Carrier for wrapping ceramide, preparation method of carrier, ceramide inclusion and preparation method and application of ceramide inclusion

Abstract

The invention provides a carrier for wrapping ceramide, a preparation method of the carrier, a ceramide inclusion and a preparation method and application of the ceramide inclusion, and relates to the technical field of chemical synthesis and cosmetics. The invention provides a carrier for wrapping ceramide. The carrier comprises hydrogenated phosphatidylcholine and fibronectin grafted on the hydrogenated phosphatidylcholine. According to the invention, the protein (fibronectin) is combined with the surface of the liposome (hydrogenated phosphatidylcholine), and the combination can enhance the targeting property and biocompatibility of the liposome, so that the liposome can better interact with cells; through protein modification, the lipidosome can obtain special biological characteristics, and the structure and function of in-vivo cells can be better simulated. The protein for modification is fibronectin, so that the delivery targeting of the drug (ceramide) can be improved, the cell absorption can be enhanced, the drug permeability can be improved, the biocompatibility can be improved, and the stability and the skin effect can be enhanced.

Description

Technical Field

[0001] The present invention relates to the technical fields of chemical synthesis and cosmetics, and in particular to a carrier encapsulating ceramide and a preparation method thereof, as well as a ceramide inclusion body and a preparation method and application thereof. Background Art

[0002] Ceramide is a type of sphingolipid composed of long-chain sphingosine bases and fatty acids. Ceramide is the primary lipid component of the stratum corneum, accounting for approximately 40-50%. Its strong ability to bind water molecules plays a vital role in retaining moisture in the stratum corneum and maintaining the skin's barrier function. It can improve dryness, scaling, and roughness, while enhancing skin elasticity and delaying skin aging. However, ceramides have poor stability and low bioavailability.

[0003] Liposomes are microscopic vesicles formed by encapsulating drugs within a lipid bilayer. The phospholipid structure within liposomes is highly similar to human skin lipids, offering advantages such as high biocompatibility and good biodegradability. Therefore, the cosmetics industry often uses liposomes as carriers to encapsulate ceramides to improve their stability and bioavailability. However, existing liposome carriers degrade in the epidermis, making it difficult for ceramides to penetrate the subcutaneous layer, resulting in poor permeability. Summary of the Invention

[0004] In view of this, the present invention aims to provide a carrier for encapsulating ceramide, a preparation method thereof, and a ceramide inclusion body, a preparation method thereof, and an application thereof. Encapsulating ceramide with the carrier provided by the present invention can significantly improve the permeability of ceramide.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a carrier for encapsulating ceramide, comprising hydrogenated phosphatidylcholine and fibronectin grafted onto the hydrogenated phosphatidylcholine.

[0007] Preferably, the fibronectin is recombinant human fibronectin.

[0008] The present invention provides a method for preparing a carrier encapsulating ceramide according to the above technical solution, comprising the following steps:

[0009] Fibronectin, hydrogenated phosphatidylcholine, a carbodiimide condensation agent and a buffer solution are mixed and subjected to a grafting reaction to obtain the carrier encapsulating ceramide; the pH value of the mixed solution obtained by the mixing is 4.5-8.5.

[0010] Preferably, the carbodiimide condensing agent includes 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

[0011] Preferably, the molar ratio of the carbodiimide condensing agent to hydrogenated phosphatidylcholine is 1:1 to 5:1, and the molar ratio of fibronectin to hydrogenated phosphatidylcholine amide is 1:400 to 1:100.

[0012] Preferably, the buffer solution includes MES buffer solution, PBS buffer solution, MOPS buffer solution, HEPES buffer solution or acetic acid / sodium acetate buffer solution.

[0013] Preferably, the temperature of the grafting reaction is 15 to 60 °C, the time is 1 to 8 h, the grafting reaction is carried out under stirring, and the stirring rate is 200 to 2000 rpm.

[0014] The present invention provides a ceramide inclusion body, which comprises the following preparation raw materials in mass percentage: 30 to 70% of glycerol, 5 to 35% of hexyl decanol, 5 to 15% of water, 2 to 8% of ceramide, 1 to 8% of cholesterol, 1 to 10% of a carrier, and the carrier is the carrier for encapsulating ceramide described in the above technical solution or the carrier for encapsulating ceramide prepared by the preparation method described in the above technical solution.

[0015] The present invention provides a preparation method of the ceramide inclusion body described in the above technical solution, which comprises the following steps:

[0016] Carry out a first mixing of ceramide, cholesterol, a carrier and hexyl decanol to obtain an oil phase;

[0017] Carry out a second mixing of glycerol and water to obtain an aqueous phase;

[0018] Add the oil phase to the aqueous phase for emulsification to obtain a mixed solution;

[0019] Carry out high-pressure microfluidic homogenization on the mixed solution to obtain the ceramide inclusion body.

[0020] The present invention provides an application of the ceramide inclusion body described in the above technical solution or the ceramide inclusion body prepared by the preparation method described in the above technical solution in the preparation of cosmetics.

[0021] The present invention provides a carrier for encapsulating ceramide, which comprises hydrogenated phosphatidylcholine and fibronectin grafted on the hydrogenated phosphatidylcholine. The present invention modifies hydrogenated phosphatidylcholine of liposome with fibronectin as a carrier for encapsulating ceramide. Compared with the prior art, the present invention has the following beneficial effects:

[0022] High efficiency targeting: Fibronectin-modified liposomes can achieve high efficiency targeting by binding to integrin receptors on the cell membrane, improve the delivery effect of active ingredients (i.e., ceramide), and significantly enhance the permeability of ceramide; the inclusion body formed by fibronectin-modified liposomes encapsulating ceramide is stable, can reach the dermis layer, is not easily completely degraded in the epidermis layer, and significantly enhances the permeability of ceramide in the subcutaneous layer;

[0023] Enhanced cell absorption: Fibronectin, as a matrix for cell growth, can accelerate cell migration, make the cell morphology and structure good, and enhance the metabolic rate, thereby promoting the absorption of active ingredients (i.e., ceramide) encapsulated in liposomes by cells;

[0024] Improved biocompatibility: Fibronectin is a natural extracellular matrix protein with good biocompatibility, reduces immune responses, and prolongs the circulation time of liposomes in vivo;

[0025] Enhanced stability: Fibronectin modification can improve the physical and chemical stability of liposomes, reduce drug leakage, and extend the storage time;

[0026] Enhanced skin efficacy: Fibronectin can promote cells to produce nutrients such as collagen and hyaluronic acid, accelerate metabolism, and help the skin maintain a youthful state. At the same time, it can also stimulate cells to secrete collagen fibers, elastin, etc., fill the sunken parts of the skin, and reduce the formation of wrinkles.

[0027] The present invention provides a preparation method of the carrier encapsulating ceramide described in the above technical solutions, including the following steps: mixing fibronectin, hydrogenated phosphatidylcholine, carbodiimide condensing agent and buffer solution, and performing a grafting reaction under the condition of a pH value of 4.5 - 8.5 to obtain the carrier encapsulating ceramide. In the present invention, the reaction principle of the grafting reaction is: the carbodiimide condensing agent reacts with the phosphate ester group in hydrogenated phosphatidylcholine to form an ester intermediate, activate the substrate, and perform a phospholipid amidation reaction with fibronectin. The present invention activates the substrate hydrogenated phosphatidylcholine through a carbodiimide condensing agent, making it easy to perform a phospholipid amidation reaction with the protein, and improving the reaction targeting and reaction efficiency.

[0028] The present invention also provides a ceramide inclusion body. The ceramide inclusion body provided by the present invention has a small micelle particle size (about 146 nm), a high encapsulation rate (~65%), and a relatively high drug loading (~2%). Compared with the ceramide monomer before encapsulation, the ceramide inclusion body provided by the present invention has a significantly improved permeability in the skin subcutaneous layer, and has a stronger human skin efficacy (promoting gene expression, reducing the expression of various inflammatory factors) and more targeting than the ceramide encapsulated by a single liposome carrier. Description of the Drawings

[0029] Figure 1 The physical picture of the ceramide inclusion body sample obtained in Example 1;

[0030] Figure 2 The fluorescence picture of DAPI staining of blank mouse epidermis;

[0031] Figure 3 The fluorescence electron microscopy picture of the transdermal absorption (penetration for 2 h) of the ceramide inclusion body sample in Example 1. Figure 3 In the left picture in the middle, it is the fluorescence picture of the active substance (ceramide) (FITC fluorescence staining, green), and in the right picture, it is the fusion image of the active substance and mouse epidermal cells;

[0032] Figure 4 The fluorescence electron microscopy picture of the transdermal absorption (penetration for 8 h) of the ceramide inclusion body sample in Example 1. Figure 4 In the left picture in the middle, it is the fluorescence picture of the active substance (ceramide) (FITC fluorescence staining, green), and in the right picture, it is the fusion image of the active substance and mouse epidermal cells;

[0033] Figure 5 The particle size test result of the ceramide inclusion body sample in Example 1. Figure 5 In the left picture in the middle, it is the number and particle size of the particles, and in the right picture, it is the volume and particle size;

[0034] Figure 6 The particle size fluorescence picture of the ceramide inclusion body sample in Example 1. Detailed implementation mode

[0035] The present invention provides a carrier for encapsulating ceramide, which comprises hydrogenated phosphatidylcholine and fibronectin grafted on the hydrogenated phosphatidylcholine.

[0036] In the present invention, the structural formula of the hydrogenated phosphatidylcholine is shown as Formula I (CAS: 97,281-48-6), and it has a characteristic phosphate ester group.

[0037]

[0038] In the present invention, the hydrogenated phosphatidylcholine is stable and not easily oxidized; the fibronectin is preferably recombinant human fibronectin; the molecular weight of the recombinant human fibronectin is preferably 17-34.9 kDa, and the number of amino acids is preferably 180-200. In the examples of the present invention, the product number of the recombinant human fibronectin used is RPA037Hu01 (supplier: Cloud-Clone Corp). In the present invention, the hydrogenated phosphatidylcholine and fibronectin are grafted together through bonding.

[0039] In the examples of the present invention, the carrier for encapsulating ceramide is also called a protein-liposome fusion carrier.

[0040] In the present invention, a protein (fibronectin) is bound to the surface of a liposome (hydrogenated phosphatidylcholine). This binding can enhance the targeting and biocompatibility of the liposome, enabling it to interact better with cells. Through protein modification, the liposome can acquire special biological properties and better mimic the structure and function of cells in vivo. The protein used for modification is fibronectin, which can improve the delivery targeting of ceramide, enhance cell uptake, increase the permeability of ceramide, improve biocompatibility, enhance stability, and enhance skin efficacy.

[0041] The present invention provides a method for preparing the carrier for encapsulating ceramide as described in the above technical solution, comprising the following steps:

[0042] Mix fibronectin, hydrogenated phosphatidylcholine, a carbodiimide condensing agent, and a buffer solution, and carry out a grafting reaction to obtain the carrier for encapsulating ceramide; the pH value of the mixed solution obtained by the mixing is 4.5 - 8.5.

[0043] In the present invention, unless otherwise specified, the raw materials involved are all well-known commercially available products in the art.

[0044] In the present invention, the hydrogenated phosphatidylcholine is the same as the hydrogenated phosphatidylcholine in the above technical solution and will not be elaborated herein. In the present invention, the fibronectin is preferably recombinant human fibronectin; the molecular weight of the recombinant human fibronectin is preferably 17 - 34.9 kDa, and the number of amino acids is preferably 180 - 200. If the molecular weight of the recombinant human fibronectin is too large, it will cause steric hindrance and affect the progress of the reaction. If the molecular weight is too small, the protein structure cannot be formed.

[0045] In the present invention, the carbodiimide condensing agent preferably includes 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCI, a water-soluble carbodiimide). In the present invention, the buffer solution preferably includes MES (2-(N-morpholino)ethanesulfonic acid) buffer solution, PBS buffer solution (phosphate buffer solution), MOPS (3-(N-morpholino)propanesulfonic acid) buffer solution, HEPES ((4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer solution, or acetic acid / sodium acetate buffer solution, and the concentration of the buffer solution is preferably 0.05 - 0.2 mol / L.

[0046] In the present invention, the molar ratio of the carbodiimide condensing agent to hydrogenated phosphatidylcholine is preferably 1:1 to 5:1, and can be 1:1, 2:1, 3:1, 4:1 or 5:1. The molar ratio of fibronectin to hydrogenated phosphatidylcholine is preferably 1:400 to 1:100, and can be 1:400, 1:333, 1:300, 1:200, 1:167, 1:133 or 1:100. When the amount of fibronectin is too large, it will affect the liposolubility of the carrier. When the amount is too small, its modification effect on hydrogenated phosphatidylcholine cannot be exerted. In the present invention, in the mixed solution obtained by mixing fibronectin, hydrogenated phosphatidylcholine, carbodiimide condensing agent and buffer, the concentration of hydrogenated phosphatidylcholine is preferably 10 mmol / L.

[0047] In the present invention, the mixing method can be to first add fibronectin and hydrogenated phosphatidylcholine, and then add carbodiimide condensing agent and buffer. In the present invention, the pH value of the obtained mixed solution is 4.5 to 8.5, and can be 4.5, 5.5, 6.0, 6.5, 7.3, preferably 5.5 to 6.5. When the pH value is lower than 3.5, the grafting reaction will not occur.

[0048] In the present invention, the temperature of the grafting reaction is preferably 15 to 60 °C, and can be 15, 20, 25, 30, 40, 50 or 60 °C. The time is preferably 1 to 8 h, and can be 1, 2, 3, 4, 5, 6, 7 or 8 h. The grafting reaction is preferably carried out under stirring, and the stirring rate is preferably 200 to 2000 rpm, and can be 300, 500, 800, 1000, 1500 or 2000 rpm.

[0049] Fibronectin is negatively charged and is a high-molecular-weight non-collagenous glycoprotein. The entire peptide chain is composed of three types (I, II, III) of internally sequence-homologous structural repeats arranged in a repetitive manner. Fibronectin plays an important role in the extracellular matrix and basement membrane and has a variety of biological activities. It can be interconnected with cell fibers and proteoglycans to play an adhesive role. The carbodiimide condensing agent catalyzes the reaction between the phosphate ester in hydrogenated phosphatidylcholine and the secondary amino group on fibronectin.

[0050] In the present invention, the reaction principle of the grafting reaction is as follows: The carbodiimide condensing agent (such as EDCI) reacts with the phosphate ester group in hydrogenated phosphatidylcholine to form an ester intermediate, activating the substrate and performing a phosphatidylation reaction with fibronectin. In the present invention, the substrate hydrogenated phosphatidylcholine is activated by the carbodiimide condensing agent, making it easy to perform a phosphatidylation reaction with the protein, thereby improving the reaction targeting and reaction efficiency.

[0051] After the graft reaction is completed, the present invention preferably adds a terminator to the obtained reaction solution, then performs column chromatography purification, and lyophilizes the purified product to obtain the carrier encapsulating ceramide.

[0052] In the present invention, the terminator is preferably Tris buffer solution; the concentration of the terminator in the reaction solution is preferably 50 mmol / L.

[0053] In the present invention, the conditions for column chromatography purification preferably include: Agilent ZORBAX SB-C18 reversed-phase C18 column, column size 10 mm×250 mm or 21.2 mm×250 mm (suitable for mg-g level samples); mobile phase A: water or 5-10 mM ammonium acetate aqueous solution, mobile phase B: acetonitrile / isopropanol (1:1, v / v); detector: ELSD (evaporative light scattering detector); gradient elution: as shown in Table 1. The present invention removes unreacted raw materials and by-products through the column chromatography purification.

[0054] Table 1 Gradient elution program

[0055] Time (min) Mobile Phase B (%) Flow Rate (mL / min) 0 20 1.0 0～10 20→80 1.0 10～15 80→100 1.0 15～20 100 1.0 20～25 100→20 1.0

[0056] The present invention provides a ceramide inclusion, comprising the following raw materials by mass percentage: glycerol 30-70%, hexyldecanol 5-35%, water 5-15%, ceramide 2-8%, cholesterol 1-8%, carrier 1-10%, and the carrier is the carrier encapsulating ceramide described in the above technical solution or the carrier encapsulating ceramide prepared by the preparation method described in the above technical solution.

[0057] In the present invention, the ceramide preferably includes one or more of ceramide NP (ceramide 3), NS, NH, NDS, NSD, AS, AP, AH, ASD, ADS, OS, OP, OH, OSD, ODS, EOP, EOS, EOH, EOSD and EODS. The fatty acid source in the structure of the ceramide can be saturated fatty acid or unsaturated fatty acid, including stearic acid, oleic acid, palmitic acid, linolenic acid, linoleic acid, etc. The fatty acid source can be of plant origin or obtained by chemical separation and synthesis. The plant oil sources include one or more of perilla seed oil, Limnanthes alba seed oil, jojoba oil, Adansonia digitata seed oil, Acer truncatum seed oil, grape seed oil, sesame oil, kiwi seed oil, walnut oil, camellia oil, rice bran oil, olive oil, safflower oil, rosehip oil, coconut oil and almond oil. The present invention has no special requirements for the source of the ceramide, and it can be obtained by using commercially available products or methods well-known in the art.

[0058] In the present invention, the mass percentage content of glycerol in the preparation raw materials can be 30%, 40%, 50%, 60% or 70%, the mass percentage content of hexyl decanol can be 10%, 20% or 30%, the mass percentage content of water can be 10%, 11%, 12%, 13%, 14% or 15%, the mass percentage content of ceramide can be 2%, 5% or 8%, the mass percentage content of cholesterol can be 1%, 5% or 8%, and the mass percentage content of the carrier can be 1%, 5% or 10%.

[0059] In the present invention, the hexyl decanol and cholesterol are used as stabilizers to reinforce the carrier structure, and glycerol and water are solvents. In the present invention, the mass ratio of the carrier to ceramide is preferably 5:1.

[0060] The present invention provides a method for preparing the ceramide inclusion complex according to the above technical solution, comprising the following steps:

[0061] Perform a first mixing of ceramide, cholesterol, the carrier and hexyl decanol to obtain an oil phase;

[0062] Perform a second mixing of glycerol and water to obtain an aqueous phase;

[0063] Add the oil phase to the aqueous phase for emulsification to obtain a mixture;

[0064] Perform high-pressure microfluidic homogenization on the mixture to obtain the ceramide inclusion complex.

[0065] In the present invention, the temperature of the first mixing is preferably 50 - 70°C, the time is preferably 10 - 15 min, and the first mixing is preferably stirring mixing. In the present invention, the operation of the first mixing is preferably: mix ceramide, cholesterol and the carrier, stir evenly; add hexyl decanol thereto, heat to 50 - 70°C, stir for 10 - 15 min to form a uniform oil phase; then keep warm.

[0066] In the present invention, the temperature of the second mixing is preferably 50 - 60°C, the second mixing is preferably stirring mixing, and the stirring time is based on mixing glycerol and water evenly; keep warm after the second mixing.

[0067] The present invention preferably adds the oil phase slowly to the aqueous phase. In the present invention, the emulsification method is preferably stirring, and the stirring speed is preferably 1000 rpm, which is rapid stirring.

[0068] In the present invention, the high-pressure microfluidic homogenization is specifically carried out in a high-pressure microfluidic homogenizer. The pressure of the high-pressure microfluidic homogenization is preferably 10,000 - 20,000 PSI (about 700 - 1400 bar), and the number of circulation treatments is preferably 3 - 5 times. The high-pressure microfluidic homogenization utilizes high pressure to form a high-speed jet of the material through a tiny nozzle, and realizes the refinement and homogenization of the material through strong impact and shear effects.

[0069] After the high-pressure microfluidic homogenization is completed, it is cooled to room temperature to obtain the ceramide inclusion body.

[0070] By encapsulating ceramide with the carrier in the present invention, the obtained ceramide inclusion body micelles have a small particle size (about 146 nm), a high encapsulation efficiency (~65%), and a relatively high drug loading (~2%). The in vitro cumulative release test shows that the micelles have a certain sustained-release effect, and the release of the active substance is mainly by Fick diffusion. In the stability analysis, it is obtained that the micelles are unstable under continuous strong light irradiation, and light exposure should be avoided. However, compared with the ceramide monomer before encapsulation, the ceramide inclusion body provided by the present invention has a significant improvement in permeability in the skin stratum corneum, and has a stronger human skin efficacy and more targeting than the ceramide encapsulated by a single liposome carrier.

[0071] The present invention provides the application of the ceramide inclusion body described in the above technical solution or the ceramide inclusion body prepared by the preparation method described in the above technical solution in the preparation of cosmetics. In the present invention, the cosmetics are specifically in the skin care field of cosmetics.

[0072] In order to further illustrate the present invention, the following examples are used to describe in detail the carrier for encapsulating ceramide, its preparation method, the ceramide inclusion body, and its application provided by the present invention, but they cannot be understood as limiting the protection scope of the present invention.

[0073] Example 1

[0074] The carrier for encapsulating ceramide is prepared as follows:

[0075] Fibronectin (Recombinant Human Fibronectin, Catalog No.: RPA037Hu01, Source: Prokaryotic expression (E. coli), Supplier: Cloud-Clone Corp) 50 μM, Hydrogenated phosphatidylcholine (the structure shown in Formula I, CAS: 97281-48-6) 10 mM, EDCI 20 mM, were added to MES buffer (pH = 6.15), the pH of the system was adjusted to 6.0, the reaction volume was 10 mL, the reaction temperature was 25 °C, and the reaction was carried out at a stirring speed of 800 rpm for 2 h; then a terminator, 50 mM Tris buffer (the above concentrations are the concentrations of the respective components in the solution system), was added, and the protein-liposome carrier (i.e., the carrier encapsulating ceramide) was separated by column chromatography (Agilent ZORBAX SB-C18 reversed-phase C18 column, column size 10 mm × 250 mm; mobile phase A: 5 mM ammonium acetate aqueous solution, mobile phase B: acetonitrile / isopropanol (1:1, v / v); detector: ELSD (Evaporative Light Scattering Detector); gradient elution: as shown in Table 1).

[0076] Ceramide inclusion, the preparation method is as follows:

[0077] The protein-liposome carrier prepared above (i.e., the carrier encapsulating ceramide) was used for ceramide encapsulation. Raw materials: The mass ratio of the above protein-liposome carrier to ceramide NP derived from the active ingredient perilla seed oil is 5:1, the mass fraction of ceramide NP is 2%, the mass fraction of glycerol is 50%, the mass fraction of hexyl decanol is 20%, the mass fraction of cholesterol is 5%, and the remaining solvent is deionized water. The preparation method of the ceramide inclusion is as follows:

[0078] Preparation of the oil phase: Ceramide NP, cholesterol, and the protein-liposome carrier were mixed, stirred evenly, hexyl decanol was added, heated to 50 °C, and stirred for 10 min to form a uniform oil phase, and kept warm;

[0079] Preparation of the water phase: Glycerol and water were mixed, heated to 50 °C, and stirred evenly to obtain the water phase, and kept warm;

[0080] Mixing and emulsification: The oil phase was slowly added to the water phase, and rapidly stirred at a speed of 1000 rpm for mixing and emulsification;

[0081] High-pressure microfluidic treatment: The emulsified mixture was transferred to a high-pressure microfluidic homogenizer, the homogenization pressure was 130 MPa, homogenized 3 times, and cooled to room temperature to obtain the ceramide inclusion.

[0082] Figure 1 It is a physical picture of the ceramide inclusion sample obtained in Example 1.

[0083] Examples 2 - 7

[0084] The conditions for preparing the carrier encapsulating ceramide are shown in Table 2. The operation of preparing the carrier encapsulating ceramide and the method for preparing the ceramide inclusion body are the same as those in Example 1.

[0085] Table 2 Condition parameters for preparing the carrier encapsulating ceramide in Examples 1 - 7

[0086]

[0087] Comparative Example 1: (Single liposome encapsulation)

[0088] Replace the protein - liposome carrier in the raw materials for preparing the ceramide inclusion body in Example 1 with hydrogenated phosphatidylcholine. Other conditions and the method for preparing the ceramide inclusion body are the same as those in Example 1.

[0089] Comparative Example 2: (Other protein, silk protein)

[0090] Replace the fibronectin in Example 1 with silk protein (molecular weight 20 kDa), and the rest is the same as Example 1.

[0091] Test the performance of the ceramide inclusion bodies prepared in the examples and comparative examples as follows:

[0092] (I) Stability test

[0093] Necessity of the test: Since ceramide NP in this system is water - insoluble at room temperature, it is necessary to consider whether there is oil - water stratification after encapsulation. Therefore, the investigation of its stability is crucial.

[0094] Test method: The high - temperature and low - temperature storage stability test methods in the laboratory include: (1) Heat - resistance stability test: Place in an electro - thermal constant - temperature incubator at 45 °C for 45 days, and observe after restoring to room temperature; (2) Cold - resistance stability test: Store at - 5 °C for 1 week and observe its stability; (3) Test method for light stability: Use a xenon arc lamp to simulate artificial light source (sunlight, non - strong light), and place for 7 and 15 days to observe stability, whether there are phenomena such as discoloration, stratification, turbidity, etc.; (4) Test method for centrifugal stability: Add the sample to a centrifuge and rotate at a speed of 10000 rpm for 12 min, and observe the liquid stratification and separation. The test results of the stability test are shown in Table 3.

[0095] Table 3 Stability test results of various example / comparative example samples

[0096] Sample Heat-resistant (45°C) Cold-resistant (-5°C) Centrifugation Light - 7 days Light - 15 days Example 1 Stable Stable Stable Stable Slightly turbid Example 2 Precipitation Stable Stable Precipitation Precipitation Example 3 Precipitation Stable Stable ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​

[0097] Table 3 results show that in the temperature, centrifugation, and 7-day simulated light stability tests, except for Example 1, Example 7, and Comparative Example 2, instability phenomena occurred in the remaining examples and comparative examples; under 15 days of long-term light exposure, slight turbidity also occurred in Example 1. This shows that the control of the content of the feed during the preparation process is relatively important, and the control of the process duration and temperature is also particularly important. Example 7 and Example 1 have the same process and the same feed ratio, only the types of ceramides are different, but it does not affect the stability. In Comparative Example 2, only the type of protein is different, the process and feed ratio are the same as those in Example 1, and the stability gap is not large.

[0098] (2) Permeation simulation: Percutaneous absorption test

[0099] Fluorescent labeling of the active substance: Since the ceramide contains a secondary amino group (-NH-), therefore, the active substance ceramide NP is fluorescently labeled with FITC. Fluorescein isothiocyanate (FITC) is a common fluorescent dye, which is commonly used in fluorescence immunoassay, fluorescence staining and other applications in the biomedical field. Fluorescein isothiocyanate (FITC) can crosslink with amino, sulfhydryl, imidazole, tyrosyl and carbonyl groups on proteins. However, only derivatives of primary and secondary amines can produce stable labeled products. The reaction is most efficient at pH 8-9 and must be carried out in an amine-free buffer such as borate or carbonate / bicarbonate. Usually, proteins react with 15 to 20 times the molar amount of FITC, which can result in several FITC molecules binding to one protein molecule. A centrifugal column filled with purified resin simplifies the steps of column equilibration, collection, and monitoring of gravity flow fractions. This system can effectively remove excess FITC, thereby accurately determining the dye-to-protein ratio (F / P) and protein concentration.

[0100] Animal skin: It is common to use animal skin in percutaneous absorption studies. It is ideal to choose the skin of Kunming mice as the percutaneous absorption research model. The permeation time is set to 2 h and 8 h, and the skin is usually taken from the back skin of mice. Apply 0.5 mL of the wrapped sample with FITC fluorescent agent evenly, then slice, stain the stratum corneum cells with DAPI, embed, observe the slice morphology under a confocal microscope, and calculate the permeation rate (fluorescent permeation amount in the skin and subcutaneous layer / total amount of 0.5 mL sample × 100%). 2 Apply 0.5 mL of the wrapped sample with FITC fluorescent agent evenly, then slice, stain the stratum corneum cells with DAPI, embed, observe the slice morphology under a confocal microscope, and calculate the permeation rate (fluorescent permeation amount in the skin and subcutaneous layer / total amount of 0.5 mL sample × 100%).

[0101] The calculated results of the permeation rates of the ceramide inclusion samples of each example and comparative example in the skin and subcutaneous layer of mice are shown in Table 4.

[0102] Table 4 Calculated results of the permeation rates of the example / comparative example samples in the skin and subcutaneous layer of mice

[0103]

[0104] According to the penetration results of various samples in Table 4 above, within 2 h and 8 h, the percutaneous penetration rate of Example 1 is the highest, which are 4.58% and 13.14% respectively; followed by Example 7. The ceramide in Example 7 is derived from sesame oil, and sesame oil is mainly composed of oleic acid and contains monounsaturated fatty acid bonds, while the ceramide in Example 1 is derived from perilla seed oil, and the main fatty acids are linolenic acid and linoleic acid, which contain conjugated double bonds, have a higher affinity with the skin and are more likely to penetrate. The stirring speed and reaction time in Examples 2-6 may be the main factors affecting encapsulation and penetration rate. Comparative Example 1 is encapsulated by single liposomes and also has a penetration rate, but it is lower than that of the examples; the affinity of silk fibroin in Comparative Example 2 with the skin is not as high as that of fibronectin, and the penetration rate of the active substance is relatively low. It can be seen that the sample of Example 1 has the best penetration rate in the skin of mice.

[0105] ​ It is the fluorescence image of DAPI staining of the blank mouse epidermis.

[0106] ​ It is the fluorescence electron microscopy image of the percutaneous absorption (penetration for 2 h) of the ceramide inclusion sample of Example 1. ​ In the left figure, it is the fluorescence image of the active substance (ceramide) (FITC fluorescence staining, green), and in the right figure, it is the fusion image of the active substance and mouse epidermal cells. It can be seen that part of the active substance penetrates into the stratum corneum and subcutaneous tissue after 2 h of penetration.

[0107] ​ It is the fluorescence electron microscopy image of the percutaneous absorption (penetration for 8 h) of the ceramide inclusion sample of Example 1. ​ In the left figure, it is the fluorescence image of the active substance (ceramide) (FITC fluorescence staining, green), and in the right figure, it is the fusion image of the active substance and mouse epidermal cells. It can be seen that more active substance (compared with 2 h) penetrates into the stratum corneum and subcutaneous tissue after 8 h of penetration.

[0108] (III) Particle size test

[0109] Testing equipment: Nano flow cytometer.

[0110] Reagents: Phosphate buffer solution with a pH value of 7.4; concentration standard product of nano flow cytometer (1.99×1010 particles / ml).

[0111] Detection steps:

[0112] 1) According to the operation instructions, perform liquid flow initialization and pipeline air bubble discharge on the nano flow cytometer.

[0113] 2) Dilute the concentration standard with ultrapure water by 100 times, perform the quality control of the nanoparticle flow cytometer according to the instructions. After adjusting the nanoparticle flow cytometer to the best detection state (the signals of both the scattering and fluorescence channels reach the strongest and are uniform), collect data for the diluted standard under the measurement parameters of the concentration standard (under the best state, the particle count range of the quality control standard is usually between 5000 and 7000).

[0114] 3) According to the instructions, wash the injection capillary with the washing solution and ultrapure water in sequence. Dilute the S16M-EXO particle size standard with ultrapure water by 100 times, and collect data for the diluted particle size standard under the measurement parameters of the S16M-EXO particle size standard.

[0115] 4) Pre-dilute the sample with clean phosphate buffer by 100 times, and collect data for the small extracellular vesicle sample under the sample measurement parameters. The accurate measurement range of the nanoparticle flow cytometer particle count is between 4000 and 8000. If the particle count of the sample is too high or too low, the dilution factor of the sample needs to be adjusted and then measured, and record the final dilution factor of the sample. In addition, it should be noted whether the baseline and automatic threshold of the scattering channel during the sample detection are consistent with those of the blank control (phosphate buffer) detection. If the baseline rises during the sample detection, a sample with higher purity needs to be prepared again.

[0116] Result analysis:

[0117] Use the NF Profession software to perform in sequence: sample threshold setting, particle size standard curve fitting, blank control particle deduction, and generation of the sample particle size distribution histogram, and finally obtain the particle size detection report of the sample to be tested. The average particle sizes of the ceramide inclusion samples in each example / control are shown in Table 5.

[0118] Table 5 Test results of the average particle sizes of the samples in each example / control

[0119] ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​

[0120] ​ For the particle size test results of the ceramide inclusion sample in Example 1, ​ The left figure in the middle is the number of particles and the particle size, and the right figure is the volume and the particle size. ​ For the particle size fluorescence map of the ceramide inclusion sample in Example 1. The average particle size of the ceramide inclusion sample in Example 1 is 146.6 nm, and the volume is about 5×10 8 nm 3 ,D 10 The particle size is 92.2 nm, D 90 The particle size is 230.8 nm.

[0121] Conclusion: In the comparison of the average particle size, the particle size of Example 1 is smaller, and the particle size is related to the properties of the carrier and the encapsulation process. Under the same encapsulation process, the particle size of Example 1 is smaller, followed by Example 7. In Example 7, the ceramide NP derived from sesame oil is mainly composed of unsaturated fatty acid oleic acid, which has a smaller degree of curl compared to linoleic acid and linolenic acid containing conjugated double bond polyunsaturated fatty acids, resulting in a nanometer particle size inferior to that of Example 1. In Comparative Example 1, the encapsulation affinity between the liposome and the active substance is inferior to that of Example 1, leading to a larger particle size than that of Example 1, but it can also form a nanostructure.

[0122] (IV) Encapsulation Efficiency Test

[0123] Method: Ultracentrifugation method. The ultracentrifugation method separates based on the gravity difference between the free drug ceramide and the carrier micelles, and calculates the encapsulation efficiency.

[0124] Specific operation and separation principle: Use a 10KDa ultrafiltration centrifugal tube to centrifuge the encapsulated sample. Since the molecular weight of monomeric ceramide is relatively low (~580Da), less than 1kDa; while the molecular weight of the carrier micelles is larger (>10kDa). Therefore, the remaining unencapsulated ceramide NP small molecules will be filtered to the bottom of the test tube, and the large molecular carrier micelles will be deposited at the bottom of the ultrafiltration centrifugal tube. The large molecular micelles in the ultrafiltration tube are ultrasonically broken with 100% amplitude and then filtered to obtain the small molecular active substances encapsulated in the micelles, and quantitative analysis by HPLC-ELSD is carried out.

[0125] HPLC-ELSD quantitative test method: 1. Instruments and reagents: Instruments: High performance liquid chromatograph (HPLC), evaporative light scattering detector (ELSD); Reagents: Acetonitrile (chromatographic grade), methanol (chromatographic grade), deionized water, ceramide standard. 2. Chromatographic conditions: Chromatographic column: C18 reverse phase chromatographic column (250mm×4.6mm, 5μm); Mobile phase: Acetonitrile:Methanol:Water (70:20:10, v / v / v); Flow rate: 1.0mL / min; Column temperature: 30℃; Injection volume: 10μL. 3. ELSD conditions: Drift tube temperature: 40℃; Gas flow rate: 2.5L / min (nitrogen); Gain: 1. 4. Sample preparation: Standard solution: Accurately weigh the ceramide standard, dissolve it with methanol and dilute to the required concentration; Sample solution: Dissolve the sample to be tested with methanol, filter and inject.

[0126] The encapsulation efficiency test results of the ceramide inclusion samples of each example / comparative example are shown in Table 6.

[0127] Table 6 Encapsulation Efficiency Test Results of Samples of Each Example / Comparative Example

[0128] ​ ​ ​ 65.75％±0.5％ ​ 54.32％±0.7％ ​ 51.39％±0.4％ ​ 56.49％±0.6％ ​ 49.80％±0.9％ ​ 53.14％±0.8％ ​ 62.93％±0.4％ ​ 50.31％±0.7％ ​ 51.02％±0.8％

[0129] As shown in the results of Table 6 above, the highest encapsulation efficiency of the sample in Example 1 was 65.75% ± 0.5%, followed by that in Example 7 with an encapsulation efficiency of 62.93% ± 0.4%. The carrier structure of the fibronectin-liposome fusion is more encapsulating and stable. The stabilities of the remaining examples and comparative examples are inferior to that of Example 1, resulting in lower encapsulation efficiencies.

[0130] (V) Efficacy test: Barrier repair gene test

[0131] Introduction of the tested barrier genes: FLG (filaggrin) is a precursor for the production of NMF (natural moisturizing factor) by stratum corneum cells. CASP14 (Caspase14) and BLMH (Bleomycin Hydrolase) are two key proteases responsible for the proteolysis of FLG protein to produce NMF. The mRNA expression levels of each gene were tested according to the qPCR method. INV (Involucrin), a key enzyme promoting skin barrier maturation, is one of the key protein components forming a stable rigid protein complex, replacing CE (cornified envelope) in the cell membrane of keratinocytes. TG (transglutaminase) is an enzyme that cross-links CE protein components such as involucrin, loricrin, and periplakin to form a tight and stable protein cell envelope. The mRNA expression levels of INV and TG-related genes were tested according to the qPCR method.

[0132] Test method: The mRNA expression levels of related genes were tested using the qPCR (Quantitative Real-time PCR) method. Human immortalized keratinocytes (Hacat) were cultured in vitro. According to the cytotoxicity results, after adding the sample at the cell-safe concentration, 1 mL of Trizol (total RNA extraction reagent) was added to extract total RNA. According to the gene primer sequences, the relative expression levels of collagen genes were analyzed using the 2-△△CT method (the relative content of the blank group was 100%).

[0133] The gene sequences are as follows: (F represents the forward primer sequence, R represents the reverse primer sequence)

[0134] FLG (F) TCG GCAAAT CCT GAA GAATCCAGA (SEQ ID NO.1)

[0135] FLG (R) GCT TGA GCCAAC TTGAATACCATCAG (SEQ ID NO.2)

[0136] CASP14 (F) GAC CTG GAT GCT CTG GAA CACA (SEQ ID NO.3)

[0137] CASP14(R)GAATCGATG GCC TGC TGGA(SEQ ID NO.4)

[0138] BLMH(F)TGT GGT TTG GCT GTGATG TT(SEQ ID NO.5)

[0139] BLMH(R)GCA CCATCC TGATCATCC TT(SEQ ID NO.6)

[0140] INV(F)CCATCA GGA GCAAAT GAAACAG(SEQ ID NO.7)

[0141] INV(R)GCT CGA CAG GCA CCT TCT G(SEQ ID NO.8)

[0142] TG(F)CCC CGG TTG GCATACACA(SEQ ID NO.9)

[0143] TG(R)GAG CGGAAG GCA GTAGAGACA(SEQ ID NO.10);

[0144] The relative expression levels of each gene in the control group were all 1.

[0145] The expression results of each barrier-related gene in the ceramide inclusion body samples of each example / comparative example are shown in Table 7.

[0146] Table 7 Expression results of each barrier-related gene in the samples of each example / comparative example

[0147]

[0148]

[0149] Conclusion: Compared with the control group, each example and the control had the effect of promoting gene expression, indicating that the active substance had a certain efficacy. Comparing the examples and the comparative examples, the expression in Example 1 was stronger, and the expression levels of each gene of FLG, CASP14, BLMH, INV, and TG were increased by 45%, 67%, 66%, 48%, and 45% respectively. There were increases in Examples 2-7 but not as much as in Example 1. There were also increases in Comparative Example 1 and Comparative Example 2, but the increases in the comparative examples were also not as much as in Example 1.

[0150] (VI) Efficacy test: Anti-inflammatory factor gene test

[0151] Method: Seed HaCaT cells in 6 / 12-well plates (adjusted according to the qPCR requirements) at a density of 5×105 cells / mL, cultured in DMEM medium containing 10% FBS until 80% confluence. Changed to serum-free medium 24 h before the experiment (to reduce the interference of serum on the inflammatory response). Establishment of the inflammation model (LPS stimulation): Added medium containing LPS (100 μg / mL) and incubated for 6 h (to induce the inflammatory response); Stimulation model control group: Only stimulated with LPS (without adding drugs); After incubation for 6 h, added 0.1 g / L of the positive control product (using dexamethasone as the positive control) and 0.1% by mass of the test sample and continued to incubate for 24 h. Subsequently, according to each ELISA kit, detected the contents of IL-1α, IL-6, and IL-8 in the cells of each group and calculated the change rate.

[0152] The content of each inflammatory factor compared with the control group: (sample group - control group) / control group × 100%.

[0153] The results of the inhibition of inflammatory factors by the ceramide inclusions of the samples in each example / control example are shown in Table 8.

[0154] Table 8 Results of the inhibition of inflammatory factors by the samples in each example / control example

[0155]

[0156] Conclusion: Compared with the control group, each example and the control have the effect of reducing the expression of each inflammatory factor, indicating that the active substance has a certain efficacy. Comparing the examples and the control examples, Example 1 has a stronger inhibition rate of inflammatory factors, and the inhibition rates of IL-1α, IL-6, and IL-8 are 34.12%, 29.42%, and 25.47% respectively. There is an anti-inflammatory effect in Examples 2 to 7, but it is not as good as Example 1. There is also an effect in Control Example 1 and Control Example 2, but the efficacy of the control examples is also not as good as Example 1.

[0157] The above is only the preferred embodiment of the present invention and does not impose any form of limitation on the present invention. It should be noted that for those of ordinary skill in the art of this technology, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A ceramide-encapsulating carrier, characterized in that, Comprising hydrogenated phosphatidylcholine and fibronectin grafted onto the hydrogenated phosphatidylcholine.

2. The ceramide-encapsulating carrier according to claim 1, wherein, The fibronectin is recombinant human fibronectin.

3. The preparation method of the carrier encapsulating ceramide according to claim 1 or 2, characterized in that, Comprising the following steps: Mix fibronectin, hydrogenated phosphatidylcholine, a carbodiimide condensing agent, and a buffer solution, and carry out a grafting reaction to obtain the carrier encapsulating ceramide; the pH value of the resulting mixed solution is 4.5 to 8.

5.

4. The preparation method according to claim 3, wherein The carbodiimide condensing agent includes 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide.

5. The preparation method according to claim 3 or 4, characterized in that, The molar ratio of the carbodiimide condensing agent to hydrogenated phosphatidylcholine is 1:1 to 5:1, and the molar ratio of fibronectin to hydrogenated phosphatidylcholine is 1:400 to 1:

100.

6. The preparation method according to claim 3, wherein The buffer solution includes MES buffer solution, PBS buffer solution, MOPS buffer solution, HEPES buffer solution, or acetic acid / sodium acetate buffer solution.

7. The preparation method according to claim 3, characterized in that The temperature of the grafting reaction is 15 to 60 °C, the time is 1 to 8 h, the grafting reaction is carried out under stirring conditions, and the stirring rate is 200 to 2000 rpm.

8. A ceramide inclusion body, characterized in that, Comprising the following raw materials for preparation by mass percentage: 30 to 70% glycerol, 5 to 35% hexyldecanol, 5 to 15% water, 2 to 8% ceramide, 1 to 8% cholesterol, 1 to 10% carrier, and the carrier is the carrier encapsulating ceramide described in claim 1 or 2 or the carrier encapsulating ceramide prepared by the preparation method described in any one of claims 3 to 7.

9. The preparation method of the ceramide inclusion body according to claim 8, characterized in that, Comprising the following steps: First mix ceramide, cholesterol, the carrier, and hexyldecanol to obtain an oil phase; Second mix glycerol and water to obtain an aqueous phase; Add the oil phase to the aqueous phase for emulsification to obtain a mixed solution; Subject the mixed solution to high-pressure microfluidic homogenization to obtain the ceramide encapsulation.

10. Use of the ceramide encapsulation described in claim 8 or the ceramide encapsulation prepared by the preparation method described in claim 9 in the preparation of cosmetics.

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