Ceramide liposome and preparation method and application thereof

Abstract

The application belongs to the technical field of liposomes, and particularly relates to a ceramide liposome, a preparation method and application thereof. The preparation method of the ceramide liposome comprises the following steps: adding hydrogenated lecithin, ceramide 3, cholestanol, cholestanol, tocopherol and polyglycerol-3 polyricinoleate into a mixed solvent of glycerol and ethoxydiglycol, and obtaining an oil phase through homogenization, heating and adding phosphorylcholine polymer; adding calcium chloride-ergothioneine solution into sodium alginate solution to obtain a water phase containing a pre-crosslinked network; adding the water phase into the oil phase to shear to form a colostrum; and obtaining the ceramide liposome through high-pressure homogenization, rapid cooling and filter membrane extrusion. The phosphorylcholine polymer is anchored on the surface to form a biomimetic hydration layer, the water phase pre-crosslinked network, the membrane softness synergistic effect of cholestanol and tocopherol and the rapid cooling fixation process are combined, and the particle size uniformity, the encapsulation efficiency, the storage stability and the transdermal absorption performance of the liposome are significantly improved.

Description

Technical Field

[0001] This invention belongs to the field of liposome technology, specifically relating to a ceramide liposome, its preparation method, and its application. Background Technology

[0002] Nanoliposomes, as delivery carriers with structures highly similar to biological membranes, can simultaneously encapsulate both water-soluble and lipid-soluble active ingredients and are widely used in the cosmetics industry. However, existing liposome technology still faces the following technical bottlenecks: First, transdermal efficiency is insufficient. Traditional liposomes mainly rely on the fusion of phospholipids and stratum corneum lipids to promote the penetration of active ingredients, and the skin delivery effect for water-soluble active ingredients is still not ideal. Second, stability needs to be improved. Liposomes are prone to phospholipid hydrolysis and oxidation, membrane structure damage, and leakage of the encapsulated material during storage. Third, water-soluble active ingredients are prone to recrystallization in the aqueous phase of liposomes. The crystal particles can puncture the liposome membrane, leading to leakage of the encapsulated material and loss of efficacy.

[0003] Chinese patent CN121868153A discloses a recombinant collagen composite nanoliposome, in which polyquaternium-51 is used as an outer coating layer. However, in this scheme, the polymer is located outside the liposome membrane rather than embedded inside the membrane layer, making it difficult to achieve fundamental modification of the membrane structure. Regarding the crystallization problem of ergothioneine, existing technologies mainly use co-crystallization technology to change the molecular arrangement to inhibit crystallization, but co-crystallization partners pose compliance risks related to new raw materials. Furthermore, in the preparation of liposomes with an aqueous phase network, it is often necessary to disperse the aqueous phase in the oil phase to form a W / O type dispersion system. However, conventional phospholipid membrane materials have high HLB values ​​and insufficient ability to stabilize the W / O interface, leading to emulsification failure or low encapsulation efficiency. Therefore, there is an urgent need for a nanoliposome and its preparation method that can simultaneously improve transdermal permeability, inhibit aqueous phase crystallization, and exhibit excellent emulsification stability. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing ceramide liposomes to solve the above-mentioned technical problems. To achieve the above-mentioned technical objectives, the technical solution of the present invention is as follows: A method for preparing ceramide liposomes includes the following steps: S1, Oil Phase: Hydrogenated lecithin, ceramide 3, cholesterol, squalane, tocopherol, and polyglycerol-3 polyricinoleate are added to a mixed solvent of glycerol and ethoxydiethylene glycol and homogenized at 45-50℃ and 3000-4000 rpm for 12-15 min to obtain a homogeneous oil phase; the homogeneous oil phase is heated to 65-75℃ at a rate of 1-2℃ / min, phosphorylcholine polymer is added, and the mixture is stirred for 15-30 min to obtain the oil phase, which is then kept at 65-75℃ for later use; S2, Aqueous phase: While stirring at 500~800 rpm, slowly add calcium chloride-ergothioneine solution dropwise to sodium alginate solution; continue stirring for 10 min to obtain aqueous phase, and keep it at 65~70℃ for later use; S3, colostrum: While the oil phase is sheared at 5000~7000 rpm, the aqueous phase is added to the oil phase at a rate of 2.5~3.5 mL / min, and shearing is continued at 5000~7000 rpm for 5~15 min to obtain the colostrum; S4. Liposome formation: Homogenize the colostrum 2-3 times at 60-65℃ and 50-80MPa, immediately and rapidly cool it to 5-10℃, pre-filter it through a 0.45μm filter membrane, and then extrude it through a 0.22μm filter membrane to obtain ceramide liposomes.

[0005] As a further improvement, in step S1, the amounts of each component by weight are as follows: 5-15 parts hydrogenated lecithin, 0.5-4 parts ceramide 3, 0.2-3 parts cholesterol, 1-5 parts squalane, 0.2-1 parts tocopherol, 0.5-2 parts polyglycerol-3 polyricinoleate, 0.5-2 parts phosphorylcholine polymer, 10-30 parts glycerol, and 1-10 parts ethoxydiethylene glycol.

[0006] As a further improvement, in step S1, the phosphorylcholine polymer is selected from one of polyquaternium-51, polyquaternium-61, and polyquaternium-65; the weight-average molecular weight of the phosphorylcholine polymer is 10,000 to 30,000 Da.

[0007] As a further improvement, in step S2, the calcium chloride-ergothioneine solution is prepared by dissolving calcium chloride in deionized water, adding ergothioneine, and stirring until dissolved. The sodium alginate solution is prepared by dissolving sodium alginate in deionized water. The calcium chloride-ergothioneine solution contains calcium chloride at a mass concentration of 0.01-0.05% and ergothioneine at a mass concentration of 1-2%; sodium alginate solution at a mass concentration of 0.2-0.4%; and the mass ratio of calcium chloride to sodium alginate is 1-3:100.

[0008] As a further improvement, in step S3, the mass ratio of the aqueous phase to the oil phase is 0.8~1.2:1.

[0009] As a further improvement, in step S4, the specific method of rapid cooling is as follows: pour the high-pressure homogenized emulsion into a stainless steel cup, stir at 200~300 rpm, place the stainless steel cup in an ice water bath, cool the emulsion to 5~10℃ within 2 minutes, and continue stirring for 2~5 minutes.

[0010] As a further improvement, in step S4, the extrusion parameters are specifically: extrusion pressure of 100~300psi and extrusion speed of 10~20mL / min.

[0011] Another object of the present invention is to provide a ceramide liposome.

[0012] Another object of the present invention is to provide the application of ceramide liposomes in the preparation of skin care products, cosmetics and medical aesthetic products.

[0013] Due to the adoption of the above technical solution, the beneficial effects of the present invention are as follows: 1. This invention anchors phosphorylcholine polymers onto the outer surface of liposome membranes, forming a stable biomimetic hydration layer that significantly enhances the anti-aggregation stability of liposomes. Furthermore, the hydrophilic phosphorylcholine side chains promote transdermal absorption of active ingredients. Through a phase change temperature control process, combined with the membrane compliance effect of squalane and the membrane stabilizing effect of tocopherol, the membrane stress generated during polymer surface anchoring is synergistically reduced, ensuring stable anchoring of the phosphorylcholine polymers to the liposome surface and overcoming the defect of easy polymer detachment in traditional physical mixing methods.

[0014] 2. This invention constructs a pre-crosslinked network of sodium alginate-calcium ions that allows flow, physically encapsulating ergothionein within the network pores. This network effectively inhibits the free migration and recrystallization of ergothionein molecules during storage through spatial confinement, solving the technical problem of easy crystallization and precipitation of water-soluble active ingredients in the aqueous phase of liposomes, without altering the molecular structure of ergothionein, thus avoiding compliance risks associated with new raw materials. Attached Figure Description

[0015] Figure 1 These are the results of in vitro transdermal experiments for Example 1 and Comparative Examples 1-5; Figure 2 These are cryo-transmission electron microscope (CTEM) images of Example 1 and Comparative Example 5; wherein, Figure a is a CTEM image of Example 1 and Figure b is a CTEM image of Comparative Example 5. Figure 3 These are the TNF-α level detection results for Example 1 and Comparative Examples 1-5; Figure 4 These are the NO level detection results for Example 1 and Comparative Examples 1-5; Figure 5 The graph shows the FLG recovery rate test results for Example 1 and Comparative Examples 1-5. Detailed Implementation

[0016] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. However, those skilled in the art will understand that the embodiments described below are some embodiments of the present invention, but not all embodiments, and are only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Where specific conditions are not specified in the embodiments, conventional conditions or manufacturer's conditions shall be followed. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0017] Example 1 A method for preparing ceramide liposomes, comprising the following steps: 1. Accurately weigh the following raw materials: 8g hydrogenated lecithin, 2g ceramide 3, 1g cholesterol, 3g squalane, 0.5g tocopherol, and 1g polyglycerol-3 polyricinoleate; in a separate beaker, add 20g glycerol and 5g ethoxydiethylene glycol, mix well to obtain a mixed solvent; add the weighed raw materials to the mixed solvent and stir evenly; homogenize at 48℃ and 3500rpm for 13min to obtain a homogeneous oil phase; heat the homogeneous oil phase to 70℃ at a rate of 1.5℃ / min, add 1g polyquaternium-51 (weight average molecular weight of 10kDa), keep warm and stir for 20min to obtain the oil phase, and keep it at 70℃ for later use; 2. At room temperature, add sodium alginate to deionized water and stir continuously until completely dissolved to prepare a sodium alginate solution with a mass concentration of 0.3%. Dissolve calcium chloride in deionized water, add ergothioneine, and stir until dissolved. The mass concentration of calcium chloride is 0.03%, and the mass concentration of ergothioneine is 1.5%, resulting in a calcium chloride-ergothioneine solution. The mass ratio of calcium chloride to sodium alginate is 1:50. Place the beaker containing the sodium alginate solution on a magnetic stirrer, set the speed to 650 rpm, and slowly add the calcium chloride-ergothioneine solution dropwise to the sodium alginate solution at a rate of 0.5 mL per minute. Keep stirring during the dropwise addition. After the dropwise addition is completed, continue stirring for 10 minutes to obtain a flowable clear sol, i.e., the aqueous phase, and keep it at 68℃ for later use. 3. The oil phase was sheared using a high-speed shear dispersion emulsifier at a speed of 6000 rpm. Under this shearing condition, the aqueous phase was slowly added dropwise to the oil phase at a rate of 3 mL / min. After the addition was completed, the shearing was continued at 6000 rpm for 10 min to ensure full dispersion and obtain the primary emulsion. The mass ratio of the aqueous phase to the oil phase was 1:1. 4. Transfer the colostrum to a high-pressure homogenizer, set the homogenization pressure to 60 MPa, and homogenize three times. During homogenization, maintain the emulsion at 62°C using a circulating water bath. After homogenization, measure the average particle size. Immediately pour the homogenized emulsion into a stainless steel cup, stir at 250 rpm, place the stainless steel cup in an ice-water bath, cool the emulsion to 8°C within 2 minutes, and continue stirring for 3 minutes to obtain a liposome emulsion. Assemble a filter membrane extruder, pre-filter the rapidly cooled liposome emulsion through a 0.45 μm filter membrane, and then extrude it through a 0.22 μm filter membrane. Control the extrusion pressure between 100 and 300 psi, and the extrusion speed at 15 mL / min. If the pressure is too high during extrusion, stop and clean the membrane before continuing extrusion. Obtain ceramide liposomes.

[0018] Example 2 A method for preparing ceramide liposomes, comprising the following steps: 1. Accurately weigh the following raw materials: 5g hydrogenated lecithin, 0.5g ceramide 3, 0.2g cholesterol, 1g squalane, 0.2g tocopherol, and 0.5g polyglycerol-3 polyricinoleate; in another beaker, add 30g glycerol and 10g ethoxydiethylene glycol, mix well to obtain a mixed solvent; add the weighed raw materials to the mixed solvent and stir evenly; homogenize at 45℃ and 4000rpm for 12min to obtain a homogeneous oil phase; heat the homogeneous oil phase to 65℃ at a rate of 1℃ / min, add 0.5g polyquaternium-61 (weight average molecular weight of 20kDa), keep warm and stir for 30min to obtain the oil phase, and keep it at 65℃ for later use; 2. At room temperature, add sodium alginate to deionized water and stir continuously until completely dissolved to prepare a sodium alginate solution with a mass concentration of 0.2%. Dissolve calcium chloride in deionized water, add ergothioneine, and stir until dissolved. The mass concentration of calcium chloride is 0.01%, and the mass concentration of ergothioneine is 1%, resulting in a calcium chloride-ergothioneine solution. The mass ratio of calcium chloride to sodium alginate is 3:100. Place the beaker containing the sodium alginate solution on a magnetic stirrer, set the speed to 500 rpm, and slowly add the calcium chloride-ergothioneine solution dropwise to the sodium alginate solution at a rate of 1 mL per minute. Keep stirring during the dropwise addition. After the dropwise addition is completed, continue stirring for 10 minutes to obtain a flowable clear sol, i.e., the aqueous phase, and keep it at 65°C for later use. 3. The oil phase was sheared using a high-speed shear dispersion emulsifier at a speed of 7000 rpm. Under this shearing condition, the aqueous phase was slowly added dropwise to the oil phase at a rate of 2.5 mL / min. After the addition was completed, the shearing was continued at 7000 rpm for 5 min to ensure sufficient dispersion and obtain the primary emulsion. The mass ratio of the aqueous phase to the oil phase was 1.2:1. 4. Transfer the colostrum to a high-pressure homogenizer, set the homogenization pressure to 50 MPa, and homogenize three times. During homogenization, maintain the emulsion at 65°C using a circulating water bath. After homogenization, measure the average particle size. Immediately pour the homogenized emulsion into a stainless steel cup, stir at 200 rpm, place the stainless steel cup in an ice-water bath, and cool the emulsion to 10°C within 2 minutes, continuing to stir for another 2 minutes to obtain a liposome emulsion. Assemble a filter membrane extruder, pre-filter the rapidly cooled liposome emulsion through a 0.45 μm filter membrane, and then extrude it through a 0.22 μm filter membrane. Control the extrusion pressure between 100 and 300 psi, and the extrusion speed at 10 mL / min. If the pressure is too high during extrusion, stop and clean the membrane before continuing extrusion. Obtain ceramide liposomes.

[0019] Example 3 A method for preparing ceramide liposomes, comprising the following steps: 1. Accurately weigh the following raw materials: 15g hydrogenated lecithin, 4g ceramide 3, 3g cholesterol, 5g squalane, 1g tocopherol, and 2g polyglycerol-3 polyricinoleate; in another beaker, add 10g glycerol and 1g ethoxydiethylene glycol, mix well to obtain a mixed solvent; add the weighed raw materials to the mixed solvent and stir evenly; homogenize at 50℃ and 3000rpm for 15min to obtain a homogeneous oil phase; heat the homogeneous oil phase to 75℃ at a rate of 2℃ / min, add 2g polyquaternium-65 (weight average molecular weight of 30kDa), keep warm and stir for 15min to obtain the oil phase, and keep warm at 75℃ for later use; 2. At room temperature, add sodium alginate to deionized water and stir continuously until completely dissolved to prepare a sodium alginate solution with a mass concentration of 0.4%. Dissolve calcium chloride in deionized water, add ergothioneine, and stir until dissolved. The mass concentration of calcium chloride is 0.05%, and the mass concentration of ergothioneine is 2%, resulting in a calcium chloride-ergothioneine solution. The mass ratio of calcium chloride to sodium alginate is 1:100. Place the beaker containing the sodium alginate solution on a magnetic stirrer, set the speed to 800 rpm, and slowly add the calcium chloride-ergothioneine solution dropwise to the sodium alginate solution at a rate of 1 mL per minute. Keep stirring during the dropwise addition. After the dropwise addition is completed, continue stirring for 10 minutes to obtain a flowable clear sol, i.e., the aqueous phase, and keep it at 70°C for later use. 3. The oil phase was sheared using a high-speed shear dispersion emulsifier at a speed of 5000 rpm. Under this shearing condition, the aqueous phase was slowly added dropwise to the oil phase at a rate of 3.5 mL / min. After the addition was completed, the shearing was continued at 5000 rpm for 15 min to ensure sufficient dispersion and obtain the primary emulsion. The mass ratio of the aqueous phase to the oil phase was 0.8:1. 4. Transfer the colostrum to a high-pressure homogenizer, set the homogenization pressure to 80 MPa, and homogenize twice. During homogenization, maintain the emulsion at 60°C using a circulating water bath. After homogenization, measure the average particle size. Immediately pour the homogenized emulsion into a stainless steel cup, stir at 300 rpm, place the stainless steel cup in an ice-water bath, cool the emulsion to 5°C within 2 minutes, and continue stirring for 5 minutes to obtain a liposome emulsion. Assemble a filter membrane extruder, pre-filter the rapidly cooled liposome emulsion through a 0.45 μm filter membrane, and then extrude it through a 0.22 μm filter membrane. Control the extrusion pressure between 100 and 300 psi, and the extrusion speed at 20 mL / min. If the pressure is too high during extrusion, stop and clean the membrane before continuing extrusion. Obtain ceramide liposomes.

[0020] Comparative Example 1: A method for preparing ceramide liposomes, differing from Example 1 in that it does not use phosphorylcholine polymer, i.e., polyquaternium-51, and includes the following steps: 1. Accurately weigh the following raw materials: 8g hydrogenated lecithin, 2g ceramide 3, 1g cholesterol, 3g squalane, 0.5g tocopherol, and 1g polyglycerol-3 polyricinoleate; in another beaker, add 20g glycerol and 5g ethoxydiethylene glycol, mix well to obtain a mixed solvent; add the weighed raw materials to the mixed solvent and stir evenly; homogenize at 48℃ and 3500rpm for 13min to obtain a homogeneous oil phase; heat the homogeneous oil phase to 70℃ at a rate of 1.5℃ / min to obtain the oil phase, and keep it at 70℃ for later use; 2. At room temperature, add sodium alginate to deionized water and stir continuously until completely dissolved to prepare a sodium alginate solution with a mass concentration of 0.3%. Dissolve calcium chloride in deionized water, add ergothioneine, and stir until dissolved. The mass concentration of calcium chloride is 0.03%, and the mass concentration of ergothioneine is 1.5%, resulting in a calcium chloride-ergothioneine solution. The mass ratio of calcium chloride to sodium alginate is 1:50. Place the beaker containing the sodium alginate solution on a magnetic stirrer, set the speed to 650 rpm, and slowly add the calcium chloride-ergothioneine solution dropwise to the sodium alginate solution at a rate of 0.5 mL per minute. Keep stirring during the dropwise addition. After the dropwise addition is completed, continue stirring for 10 minutes to obtain a flowable clear sol, i.e., the aqueous phase, and keep it at 68℃ for later use. 3. The oil phase was sheared using a high-speed shear dispersion emulsifier at a speed of 6000 rpm. Under this shearing condition, the aqueous phase was slowly added dropwise to the oil phase at a rate of 3 mL / min. After the addition was completed, the shearing was continued at 6000 rpm for 10 min to ensure full dispersion and obtain the primary emulsion. The mass ratio of the aqueous phase to the oil phase was 1:1. 4. Transfer the colostrum to a high-pressure homogenizer, set the homogenization pressure to 60 MPa, and homogenize three times. During homogenization, maintain the emulsion at 62°C using a circulating water bath. After homogenization, measure the average particle size. Immediately pour the homogenized emulsion into a stainless steel cup, stir at 250 rpm, place the stainless steel cup in an ice-water bath, cool the emulsion to 8°C within 2 minutes, and continue stirring for 3 minutes to obtain a liposome emulsion. Assemble a filter membrane extruder, pre-filter the rapidly cooled liposome emulsion through a 0.45 μm filter membrane, and then extrude it through a 0.22 μm filter membrane. Control the extrusion pressure between 100 and 300 psi, and the extrusion speed at 15 mL / min. If the pressure is too high during extrusion, stop and clean the membrane before continuing extrusion. Obtain ceramide liposomes.

[0021] Comparative Example 2: A method for preparing ceramide liposomes, differing from Example 1 in that sodium alginate and calcium chloride are not added, and includes the following steps: 1. Accurately weigh the following raw materials: 8g hydrogenated lecithin, 2g ceramide 3, 1g cholesterol, 3g squalane, 0.5g tocopherol, and 1g polyglycerol-3 polyricinoleate; in another beaker, add 20g glycerol and 5g ethoxydiethylene glycol, mix well to obtain a mixed solvent; add the weighed raw materials to the mixed solvent and stir evenly; homogenize at 48℃ and 3500rpm for 13min to obtain a homogeneous oil phase; heat the homogeneous oil phase to 70℃ at a rate of 1.5℃ / min, add 1g polyquaternium-51 (weight average molecular weight of 10kDa), keep warm and stir for 20min to obtain the oil phase, and keep it at 70℃ for later use; 2. At room temperature, add ergothioneine to deionized water and stir until completely dissolved to prepare an aqueous solution with a 1.5% ergothioneine mass concentration (i.e., the aqueous phase). Keep it at 68℃ for later use. 3. The oil phase was sheared using a high-speed shear dispersion emulsifier at a speed of 6000 rpm. Under this shearing condition, the aqueous phase was slowly added dropwise to the oil phase at a rate of 3 mL / min. After the addition was completed, the shearing was continued at 6000 rpm for 10 min to ensure full dispersion and obtain the primary emulsion. The mass ratio of the aqueous phase to the oil phase was 1:1. 4. Transfer the colostrum to a high-pressure homogenizer, set the homogenization pressure to 60 MPa, and homogenize three times. During homogenization, maintain the emulsion at 62°C using a circulating water bath. After homogenization, measure the average particle size. Immediately pour the homogenized emulsion into a stainless steel cup, stir at 250 rpm, place the stainless steel cup in an ice-water bath, cool the emulsion to 8°C within 2 minutes, and continue stirring for 3 minutes to obtain a liposome emulsion. Assemble a filter membrane extruder, pre-filter the rapidly cooled liposome emulsion through a 0.45 μm filter membrane, and then extrude it through a 0.22 μm filter membrane. Control the extrusion pressure between 100 and 300 psi, and the extrusion speed at 15 mL / min. If the pressure is too high during extrusion, stop and clean the membrane before continuing extrusion. Obtain ceramide liposomes.

[0022] Comparative Example 3: A method for preparing ceramide liposomes, differing from Example 1 in that it does not contain squalane and tocopherol, and includes the following steps: 1. Accurately weigh the following raw materials: 8g hydrogenated lecithin, 2g ceramide 3, 1g cholesterol, and 1g polyglycerol-3 polyricinoleate; in another beaker, add 20g glycerol and 5g ethoxydiethylene glycol, mix well to obtain a mixed solvent; add the weighed raw materials to the mixed solvent and stir evenly; homogenize at 48℃ and 3500rpm for 13min to obtain a homogeneous oil phase; heat the homogeneous oil phase to 70℃ at a rate of 1.5℃ / min, add 1g polyquaternium-51 (weight average molecular weight of 10kDa), keep warm and stir for 20min to obtain the oil phase, and keep it at 70℃ for later use; 2. At room temperature, add sodium alginate to deionized water and stir continuously until completely dissolved to prepare a sodium alginate solution with a mass concentration of 0.3%. Dissolve calcium chloride in deionized water, add ergothioneine, and stir until dissolved. The mass concentration of calcium chloride is 0.03%, and the mass concentration of ergothioneine is 1.5%, resulting in a calcium chloride-ergothioneine solution. The mass ratio of calcium chloride to sodium alginate is 1:50. Place the beaker containing the sodium alginate solution on a magnetic stirrer, set the speed to 650 rpm, and slowly add the calcium chloride-ergothioneine solution dropwise to the sodium alginate solution at a rate of 0.5 mL per minute. Keep stirring during the dropwise addition. After the dropwise addition is completed, continue stirring for 10 minutes to obtain a flowable clear sol, i.e., the aqueous phase, and keep it at 68℃ for later use. 3. The oil phase was sheared using a high-speed shear dispersion emulsifier at a speed of 6000 rpm. Under this shearing condition, the aqueous phase was slowly added dropwise to the oil phase at a rate of 3 mL / min. After the addition was completed, the shearing was continued at 6000 rpm for 10 min to ensure full dispersion and obtain the primary emulsion. The mass ratio of the aqueous phase to the oil phase was 1:1. 4. Transfer the colostrum to a high-pressure homogenizer, set the homogenization pressure to 60 MPa, and homogenize three times. During homogenization, maintain the emulsion at 62°C using a circulating water bath. After homogenization, measure the average particle size. Immediately pour the homogenized emulsion into a stainless steel cup, stir at 250 rpm, place the stainless steel cup in an ice-water bath, cool the emulsion to 8°C within 2 minutes, and continue stirring for 3 minutes to obtain a liposome emulsion. Assemble a filter membrane extruder, pre-filter the rapidly cooled liposome emulsion through a 0.45 μm filter membrane, and then extrude it through a 0.22 μm filter membrane. Control the extrusion pressure between 100 and 300 psi, and the extrusion speed at 15 mL / min. If the pressure is too high during extrusion, stop and clean the membrane before continuing extrusion. Obtain ceramide liposomes.

[0023] Comparative Example 4: A method for preparing ceramide liposomes, differing from Example 1 in that it omits the rapid cooling step and includes the following steps: 1. Accurately weigh the following raw materials: 8g hydrogenated lecithin, 2g ceramide 3, 1g cholesterol, 3g squalane, 0.5g tocopherol, and 1g polyglycerol-3 polyricinoleate; in another beaker, add 20g glycerol and 5g ethoxydiethylene glycol, mix well to obtain a mixed solvent; add the weighed raw materials to the mixed solvent and stir evenly; homogenize at 48℃ and 3500rpm for 13min to obtain a homogeneous oil phase; heat the homogeneous oil phase to 70℃ at a rate of 1.5℃ / min, add 1g polyquaternium-51 (weight average molecular weight of 10kDa), keep warm and stir for 20min to obtain the oil phase, and keep it at 70℃ for later use; 2. At room temperature, add sodium alginate to deionized water and stir continuously until completely dissolved to prepare a sodium alginate solution with a mass concentration of 0.3%. Dissolve calcium chloride in deionized water, add ergothioneine, and stir until dissolved. The mass concentration of calcium chloride is 0.03%, and the mass concentration of ergothioneine is 1.5%, resulting in a calcium chloride-ergothioneine solution. The mass ratio of calcium chloride to sodium alginate is 1:50. Place the beaker containing the sodium alginate solution on a magnetic stirrer, set the speed to 650 rpm, and slowly add the calcium chloride-ergothioneine solution dropwise to the sodium alginate solution at a rate of 0.5 mL per minute. Keep stirring during the dropwise addition. After the dropwise addition is completed, continue stirring for 10 minutes to obtain a flowable clear sol, i.e., the aqueous phase, and keep it at 68℃ for later use. 3. The oil phase was sheared using a high-speed shear dispersion emulsifier at a speed of 6000 rpm. Under this shearing condition, the aqueous phase was slowly added dropwise to the oil phase at a rate of 3 mL / min. After the addition was completed, the shearing was continued at 6000 rpm for 10 min to ensure full dispersion and obtain the primary emulsion. The mass ratio of the aqueous phase to the oil phase was 1:1. 4. Transfer the colostrum to a high-pressure homogenizer, set the homogenization pressure to 60 MPa, and homogenize three times. During homogenization, maintain the emulsion at 62°C using a circulating water bath. After homogenization, measure the average particle size. Allow the homogenized emulsion to cool to room temperature to obtain a liposome emulsion. Assemble a membrane extruder, pre-filter the rapidly cooled liposome emulsion through a 0.45 μm membrane, and then extrude it through a 0.22 μm membrane. Control the extrusion pressure between 100 and 300 psi and the extrusion speed at 15 mL / min. If the pressure is too high during extrusion, stop and clean the membrane before continuing extrusion. Obtain ceramide liposomes.

[0024] Comparative Example 5: A method for preparing ceramide liposomes, differing from Example 1 in that the phosphorylcholine polymer is added after the formation of the colostrum, comprising the following steps: 1. Accurately weigh the following raw materials: 8g hydrogenated lecithin, 2g ceramide 3, 1g cholesterol, 3g squalane, 0.5g tocopherol, and 1g polyglycerol-3 polyricinoleate; in another beaker, add 20g glycerol and 5g ethoxydiethylene glycol, mix well to obtain a mixed solvent; add the weighed raw materials to the mixed solvent and stir evenly; homogenize at 48℃ and 3500rpm for 13min to obtain a homogeneous oil phase; heat the homogeneous oil phase to 70℃ at a rate of 1.5℃ / min, stir for 20min to obtain the oil phase, and keep it at 70℃ for later use; 2. At room temperature, add sodium alginate to deionized water and stir continuously until completely dissolved to prepare a sodium alginate solution with a mass concentration of 0.3%. Dissolve calcium chloride in deionized water, add ergothioneine, and stir until dissolved. The mass concentration of calcium chloride is 0.03%, and the mass concentration of ergothioneine is 1.5%, resulting in a calcium chloride-ergothioneine solution. The mass ratio of calcium chloride to sodium alginate is 1:50. Place the beaker containing the sodium alginate solution on a magnetic stirrer, set the speed to 650 rpm, and slowly add the calcium chloride-ergothioneine solution dropwise to the sodium alginate solution at a rate of 0.5 mL per minute. Keep stirring during the dropwise addition. After the dropwise addition is completed, continue stirring for 10 minutes to obtain a flowable clear sol, i.e., the aqueous phase, and keep it at 68℃ for later use. 3. The oil phase was sheared using a high-speed shear dispersion emulsifier at a speed of 6000 rpm. Under this shearing condition, the aqueous phase was slowly added dropwise to the oil phase at a rate of 3 mL / min. After the addition was completed, the shearing was continued at 6000 rpm for 10 min to ensure full dispersion and obtain the primary emulsion. The mass ratio of the aqueous phase to the oil phase was 1:1. 4. Add 1g of polyquaternium-51 (weight average molecular weight of 10kDa) to the colostrum and stir at 3000rpm for 5min to initially disperse the polymer; transfer to a high-pressure homogenizer, set the homogenization pressure to 60MPa, and homogenize 3 times. During homogenization, use a circulating water bath to maintain the emulsion at 62℃; after homogenization, measure the average particle size; immediately pour the homogenized emulsion into a stainless steel cup, stir at 250rpm, place the stainless steel cup in an ice water bath, cool the emulsion to 8℃ within 2min, and continue stirring for 3min to obtain a liposome emulsion; assemble a filter membrane extruder, pre-filter the rapidly cooled liposome emulsion through a 0.45μm filter membrane, and then extrude it through a 0.22μm filter membrane. Control the extrusion pressure between 100~300psi and the extrusion speed at 15mL / min. If the pressure is too high during extrusion, stop and clean the membrane before continuing extrusion; obtain ceramide liposomes.

[0025] Performance testing 1. Average particle size, PDI The average particle size and polydispersity index (PDI) of the samples prepared in Examples 1-3 and Comparative Examples 1-5 were determined to evaluate the dispersibility and uniformity of liposomes.

[0026] Take 1 mL of each group of samples and dilute it 100 times with deionized water. The turbidity of the diluted samples should meet the requirements of the instrument. Use a dynamic light scattering particle size analyzer to measure the particle size at a temperature of 25℃. Measure each group of samples three times and take the average value as the final result. Record the average particle size and polydispersity index (PDI). The test results are shown in Table 1: Table 1. Average particle size and PDI results As can be seen from Table 1, the average particle size of the ceramide liposomes prepared in Examples 1 to 3 is similar, and the PDI is between 0.19 and 0.22, indicating that the liposomes prepared by the present invention have uniform particle size and good dispersibility, which meets the quality requirements of nanoliposomes.

[0027] Compared to the examples, Comparative Example 1 showed a larger average particle size, higher PDI, and wider particle size distribution. Due to the lack of polymer surface anchoring in Comparative Example 1, the liposomes lacked the steric hindrance protection of the hydration layer, making them prone to aggregation and fusion during preparation and storage. Comparative Example 2 had similar average particle size and PDI to the examples, indicating that the addition of sodium alginate and calcium chloride had little effect on liposome particle size and dispersibility. Comparative Example 3 showed a significantly increased average particle size and a noticeably wider particle size distribution. Due to the lack of membrane compliance effect from squalane, membrane stress could not be effectively released during polymer surface anchoring, leading to membrane bridging. The structure was disordered; at the same time, the lack of tocopherol reduced the stability of the membrane interface, causing liposomes to aggregate and the particle size to increase significantly; in Comparative Example 4, no rapid cooling method was used. During the natural cooling process, the liposome membrane slowly changed from the liquid crystal state to the gel state. The anchored polymer chains partially desorbed or rearranged, resulting in the membrane structure not being firmly locked, some liposomes aggregated, and the particle size was not uniform; in Comparative Example 5, since the polymer did not undergo an anchoring process and only existed in a physical mixing manner, some polymers were free or loosely adsorbed on the surface of the liposomes, failing to form a uniform and firm biomimetic hydration layer, resulting in a wider particle size distribution.

[0028] 2. Encapsulation efficiency of ergothioneine The encapsulation efficiency of ergothionein in the samples prepared in Examples 1-3 and Comparative Examples 1-5 was determined.

[0029] Take 1 mL of each group of liposome samples and add them to an ultrafiltration centrifuge tube with a molecular weight cutoff of 10 kDa. Centrifuge at 4000 rpm for 30 min, collect the throttling liquid in the ultrafiltration tube, rinse the ultrafiltration tube with deionized water, combine the rinsing liquid with the throttling liquid, and make up to 1 mL. Add an equal volume of ethanol, vortex for 1 min, and sonicate for 10 min to obtain the demulsified throttling liquid. Ergothioneine content was determined by high performance liquid chromatography (HPLC). Chromatographic conditions: C18 column (4.6 mm × 250 mm, 5 μm), mobile phase: methanol:water (containing 0.1% formic acid) = 5:95, flow rate 1.0 mL / min, detection wavelength 254 nm, column temperature 30 ℃; ergothioneine standard solutions (1~100 μg / mL) were prepared and a standard curve was plotted.

[0030] The concentration of ergothioneine in the retrieval fluid after demulsification was determined by HPLC, and the ergothioneine content was calculated and recorded as the ergothioneine encapsulation amount. In addition, 1 mL of liposome samples from each group were demulsified in the same way, and the ergothioneine content was determined by HPLC and recorded as the total ergothioneine. Calculate the encapsulation ratio: Encapsulation percentage (%) = (Ergothioneine encapsulation amount / Total ergothioneine) × 100% The experimental results are shown in Table 2: Table 2. Encapsulation efficiency test results of ergothioneine As can be seen from Table 2, the ergothioneine encapsulation efficiency of the ceramide liposomes prepared in Examples 1-3 was between 92.4% and 93.6%, which was higher than that of all comparative examples, indicating that the liposomes prepared in this invention have excellent encapsulation capacity for ergothioneine.

[0031] The encapsulation efficiency of Comparative Example 1 was lower than that of Example 1, indicating that the absence of polymers leads to decreased liposome membrane stability, resulting in leakage of some loaded material during homogenization and ultrafiltration, and a reduced encapsulation efficiency. In Comparative Example 2, the absence of the crosslinking network in the preparation method resulted in ergothioneine existing only in a free state within the aqueous phase of the liposomes, making it prone to leakage during ultrafiltration and centrifugation, leading to a significant decrease in encapsulation efficiency. Comparative Example 3 lacked squalane and tocopherol, resulting in decreased membrane flexibility and stability. When the polymer was anchored on the surface, membrane stress could not be released, leading to defects in the membrane structure and leakage of some loaded material. In Comparative Example 4, the membrane structure was not firmly locked during natural cooling, and some anchored polymer chains desorbed, resulting in decreased liposome membrane integrity and a reduced encapsulation efficiency. In Comparative Example 5, the phosphorylcholine polymer existed only physically mixed on the outside of the liposomes and could not be effectively anchored to the liposome membrane surface, causing membrane structure disturbance during homogenization and ultrafiltration, resulting in significant leakage of the loaded material.

[0032] 3. Storage stability The liposome samples prepared in Examples 1-3 and Comparative Examples 1-5 were aliquoted into 5mL light-proof glass bottles, with 3mL in each bottle. The bottles were placed at 55°C for accelerated stability testing. Samples were taken on day 60 for visual and optical microscopic observation.

[0033] Visual observation: At each sampling time point, place the sample bottle against a white or black background and observe it directly under light. Record whether there is any precipitation, crystals or turbidity.

[0034] Optical microscopy observation: 10 μL of samples from each time point were dropped onto a glass slide, covered with a coverslip, and observed under a 400x optical microscope. The presence of ergothioneine crystals was observed and recorded. Three fields of view were observed for each sample. The experimental results are shown in Table 3. Table 3. Observation results of storage stability As can be seen from Table 3, after 60 days of storage at 55°C under accelerated conditions, Examples 1-3 were all clear and transparent, without precipitation or turbidity, as observed visually. The field of view was clean and there were no ergothionein crystals, indicating that the ceramide liposomes prepared by this invention have excellent storage stability.

[0035] Comparative Example 1 showed slight turbidity, with no crystals visible under the microscope but liposome aggregation. Due to the lack of polymer surface anchoring, the liposome surface lacked steric hindrance protection, making it prone to aggregation at high temperatures. Comparative Example 2 showed a large amount of precipitation, with numerous crystal aggregations visible under the microscope. This was because the absence of the cross-linking network caused ergothioneine to recrystallize rapidly at high temperatures, forming numerous visible crystal aggregations. Comparative Example 3 showed slight turbidity, with partial rupture of liposomes visible under the microscope. The loss of the membrane compliance effect of squalane and the membrane stabilizing effect of tocopherol led to an increase in membrane rigidity or a decrease in stability. The temperature drop caused the liposomes to rupture at high temperatures; in Comparative Example 4, a small amount of precipitation was observed, and a few fine crystals were visible under a microscope. During natural cooling, the membrane structure was not firmly locked, and the aqueous pre-crosslinked network partially dissociated, resulting in the precipitation of trace amounts of ergothioneine crystals after long-term storage; Comparative Example 5 showed a clear and transparent appearance, without precipitation, turbidity, or crystals. This was because Comparative Example 5 retained the sodium alginate-calcium ion pre-crosslinked network in the aqueous phase, which effectively inhibited the molecular migration and recrystallization of ergothioneine, and the membrane structure was relatively intact, thus producing the same observation results as the examples.

[0036] 4. In vitro transdermal test The ceramide liposome samples prepared in Example 1 and Comparative Examples 1-5 were subjected to in vitro transdermal tests.

[0037] Fresh pig ear skin was used as the experimental subject. Subcutaneous fat was removed, and the skin was washed with physiological saline. The pig ear skin was fixed in a Franz diffusion cell with the stratum corneum facing upwards. 6 mL of receiving solution (pH 7.4 phosphate buffer containing 0.1% sodium azide) preheated to 32°C was added to the receiving cell. During the experiment, the receiving cell was maintained at 32°C and magnetically stirred at 600 rpm. 0.5 mL of each sample was taken and evenly spread on the stratum corneum of the pig ear skin. The supply cell was then covered with sealing film. At 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h, 1 mL of the receiving cell was collected, and 1 mL of freshly prepared receiving solution was added. After sampling, the samples were centrifuged at 12000 rpm for 10 min at 4°C. The supernatant was filtered through a 0.22 μm filter membrane, and the ergothioneine content was determined using high-performance liquid chromatography (HPLC). The ergothioneine permeate at each time point was calculated, and a line graph was plotted.

[0038] The results are as follows Figure 1 As shown, from Figure 1As can be seen, the cumulative permeation of the ceramide liposomes prepared in Example 1 was significantly higher than that of the comparative examples at each time point. Comparative Example 1 had the worst transdermal performance because it lacked phosphorylcholine polymer and could not form a biomimetic hydration layer. Comparative Example 5 also had limited transdermal promotion effect because the polymer did not undergo a surface anchoring process. Although Comparative Examples 2-4 retained phosphorylcholine polymer and had a higher transdermal effect than Comparative Examples 1 and 5, their transdermal performance was lower than that of Example 1 because they lacked a pre-crosslinked network, membrane compliance components, or rapid cooling fixation process.

[0039] The results of this experiment show that the biomimetic hydration layer formed by anchoring phosphorylcholine polymer surface in this invention can significantly promote transdermal absorption of ergothionein, and this effect depends on the combined effect of multiple technical features such as anchoring process, membrane compliance synergy and rapid cooling fixation.

[0040] 5. Dialysis-bound stability test of phosphorycholine polymer The ceramide liposome samples prepared in Example 1 and Comparative Examples 3-5 were subjected to a phosphorylcholine polymer dialysis binding stability test.

[0041] Dialysis was performed using a regenerated cellulose dialysis bag with a molecular weight cutoff of 100 kDa. 10 mL of each of the samples from Example 1 and Comparative Examples 3-5 were added to the pretreated dialysis bags, sealed, and placed in 500 mL of dialysis buffer (pH 7.4 phosphate buffer containing 0.02% sodium azide). The dialysis buffer was stirred at 200 rpm at room temperature, and the dialysis buffer was replaced with fresh dialysis buffer at 4 h, 8 h, 12 h, 16 h, and 24 h after dialysis. The total dialysis time was 24 h.

[0042] After dialysis, the throttled liquid in the bag was taken and centrifuged at 12,000 rpm for 10 min. The supernatant was taken as the experimental group. The undialyzed samples of Example 1 and Comparative Examples 3-5 were taken as the control group. The polyquaternium-51 content of the experimental group and the control group samples were determined by high performance liquid chromatography (HPLC). Each sample was measured in parallel 3 times, and the average value was taken as the final result.

[0043] Calculate the retention rate of phosphorylcholine polymer: Retention rate (%) = (content of polyquaternium-51 in experimental group / content of polyquaternium-51 in control group) × 100%; The test results are shown in Table 4.

[0044] Table 4. Results of dialysis binding stability experiments of phosphorycholine polymers As shown in Table 4, the phosphorylcholine polymer retention rate of Example 1 was 92.5%, significantly higher than that of the comparative examples. This indicates that in the ceramide liposomes prepared by this invention, polyquaternium-51 is anchored on the liposome surface, and most of the polymer is retained after dialysis. In Comparative Example 3, the absence of squalane and tocopherol led to a decrease in membrane flexibility and stability, resulting in ineffective release of membrane stress during polymer anchoring and weak polymer bonding. In Comparative Example 4, natural cooling treatment caused the liposome membrane to slowly transition from a liquid crystal state to a gel state, resulting in partial desorption or rearrangement of the anchored polymer chains and a decrease in retention rate after dialysis. Comparative Example 5 showed the lowest retention rate, as the polymers did not undergo the phase change temperature-controlled anchoring process during the oil phase heating stage and existed only in a physical mixing manner or formed loose adsorption on the liposome surface. Most of the polymers were removed during dialysis, demonstrating that the method in Comparative Example 5 could not achieve effective polymer anchoring.

[0045] 6. Cryo-transmission electron microscopy To confirm the anchoring state of the phosphorylcholine polymer and its effect on the surface structure of liposomes, cryo-transmission electron microscopy (Cryo-TEM) was performed on the samples of Example 1 and Comparative Example 5.

[0046] Experimental results are as follows Figure 2 As shown, Figure a is an image of Example 1, and Figure b is an image of Comparative Example 5; from Figure 2 As can be seen, the liposomes of Example 1 exhibit a regular spherical vesicle morphology, and the lipid bilayer presents a clear, continuous, and smooth thin film structure; that is, a thin and uniform halo (red arrow) is visible on the surface of the liposomes. This halo is continuously and uniformly spread on the outer surface of the liposomes, with a consistent thickness and no local thickening or exposed areas. This morphological feature indicates that the phosphorylcholine polymer is stably inserted into the liposome membrane, and its hydrophilic phosphorylcholine side chains extend outward, forming a biomimetic hydration layer with a regular structure, uniform thickness, and relatively thin thickness on the surface of the liposomes.

[0047] In contrast, the hydration layer on the surface of the liposomes in Comparative Example 5 was significantly thicker and uneven in thickness, exhibiting a fluffy morphology. The thickness of the hydration layer (red arrow) was significantly thicker than that in Example 1. Polyquaternium-51 was added only after the formation of the colostrum through physical mixing. The polymer could not be effectively inserted into the liposome membrane. Most of the polymer existed on the outside of the liposomes in a free or loosely adsorbed form. Therefore, it was possible to see that these loosely attached polymer chains formed a thick and uneven accumulation layer on the surface of the liposomes.

[0048] 7. Soothing Efficacy Test The soothing efficacy of the ceramide liposomes prepared in Example 1 and Comparative Examples 1-5 was tested.

[0049] The soothing effect was assessed using a keratinocyte (HaCaT) UVB inflammation model. The levels of tumor necrosis factor-α (TNF-α) in the cell supernatant were detected using an enzyme-linked immunosorbent assay (ELISA) kit, and the NO levels were detected using a detection kit.

[0050] HaCaT cells were used at a rate of 1×10 5 Cells were seeded at a density of cells / well in 24-well plates and cultured at 37°C and 5% CO2 for 24 h. Experimental groups included: normal control group (0 mJ / cm² + culture medium), model control group (80 mJ / cm² + culture medium), positive control group (80 mJ / cm² + 1 μM dexamethasone), and sample group (80 mJ / cm² + each sample, 1% v / v). Each group had 3 replicates. The original culture medium was discarded, and each well was washed once with 500 μL PBS. The normal control group was protected from light by wrapping it in aluminum foil, while the other groups were irradiated with UVB. The UVB lamps were preheated for 15 min, and the irradiation intensity was adjusted to approximately 0.5 mW / cm², with a total irradiation dose of 80 mJ / cm². After irradiation, the PBS was discarded. The normal and model control groups were added to DMEM medium containing 1% FBS, the positive control group was added to medium containing 1 μM dexamethasone, and the sample groups were added to medium containing the corresponding samples. Cultures were continued for 24 h. After culture, cell supernatants from each group were collected, centrifuged at 1000g for 10 min at 4℃, and the supernatants were aliquoted and stored at -80℃ for later analysis. The TNF-α content in the cell supernatant was detected using a human TNF-α ELISA kit, and the NO content in the cell supernatant was detected using a nitric oxide (NO) detection kit.

[0051] The results are as follows Figure 3 and Figure 4 As shown, where, Figure 3 This is a graph showing the results of TNF-α level detection; Figure 4 This is a graph showing the NO level detection results; from Figure 3 and Figure 4 As can be seen, compared with the normal control group, the levels of TNF-α and NO in the model control group were significantly increased, indicating that the UVB inflammation model was successfully established; among the sample groups, Example 1 showed the most significant inhibitory effect on TNF-α and NO, with an inhibitory effect comparable to that of the positive control group.

[0052] Comparative Example 1 lacked phosphorylcholine polymer and had no biomimetic hydration layer, resulting in ineffective transdermal delivery of the active ingredient to cells. Consequently, its inhibitory effect on TNF-α and NO was poor, indicating that the surface anchoring of phosphorylcholine polymer is crucial for the soothing efficacy of liposomes. Comparative Example 2 showed weaker inhibitory effects on TNF-α and NO than Example 1, demonstrating that the cross-linking network used in this invention can stabilize the active ingredient, thereby enhancing the soothing effect. In Comparative Example 3, the absence of squalane and tocopherol led to decreased liposome membrane flexibility and stability, making the membrane structure regularity easily disrupted. This, in turn, affected the fusion of liposomes with cells and the intracellular delivery of the active ingredient, thus impacting the soothing efficacy. Comparative Example 4 used a natural cooling process, which reduced the structural integrity of the liposomes and decreased the delivery efficiency of the active ingredient. In Comparative Example 5, the phosphorylcholine polymer existed outside the liposome membrane in a physically mixed manner, failing to form a uniform biomimetic hydration layer. This limited the transdermal delivery efficiency and cell protection, affecting the soothing efficacy.

[0053] 8. Repairing effects The repair efficacy of the ceramide liposomes prepared in Example 1 and Comparative Examples 1-5 was tested.

[0054] The repair efficacy was assessed using a keratinocyte (HaCaT) SDS damage model, and the expression level of filaggrin (FLG) in the cells was detected by an ELISA kit.

[0055] HaCaT cells were used at a rate of 2 × 10 5 Cells were seeded at a density of [number] cells / well in 6-well plates and cultured at 37°C and 5% CO2 for 24 h. Experimental groups included: normal control group (no SDS, culture medium added), model control group (treated with 0.005% SDS for 2 h followed by culture medium added), positive control group (treated with SDS followed by 1% ceramide repair cream), and sample group (treated with SDS followed by each sample, 1% by volume). Each group had 3 replicates. The original culture medium was discarded. Except for the normal control group, all other groups were treated with culture medium containing 0.005% SDS and cultured at 37°C and 5% CO2 for 2 h to establish a cell damage model. The SDS culture medium was discarded, and the cells were washed twice with PBS. Fresh culture medium was added to the normal and model control groups, culture medium containing the corresponding sample was added to the sample group, and culture medium containing 1% ceramide repair cream was added to the positive control group. Cultures were continued for 48 h. After culture, the culture medium was discarded, and the cells were washed twice with PBS. 200 μL of cell lysis buffer (containing protease inhibitors) was added to each well, and cells were lysed on ice for 30 min, gently shaking every 10 min during lysis. Cells were scraped off with a cell scraper, and the lysis buffer was collected. The buffer was centrifuged at 12000g for 15 min at 4°C, and the supernatant was collected, aliquoted, and stored at -80°C for later analysis. The FLG content in the cell lysis buffer was detected using a human FLG ELISA kit, and the FLG recovery rate was calculated using the following formula: FLG recovery rate (%) = (FLG expression level in the sample group - FLG expression level in the model control group) / (FLG expression level in the normal control group - FLG expression level in the model control group) × 100% The results are as follows Figure 5 As shown, from Figure 5 As can be seen, after 48 hours of treatment, the relative expression level of FLG in the Example 1 group was significantly restored, and the restoration effect was better than that of the positive control group. This result indicates that the ceramide liposomes prepared in Example 1 of this invention have excellent repair effects, can effectively promote the upregulation of filaggrin expression in HaCaT cells after SDS damage, and accelerate the repair of skin barrier function.

[0056] The repair effects of Comparative Examples 1-5 were significantly weaker than those of Example 1. Specifically, Comparative Example 1 lacked phosphorylcholine polymer, resulting in the inability of the active ingredient to be effectively delivered to cells, and the liposome's ability to protect cells and deliver active ingredients was greatly reduced, severely weakening the repair efficacy. Comparative Example 2 lacked a cross-linking network, causing ergothioneine to easily recrystallize during liposome storage and delivery, reducing the stability of the active ingredient and weakening the repair efficacy. In Comparative Example 3, the lack of squalane and tocopherol led to decreased membrane stability, affecting the fusion efficiency of liposomes with cells and the intracellular delivery of active ingredients, thus affecting the repair efficacy. In Comparative Example 4, the liposome structure was not intact, reducing the efficiency of active ingredient delivery and affecting the repair efficacy. In Comparative Example 5, because the phosphorylcholine polymer failed to be effectively anchored on the outer membrane of the liposome, it could not achieve the same optimal repair level as Example 1.

[0057] The specific embodiments of the present invention described above do not constitute a limitation on the scope of protection of the present invention. Any other corresponding changes and modifications made in accordance with the technical concept of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing ceramide liposomes, characterized in that, Includes the following steps: S1, Oil Phase: Hydrogenated lecithin, ceramide 3, cholesterol, squalane, tocopherol, and polyglycerol-3 polyricinoleate are added to a mixed solvent of glycerol and ethoxydiethylene glycol and homogenized at 45-50℃ and 3000-4000 rpm for 12-15 min to obtain a homogeneous oil phase; the homogeneous oil phase is heated to 65-75℃ at a rate of 1-2℃ / min, phosphorylcholine polymer is added, and the mixture is stirred for 15-30 min to obtain the oil phase, which is then kept at 65-75℃ for later use; S2, Aqueous phase: While stirring at 500~800 rpm, slowly add calcium chloride-ergothioneine solution dropwise to sodium alginate solution; Continue stirring for 10 minutes to obtain the aqueous phase, and keep it at 65~70℃ for later use; S3, colostrum: While the oil phase is sheared at 5000~7000 rpm, the aqueous phase is added to the oil phase at a rate of 2.5~3.5 mL / min, and shearing is continued at 5000~7000 rpm for 5~15 min to obtain the colostrum; S4. Liposome formation: Homogenize the colostrum 2-3 times at 60-65℃ and 50-80MPa, immediately and rapidly cool it to 5-10℃, pre-filter it through a 0.45μm filter membrane, and then extrude it through a 0.22μm filter membrane to obtain ceramide liposomes.

2. The method for preparing ceramide liposomes according to claim 1, characterized in that, In step S1, the amounts of each component by weight are as follows: 5-15 parts hydrogenated lecithin, 0.5-4 parts ceramide 3, 0.2-3 parts cholesterol, 1-5 parts squalane, 0.2-1 parts tocopherol, 0.5-2 parts polyglycerol-3 polyricinoleate, 0.5-2 parts phosphorylcholine polymer, 10-30 parts glycerol, and 1-10 parts ethoxydiethylene glycol.

3. The method for preparing ceramide liposomes according to claim 1, characterized in that, In step S1, the phosphorylcholine polymer is selected from one of polyquaternium-51, polyquaternium-61, and polyquaternium-65; the weight-average molecular weight of the phosphorylcholine polymer is 10,000 to 30,000 Da.

4. The method for preparing ceramide liposomes according to claim 1, characterized in that, In step S2, the calcium chloride-ergothioneine solution is prepared by dissolving calcium chloride in deionized water, adding ergothioneine, and stirring until dissolved. The sodium alginate solution is prepared by dissolving sodium alginate in deionized water. The calcium chloride-ergothioneine solution contains calcium chloride at a mass concentration of 0.01-0.05% and ergothioneine at a mass concentration of 1-2%; sodium alginate solution at a mass concentration of 0.2-0.4%; and the mass ratio of calcium chloride to sodium alginate is 1-3:

100.

5. The method for preparing ceramide liposomes according to claim 1, characterized in that, In step S3, the mass ratio of the aqueous phase to the oil phase is 0.8~1.2:

1.

6. The method for preparing ceramide liposomes according to claim 1, characterized in that, In step S4, the specific method for rapid cooling is as follows: pour the high-pressure homogenized emulsion into a stainless steel cup, stir at 200~300 rpm, place the stainless steel cup in an ice water bath, cool the emulsion to 5~10℃ within 2 minutes, and continue stirring for 2~5 minutes.

7. The method for preparing ceramide liposomes according to claim 1, characterized in that, In step S4, the extrusion parameters are specifically: extrusion pressure of 100~300psi and extrusion speed of 10~20mL / min.

8. A ceramide liposome, characterized in that, The ceramide liposomes are prepared by the preparation method according to any one of claims 1 to 7.

9. The use of the ceramide liposomes according to claim 8 in the preparation of skin care products, cosmetics and medical aesthetic products.

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