A novel artificial skin model and its preparation method

Abstract

The present invention provides a novel artificial skin model and a preparation method thereof, belonging to the technical field of skin models. The skin model comprises a porous support membrane, a lipid barrier is arranged on one side of the porous support membrane, and a water barrier is arranged on the other side of the porous support membrane. Since the skin model comprises both the lipid barrier and the water barrier, the skin permeation of highly lipid-soluble drugs has a better correlation with real skin, has the advantage of obtaining an effect closer to real skin, and can provide a new tool for drug transdermal research.

Description

Technical Field

[0001] The invention belongs to the technical field of skin models, and in particular relates to a novel artificial skin model, and also relates to a preparation method of the skin model. Background Art

[0002] There are two main pathways for drug transdermal absorption: the first is that the drug is absorbed through the skin appendages (hair follicles, sebaceous glands, sweat glands, etc.) to reach the dermis and then enter the systemic circulation. Since the skin appendages account for only 0.1% of the entire skin surface area, this pathway is not the main pathway for drug transdermal absorption; the second is that the drug enters the dermis through the lipophilic stratum corneum and the hydrophilic active epidermis, which is the main pathway for fat-soluble, non-polar drugs to enter the systemic circulation. For the lipophilic stratum corneum, the drug delivery system can increase the oil-water partition coefficient of the drug, which helps the drug penetrate the stratum corneum. However, when the oil-water partition coefficient is too high, the hydrophilic active epidermis will significantly hinder the diffusion of the drug. Therefore, the relationship between the oil-water partition coefficient and the skin permeation of the drug is not a linear relationship, but a parabolic relationship. This requires that the artificial skin membrane that replaces the real skin must not only have both a lipid barrier and a water barrier, but also the blocking balance of the two barriers to the drug must be achieved to fully simulate the real skin, so as to improve the correlation between the results of the artificial skin membrane and the results of the real skin experiment.

[0003] At present, a variety of artificial skin models have been developed at home and abroad. For example, the invention patent with patent number CN201510818671.3 provides a liposome artificial skin membrane and its preparation method and application in topical preparations. It belongs to the stratum corneum substitute model (SCS), which is to cover a lipid mixture on a porous material to simulate the lipid structure of the stratum corneum. The steady-state flow of model drugs with moderate hydrophilicity and lipophilicity in the SCS model and the human stratum corneum is very similar. Kansy et al. coated an organic solution of lecithin on polyvinylidene fluoride or polycarbonate membranes and established the PAMPA model (Parallel Artificial Membrane Permeability Assay). Based on the PAMPA model, Ottaviani et al. used a mixture of dimethyl silicone oil and isopropyl myristate as the lipid layer of PAMPA to simulate drug skin absorption, established a skin-PAMPA model, and systematically compared the permeability coefficient of the model with the permeability coefficient of human skin, and screened out the mixed lipid composition with the highest correlation. Hui Zhang et al. prepared a liposome-based artificial skin membrane (LASM), which consists of a tight layer of liposomes fixed on a filter, and demonstrated that the permeation of five active substances, ferulic acid, paeoniflorin, alocasia, tetrahydropalmatine and tetrahydropalmatine, in the artificial skin membrane had a good correlation with the permeability coefficient of pig ear skin (r2=0.9743).

[0004] Synthetic membrane Strat-M TM It is an artificial skin membrane that has been commercially produced. It consists of different polyester sulfone layers and an attached polyolefin layer. It has a morphology similar to human skin and a very tight surface layer. Uchida et al. demonstrated that Strat-M TM It can be used to determine the permeability of low molecular weight (152-289Da) drugs, such as lidocaine, caffeine, aminopyrine, isosorbide dinitrate, etc. Joshi et al. also demonstrated that it can be used as a surrogate model of human skin for permeation studies of some other low molecular weight drugs (aspirin and nicotine).

[0005] Although the above studies or commercial products have prepared different artificial skin models using various ingredients, the above models all have the following problems:

[0006] First, the above model only includes the lipid barrier and ignores the aqueous barrier function of the active epidermis during skin permeation.

[0007] Second, when the lipid solubility of the drug is the rate-limiting factor for transdermal penetration, the above model may have an acceptable in vitro and in vivo correlation. However, when the lipid solubility of the drug is too high and the aqueous barrier of the active epidermis becomes the rate-limiting factor for drug penetration, the predicted results of the above model may deviate from the actual results. Summary of the invention

[0008] Based on the above technical requirements and analysis of domestic and foreign research progress, we propose to establish a new artificial skin model based on the existing model, which includes a porous support membrane, one side of the porous support membrane is provided with a lipid barrier, and the other side of the porous support membrane is provided with an aqueous barrier. It is an artificial skin membrane that contains both a lipid barrier and an aqueous barrier, which can be used to improve the applicability of the evaluation of the skin penetration behavior of drugs with a high oil-water partition coefficient.

[0009] Preferably, the porous support membrane is a water-based nylon filter membrane with a pore size of 0.15-0.25 μm.

[0010] Preferably, the porous support membrane is a polycarbonate filter membrane with a pore size of 0.2 μm; the lipid barrier is constructed by centrifugal sedimentation of two liposomes of different particle sizes, wherein the particle size of the small-particle liposome is 150nm-200nm, and the particle size of the large-particle liposome is 250nm-500nm, the small-particle liposome fills the pores of the porous support membrane, and the large-particle liposome is flat on the surface of one side of the porous support membrane.

[0011] Preferably, the components of the liposome include cholesterol and lecithin, and the content ratio is cholesterol: lecithin = 1:2, and the lecithin includes one or a mixture of several of soybean lecithin, hydrogenated lecithin or egg lecithin; the components are mixed to form liposome colostrum, and the liposome colostrum is sheared to form large-size liposomes and small-size liposomes.

[0012] Preferably, the aqueous barrier is a sodium alginate gel prepared by cross-linking a sodium alginate solution and a calcium chloride solution.

[0013] Preferably, the mass concentration of the calcium chloride solution is 0.5%, and the mass concentration of the sodium alginate solution is 4%.

[0014] The present invention also provides a method for preparing a novel artificial skin model, which comprises the following detailed steps:

[0015] Step S1, fixing the porous support membrane to the support frame, the porous support membrane and the support frame cooperate to form a container on one side of the porous support membrane;

[0016] Step S2, adding a small-particle liposome solution into the container, centrifuging, turning the container over and centrifuging again, removing the supernatant, repeating the above operation of this step once, drying the water, and naturally cooling to room temperature;

[0017] Step S3, adding a large-size liposome solution into the container, centrifuging, turning the container over and centrifuging again, removing the supernatant, repeating the above operation of this step once, freezing at low temperature, thawing in vacuum, repeating freezing and thawing multiple times, that is, forming a lipid barrier on one side of the porous support membrane;

[0018] Step S4, firstly adhering the other side of the porous support membrane relative to the container to the sodium alginate solution, and then cross-linking the sodium alginate solution with the calcium chloride solution, that is, forming an aqueous barrier on the other side of the porous support membrane.

[0019] Preferably, the porous support membrane is a water-based nylon filter membrane with a pore size of 0.15-0.25 μm, and the support frame is a transwell chamber of a 24-well plate; the centrifugation conditions in step S2 and step S3 are 4°C, 600 rpm centrifugation for 5 min; the drying temperature in step S2 is 50°C; the low-temperature freezing condition in step S3 is freezing at -80°C for 1 h; the vacuum thawing condition in step S3 is vacuum thawing at 45°C for 0.5 h.

[0020] Preferably, the "fixing the porous support membrane to the support frame" in step S1 is to stick a polycarbonate membrane with a pore size of 0.2 μm to the bottom of the transwell chamber of a 24-well plate on a 150°C hot plate; the mass concentration of the sodium alginate solution in step S4 is 4%, and the calcium chloride solution is a calcium chloride solution with a mass concentration of 0.5% prepared using anhydrous calcium chloride, and the cross-linking time of the sodium alginate solution and the calcium chloride solution is 5 minutes.

[0021] Preferably, the preparation method of the small-particle liposome solution in step S2 and the large-particle liposome solution in step S3 is as follows: cholesterol and soybean lecithin are weighed respectively in a mass ratio of cholesterol: lecithin = 1:2, mixed and dissolved using dichloromethane, diluted with pure water and sheared to form colostrum, and then the colostrum is sheared into a small-particle liposome solution and a large-particle liposome solution, wherein the particle size of the small-particle liposome is 150nm-200nm, and the particle size of the large-particle liposome is 250nm-500nm.

[0022] The beneficial effects of the present invention are: providing a new artificial skin model, which contains both a lipid barrier and an aqueous barrier, has a better correlation with real skin, especially for the skin penetration of highly lipid-soluble drugs, obtains an effect closer to real skin, and can provide a new tool for drug transdermal research. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is a schematic diagram of the preparation process of Example 2.

[0024] Figure 2 This is a graph showing the correlation of the permeation results of the three drugs in Example 3 on the aqueous barrier artificial skin membrane-mouse skin.

[0025] Figure 3 This is a graph showing the correlation of the permeation results of the three drugs in Example 3 on the lipid barrier artificial skin membrane-mouse skin.

[0026] Figure 4 This is a graph showing the correlation of the permeation results of the three drugs in Example 3 on the complete artificial skin membrane-mouse skin.

[0027] Figure 5 This is a graph showing the effect of support membranes with different pore sizes on the permeation of calcein in Example 4.

[0028] Figure 6 This is a graph showing the effect of the number of liposome layers on calcein permeability in Example 5.

[0029] Figure 7 This is a graph showing the effect of the number of large liposome layers on calcein permeation in Example 5.

[0030] Figure 8 This is an optical microscope image of the aqueous barrier in Example 6.

[0031] The technical solution of the present invention is described below in conjunction with the accompanying drawings and specific embodiments.

[0032] Embodiment 1: A novel artificial skin model.

[0033] The present embodiment provides a novel artificial skin model, which includes a porous support membrane, wherein the porous support membrane is a water-based nylon filter membrane with a pore size of 0.15-0.25 μm, preferably a polycarbonate filter membrane with a pore size of 0.2 μm, and a lipid barrier is arranged on one side of the polycarbonate filter membrane, wherein the lipid barrier is constructed by a centrifugal precipitation method using two liposomes of different particle sizes, wherein the components of the liposomes include cholesterol and soybean lecithin, and the content ratio is cholesterol: soybean lecithin = 1:2, wherein the components are mixed to form liposome colostrum, and the liposome colostrum is sheared to form large-particle-size liposomes and small-particle-size liposomes, wherein The particle size of the small-size liposome is 150nm-200nm, and the particle size of the large-size liposome is 250nm-500nm. The small-size liposome fills the pores of the porous support membrane, and the large-size liposome is spread on the surface of one side of the porous support membrane. Hydrogenated lecithin is difficult to dissolve in dichloromethane because its phase transition temperature is higher than that of dichloromethane. Soy lecithin is easier to obtain than egg lecithin, so soybean lecithin is preferably used as the main component of the liposome; an aqueous barrier is arranged on the other side of the porous support membrane, and the aqueous barrier is sodium alginate gel, which is made by cross-linking a sodium alginate solution with a mass concentration of 4% and a calcium chloride solution with a mass concentration of 0.5%.

[0034] Embodiment 2: A method for preparing a novel artificial skin model.

[0035] As attached Figure 1 The method for preparing an artificial skin model shown includes the following detailed steps:

[0036] Step S1, a polycarbonate membrane with a pore size of 0.2 μm is adhered to the bottom of the transwell chamber of a 24-well plate on a hot plate at 150° C. The polycarbonate membrane and the transwell chamber are matched on one side of the polycarbonate membrane to form a cylindrical container with the transwell chamber as the side wall and the polycarbonate membrane as the bottom, and the transwell chamber is installed back on the 24-well plate;

[0037] In addition, 90 mg of soybean lecithin and 45 mg of cholesterol were weighed and added to a 5 mL Ep tube, and then 1.2 mL of dichloromethane was added to the Ep tube, vortexed and mixed evenly, and then 0.4 mL of pure water was added to the Ep tube, and a handheld shearing machine (30,000 rpm) was used to shear for 5 minutes to form colostrum, and the colostrum was poured into a 50 mL beaker filled with 20 mL of pure water to obtain diluted milk, and then the diluted milk was sheared with a handheld shearing machine (30,000 rpm) for 5 minutes to obtain a small-particle liposome solution, and the particle size of the small-particle liposome is 150 nm-200 nm; the diluted milk was obtained by the same method, and the diluted milk was sheared with a handheld shearing machine (30,000 rpm) for 3 minutes to obtain a large-particle liposome solution, and the particle size of the large-particle liposome is 250 nm-500 nm.

[0038] Step S2, add 200 μL of small-particle liposome solution into the transwell chamber, centrifuge the entire 24-well plate for 5 min (4°C, 600 rpm), flip the 24-well plate symmetrically and centrifuge again for 5 min (4°C, 600 rpm) to ensure uniform distribution of the liposomes, carefully remove the supernatant, repeat the above operation of this step once, then dry the water in the transwell chamber in an oven at 50°C, and cool naturally to room temperature;

[0039] Step S3, add 200 μL of large-particle liposome solution into the transwell chamber, centrifuge the entire 24-well plate for 5 min (4°C, 600 rpm), flip the 24-well plate symmetrically and centrifuge it again for 5 min (4°C, 600 rpm) to ensure that the liposomes are evenly distributed, carefully remove the supernatant, repeat the above operation of this step once, then freeze the 24-well plate at -80°C for 1 h, thaw it in vacuum at 45°C for 0.5 h, and perform three freeze-thaw cycles to form a lipid barrier on one side of the porous support membrane;

[0040] Step S4, the transwell chamber is disassembled, and then 0.05 g of 4% (W / V) sodium alginate solution is adhered to the bottom of the transwell chamber containing the lipid barrier, and cross-linked with an excess of 0.5% calcium chloride solution for 5 minutes, that is, an aqueous barrier is formed on the other side of the porous support membrane, and finally a complete artificial skin model is prepared.

[0041] Each transwell chamber forms a supply chamber and a receiving chamber separated by the artificial skin model with the 24-well plate. That is, after the separation of the artificial skin model, a supply chamber is formed in the transwell chamber, and the remaining space of the artificial skin model and the 24-well plate forms a receiving chamber. The substance in the supply chamber passes through the artificial skin model and enters the receiving chamber, which simulates the passive transport of drugs in vitro.

[0042] Example 3: Drug penetration correlation test on artificial skin model - mouse skin.

[0043] Three artificial skin models were taken, namely, an artificial skin model containing only an aqueous barrier (referred to as aqueous barrier), an artificial skin model containing only a lipid barrier (referred to as lipid barrier), and an artificial skin model containing both an aqueous barrier and a lipid barrier (the skin model in Example 1, referred to as a complete artificial skin membrane). The three artificial skin models and mouse skin were simultaneously subjected to permeation tests of three drugs (prilocaine, benzocaine and propofol) to verify the permeation correlation of the three drugs in the three skin models and mouse skin. The specific operation steps are as follows:

[0044] Four test objects were placed in a 24-well plate to form 12 experimental groups. 200 μL of the corresponding drug solution was added to the supply chamber at the upper part of the test object, and 1 mL of phosphate buffered saline solution (pH=6.8) containing 20% ​​ethanol was added to the receiving chamber at the lower part of the test object as the receiving solution. 1 ml of the receiving solution was sampled from the receiving chamber at 15 min, 30 min, 60 min and 120 min, and 1 ml of blank receiving solution was added to the receiving chamber at the same time. The sample solution was filtered through a 0.22 μm microporous filter membrane and then HPLC was used to determine the content of the three drugs in the sample solution. The concentration was calculated according to the standard curve. The fitting of the drug content and the standard curve is shown in the following table:

[0045] Table 1 Permeation correlation results of three drugs on artificial skin membrane-mouse skin.

[0046]

[0047] Please see the attached for specific drug content determination results. Figure 2-4 , it can be seen that the permeability of the three drugs in the complete artificial skin membrane and the permeability in mouse skin have the highest fit, that is, the complete artificial skin membrane is closest to the real skin.

[0048] Example 4: Experiment on the effect of support membranes with different pore sizes on the permeation of calcein.

[0049] Select support membranes with different pore sizes and use calcein permeation as the evaluation index to determine the optimal pore size of the support membrane. The specific steps are as follows:

[0050] Polycarbonate filter membranes with pore sizes of 0.2 μm and 0.45 μm were selected to prepare two artificial skin models according to the method of Example 2. 200 μL of 11 mM calcein solution was taken and added to the supply chambers of the two artificial skin models. 1 mL of PBS solution containing 20% ​​ethanol was filled in the lower receiving chamber as the receiving solution. Samples were taken at 15 min, 30 min, 60 min and 120 min, respectively. The fluorescence intensity of calcein in the receiving solution was measured by an ELISA instrument. The results are shown in FIG. Figure 5As shown, it can be seen from the results that the artificial skin model prepared by the 0.2 μm polycarbonate filter membrane can form a more stable permeation barrier, so the pore support membrane is preferably a polycarbonate filter membrane with a pore size of 0.2 μm.

[0051] Example 5: Experiment on the effect of liposome size and number of liposomes on calcein permeation.

[0052] Select skin models with different numbers of liposome layers and use calcein permeation as the evaluation index to determine the optimal number of liposome layers. The specific steps are as follows:

[0053] Eight artificial skin models were prepared according to the method of Example 2. The variable of the eight artificial skin models was the number of liposome layers (ie, the number of repetitions in steps S2 and S3), including 1-5 layers of small liposomes and 1-3 layers of large liposomes.

[0054] 200 μL of 11 mM calcein solution was taken and added to the supply chambers of the eight artificial skin models. 1 mL of PBS solution containing 20% ​​ethanol was placed in the lower receiving chamber as the receiving solution. Samples were taken at 15 min, 30 min, 60 min and 120 min, respectively. The fluorescence intensity of calcein in the receiving solution was measured by an ELISA instrument. The results are shown in Figure 2. Figure 6 and 7 As shown, when the number of small liposome layers is filled to the second layer, the permeability is the smallest. When it is filled to the third and fourth layers, the calcein permeability increases and there is no significant difference from the first layer. As the number of large liposome layers increases, the permeability of calcein decreases, and there is no significant difference in the permeability of calcein between 2 and 3 layers. That is, the optimal number of layers for filling small liposomes and the optimal number of layers for tiling large liposomes are both 2 layers.

[0055] Example 5: Microscopic observation of aqueous barriers.

[0056] (1) The peeled aqueous barrier was embedded in OCT and stored at -80°C until use.

[0057] (2) When slicing, fix the aqueous barrier on a slice holder and cut it into slices with a thickness of 20 μm. Use a brush to unfold the slices and adsorb them onto a glass slide.

[0058] (3) The cut aqueous barrier was placed in a slice box and stored at -20°C.

[0059] (4) Place the slice under a fluorescence microscope to observe and collect images

[0060] The results are as follows Figure 8 As shown in the figure, it can be seen that the aqueous barrier stained with rhodamine B has uniform red fluorescence, indicating that a complete structure of the aqueous barrier has been formed.

[0061] The present invention provides the above embodiments only to illustrate rather than limit the technical solutions of the present invention. Although the above embodiments describe the present invention in detail, relevant technical personnel in the field should understand that the present invention can be modified or equivalently replaced, but any modification and partial replacement that does not depart from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A novel artificial skin model, characterized in that: The invention comprises a porous support membrane, wherein a lipid barrier is arranged on one side of the porous support membrane, and a water barrier is arranged on the other side of the porous support membrane.

2. A novel artificial skin model according to claim 1, characterized in that: The porous support membrane is a water-based nylon filter membrane with a pore size of 0.15-0.25 μm.

3. A novel artificial skin model according to claim 2, characterized in that: The porous support membrane is a polycarbonate filter membrane with a pore size of 0.2 μm; the lipid barrier is constructed by two liposomes of different particle sizes through a centrifugal sedimentation method, wherein the particle size of the small-size liposome is 150nm-200nm, and the particle size of the large-size liposome is 250nm-500nm. The small-size liposomes fill the pores of the porous support membrane, and the large-size liposomes are flat on the surface of one side of the porous support membrane.

4. A novel artificial skin model according to claim 3, characterized in that: The components of the liposome include cholesterol and lecithin, and the content ratio is cholesterol:lecithin=1:

2. The lecithin includes one or a mixture of several of soybean lecithin, hydrogenated lecithin or egg lecithin. The components are mixed to form liposome colostrum, and the liposome colostrum is sheared to form large-size liposomes and small-size liposomes.

5. A novel artificial skin model according to claim 1, characterized in that: The aqueous barrier is a sodium alginate gel prepared by cross-linking a sodium alginate solution and a calcium chloride solution.

6. A novel artificial skin model according to claim 5, characterized in that: The mass concentration of the calcium chloride solution is 0.5%, and the mass concentration of the sodium alginate solution is 4%.

7. A method for preparing a novel artificial skin model, characterized in that: It includes the following detailed steps: Step S1, fixing the porous support membrane to the support frame, the porous support membrane and the support frame cooperate to form a container on one side of the porous support membrane; Step S2, adding a small-particle liposome solution into the container, centrifuging, turning the container over and centrifuging again, removing the supernatant, repeating the above operation of this step once, drying the water, and naturally cooling to room temperature; Step S3, adding a large-size liposome solution into the container, centrifuging, turning the container over and centrifuging again, removing the supernatant, repeating the above operation of this step once, freezing at low temperature, thawing in vacuum, repeating freezing and thawing multiple times, that is, forming a lipid barrier on one side of the porous support membrane; Step S4, firstly adhering the other side of the porous support membrane relative to the container to the sodium alginate solution, and then cross-linking the sodium alginate solution with the calcium chloride solution, that is, forming an aqueous barrier on the other side of the porous support membrane.

8. The method for preparing a novel artificial skin model according to claim 7, characterized in that: The porous support membrane is a water-based nylon filter membrane with a pore size of 0.2 μm, and the support frame is a transwell chamber of a 24-well plate; the centrifugation conditions in steps S2 and S3 are 4°C, 600 rpm centrifugation for 5 min; the drying temperature in step S2 is 50°C; the low-temperature freezing condition in step S3 is freezing at -80°C for 1 h; the vacuum thawing condition in step S3 is vacuum thawing at 45°C for 0.5 h.

9. The method for preparing a novel artificial skin model according to claim 8, characterized in that: The "fixing the porous support membrane to the support frame" in step S1 is to stick a polycarbonate membrane with a pore size of 0.2 μm to the bottom of the transwell chamber of a 24-well plate on a 150°C hot plate; the mass concentration of the sodium alginate solution in step S4 is 4%, and the calcium chloride solution is a calcium chloride solution with a mass concentration of 0.5% prepared using anhydrous calcium chloride, and the cross-linking time of the sodium alginate solution and the calcium chloride solution is 5 minutes.

10. The method for preparing a novel artificial skin model according to claim 9, characterized in that: The preparation method of the small-particle liposome solution in step S2 and the large-particle liposome solution in step S3 is as follows: cholesterol and soybean lecithin are weighed respectively in a mass ratio of cholesterol: lecithin = 1:2, mixed and dissolved with dichloromethane, diluted with pure water and sheared to form colostrum, and then the colostrum is sheared into a small-particle liposome solution and a large-particle liposome solution, wherein the particle size of the small-particle liposome is 150nm-200nm, and the particle size of the large-particle liposome is 250nm-500nm.

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