Method for preparing a biomimetic cuticle and a biomimetic skin having a tissue-like differentiated structure

Abstract

The preparation method of the biomimetic stratum corneum and the biomimetic skin with tissue-like differentiation structure belongs to the technical field of transdermal drug delivery. Polymer microspheres are dispersed in a lipid mixture to obtain lipid-coated microspheres, and then a polymer-lipid composite film with a brick-mortar-like structure is formed by hot pressing, thereby obtaining a biomimetic stratum corneum highly consistent with the structure of human stratum corneum. The biomimetic stratum corneum not only has anisotropic ultrastructure in the natural stratum corneum, but also shows a permeation barrier function highly consistent with the ex vivo human skin. The biomimetic stratum corneum is co-cultured with an artificial tissue culture skin to prepare a biomimetic skin, so as to introduce skin metabolic function and / or serve as a hair follicle stem cell carrier, and construct a biomimetic skin containing a hair follicle structure. The present application is suitable for in vitro transdermal penetration test and evaluation of drugs and cosmetics, and provides reliable and efficient alternative materials and methods to solve the problems of poor predictability and large individual differences of existing animal skin models.

Description

Technical Field

[0001] This invention belongs to the field of transdermal drug delivery technology, specifically relating to a method for preparing a biomimetic stratum corneum and biomimetic skin with a tissue differentiation-like structure. This material can replace ex vivo human or animal skin for use in in vitro experiments and evaluations. Background Technology

[0002] In the development of local / transdermal drug delivery systems and cosmetic formulations, quantitatively assessing the transdermal absorption and penetration behavior of drugs is a crucial preclinical step. In vitro permeation testing (IVPT), ​​as a mature method, has become an important tool for evaluating the safety and efficacy of formulations.

[0003] Currently, skin models used for transdermal research mainly fall into two categories: cellular constructs and cell-free membranes. While cellular models are recommended by OECD guidelines, their incomplete lipid structure and defects in tight junctions result in weak barrier function and high permeability, making them unsuitable for in vitro transdermal permeation experiments. Cell-free membranes, although possessing advantages such as high stability and ease of industrial application, suffer from a homogeneous porous structure that cannot mimic the unique brick-and-mortar arrangement of the human stratum corneum, leading to isotropic permeation behavior. This fails to accurately reflect the differential permeation of lipophilic / hydrophilic compounds in real skin and makes in vitro skin metabolism and binding experiments impossible. Therefore, developing human skin substitutes with structural biomimetic features is crucial for improving in vitro testing and predictive capabilities. Summary of the Invention

[0004] The purpose of this invention is to address the structural and functional deficiencies of existing skin models used for in vitro permeation experiments, and to combine the advantages of natural skin structure. The first step is to construct a biomimetic stratum corneum preparation method based on polymers and possessing a biomimetic skin differentiation structure. In the prepared biomimetic stratum corneum, polymer microspheres serve as the building blocks, and lipids serve as the slurry. Through processes such as heat-assisted compression molding, an artificial membrane with the anisotropic structure of the human stratum corneum was successfully constructed.

[0005] To achieve the goal of constructing a biomimetic stratum corneum, the technical solution used in this invention is as follows: a method for preparing a biomimetic stratum corneum with a tissue differentiation-like structure, the method comprising the following steps:

[0006] Step 1: Prepare polymer microspheres with a diameter of 1-50 μm;

[0007] Step 2: Disperse the above microspheres in a solution containing lipids to prepare lipid-encapsulated microspheres;

[0008] Step 3: Transfer the lipid-encapsulated microspheres to a mold and hot-press them to obtain a polymer-lipid composite film with a brick-slurry structure;

[0009] Step 4: Spin-coat the obtained polymer-lipid composite membrane with lipids and dry it under vacuum to obtain a biomimetic stratum corneum.

[0010] Furthermore, the microspheres described in step 1 are prepared by an emulsion method, in which the oil phase is added to an aqueous phase containing 0.5-5 wt% emulsifier and stirred continuously for 6-12 h to obtain microspheres;

[0011] The emulsifier has an HLB value of 8-18, preferably selected from one or more of polyvinyl alcohol, sodium dodecyl sulfonate, sodium dodecyl sulfate, lecithin, and Tween; for example, the aqueous phase is an aqueous solution containing 2 wt% polyvinyl alcohol; an aqueous solution containing 2 wt% sodium dodecyl sulfonate; an aqueous solution containing 2 wt% lecithin; an aqueous solution containing 2 wt% Tween; or an aqueous solution containing 2 wt% polyvinyl alcohol.

[0012] The oil phase is a solution obtained by dissolving a high molecular weight polymer in an organic solvent, with a concentration of 0.1-20 wt%.

[0013] The polymer is one or more selected from polycaprolactone, polymethyl methacrylate, polylactic acid, polycarbonate, polyhydroxyalkanoates, polyurethane, and polysiloxane. The organic solvent is one or more selected from dichloromethane, chloroform, ethyl acetate, toluene, acetone, and tetrahydrofuran. For example, the oil phase is a chloroform solution of 10 wt% polycaprolactone; a toluene solution of a mixture of 20 wt% polycaprolactone, polylactic acid, and polyhydroxyalkanoates; a tetrahydrofuran solution of a mixture of 0.5 wt% polycaprolactone, polymethyl methacrylate, and polyurethane; an ethyl acetate-chloroform mixture of a mixture of 7.5 wt% polycaprolactone and polycarbonate; and an acetone-chloroform mixture of a mixture of 10 wt% polycaprolactone and polysiloxane.

[0014] Furthermore, the lipids mentioned in step 2 are selected from one or more of egg yolk lecithin, soybean lecithin, cholesterol, ceramide, stearic acid and sodium cholesterol sulfate, and the solution containing lipids is dissolved in one or more of methanol, chloroform, dichloromethane, petroleum ether and n-hexane, with a concentration of 0.2-20 mg / mL.

[0015] For example, solutions containing lipids include: 1 wt% soybean lecithin in methanol-n-hexane; 1 wt% egg yolk lecithin in methanol-chloroform; 1 wt% egg yolk lecithin, ceramide, and cholesterol in methanol-petroleum ether; 1 wt% egg yolk lecithin, ceramide, cholesterol, stearic acid, and sodium cholesterol sulfate in methanol-dichloromethane; and 1 wt% egg yolk lecithin and cholesterol in methanol-petroleum ether.

[0016] Furthermore, the hot pressing process described in step 3 has a temperature of 50-70℃ and a pressure of 10-500 MPa.

[0017] Furthermore, the lipids mentioned in step 4 are selected from one or more of egg yolk lecithin, soybean lecithin, cholesterol, ceramide, stearic acid, and sodium cholesterol sulfate.

[0018] Preferably, the lipids in step 2 are the same as those in step 4.

[0019] A biomimetic stratum corneum with a tissue differentiation-like structure was prepared using the above-described method.

[0020] A biomimetic skin, wherein the biomimetic skin is co-cultured with artificial tissue cultured skin using the biomimetic stratum corneum as a substrate to obtain biomimetic skin with metabolic activity.

[0021] A biomimetic skin, wherein the biomimetic skin uses the aforementioned biomimetic stratum corneum as a substrate and cell carrier, and hair follicle stem cells are cultured using microneedles to obtain biomimetic skin with hair follicle structure.

[0022] The aforementioned biomimetic stratum corneum and biomimetic skin can replace ex vivo human or animal skin to conduct in vitro transdermal penetration and other experiments.

[0023] Compared to existing technologies, the beneficial effects of this invention are as follows: This invention focuses on overcoming the limitations of traditional structures that merely fill porous polymer substrates with lipids, fundamentally resolving the core contradiction of integrating structure and function. To address the aforementioned problems, this invention proposes a biomimetic stratum corneum with a biomimetic differentiation structure. Its core innovation lies in realistically replicating the brick-and-mortar ultrastructure of the human stratum corneum, thereby systematically simulating the barrier and permeation blocking behavior of the natural skin stratum corneum. This material can not only serve as a high-performance in vitro testing and evaluation carrier, but can also be further integrated with artificially cultured skin or hair follicle stem cells to construct biomimetic skin models with metabolic functions or hair follicle structures. This provides a structurally biomimetic, functionally consistent, scalable, and animal-substitute-compliant in vitro testing and evaluation method for drug screening and safety evaluation.

[0024] 1) The artificial skin obtained by this invention has a highly biomimetic structure, accurately simulating the skin barrier function. Existing models, lacking a realistic brick-and-mortar microstructure, exhibit isotropic permeation behavior, failing to replicate the key characteristics of lipophilic / hydrophilic compounds diffusing along different pathways in human skin. This study successfully constructed a biomimetic structure with a tortuous diffusion path by embedding polymer microspheres (bricks) of size matching keratinocytes into a lipid matrix (mortar) and employing a hot-pressing molding process. This structure forces the permeate to experience alternating hydrophilic-hydrophobic environments similar to those of real skin. Consequently, in tests with various model drugs exhibiting different physicochemical properties, its permeation curve and permeation coefficient showed a significant correlation with ex vivo human skin (Pearson's r = 0.999), demonstrating the innovation and advancement of this invention in skin biomimetic design principles, structure, and function.

[0025] 2) This invention possesses outstanding practicality and scalability. Compared to the limitations of tissue-cultured cell models, such as weak barrier function and large batch-to-batch variability, as well as the ethical constraints and short shelf life of ex vivo human skin, this invention employs stable synthetic materials and engineered preparation methods, without biological cells or tissues, exhibiting excellent batch consistency and storage stability (barrier performance shows no significant change after one month of storage at room temperature). Simultaneously, this biomimetic stratum corneum provides a platform for subsequent functional integration. For example, it can be co-cultured with epidermal tissue from artificially cultured skin to introduce biological metabolic functions, or used as a carrier for hair follicle stem cells to construct complex skin models containing hair follicle units. This provides a more reliable, predictable, and standardized experimental and evaluation tool that conforms to animal substitution principles for the development and evaluation of transdermal formulations, demonstrating the practicality and scalability of this invention.

[0026] In summary, this invention constructs a biomimetic stratum corneum and biomimetic skin that highly mimics the tissue differentiation structure of human skin in both structure and function. The successful implementation of this project will effectively solve the core technical problems of poor predictability and large data variability in current transdermal drug delivery research and development, significantly reduce the dependence on scarce human skin and experimental animals, and provide stable, reliable, green, and efficient materials and tools for in vitro transdermal testing and evaluation. Attached Figure Description

[0027] Figure 1 A schematic diagram of the preparation of a biomimetic stratum corneum with a tissue differentiation structure.

[0028] Figure 2 This is a physical image of a biomimetic stratum corneum with a tissue-differentiation-like structure.

[0029] Figure 3 To compare the transdermal resistance values ​​of the biomimetic stratum corneum with a tissue-differentiation-like structure with those of isolated human epidermis (repeated experiments n = 3).

[0030] Figure 4 The permeation curves of lidocaine through ex vivo human skin and biomimetic keratinocytes (number of repeated experiments n = 4).

[0031] Figure 5 The permeation curves of naproxen through isolated human skin and biomimetic keratinocytes (number of repeated experiments n = 4).

[0032] Figure 6 The permeation curves of granisetron through ex vivo human skin and biomimetic keratin (number of repeated experiments n = 4).

[0033] Figure 7 The permeation curves of antipyrine through ex vivo human skin and biomimetic stratum corneum (number of repeated experiments n = 4).

[0034] Figure 8Point-to-point correlation diagram of lidocaine permeation behavior through ex vivo human skin and biomimetic stratum corneum. Detailed Implementation

[0035] The following specific embodiments are provided to further illustrate the content of the present invention. It should be noted that the description of these embodiments is for understanding the present invention and should not be construed as limiting the present invention in any way.

[0036] A method for preparing a biomimetic stratum corneum with a tissue-differentiation-like structure includes the following steps:

[0037] Step 1: Obtain polymer microspheres in an oil-in-water emulsion using the emulsion method;

[0038] Step 2: Disperse the microspheres thoroughly in an organic solvent containing lipids to prepare lipid-encapsulated microspheres;

[0039] Step 3: Transfer the lipid-encapsulated microspheres to a mold and hot-press them to obtain a polymer-lipid composite film with a brick-slurry structure;

[0040] Step 4: Spin-coat the obtained polymer-lipid composite membrane with a lipid mixture, and then vacuum dry to obtain a biomimetic stratum corneum with a tissue differentiation structure.

[0041] In the preparation of the biomimetic stratum corneum, the emulsifier in step 1 has an HLB value of 8 to 18, preferably one or more of polyvinyl alcohol, sodium dodecyl sulfonate, sodium dodecyl sulfate, lecithin, and Tween; the oil phase is one or more of polycaprolactone, polymethyl methacrylate, polylactic acid, polycarbonate, polyhydroxyalkanoate, polyurethane, and polysiloxane, and is dissolved in one or more solvents of dichloromethane, chloroform, ethyl acetate, toluene, acetone, and tetrahydrofuran, with a concentration of 0.1-20 wt%; the oil phase is added to the aqueous phase containing the emulsifier for emulsification and stirred continuously for 6-12 h to obtain microspheres;

[0042] Further, in step 2, the lipid is preferably one or more of egg yolk lecithin, soybean lecithin, cholesterol, ceramide, stearic acid, and sodium cholesterol sulfate, and is dissolved in an organic solvent including one or more of methanol, chloroform, dichloromethane, petroleum ether, and n-hexane.

[0043] Furthermore, in step 3, the hot pressing process is carried out at a temperature of 50-70℃ and a pressure of 10-500 MPa.

[0044] Further, in step 4, the lipids are preferably one or more of egg yolk lecithin, soybean lecithin, cholesterol, ceramide, stearic acid, and sodium cholesterol sulfate.

[0045] Furthermore, in order to endow the biomimetic skin with metabolic functions, the biomimetic stratum corneum was used as a substrate and co-cultured with artificial tissue cultured skin to obtain biomimetic skin with metabolic activity.

[0046] Furthermore, in order to endow the bionic skin with appendages similar to those of natural skin, the bionic stratum corneum is used as a substrate and cell carrier, and hair follicle stem cells are cultured using microneedles to obtain bionic skin with hair follicle structure.

[0047] A biomimetic stratum corneum and biomimetic skin with a tissue differentiation structure were prepared by the above-described method.

[0048] The aforementioned biomimetic stratum corneum and biomimetic skin are applied in the field of in vitro transdermal penetration technology.

[0049] Example 1: Preparation of a biomimetic stratum corneum with a tissue differentiation-like structure.

[0050] Preparation of biomimetic stratum corneum with tissue differentiation structure, such as Figure 1 As shown, 10 mL of chloroform solution containing 10 wt% polycaprolactone was emulsified with 100 mL of aqueous solution containing 2 wt% polyvinyl alcohol (HLB value approximately 8-9). The emulsion was continuously stirred for 8 hours. Subsequently, the obtained polymer microspheres were washed with a water-methanol solution. The washed microspheres were dispersed in a methanol-n-hexane solution containing 20 mg / mL soybean lecithin and stirred continuously until the solvent evaporated. The microspheres were transferred to a mold and hot-pressed at 60 °C (10 MPa, 10 min). 0.2 mg of soybean lecithin was spin-coated onto the composite film. The final result was a biomimetic stratum corneum with a tissue-differentiation-like structure, denoted as biomimetic stratum corneum 1.

[0051] Example 2: Preparation of a biomimetic stratum corneum with a tissue differentiation-like structure.

[0052] A 10 mL toluene solution containing a mixture of 20 wt% polycaprolactone and polysiloxane was emulsified with 100 mL of an aqueous solution (HLB value approximately 11-12) containing 2 wt% sodium dodecyl sulfate and lecithin. The emulsion was continuously stirred for 10 hours. The resulting polymer microspheres were then washed with a water-methanol solution. The washed microspheres were dispersed in a methanol-chloroform solution containing 2 mg / mL egg yolk lecithin and stirred continuously until the solvent evaporated. The microspheres were transferred to a mold and hot-pressed at 50 °C (300 MPa, 10 min). 0.2 mg of egg yolk lecithin was spin-coated onto the composite film. This resulted in a biomimetic stratum corneum with a tissue-differentiated structure, denoted as biomimetic stratum corneum 2.

[0053] Example 3: Preparation of a biomimetic stratum corneum with a tissue differentiation-like structure.

[0054] A 10 mL tetrahydrofuran solution containing a mixture of 0.5 wt% polycaprolactone, polylactic acid, and polyurethane was emulsified with 100 mL of an aqueous solution containing 2 wt% lecithin. The emulsion was continuously stirred for 8 hours. The resulting polymer microspheres were then washed with a water-methanol solution. The washed microspheres were dispersed in a methanol-petroleum ether solution containing 0.2 mg / mL egg yolk lecithin, ceramide, and cholesterol, and stirred continuously until the solvent evaporated. The microspheres were transferred to a mold and hot-pressed at 70°C (500 MPa, 10 min). A composite film containing 0.2 mg of egg yolk lecithin, ceramide, and cholesterol was spin-coated. This resulted in a biomimetic stratum corneum with a tissue-differentiated structure, denoted as biomimetic stratum corneum 3.

[0055] Example 4: Preparation of a biomimetic stratum corneum with a tissue differentiation-like structure.

[0056] A mixture of 10 mL ethyl acetate and chloroform containing 7.5 wt% polycaprolactone and polycarbonate was emulsified with 100 mL of an aqueous solution containing 2 wt% Tween (HLB value approximately 15-16). The emulsion was continuously stirred for 8 hours. The resulting polymer microspheres were then washed with a water-methanol solution. The washed microspheres were dispersed in a methanol-dichloromethane solution containing a mixture of 12 mg / mL egg yolk lecithin, ceramide, cholesterol, stearic acid, and sodium cholesterol sulfate, under continuous stirring until the solvent evaporated. The microspheres were transferred to a mold and hot-pressed at 60 °C (75 MPa, 10 min). A composite film was spin-coated with a mixture of 0.2 mg egg yolk lecithin, ceramide, cholesterol, stearic acid, and sodium cholesterol sulfate. This resulted in a biomimetic stratum corneum with a tissue-differentiated structure, denoted as biomimetic stratum corneum 4.

[0057] Example 5: Preparation of a biomimetic stratum corneum with a tissue differentiation-like structure.

[0058] A mixture of 10 mL acetone and chloroform containing 10 wt% polycaprolactone, polymethyl methacrylate, and polyhydroxyalkanoates was emulsified with 100 mL of an aqueous solution (HLB value approximately 17-18) containing 2 wt% polyvinyl alcohol and sodium dodecyl sulfate. The emulsion was continuously stirred for 8 hours. The resulting polymer microspheres were then washed with a water-methanol solution. The washed microspheres were dispersed in a methanol-petroleum ether solution containing a mixture of 10 mg / mL egg yolk lecithin and cholesterol, and stirred continuously until the solvent evaporated. The microspheres were transferred to a mold and hot-pressed at 65°C (200 MPa, 10 min). A composite film was then spin-coated with a mixture of 0.2 mg egg yolk lecithin and cholesterol. This resulted in a biomimetic stratum corneum with a tissue-differentiated structure, denoted as biomimetic stratum corneum 5.

[0059] Example 6: Preparation of a biomimetic stratum corneum with a tissue differentiation-like structure.

[0060] A 10 mL chloroform mixture containing 10 wt% polycaprolactone, polymethyl methacrylate, and polyhydroxyalkanoates was emulsified with 100 mL of an aqueous solution containing 2 wt% polyvinyl alcohol (HLB value approximately 8-10). The emulsion was continuously stirred for 8 hours. The resulting polymer microspheres were then washed with a water-methanol solution. The washed microspheres were dispersed in a methanol-petroleum ether solution containing a mixture of 10 mg / mL egg yolk lecithin and cholesterol, and stirred continuously until the solvent evaporated. The microspheres were transferred to a mold and hot-pressed at 65°C (200 MPa, 10 min). A composite film was spin-coated with a mixture of 0.2 mg ceramide, egg yolk lecithin, stearic acid, cholesterol, and sodium cholesterol sulfate. This resulted in a biomimetic stratum corneum with a tissue-differentiated structure, denoted as biomimetic stratum corneum 6.

[0061] Example 7: Preparation of biomimetic skin with metabolic activity based on biomimetic stratum corneum.

[0062] A photoinitiator and an 8% methacrylamide gelatin solution were added to the permeable membrane of the Transwell chamber at a concentration of 5 × 10⁻⁶. 6 Add dermal fibroblasts at a density of 5 mW / cm³. 2 Gel formation occurred after 1 minute of UV light irradiation. Primary human keratinocytes were injected at a concentration of 5 × 10⁻⁶ mcg. 6 The cells / mL were seeded onto the dermal surface of artificially cultured skin and then covered with the biomimetic stratum corneum prepared in Example 5 above. The sample was immediately transferred to skin cell culture medium and cultured at 37°C and 5% CO2 for 2 days. Then, the active epidermal portion was cultured at the air-liquid interface for 8 days, with the culture medium changed daily. Finally, a biomimetic skin with metabolic activity based on a biomimetic stratum corneum was formed.

[0063] Example 8: Preparation of biomimetic skin with hair follicle structure based on biomimetic stratum corneum.

[0064] At the bottom of the Transwell cell, at 5 × 10 6 Dermal fibroblasts were seeded at a density of cells / mL as a feeder layer, and a biomimetic keratinocyte was placed on a permeable membrane within the chamber. Primary human hair follicle dermal papilla cells were pre-cultured in ultra-low adsorption plates for 2-3 days, spontaneously aggregating into spherical aggregates. These spherical aggregates were carefully seeded at predetermined locations on the biomimetic keratinocyte using microneedles to simulate the density distribution of hair follicles. Primary hair follicle epithelial stem cells were then seeded at a density of 5 × 10⁶ cells / mL. 6Cells were seeded at a density of cells / mL onto the biomimetic stratum corneum prepared in Example 5 above, covering and encapsulating the spherical aggregates. The cells were statically cultured at 37°C and 5% CO2 for 2 days. After the cells fused into a monolayer on the membrane surface, the culture system was transferred to an air-liquid interface for 14-28 days, with the culture medium changed every 2-3 days. This ultimately resulted in a biomimetic skin with hair follicle structure based on the biomimetic stratum corneum.

[0065] Example 9: Measurement of transcutaneous resistance.

[0066] Prior to the permeation experiments, the integrity and similarity between the biomimetic stratum corneum with its biomimetic skin differentiation structure and the ex vivo human epidermis were verified by transcutaneous electrical resistance (TEER). After equilibrating the test samples in PBS for 2 h, the TEER values ​​were measured using a Millicell ERS-2 cellular electrical resistance meter. All experiments were repeated three times, and measurements were expressed in Ω·cm. 2 Normalization. The result is as follows: Figure 3 As shown, the biomimetic stratum corneum resistance value prepared in Example 5 of the present invention is very close to or even higher than the resistance value of ex vivo human epidermis.

[0067] Example 10: Application of biomimetic stratum corneum with tissue differentiation structure in in vitro permeation test.

[0068] In vitro permeation tests were conducted using a horizontal diffusion cell, and the effective diffusion area was 0.79 cm². 2 Skin samples (ex vivo human skin and the biomimetic stratum corneum from Example 5) were thawed overnight at 4°C and placed between the supply and receiving tanks, with the epidermal base facing the receiving tank containing degassed PBS (pH 7.4). Four model drugs with different physicochemical properties were selected, including lidocaine, naproxen, granisetron, and antipyrine. For lidocaine, naproxen, and granisetron, supersaturated suspensions containing a 20% excess of drug were prepared to maintain saturated concentrations. For antipyrine, based on its very high solubility (>1 g / mL), a 50 mg / mL drug solution was prepared, and the actual concentration was verified by HPLC. After equilibration for 30 min, the model drug solutions were loaded into the supply tank with initial concentrations of lidocaine 3.27 mg / mL. -1 Naproxen 1.24 mg mL -1 Granisetron 0.90 mg mL -1 Antipyrine 50.06 mg mL -1This concentration ensured minimal permeability variation throughout the permeation study. Both chambers were kept at a constant temperature of 32 ± 1 °C and continuously stirred at 600 rpm. At regular intervals (0.5, 1, 2, 4, 6, 8, 10, 12, 24, 28, 32, 36, 48 h), an equal volume of solution (0.6 mL) was withdrawn from the receiver chamber and replaced with an equal volume of fresh PBS. All samples were analyzed using established high-performance liquid chromatography (HPLC) methods.

[0069] Four repeated measurements were performed, and the results were reported as mean ± standard deviation (SD). The results of the in vitro permeation experiment are shown in [link to results]. Figures 4 to 7 The horizontal axis represents penetration time, and the vertical axis represents cumulative penetration per unit area. These four graphs show a comparison of the 24-hour cumulative penetration of lidocaine, naproxen, granisetron, and antipyrine between biomimetic stratum corneum and ex vivo human skin. Figure 8 This study investigated the point-to-point correlation of lidocaine permeation behavior through ex vivo human skin and biomimetic stratum corneum. The specific method is as follows: The cumulative permeation amount per unit area (μg / cm²) of ex vivo human skin at each time point was used as the basis for the correlation. 2 Using X as the independent variable and the corresponding value of the biomimetic stratum corneum at the same time point as the dependent variable, a linear regression analysis was performed using the least squares method to calculate the linear regression equation and R². 2 Among them, R 2 A number between 0 and 1, used to measure the strength of the relationship between the model and the dependent variable; the closer to 1, the stronger the correlation. For example... Figure 8 As shown, the permeability data points of the biomimetic stratum corneum and ex vivo human skin are highly clustered on both sides of the fitted line (Y = 1.008X - 0.214), R 2 The correlation coefficient was 0.984, indicating a good correlation. Overall, the permeability of this biomimetic stratum corneum is superior to or matches that of ex vivo human skin, and can be used as an alternative model for skin permeability studies.

[0070] Example 11: Comparison of transdermal permeability parameters of biomimetic stratum corneum with tissue differentiation structure and other ex vivo skin.

[0071] Steady-state permeation flux (J) ss μg cm -2 h -1 The permeability coefficient (K) is derived from the slope of the linear region in a graph showing the relationship between cumulative permeability and time. p , cm s -1 The concentration of the model drug was calculated using the formula Kp = Jss / c0, where c0 is the initial concentration of the model drug. Regression analysis was performed on the measured percutaneous permeation parameters of lidocaine and granisetron, as shown in Table 1. The parameters measured in the biomimetic stratum corneum prepared in this invention are closer to those of isolated human skin than those of isolated rat skin and isolated nude mouse skin.

[0072] Table 1. Permeation parameters of lidocaine and granisetron in various skin models

[0073]

[0074] Specifically: For lidocaine, the Jss and Kp values ​​of the biomimetic stratum corneum (28.877 ± 0.379 μg·cm⁻¹) -2 ·h -1 , 24.530 ± 0.322 × 10 -6 cm·s -1 ) and isolated human skin (23.907 ± 0.262 μg·cm) -2 ·h -1 20.308 ± 0.223 × 10 -6 cm·s -1 The results showed a high degree of agreement, with numerical differences far smaller than those between isolated rat skin and isolated nude rat skin.

[0075] For granisetron, the biomimetic stratum corneum permeability parameters were (0.970 ± 0.009 μg·cm). -2 ·h -1 , 2.994± 0.028 × 10 -6 cm·s -1 It is also closest to extracted human skin (0.609 ± 0.030 μg·cm). -2 ·h -1 , 1.880± 0.093 × 10 -6 cm·s -1 In contrast, the permeability parameters of the two commonly used animal skin models were significantly higher (the Kp value of isolated rat skin was about 29 times higher than that of human skin), indicating that the barrier function of animal skin to granisetron is much weaker than that of human skin, which would seriously overestimate the permeability of the drug.

[0076] The data in Table 1 fully demonstrate that the biomimetic stratum corneum constructed in this invention is significantly superior to traditionally used isolated rat and nude mouse skin in terms of permeability barrier function, and its permeability parameters have the highest similarity to isolated human skin. This solves the core problem of poor predictability and large variability in permeability data caused by species differences in existing animal models, and provides a standardized in vitro evaluation tool with reliable data that is closer to the real human condition for transdermal drug delivery research.

[0077] For anyone skilled in the art, many possible variations and modifications can be made to the technical solutions of this invention, or equivalent embodiments can be modified based on the disclosed technical content, without departing from the scope of the technical solutions of this invention. Therefore, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of this invention without departing from the content of the technical solutions of this invention should still fall within the protection scope of the technical solutions of this invention.

Claims

1. A method for preparing a biomimetic stratum corneum with a tissue-differentiation-like structure, characterized in that, The method includes the following steps: Step 1: Prepare polymer microspheres with a diameter of 1-50 μm; The microspheres are prepared by an emulsion method, in which the oil phase is added to an aqueous phase containing 0.5-5 wt% emulsifier and stirred continuously for 6-12 h to obtain microspheres; the polymer is one or more of polycaprolactone, polymethyl methacrylate, polylactic acid, polycarbonate, polyhydroxyalkanoate, polyurethane, and polysiloxane. Step 2: Disperse the above microspheres in a solution containing lipids to prepare lipid-encapsulated microspheres; The lipids are selected from one or more of egg yolk lecithin, soybean lecithin, cholesterol, ceramide, stearic acid, and sodium cholesterol sulfate. Step 3: Transfer the lipid-encapsulated microspheres to a mold and hot-press them to obtain a polymer-lipid composite film with a brick-slurry structure; Step 4: Spin-coat the obtained polymer-lipid composite membrane with lipids and dry it under vacuum to obtain a biomimetic stratum corneum.

2. The preparation method according to claim 1, characterized in that, The emulsifier in step 1 has an HLB value of 8 to 18 and is selected from one or more of polyvinyl alcohol, sodium dodecyl sulfonate, sodium dodecyl sulfate, lecithin, and Tween; the oil phase is a solution obtained by dissolving a polymer in an organic solvent with a concentration of 0.1-20 wt%; the organic solvent is one or more of dichloromethane, chloroform, ethyl acetate, toluene, acetone, and tetrahydrofuran.

3. The preparation method according to claim 1, characterized in that, The solvent for the lipid-containing solution in step 2 is one or more of methanol, chloroform, dichloromethane, petroleum ether, and n-hexane, with a concentration of 0.2-20 mg / mL.

4. The preparation method according to claim 1, characterized in that, The hot pressing process described in step 3 involves a temperature of 50-70℃ and a pressure of 10-500 MPa.

5. The preparation method according to claim 1, characterized in that, The lipids mentioned in step 4 are selected from one or more of egg yolk lecithin, soybean lecithin, cholesterol, ceramide, stearic acid, and sodium cholesterol sulfate.

6. A biomimetic stratum corneum with a tissue-differentiation-like structure, characterized in that: It is prepared by the preparation method according to any one of claims 1-5.

7. A bionic skin, characterized in that: The bionic skin is obtained by co-culturing the bionic stratum corneum as described in claim 6 with artificial tissue cultured skin to obtain bionic skin with metabolic activity.

8. A bionic skin, characterized in that: The bionic skin uses the bionic stratum corneum as a substrate and cell carrier as described in claim 6, and hair follicle stem cells are cultured using microneedles to obtain bionic skin with hair follicle structure.

9. The application of the biomimetic stratum corneum as described in claim 6, characterized in that: The biomimetic stratum corneum is used for in vitro transdermal penetration testing and evaluation.

10. The application of the bionic skin as described in claim 7, characterized in that: The aforementioned biomimetic skin is used in in vitro transdermal permeation tests and evaluations.

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