Preparation method and application of ceramide E-containing nano-liposome

Abstract

The invention provides a preparation method and application of a ceramide E-containing nano-liposome. According to the preparation method, an innovative molecular rearrangement process is adopted, a nano-carrier with uniform particle size, good water solubility and high stability is constructed through an environment-friendly low-consumption pretreatment process, and the use of a toxic organic solvent is avoided. In addition, the ceramide E can penetrate through the skin more easily due to the small size of the ceramide E, the retention amount in the skin is increased, the skin barrier is repaired, and the effect of the ceramide E is better exerted. The composition comprises the following components: 3-8% of ceramide E, 2.5-5% of phospholipid, 0.5-1% of cholesterol and the like. CNLC has excellent skin permeability and retention capacity, can effectively repair skin barriers, and improves the efficacy of cosmetics in the aspect of skin injury repair. According to the invention, the crystallinity of ceramide E is obviously reduced, and the content and water dispersibility are improved. The technology is expected to promote wide application in the field of medical cosmetology.

Description

Technical Field

[0001] The present invention relates to the technical field of ceramide E-containing nanolipid carriers, and in particular to a preparation method and application of ceramide E-containing nanoliposomes. Background Art

[0002] The skin barrier is a physical permeability barrier that prevents water loss and protects the body from the external environment. It is formed by the stratum corneum, a defensive structure primarily composed of corneocytes interwoven with a complex lipid mixture. This lipid mixture includes equimolar concentrations of cholesterol, ceramides, and free fatty acids. The composition and structure of the lipid sheets arranged in a multilayer membrane are crucial to the permeability barrier function. Ceramide, as the main component of the lipids in the human epidermis, together with other lipids, forms the stratum corneum protective barrier between the human body and the external environment. This barrier mainly functions to retain moisture and protect against external stimuli.

[0003] Ceramide (Cer) is a class of lipid molecules with a ceramide backbone. Ceramides make up 40% to 50% of the lipids in the stratum corneum. As a crucial component of the skin's outermost layer, the stratum corneum, ceramides provide mechanical protection from environmental influences, regulate water permeation, prevent water loss from the skin, and block the entry of toxins, allergens, and microbial pathogens. They play a significant role in skin repair, moisturizing, and delaying skin aging, resulting in excellent cosmetic effects. Depending on their source, ceramides can be natural or chemically synthesized. Natural ceramides are primarily extracted from plants or animals, requiring complex extraction processes and resulting in relatively low concentrations, making them very expensive. Ceramide E (cetyl-PG hydroxyethyl palmitamide) is a chemically synthesized ceramide analog with a similar structure and efficacy to ceramides. It offers a high cost-effectiveness. As an analog of the intercellular lipid component ceramide, it can improve skin water retention and enhance skin barrier repair. However, ceramide E has a high degree of crystallinity, poor water solubility, and is prone to crystallization or flocculation in formulations, resulting in poor product stability. In addition, ceramide E has poor transdermal absorption, which significantly affects its skin biological efficacy.

[0004] Nanolipid carriers are colloidal dispersion systems composed of a biocompatible solid lipid or a mixture of solid and liquid lipids, stabilized by surfactants, into which drugs are loaded by encapsulation or adsorption. Nanolipid carriers offer advantages such as high physical stability, good biocompatibility, low toxicity, and suitability for large-scale industrial production. Furthermore, their extremely small size not only increases the contact area between the surfactant and the site of action but also enhances the tissue penetration and permeability of the active substance, making it easier for the active substance to penetrate the skin and improving transdermal absorption. Therefore, preparing ceramide E as a nanolipid carrier can effectively solve the problem of ceramide E crystallization, prevent its precipitation in the formulation, and improve its water dispersibility, stability, and skin care effects.

[0005] Currently, nanolipid carriers are widely used in active ingredient delivery systems due to their excellent biocompatibility, high physical stability, and superior transdermal penetration. The preparation of ceramide E as a nanolipid carrier is expected to effectively improve its solubility, dispersibility, and skin retention, thereby enhancing its skincare efficacy. Therefore, it is necessary to develop a method for preparing efficient and stable nanolipid carriers containing ceramide E to overcome the shortcomings of existing technologies and expand their applications in cosmetics and medical aesthetics. Therefore, we propose a method for preparing nanoliposomes containing ceramide E and its application. Summary of the Invention

[0006] The present invention aims to address the problems raised by the existing background technology. To achieve the above-mentioned invention objectives, the present invention provides the following technical solution: a nano-lipid carrier containing ceramide E, wherein the nano-lipid carrier mainly comprises an oil phase and an aqueous phase, wherein the components and their contents of the oil phase and the aqueous phase are as follows, based on the total weight of the nano-lipid carrier: ceramide E / NP: 3-8%; phospholipid: 2.5-5%; cholesterol: 0.5-1%; emulsifier: 5-13%; co-emulsifier: 2.5-5%; preservative: 0.5-1%; deionized water: up to 100%.

[0007] As a preferred technical solution of the present invention, the phospholipid is selected from at least one of soybean lecithin and hydrogenated lecithin.

[0008] As a preferred technical solution of the present invention, the emulsifier is at least one of sodium lauroyl lactylate, sodium methyl cocoyl taurate, sodium di(lauryl glutamine) lysine, and polyglyceryl-10 myristate.

[0009] As a preferred technical solution of the present invention, the co-emulsifier is two or more of 1,3-propylene glycol, caprylic / capric triglyceride, and triolein.

[0010] As a preferred technical solution of the present invention, the preservative is at least one of 1,2-hexanediol, p-hydroxyacetophenone, and octanoylhydroxamic acid.

[0011] As a preferred technical solution of the present invention, the nano-lipid carrier is a white to light yellow opalescent liquid with no odor and an average particle size of 150 to 300 nm.

[0012] As a preferred technical solution of the present invention, the nano-lipid carrier does not show any abnormal phenomena such as stratification, particle precipitation, coarsening, color change, or taste change when placed under 45°C, 25°C, 4°C, and natural light conditions for 90 days. The nano-lipid carrier is compatible with cosmetic aqueous solutions, polyols, and different thickening stabilizers without any abnormalities.

[0013] A method for preparing ceramide E-containing nanoliposomes mainly comprises the following steps:

[0014] Step 1: Heat the oil phase to 80°C and stir until dissolved;

[0015] Step 2: Heat the aqueous phase to 80°C and stir until dissolved;

[0016] Step 3: Slowly pour the oil phase into the water phase while stirring at 1000 rpm and mix well;

[0017] Step 4: The mixed sample was sheared at 10,000 rpm for 3 minutes and ultrasonicated at 500 W for 5 minutes to prepare the ceramide E nanolipid carrier.

[0018] A nano-lipid carrier composition comprises the composite ceramide E nano-lipid carrier composition and cosmetic excipients.

[0019] Application of a composite ceramide E nanolipid carrier composition in cosmetics with moisturizing and repairing effects.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The CNLC provided by the present invention is a white to pale yellow opalescent liquid with no odor. The average particle size of the nanolipid carrier is 150 to 300 nm. When the CNLC was stored at 45°C, 25°C, and 4°C, as well as under natural light for 90 days, it showed no delamination, particle precipitation, or coarsening, nor did it exhibit discoloration, taste change, or other abnormalities. Furthermore, compatibility tests of the prepared CNLC with cosmetic aqueous solutions, polyols, and various commonly used thickening and stabilizing agents revealed no abnormalities, demonstrating the CNLC's good stability and compatibility with aqueous solutions, polyols, and thickeners.

[0022] The present invention provides a stable nanolipid carrier. Because the ceramide E molecule is composed of two fatty acid alkyl chains with different carbon chains, two hydroxyl groups, and an amide group, it has a bimolecular structure, making it both hydrophilic and lipophilic, but lacking emulsifying properties. The fatty acid ester can be inserted into the interstices of the ceramide E molecules, forming a dense and ordered viscoelastic interfacial membrane at the oil / water interface, preventing the precipitation of ceramide E and improving its stability.

[0023] 2. This invention provides a novel nanolipid carrier preparation technology—an innovative molecular rearrangement process. This technology utilizes an environmentally friendly, low-cost pretreatment process, labeling agents to achieve initial molecular rearrangement, an intelligent thermodynamic circulation system, and molecular-level energy activation technology to achieve tertiary molecular rearrangement to construct CNLC. This technology offers the advantages of a simple and controllable process flow, no risk of toxic organic solvent residues, and a flexible and efficient production model.

[0024] 3. The CNLC provided by the present invention is a white to pale yellow opalescent liquid with good fluidity and water dispersibility, and can be compounded with cosmetic formulas at room temperature. It has a small particle size, and the average particle size of the nanolipid carrier is 150 to 300 nm. The small size facilitates the penetration of ceramide E through the nanolipid carrier structure through the skin surface, thereby achieving efficient moisturizing and water lock, repairing the skin barrier, and delaying aging. It also has good affinity with the skin, making the skin more hydrated, smooth, and firm. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 (A, B) Particle size, PDI, and Zeta potential of CNLC1-7; (C) Particle size distribution of CNLC-3, CNLC-6, and CNLC-7; (D) Transmission electron microscopy photography to detect the morphology and size of CNLC-3, CNLC-6, and CNLC-7 (scale = 100 nm); (E, F, G) Particle size (E), PDI (F), and Zeta potential (G) of CNLC-3 at 25°C, 4°C, and 45°C.

[0026] Figure 2 This is the cytotoxicity result of CNLC on HaCat cells

[0027] Figure 3 The effect of CNLC on the blood vessels of the chick embryo chorioallantoic membrane

[0028] Figure 4 This is the result of the retention of Cer-E in the skin in CNLC

[0029] Figure 5 The distribution of CNLC in the skin

[0030] Figure 6 (A) is the appearance of damaged skin after CNLC repair. Figure 6 (B) is the observation of the tissue morphology of the damaged skin after repair

[0031] Figure 7 This is the result diagram of the effect of CNLC on the water content of human skin. DETAILED DESCRIPTION

[0032] To make the purpose, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiment is a specific implementation of the present invention and is not limited to all embodiments.

[0033] Therefore, the following detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely represents some embodiments of the present invention. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort shall fall within the scope of protection of the present invention.

[0034] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features and technical solutions therein can be combined with each other. It should be noted that similar numbers and letters represent similar items in the following drawings. Therefore, once an item is defined in one drawing, it does not need to be further defined and explained in subsequent drawings.

[0035] Example: A nanolipid carrier containing ceramide E, the nanolipid carrier mainly comprises an oil phase and an aqueous phase. Based on the total weight of the nanolipid carrier, the components contained in the oil phase and the aqueous phase and their contents are as follows: ceramide E / NP: 3-8%; phospholipid: 2.5-5%; cholesterol: 0.5-1%; emulsifier: 5-13%; co-emulsifier: 2.5-5%; preservative: 0.5-1%; deionized water: up to 100%.

[0036] The phospholipid is selected from at least one of soybean lecithin and hydrogenated lecithin.

[0037] The emulsifier is at least one of sodium lauroyl lactylate, sodium methyl cocoyl taurate, sodium di(lauryl glutamine) lysine, and polyglyceryl-10 myristate.

[0038] The co-emulsifier is two or more of 1,3-propylene glycol, caprylic / capric triglyceride, and triolein.

[0039] The preservative is at least one of 1,2-hexanediol, p-hydroxyacetophenone, and octanoylhydroxamic acid.

[0040] Nanolipid carriers are white to light yellow opalescent liquids with no odor and an average particle size of 150 to 300 nm.

[0041] The nanolipid carrier does not show any abnormal phenomena such as stratification, particle precipitation, coarsening, color change or taste change when placed at 45°C, 25°C, 4°C and natural light for 90 days. The nanolipid carrier is compatible with cosmetic aqueous solutions, polyols and different thickening stabilizers without any abnormalities.

[0042] A method for preparing ceramide E-containing nanoliposomes mainly comprises the following steps:

[0043] Step 1: Heat the oil phase to 80°C and stir until dissolved;

[0044] Step 2: Heat the aqueous phase to 80°C and stir until dissolved;

[0045] Step 3: Slowly pour the oil phase into the water phase while stirring at 1000 rpm and mix well;

[0046] Step 4: The mixed sample was sheared at 10,000 rpm for 3 minutes and ultrasonicated at 500 W for 5 minutes to prepare the ceramide E nanolipid carrier.

[0047] A nano-lipid carrier composition comprises a composite ceramide E nano-lipid carrier composition and cosmetic excipients.

[0048] Application of a composite ceramide E nanolipid carrier composition in cosmetics with moisturizing and repairing effects.

[0049] Test example: 1. Reagents and instruments:

[0050] 1.1 Reagents:

[0051] Soy lecithin, cholesterol, ceramide E, 1,3-propylene glycol, caprylic / capric triglyceride, polyglyceryl-10 myristate, sodium methyl stearoyl taurate, sodium lauroyl lactylate, sodium di(lauryl glutamine) lysine, deionized water, saline, methanol, sodium lauryl sulfate.

[0052] 1.2 Instruments:

[0053] Magnetic stirrer, stirring bar, electronic balance, constant temperature water bath, heating stirrer, high-pressure homogenizer, vortexer, high-speed shear, intelligent high-efficiency transdermal instrument, high-performance liquid chromatograph, Brookhaven nanoparticle size potentiometer, vacuum concentrator, tissue homogenizer, skin moisture content tester Corneometer CM825, skin elasticity tester MPA580.

[0054] 2. Preparation and Characterization of CNLC

[0055] 2.1 Preparation of CNLC (The following is the preferred CNLC formulation and preparation method)

[0056] Test Example 1 (CNLC-1)

[0057] A nano-lipid carrier containing ceramide E, comprising the following components based on the total weight of the composition:

[0058] Oil phase: Ceramide E: 3.0%, Soy lecithin: 2.5%, Cholesterol: 0.5%, 1,3-propylene glycol: 1.25%, Polyglyceryl-10 myristate: 2%, Triolein: 1.25%, Caprylhydroxamic acid: 0.5%.

[0059] Aqueous phase: Sodium lauroyl lactylate: 3%, the balance is water.

[0060] Test Example 2 (CNLC-2)

[0061] A nano-lipid carrier containing ceramide E, comprising the following components based on the total weight of the composition:

[0062] Oil phase: Ceramide E: 5.0%, Soy Lecithin: 2.5%, Cholesterol: 1%, 1,3 Propanediol: 2.5%, Polyglyceryl-10 Myristate: 4%, Triolein: 2.5%, Parahydroxyacetophenone: 0.5%, 1,2 Hexanediol: 0.5%.

[0063] Aqueous phase: Sodium lauroyl lactylate: 5%, the balance is water.

[0064] Test Example 3 (CNLC-3)

[0065] A nano-lipid carrier containing ceramide E, comprising the following components based on the total weight of the composition:

[0066] Oil phase: Ceramide E: 5.0%, Soy Lecithin: 5.0%, Cholesterol: 1%, 1,3 Propanediol: 1.25%, Polyglyceryl-10 Myristate: 4%, Triolein: 1.25%, Parahydroxyacetophenone: 0.5%, 1,2 Hexanediol: 0.5%.

[0067] Aqueous phase: Sodium lauroyl lactylate: 5%, the balance is water.

[0068] Test Example 4 (CNLC-4)

[0069] A nano-lipid carrier containing ceramide E, comprising the following components based on the total weight of the composition:

[0070] Oil phase: Ceramide E: 5.0%, Soy Lecithin: 5.0%, Cholesterol: 1%, 1,3 Propanediol: 2.5%, Polyglyceryl-10 Myristate: 6%, Caprylic / Capric Triglyceride: 2.5%, Parahydroxyacetophenone: 0.5%, 1,2 Hexanediol: 0.5%.

[0071] Aqueous phase: Sodium lauroyl lactylate: 5%, the balance is water.

[0072] Test Example 5 (CNLC-5)

[0073] Oil phase: Ceramide E: 8.0%, Hydrogenated Lecithin: 5.0%, Cholesterol: 1%, 1,3 Propanediol: 2.5, Polyglyceryl-10 Myristate: 6%, Triolein: 2.5%, Parahydroxyacetophenone: 0.5%, 1,2 Hexanediol: 0.5%.

[0074] Aqueous phase: Sodium lauroyl lactylate: 7%, the balance is water.

[0075] Test Example 6 (CNLC-6)

[0076] A nano-lipid carrier containing ceramide E, comprising the following components based on the total weight of the composition:

[0077] Oil phase: Ceramide E: 5.0%, Soy Lecithin: 5.0%, Cholesterol: 1%, 1,3 Propanediol: 1.25%, Polyglyceryl-10 Myristate: 4%, Triolein: 1.25%, Parahydroxyacetophenone: 0.5%, 1,2 Hexanediol: 0.5%.

[0078] Aqueous phase: sodium di(laurylamideglutamine)lysine: 5%, the balance is water.

[0079] Test Example 7 (CNLC-7)

[0080] A nano-lipid carrier containing ceramide E, comprising the following components based on the total weight of the composition:

[0081] Oil phase: Ceramide E: 5.0%, Soy Lecithin: 5.0%, Cholesterol: 1%, 1,3 Propanediol: 1.25%, Polyglyceryl-10 Myristate: 4%, Triolein: 1.25%, Parahydroxyacetophenone: 0.5%, 1,2 Hexanediol: 0.5%.

[0082] Aqueous phase: sodium methyl cocoyl taurate: 5%, the balance is water.

[0083] The preparation method of the nano-lipid carrier described in Examples 1-7 of the present invention is as follows:

[0084] Heat the oil phase to 80°C and stir until dissolved;

[0085] Heat the aqueous phase to 80°C and stir until dissolved;

[0086] Slowly pour the oil phase into the water phase while stirring at 1000 rpm and mix well;

[0087] The mixed sample was sheared at 10,000 rpm for 3 minutes and ultrasonicated with a 500 W probe for 5 minutes to prepare CNLC.

[0088] 2.2 Characterization of CNLC

[0089] 2.2.1 Formulation Characterization and Stability

[0090] The particle size, polydispersity index (PDI), and zeta potential of CNLC were measured using a NanoBrook 90Plus PALS (Brookhaven, New York, USA). Before testing, 20 μL of the prepared CNLC was dispersed in 2 mL of deionized water. The average particle size, polydispersity index (PDI), and zeta potential were obtained by DLS, with each measurement repeated three times. The CNLC was then stored at 4°C, 25°C, and 45°C for 90 days, and the samples were observed for precipitation, flocculation, and stratification. The particle size, PDI, and zeta potential were measured weekly using a NanoBrook 90Plus PALS to investigate storage stability.

[0091] The CNLC provided by the present invention is a white to light yellow opalescent liquid. Figure 1 As shown in AC, A is the particle size and PDI, B is the Zeta potential, and C is the particle size distribution. The particle size of Examples 1 to 7 is between 150 and 300 nm, the PDI is less than 0.3, the surface potentials are all negative, and the particle size distribution is relatively narrow. Preferably, the ceramide E content in the above Example 3 (CNLC-3) is 5%, the prepared CNLC has a particle size of 186.69±2.52 nm, a PDI of 0.21±0.02, and a Zeta potential of -36.58±1.90. After the sample of Example 3 was placed at 4°C, 25°C, and 45°C for 90 days, the CNLC was still able to remain stable when loaded with a high content of ceramide E, no crystallization of ceramide E was found, and no stratification or oil-water separation was observed. Figure 1 Figures E, F, and G show the changes in particle size, PDI, and potential of CLNC-3 after being stored at 4°C, 25°C, and 45°C for 90 days. The results show that the particle size, PDI, and potential did not change significantly, indicating that the CNLC prepared according to the components and contents used in the present invention has good stability.

[0092] 2.2.2 Transmission electron microscopy (TEM)

[0093] The CNLC morphology was observed using a negative staining method and transmission electron microscopy (TEM). Briefly, a CNLC sample was diluted 100-fold in a buffer solution and placed on a carbon-polyformaldehyde-coated copper grid. After 20 minutes of spontaneous evaporation, the remaining buffer solution was removed. The dried grid was then stained with 1% uranyl acetate solution for 6 minutes at an accelerating voltage of 80 kV, and its morphology was observed using a microscope.

[0094] like Figure 1 As shown in Figure 4D, the transmission electron microscopy image shows that CNLC is nearly spherical. We can see that CNLC exhibits uniform brightness throughout the system, showing a dark band surrounding the periphery of the particle, and the structure is similar to that of nanoemulsion, which means that ceramide E has a thin but strong and elastic molecular layer on the particle surface.

[0095] 2.3 Safety and efficacy evaluation

[0096] 2.3.1 Cytotoxicity assay

[0097] The cytotoxicity assay used the 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonatophenyl)-2H-tetrazolium monosodium salt (CCK-8) cytotoxicity assay, which is based on the reduction of CCK-8 by mitochondrial dehydrogenases to highly water-soluble yellow formazan crystals. Briefly, HaCaT keratinocytes were cultured at 37°C and 5% CO₂. After reaching the optimal cell density, cells were seeded at 7 × 10⁺ cells / well in 96-well plates and treated with a gradient of 1% CNLC (100%, 50%, 25%, 12.5%, 6.25%, and 3.12%) for 24 hours. After addition of 10% CCK-8 reagent and incubation in an incubator for 1–4 hours, the absorbance (OD) was measured at 450 nm using a microplate reader, maintaining an OD value around 1.0.

[0098] To investigate the safety of CNLC, the toxicity of CNLC to HaCat cells was evaluated using CCK-8. Figure 2 As shown in the results, when the concentration of Cer-E in CNLC was 0-125 μg / mL, the cell viability was higher than 95%. When the concentration of Cer-E was 250 μg / mL or 500 μg / mL, the cell viability decreased, indicating that Cer-E in CLNC has a high safety in the concentration range of 0-125 μg / mL.

[0099] 2.3.2 Chicken embryo chorioallantoic membrane test (HET-CAM)

[0100] The chick chorioallantoic membrane test is an earlier in vitro method for evaluating eye irritation. It is an alternative method developed by leveraging the structural similarities between the chick chorioallantoic membrane and the human conjunctiva. The chorioallantoic membrane (CAM) is a respiratory membrane that surrounds the chick embryo. This test utilizes the integrity, clarity, and transparency of the chorioallantoic membrane vascular system in mid-incubation chick embryos. The test substance is directly exposed to the CAM membrane. After 5 minutes, the chorioallantoic membrane toxicity effect indicators are observed, primarily for bleeding, coagulation, and vascular melting. These indicators reflect changes in the morphology, structure, color, and permeability of the blood vessels and vascular network, as well as the denaturation and degree of damage of chorioallantoic membrane proteins. These indicators are then combined to generate a score used to assess the eye irritation potential of the test substance.

[0101] Scoring criteria:

[0102] Table 1 HET-CAM score table

[0103]

[0104] Irritation evaluation:

[0105] Endpoint score (ES): score for each embryo = the sum of the degree of bleeding, coagulation and vascular lysis observed in each embryo; Endpoint score (ES) = the sum of the scores of 6 embryos

[0106] Table 2. Endpoint scoring method evaluation results

[0107] Stimulus classification No / mild irritation Moderate irritation Severe irritation / corrosiveness End point score ES≤12 12<ES<16 ES≥16

[0108] Table 3 Chicken embryo endpoint score (ES)

[0109] Group Endpoint score (ES) Stimulus classification 25% CLNC 0 Non-irritating 50% CLNC 0 Non-irritating 100% CLNC 0 Non-irritating

[0110] like Figure 3 As shown in Table 3, at the beginning of the test (0 min), the chorioallantoic membrane blood vessels of all groups were intact and in good condition. At the 5th minute, the chorioallantoic membrane blood vessels treated with 0.1 mol / L NaOH showed severe rupture and heavy bleeding, indicating that the positive control was effective. However, the blood vessels in the 0.9% NaCl group did not show rupture or bleeding, and remained consistent with the initial state, indicating that the negative control was effective. Observing the state of the chicken embryo chorioallantoic membrane blood vessels of 25% CLNC, 50% CLNC, and 100% CLNC, the blood vessels of each concentration of CLNC all showed no bleeding, coagulation, or vascular melting. The endpoint scores are shown in Table 3, and the ES score of each concentration group was all 0, indicating that 25% CLNC, 50% CLNC, and 100% CLNC were non-irritating to the fragile chicken embryo chorioallantoic membrane.

[0111] 2.4 Skin penetration and barrier repair

[0112] 2.4.1 Determination of skin retention

[0113] Skin retention was determined using a vertical, jacketed Franz diffusion apparatus with a surface area of ​​1.77 cm² and a receiving chamber volume of 10 mL. Male albino rats weighing 250-275 g were sacrificed, their backs shaved, and dorsal skin samples obtained. The skin was rinsed with cold water and stored at -20°C until use. On the day of the experiment, the skin was thawed, cut to an appropriate size, and secured between the receiving and donor chambers of a diffusion cell, with the stratum corneum facing upward. The samples were allowed to equilibrate for 1 h. 500 mg each of free ceramide cream, Example 3, Example 6, and Example 7 were placed in the donor chamber and diffused in a receiving solution of 2% SDS (w / v)-20% ethanol (v / v)-normal saline. The samples were stirred at 32°C and 350 rpm. The upper portion of the donor chamber and the receiving chamber were sealed with parafilm to prevent evaporation of the solution. After 24 hours, the excess sample in the skin sample was carefully removed with ultrapure water, the effective administration site was taken and cut into pieces, methanol was added and homogenized, vortex mixed, and centrifuged at 10,000×g for 10 minutes. The supernatant was taken, the organic solvent was evaporated by a vacuum concentrator, and the mobile phase was added for re-dissolution. After water bath sonication, the supernatant was taken for HPLC analysis to calculate the retention amount of ceramide E in the skin.

[0114] The retention of ceramide E in the skin was measured, and the results were as follows: Figure 4 The skin retention of ceramide E in the free ceramide E cream was 9.55±1.54μg / cm2, the skin retention of CNLC-3 was 28.78±1.28μg / cm2, the skin retention of CNLC-6 was 21.56±0.29μg / cm2, and the skin retention of CNLC-7 was 17.06±1.10μg / cm2. Compared with the free ceramide E cream, the skin retention of CNLC-3, CNLC-6, and CNLC-7 with the same ceramide E mass fraction was 3.0 times, 2.3 times, and 1.8 times that of the free ceramide E cream, respectively. This indicates that the prepared CNLC can effectively promote high-concentration retention of ceramide E in the skin.

[0115] 2.4.2 Intradermal distribution

[0116] KM mice were anesthetized and the hair covering the abdominal skin area was removed using a shaver. 100 μL of each coumarin 6 (C6)-encapsulated test sample was applied to 1 cm² nonwoven fabric of the same size and fixed to the depilated mouse skin. After 15, 30, 60, and 120 minutes of exposure to each sample, approximately 1 cm² of dorsal skin was sampled, fixed with 4% paraformaldehyde, embedded in OCT, and cryosectioned to a thickness of 10 μm. Fluorescence distribution was observed under a fluorescence microscope.

[0117] like Figure 5 As shown, at 15 minutes, almost all of the fluorescent material in CNLC-3, CNLC-6, and CNLC-7 remained on the outer side of the epidermis, with only a very small amount of fluorescence observed within the epidermis. This indicates that at this point, very little fluorescein has begun to undergo transdermal absorption, while the vast majority has not yet entered the skin. Only in Free Ceramide-E can some fluorescence be observed penetrating deeper into the skin. At 30 minutes, the fluorescence intensity in the deeper layers of the skin was higher in the Free Ceramide-E group. The fluorescence intensities in the CNLC-3, CNLC-6, and CNLC-7 groups were lower than those in the Free Ceramide-E group and were almost at the same level. This indicates that a small amount of fluorescence had already appeared deeper beneath the epidermis by 30 minutes, indicating that the rate of transdermal absorption of some fluorescein had begun to accelerate. At 60 minutes, almost no fluorescence was observed in the Free Ceramide-E group, while fluorescence was evenly distributed throughout the epidermis and dermis in the CNLC-3, CNLC-6, and CNLC-7 groups, with the highest fluorescence intensity in CNLC-3. This indicates that after this brief period of stagnation, transdermal absorption of fluorescein has reached a relatively rapid rate. At 120 minutes, almost no fluorescence was observed in the Free Ceramide-E group, while CNLC-3, CNLC-6, and CNLC-7 showed higher fluorescence intensity in the skin, with some accumulation at the base. In summary, before 30 minutes, Free Ceramide-E entered the skin faster than the preferred formulation, but it was unable to remain in the skin, resulting in a lower skin retention rate. The preferred CNLC formulation remained in the skin for a longer period of time.

[0118] 2.4.3 Skin Barrier Repair

[0119] KM mice were anesthetized and fixed, and the hair on the skin on the back of the neck was removed with a shaver (about 3cm×3cm). The skin surface was cleaned with warm water and dried, and the mice were raised naturally overnight. A skin injury model was established by tape stripping. A transparent tape was pressed on the skin with a customized weight (225g / cm2), and pressure was applied to each tape for 10 seconds to ensure that the tape was completely adhered to the skin. The tape was torn off at a stable high speed, and the direction of the tape was changed after each tape was torn off to ensure uniform damage. The skin area was stripped 10 times to obtain damaged skin. After the model was successfully established, the above samples were taken and applied to the damaged skin of the mice (0.5g / mouse), and the drug was administered once a day in the morning and evening (12h apart) for 3 consecutive days. After the last administration, the mice were depilated and then killed by cervical dislocation. The back skin of about 1 cm2 was cut and fixed in 4% paraformaldehyde solution. Then, the skin was dehydrated, embedded, sectioned, stained with hematoxylin-eosin (HE), and sealed. Finally, the changes in skin tissue structure were observed under a microscope.

[0120] like Figure 6 As shown, Figure 6 A shows the skin appearance of the different groups three days after treatment. The skin of the control group mice was smooth, lustrous, and delicate; the skin of the model group was rough, dry, dark, red, swollen, and scabby, indicating successful modeling. The skin of the Free Cer-E group was smoother than the model group, but with more severe scabby. In the CLNC group, increasing CLNC concentration reduced skin redness, swelling, and scabby, showing significant improvements in smoothness, lustrousness, and roughness of the injured mouse skin. Skin treated with 100% CLNC was significantly smoother, and redness, swelling, and scabby were nearly eliminated.

[0121] After the last administration, the mice were depilated and then killed by cervical dislocation. The back skin of about 1 cm2 was cut and fixed in 4% paraformaldehyde solution. Then, the skin was dehydrated, embedded, sectioned, stained with hematoxylin-eosin (HE), and sealed. Finally, the skin was imaged under a microscope to observe the changes in the microstructure of the skin tissue. Figure 6 B shows the skin histology of the different treatment groups after H&E staining three days after administration. The skin structure of mice in the control group was intact, with uniform epidermal thickness. The epidermal structure of mice in the model group was incomplete, with uneven thickness and a missing stratum corneum. The Free Cer-E group was similar to the model group, but with a thicker stratum corneum. The epidermal structure of mice in the CLNC group was relatively intact, with uniform thickness. The stratum corneum thickened with increasing CLNC concentration, reaching a thickness comparable to that of the control group in the 100% CLNC group. In summary, Cer-E can improve the appearance and micromorphology of damaged skin, and the improvement effect is even greater when ceramide is prepared as a ceramide E nanolipid carrier, demonstrating enhanced efficacy in promoting skin damage repair.

[0122] 2.4.3 Skin irritation test

[0123] Twenty volunteers (12 women and 8 men) were randomly divided into four groups (5 participants per group). Participants met the inclusion criteria, had no history of allergic dermatitis, and had not used irritating products in the previous month. Approximately 0.020 to 0.025 mL of the prepared test sample from each group was applied to a patch tester (Beijing Science and Technology Instrument Factory) and then applied to the subject's back with a special adhesive tape. After 24 hours, the patch tester was removed, and the remaining preparation was gently wiped off with a moistened paper towel. The irritation was scored based on the degree of skin reaction at 0.5, 24, and 48 hours.

[0124] Table 3 Skin irritation reaction scores

[0125] class Identification and characterization 0 No irritation, no erythema 1 Mild erythema 2 erythema 3 Erythema, papules, blisters 4 Severe edema and bullae

[0126] Skin irritation test results: Subjects showed no adverse reactions to the ceramide E nanolipid carriers prepared in Examples 1-7 at 0.5, 24, or 48 hours, including no erythema, burning, or itching. This indicates that the ceramide E nanolipid carriers prepared in this invention are non-irritating.

[0127] 2.4.4 Skin moisturizing evaluation

[0128] 1. Test principle: The dielectric constant of water in the skin surface is much higher than that of other substances. The change in the dielectric constant of the skin surface is mainly caused by the change in the water content of the skin surface. The different water content of the skin stratum corneum will result in different measured skin capacitance values. By measuring the dielectric constant of the skin surface, the water content of the skin surface can be analyzed.

[0129] 2. Volunteer requirements: 20 volunteers with an average age of 16 to 65 years old were selected and randomly divided into 4 groups, with 5 volunteers in each group. Volunteer requirements: the basic value of the skin moisture measurement significance of the forearm test area by capacitance method is between 15-100, no serious systemic diseases, no immunodeficiency or autoimmune diseases; no active allergic diseases; no history of severe allergies to skin care cosmetics; no use of hormonal drugs and immunosuppressants in the past month; participants in other clinical trials; use of the test drugs as required and complete information; all volunteers should fill out the informed consent form before the test.

[0130] 3. Test Method: Mark a 3cm x 3cm area on the curved side of the arm as the test area. Apply 2.0±0.1mg / cm² of the product evenly to the test area using latex gloves. Use a Corneometer CM825 skin moisture meter to measure the skin's moisture content before application of the cosmetic, and 1, 2, and 4 hours after application. Repeat five times, calculate the average value, and then calculate the percentage increase in moisture content. During the test, subjects must not apply any other cosmetics to the test area.

[0131] Skin moisture content growth rate MWV (%) = (Wn-W0) / W0×100%;

[0132] W0: skin moisture content before use;

[0133] Wn: Skin moisture content after n hours of use.

[0134] The results are shown in Table 1 and Figure 7

[0135] Table 4 Skin water content growth rate MWV (%) in each time period

[0136]

[0137]

[0138] From Table 4 and Figure 7 It can be seen that the ceramide E nanolipid carriers prepared by CNLC-3, CNLC-6, and CNLC-7 can significantly increase the moisture content of the skin. After 1 hour of use, the skin moisture content growth rate is as high as 28% or more, indicating that the addition of the nanolipid carrier of the present invention has excellent moisturizing and water-locking ability, among which the cosmetics prepared by CNLC-3 have the best moisturizing effect. After using the free ceramide E cream, the skin moisture content growth rate is less than 10%, which is worse than the moisturizing ability of the samples prepared by CNLC-3, CNLC-6, and CNLC-7. This shows that the preparation of ceramide E into ceramide E nanolipid carrier can significantly improve the moisturizing and water-locking ability of ceramide E.

[0139] The above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention. Although this specification has described the present invention in detail with reference to the above embodiments, the present invention is not limited to the above specific implementation methods. Therefore, any modification or replacement of the present invention; and all technical solutions and improvements thereof that do not depart from the spirit and scope of the invention are included in the scope of the claims of the present invention.

Claims

1. A nano lipid carrier containing ceramide E, characterized in that: The nano lipid carrier mainly comprises an oil phase and an aqueous phase. Based on the total weight of the nano lipid carrier, the components and contents of the oil phase and the aqueous phase are as follows: ceramide E / NP: 3-8%; Phospholipids: 2.5-5%; Cholesterol: 0.5-1%; Emulsifier: 5-13%; Co-emulsifier: 2.5-5%; Preservative: 0.5-1%; Deionized water: up to 100%.

2. The ceramide E-containing nanolipid carrier according to claim 1, characterized in that The phospholipid is selected from at least one of soybean lecithin and hydrogenated lecithin.

3. The ceramide E-containing nanolipid carrier according to claim 1, characterized in that The emulsifier is at least one of sodium lauroyl lactylate, sodium methyl cocoyl taurate, sodium di(lauryl glutamine) lysine, and polyglyceryl-10 myristate.

4. The ceramide E-containing nanolipid carrier according to claim 1, characterized in that The auxiliary emulsifier is two or more of 1,3-propylene glycol, caprylic / capric triglyceride, and triolein.

5. The ceramide E-containing nanolipid carrier according to claim 1, characterized in that The preservative is at least one of 1,2-hexanediol, p-hydroxyacetophenone, and octanoylhydroxamic acid.

6. The ceramide E-containing nanolipid carrier according to any one of claims 1 to 5, characterized in that: The nano lipid carrier is a white to light yellow opalescent liquid with no odor and an average particle size of 150 to 300 nm.

7. The ceramide E-containing nanolipid carrier according to any one of claims 1 to 5, characterized in that The nanolipid carrier does not show abnormal phenomena such as stratification, particle precipitation, coarsening, color change, and taste change when placed at 45°C, 25°C, and 4°C, as well as under natural light conditions for 90 days. The nanolipid carrier is compatible with cosmetic aqueous solutions, polyols, and different thickening stabilizers without any abnormalities.

8. A method for preparing nanoliposomes containing ceramide E, characterized in that: The main steps include: Step 1: Heat the oil phase to 80°C and stir until dissolved; Step 2: Heat the aqueous phase to 80°C and stir until dissolved; Step 3: Slowly pour the oil phase into the water phase while stirring at 1000 rpm and mix well; Step 4: The mixed sample was sheared at 10,000 rpm for 3 minutes and ultrasonicated at 500 W for 5 minutes to prepare the ceramide E nanolipid carrier.

9. A nano lipid carrier composition, characterized in that The invention comprises the composite ceramide E nano-lipid carrier composition according to any one of claims 1 to 5 and cosmetic excipients.

10. Application of a composite ceramide E nanolipid carrier composition in cosmetics with moisturizing and repairing effects.

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