Skin barrier model for detecting permeability of cosmetics and application

Abstract

The invention discloses a skin barrier model for detecting permeability of cosmetics and application. The invention provides a skin barrier model for detecting permeability of cosmetics. The permeability of the skin barrier model is obviously similar to that of pigskin detection. Therefore, the skin barrier model provided by the invention can be used for replacing pigskin in vitro for permeability test.

Description

Technical Field

[0001] The present invention belongs to the field of cosmetics, and particularly relates to a skin barrier model for detecting the penetration rate of cosmetics and its application. Background Art

[0002] Cosmetics, as an effective tool for consumers to pursue beauty and achieve a beautiful makeup, play an increasingly important role in daily consumption. With the improvement of living standards, consumers' lifestyles and habits have changed greatly. More and more consumers are paying more attention to quality, health, and hygiene issues, and their choices of skin care products are not only limited to brand, efficacy, and cost performance, but they also attach more importance to safety and effectiveness.

[0003] In vitro evaluation models are the most important and commonly used laboratory methods for evaluating the safety and efficacy of cosmetics and their raw materials. In order to develop a skin barrier model for detecting the penetration rate of cosmetics, the present invention is specifically proposed. Summary of the Invention

[0004] The purpose of the present invention is to provide a skin barrier model for detecting the penetration rate of cosmetics and its application.

[0005] The above object of the present invention is achieved by the following technical solutions:

[0006] A skin barrier model for detecting the penetration rate of cosmetics is prepared by the following method:

[0007] Absorb 100 μL of liposome LUV, add it to the Transwell chamber, and place the Transwell chamber in a 24-well plate, and centrifuge at 2030 rpm for 5 min; rotate the well plate horizontally by 180°, add another 100 μL of liposome LUV and 50 μL of HaCaT cell resuspension, blow and mix evenly, then centrifuge at 1500 rpm for 5 min. Take out the Transwell chamber and fuse it at 50 °C for 45 min. After that, add 50 μL of liposome MLV, dry it at 50 °C for 40 min, add another 50 μL of liposome MLV, dry it at 50 °C for 60 min, transfer it to -80 °C and maintain it for more than 4 h, take it out and fuse it at 50 °C for 2 hours, maintain it at room temperature for 15 min, and store it at 4 °C;

[0008] Wherein:

[0009] The original bottom carrier membrane of the Transwell chamber is replaced with a carrier membrane made of MCE microporous membrane, and the Transwell chamber and the carrier membrane made of MCE microporous membrane are bonded with an EVA adhesive ring to form a skin barrier model support;

[0010] The preparation methods of the liposome LUV and liposome MLV are as follows: Weigh lecithin and cholesterol according to a mass ratio of 77:23 and add them to a container. Dissolve them with a chloroform-methanol mixed solvent with a volume ratio of 2:1, and spin-dry under vacuum to remove trace solvents to obtain a thin film. In the container containing the thin film, add 10% EtOH-PBS according to the ratio of adding 1 mL of solvent for every 20 mg of the sum of the masses of lecithin and cholesterol, slowly mix evenly to dissolve the thin film, and stir and hydrate at 40 °C in a water bath at a rotation speed of 100 rpm for 1.5 hours and anneal for 0.5 hour to obtain a liposome dispersion. Use a filter membrane with a pore size of 800 nm to continuously filter the liposome dispersion 3 times to obtain liposome MLV, and use a filter membrane with a pore size of 450 nm to continuously filter the liposome dispersion 5 times to obtain liposome LUV;

[0011] The cell density of the HaCaT cell resuspension is 10,000 cells / μL, and the solvent is PBS.

[0012] Preferably, the pore size of the MCE microporous filter membrane is Φ650 nm.

[0013] Preferably, use a puncher to cut the MCE microporous filter membrane into a circular piece with a diameter adapted to the bottom of the Transwell chamber as the carrier membrane of the model. Use a puncher to cut the EVA film into a ring adapted to the bottom of the Transwell chamber. Place the MCE microporous filter membrane, the EVA adhesive ring, and the Transwell chamber with the original bottom carrier membrane removed on a heating plate in alignment, heat at 90 °C for 5 minutes, and press the chamber tightly to make the three adhere closely to obtain the skin barrier model support.

[0014] A method for recycling and reusing the above skin barrier model support, which is characterized in that: the MCE microporous filter membrane and the EVA adhesive ring at the bottom of the model can be peeled off from the Transwell chamber after the detection experiment. First, wash the Transwell chamber with the solvent of the sample measured before recycling, and then ultrasonically wash with 95% ethanol and water in sequence, and dry. The cleaned Transwell chamber can be adhered to the MCE carrier membrane in the same way to prepare the support.

[0015] The application of the above skin barrier model in the detection of the penetration rate of cosmetics.

[0016] Beneficial effects:

[0017] The present invention provides a skin barrier model for the detection of the penetration rate of cosmetics, and this model has a significant similarity to pig skin. Therefore, the skin barrier model provided by the present invention can be used to replace pig skin for permeability testing in vitro. Description of the drawings

[0018] Figure 1 It is a flow chart for the preparation of the model of the present invention. Specific Embodiments

[0019] The following specifically introduces the substantial content of the present invention in combination with embodiments, but does not limit the protection scope of the present invention thereby.

[0020] I. Model Construction Materials

[0021] 1. Instruments

[0022] Electronic heating plate, -20°C refrigerator, ten-thousandth electronic balance, rotary evaporator, centrifuge, oven, -80°C oven, microplate reader, CO 2 Constant temperature cell culture incubator.

[0023] 2. Materials

[0024] 8mm / 6mm punching tool bit, puncher, forceps, ep tube, pipette, Φ650nm MCE microporous membrane (mixed cellulose microporous membrane), EVA film (polyethylene-polyvinyl acetate copolymer film), Transwell chamber, 24-well plate, 1000mL eggplant-shaped flask, disposable syringe, needle filter membrane (450nm, 800nm), test tube rack, cryopreservation tube, cell culture dish.

[0025] 3. Reagents

[0026] Pure water, absolute ethanol, chloroform, methanol, soy lecithin, cholesterol, PBS (pH 7.4) solution, HCl-PBS solution, NaOH-PBS solution, trypsin, DMEM medium, FBS.

[0027] II. Model Construction Method and Application

[0028] 1. Cleaning of Transwell Chamber

[0029] Before the experiment, a clean and dry Transwell chamber should be prepared for use: 1) When using the chamber for cell experiments, remove the bottom carrier membrane with forceps and soak it in 75% alcohol and ultrasonically wash the residue for 2 hours (if necessary, soak it in trypsin to remove the adhered protein), ultrasonically wash it with pure water for 2 hours and then dry it in the oven for use. 2) When using the chamber after the permeation experiment, carefully tear off the bottom MCE microporous membrane and the EVA bonding ring with forceps, first wash it with the solvent of the test substance, and then ultrasonically wash it with 95% ethanol and water for 2 hours in sequence and then dry it for use.

[0030] 2. Preparation of Model Support

[0031] Use a punch to cut the MCE microporous membrane into circular pieces with a diameter of 8 mm as the carrier membrane of the model. Use a punch to cut the EVA film into a ring with a size of 8 mm / 6 mm (i.e., the EVA bonding ring). Place the MCE microporous membrane, the EVA bonding ring, and a clean and dry Transwell chamber on the heating plate in sequence and align them. Heat at 90 °C for 5 min, and manually press the chamber tightly during this period to make the three bond closely for later use.

[0032] 3. Liposome preparation

[0033] Weigh a total of 400 mg of lecithin and cholesterol (77:23, m / m), transfer them to a 1000 mL eggplant-shaped flask, dissolve them with a chloroform-methanol solution (2:1, v / v), and spin-dry under vacuum for 2 hours to remove trace solvents, obtaining a film. Add 20 mL of 10% EtOH-PBS (PBS solution containing ethanol, with the volume percentage of ethanol being 10%) to the eggplant-shaped flask containing the film, slowly mix evenly to dissolve the film, and perform a hydration reaction at 100 rpm for 1.5 hours in a 40 °C water bath, and anneal for 0.5 hour to obtain a 2% liposome dispersion.

[0034] Preparation of MLV: Use a needle filter with a pore size of 800 nm to pass the liposome dispersion through the membrane continuously for 3 times.

[0035] Preparation of LUV: Use a needle filter with a pore size of 450 nm to pass the liposome dispersion through the membrane continuously for 5 times.

[0036] 4. Cell culture and collection

[0037] Resuscitation of HaCaT cells:

[0038] The seed cells are stored in a -80 °C ultra-low temperature refrigerator. When in use, take out the cryopreservation tube, thaw it in a 37 °C water bath, centrifuge at 1000 rpm for 5 min, discard the supernatant, resuspend it with DMEM complete medium (containing 10% FBS), transfer it to a culture dish for dilution, and culture it in a 37 °C incubator, changing the medium every 12 h.

[0039] Digestion and collection of HaCaT cells:

[0040] After culturing the cells for 2 - 5 days until they cover the bottom of the dish, discard the medium, wash with PBS, add trypsin to digest for 5 - 10 min, and then terminate the digestion with complete medium. Centrifuge at 1000 rpm for 5 min, discard the supernatant, dilute it with PBS at a low dilution factor, and count it using a hemocytometer. According to the counting result, dilute the resuspension to 10 7 / mL, and store it in a -80 °C refrigerator for later use. For subculture, after centrifugation, dilute it with complete medium and inoculate it into a culture dish at a ratio of 1:3 for culture.

[0041] 5. Model preparation

[0042] Absorb 100 μL of LUV, add it into the insert, and place the insert in a 24-well plate. Centrifuge at 2030 rpm for 5 min. Horizontally rotate the plate 180°. Add another 100 μL of LUV and 50 μL of resuspended HaCaT cell solution (containing 5×10 5 ) cells. After pipetting and mixing evenly, centrifuge at 1500 rpm for 5 min. Take out the insert and place it at 50 °C for 45 min to fuse. After that, add 50 μL of MLV, dry at 50 °C for 40 min, add another 50 μL of MLV, and dry at 50 °C for 60 min. Transfer it to a -80 °C refrigerator overnight (>4 h), take it out, fuse at 50 °C for 2 h, recover at room temperature for 15 min, and store it in a 4 °C refrigerator for later use.

[0043] 6. Preparation of the test solution

[0044] Weigh the drug to be tested and dissolve it in PBS. Adjust the pH with HCl / NaOH if necessary until it is completely dissolved. The concentration of the test solution should be high enough (≥5 mmol / L) to facilitate detection in the receiving solution.

[0045] 7. Preparation of the standard curve

[0046] Gradiently dilute the test solution. Pipette 200 μL of the diluted solution into a 96-well plate and measure the OD value at the maximum absorption wavelength. Plot the standard curve of OD value versus concentration, and use the linear part (r 2 >0.99).

[0047] 8. Permeation detection

[0048] Model detection method: Add 1 mL of PBS solution into a 24-well plate as the receiving solution. Add 100 μL of the test solution into the insert. Every 1 hour, transfer the insert into a well containing fresh PBS. Perform permeation for 8 hours. After that, pipette 200 μL from each well and add it into a 96-well plate, and measure the OD value at the maximum absorption wavelength. Each drug is in triplicate.

[0049] Porcine skin detection method: Install the porcine skin between the dosing chamber and the receiving chamber of the FRANZ diffusion cell according to the OECD Test Guideline 428 method. The receiving chamber contains 8 mL of PBS solution. Add 1 mL of the test solution into the dosing chamber. Every 1 hour, pipette 1 mL of the receiving solution from the receiving chamber and replenish it with an equal volume of fresh PBS solution. Perform permeation for 8 hours. After that, measure the OD value of the pipetted solution at each time point. Each is in triplicate.

[0050] III. Results and data analysis

[0051] Calculate the apparent permeability according to the following formula and report the permeation results:

[0052]

[0053] Wherein:

[0054] J is the cumulative permeation rate over time (mmol / s), calculated by calculating the permeation amount per hour through the standard curve, and calculating the cumulative amount over time as the ordinate and time as the abscissa, and obtained by calculating the slope of the linear part of the stable flux;

[0055] A is the permeation area (model: 0.283 cm 2 ; porcine skin: 1.13 cm 2 );

[0056] C 0 is the concentration of the test product (mmol / L).

[0057] The comparison of the drug permeation rates of the model and porcine skin is shown in the following table. After verification, the model has a significant similarity to porcine skin and can be used to replace porcine skin for permeability testing in vitro.

[0058] Table 1 Detection results of the permeation rates of the model and porcine skin

[0059]

[0060] Table 2 Results of Pearson correlation analysis of the apparent permeation rates of the model and porcine skin (****p < 0.001)

[0061]

[0062] In summary, the present invention provides a skin barrier model for detecting the permeability of cosmetics, and the model has a significant similarity to porcine skin. Therefore, the skin barrier model provided by the present invention can be used to replace porcine skin for permeability testing in vitro.

[0063] The role of the above embodiments is to specifically introduce the substantive content of the present invention, but those skilled in the art should know that the protection scope of the present invention should not be limited to this specific embodiment.

Claims

1. A skin barrier model for cosmetic permeability detection, characterized in that: Prepared as follows: 100 μL of liposome LUV was taken out and added to the Transwell chamber, and the Transwell chamber was placed in a 24-well plate and centrifuged at 2030 rpm for 5 min. The well plate was rotated horizontally 180°, and another 100 μL of liposome LUV and 50 μL of HaCaT cell resuspension were added. After mixing evenly, the mixture was centrifuged at 1500 rpm for 5 min. The Transwell chamber was taken out and fused at 50°C for 45 min. After the mixture was fused, 50 μL of liposome MLV was added and dried at 50°C for 40 min. Another 50 μL of liposome MLV was added and dried at 50°C for 60 min. The mixture was transferred to -80°C and maintained for more than 4 h. The mixture was taken out and fused at 50°C for 2 hours, maintained at room temperature for 15 min, and stored at 4°C. in: The Transwell chamber uses a carrier membrane made of an MCE microporous filter membrane to replace the original bottom carrier membrane, and an EVA adhesive ring is used to bond the Transwell chamber and the carrier membrane made of the MCE microporous filter membrane to form a skin barrier model scaffold; The preparation method of the liposome LUV and liposome MLV is as follows: weighing lecithin and cholesterol in a mass ratio of 77:23 and adding them to a container, dissolving them in a chloroform-methanol mixed solvent in a volume ratio of 2:1, vacuum drying, removing trace solvents, and obtaining a film; adding 10% EtOH-PBS to the container containing the film in a ratio of 1 mL of solvent per 20 mg of the sum of the mass of lecithin and cholesterol, slowly mixing to dissolve the film, stirring at a speed of 100 rpm for hydration reaction at 40° C. in a water bath for 1.5 hours, annealing for 0.5 hours, and obtaining a liposome dispersion; using a filter membrane with a pore size of 800 nm to continuously pass the liposome dispersion 3 times to obtain the liposome MLV, and using a filter membrane with a pore size of 450 nm to continuously pass the liposome dispersion 5 times to obtain the liposome LUV; The cell density of the HaCaT cell resuspension is 10,000 cells / μL, and the solvent is PBS.

2. The skin barrier model according to claim 1, characterized in that: The pore size of the MCE microporous filter membrane is Φ650nm.

3. The skin barrier model according to claim 1, characterized in that: Use a puncher to cut the MCE microporous filter membrane into discs with a diameter that fits the bottom of the Transwell chamber as the carrier membrane of the model. Use a puncher to cut the EVA film into a ring that fits the bottom of the Transwell chamber. Place the MCE microporous filter membrane, EVA adhesive ring and the Transwell chamber with the original bottom carrier membrane removed on the heating plate in sequence and align them. Heat at 90°C for 5 minutes, press the chamber to make the three tightly bonded, and then obtain the skin barrier model scaffold.

4. A method for recycling the skin barrier model scaffold according to claim 3, characterized in that: The MCE microporous filter membrane and EVA adhesive ring at the bottom of the model can be peeled off from the Transwell chamber after the test experiment is completed. The Transwell chamber is first cleaned with the solvent of the sample measured before recovery, and then ultrasonically washed with 95% ethanol and water in sequence, and dried. The clean Transwell chamber can be bonded to the MCE carrier membrane in the same way to prepare a scaffold.

5. Use of the skin barrier model according to any one of claims 1 to 3 in the detection of cosmetic permeability.

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