostatic repulsion between silk fibroin molecules and tighter intermolecular interactions, making the solution unstable and prone to gelation. Therefore, cross-linked SF fibers at concentrations of 11% to 21% by weight are used, corresponding to zeta potentials of -4.5 mV to -1.5 mV.

[0111] The concentrated solution in step (a) above can be provided through the following steps: (ai) Provides a solution comprising cross-linked silk fibroin (SF) fibers, said solution having been filtered to remove suspended solids, and the concentration of cross-linked SF fibers being less than 7% by weight of the total weight of said solution; and (aii) The solution is concentrated to provide the concentrated solution by keeping the solution at a high temperature for a period of time, optionally the high temperature being 55°C to 70°C, for example about 60°C, and the period of time being 1 hour to 5 hours, for example about 3 hours.

[0112] The solution described in step (ai) above can be provided through the following steps: (bi) A cross-linking agent is added to a silk fibroin fiber solution, wherein the concentration of the silk fibroin fiber is less than 7% by weight of the total weight of the solution, and the solution is allowed to react for a period of time to provide a cross-linked silk fibroin solution; and (bii) The solution is filtered to remove suspended solids (e.g., one or both of unreacted crosslinking agent and unreacted silk fibroin) to provide a solution containing silk fibroin (SF) fibers crosslinked by the crosslinking agent.

[0113] Any suitable weight ratio of SF fiber to crosslinking agent may be used in this document. For example, the weight ratio of SF fiber to crosslinking agent may be from 20:1 to 5:1, such as about 10:1.

[0114] This article also discloses a method for forming artificial skin, including the following steps: (ci) provides multiple sensory skins as described herein; and (cii) The plurality of sensory skins are attached together to provide a single stack that can mimic the absorption behavior of human skin (absorbent skin).

[0115] Absorbing skin may comprise a single stack of 2 to 10 layers, such as 3 to 5 layers of sensory skin, which is formed by the method described above.

[0116] Any suitable attachment method can be used to provide material suitable for deposition testing. For example, attachment can be performed using hot pressing.

[0117] Other aspects and embodiments of the invention are provided in the following numbered statements.

[0118] 1. An artificial human skin comprising: 1.1. Modified silk fibroin (SF) membrane; 1.2. Wherein the SF membrane is modified with fatty acids or fatty amines (ranging from C14 to C20) having -NH2 and -COOH functional groups; or SF6 membrane is modified with polyethylene glycol (PEG) derivatives; Preferably, the modified SF film has a structure similar to human skin.

[0119] 2. The artificial human skin according to Statement 1, wherein the SF membrane is modified with a fatty acid or fatty amine, wherein the fatty acid or fatty amine is hexadecylamine.

[0120] 3. The artificial human skin as described in Statements 1 and 2, wherein the SF membrane modified with fatty acids or fatty amines simulates the deposition behavior of human skin (deposited skin).

[0121] 4. The artificial human skin according to Statement 3, wherein the deposited skin has a brick-wall structure that mimics the stratum corneum structure in the human epidermis.

[0122] 5. The artificial human skin as described in Statement 3, wherein the thickness of the deposited skin is from 20 μm to 400 μm.

[0123] 6. The artificial human skin according to Statement 1, wherein the PEG-modified SF membrane mimics the sensory behavior of human skin (sensory skin).

[0124] 7. The artificial human skin according to Statement 6, wherein the thickness of the sensory skin is 60 μm to 200 μm.

[0125] 8. The artificial human skin according to Statement 6, wherein several layers of sensory skin are stacked to mimic the absorption behavior of human skin (absorbent skin).

[0126] 9. The artificial human skin as described in Statement 8, wherein the absorbent skin comprises 2 to 20 layers of sensory skin.

[0127] 10. The artificial human skin according to Statement 1, wherein an example of a PEG derivative is polyethylene glycol diglycidyl ether (PEGDE).

[0128] 11. The artificial human skin according to claims 1 to 3, wherein a surfactant and a suspended solid are added to an SF membrane modified with fatty acids or fatty amines, and the membrane is coated with artificial sebum.

[0129] 12. The artificial human skin according to Statement 1, wherein the SF membrane modified with a polyethylene glycol (PEG) derivative has different water contact angles to simulate different skin types.

[0130] 13. The artificial human skin according to Statement 6, wherein the water contact angle of oily skin is 60° to 85°, that of neutral skin is 80° to 90°, and that of dry skin is 90° to 135°.

[0131] Other aspects and embodiments of the invention will now be discussed with reference to the following non-limiting examples.

[0132] Example

[0133] method

[0134] Preparation of deposited artificial skin, namely D-skin (SH artificial skin): Take 300 ml of silk fibroin (5%) solution, add 20-40 ml of MES solution, 600-1000 mg of EDC, 0-300 mg of NHS, and 5-15 ml of cetylamine solution (dissolved in acetaldehyde, 75 mg / ml). Heat at 50-80℃ for 2 to 4 hours. The MES solution is prepared by dissolving 2.93 mg of MES hydrate in 60 ml of deionized water and 40 ml of acetaldehyde, and then adding 1.25 ml of 100 mg / ml NaOH solution.

[0135] Preparation of D-skin (SHSS artificial skin): Take 300 ml of silk fibroin (5%) solution, add 20-40 ml of MES solution, 600-1000 mg of EDC, 0-300 mg of NHS, 5-15 ml of cetylamine solution (dissolved in acetaldehyde, 75 mg / ml), and 5-15 ml of sodium dodecylbenzenesulfonate solution (100 mg / ml). Heat at 50-80℃ for 2 to 4 hours.

[0136] Preparation of D-skin (SHSSs artificial skin): SHSSs artificial skin is prepared by spin-coating artificial sebum onto SHSS artificial skin. The proportion of artificial sebum is 0.5% to 8%. The artificial sebum is dissolved in ethanol at a concentration of about 50 mg / ml, wherein the weight ratio of triglycerides:paraffin:free fatty acids (palmitic acid):squalene:cholesterol is 8:5:5:3:1.

[0137] Preparation of artificial skin (i.e., S-skin): Take 300 ml of silk fibroin (5%) solution and crosslink it with 1.5 g of PEGDE at 60°C for 3 hours. Then centrifuge and filter the solution. Subsequently, concentrate the solution to 12% to 25% by heating in a water bath. This concentrated solution is used to prepare films by slot coating or doctor blade coating in roll-to-roll or sheet-to-sheet systems.

[0138] Preparation of absorbable artificial skin (i.e., Skin A): Several pieces of sensory artificial skin (i.e., S-skin) are stacked together and fed into a hot press, where they are hot-pressed for 1 to 10 minutes at a temperature of 100°C to 200°C and a pressure of 1 MPa to 10 MPa.

[0139] Example 1: Selection of Silk Fibroin (SF)

[0140] To select the most suitable raw material for artificial skin, protein-based biomaterials were considered. Silk fibroin (SF), wool, feathers, and human hair were chosen because they are widely used and belong to keratin or silk fibroin materials. Their amino acid compositions were compared with those of the keratinized envelope (CE) to determine their relative similarity (Table 1). The weighted sum of squares (WSS) values ​​of these materials were calculated according to the following formula.

[0141]

[0142] Table 1: Comparison of amino acid composition among CE, SF, wool, feathers and hair [1-4] .

[0143]

[0144] [1] Wang, B., Yang, W., McKittrick, J., & Meyers, MA Progress in Material Science. 2016, 76, 229-318. [2] Alasdair C. Steven, Peter M.Steinert. J Cell Sci. 1994, 107(2), 693–700. [3] Vasconcelos A, Freddi G, Cavaco-Paulo A . 2009, 10 (4), 1019. [4] Wolfram LJ. J Am Acad Dermatol. 2003, (6 Suppl), 06-14.

[0145] Results and Discussion: from Figure 1 It is clear that SF is most similar to CE in terms of amino acid composition, and its WSS value is much smaller than that of feathers, wool, and hair. Therefore, SF was chosen as the raw material for preparing protein-based artificial skin.

[0146] Besides their similar amino acid compositions, SF and CE share several other structural and functional similarities. For example, both SF and CE are composed of proteins whose synthesis and assembly involve filaggrins, which play a crucial role in the aggregation and orderly arrangement of intermediate filaments into tight bundles. Furthermore, the structures of both SF and CE are stabilized and reinforced by disulfide bonds, which provide additional mechanical stability and resistance to chemical and enzymatic degradation. These disulfide bonds form crosslinks between protein chains, improving the overall durability and robustness of the materials (see Kalinin AE, Kajava AV, Steinert PM). Bioessays. 2002, 24 (9), 789-800. Koh, LD, Cheng, Y., Teng,CP, Khin, YW, Loh, XJ, Tee, SY, Low, M., Ye, E., Yu, H., Zhang, Y., &Han, M. Progress in Polymer Science. 2015, 46, 86-110).

[0147] According to the cosmetics industry, the following aspects are important when a product is applied to the skin: deposition, sensation, and absorption. Figure 2 The following questions need to be considered: 1) What will remain on the skin? 2) What will be absorbed by the skin? 3) How will the skin feel? Therefore, D-skin, S-skin, and A-skin are used to answer these questions respectively. D-skin is used for deposition testing to simulate the deposition of a product (such as perfume) on human skin after use. S-skin is a sensation-oriented artificial skin that reflects how consumers feel when applying a product to their skin. A-skin is used to evaluate the absorption of specific molecules of a product (such as niacinamide) when applied to the skin.

[0148] Example 2: Preparation of S-skin (CPSF film)

[0149] As mentioned above, S-skin aims to simulate the feel of different skin types after treatment with the product. Here, contact angle is used to define different skin types, and artificial skin for each skin type (dry, oily, and normal skin) is prepared by varying the manufacturing method. For cosmetic products, the feel of the product on the skin is quite important, as many consumers choose products based on this tactile feedback. Two factors are used to evaluate the product's effect on the artificial skin: contact angle, which describes the product's spreadability on human skin; and coefficient of friction, which describes the smoothness of the skin after treatment with the product. Here, PEGDE is used as a crosslinking agent to crosslink the silk fibroin solution (see reference Y. Cui). et al. Adv. Mater (2021, 33, 2100221). Unlike the conventional film-forming process using solvent casting, we employed an alternative method. Here, we concentrated the solution and then formed a film using a blade coating method, which accelerates the film-forming process (blade coating takes 8 hours, while solvent casting takes 3 to 6 days) and achieves better thickness uniformity. For different skin types, we used different molds to modify the contact angle of the artificial skin, as different skin types have different contact angles. The contact angle for oily skin is 60°–85°, for normal skin it is 80°–90°, and for dry skin it is 90°–110°. From Figure 3 and Figure 4 As can be seen, the S-skin is similar to human skin in terms of contact angle and coefficient of friction, demonstrating the sensory effect of the product after it is applied to the skin.

[0150] To obtain concentrated polyethylene glycol-modified silk fibroin (CPSF) films, the process involves three main steps, such as... Figure 5 As shown. First, an SF solution was prepared using a classical method, with a concentration of approximately 5% to 6% by weight. Next, the SF solution was reacted with polyethylene glycol diglycidyl ether (PEGDE) at a ratio of 10:1 at 60°C for 3 hours. This reaction produced a polyethylene glycolated SF (PSF) solution, which was then filtered to remove suspended solids. The next step was to concentrate the low-concentration PSF solution to 15% by weight by heating at 50°C. After centrifugation, the concentrated polyethylene glycolated SF (CPSF) solution was obtained, which was then cast onto aluminum foil and spread using a doctor blade coating technique to achieve a solution height of approximately 400 μm. After air drying at 25°C and 60% relative humidity for 8 hours, a CPSF film with a thickness of approximately 120 μm was obtained.

[0151] The fundamental principle behind concentrating polyethylene glycol-modified silk fibroin solutions is that low-concentration solutions cannot be used to produce films of the desired thickness. In this case, the target thickness is approximately 120 μm, a requirement in the cosmetics industry. Using an initial concentration of polyethylene glycol-modified silk fibroin solution, a higher solution height is needed to achieve this thickness. However, such a higher height is impractical for blade coating because the solution would overspread. By concentrating the solution, the required height is reduced, allowing the target thickness to be achieved without the need for a three-dimensional mold with walls.

[0152] Results and Discussion: (a) Characterization of CPSF solution Ideally, increasing the concentration of the CPSF solution would produce a thicker film. However, higher concentrations can make the solution unstable and prone to gelation. Different concentrations of CPSF were prepared by solvent casting at 50°C, yielding solutions with concentrations of 12.0 wt%, 12.7 wt%, 15.4 wt%, 16.5 wt%, 18.0 wt%, and 20.7 wt%, as well as a 5.70 wt% original sample polyethylene glycol-modified CPSF solution.

[0153] As shown in Figure 6c, the turbidity of the solution increases with increasing concentration, rising from 60 NTU to nearly 100 NTU. This indicates a higher concentration of suspended solids, implying more significant aggregation of silk fibroin molecules. High turbidity indicates solution instability, as larger aggregates accelerate the gelation process. Furthermore, the zeta potential of the solution decreases with increasing concentration. This decrease in zeta potential indicates reduced electrostatic repulsion between silk fibroin molecules, promoting tighter intermolecular interactions and facilitating the transition from a liquid to a gel state.

[0154] As shown in Figure 6d, the transition of the CPSF solution to the gel state is marked by the change from a transparent solution to an opaque, milky-white gel. The gelation time is influenced by both turbidity and zeta potential, further highlighting the stability of the solution. With increasing concentration, the gelation time shortens, indicating increased instability. The original sample, with a lower concentration of polyethylene glycol-modified CPSF, remained stable for approximately 12 days before gelation, while the 20.7% CPSF solution gelled in approximately 5 days. Based on these observations, a concentration of approximately 15% was chosen for subsequent applications, as it strikes a balance between sufficient film thickness requirements and adequate solution stability requirements.

[0155] (b) Characterization of CPSF thin films

[0156] like Figure 7As shown, the solvent-cast PSF film exhibits a fairly wide thickness distribution (117 ± 30 μm), indicating significant inhomogeneity. In contrast, the CPSF film prepared by blade coating has a much more uniform thickness distribution (115 ± 4.6 μm). This demonstrates that the CPSF film prepared by blade coating is significantly superior in terms of thickness uniformity compared to the classic solvent casting method.

[0157] (c) Mold-based surface free energy (SFE) modification technology for S-skin

[0158] A convenient method for achieving surface finish (SFE) of a modified CPSF film is to apply a CPSF solution via a doctor blade onto different mold materials. Here, the SFE of these mold materials differs. For the CPSF film, there are two sides: an air-facing side and a mold-facing side. Figure 8 Only the mold-facing side is affected by the mold material. Therefore, the results for CPSF films are relative to the mold-facing side. These materials include silicone, aluminum foil, polystyrene (PS), polyvinyl chloride (PVC), and glass, each exhibiting a different SFE.

[0159] A 15.0 wt% CPSF solution was doctor-coated onto various mold materials, including silicone, aluminum foil, PS, PVC, and glass. The coated solution was allowed to air dry at 25 °C and 60% relative humidity, with the doctor blade height maintained at 400 μm. CPSF films were obtained on different molds over approximately 8 hours. The corresponding SFE values ​​were 15.43 ± 1.08 mN / m, 28.55 ± 2.51 mN / m, 41.33 ± 1.73 mN / m, 49.93 ± 7.44 mN / m, and 61.53 ± 1.67 mN / m, respectively. By selecting different mold materials, the SFE of the CPSF films can be easily modified to obtain the desired properties.

[0160] Figure 9 The contact angle measurements of CPSF films prepared using different molds are shown, with water on the left and diiodomethane on the right. a) CPSF film on silicone. b) CPSF film on aluminum foil. c) CPSF film on polystyrene (PS). d) CPSF film on polyvinyl chloride (PVC). e) CPSF film on glass. In summary, the contact angle data of CPSF films prepared on different molds show significant differences, indicating diverse surface properties. Films obtained from some molds have similar contact angles, highlighting the influence of the mold material on the wettability and surface characterization of the resulting films.

[0161] Furthermore, the surface finish (SFE) of the CPSF film was calculated using a moving surface analyzer (KRUSS), which takes into account the contact angles of water and diiodomethane. Figure 10As shown in figure a, an increase in SFE indicates a more hydrophilic surface, meaning the surface has higher wettability and is more likely to interact with liquids. Conversely, a lower SFE indicates a more hydrophobic surface, repelling water and having lower wettability. This result validates the effectiveness of mold-based SFE modification, demonstrating its convenient method for producing CPSF films with different SFEs.

[0162] The skin surface area (SFE) of human skin also varies depending on skin type and location. For example, the SFE of forearm skin is typically between 30 mN / m and 40 mN / m (see Mavon, A., Zahouani, H., Redoules, D., Agache, PG, Gall, Y., & Humbert, PG). Colloids and Surfaces B: Biointerfaces , 1997, 8, 147-155.Krawczyk J. Skin Res Technol. 2015, 21(2), 214-23). ​​The presence of sebum and stratum corneum lipids increases the surface free energy of human skin. Therefore, CPSF films with an SFE of approximately 30 mN / m fabricated on aluminum foil can be used to simulate forearm skin in various applications. This demonstrates the practical application of SFE modification in creating films capable of accurately simulating different types of human skin.

[0163] To understand the mechanism behind mold-based SFE modification, the SFE of the mold material was measured and compared with that of the corresponding CPSF film, such as... Figure 10 As shown in b. The results show that the SFE of the CPSF film is very well matched with the SFE of the mold material, from silicone to PVC. For example, the mold-facing side of the CPSF film fabricated on silicone and the silicone itself both exhibit an SFE of approximately 18 mN / m. This phenomenon also occurs when the mold is changed to aluminum foil (SFE approximately 24 mN / m) and PS (SFE approximately 39 mN / m). For PVC, there is a difference between the mold and the CPSF film, with the CPSF film having an SFE of approximately 43 mN / m and PVC approximately 49 mN / m. This difference becomes more pronounced when the material is changed to glass, with the CPSF film having an SFE of approximately 44 mN / m, instead of increasing to approximately 60 mN / m as expected.

[0164] The observed correlation between the surface finish (SFE) of CPSF films and the mold material has two main reasons. First, as mentioned earlier, the SFE is largely dependent on the surface composition (see Chaudhury, Manoj & Whitesides, George). Science.(1992, 255, 1230-2). Therefore, using a mold with a lower SFE will result in a self-assembled structure with a more hydrophobic surface during film formation. Conversely, a mold with a higher SFE will result in a self-assembled structure with a more hydrophilic surface. By definition, a surface with a smaller SFE has more hydrophobic components; therefore, hydrophobic interactions are promoted to form a self-assembled film.

[0165] To clarify this, CPSF films cast on different molds were analyzed by XPS to determine the chemical environment of the surface carbon. Figure 11 As shown, the chemical composition of CPSF films cast on different molds varies considerably. For example, CPSF films made on silicone have a carbon content (CC) higher than 90%, indicating that aliphatic carbon is the dominant form on the surface. This high proportion of aliphatic carbon explains why CPSF films made on silicone have the lowest surface efficiency (SFE). A similar trend was observed in CPSF films made on aluminum foil, which contain approximately 60% aliphatic carbon, with the remaining percentage of carbon present as CO and C=O groups. The differences between CPSF films cast on PS, PVC, and glass are less pronounced, likely because their SFE differences are not significant. Figure 10 As shown in b, the CPSF films cast on these three molds exhibit approximately 50% CC, reflecting relatively small deviations in their surface composition.

[0166] Second, SFE is also affected by surface morphology (see references Z. Hong, X. Yu, H. Jiang, M. Xue, S. Peng, Y. Luo, Z. Yin, C. Xie, J. Appl. Polym. Sci. 2022, 139 (17), e52005). CPSF films tend to replicate the surface morphology of the mold on which the film is formed. Therefore, if the mold surface has a specific texture or pattern, the CPSF film will exhibit a similar morphology, further affecting its SFE.

[0167] Because SF contains both hydrophilic and hydrophobic segments in its molecular structure, SF-based materials can be used to generate thin films with different surface areas of force (SFE) (see Foo, C., Bini, E., Hensman, J.). et al. Appl. Phys. A.(2006, 82, 223–233). However, each material has an inherent SFE limit. This means that even if the formed film has a higher concentration of hydrophilic segments on its surface, there is a maximum SFE that the film can achieve. This concept helps explain the behavior of CPSF films formed on glass. Glass has a very high SFE, exceeding the inherent SFE limit of SF materials. Therefore, CPSF films formed on glass do not exhibit the expected high SFE. The SFE limit of CPSF films appears to be about 43 mN / m. Therefore, although CPSF can adjust its SFE to some extent by forming films with different surface compositions, it cannot match the extremely high SFE of glass.

[0168] These findings highlight the importance of both surface composition and morphology in determining the surface finish (SFE) of CPSF films. This mold-based SFE modification technique allows for the convenient production of CPSF films with customized SFEs, enabling their use in a variety of applications requiring specific surface properties.

[0169] Example 3: Preparation of Skin A (PraE)

[0170] Absorption is another important issue in the research of cosmetic and skincare products. For skincare products such as whitening, moisturizing, and anti-aging products, researchers and consumers are concerned about how much of the active molecules (including niacinamide, hyaluronic acid, and ascorbic acid) can be absorbed by the skin.

[0171] Figure 35 This is a schematic diagram illustrating the step-by-step preparation of Skin A, demonstrating precise control over protein structure at the molecular level, starting from the silkworm cocoon. First, sericin is removed by degumming to extract silk fibroin. Then, the silk fibroin is regenerated into a protein solution by disrupting internal hydrogen bonds. Subsequently, a protein film is formed through self-assembly, and then multiple thinner films are processed by hot pressing to form a single film (Skin A), thereby significantly altering its secondary structure.

[0172] As described above, to obtain the protein-reconstructed artificial skin (PraE), 3 to 5 CPSF films of the same size and approximately 120 μm in thickness were aligned and stacked. These stacks were then hot-pressed for approximately 3 minutes at different temperatures of 60°C, 80°C, 100°C, 120°C, 140°C, 160°C, and 180°C, and pressures of 3 MPa, 4 MPa, 5 MPa, and 10 MPa. Figure 12 After the hot pressing process, PraE is obtained and allowed to cool naturally to room temperature.

[0173] Results and Discussion: (a) PraE (A skin) prepared at different temperatures After hot-pressing CPSF films into PraE, it was observed that not all temperatures at 4 MPa and 3 minutes could effectively integrate multiple CPSF film layers into a uniformly thick PraE. Figure 13 As shown. Specifically, under conditions of 60°C and 80°C, 4 MPa, and 3 minutes, these CPSF films were independent, indicating insufficient integration. However, this effect changed when the temperature exceeded 100°C. As the temperature increased, the integration effect appeared to improve. With further increases in temperature, the color of PraE changed from almost transparent to light yellow, and then to dark yellow.

[0174] To verify the seamless fusion of the CPSF film into a single PraE, cross-sectional scanning electron microscope (SEM) images were obtained, such as... Figure 14 As shown in Figure a, the image reveals that the CPSF film hot-pressed at 100°C exhibits significant gaps, indicating that this temperature is insufficient to obtain a uniform, thick film. In contrast, no gaps were observed in the cross-section of films hot-pressed within the temperature range of 120°C to 180°C, exhibiting a uniform morphology with indistinguishable CPSF layers, indicating perfect integration. This can be attributed to the decrease in glass transition temperature caused by pressure. The glass transition temperature of silk fibroin has been reported to be approximately 175°C (see Magoshi, J. and Nakamura, S.). J. Appl. Polym. Sci. 1975, 19, 1013-1015. Motta, A., Fambri, L.and Migliaresi, C. Macromol. Chem. Phys. 2002, 203, 1658-1665. In some cases, this temperature may be lower due to the influence of water, as the bound water in the silk film acts as a plasticizer; this is also known as the water-induced glass transition temperature (see Hu, Xiao & Kaplan, David & Cebe, Peggy). Thermochimica Acta. (2007, 461, 137-144). Temperatures above 200°C were not considered in this study because silk fibroin begins to degrade at such higher temperatures.

[0175] (b) Characterization of PraE

[0176] Quantitative analysis showed that as temperature increased, the proportion of random coils decreased from 40% to below 10%, while the proportion of β-folds / β-turns increased from 40% to 70%. Figure 15 The α-helical content showed no significant change.

[0177] To determine the driving force behind the structural changes observed during hot pressing, a control experiment was conducted in which CPSF films were subjected to different heating temperatures and pressures. Figure 16As shown, both temperature and pressure contribute to changes in the secondary structure, but the effect of temperature appears to be more pronounced.

[0178] Next, the PraE prepared by hot pressing was characterized using XRD. Because β-sheets are crystalline structures, they produce a different signal in the XRD spectrum compared to random coils. Figure 17 As shown, the peak near 2θ = 20° corresponds to the (120) crystal plane, where the d-interval is 4.27 Å. This peak becomes more prominent with increasing temperature, indicating an increase in the crystalline region (see Warwicker Jo). J Mol Biol. (1960, 2, 350-62). Quantitative analysis showed that the crystallinity of PraE increased from 25% to 50% with increasing temperature, consistent with the results obtained by FTIR. Therefore, higher temperatures lead to higher crystallinity of the structure in PraE, which is mainly in the β-sheet form.

[0179] The trend towards a more crystalline structure with increasing temperature is further confirmed by the observed increase in density, such as... Figure 18 As shown, the density of PraE increases significantly with increasing hot-pressing temperature; the density of the original CPSF sample is approximately 1.3 g / cm³. 3 The density of PraE prepared at 180℃ increased to 1.8 g / cm³. 3 The significant increase in density indicates the formation of a denser protein structure. The densification process is attributed to enhanced molecular packing and reduced porosity within the material, which is facilitated by higher temperatures promoting the transition from amorphous regions to a more ordered crystalline β-sheet structure.

[0180] To determine whether PraE underwent significant degradation or oxidation during hot pressing, XPS and [other methods were used]. 1 ¹H NMR was used to examine the chemical environment of carbon and hydrogen. In the C1s spectrum, three distinct peaks representing the CCC, CO, and C=O carbon states were identified. Figure 19 As shown, no significant differences were observed between the original CPSF film and the PraE samples prepared at different temperatures, indicating that no significant degradation or oxidation occurred during the hot pressing process.

[0181] This conclusion is obtained 1 Further support from H NMR spectroscopy, in which Figure 20 None of the peaks showed shift, indicating that the chemical environment of hydrogen atoms remained unchanged during hot pressing. This consistency between the spectra suggests that while hot pressing significantly alters the secondary structure of the material, its chemical composition or environment remains largely unchanged. Therefore, it can be concluded that hot pressing induces structural modification of PraE without causing significant chemical changes.

[0182] Even for thicker films, hot pressing exhibits consistent thickness uniformity. For example, films with a thickness of approximately 295 ± 8.0 μm prepared by hot pressing show significantly better uniformity than films prepared by solvent casting and doctor blade coating. Figure 21 As the thickness increases, the change in thickness uniformity remains minimal, indicating that hot pressing is effective for producing thicker films with uniform thickness. Therefore, hot pressing becomes a very promising method for preparing PraE films with uniform thickness, making it suitable for applications requiring precise and consistent film properties.

[0183] Example 4: Skin A (PraE) and Skin A containing artificial lipids (PLraE) for transdermal permeability testing

[0184] The outermost layer of human skin contains both proteins and lipids, which are crucial for the skin's barrier function. To improve the similarity to human skin, it is essential to add lipids that mimic those found in human skin. The lipid composition in the stratum corneum is a complex mixture of free fatty acids, neutral lipids, polar lipids, and cholesterol sulfates. To replicate this composition, triglycerides (trioleic acid esters, purchased from Sigma-Aldrich), free fatty acids (palmitic acid, purchased from Sigma-Aldrich), free sterols (cholesterol, purchased from Sigma-Aldrich), and squalene (purchased from Sigma-Aldrich) were combined in a 5:4:2:1 ratio (see reference MA Lampe, AL Burlingame, J Whitney, ML Williams, B EBrown, E Roitman, PM Elias). Journal of Lipid Research . 1983, 24 (2), 120-30). The mixture was melted at 72°C and then coated onto a protein-reconstructed artificial skin (PraE), wherein the lipid mixture comprised 5% by weight of the total mass ( Figure 22 ).

[0185] Results and Discussion: (a) Permeation rate of PraE prepared at different temperatures Transdermal penetration involves the release of the permeate from the dosage form, its diffusion through the stratum corneum, and its distribution into the epidermal environment. These processes are influenced by the physicochemical properties of the permeate and the specific characteristics of the skin at the application site. The rate of penetration of different compounds can be described using steady-state flux (J) or Fick's law of diffusion, which quantifies the cumulative mass (m) of drug per unit area through the skin in a given time, as shown in the following equation, where D represents the diffusion coefficient, ΔC is the concentration gradient, and h is the thickness of the skin or artificial skin (see Barry BW. Eur...). J Pharm Sci . 2001,14 (2), 101-14).

[0186]

[0187] According to the equation above, the steady-state flux (J) (which quantifies the permeation rate) is inversely proportional to the thickness of the skin or artificial skin. To verify the applicability of this equation in permeation rate testing using our method in a Franz diffusion cell, PraE samples prepared under the same conditions but with different thicknesses were tested using a caffeine solution. Figure 23 As shown in b, the secondary structures of PraE samples of different thicknesses are very similar, indicating that the diffusion coefficients remain consistent. With careful control of the caffeine concentration gradient in each experiment, the main variable was thickness. Figure 23 c indicates that the permeation rate (J) and thickness (h) are inversely proportional, confirming that the equation is applicable to quantifying the permeation rate.

[0188] The permeation rate of PraE samples prepared at different temperatures (120℃ (PraE-120), 140℃ (PraE-140), 160℃ (PraE-160), and 180℃ (PraE-180)) was tested using caffeine solution. Figure 24 As shown in Figure a, the amount of caffeine permeating through PraE decreases with increasing preparation temperature, indicating a slower permeation rate. Furthermore, the curve tends to be linear over time, indicating the attainment of steady-state flux, with the permeation rate represented by the slope of the curve. This data, along with thickness measurements of different PraE samples (indicating that PraE prepared at higher temperatures is denser and therefore thinner), were used to calculate the diffusion coefficient. Therefore, the diffusion coefficients of PraE-120, PraE-140, PraE-160, and PraE-180 were calculated using the above equation. The results clearly show that the higher the temperature at which PraE is hot-pressed, the smaller the diffusion coefficient, indicating a slower permeation rate.

[0189] To explore the relationship between permeation rate and different previously measured secondary structures, the diffusion coefficient for each type of secondary structure was calculated using the following equation. In the context, D x Denotes the diffusion coefficient, and x It represents the ratio of a certain part x of the secondary structure, where x can be β-fold / β-turn, α-helix, and random coil.

[0190]

[0191] By combining diffusion coefficient data with secondary structure composition, linear programming was used to calculate the diffusion coefficient of caffeine with different secondary structures, such as... Figure 25As shown in b, the diffusion coefficient of the random coil is significantly larger, approximately ten times that of the β-sheet / β-turn and α-helical structures. In this context, the effects of the β-sheet / β-turn and α-helical structures on the permeation rate are negligible because the random coil acts as the primary channel for compound permeation through PraE. Therefore, the amount of caffeine permeating through the β-sheet / β-turn and α-helical structures is minimal and can be disregarded. This finding highlights the crucial role of the random coil in facilitating transdermal permeation through PraE.

[0192] Furthermore, this discovery elucidates the mechanism by which small molecules permeate through dense protein-based materials such as PraE, highlighting that random coils act as the primary channel for this process, such as... Figure 26 As shown in the figure. This mechanism suggests that fewer ordered flexible regions in protein structures facilitate molecular diffusion, providing more accessible pathways compared to rigid crystalline β-sheets / β-turns and α-helical structures.

[0193] Furthermore, this insight extends to understanding how compounds penetrate the stratum corneum (the outermost layer of human skin). The presence of random coils within the stratum corneum can similarly facilitate the permeability of small molecules, assisting them through a lipid-rich environment dense with keratinocytes. This understanding can inform the design of more efficient transdermal drug delivery systems and improve predictions of compound absorption based on interactions between compounds and specific protein structures.

[0194] To determine the optimal temperature for PraE preparation, the caffeine penetration rates of PraE-120, PraE-140, PraE-160, and PraE-180 were compared with those obtained through human skin. Figure 27 As shown in the figure. The results indicate that the penetration rates of PraE-160 and PraE-180 are most similar to those of human skin, with PraE-180 showing the closest match at approximately 2.3 µg / cm³. 2 / h (see van de Sandt JJ, van Burgsteden JA, Cage S, Carmichael PL, Dick I, Kenyon S, Korinth G, Larese F, Limasset JC, Maas WJ, Montomoli L, Nielsen JB, Payan JP, Robinson E, Sartorelli P, Schaller KH, Wilkinson SC, Williams FM. Regul Toxicol Pharmacol2004, 39(3), 271-81). In contrast, other PraE samples showed faster penetration rates. Therefore, PraE-180 was chosen for transdermal penetration testing because its penetration properties are highly consistent with those of human skin.

[0195] (b) Permeation rate test of different compounds

[0196] Although PraE-180 exhibits a caffeine penetration rate very similar to that of human skin, testing a single compound is insufficient to conclude its applicability in transdermal penetration assays. To verify its suitability, testing with other compounds is necessary. Three additional compounds were chosen: salicylic acid, nicotinamide, and rhodamine B, as other studies have reported their penetration rates in human skin. Therefore, the penetration rates of these three molecules, along with caffeine, were tested on PraE-180, PLraE-180, Episkin, and Strat-M. The results were then compared with those obtained from the literature on human skin.

[0197] like Figure 28 As shown, the rate of caffeine penetration through human skin is approximately 2.3 µg / cm³. 2 / h. The results for PLraE and PraE were very similar to those for human skin, with PLraE showing slightly better matching and PraE showing a slightly faster penetration rate of approximately 2.4 µg / cm. 2 / h. In comparison, Strat-M (a commercially available artificial membrane used for transdermal penetration testing) also showed some similarity to human skin, with a penetration rate of approximately 3.2 µg / cm. 2 / h. Episkin (a skin-reconstructing agent for transdermal penetration) exhibited a significantly faster penetration rate of approximately 24 µg / cm³. 2 The permeation rate is approximately ten times that of human skin. This increased permeation rate is consistent with previous observations regarding reconstructed skin, likely due to its thinner structure (typically less than 100 μm), which could be a cost-effective measure. Therefore, in caffeine permeation rate testing, both PLraE and PraE exhibit permeation rates that closely match those of human skin, outperforming Strat-M in accuracy and significantly surpassing Episkin.

[0198] For salicylic acid and nicotinamide, PraE(TEST-180) and PLraE(TEST-L-180) also showed better similarity (see N. Leveque et al.). International Journal of Pharmaceutics. 269(2004) 323–328; GB Kasting et al. Journal of Pharmaceutical Sciences, 111(2022) 727-733).

[0199] Finally, Rhodamine B is a synthetic dye widely used in various scientific applications, particularly in histology and fluorescence microscopy. Known for its bright pink color and strong fluorescence, Rhodamine B is ideal for staining and visualizing biological tissues, cells, and molecular structures. Furthermore, Rhodamine B is used in water tracing studies to track water flow and distribution in environmental and hydrological research. Its vibrant fluorescence and stability make it a valuable tool in biological and environmental sciences. However, this molecule is known not to be absorbed by human skin within 8 hours, so it was used for testing. Both PLraE and PraE are skin-matched, showing no absorption of the molecule. However, Strat-M does show some permeation, at a rate of approximately 0.8 µg / cm³. 2 / h, as indicated by a slight reddening of the receptor solution ( Figure 29 c). Episkin exhibits a faster osmosis rate, resulting in a deep red color in the receptor solution at a rate of approximately 2.4 µg / cm³. 2 / h ( Figure 29 d).

[0200] (c) Correlation analysis between permeation rate and physicochemical properties

[0201] In addition to the four molecules mentioned above, other compounds, including nicotine, methylparaben, ninhydrin, tyrosine, Congo red, methylene blue, and bromothymol blue, were tested using the same method. The permeation rate was analyzed in relation to various physicochemical properties, including molecular weight (MW), logarithm of octanol-water partition coefficient (log P), and logarithm of water solubility (log S). aq The relationship between ) ).

[0202] The permeation rate of PLraE was analyzed in relation to the molecular weight (MW) and log S of compounds in aqueous solution. aq The correlation between them, such as Figure 30 As shown. log S aq Similar compounds exhibit higher permeation rates as MW decreases, while similar compounds at MW show higher permeation rates at logS. aq Increased water solubility results in a higher penetration rate. On one hand, smaller molecules penetrate the dense, semi-permeable barrier of the skin more easily than larger molecules, encountering less resistance along their path. On the other hand, higher water solubility ensures that the compound remains dissolved in the skin's aqueous environment, promoting its movement through the skin layers. Hydrophilic compounds with high water solubility can diffuse more easily through the aqueous channels in the skin.

[0203] Traditionally, log P has been considered a crucial factor influencing the permeation rate of compounds, with an optimal range between 1 and 3. This is likely because compounds with higher log P values ​​typically exhibit poorer solubility in aqueous solutions, leading to the use of organic solvents such as ethanol, propylene glycol, and acetone as carriers (see Roberts MS, Cheruvu HS, Mangion SE, Alinaghi A, Benson HAE, Mohammed Y, Holmes A, van der Hoek J, Pastore M, Grice JE). Adv Drug Deliv Rev. 2021, 177, 113929. These solvents act as chemical promoters, increasing the permeation rate of such molecules (see Lane ME). Int J Pharm. 2013, 447, 12-21). A recent update to the database of human epidermal permeability coefficients for drugs, exogenous substances, and other solutes administered in aqueous solutions examined the permeation rate in relation to various physicochemical properties (including MW, log P, log S). aq Melting point (MP) and hydrogen bond donor (H) d ) and receptor (H a The relationship between the quantity of ) and (see the literature Cheruvu HS, Liu X, Grice JE, Roberts MS). Data Brief. 2022, 42, 108242). These extensive data indicate that when applied in aqueous solution, log S aq The effect on the permeation rate is more significant than log P, and the log S is larger. aq It will exhibit a faster permeation rate (see Pereira R, SilvaSG, Pinheiro M, Reis S, Vale MLD. 2021 May, 11 (5), 343).

[0204] These observations further support the conclusion that PraE and PLraE mimic human skin behavior in terms of permeability. To achieve better permeation rates, compounds must possess low molecular weight (MW) and high water solubility, thus making these models relevant and effective in testing permeability via the transdermal route.

[0205] Example 5: Mass production of PraE and CPSF films

[0206] To assess the industrialization potential of this technology, scaling up the production of PraE and CPSF films is crucial. In the current approach, the key step in preparing both PraE and CPSF films is the blade coating process. For industrial-scale production, this step needs to be adapted to a roll-to-roll (R2R) method (see Krebs, FC, Tromholt, T., & Jørgensen, M). Nanoscale. 2010, 2 (6), 873-86). Implementing the R2R method can significantly accelerate the process by effectively drying the CPSF solution. Furthermore, the R2R method allows for continuous and automated production, which is crucial for achieving industrial scalability. The transition from laboratory-scale to industrial-scale production is essential to meeting the demands of large-scale manufacturing and ensuring the feasibility of this technology for commercial applications.

[0207] For R2R systems, there are several coating methods, namely dip coating, edge coating, slot coating, gravure coating, and spray coating (see Park, Janghoon & Shin, Keehyun & Changwoo, Lee). International Journal of Precision Engineering and Manufacturing. 2016, 17, 537-550). Here, slot coating (commonly used for multilayer and stripe coating) is chosen for film production due to its suitability for producing thicker films and its convenience. A slot coating apparatus includes a slot die with a narrow opening from which the coating liquid flows out, and the material is uniformly distributed across the width of the substrate. Figure 31 (b) Material is pumped from a storage tank in the feeding system to the slit die, where a precision pump controls the material flow rate at a predetermined rate to achieve a uniform film thickness. A substrate handling system moves the substrate under the slit die at a constant speed, maintaining uniform motion. After coating, the film is dried using drying equipment such as a heater or UV lamp. The entire process is guided by a monitoring system that acquires and adjusts settings while controlling real-time parameters to obtain a precise and uniformly coated film.

[0208] Results and Discussion: The following are some preliminary results of thin films produced using the R2R system, such as Figure 31 As shown in c. To control the thickness of the CPSF film, the following equation was applied, which can be used to predict the actual coating thickness of high-viscosity solutions (see references Park, J., Shin, K., and Lee, C.). Int. J. Precis. Eng. Manuf. 2015, 16 (5), 937-943). In this equation, t 干燥 f represents the thickness of the film after drying. r ρ is the liquid flow rate. t Let ρ represent the solution density, W be the width of the coating area, V be the speed of the web movement, and ρ be the density of the solution.s This represents the density of the dried film.

[0209]

[0210] Clearly, the film thickness can be adjusted by controlling the web movement speed or the liquid flow rate. Here, with a fixed web movement speed of 0.05 m / min, the thickness can be precisely controlled by changing the liquid flow rate, such as... Figure 31 As shown in d. Currently, laboratory-scale R2R systems are more suitable for producing thinner films because the length of the drying heater is insufficient to accommodate thicker films, resulting in insufficient drying time.

[0211] Example 6: Preparation of D-skin

[0212] Here, the synthesis of skin D is inspired by the structure of the stratum corneum, in which proteins and lipids are linked by ester bonds. In the stratum corneum, ester bonds originate from the carboxyl groups in proteins and the hydroxyl groups in lipids (ceramides). Figure 32 The lipid component can be selected from fatty acids and aliphatic amines, with carbon chain lengths ranging from C14 to C20, containing -NH2 and -COOH functional groups. After testing various fatty acids and aliphatic amines, we selected hexadecylamine as the ideal lipid component for interaction with silk fibroin. This selection was based on the following considerations: the reaction between -COOH and -NH2 groups is easier than the reaction between -COOH and -OH groups, and its aliphatic tail chain length is also more suitable. Figure 32 Based on the amino acid composition of silk fibroin, some amino acids with carboxyl groups exist, such as aspartic acid and glutamic acid. Through this reaction, the first type of D skin was obtained and named SH artificial skin.

[0213] Results and Discussion: To assess the similarity between human skin and the SH artificial skin, a deposition test was performed using Safeguard (a type of disinfectant soap). Safeguard was applied to both human palm skin and the artificial skin, then washed off, and the remaining fragrance molecules were tested and analyzed. Correlation coefficient curves for the ratio of each fragrance molecule were plotted. Figure 33 It is evident that SH artificial skin is highly suitable for fragrance deposition testing, as evidenced by the R-value of the correlation coefficient curve. 2 It is approximately 0.95. In fact, for palm skin from different individuals, R... 2 The range is from 0.9 to 0.96. Therefore, SH artificial skin can replace palm skin for this test.

[0214] However, this SH artificial skin does not resemble human forearm skin. To obtain human forearm skin, we first added surfactants and suspended solids to an SH solution to obtain SHSS artificial skin. Then, we coated the SHSS artificial skin with artificial sebum to obtain SHSSs artificial skin. Figure 34 It can be seen that this method improves the similarity to forearm skin (R0). 2 (Approximately 0.8).

Claims

1. An artificial human skin comprising a modified silk fibroin (SF) membrane, the artificial human skin having a first surface and a second surface, wherein the SF membrane is modified by one or both of the following: A crosslinking agent that forms crosslinks between SF fibers, wherein when the crosslinking agent is used alone, the artificial human skin has the following characteristics: The thickness ranges from 105 μm to 130 μm; and The surface free energy of the first surface is 10 mN / m to 65 mN / m; as well as Lipid mimics, selected from one or more of the following: fatty alcohols; more particularly, fatty acids and fatty amines, wherein: The fatty alcohol forms an ester bond with the carboxylic acid functional group in the SF; The fatty acid forms an ester bond with the hydroxyl functional group in the SF; and The fatty amine forms an amide bond with the carboxylic acid functional group in the SF.

2. The artificial human skin according to claim 1, wherein the modified SF film has a first surface shaped to mimic the structure of human skin.

3. The artificial human skin according to claim 1 or 2, wherein the crosslinking agent is a crosslinking agent derived from polyethylene glycol (PEG).

4. The artificial human skin according to claim 3, wherein the crosslinking agent is selected from one or more of the group consisting of: O'O-bis[2-(N-succiniminosuccinamido)ethyl]polyethylene glycol (NHSP), polyethylene glycol dithiol, 4-arm PEG-thiol, 4-arm PEG-epoxide, 4-arm PEG-isocyanate, isocyanate PEG isocyanate and polyethylene glycol diglycidyl ether (e.g. polyethylene glycol dithiol, 4-arm PEG-thiol, 4-arm PEG-epoxide, 4-arm PEG-isocyanate, isocyanate PEG isocyanate and polyethylene glycol diglycidyl ether).

5. The artificial human skin according to claim 4, wherein the crosslinking agent is polyethylene glycol diglycidyl ether.

6. The artificial human skin according to claim 4 or 5, wherein the number average molecular weight of the crosslinking agent is from 150 Daltons to 4,000 Daltons, for example from 150 Daltons to 1,000 Daltons, for example from 200 Daltons to 700 Daltons, for example from 250 Daltons to 500 Daltons.

7. The artificial human skin according to any one of claims 3 to 6, wherein when the SF membrane is modified by crosslinking, it mimics the sensory behavior of human skin (sensory skin).

8. The artificial human skin according to claim 7, wherein the thickness of the sensory skin is about 120 μm.

9. The artificial human skin according to any one of claims 3 to 8, wherein the surface free energy of the first surface of the artificial human skin is: 10mN / m to 20mN / m; 25mN / m to 35mN / m; 30mN / m to 40mN / m; 39mN / m to 45mN / m; 42mN / m to 58mN / m; or 59mN / m to 65mN / m.

10. The artificial human skin according to any one of claims 9, wherein the surface free energy of the first surface of the artificial human skin is about 30 mN / m.

11. The artificial human skin according to any one of claims 7 to 10, wherein 2 to 20 layers of sensory skin are formed as a single stack to mimic the absorption behavior of human skin (absorbent skin).

12. The artificial human skin of claim 11, wherein the absorbent skin comprises 2 to 10 layers, for example 3 to 5 layers, of the sensory skin.

13. The artificial human skin according to claim 11 or 12, wherein the absorbent skin has one or more of the following characteristics: (aa) In the absorbent skin, more than 50% of the silk fibroin is in a β-sheet / β-turn conformation, less than 30% of the silk fibroin is in a random coil, and if the remaining silk fibroin is present, it is in an α-helix form (e.g., in the absorbent skin, 51% to 75% of the silk fibroin is in a β-sheet / β-turn conformation, 3% to 25% of the silk fibroin is in a random coil, and the remaining silk fibroin is in an α-helix form). (ab) Crystallinity value greater than 25%, for example, 27% to 50%; (ac) has a caffeine permeation rate of 1 μg / cm 2 / h to 3μg / cm 2 / h, for example, about 2.5 μg / cm 2 / h; (ad) The permeation rate of nicotinamide was 7 μg / cm. 2 / h to 15μg / cm 2 / h, for example, about 10 μg / cm 2 / h; and (ae) has a permeation rate of 2 μg / cm for salicylic acid. 2 / h to 7μg / cm 2 / h, for example, about 5 μg / cm 2 / h.

14. The artificial human skin according to any one of the preceding claims, wherein the lipid mimic has 14 to 20 carbon atoms arranged in a branched chain, or more particularly in a straight chain.

15. The artificial human skin according to any one of the preceding claims, wherein when the SF membrane is modified with the lipid mimicry, the lipid mimicry is hexadecylamine.

16. The artificial human skin according to any one of the preceding claims, wherein when the SF membrane is modified with the lipid mimicry, the artificial human skin mimics the deposition behavior of human skin (deposited skin).

17. The artificial human skin according to claim 16, wherein the deposited skin has a brick-wall structure that mimics the stratum corneum structure in human epidermis.

18. The artificial human skin according to claim 16 or 17, wherein the thickness of the deposited skin is from 20 μm to 400 μm.

19. The artificial human skin according to any one of the preceding claims, wherein when the SF membrane is modified with the lipid mimicry, the SF membrane further comprises one or both of the following: a surfactant and a suspended solid material comprising the unreacted lipid mimicry.

20. The artificial human skin according to any one of the preceding claims, wherein the artificial human skin is coated with sebum, optionally wherein the sebum is artificial sebum, and further optionally wherein one or more of the following are applicable: (a) The artificial sebum comprises triglycerides, paraffin, free fatty acids, squalene, and cholesterol, optionally wherein the weight ratio of triglycerides:paraffin:free fatty acids:squalene:cholesterol is 8:5:5:3:1; and (b) The sebum content ratio is 0.5% to 10% by weight, for example, 0.6% to 8% by weight, relative to the weight of the modified SF film.

21. The artificial human skin according to any one of the preceding claims, wherein the SF membrane is modified only by one or more lipid mimics selected from: fatty acids; and fatty amines.

22. The artificial skin according to any one of the preceding claims, wherein the artificial skin is configured to have a water contact angle of 60° to 135° before the test material is applied to the artificial skin to simulate one of oily skin, normal skin or dry skin.

23. The artificial skin according to claim 22, wherein the artificial skin comprises: (i) A water contact angle of 60° to 85° to simulate oily skin; (ii) A water contact angle of 80° to 90° to simulate neutral skin; or (iii) A water contact angle of 90° to 135° to simulate dry skin.

24. The artificial skin according to claim 22 or 23, wherein, when subordinate to claim 7, the artificial skin is sensory skin.

25. A method for manufacturing artificial skin, the method comprising the following steps: (a) Providing a concentrated solution comprising cross-linked silk fibroin (SF) fibers, wherein the concentration of the cross-linked SF fibers is from 11% to 21% by weight of the total weight of the solution; and (b) The concentrated solution is poured onto a substrate, and the poured material is spread out using a doctor blade coating technique to achieve a solution height of approximately 350 μm to 450 μm. The resulting material is then dried to provide the artificial skin, wherein the artificial skin has a second surface and a first surface in contact with the substrate, and has the following characteristics: The thickness ranges from 105 μm to 130 μm; and The surface free energy of the first surface is between 10 mN / m and 65 mN / m.

26. The method of claim 25, wherein the crosslinking agent is a crosslinking agent derived from polyethylene glycol (PEG).

27. The method of claim 25, wherein the crosslinking agent is selected from one or more of the group consisting of: O'O-bis[2-(N-succiniminosuccinamido)ethyl]polyethylene glycol (NHSP), polyethylene glycol dithiol, 4-arm PEG-thiol, 4-arm PEG-epoxide, 4-arm PEG-isocyanate, isocyanate PEG isocyanate, and polyethylene glycol diglycidyl ether (e.g., polyethylene glycol dithiol, 4-arm PEG-thiol, 4-arm PEG-epoxide, 4-arm PEG-isocyanate, isocyanate PEG isocyanate, and polyethylene glycol diglycidyl ether).

28. The method according to claim 27, wherein the crosslinking agent is polyethylene glycol diglycidyl ether.

29. The method of claim 27 or 28, wherein the number average molecular weight of the crosslinking agent is from 150 Daltons to 4,000 Daltons, for example from 150 Daltons to 1,000 Daltons, for example from 200 Daltons to 700 Daltons, for example from 250 Daltons to 500 Daltons.

30. The method according to any one of claims 25 to 29, wherein the thickness of the artificial skin is about 120 μm.

31. The method according to any one of claims 25 to 29, wherein the surface free energy of the first surface of the artificial human skin is: 10mN / m to 20mN / m; 25mN / m to 35mN / m; 30mN / m to 40mN / m; 39mN / m to 45mN / m; 42mN / m to 58mN / m; or The surface free energy of the first surface of the artificial human skin is 59 mN / m to 65 mN / m, optionally with a surface free energy of about 30 mN / m.

32. The method according to any one of claims 25 to 31, wherein the concentrated solution in step (a) of claim 25 is provided by the following steps: (ai) Provides a solution comprising cross-linked silk fibroin (SF) fibers, said solution having been filtered to remove suspended solids, and the concentration of cross-linked SF fibers being less than 7% by weight of the total weight of said solution; and (aii) The solution is concentrated to provide the concentrated solution by keeping the solution at a high temperature for a period of time, optionally wherein the high temperature is 55°C to 70°C, for example about 60°C, and the period of time is 1 hour to 5 hours, for example about 3 hours.

33. The method of claim 32, wherein the solution in step (ai) of claim 32 is provided by the following steps: (bi) A cross-linking agent is added to a silk fibroin fiber solution, wherein the concentration of the silk fibroin fiber is less than 7% by weight of the total weight of the solution, and the solution is allowed to react for a period of time to provide a cross-linked silk fibroin solution; and (bii) The solution is filtered to remove suspended solids (e.g., one or both of unreacted crosslinking agent and unreacted silk fibroin) to provide a solution containing silk fibroin (SF) fibers crosslinked by the crosslinking agent.

34. The method of claim 33, wherein the weight ratio is from 20:1 to 5:1, for example about 10:

1.

35. A method for forming artificial skin, comprising the following steps: (ci) Provides a plurality of sensory skins as described in claim 7 and as described in claims 8 to 24 which are dependent on claim 7; as well as (cii) The plurality of sensory skins are attached together to provide a single stack to mimic the absorption behavior of human skin (absorbent skin).

36. The method of claim 35, wherein the absorbent skin comprises 2 to 10 layers, for example 3 to 5 layers of the sensory skin.

37. The method of claim 35 or 36, wherein the attachment is performed using hot pressing.

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