Artificial human skin and method of manufacturing thereof

Abstract

The invention provides an artificial human skin, comprising a modified silk fibroin (SF) membrane having a first surface and a second surface, wherein the SF membrane is modified with one or both of: a crosslinking agent, which forms a crosslink between SF fibres, wherein when the crosslinking agent is used alone, the artificial human skin is provided with: a thickness of from 105 to 130 μm; and the first surface has a surface free energy of from 10 to 65 mN / m; and a lipid mimic selected from one or more of: a fatty alcohol; and, more particularly, a fatty acid and a fatty amine, where: the fatty alcohol forms ester bonds with carboxylic acid functional groups in the SF; the fatty acid forms ester binds with hydroxyl functional groups in the SF; and the fatty amine forms amide bonds with carboxylic acid functional groups in the SF. The invention also provides a method of manufacturing thereof.

Description

[0001] ARTIFICIAL HUMAN SKIN AND METHOD OF MANUFACTURING THEREOF

[0002] FIELD OF INVENTION

[0003] The present invention provides an artificial human skin, more particularly, an artificial human skin comprising a modified silk fibroin (SF) membrane. That is, the present invention may relate to a protein reconstruction of human skin.

[0004] BACKGROUND

[0005] The listing or discussion of a prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

[0006] An increasing number of countries have enacted policies to ban animal experiments, and many others have decided to join them in the future. Therefore, the non-animal alternative testing market is becoming increasingly attractive. In fact, the market scale of non-animal experiments is estimated to be around $29.4 billion by 2030.

[0007] Current alternatives to animal testing, such as human skin cell-based models and synthetic polymer-based skins, face major limitations. Human skin cell models are highly accurate but prohibitively expensive and difficult to produce at scale, while polymer-based models are more affordable but fail to replicate the biological complexity of real skin. This leaves a significant gap in the market for a solution that combines the affordability of polymer models with the accuracy of human skin cells.

[0008] Cosmetic companies have an average R&D intensity of around 2.2%, which refers to the percentage of revenue spent on research and development. This is significantly lower compared to the health industries, including pharmaceuticals, which average around 20.9%. Consequently, cosmetic companies are more sensitive to price than pharmaceutical companies. As such, the most suitable artificial skin for the market is one that is cheap and has a moderate level of similarity with human skin.

[0009] Thus, there is a need for alternative and / or improved artificial human skin suitable for various applications and methods of manufacturing thereof.

[0010] SUMMARY Aspects and embodiments of the current invention will now be described by reference to the following numbered clauses.

[0011] 1. An artificial human skin, comprising a modified silk fibroin (SF) membrane having a first surface and a second surface, wherein the SF membrane is modified with one or both of: a crosslinking agent, which forms a crosslink between SF fibres, wherein when the crosslinking agent is used alone, the artificial human skin is provided with: a thickness of from 105 to 130 pm; and the first surface has a surface free energy of from 10 to 65 mN / m; and a lipid mimic selected from one or more of: a fatty alcohol; and, more particularly, a fatty acid and a fatty amine, where: the fatty alcohol forms ester bonds with carboxylic acid functional groups in the SF; the fatty acid forms ester binds with hydroxyl functional groups in the SF; and the fatty amine forms amide bonds with carboxylic acid functional groups in the SF.

[0012] 2. The artificial human skin according to Clause 1 , wherein the modified SF membrane has a first surface that has been shaped to mimic human skin structures.

[0013] 3. The artificial human skin according to Clause 1 or Clause 2, wherein the crosslinking agent is a crosslinking agent derived from a polyethylene glycol) (PEG).

[0014] 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-succinimidyl succinylamino) ethyl]polyethylene glycol (NHSP), poly(ethylene glycol) dithiol, a 4-arm PEG-thiol, a 4-arm PEG-epoxide, a 4-arm PEG isocyanate, isocyanate PEG isocyanate, and polyethylene glycol) diglycidyl ether (e.g. polyethylene glycol) dithiol, a 4-arm PEG-thiol, a 4-arm PEG- epoxide, a 4-arm PEG isocyanate, isocyanate PEG isocyanate, and poly(ethylene glycol) diglycidyl ether).

[0015] 5. The artificial human skin according to Clause 4, wherein the crosslinking agent is polyethylene glycol) diglycidyl ether. 6. The artificial human skin according to Clause 4 or Clause 5, wherein the crosslinking agent has a number average molecular weight of from 150 to 4,000 Daltons, such as from 150 to 1 ,000 Daltons, such as from 200 to 700 Daltons, such as from 250 to 500 Daltons.

[0016] 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 human skin sensation behaviour (Sensation skin).

[0017] 8. The artificial human skin according to Clause 7, wherein the thickness of the Sensation skin is about 120 gm.

[0018] 9. The artificial human skin according to any one of Clauses 3 to 8, wherein the first surface of the artificial human skin has a surface free energy of: from 10 to 20 mN / m; from 25 to 35 mN / m; from 30 to 40 mN / m; from 39 to 45 mN / m; from 42 to 58 mN / m; or from 59 to 65 mN / m.

[0019] 10. The artificial human skin according to any one of Clause 9, wherein the first surface of the artificial human skin has a surface free energy of about 30 mN / m.

[0020] 11 . The artificial human skin according to any one of Clauses 7 to 10, wherein two to twenty, layers of Sensation skin are formed into a unitary stack to mimic human skin absorption behaviour (Absorption skin).

[0021] 12. The artificial human skin according to Clause 11 , wherein the Absorption skin comprises from 2 to 10, such as 3 to 5 layers of the Sensation skin.

[0022] 13. The artificial human skin according to Clause 1 1 or Clause 12, wherein the Absorption skin has one or more of the following properties:

[0023] (aa) greater than 50% of the silk fibroin in the Absorption skin is present in a p-sheet / p-turn configuration, less than 30% of the silk fibroin is present as random coils, with the remainder of the silk fibroin, if any, is present in a-helix form (e.g. from 51 to 75% of the silk fibroin in the Absorption skin is present in a p-sheet / p-turn configuration, from 3 to 25% of the silk fibroin is present as random coils, with the remainder of the silk fibroin in a-helix form);

[0024] (ab) a crystallinity value of greater than 25%, such as from 27 to 50%;

[0025] (ac) a permeability speed of from 1 to 3 pg / cm2 / h, such as about 2.5 pg / cm2 / h for caffeine;

[0026] (ad) a permeability speed of from 7 to 15 pg / cm2 / h, such as about 10 pg / cm2 / h for nicotinamide; and

[0027] (ae) a permeability speed of from 2 to 7 pg / cm2 / h, such as about 5 pg / cm2 / h for salicylic acid.

[0028] 14. The artificial human skin according to any one of the preceding clauses, wherein the lipid mimic has from 14 to 20 carbon atoms, which are presented in a branched or, more particularly, linear arrangement.

[0029] 15. The artificial human skin according to any one of the preceding clauses, wherein when the SF membrane is modified with the lipid mimic, the lipid mimic is hexadecylamine.

[0030] 16. The artificial human skin according to any one of the preceding clauses, wherein when the SF membrane is modified with the lipid mimic, the artificial human skin mimics human skin deposition behaviour (Deposition skin).

[0031] 17. The artificial human skin according to Clause 16, wherein the Deposition skin has a brick-mortar structure mimicking human stratum corneum structure in epidermis.

[0032] 18. The artificial human skin according to Clause 16 or Clause 17, wherein the thickness of the Deposition skin is from 20 to 400 pm.

[0033] 19. The artificial human skin according to any one of the preceding clauses, wherein when the SF membrane is modified with the lipid mimic, the SF membrane further comprises one or both of a surfactant and a suspended solid material comprising unreacted lipid mimic.

[0034] 20. The artificial human skin according to any one of the preceding clauses, wherein the artificial human skin is coated with a sebum, optionally wherein the sebum is an artificial sebum, further optionally wherein one or more of the following apply:

[0035] (a) the artificial sebum comprises a triglyceride, a paraffin, a free fatty acid, squalene and cholesterol, optionally wherein a weight to weight ratio of the triglyceride: the paraffin: the free fatty acid: the squalene: the cholesterol is 8:5:5:3:1 ; and (b) the sebum is provided at a ratio of from 0.5 to 10 wt%, such as from 0.6 to 8 wt% relative to the weight of the modified SF membrane..

[0036] 21 . The artificial human skin according to any one of the preceding clauses, wherein the SF membrane is only modified by a lipid mimic selected from one or more of: a fatty acid; and a fatty amine.

[0037] 22. The artificial skin according to any one of the preceding clauses, wherein the artificial skin is configured to have a water contact angle before application of a test material to the artificial skin that is from 60 to 135B, so as to mimic one of an oily skin, a normal skin or a dry skin.

[0038] 23. The artificial skin according to Clause 22, wherein the artificial skin has:

[0039] (i) a water contact angle of from 60 to 85° so as to mimic oily skin;

[0040] (ii) a water contact angle of from 80 to 90° so as to mimic normal skin; or

[0041] (iii) a water contact angle of from 90 to 135° so as to mimic dry skin.

[0042] 24. The artificial skin according to Clause 22 or Clause 23, as dependent upon Clause 7, wherein the artificial skin is a sensation skin.

[0043] 25. A method of manufacturing an artificial skin, the method comprising the steps of:

[0044] (a) providing a concentrated solution comprising silk fibroin (SF) fibres crosslinked by a crosslinking agent, where the SF fibres crosslinked by a crosslinking agent has a concentration of from 1 1 to 21 wt% of the total weight of the solution; and

[0045] (b) pouring the concentrated solution onto a substrate material and spreading the poured material using a blade coating technique to achieve a solution height of around 350 to 450 pm and allowing the resulting material to dry to provide the artificial skin, wherein the artificial skin has a first surface contacted by the substrate and a second surface and has: a thickness of from 105 to 130 pm; and the first surface has a surface free energy of from 10 to 65 mN / m.

[0046] 26. The method according to Clause 25, wherein the crosslinking agent is a crosslinking agent derived from a polyethylene glycol) (PEG).

[0047] 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-succinimidyl succinylamino) ethyl]polyethylene glycol (NHSP), polyethylene glycol) dithiol, a 4-arm PEG-thiol, a 4-arm PEG-epoxide, a 4-arm PEG isocyanate, isocyanate PEG isocyanate, and polyethylene glycol) diglycidyl ether (e.g. polyethylene glycol) dithiol, a 4-arm PEG-thiol, a 4-arm PEG- epoxide, a 4-arm PEG isocyanate, isocyanate PEG isocyanate, and polyethylene glycol) diglycidyl ether).

[0048] 28. The method according to Clause 27, wherein the crosslinking agent is polyethylene glycol) diglycidyl ether.

[0049] 29. The method according to Clause 27 or Clause 28, wherein the crosslinking agent has a number average molecular weight of from 150 to 4,000 Daltons, such as from 150 to 1 ,000 Daltons, such as from 200 to 700 Daltons, such as from 250 to 500 Daltons.

[0050] 30. The method according to any one of Clauses 25 to 29, wherein the thickness of the artificial skin is about 120 pm.

[0051] 31 . The method according to any one of Clauses 25 to 29, wherein the first surface of the artificial human skin has a surface free energy of: from 10 to 20 mN / m; from 25 to 35 mN / m; from 30 to 40 mN / m; from 39 to 45 mN / m; from 42 to 58 mN / m; or from 59 to 65 mN / m, optionally wherein the first surface of the artificial human skin has a surface free energy of about 30 mN / m.

[0052] 32. The method according to any one of Clauses 25 to 31 , wherein the concentrated solution of step (a) in Clause 25 is provided by the steps of:

[0053] (al) providing a solution comprising silk fibroin (SF) fibres crosslinked by a crosslinking agent that has been filtered to remove suspended solids and the SF fibres crosslinked by a crosslinking agent has a concentration of less than 7 wt% of the total weight of the solution; and

[0054] (aii) concentrating the solution to provide the concentrated solution by subjecting it to an elevated temperature for a period of time, optionally wherein the elevated temperature is from 55 to 70SC, such as about 60eC, and the period of time is from 1 to 5 hours, such as about 3 hours. 33. The method according to Clause 32, wherein the solution of step (ai) in Clause 32 is provided by the steps of:

[0055] (bi) adding a crosslinking agent to a solution of silk fibroin fibres having a concentration of less than 7 wt% of the total weight of the solution and allowing to react for a period of time to provide a crosslinked silk fibroin solution; and

[0056] (bii) subjecting the solution to filtering to remove suspended solids (e.g. one or both of an unreacted crosslinking agent and unreacted silk fibroin) to provide the solution comprising silk fibroin (SF) fibres crosslinked by a crosslinking agent.

[0057] 34. The method according to Clause 33, wherein the weight to weight ratio is from 20:1 to 5:1 , such as about 10:1 .

[0058] 35. A method of forming an artificial skin comprising the steps of:

[0059] (ci) providing a plurality of sensation skins according to Clause 7 and Clauses 8 to 24 as dependent upon Clause 7; and

[0060] (cii) attaching the plurality of sensation skins together to provide a unitary stack to mimic human skin absorption behaviour (Absorption skin).

[0061] 36. The method according to Clause 35, wherein the Absorption skin comprises from 2 to 10, such as 3 to 5 layers of the Sensation skin.

[0062] 37. The method according to Clause 35 or Clause 36, wherein the attaching is conducted using hot-pressing.

[0063] BRIEF DESCRIPTION OF DRAWINGS

[0064] FIG. 1 depicts the WSS value between different materials (i.e., silk fibroin (SF), wool, feather and hair) and cornified envelope (CE).

[0065] FIG. 2 depicts three types of artificial skin (i.e., D skin, S skin and A skin), with their functions and application cases.

[0066] FIG. 3 depicts the water contact angle of human forearm skin and S artificial skin after different skincare product treatments. FIG. 4 depicts the friction coefficient of human cheek skin and S artificial skin after different skincare product treatments. A) human cheek skin. B) S artificial skin.

[0067] FIG. 5 depicts the fabrication of concentrated PEGylated silk fibroin (CPSF) films, a, Schematic illustration of the fabrication procedures for CPSF films, b, Photograph of dry CPSF film, c, Photograph of hydrated CPSF film.

[0068] FIG. 6 depicts the characteristics of CPSF Solution at Different Concentrations, a, Turbidity of CPSF solution at various concentrations. The data is expressed as the average ± standard deviation, based on three separate measurements, b, Zeta potential and gelation time of CPSF solution at different concentrations. Gelation condition: 25 °C. The data is expressed as the average ± standard deviation, based on three separate measurements, c, Photograph of CPSF solution at various concentrations, alongside two standard turbidity solutions. The solution on the far left is a standard with 0 NTU, while the solution on the far right is a standard with 100 NTU. D, Photograph showing the gelation of CPSF solution after being stored at room temperature for 13 days.

[0069] FIG. 7 depicts the characteristics of thickness uniformity of CPSF film made by blade coasting with PSF film made by solvent casting. The data is expressed as the average ± standard deviation, based on thirteen separate measurements.

[0070] FIG. 8 depicts the mold-based SFE modification technique, a, Schematic illustration of different materials, including silicone, aluminum foil, PS, PVC and glass, used for blade coating, b, Schematic illustration showing that the face-mold side of the CPSF film is in contact with the mold materials.

[0071] FIG. 9 depicts the water contact angle measurements for CPSF films made using different molds, with water (left) and diiodomethane (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.

[0072] FIG. 10 depicts the SFE of various mold materials and CPSF films made using these molds, a, Water contact angle measurements (water and diiodomethane) and SFE data for CPSF films produced on different mold materials, including silicone, aluminum foil, PS, PVC, and glass. The data is expressed as the average ± standard deviation, based on six separate measurements, b, Comparison of SFE data between different mold materials and the CPSF films created on these molds.

[0073] FIG. 11 depicts the XPS characteristics of CPSF films made using different molds a, XPS spectra of CPSF films made using different molds, b, Quantitative analysis of chemical environment of the carbon atom of CPSF films made using different molds.

[0074] FIG. 12 is a schematic illustration of hot pressing CPSF films into PraE.

[0075] FIG. 13 is a photograph of CPSF films hot pressed under different temperatures.

[0076] FIG. 14 includes cross-sectional SEM images of PraE fabricated via hot pressing at various temperatures, a, SEM cross-sectional images at 70x magnification for PraE fabricated under different temperatures, b, SEM cross-sectional images at 10,000x magnification for PraE fabricated under different temperatures, c, SEM cross-sectional images at 20,000x magnification for PraE fabricated under different temperatures.

[0077] FIG. 15 depicts the FTIR characteristics of PraE fabricated by hot pressing at different temperatures, a, FTIR spectra of PraE produced by hot pressing at various temperatures under 4 MPa for 3minutes. b, Deconvolution of amide I peak from FTIR spectra to quantitively calculate the percentage of different secondary structures, including a-helix, p-sheet / p-turn, and random coil, c, Quantitative analysis of secondary structure of PraE fabricated at different temperature. The data is expressed as the average ± standard deviation, based on five / six separate measurements.

[0078] FIG. 16 depicts the control experiments for CPSF films subjected to heating or pressing

[0079] FIG. 17 depicts the XRD characteristics of PraE fabricated by hot pressing at different temperatures, a, XRD spectra of PraE produced by hot pressing at various temperatures under 4 MPa for 3minutes. b, Deconvolution of XRD spectra to quantitively calculate the percentage of crystallinity by calculating the crystalline peak (blue dashed line) and amorphous peak (red dashed line), c, Quantitative analysis of crystallinity of PraE fabricated at different temperature.

[0080] FIG. 18 depicts the density of PraE fabricated by hot pressing at different temperatures. FIG. 19 depicts the XPS characteristics of PraE fabricated by hot pressing at different temperatures, a, XPS spectra of PraE produced by hot pressing at various temperatures under 4 MPa for 3minutes. b, Deconvolution of the C1 s spectra to quantitively analyze the chemical environment of the carbon atom by calculating the C-C peak (blue dashed line), C-0 peak (green dashed line) and C=O (red dashed line), c, Quantitative analysis of chemical environment of the carbon atom of PraE fabricated at different temperature.

[0081] FIG. 20 is a1H NMR spectra of CPSF film (pristine) and PraE fabricated at different temperatures. These films were dissolved in 9.3M Li Br for 4 hours at 60BC, followed by dialysis in a tube (molecular weight cut-off: 3.5k) for 2 days to remove LiBr. The solution was then freeze-dried and redissolved in D2O for1H NMR analysis.

[0082] FIG. 21 depicts different methods including solvent casting, blade coating and hot pressing, used to fabricate films with different thickness (fom 60um to 600um). a, Thickness uniformity distribution of films with different thickness fabricated by solvent casting, blade coating and hot pressing. The data is expressed as the average ± standard deviation, based on fifteen separate measurements, b, Schematic illustration of solvent casting, blade coating and hot pressing.

[0083] FIG. 22 depicts the coating artificial lipid onto PraE. a, schematic illustration of the process for coating artificial lipid onto PraE at 72 °C to obtain PLraE. b, Photograph of PraE and PLraE.

[0084] FIG. 23 depicts the rational relationship between thickness and permeability speed, a, Illustration and calculation formular of steady-state permeability speed, influenced by the concentration gradient, path length and diffusion coefficient, b, Secondary structure of PraE- 180 with varying thickness fabricated by altering the number of CPSF films and the thickness of individual CPSF films (from 200 urn to 650 urn), c, Fit-curve analysis to verify the relationship between the permeability speed and path length.

[0085] FIG. 24 depicts the caffeine permeability speed testing for PraE. a, Cumulative amounts of caffeine permeating into PraE fabricated via hot pressing under 120eC, 140eC, 160SC, 180SC. The data is expressed as the average ± standard deviation, based on three separate measurements, b, Diffusion coefficients of PraE fabricated via hot pressing under 120SC, 140eC, 160SC, 180SC. The data is expressed as the average ± standard deviation, based on three separate measurements, c, Photograph of PraE fabricated via hot pressing under 120fiC, 140fiC, 160fiC, 180BC. d, Photograph of PraE fixed in Franz cell for transdermal permeation testing.

[0086] FIG. 25 depicts the caffeine distribution coefficient of different secondary structure in PraE. a, Quantitative analysis of secondary structure of PraE fabricated at different temperature. The data is expressed as the average ± standard deviation, based on five / six separate measurements, b, Diffusion coefficients of different secondary structure including p-sheet / - turn, a-helix and random coil. The data is expressed as the average ± standard deviation, based on three separate measurements.

[0087] FIG. 26 depicts the mechanism of small compounds (caffeine) permeate through PraE.

[0088] FIG. 27 depicts the caffeine permeability speed of PraE fabricated at different temperatures compared to human skin. The data is expressed as the average ± standard deviation, based on three separate measurements.

[0089] FIG. 28 depicts the comparison of permeability speeds for four compounds among Human skin, PraE, PLraE, Strat-M, and Episkin, a, Caffeine permeability speed across different skin types, b, Nicotinamide permeability speed across different skin types, c, Salicylic acid permeability speed across different skin types, d, Rhodamine B permeability speed across different skin types. The data is expressed as the average ± standard deviation, based on three separate measurements. The permeability speed of human skin for each molecule was calculated by averaging the data obtained from published studies.

[0090] FIG. 29 are photographs of Franz cells after an 8-hour Rhodamine B permeation test by different skin type, a, PLraE. b, PraE. c, Strat-M. d, Episkin.

[0091] FIG. 30 depicts the correlation of permeability speed in PLraE and PraE with MW and log Saqof compounds from aqueous solutions.

[0092] FIG. 31 depicts the R2R system utilizing slot-die coating for film production, a, Schematic representation of R2R system with slot-die coating, illustrating the film-making process, including coating, drying, and web motion, b, Structure details of slot-die coating part, c, Photograph of CPSF film produced by R2R system, d, Thickness of CPSF films produced by the R2R system at different flow rates. FIG. 32 depicts the structure of the connection between proteins and lipids in stratum corneum (SC) and D skin. A) structure of SC, B) schematic of synthesis of D skin and its structure.

[0093] FIG. 33 includes a schematic of the perfume deposition test and the comparison of the fragrance molecules’ ratio on D skin (SH artificial skin) and on the human palm of three different people (i.e., tester 1 , tester 2 and tester 3).

[0094] FIG. 34 includes a schematic of the fabrication of SHSSs artificial skin and the comparison of the fragrance molecules’ ratio on both the SHSS artificial skin and SHSSs artificial skin and human forearm skin.

[0095] FIG. 35 includes a, Schematic illustration of the stepwise fabrication of A Skin, demonstrating precise control over protein structure at the molecular level, originating from silk cocoons. Initially, silk fibroin is extracted by removing sericin through degumming. The silk fibroin is then regenerated into a protein solution via the disruption of internal hydrogen bonds. Subsequently, a protein film forms through self-assembly, which is further processed by hot-pressing multiple thin films into a consolidated film (A Skin), resulting in significant alterations in the secondary structure, b, Selective transdermal permeation is characterized by three key principles: (i) diverse compounds penetrate the protein matrix through random coil regions; (ii) smaller molecules penetrate more rapidly compared to larger ones; (ill) compounds with higher aqueous solubility exhibit faster permeation rates, c, Schematic representation of the transdermal permeation process, showing how therapeutic agents penetrate though the epidermis and dermis layers. The illustration highlights that certain compounds can penetrate the skin, why others cannot.

[0096] DESCRIPTION

[0097] It has been surprisingly found that silk fibroin can be treated in a number of ways to make skin test platforms that more closely mimic the performance of real human skin types. Thus, in a first aspect of the invention, there is provided an artificial human skin, comprising a modified silk fibroin (SF) membrane having a first surface and a second surface, wherein the SF membrane is modified with one or both of: a crosslinking agent, which forms a crosslink between SF fibres, wherein when the crosslinking agent is used alone, the artificial human skin is provided with: a thickness of from 105 to 130 pm; and the first surface has a surface free energy of from 10 to 65 mN / m; and a lipid mimic selected from one or more of: a fatty alcohol; and, more particularly, a fatty acid and a fatty amine, where: the fatty alcohol forms ester bonds with carboxylic acid functional groups in the SF; the fatty acid forms ester binds with hydroxyl functional groups in the SF; and the fatty amine forms amide bonds with carboxylic acid functional groups in the SF.

[0098] For the avoidance of doubt, this aspect list three possible types of artificial skins. These are:

[0099] (a) a skin formed by SF and a crosslinking agent;

[0100] (b) a skin formed by SF and a lipid mimic, which forms covalent bonds with the SF; and

[0101] (c) a skin formed by SF and a crosslinking agent and a lipid mimic.

[0102] In embodiments herein, the word “comprising” may be interpreted as requiring the features mentioned, but not limiting the presence of other features. Alternatively, the word “comprising” may also relate to the situation where only the components / features listed are intended to be present (e.g. the word “comprising” may be replaced by the phrases “consists of” or “consists essentially of”). It is explicitly contemplated that both the broader and narrower interpretations can be applied to all aspects and embodiments of the present invention. In other words, the word “comprising” and synonyms thereof may be replaced by the phrase “consisting of” or the phrase “consists essentially of’ or synonyms thereof and vice versa.

[0103] The phrase, “consists essentially of’ and its pseudonyms may be interpreted herein to refer to a material where minor impurities may be present. For example, the material may be greater than or equal to 90% pure, such as greater than 95% pure, such as greater than 97% pure, such as greater than 99% pure, such as greater than 99.9% pure, such as greater than 99.99% pure, such as greater than 99.999% pure, such as 100% pure.

[0104] As will be appreciated, the modified SF membrane may have a first surface that has been shaped to mimic human skin structures. Said human skin structures may be provided by the interaction of (a) to (c) above with a mould surface, which may impart differing properties to the resulting first surface, or through the chemical structures of the SF and the lipid mimics and / or the crosslinking agents. Any suitable crosslinking agent may be used herein. For example, the crosslinking agent may be one that provides ionic crosslinking or covalent crosslinking between SF fibres. More particularly, the crosslinking may be by covalent crosslinking. For example, in certain embodiments that may be mentioned herein, the crosslinking agent may be a crosslinking agent derived from a polyethylene glycol) (PEG). As such, any suitable PEG-derived crosslinking agent may be used herein, provided that it contains functionality that enables crosslinking to occur. The crosslinking agent may be bi- tri-or tetra-dentate in terms of the functional groups available to form crosslinking with the SF. In yet more particular embodiments of the invention that may be mentioned herein, the crosslinking agent may be selected from one or more of the group consisting of O'O-bis[2-(N-succinimidyl succinylamino) ethyl]polyethylene glycol (NHSP), polyethylene glycol) dithiol, a 4-arm PEG-thiol, a 4-arm PEG-epoxide, a 4-arm PEG isocyanate, isocyanate PEG isocyanate, and polyethylene glycol) diglycidyl ether (e.g. poly(ethylene glycol) dithiol, a 4-arm PEG-thiol, a 4-arm PEG- epoxide, a 4-arm PEG isocyanate, isocyanate PEG isocyanate, and poly(ethylene glycol) diglycidyl ether). In particular embodiments of the invention, when a crosslinking agent is present in the artificial skin, it may be poly(ethylene glycol) diglycidyl ether. For the avoidance of doubt, while the crosslinking agents are mentioned above in relation to the unreacted material, it will be appreciated that the crosslinkable functional groups therein will be covalently bonded to SF fibres in the artificial skins discussed herein.

[0105] Any suitable the weight to weight ratio of the SF fibres to crosslinking agent may be used herein. For example, the weight to weight ratio of the SF fibres to crosslinking agent may be from 20:1 to 5:1 , such as about 10:1 . As will be appreciated, these values relate to the relative amounts of the chemical components derived from silk fibroin and the crosslinking agent in the final product (where the SF fibres are crosslinked by way of the crosslinking agent).

[0106] The crosslinking agent may have any suitable molecular weight. For example, the crosslinking agent may have a number average molecular weight of from 150 to 4,000 Daltons, such as from 150 to 1 ,000 Daltons, such as from 200 to 700 Daltons, such as from 250 to 500 Daltons.

[0107] As noted above, the crosslinking agent may be used alone and not in combination with the lipid mimic. In such circumstances, the artificial skin may be provided with a thickness of from 105 to 130 pm and the first surface may have a surface free energy of from 10 to 65 mN / m. This may allow the artificial skin to be suitable for use as a sensation (or S) skin. S skin is aimed at mimicking the sensation of different type skin after treatment of cosmetics products. In such skins, the water contact angle may be used to define different types of skin (dry skin, oily skin and normal skin) is made by changing different method. For cosmetic products, the feeling after spreading products onto skin is quite important, since many consumers will choose products according to the feeling of the product on their skin. As such, S skin is made to mimick human skin’s sensation when products are applied to them. Here, two factors can be used to assess the effects of artificial skin: one is water contact angle, which describes the spreadability on human skin of products; another is friction coefficient, describing the smoothness of skin after treatment with a product.

[0108] In embodiments where the artificial skin is S skin, it may have a thickness of from 105 to 130 pm, such as from 110 to 125 pm, such as about 120 pm.

[0109] In embodiments the artificial skin may have a water contact angle of from 60 to 135B, so as to mimic one of an oily skin, a normal skin or a dry skin. While this may be a general property of the artificial skins made herein, it may be particularly suited for S skin material. More particularly, the artificial skins disclosed herein may have:

[0110] (i) a water contact angle of from 60 to 85° so as to mimic oily skin;

[0111] (ii) a water contact angle of from 80 to 90° so as to mimic normal skin; or

[0112] (iii) a water contact angle of from 90 to 135° so as to mimic dry skin.

[0113] It is noted that S skins having the above properties closely mimic the properties of real human skin in sensation testing - both in terms of water contact angle of the various skin types, but also in terms of the friction coefficients generated by cosmetic products after application, as shown in the examples hereinbelow.

[0114] In embodiments of the invention making use of a crosslinking agent derived from a polyethylene glycol) (PEG), the first surface of the artificial human skin may be confuigured to have any suitable surface free energy in the range of from 10 to 65 mN / m. For example, the first surface of the artificial human skin has a surface free energy of: from 10 to 20 mN / m; from 25 to 35 mN / m; from 30 to 40 mN / m; from 39 to 45 mN / m; from 42 to 58 mN / m; or from 59 to 65 mN / m. In particular embodiments of the invention that may be mentioned herein, the first surface of the artificial human skin may have a surface free energy of about 30 mN / m.

[0115] This may be achieved through the use of differing materials in the moulds used to prepare the artificial skins, as discussed in more detail below.

[0116] While S skin is useful in and of itself, it may also be used to form an Absorption skin. Absorption is another important issue for the research of cosmetic and skincare products. For skincare products, such as whitening, moisturizing, and anti-aging products, both researchers and consumers care about how much the effective molecules, including nicotinamide, hyaluronic acid and ascorbic acid, can be absorbed by the skin. It has been surprisingly found that forming from two to twenty, layers of Sensation skin into a unitary stack results in a product that mimics human skin absorption behaviour (Absorption (or A) skin). For example, the Absorption skin may comprise from 2 to 10, such as 3 to 5 layers of the Sensation skin. This unification may be achieved through hot-pressing of layers of S skin together to form a unified whole.

[0117] Such an Absorption skin may have one or more of the following properties:

[0118] (aa) greater than 50% of the silk fibroin in the Absorption skin is present in a p-sheet / p-turn configuration, less than 30% of the silk fibroin is present as random coils, with the remainder of the silk fibroin, if any, is present in a-helix form (e.g. from 51 to 75% of the silk fibroin in the Absorption skin is present in a p-sheet / p-turn configuration, from 3 to 25% of the silk fibroin is present as random coils, with the remainder of the silk fibroin in a-helix form);

[0119] (ab) a crystallinity value of greater than 25%, such as from 27 to 50%;

[0120] (ac) a permeability speed of from 1 to 3 pg / cm2 / h, such as about 2.5 pg / cm2 / h for caffeine;

[0121] (ad) a permeability speed of from 7 to 15 pg / cm2 / h, such as about 10 pg / cm2 / h for nicotinamide; and

[0122] (ae) a permeability speed of from 2 to 7 pg / cm2 / h, such as about 5 pg / cm2 / h for salicylic acid.

[0123] It is noted that the permeability speed above are close to that of real human skin. In addition, it is noted that the A skins show no permeability to Rhodamine B and therefore more closely correlates to the performance of real human skin than other test platforms.

[0124] In embodiments of the invention where a lipid mimic is provided, the artificial human skin may mimic human skin deposition behaviour (Deposition (or D) skin). For example, when the lipid mimic is used with or without (e.g. without) the crosslinking agent. As noted above, the lipid mimic may be one or more of a fatty alcohol; and, more particularly, a fatty acid and a fatty amine, where: the fatty alcohol forms ester bonds with carboxylic acid functional groups in the SF; the fatty acid forms ester binds with hydroxyl functional groups in the SF; and the fatty amine forms amide bonds with carboxylic acid functional groups in the SF.

[0125] The amount of lipid in the SF may be from 5 to 20 wt% of the total weight of the SF.

[0126] D skin is used for a deposition test, for example deposition of a substance to be applied to the skin (e.g. perfumes, cosmetics, sunscreens, antibacterial treatments and cleansers, etc.) on D skin to mimic the effects of perfume deposition on human skin.

[0127] The lipid mimic may be chosen to provide properties that closely mimic the properties of human skin for deposition characteristics. For example, the lipid mimic may have from 14 to 20, such as from 15 to 18, such as 16 carbon atoms which are presented in a branched or, more particularly, linear arrangement. In particular embodiments that may be mentioned herein, the lipid mimic may be hexadecylamine.

[0128] Without wishing to be bound by theory, is believed that the use of lipid mimics enables the resulting artificial skin to more closely resemble that of real skin. For example, the Deposition skin may have a brick-mortar structure mimicking human stratum corneum structure in epidermis. Without wishing to be bound by theory, it is believed that this structure is obtained due to the fact that the artificial skin contains both protein part (SF) and lipid parts that mimic the bonding pattern found in real human skin’s stratum corneum, which portion of the skin contains ester bonds between skin proteins and ceramides (lipids).

[0129] D skin may have any suitable thickness. For example the thickness of Deposition skin may be from 20 to 400 pm.

[0130] In embodiments of the invention where the when the SF membrane is modified with the lipid mimic, the SF membrane may further comprises one or both of a surfactant and a suspended solid material. It is noted that the suspended solid material may simply be unreacted (i.e. free) lipid mimic (e.g. unreacted hexadecylamine). Any suitable surfactant may be used herein - e.g. those mentioned in the examples below. In certain embodiments, the artificial human skin may be coated with a sebum. While any suitable sebum may be used herein (e.g. human, animal or artificial), the sebum may in certain embodiments be an artificial sebum. In embodiments where a sebum is present, one or more of the following may apply:

[0131] (a) when the sebum is an artificial sebum it may comprise a triglyceride, a paraffin, a free fatty acid, squalene and cholesterol, optionally wherein a weight to weight ratio of the triglyceride: the paraffin: the free fatty acid: the squalene: the cholesterol is 8:5:5:3:1 ; and

[0132] (b) the sebum may be provided at a ratio of from 0.5 to 10 wt%, such as from 0.6 to 8 wt% relative to the weight of the modified SF membrane.

[0133] In certain embodiments that may be mentioned herein, the SF membrane is only modified by a lipid mimic selected from one or more of: a fatty acid; and a fatty amine. In which case, the resulting artificial skin may be a Deposition skin.

[0134] As noted in the examples, D skin is a good replacement for human palm skin in a deposition test.

[0135] In a further aspect of the invention, there is provided a method of manufacturing an artificial skin, the method comprising the steps of:

[0136] (a) providing a concentrated solution comprising silk fibroin (SF) fibres crosslinked by a crosslinking agent, where the SF fibres crosslinked by a crosslinking agent has a concentration of from 1 1 to 21 wt% of the total weight of the solution; and

[0137] (b) pouring the concentrated solution onto a substrate material and spreading the poured material using a blade coating technique to achieve a solution height of around 350 to 450 pm and allowing the resulting material to dry to provide the artificial skin, wherein the artificial skin has a first surface contacted by the substrate and a second surface and has: a thickness of from 105 to 130 pm; and the first surface has a surface free energy of from 10 to 65 mN / m.

[0138] The crosslinking agent may be as described hereinbefore. The resulting artificial human skin may have the properties described hereinbefore.

[0139] As demonstrated in the Examples disclosed herein, the zeta potential of the concentrated solution comprising the crosslinked silk fibroin fibres becomes less negative with increasing concentration, indicating reduced electrostatic repulsion and closer molecular interactions between the silk fibroin molecules, rendering the solution unstable and prone to gelation. As such, a concentration of 1 1 to 21 wt% of the crosslinked SF fibres is used, which corresponds to a zeta potential of -4.5 mV to -1 .5 mV.

[0140] The concentrated solution of step (a) above may be provided by the steps of:

[0141] (al) providing a solution comprising silk fibroin (SF) fibres crosslinked by a crosslinking agent that has been filtered to remove suspended solids and the SF fibres crosslinked by a crosslinking agent has a concentration of less than 7 wt% of the total weight of the solution; and

[0142] (aii) concentrating the solution to provide the concentrated solution by subjecting it to an elevated temperature for a period of time, optionally wherein the elevated temperature is from 55 to 70SC, such as about 60SC, and the period of time is from 1 to 5 hours, such as about 3 hours.

[0143] The solution of step (ai) above may be provided by the steps of:

[0144] (bi) adding a crosslinking agent to a solution of silk fibroin fibres having a concentration of less than 7 wt% of the total weight of the solution and allowing to react for a period of time to provide a crosslinked silk fibroin solution; and

[0145] (bii) subjecting the solution to filtering to remove suspended solids (e.g. one or both of an unreacted crosslinking agent and unreacted silk fibroin) to provide the solution comprising silk fibroin (SF) fibres crosslinked by a crosslinking agent.

[0146] Any suitable the weight to weight ratio of the SF fibres to crosslinking agent may be used herein. For example, the weight to weight ratio of the SF fibres to crosslinking agent may be from 20:1 to 5:1 , such as about 10:1 .

[0147] Also disclosed herein is a method of forming an artificial skin comprising the steps of:

[0148] (ci) providing a plurality of sensation skins as described herein; and

[0149] (cii) attaching the plurality of sensation skins together to provide a unitary stack that can mimic human skin absorption behaviour (Absorption skin).

[0150] The Absorption skin may comprise from 2 to 10, such as 3 to 5 layers of the Sensation skin in the unitary stack so formed by the above method.

[0151] Any suitable means of attachment may be used to provide a material suitable for deposition testing. For example, the attaching may be conducted using hot-pressing.

[0152] Further aspects and embodiments of the invention are provided in the following numbered statements. 1. An artificial human skin, comprising:

[0153] 1 .1 . A modified silk fibroin (SF) membrane;

[0154] 1.2. wherein the SF membrane is modified with fatty acid or fatty amines with -NH2 and - COOH functional groups (ranging from C14 to C20); or the SF membrane is modified with derivatives of polyethylene glycol) (PEG); preferably the modified SF membrane has human skin structures.

[0155] 2. An artificial human skin according to Statement 1 , wherein the SF membrane is modified with the fatty acid or fatty amines, the fatty acid or fatty amine is hexadecylamine.

[0156] 3. An artificial human skin according to Statement 1 and 2, wherein the SF membrane modified with the fatty acid or fatty amines mimics human skin deposition behaviour (Deposition skin).

[0157] 4. An artificial human skin according to Statement 3, wherein the Deposition skin has a brickmortar structure miming human stratum corneum structure in epidermis.

[0158] 5. An artificial human skin according to Statement 3, wherein the thickness of the Deposition skin is 20 to 400 pm.

[0159] 6. An artificial human skin according to Statement 1 , wherein the SF membrane modified with PEG mimics human skin sensation behaviour (Sensation skin).

[0160] 7. An artificial human skin according to Statement 6, wherein the thickness of the Sensation skin is 60 to 200 pm.

[0161] 8. An artificial human skin according to Statement 6, wherein few layers of the Sensation skin are stacked to mimic human skin absorption behaviour (Absorption skin).

[0162] 9. An artificial human skin according to Statement 8, wherein the Absorption skin comprises 2 to 20 layers of the Sensation skin.

[0163] 10. An artificial human skin according to Statement 1 , wherein an example of the derivatives of PEG is polyethylene glycol) diglycidyl ether (PEGDE). 11 . An artificial human skin according to Statements 1 to 3, wherein surfactant and suspended solids are added to the SF membrane modified with the fatty acid or fatty amines and artificial sebum is used to coat the membrane.

[0164] 12. An artificial human skin according to Statement 1 , wherein the SF membrane modified with derivatives of polyethylene glycol) (PEG) has various water contact angles to mimic various skin types.

[0165] 13. An artificial human skin according to Statement 6, wherein the water contact angle is 60- 85° for oily skin, 80-90° for normal skin and 90-135° for dry skin.

[0166] Further aspects and embodiments of the invention will now be discussed by reference to the following non-limiting examples.

[0167] EXAMPLES

[0168] Methods

[0169] Fabrication of Deposition artificial skin i.e., D Skin (SH artificial skin):

[0170] 300 ml silk fibroin (5%) solution, with 20-40 ml MES solution, 600 - 1000 mg EDC, 0-300 mg NHS, 5-15 ml hexadecylamine solution (resolved in ethanal, 75 mg / ml). Heating them under 50-80 degree for 2 to 4 hours. MES solution is made by dissolving 2.93mg MES hydrate in 60 ml DI water and 40 ml ethanal. Then adding 1.25ml of NaOH solution of 100mg / ml.

[0171] Fabrication of D Skin (SHSS artificial skin):

[0172] 300 ml silk fibroin (5%) solution, with 20-40 ml MES solution, 600 - 1000 mg EDC, 0-300 mg NHS, 5-15 ml hexadecylamine solution (resolved in ethanal, 75 mg / ml), 5-15 ml sodium dodecyl benzene sulfonate solution (100 mg / ml). Heating them under 50-80 degree for 2 to 4 hours.

[0173] Fabrication of D Skin (SHSSs artificial skin):

[0174] SHSSs artificial skin is made by spin coating artificial sebum onto SHSS artificial skin. The ratio of artificial sebum is from 0.5% to 8%. Artificial sebum is dissolved in ethanol and around 50 mg / ml, the ratio of triglyceride: paraffin: free fatty acid (Palmitic acid): squalene: cholesterol is 8:5:5:3:1. Fabrication of Sensation artificial skin i.e., S Skin

[0175] 300 ml silk fibroin (5%) solution is crosslinked by PEGDE of 1 .5 g under 60 degree for 3 hours. This solution, then, sent to centrifuge and be filtered. After which, this solution is concentrated to 12% to 25% by bath heating. This concentrated solution is used for film-making by slot-die or blade coating in roll-to-roll or sheet-to-sheet system.

[0176] Fabrication of Absorption artificial skin i.e., A Skin

[0177] Several pieces of sensation artificial skin (i.e., S Skin) are stacked together and sent to a hot- pressing machine, where the temperature is from 100 to 200 degree and the pressure is 1 Mpa to 10 Mpa, for 1 to 10 minutes.

[0178] Example 1 : Selection of silk fibroin (SF)

[0179] To select the most suitable ingredient for an artificial skin, protein-based biological materials were considered. Silk fibroin (SF), wool, feathers, and human hair were chosen due to their common usage and classification as keratin or fibroin materials. Their amino acid compositions were compared to that of the cornified envelope (CE) to determine their relative similarity (Table 1). The weighted sum of squares (WSS) values for the materials were also calculated based on the equation below.

[0180] Table 1 : Comparison of amino acid composition among CE, SF, wool, feather, and hair.

[0014]

[0181] Amino acid ratio (%) CE SF Wool Feather Hair

[0182] Asx 1.8 0 6.5 5.6 5.43

[0183] Thr 2.2 0.96 6.1 4.1 7.13

[0184] Ser 20.2 11.48 9.6 14.1 11.67

[0185] Glx 9.2 1.37 11.3 6.9 11.96

[0186] Gly 35.3 43.68 8.8 13.7 5.93

[0187] Ala 3.6 29.34 5.5 8.7 4.98

[0188] Vai 3.8 2.23 5.9 7.8 5.77

[0189] Cys 4.7 0.1 11.4 7.8 16.33

[0190] Xie 3.6 0.66 11.2 11.5 6.26

[0191] Phe 2.5 0.72 2.5 3.1 1.74

[0192] Tyr 2.3 5.3 4.1 1.4 2.01

[0193] Lys 5 0.33 3 0.6 2.43

[0194] His 0.7 0.2 0.8 0.2 0.78

[0195] Arg 1.9 0.62 6.6 3.8 6.07

[0196] Others 3.2 3.01 6.7 10.7 11.52 [1] Wang, B., Yang, W., McKittrick, J., & Meyers, M.A. Progress in Materials 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.

[0197] Results and Discussion:

[0198] From FIG. 1 , it is clear that SF is most similar with CE in terms of amino acid compositions, whose WSS is much smaller than that of feather, wool and hair. Therefore, SF is chosen as the ingredient to fabricating protein-based artificial epidermis.

[0199] In addition to their comparable amino acid compositions, SF and CE share several other structural and functional similarities. For instance, both SF and CE are composed of proteins that are synthesized and assembled in a manner that involves the protein filaggrin, which plays a crucial role in the aggregation and alignment of intermediate filaments into tight bundles. Furthermore, both SF and CE structures are stabilized and strengthened by disulfide bonds, which provide additional mechanical stability and resistance to chemical and enzymatic degradation. These disulfide bonds form crosslinks between protein chains, enhancing the overall durability and robustness of the material (Kalinin AE, Kajava AV, Steinert PM. Bioessays. 2002, 24 (9), 789-800. Koh, L.D., Cheng, Y., Teng, C.P., Khin, Y.W., Loh, X.J., Tee, S.Y., Low, M., Ye, E., Yu, FL, Zhang, Y., & Han, M. Progress in Polymer Science. 2015, 46, 86-1 10.)

[0200] According to the cosmetic industry, the following aspects of a product are important when applied to skin: deposition, sensation and absorption (FIG. 2). These questions are considered: 1 ) what will be left on the skin? 2) what will be absorbed by the skin? 3) how is the sensation of the skin? Therefore, D skin, S skin and A skin are made for answering these questions. D skin is used for the deposition test to mimic the deposition of a product (such as a perfume) on human skin after usage. S skin is sensation-targeted artificial skin, which reflects how consumers feel when a product is applied on the skin. A skin is made to assess the absorption effects of certain molecules when a product (such as nicotinamide) is spread onto skin.

[0201] Example 2: Fabrication of S Skin (CPSF film) As mentioned above, S Skin is aimed at mimicking the sensation of different skin types after treatment with a product. Here, contact angle is used to define different skin types, and artificial skin of each skin type (dry skin, oily skin and normal skin) is made by changing the method of manufacturing. For cosmetic products, the feeling after spreading products onto skin is quite important, since many consumers will choose products according to those feelings. Two factors are used to assess the effects of a product on the artificial skin: one is contact angle, which describes the spreadability on human skin of products; another is friction coefficient, which describes the smoothness of the skin after treatment with the product. Here, we use PEGDE as a crosslinker to cross link a silk fibroin solution (Y. Cui et al. Adv. Mater. 2021 , 33, 2100221). Different from conventional film-making process, where solvent casting is used for film making, we apply another method. Here, we concentrate this solution and then make it a film by blade coating method, which could speed up this film-making process (8h by blade coating method compared to 3 to 6 days by solvent casting method) and obtain better thickness uniformity. As for different skin types, we used different mold to change the contact angle of artificial skin, since different skin types shares different contact angle. The contact angle is 60 - 85° for oily skin, 80 - 90° for normal skin and 90 - 1 10° for dry skin. From FIG. 3 and FIG. 4, S Skin is similar to human skin in terms of contact angle and friction coefficient, which exhibit the sensation after products applied onto skin.

[0202] To obtain concentrated PEGylated silk fibroin (CPSF) films, the process involves three major steps as illustrated in FIG. 5. Firstly, the classical method is used to prepare the SF solution, achieving a concentration of approximately 5-6 wt%. Next, the SF solution is reacted with Polyethylene Glycol Diglycidyl Ether (PEGDE) at a ratio of 10:1 , maintained at 60 °C for 3 hours. This reaction results in PEGylated SF (PSF) solution, which is then filtered to remove suspended solids. Next step is to concentrate PSF solution with low concentration to 15 wt% by heating it at 50 °C. After centrifugation, the concentrated PEGylated SF (CPSF) solution is obtained, which is then poured onto aluminium foil and spread using a blade coating technique to achieve a solution height of approximately 400 pm. Following 8 hours of natural drying at 25 °C and 60% relative humidity, the CPSF film is obtained with around 120 pm.

[0203] The rationale for concentrating the PEGylated silk fibroin solution is that a low-concentration solution cannot be used to generate films with the desired thickness. In this case, the target thickness is around 120 pm, a requirement in the cosmetic industry. Using the PEGylated silk fibroin solution at its initial concentration would necessitate a solution with greater height to achieve this thickness. However, this greater height is impractical for blade coating as the solution would spread excessively. By concentrating the solution, the required height is reduced, making it feasible to achieve the target thickness without the need for a three- dimensional mold with walls.

[0204] Results and Discussion:

[0205] (a) Characterization of CPSF solution

[0206] Ideally, increasing the concentration of CPSF solution would enable the generation of films with greater thickness. However, higher concentrations can render the solution unstable and prone to gelation. Different concentrations of CPSF were prepared by solvent casting at 50 °C, resulting in solutions with concentrations of 12.0, 12.7, 15.4, 16.5, 18.0, and 20.7 wt%, alongside the pristine PEGylated SF solution at 5.70 wt%.

[0207] As illustrated in FIG. 6c, the turbidity of the solution increases with concentration, rising from 60 NTU to nearly 100 NTU. This indicates a higher presence of suspended solids, suggesting more substantial aggregation of silk fibroin molecules. High turbidity is indicative of instability in the solution, as larger aggregates would speed up gelation process. Moreover, the zeta potential of the solution becomes less negative with increasing concentration. A less negative zeta potential indicates reduced electrostatic repulsion between the silk fibroin molecules, promoting closer molecular interactions and facilitating the transition from a liquid to a gel state.

[0208] As illustrated in FIG. 6d, the transition of the CPSF solution into a gel state is marked by a change from a transparent solution to an opaque and milky gel. The gelation time, influenced by both turbidity and zeta potential, further underscores the stability of the solution. As the concentration increases, the gelation time decreases, indicating greater instability. The pristine PEGylated SF solution, with its lower concentration, remains stable for approximately 12 days before gelation, whereas the 20.7% CPSF solution gels in around 5 days. Given these observations, a concentration of approximately 15% is chosen for future applications, as it balances the need for adequate film thickness with sufficient solution stability.

[0209] (b) Characterization of CPSF film

[0210] As illustrated in FIG. 7, the thickness distribution of the solvent-cast PSF film is quite large (117 ± 30 pm), indicating significant variability. In contrast, the thickness distribution of the CPSF film made by blade coating is much more uniform (115 ± 4.6 pm). This demonstrates that blade coating CPSF films results in significantly better thickness uniformity compared to the classical solvent casting method.

[0211] (c) Mold-based surface free energy (SFE) modification technique for S Skin

[0212] A convenient method to modify the SFE of CPSF films involves blade coating the CPSF solution on different mold materials. Here, these mold materials are different in their SFE. For CPSF films, there are two sides: the face-air side and the face-mold side (FIG. 8). Only the face-mold side is influenced by the mold materials. Therefore, the results of CPSF films are about the face-mold side. These materials include silicone, aluminum foil, polystyrene (PS), polyvinyl chloride (PVC), and glass, each exhibiting distinct SFEs.

[0213] The 15.0 wt% CPSF solution was blade-coated onto various mold materials, including silicone, aluminum foil, PS, PVC, and glass. The coated solution was left to dry naturally at 25°C and 60% relative humidity, with the blade height maintained at 400 pm. Over the course of approximately 8 hours, this process yielded CPSF films on the different molds. The corresponding SFEs are 15.43 ± 1 .08, 28.55 ± 2.51 , 41 .33 ± 1 .73, 49.93 ± 7.44, and 61 .53 ± 1 .67 mN / m, respectively. By selecting different mold materials, the SFE of the CPSF films can be easily modified to achieve the desired properties.

[0214] FIG. 9 depicts the contact angle measurements for CPSF films made using different molds, with water (left) and diiodomethane (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, there is a significant variation in contact angle data for CPSF films produced on different molds, indicating diverse surface properties. Some molds yield films with similar contact angles, highlighting the influence of mold material on the resulting film's wettability and surface characteristics.

[0215] Moreover, the SFE of the CPSF films was calculated using a mobile surface analyzer (KRUSS), which takes into account the contact angles of both water and diiodomethane. As illustrated in FIG. 10a, an increase in SFE indicates a more hydrophilic surface, meaning the surface has higher wettability and interacts more readily with liquids. Conversely, a lower SFE suggests a more hydrophobic surface, which repels water and has lower wettability. This result validates the effectiveness of mold-based SFE modification, demonstrating that it is a convenient method for producing CPSF films with different SFEs. The SFE of human skin also varies depending on skin type and location. For instance, the SFE of forearm skin typically ranges between 30 to 40 mN / m (Mavon, A., Zahouani, FL, Redoules, D., Agache, P.G., Gall, Y., & Humbert, P.G. 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 made on aluminum foil, which have an SFE around 30 mN / m, can be used to mimic forearm skin in various applications. This demonstrates the practical application of SFE modification in creating films that accurately simulate different types of human skin.

[0216] To understand the mechanism behind mold-based SFE modification, the SFE of the mold materials was measured and compared with the corresponding CPSF films, as illustrated in FIG. 10b. The results show that from silicone to PVC, the SFE of the CPSF films closely matches that of the mold materials. For instance, the face-mold side of CPSF film made on silicone and silicone both display SFE around 18 mN / m. This phenomenon also appears when the mold is changed to aluminum foils, exhibiting SFE around 24 mN / m, and PS, with SFE around 39 mN / m. As for PVC, there are differences between mold and CPSF films, where the SFE of CPSF film is around 43 mN / m and PVC is around 49 mN / m. This difference becomes more significant when material changes into glass, where the SFE of the CPSF films, which is around 44 mN / m, does not increase as expected to around 60 mN / m.

[0217] There are two main reasons for the observed correlation of the SFE of CPSF films with the mold materials. The first is that the SFE, as pointed out, largely depends on the surface composition (Chaudhury, Manoj & Whitesides, George. Science. 1992, 255, 1230-2). Using the molds with lower SFE will therefore give rise, during film formation, to self-assembly with a more hydrophobic surface. On the contrary, the molds with higher SFE will lead to selfassembly with a more hydrophilic surface. By definition, the surfaces with the smaller SFE have more hydrophobic components; thus, hydrophobic interactions are promoted to form self-assembled films.

[0218] To clarify this point, CPSF films cast on different molds were analyzed by XPS to determine the chemical environment of the carbon at the surface. As can be observed in FIG. 11 , there is quite a big difference in chemical composition for CPSF films cast on different molds. For instance, the C-C ratio of the CPSF film made on silicone is above 90%, indicating that aliphatic carbon is the major form on the surface. This high proportion of aliphatic carbon may account for why the CPSF film made on silicone had the lowest SFE. The same trend is observed for the case of CPSF film made on aluminum foil, which contains about 60% of aliphatic carbon, and the remaining percentage of the carbon is present in the form of C-O and C=O groups. The deviations among CPSF films cast on PS, PVC, and glass are not very clear most probably because they differ insignificantly in their SFEs, as shown in FIG. 10b. CPSF films cast on these three molds showed around 50% C-C, reflecting less deviation in their surface composition.

[0219] Secondly, the SFE is also affected by surface morphology (Z. Hong, X. Yu, H. Jiang, M. Xue, S. Peng, Y. Luo, Z. Yin, C. Xie, J. Appl. Polym. Sci. 2022, 139 (17), e52005). The CPSF films tend to replicate the surface morphology of the molds on which they are formed. Thus, if the mold surface has a specific texture or pattern, the CPSF film will exhibit a similar morphology, further influencing its SFE.

[0220] Since SF contains both hydrophilic and hydrophobic segments within its molecular structure, SF-based materials can generate films with varying SFE (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 film forms with a higher concentration of hydrophilic segments on its surface, there is a maximum SFE it can achieve. This concept helps explain the behaviour of CPSF films made on glass. Glass has a very high SFE, which exceeds the inherent SFE limit of SF materials. As a result, the CPSF film formed on glass does not exhibit the expected high SFE. The SFE limit for CPSF films appears to be around 43 mN / m. Thus, while CPSF can adjust its SFE to some extent by forming films with different surface compositions, it cannot match the exceptionally high SFE of glass.

[0221] These findings highlight the importance of both surface composition and morphology in determining the SFE of CPSF films. This mold-based SFE modification technique allows for the convenient production of CPSF films with tailored SFEs, enabling their use in various applications that require specific surface properties.

[0222] Absorption is another important issue for the research of cosmetic and skincare products. For skincare products, such as whitening, moisturizing, and anti-aging products, both researchers and consumers care about how much the effective molecules, including nicotinamide, hyaluronic acid and ascorbic acid, could be absorbed by the skin. FIG. 35 is a schematic illustration of the stepwise fabrication of A Skin, demonstrating precise control over protein structure at the molecular level, originating from silk cocoons. Initially, silk fibroin is extracted by removing sericin through degumming. The silk fibroin is then regenerated into a protein solution via the disruption of internal hydrogen bonds. Subsequently, a protein film forms through self-assembly, which is further processed by hot-pressing multiple thin films into a consolidated film (A Skin), resulting in significant alterations in the secondary structure.

[0223] As mentioned above, to obtain protein-reconstructed artificial epidermis (PraE), three to five pieces of CPSF films, each approximately 120 pm thick and of the same size, are aligned and stacked. These stacks are then subjected to hot pressing at varying 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 for about three minutes. (FIG. 12) After the hot-pressing process, the PraE is obtained and let it cool naturally to room temperature.

[0224] Results and Discussion:

[0225] (a) PraE (A skin) fabricated at various temperature

[0226] After hot pressing CPSF films into PraE, it is observed that not all temperatures under 4 MPa for 3 minutes are effective for integrating several CPSF films into a homogeneous thick PraE, as shown in FIG. 13. Specifically, at 60 °C and 80 °C under 4 MPa for 3 minutes, these CPSF films are independent, indicating the integration is insufficient. However, this effect changes after temperature reaches more than 100 °C. As temperature increases, it looks the integration effect becomes better. As the temperature increases further, the color of the PraE transitions from nearly transparent to light yellow and then to dark yellow.

[0227] To verify the seamless merging of CPSF films into a single PraE, cross-sectional scanning electron microscopy (SEM) images were obtained, as shown in FIG. 14a. The images reveal that CPSF films hot pressed at 100 °C display noticeable gaps, indicating that this temperature is insufficient for achieving a homogeneous thick film. In contrast, no gaps are observed in the cross-sections of films hot pressed at temperatures ranging from 120 °C to 180 °C, exhibiting a uniform morphology with individual CPSF layers indistinguishable, indicating perfect integration. This may be attributed to the decreased glass transition temperature facilitated by the application of pressure. The glass transition temperature of silk fibroin is reported to be around 175 °C (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, it would be less than this temperature due to water influence, since the bound water in the silk film acts as a plasticizer, which is also termed as water-induced glass transition temperature (Hu, Xiao & Kaplan, David & Cebe, Peggy. Thermochimica Acta. 2007, 461 , 137- 144). In this study, temperatures above 200 °C are not considered because silk fibroin begins to undergo degradation at these higher temperatures.

[0228] (b) Characterization of PraE

[0229] Quantitative analysis reveals that as the temperature rises, the proportion of random coils decreases from 40% to less than 10%, while the proportion of p-sheets / p-turns increases from 40% to 70% (FIG. 15). There is no significant change in the a-helix content.

[0230] To determine the driving force behind the structural changes observed during hot pressing, control experiments were conducted in which CPSF films were subjected to different heating temperature and pressure. As shown in FIG. 16, both temperature and pressure contribute to the change of secondary structure, while temperature seems influence more obviously.

[0231] Next, XRD was used to characterize PraE fabricated by hot pressing, as p-sheets are crystalline structures that produce distinct signals in XRD spectra compared to random coils. As shown in FIG. 17, the peak around 20 = 20°, corresponding to the (120) plane with a d- spacing of 4.27 A, becomes increasingly prominent with rising temperatures, indicating an increase in crystalline regions (Warwicker Jo. J Mol Biol. 1960, 2, 350-62). Quantitative analysis reveals that the crystallinity of PraE increases from 25% to 50% as the temperature rises, which aligns with the results obtained from FTIR. Thus, higher temperatures lead to a more crystalline structure in PraE, predominantly in the form of p-sheets.

[0232] The trend towards a more crystalline structure with increasing temperature is further confirmed by the observed increase in density, as illustrated in FIG. 18. As the temperature for hot pressing rises, the density of PraE increases significantly, from approximately 1.3 g / cm3for pristine CPSF to 1.8 g / cm3for PraE fabricated at 180°C. This notable increase in density indicates the formation of a denser protein structure. The densification process is attributed to the enhanced molecular packing and reduced voids within the material, resulting from higher temperatures promoting the transition from amorphous regions to more ordered, crystalline p- sheet structures. To determine whether there is significant degradation or oxidation of PraE during the hot- pressing process, XPS and1H NMR were employed to examine the carbon and hydrogen chemical environments. In the C1s spectra, three distinct peaks representing C-C, C-O, and C=O carbon states were identified. As illustrated in FIG. 19, no noticeable differences were observed among the pristine CPSF film and PraE samples fabricated at different temperatures, indicating the absence of significant degradation or oxidation during hot pressing.

[0233] This conclusion is further supported by the1H NMR spectra, where all the peaks in FIG. 20 show no shifts, which means the chemical environment of hydrogen atoms are not changed during the hot-pressing process. This consistency across the spectra suggests that while there are substantial alterations in the secondary structure of the material due to the hot pressing, there are no significant changes in the chemical composition or environment. Thus, it can be concluded that the hot-pressing process induces structural modifications in PraE without causing obvious chemical changes.

[0234] Hot pressing demonstrates consistent thickness uniformity even for thicker films. For example, films produced by hot pressing at a thickness of approximately 295 ± 8.0 pm exhibit significantly better uniformity than those made by solvent casting and blade coating (FIG. 21). As the thickness increases, the variance in thickness uniformity remains minimal, indicating that hot pressing is effective for producing thicker films with uniform thickness. Thus, hot pressing emerges as a highly promising method for fabricating PraE with uniform thickness, making it suitable for applications requiring precise and consistent film properties. and A skin with artificial li for transdermal

[0235] The outermost layer of human skin contains both proteins and lipids, which are crucial for the skin's barrier function. To enhance the similarity to human skin, adding lipids that mimic those found in human skin is essential. The lipid composition in the stratum corneum is a complex mixture of free fatty acids, neutral lipids, polar lipids, and cholesterol sulfate. To replicate this composition, triglyceride (glyceryl trioleate, purchased from Sigma-Aldrich), free fatty acid (palmitic acid, purchased from Sigma-Aldrich), free sterol (cholesterol, purchased from Sigma- Aldrich) and squalene (purchased from Sigma-Aldrich) are combined at the ratio of 5:4:2: 1 (M A Lampe, A L Burlingame, J Whitney, M L Williams, B E Brown, E Roitman, P M Elias. Journal of Lipid Research. 1983, 24 (2), 120-30). This mixture is melted at 72°C and then coated onto the protein-reconstructed artificial epidermis (PraE), with the lipid mixture constituting 5 wt% of the total mass (FIG. 22).

[0236] Results and Discussion:

[0237] (a) Permeability speed of PraE fabricated in different temperature

[0238] The transdermal permeation processes involved the release of the permeant from the dosage form, its diffusion into and through the stratum corneum, and its partitioning into the epidermal environment. These processes are influenced by the physicochemical characteristics of the permeant as well as the specific characteristics of the skin at the application site. The rate of different compound permeation can be described using the steady-state flux (J) or Fick’s law of diffusion, which quantifies the cumulative mass of the drug passing per unit area through the skin (m) over a given time, as illustrated in below equation, where D denotes the diffusion coefficient, AC is the concentration gradient and h is the thickness of the skin or artificial skin (Barry BW. Eur J Pharm Sci. 2001 , 14 (2), 101 -14.).

[0239] From the above equation, the steady-state flux (J), which quantifies the permeability speed, is inversely proportional to the thickness of the skin or artificial skin. To validate the applicability of this equation for permeability speed tests using our method via Franz cells, PraE samples fabricated under identical conditions but with varying thicknesses were tested with caffeine solution. As shown in FIG. 23b, the secondary structures of each PraE sample with different thicknesses are quite similar, suggesting that the diffusion coefficient remains consistent. With the caffeine concentration gradient carefully controlled for each experiment, the primary variable is the thickness. FIG. 23c demonstrates that the permeability speed (J) and thickness (h) exhibit an inversely proportional relationship, confirming that this equation is appropriate for quantifying permeability speed.

[0240] The caffeine solution was used to test the permeability speed of PraE samples fabricated at different temperatures: 120eC (PraE-120), 140SC (PraE-140), 160eC (PraE-160), and 180SC (PraE-180). As shown in FIG. 24a, the amount of caffeine permeating through the PraE decreases as the fabrication temperature increases, indicating a slower permeability speed. Additionally, the curve tends to become linear over time, suggesting that a steady-state flux is achieved, with the permeability speed represented by the slope of the curve. This data, along with the thickness measurements of the different PraE samples — which show that PraE fabricated at higher temperatures are denser and consequently thinner — are used to calculate the diffusion coefficient. Therefore, the diffusion coefficients of PraE-120, PraE-140, PraE-160, and PraE-180 were calculated using equation shown above. The results clearly demonstrate that the higher the temperature at which PraE is hot pressed, the smaller the diffusion coefficient, indicating a slower permeability speed.

[0241] To explore the relationship between the permeability speed and the different secondary structures measured earlier, the below equation is employed for to calculate the diffusion coefficient for each type of secondary structure correspondingly. In this context, Dxdenotes the diffusion coefficient and xxdenotes the ratio of x, a part of secondary structure, where x could be p-sheet / p-turn, a-helix and random coil.

[0242] D = xaDa+ XpDp + xrDr

[0243] By combining the diffusion coefficient data with the secondary structure composition, the caffeine diffusion coefficients for different secondary structures are calculated using linear programming, as illustrated in FIG. 25b. It is evident that the diffusion coefficient for random coils is significantly larger — approximately ten times greater — than that of p-sheet / p-turn and a-helix structures. In this context, the influence of p-sheet / p-turn and a-helix structures on permeability speed can be considered negligible, as random coils serve as the primary channels for compounds to penetrate PraE. Consequently, the amount of caffeine permeating through p-sheet / p-turn and a-helix structures is minimal and can be disregarded. This finding highlights the critical role of random coils in facilitating transdermal permeation through PraE.

[0244] Moreover, this finding elucidates the mechanism by which small molecules permeate through dense protein-based materials such as PraE, highlighting that random coils serve as the primary channels for this process, as illustrated in FIG. 26. This mechanism suggests that the less ordered, flexible regions of the protein structure facilitate the diffusion of molecules, providing pathways that are more accessible compared to the rigid, crystalline p-sheet / p-turn and a-helix structures.

[0245] Additionally, this insight may extend to understanding how compounds penetrate the stratum corneum, the outermost layer of human skin. In the stratum corneum, the presence of random coils could similarly contribute to the permeability of small molecules, assisting in their passage through the lipid-rich and keratinocyte-dense environment. This understanding can inform the design of more effective transdermal drug delivery systems and improve predictions of compound absorption based on their interactions with specific protein structures.

[0246] To determine the optimal temperature for fabricating PraE, the caffeine permeability speed of PraE-120, PraE-140, PraE-160, and PraE-180 was compared to that of human skin, as illustrated in FIG. 27. The results indicate that PraE-160 and PraE-180 exhibit permeability speeds most similar to human skin, with PraE-180 showing the closest match at approximately 2.3 pg / cm2 / h (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 Pharmacol. 2004, 39 (3), 271-81 ). In contrast, the other PraE samples display faster permeability speeds. Consequently, PraE-180 is selected for transdermal permeation testing due to its closely aligned permeability characteristics with human skin.

[0247] (b) Permeability speed testing for different compounds

[0248] Although PraE-180 exhibits a caffeine permeation speed that is quite similar to that of human skin, this single compound test is insufficient to conclude its suitability for transdermal permeation testing. To validate its applicability, additional compounds need to be tested. Here, three other compounds — salicylic acid, nicotinamide, and rhodamine B — were selected for their documented permeation speeds into human skin as reported in other studies. Therefore, the permeation speeds of these three molecules, along with caffeine, were tested on PraE- 180, PLraE-180, Episkin, and Strat-M. The results were then compared to those for human skin, which can be found in the literature.

[0249] As illustrated in FIG. 28, the caffeine permeability speed of human skin is approximately 2.3 pg / cm2 / h. The results for PLraE and PraE are very similar to human skin, with PLraE demonstrating a slightly better match and PraE exhibiting a marginally faster permeability speed of around 2.4 pg / cm2 / h. In contrast, Strat-M, a commercial artificial membrane used for transdermal permeation testing, also shows some similarity to human skin with a permeability speed of approximately 3.2 pg / cm2 / h. Episkin, a type of reconstructed skin used for transdermal permeation, exhibits a significantly faster permeability speed of around 24 pg / cm2 / h, which is roughly ten times that of human skin. This increased permeation speed aligns with prior observations regarding reconstructed skin, likely due to its thinner structure, often less than 100 pm, which may be a cost-saving measure. Therefore, in the case of caffeine permeability speed testing, both PLraE and PraE demonstrate permeability speeds closely matching human skin, outperforming Strat-M and significantly surpassing Episkin in accuracy.

[0250] Moving on to salicylic acid and nicotinamide, PraE (TEST-180) and PLaE (TEST-L-180) also exhibit better similarity (N. Leveque et al. International Journal of Pharmaceutics. 269 (2004) 323-328; G.B. Kasting et al. Journal of Pharmaceutical Sciences, 111 (2022) 727-733).

[0251] Lastly, Rhodamine B, a synthetic dye widely used in various scientific applications, particularly in the fields of histology and fluorescence microscopy. It is known for its bright pink color and strong fluorescence, making it ideal for staining and visualizing biological tissues, cells, and molecular structures. Additionally, Rhodamine B is utilized in water tracing studies to track the flow and distribution of water in environmental and hydrological research. Its vibrant fluorescence and stability make it a valuable tool in both biological and environmental sciences. However, this molecule is known not to be absorbed through human skin within an 8-hour period, is used for testing. Both PLraE and PraE match human skin by showing no absorption of this molecule. However, Strat-M does show some degree of permeation, with a permeability speed of around 0.8 ng / cmz / h, as indicated by the slight redness in the receptor solution. (FIG. 29c) Episkin exhibits a much faster permeability speed, resulting in a dark red receptor solution with a speed of approximately 2.4 pg / cm2 / h (FIG. 29d).

[0252] (c) Correlation analysis between permeability speed with physicochemical properties

[0253] Except from the above four molecules, other compounds including nicotine, methylparaben, ninhydrin, tyrosine, Congo red, methylene blue and bromothymol blue, have also been tested by same methodology. The relationship between permeability speed and various physicochemical properties, including molecular weight (MW), logarithm of the octanol-water partition coefficient (log P), and logarithm of the aqueous solubility (log Saq), has been analyzed.

[0254] The correlation between permeability speed in PLraE with MW and log Saqof compounds from aqueous solutions has been analyzed, as illustrated in FIG. 30. that compounds of similar log Saq have higher permeability rates when they have MW decreased, while compounds of similar MW have higher permeability rates when they have log Saqincreased. For one thing, smaller molecules can navigate through the dense, semi-permeable barriers of the skin more easily than larger ones, encountering less resistance in their path. For another, higher aqueous solubility ensures that a compound remains dissolved in the aqueous environment of the skin, facilitating its movement across the skin layers. Hydrophilic compounds with high aqueous solubility can diffuse more readily through the aqueous channels in the skin.

[0255] Traditionally, log P is considered an important factor influencing the permeability speed of a compound, with an optimal range between 1 and 3. This might be because compounds with larger log P values often have poor solubility in aqueous solutions, leading to the use of organic solvents like ethanol, propylene glycol and acetone as vehicles (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 enhancers, increasing the permeability speed of such molecules (Lane ME. IntJ Pharm. 2013, 447, 12-21 ). Recent updates to the database of human epidermal permeability coefficients for drugs, xenobiotics, and other solutes applied in aqueous solutions have examined the relationship between permeability speed and various physicochemical properties, including MW, log P, log Saq, melting point (MP), and the number of hydrogen bond donors (Hd) and acceptors (Ha) (Cheruvu HS, Liu X, Grice JE, Roberts MS. Data Brief. 2022, 42, 108242). This extensive data indicates that log Saq has a more significant influence on permeability speed than log P when applied in aqueous solutions, and larger log Saq will exhibit faster permeability speed (Pereira R, Silva SG, Pinheiro M, Reis S, Vale MLD. 2021 May, 1 1 (5), 343).

[0256] These observations further support that both PraE and PLraE simulate the behavior of human skin in terms of permeability. Compounds have to show lower MW as well as higher aqueous solubility for better permeation rates, thus making the models relevant and effective in testing permeation across the transdermal route.

[0257] Example 5: Mass production of PraE and CPSF films

[0258] To evaluate the industrialization potential of this technology, upscaling the production of PraE and CPSF films is essential. A critical step in the current protocol for fabricating 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) approach (Krebs, F.C., Tromholt, T., & Jorgensen, M. Nanoscale. 2010, 2 (6), 873-86). Implementing the R2R method can significantly accelerate the process by efficiently drying the CPSF solution. Additionally, the R2R approach allows for continuous and automated production, which is crucial for achieving industrial scalability. This transition from laboratory-scale to industrial-scale production is vital to meet the demands of large-scale manufacturing and ensure the feasibility of this technology for commercial applications. As for R2R system, there are some coating methods, namely dip coating, knife-over-edge coating, slot-die coating, gravure coating and spray coating (Park, Janghoon & Shin, Keehyun & Changwoo, Lee. International Journal of Precision Engineering and Manufacturing. 2016, 17, 537-550). Here, slot-die coating, frequently applied for multi-layer and stripe coating, is selected for film-making due to its suitability and convenience to generate thicker films. Consisting of a slot-die head with a narrow opening from which the coating flows, a material is evenly distributed over the width of a substrate (FIG. 31b). Materials are pumped from a feed-system reservoir to a slot-die head, wherein a precision pump controls material flow at a predetermined rate to achieve even thickness in the film. The substrate handling system moves the substrate at a constant speed under the slot-die head, maintaining uniform motion. After the process of coating, the film is dried by drying equipment, such as heaters or UV lights. The entire process is directed by a supervising system that acquires and adjusts settings while controlling real-time parameters to achieve precision and uniformly coated films.

[0259] Results and Discussion:

[0260] Here are some preliminary results of films produced using the R2R system, as shown in FIG. 31c. To control the thickness of CPSF films, equation below is applied, which can be used for predicting the actual coating thickness of high-viscosity solutions (Park, J., Shin, K., and Lee, C. Int. J. Precis. Eng. Manuf. 2015, 16 (5), 937-943). In this equation, tdrydenotes the thickness of the film after drying, fris flow rate of the liquid, ptsignifies the density of the solution, W is width of coating area, V represents web motion speed, and psis density of the dry film.

[0261] It is evident that by controlling either the web motion speed or the flow rate of the liquid, the thickness of the film can be adjusted. Here, with a fixed web motion speed of 0.05 m / min, the thickness can be precisely controlled by varying the flow rate of the liquid, as demonstrated in FIG. 31 d. Currently, the laboratory-sized R2R system is more suitable for producing thin films because the drying heater is not long enough to accommodate thicker films, resulting in insufficient drying time.

[0262] Example 6: Fabrication of D Skin Here, the synthesis of D Skin is inspired by the structure of stratum corneum, where proteins and lipids are connected by ester bonds. In stratum corneum, the ester bonds stem from carboxyl group from proteins and hydroxyl group from lipids (ceramides) (FIG. 32). The lipid component could be selected from fatty acid and fatty amines, ranging from C14 to C20, containing -NH2and -COOH functional groups. After testing various fatty acids and fatty amines, we opted for hexadecylamine as the ideal lipid component to interact with silk fibroin. This choice was based on the ease of reaction between -COOH and -NH2groups compared to that of -COOH and -OH groups, alongside the suitable length of its aliphatic tail. (FIG. 32) According to the amino acid composition of silk fibroin, there are some amino acids with carboxyl groups, such as aspartic acid and glutamic acid. By this reaction, a first type of D Skin is obtained and named as SH artificial skin.

[0263] Results and Discussion:

[0264] In order to assess the similarity between human skin and this SH artificial skin, Safeguard, a disinfectant soap, is used for the deposition test. Both human palm skin and artificial skin are applied with Safeguard, after which it is washed away and the fragrance molecules left are tested and analysed. Correlation curve for every fragrance molecule’s ratio is made. As seen from FIG. 33, it is obvious that SH artificial skin is quite good for perfume deposition test, since the R2of the correlation curve is around 0.95. In fact, for palm skin from different people, R2could range from 0.9 to 0.96. Therefore, SH artificial skin could replace palm skin for this test.

[0265] However, this SH artificial skin is not similar to human forearm skin. In order to create forearm artificial skin, we, at first, add surfactant and suspended solids into SH solution to get SHSS artificial skin. And then, we used artificial sebum to coat SHSS artificial skin to get SHSSs artificial skin. As seen from FIG. 34, this approach improves the similarity with forearm skin (R2is around 0.8).

Claims

CLAIMS1. An artificial human skin, comprising a modified silk fibroin (SF) membrane having a first surface and a second surface, wherein the SF membrane is modified with one or both of: a crosslinking agent, which forms a crosslink between SF fibres, wherein when the crosslinking agent is used alone, the artificial human skin is provided with: a thickness of from 105 to 130 pm; and the first surface has a surface free energy of from 10 to 65 mN / m; and a lipid mimic selected from one or more of: a fatty alcohol; and, more particularly, a fatty acid and a fatty amine, where: the fatty alcohol forms ester bonds with carboxylic acid functional groups in the SF; the fatty acid forms ester binds with hydroxyl functional groups in the SF; and the fatty amine forms amide bonds with carboxylic acid functional groups in the SF.

2. The artificial human skin according to Claim 1 , wherein the modified SF membrane has a first surface that has been shaped to mimic human skin structures.

3. The artificial human skin according to Claim 1 or Claim 2, wherein the crosslinking agent is a crosslinking agent derived from a 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-succinimidyl succinylamino) ethyl]polyethylene glycol (NHSP), polyethylene glycol) dithiol, a 4-arm PEG-thiol, a 4-arm PEG-epoxide, a 4-arm PEG isocyanate, isocyanate PEG isocyanate, and poly(ethylene glycol) diglycidyl ether (e.g. poly(ethylene glycol) dithiol, a 4-arm PEG-thiol, a 4-arm PEG- epoxide, a 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 Claim 5, wherein the crosslinking agent has a number average molecular weight of from 150 to 4,000 Daltons, such as from 150 to 1 ,000 Daltons, such as from 200 to 700 Daltons, such as from 250 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 human skin sensation behaviour (Sensation skin).

8. The artificial human skin according to Claim 7, wherein the thickness of the Sensation skin is about 120 gm.

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

10. The artificial human skin according to any one of Claim 9, wherein the first surface of the artificial human skin has a surface free energy of about 30 mN / m.11 . The artificial human skin according to any one of Claims 7 to 10, wherein two to twenty, layers of Sensation skin are formed into a unitary stack to mimic human skin absorption behaviour (Absorption skin).

12. The artificial human skin according to Claim 1 1 , wherein the Absorption skin comprises from 2 to 10, such as 3 to 5 layers of the Sensation skin.

13. The artificial human skin according to Claim 1 1 or Claim 12, wherein the Absorption skin has one or more of the following properties:(aa) greater than 50% of the silk fibroin in the Absorption skin is present in a p-sheet / p-turn configuration, less than 30% of the silk fibroin is present as random coils, with the remainder of the silk fibroin, if any, is present in a-helix form (e.g. from 51 to 75% of the silk fibroin in theAbsorption skin is present in a p-sheet / p-turn configuration, from 3 to 25% of the silk fibroin is present as random coils, with the remainder of the silk fibroin in a-helix form);(ab) a crystallinity value of greater than 25%, such as from 27 to 50%;(ac) a permeability speed of from 1 to 3 pg / cm2 / h, such as about 2.5 pg / cm2 / h for caffeine;(ad) a permeability speed of from 7 to 15 pg / cm2 / h, such as about 10 pg / cm2 / h for nicotinamide; and(ae) a permeability speed of from 2 to 7 pg / cm2 / h, such as about 5 pg / cm2 / h for salicylic acid.

14. The artificial human skin according to any one of the preceding claims, wherein the lipid mimic has from 14 to 20 carbon atoms, which are presented in a branched or, more particularly, linear arrangement.

15. The artificial human skin according to any one of the preceding claims, wherein when the SF membrane is modified with the lipid mimic, the lipid mimic 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 mimic, the artificial human skin mimics human skin deposition behaviour (Deposition skin).

17. The artificial human skin according to Claim 16, wherein the Deposition skin has a brick-mortar structure mimicking human stratum corneum structure in epidermis.

18. The artificial human skin according to Claim 16 or Claim 17, wherein the thickness of the Deposition skin is from 20 to 400 pm.

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

20. The artificial human skin according to any one of the preceding claims, wherein the artificial human skin is coated with a sebum, optionally wherein the sebum is an artificial sebum, further optionally wherein one or more of the following apply:(a) the artificial sebum comprises a triglyceride, a paraffin, a free fatty acid, squalene and cholesterol, optionally wherein a weight to weight ratio of the triglyceride: the paraffin: the free fatty acid: the squalene: the cholesterol is 8:5:5:3:1 ; and(b) the sebum is provided at a ratio of from 0.5 to 10 wt%, such as from 0.6 to 8 wt% relative to the weight of the modified SF membrane.21 . The artificial human skin according to any one of the preceding claims, wherein the SF membrane is only modified by a lipid mimic selected from one or more of: a fatty acid; and a fatty amine.

22. The artificial skin according to any one of the preceding claims, wherein the artificial skin is configured to have a water contact angle before application of a test material to the artificial skin that is from 60 to 135B, so as to mimic one of an oily skin, a normal skin or a dry skin.

23. The artificial skin according to Claim 22, wherein the artificial skin has:(i) a water contact angle of from 60 to 85° so as to mimic oily skin;(ii) a water contact angle of from 80 to 90° so as to mimic normal skin; or(iii) a water contact angle of from 90 to 135° so as to mimic dry skin.

24. The artificial skin according to Claim 22 or Claim 23, as dependent upon Claim 7, wherein the artificial skin is a sensation skin.

25. A method of manufacturing an artificial skin, the method comprising the steps of:(a) providing a concentrated solution comprising silk fibroin (SF) fibres crosslinked by a crosslinking agent, where the SF fibres crosslinked by a crosslinking agent has a concentration of from 1 1 to 21 wt% of the total weight of the solution; and(b) pouring the concentrated solution onto a substrate material and spreading the poured material using a blade coating technique to achieve a solution height of around 350 to 450 pm and allowing the resulting material to dry to provide the artificial skin, wherein the artificial skin has a first surface contacted by the substrate and a second surface and has: a thickness of from 105 to 130 pm; and the first surface has a surface free energy of from 10 to 65 mN / m.

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

27. The method according to Claim 25, wherein the crosslinking agent is selected from one or more of the group consisting of O'O-bis[2-(N-succinimidyl succinylamino)ethyl]polyethylene glycol (NHSP), polyethylene glycol) dithiol, a 4-arm PEG-thiol, a 4-arm PEG-epoxide, a 4-arm PEG isocyanate, isocyanate PEG isocyanate, and polyethylene glycol) diglycidyl ether (e.g. polyethylene glycol) dithiol, a 4-arm PEG-thiol, a 4-arm PEG- epoxide, a 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 according to Claim 27 or Claim 28, wherein the crosslinking agent has a number average molecular weight of from 150 to 4,000 Daltons, such as from 150 to 1 ,000 Daltons, such as from 200 to 700 Daltons, such as from 250 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 pm.31 . The method according to any one of Claims 25 to 29, wherein the first surface of the artificial human skin has a surface free energy of: from 10 to 20 mN / m; from 25 to 35 mN / m; from 30 to 40 mN / m; from 39 to 45 mN / m; from 42 to 58 mN / m; or from 59 to 65 mN / m, optionally wherein the first surface of the artificial human skin has 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 of step (a) in Claim 25 is provided by the steps of:(al) providing a solution comprising silk fibroin (SF) fibres crosslinked by a crosslinking agent that has been filtered to remove suspended solids and the SF fibres crosslinked by a crosslinking agent has a concentration of less than 7 wt% of the total weight of the solution; and(aii) concentrating the solution to provide the concentrated solution by subjecting it to an elevated temperature for a period of time, optionally wherein the elevated temperature is from 55 to 70SC, such as about 60eC, and the period of time is from 1 to 5 hours, such as about 3 hours.

33. The method according to Claim 32, wherein the solution of step (ai) in Claim 32 is provided by the steps of:(bi) adding a crosslinking agent to a solution of silk fibroin fibres having a concentration of less than 7 wt% of the total weight of the solution and allowing to react for a period of time to provide a crosslinked silk fibroin solution; and(bii) subjecting the solution to filtering to remove suspended solids (e.g. one or both of an unreacted crosslinking agent and unreacted silk fibroin) to provide the solution comprising silk fibroin (SF) fibres crosslinked by a crosslinking agent.

34. The method according to Claim 33, wherein the weight to weight ratio is from 20:1 to 5:1 , such as about 10:1 .

35. A method of forming an artificial skin comprising the steps of:(ci) providing a plurality of sensation skins according to Claim 7 and Claims 8 to 24 as dependent upon Claim 7; and(cii) attaching the plurality of sensation skins together to provide a unitary stack to mimic human skin absorption behaviour (Absorption skin).

36. The method according to Claim 35, wherein the Absorption skin comprises from 2 to 10, such as 3 to 5 layers of the Sensation skin.

37. The method according to Claim 35 or Claim 36, wherein the attaching is conducted using hot-pressing.

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