Hyaluronic acid ceramide nano raw material as well as preparation method and application thereof

Abstract

The invention provides a hyaluronic acid ceramide nano raw material applied to the fields of cosmetics and medicines and a preparation method of the hyaluronic acid ceramide nano raw material. The nano raw material disclosed by the invention is characterized in that ceramide and a hyaluronic acid derivative are self-assembled to form 10-1000nm nanoparticles, wherein the drug loading capacity of ceramide is 2.5-50%. The nano raw material can improve the dispersity and stability of ceramide and enhance the skin permeability of the ceramide. The hyaluronic acid ceramide nano raw material disclosed by the invention can be used for preparing cosmetics and medical products for moisturizing, resisting aging, whitening, controlling oil, removing acnes, preventing alopecia and repairing skin barriers.

Description

Technical Field

[0001] The present invention relates to a hyaluronic acid ceramide nano raw material applied to the fields of cosmetics and medicine and a preparation method thereof. Background Art

[0002] Ceramide is a class of compounds formed by the combination of sphingosine and long-chain fatty acids through amide bonds. It is synthesized and secreted by lamellar bodies and constitutes intercellular lipids together with cholesterol and free fatty acids, participating in maintaining the skin barrier function. There are 16 different subtypes of ceramide in the stratum corneum of the skin. Except for 4 subtypes that are connected to the cell cornified envelope, the remaining 12 subtypes are free subtypes and can be extracted separately, including ceramide NP (also known as ceramide 3, accounting for 22.1%), NH (also known as ceramide 8, accounting for 14.5%), AH (also known as ceramide 7, accounting for 10.8%), NDS (also known as ceramide 10, accounting for 9.8%), AS (also known as ceramide 5, accounting for 9.6%), AP (also known as ceramide 6, accounting for 8.8%), NS (also known as ceramide 2, accounting for 7.4%), EOS (also known as ceramide 1, accounting for 6.5%), EOH (also known as ceramide 4, accounting for 4.3%), ADS (also known as ceramide 11, accounting for 1.6%), EOP (also known as ceramide 9, accounting for 1.1%) and EODs (accounting for 0.4%).

[0003] Ceramide, as the main component of the intercellular lipids in the stratum corneum of the skin, not only plays an important role in the formation of the epidermal stratum corneum, but also is the central molecule of the sphingomyelin signaling pathway. As a second messenger, it regulates signal processes such as apoptosis, proliferation, differentiation, and growth arrest, and has functions such as maintaining the skin barrier, moisturizing, anti-aging, whitening, and disease treatment [Wei Jing, et al. Physiological effects of ceramide on the skin and its application in cosmetics. Cereals and Oils, 2007, (1): 21-24]. However, due to its strong hydrophobicity, poor solubility, easy crystallization and precipitation, and poor transdermal performance, traditional formulations are difficult to effectively deliver to the deep layer of the skin, affecting the exertion of its efficacy, which greatly limits its application in cosmetics and drugs. Nanopreparations can significantly improve the solubility, stability, and transdermal performance of ceramide, and increase its retention amount and bioavailability in the skin. Ceramide nanopreparations can effectively treat skin diseases such as xerosis, eczema, and psoriasis, relieve symptoms, and promote skin repair and regeneration. The currently disclosed ceramide nanotechnologies (including nanoemulsions and liposomes) have preparation methods including high-pressure homogenization, emulsification, and self-assembly methods, etc. Among them, the liposome preparation technology (such as CN201811293278.7 and CN201810215403.6) is used to prepare water-soluble ceramide liposomes and add them to products. However, its preparation method still has problems such as difficult particle size control, high cost, and complex large-scale production processes. In addition, since additional surfactants need to be added to such nano raw materials, it increases the risk of skin irritation and limits its further popularization and application.

[0004] Hyaluronic acid (HA), also known as hyaluronan and sodium hyaluronate, is one of the main components of the extracellular matrix of the skin. HA has strong water absorption and can absorb 1000 times its own weight of water. It is considered an ideal natural moisturizing factor when applied to the skin surface. Small molecule HA also has functions such as regulating the differentiation and migration of epidermal cells and scavenging free radicals. Therefore, in addition to its moisturizing effect, it also has functions such as skin nutrition, sunscreen repair, lubrication, film formation, and thickening.

[0005] Natural HA is a chain polyanionic mucopolysaccharide composed of repeating units of (1-β-4) D-glucuronic acid and (1-β-3) N-acetyl-D-glucosamine disaccharide units. Due to having multiple hydroxyl and carboxyl groups with strong hydrophilicity, it easily forms hydrogen bonds with water molecules. In a solution with a lower concentration, it is a curled linear molecule, and in a solution with a higher concentration, it is a network structure. Since it lacks hydrophobic groups, it cannot form spherical molecules. Appropriate modification of the HA macromolecule with small molecule compounds containing hydrophobic groups to form partial hydrophobic regions on the HA molecule can obtain modified HA derivatives (such as esterified derivatives of HA), making it possible for its molecules to form spheres in solution.

[0006] Diseases such as acne, seborrheic alopecia, seborrheic dermatitis, benign prostatic hyperplasia and prostate cancer are all related to excessive levels of androgen in the body. Excessive levels of androgen in the body are the most important cause of sebaceous gland hyperfunction, abnormal metabolism and secretion of skin fat cells, and excessive proliferation of stratum corneum cells, which in turn lead to acne. Increased levels of dihydrotestosterone in different parts of the body are the main endogenous hormones that cause these diseases. 5α-reductase is mainly distributed in the liver, skin, sebaceous glands and hair follicles (type I enzyme) and prostate and reproductive tissues (type II enzyme). It is a membrane protease located in microsomes and cell nuclei. It uses nicotinamide adenine dinucleotide (NADPH) as a substrate hydrogen donor to catalyze the conversion of physiologically active androgen testosterone (Testosterone, T) in the blood into more active dihydrotestosterone (DHT). Therefore, effectively reducing the level of dihydrotestosterone in the body is one of the effective methods to prevent and treat such diseases.

[0007] It is reported that compounds including vitamin A and isotretinoin, as well as lauric acid, oleic acid, myristic acid, linoleic acid, palmitic acid, etc., all have a certain degree of inhibitory effect on 5α-reductase [Reference: Yao Xincheng et al. Research progress of natural plants with 5α-reductase inhibitory effect. Natural Product Research and Development, 2014, 26(5): 800-805]. Ester derivatives modified with such compounds for HA have moisturizing, inhibiting sebum secretion and acne bacteria growth, as well as exfoliating, anti-inflammatory, anti-aging, repairing and whitening effects when applied to the skin [ZL202411907798.8].

[0008] The occurrence of acne is closely related to excessive sebum secretion and Propionibacterium acnes. Excessive sebum secretion is not caused by the single reason of active sebaceous glands, but is related to the health of the skin barrier. Using only a single oil control method cannot fundamentally solve the problem. Due to the damage of the barrier, the skin becomes dry and dehydrated, and bacteria including Propionibacterium acnes invade. The proliferation of bacteria further stimulates the secretion of sebum. The vicious cycle aggravates the damage of the skin barrier and causes the formation of acne. Therefore, skin moisturizing and barrier repair are important means to control oil, prevent acne formation and restore skin health.

[0009] In order to overcome the defects of the prior art, the present invention adopts a self-assembly method to combine HA ester derivatives with ceramide to develop a nano raw material with multiple functions. In addition to having a nanostructure that can improve the solubility, stability, transdermal performance and bioavailability of ceramide, the nano raw material of the present invention can also significantly enhance the moisturizing and skin barrier repair effects of the product, and can be widely used in the preparation of cosmetics and medical products with moisturizing, anti-aging, whitening, oil control, acne removal, anti-shedding and skin barrier repair effects, providing a broader space for the application of ceramide. Summary of the invention

[0010] The objective of the present invention is to provide a hyaluronic acid ceramide nano-material with oil control, acne treatment, and skin barrier repair effects, its preparation method, and their applications in the fields of cosmetics and medicine.

[0011] A hyaluronic acid ceramide nano-material of the present invention is characterized in that ceramide and a hyaluronic acid derivative self-assemble to form nanoparticles with an average particle size of 10 - 1000 nm. The nanoparticles formed by the self-assembly of ceramide and the hyaluronic acid derivative in the nano-material of the present invention are spherical, and the average particle size range of the spherical nanoparticles is 10 - 1000 nm, preferably 30 - 500 nm, more preferably 80 - 120 nm.

[0012] The nano-material of the present invention is characterized in that the drug loading amount of the ceramide, calculated by mass, is 2.5% - 50%, preferably 5% - 25%, more preferably 8% - 12%. In other words, the ratio of ceramide to the hyaluronic acid derivative is 1:1 - 39, preferably 1:3 - 19, more preferably 1:7 - 12. The drug loading amount is the percentage ratio of the mass of ceramide in the nano-material to the total mass of ceramide and the hyaluronic acid derivative.

[0013] The nano-material of the present invention can be embodied in the form of a solid powder or a nano-solution. In the case of being embodied in the form of a nano-solution, preservatives commonly used in liquid preparations can be added to the solution, such as methyl paraben, ethyl paraben, propyl paraben, phenethyl alcohol, p-methylbenzyl alcohol, phenoxyethanol, 1,2-hexanediol, 1,2-pentanediol, propylene glycol, butylene glycol, 2-methyl-1,3-propanediol, 3-[2-(ethylhexyl)oxy]-1,2-propanediol, and octyldecanol, etc., and one or a mixture of two or more of them can be selected.

[0014] A preparation method of a hyaluronic acid ceramide nano-material of the present invention is characterized by including the following operation steps:

[0015] a) Dissolve the hyaluronic acid derivative in an aqueous solution to obtain solution A;

[0016] b) Dissolve ceramide in an organic solvent to obtain solution B;

[0017] c) Mix and homogenize solution A and solution B to obtain solution C;

[0018] d) Purify solution C to remove the organic solvent.

[0019] The preparation method of the present invention is further characterized in that the purified solution obtained in step d) is freeze-dried to obtain a powdered solid nano-material.

[0020] The characteristics of the preparation method of the present invention also lie in adding a preservative to the purified liquid obtained in step d) to obtain a liquid nano raw material containing hyaluronic acid ceramide. The preservative is a preservative commonly used in liquid preparations, such as methyl paraben, ethyl paraben, propyl paraben, phenethyl alcohol, p-methylbenzyl alcohol, phenoxyethanol, 1,2-hexanediol, 1,2-pentanediol, propylene glycol, butylene glycol, 2-methyl-1,3-propanediol, 3-[2-(ethylhexyl)oxy]-1,2-propanediol, and octyldecanol, etc., and one or a mixture of two or more of them can be selected.

[0021] The characteristics of the preparation method of the present invention also lie in filtering with a microporous membrane before purifying the liquid C in step d).

[0022] The characteristics of the preparation method of the present invention lie in that the organic solvent described in step b) is selected from one or a combination of two or more of methanol, ethanol, acetone, tetrahydrofuran, dimethyl sulfoxide, and / or dimethylformamide.

[0023] The characteristics of the preparation method of the present invention lie in that the mixing and homogenization method described in step c) can adopt ordinary mechanical stirring, or methods such as high-pressure homogenization, microjet homogenization, or microfluidic homogenization, etc., so as to obtain nano-particles with smaller size and more uniform size.

[0024] The characteristics of the preparation method of the present invention lie in that the purification method described in step d) can adopt one method or two or more methods of reduced-pressure rotary evaporation, ultrafiltration, and / or dialysis successively. For example, the purified nano-solution can be obtained by separately adopting one method of reduced-pressure rotary evaporation, ultrafiltration, or dialysis, or by first performing reduced-pressure rotary evaporation and then ultrafiltration; or by first performing dialysis and then reduced-pressure rotary evaporation; or by first performing ultrafiltration and then reduced-pressure rotary evaporation; or by first performing dialysis, then ultrafiltration, and finally reduced-pressure rotary evaporation.

[0025] In the nano raw materials of the present invention and the preparation method of the present invention, the hyaluronic acid derivative is selected from one or a combination of two or more of sodium retinoate hyaluronate, sodium octanoyl hyaluronate, sodium decanoyl hyaluronate, sodium lauroyl hyaluronate, sodium myristoyl hyaluronate, sodium palmitoyl hyaluronate, sodium oleoyl hyaluronate, sodium linoleoyl hyaluronate and / or sodium stearoyl hyaluronate. Among them, sodium retinoate hyaluronate is an ester formed by the combination of retinoic acid and the hydroxyl group of sodium hyaluronate. Sodium octanoyl hyaluronate, sodium decanoyl hyaluronate, sodium lauroyl hyaluronate, sodium myristoyl hyaluronate, sodium palmitoyl hyaluronate, sodium oleoyl hyaluronate, sodium linoleoyl hyaluronate and / or sodium stearoyl hyaluronate are medium- and long-chain fatty acids, that is, octanoic acid with 8 carbons, decanoic acid with 10 carbons, lauric acid with 12 carbons, myristic acid with 14 carbons, palmitic acid with 16 carbons, oleic acid with 18 carbons, linoleic acid or stearic acid and the hydroxyl group of sodium hyaluronate form an ester.

[0026] In the nano raw materials of the present invention and the preparation method of the present invention, the average molecular weight of the hyaluronic acid derivative is 2 kDa to 500 kDa, preferably 5 kDa to 300 kDa, more preferably 10 kDa to 100 kDa. The average molecular weight is the weight-average molecular weight obtained by gel permeation chromatography (GPC) and / or laser scattering-gel permeation chromatography (LLS-GPC).

[0027] In the nano raw materials of the present invention and the preparation method of the present invention, the degree of substitution (also known as the grafting rate or esterification degree) of the hyaluronic acid derivative ranges from 1% to 20%, preferably 5% to 12%. The degree of substitution is the ratio of the retinoic acid molecule or medium- and long-chain fatty acid molecule combined with the hydroxyl group of sodium hyaluronate to the hyaluronic acid disaccharide molecule. Among them, the number of retinoic acid and medium- and long-chain fatty acid molecules is obtained by conversion after determining the content by high performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (HPLC-MS) after hydrolyzing the hyaluronic acid derivative, and the number of hyaluronic acid disaccharide molecules is obtained by conversion after determining the content of glucuronic acid by the carbazole method.

[0028] In the nano raw materials of the present invention and the preparation method of the present invention, the ceramide is one and / or a combination of two or more of 12 extractable subtype ceramides. For example, ceramide EOS (ceramide 1), NS (ceramide 2), NP (ceramide 3), EOH (ceramide 4), AS (ceramide 5), AP (ceramide 6), AH (ceramide 7), NH (ceramide 8), EOP (ceramide 9), NDS (ceramide 10), ADS (ceramide 11) and ceramide EODs, and one or a combination of two or more of them can be selected.

[0029] The technical solution of the present invention uses HA ester derivatives as carriers and prepares ceramide nano raw materials by self-assembly method, which has the advantages of nano preparations: ① Significantly improving the dispersibility and solubility of ceramide in water, restricting the crystallization of ceramide, and enhancing its stability in the preparation. ② Protecting ceramide from the influence of external environments such as enzymatic degradation and oxidation, and delivering ceramide to the action site through the targeting effect of the nano carrier to exert its physiological function and improve its bioavailability in vivo. ③ The nanoparticles carrying ceramide can better penetrate the stratum corneum of the skin, improve its permeability and retention amount in the skin, thereby enhancing its moisturizing and repair effects.

[0030] In addition, the technical solution of the present invention also has the following advantages: ① HA provides moisture for the skin to keep the skin moist, while ceramide forms a protective film on the skin surface to prevent moisture loss; after ceramide repairs the skin barrier, the absorption ability of the skin is enhanced, and HA can better play its moisturizing role and promote the rapid recovery of the skin barrier. The two work together to enhance the moisturizing ability and barrier repair function of the skin. ② HA and ceramide combine to form a protective film on the skin surface to repair the damaged skin barrier, prevent the intrusion of external harmful substances, reduce the loss of moisture at the same time, restore the normal function of the skin, and can effectively control sebum secretion and acne occurrence. ③ Since HA is an inherent substance of the body and has good tissue compatibility, the ceramide nano raw materials prepared by using HA as a carrier and the self-assembly method do not need to add surfactants, thus avoiding skin irritation caused thereby.

[0031] Therefore, the nano raw materials with the technical features of the present invention and prepared by the preparation method of the present invention can not only improve the dispersibility and stability of ceramide in the solution, but also significantly enhance its skin permeability and effectively play the skin barrier repair role of ceramide, and can be widely used in the preparation of cosmetics and medical products with moisturizing, anti-aging, whitening, oil control, acne removal, hair loss prevention and skin barrier repair functions.

[0032] The nano raw materials with the technical features of the present invention and prepared by the preparation method of the present invention can be embodied in the form of solid powder or nano solution. The nano solution raw materials and solid powder nano raw materials can be dispersed, dissolved and diluted with water and used directly, or can be formulated with common components of cosmetics to be prepared into various forms such as solutions, gels, pastes, creams or lotions for use. For example, repair moisturizing lotion, repair moisturizing spray, balance repair mask, oil control toner, lotion, softener, exfoliating gel, acne removal gel, balance moisturizing and whitening milk, cream, body cream, hand cream, foot cream, repair essence, moisturizing paste, repair milk, scar repair cream, scalp care liquid, etc. Brief Description of the Drawings

[0033] Figure 1The shape and size of the nanoparticles of the hyaluronic acid ceramide nano raw material (Sample 1-1) of the present invention;

[0034] Figure 2 Results of the cell phagocytosis test of the hyaluronic acid ceramide nano raw material (B.F is the cell bright field, DAPI is the stained cell nucleus, NR is Nile red, the left Merge is the combined graph of DAPI and NR, and the right Merge is the combined graph of B.F, DAPI and NR);

[0035] Figure 3 Results of the in vitro skin penetration test of the hyaluronic acid ceramide nano raw material [Group I: free Nile red 0.625 μg / ml; Group II: the hyaluronic acid ceramide nano raw material of the present invention (Sample 1-1 of Example 1) 1 mg / ml and Nile red 0.625 μg / ml; Group III: the hyaluronic acid ceramide nano raw material of the present invention (Sample 1-2 of Example 1) 1 mg / ml and Nile red 0.625 μg / ml];

[0036] Figure 4 Results of the determination of the hemolysis rate of rabbit red blood cells by the hyaluronic acid ceramide nano raw material;

[0037] Figure 5 Results of the determination of the hemolysis inhibition rate of rabbit red blood cells by the hyaluronic acid ceramide nano raw material. Detailed implementation manners

[0038] The following examples are for better illustrating the present invention and do not limit the present invention.

[0039] Example 1 Hyaluronic acid ceramide nano solid raw material

[0040] The composition of the hyaluronic acid ceramide nano solid raw material of the present invention is shown in Table 1.

[0041] Table 1 Hyaluronic acid ceramide nano solid raw material

[0042]

[0043] Method for determining the content of ceramide [C / %(g / g)]:

[0044] Take the solid nano raw material of the present invention, add an appropriate amount of water to dissolve it, then add a methanol solution, and determine it by HPLC method. Using ceramide as the reference substance, adopt an octadecylsilane-bonded silica gel chromatographic column and an ultraviolet detector (or a mass spectrometer detector), and the mobile phase is methanol∶acetonitrile = 65:35. Calculate the content of ceramide [C / %(g / g)] by the external standard method based on the peak area.

[0045] Method for determining the content of water [h / %(g / g)]:

[0046] Weigh about 0.5 - 2 g of the present invention (W1) and place it in a flat weighing bottle (W2) that has been dried to a constant weight in an oven at 105°C. Then, dry it to a constant weight in the oven at 105°C, and after that, place it in a desiccator to cool to room temperature, and weigh the mass (W3). The water content is calculated by loss on drying:

[0047]

[0048] Calculation of drug loading L / % (g / g):

[0049] L = C / (1 - h) * 100%

[0050] Example 2 Raw materials of hyaluronic acid ceramide nano - liquid

[0051] The composition of the raw materials of the hyaluronic acid ceramide nano - liquid of the present invention is shown in Table 2.

[0052] Table 2 Raw materials of hyaluronic acid ceramide nano - liquid

[0053]

[0054] Determination method for the content of ceramide [C / % (g / g)]:

[0055] Take the liquid nano - raw material of the present invention, add an appropriate amount of water for dilution and then add a methanol solution, and determine it by HPLC method. Using ceramide as the reference substance, adopt an octadecylsilane - bonded silica gel chromatographic column and an ultraviolet detector (or mass spectrometry detector), and the mobile phase is methanol∶acetonitrile = 65:35. Calculate the content of ceramide [C / % (g / g)] by the external standard method based on the peak area.

[0056] Determination method for the content of glucuronic acid [T / % (g / g)]:

[0057] According to the literature [Bitter T, Muir H M. A modif ied uronic acid carbarbazolereationl J J. Anal Biochem. 1962, 4:330 - 333.], adopt the modified carbazole method to determine the glucuronic acid content T / % (g / g) of the sample.

[0058] Calculation of drug loading L' / % (g / g):

[0059] L' = C / (T×401 / 194.14 + C) = C / (2.06*T + C)

[0060] Example 3 Preparation method of hyaluronic acid ceramide nano - raw material

[0061] The preparation method of the hyaluronic acid ceramide nano - raw material of the present invention is as follows:

[0062] (1) Take an appropriate amount of hyaluronic acid derivative according to Table 1 and add it to water, stir to dissolve it into a solution with a concentration of 0.05 - 5%, and obtain Solution A;

[0063] (2) Take an appropriate amount of ceramide according to Table 1, add it to an organic solvent according to Table 3, stir to dissolve it into a solution with a concentration of 0.05 - 1%, and obtain Solution B;

[0064] (3) Mix and homogenize Solution A and Solution B according to the method in Table 3 to obtain Solution C;

[0065] (4) After filtering Solution C through a microporous filter membrane with a pore size of 0.22 - 2 μm, purify Solution C according to the method in Table 3 to remove the organic solvent;

[0066] (5) Lyophilize the solution purified in the above step (4) to obtain a light yellow, yellow or white powder, which is the solid nano - raw material of the present invention (Samples 1 - 1 to 1 - 12).

[0067] (6) Adjust the concentration of the solution purified in the above step (4), and add a preservative according to Table 2 to obtain the liquid nano - raw material of the present invention (Samples 2 - 1 to 2 - 10).

[0068] Table 3 Preparation method of hyaluronic acid - ceramide nano - raw material

[0069]

[0070] Calculation of encapsulation efficiency R / % (g / g):

[0071] R = C * P / Q * 100%

[0072] Where C / % (g / g) is the content of ceramide, P (g) is the yield of the nano - raw material, and Q (g) is the input amount of ceramide.

[0073] Taking Method II as an example, prepare a 0.2% solution of sodium retinoil hyaluronate (prepared according to Example 1 of ZL202411907798.8, substitution degree 8.0, K value 2.3, molecular weight 8.1 kDa) as Solution A, and prepare a 0.2% solution of ceramide NP as Solution B. Under the condition of a mechanical stirring speed of 100 - 1000 rpm, slowly add 250 ml of Solution B to 1000 ml of Solution A, continuously stir for 30 - 60 min, filter through a 0.45 - μm filter membrane, and rotary evaporate under reduced pressure at 35 - 65 °C until the remaining solution is 100 - 500 ml. After lyophilization, a light yellow to yellow powder is obtained, which is the solid nano - raw material sample 1 - 1 of the present invention. It is measured that the drug - loading amount of ceramide NP is 19.7% and the encapsulation efficiency is 96.0%.

[0074] The solution filtered through the filter membrane above was rotary evaporated under reduced pressure until the weight of the remaining solution was 500 g. Preservatives were added according to Table 2 and stirred until dissolved uniformly, thus obtaining the solid nano raw material sample 2-1 of the present invention. After measurement, the drug loading of ceramide NP was 19.5% and the encapsulation efficiency was 96.5%.

[0075] The solution filtered through the filter membrane above was rotary evaporated under reduced pressure until the weight of the remaining solution was 100 g. Preservatives were added according to Table 2 and stirred until dissolved uniformly, thus obtaining the solid nano raw material sample 2-9 of the present invention. After measurement, the drug loading of ceramide NP was 20.1% and the encapsulation efficiency was 96.2%.

[0076] By changing the concentrations and ratios of Liquid A and Liquid B, nano raw material samples with a drug loading of 2.5 - 50% and an encapsulation efficiency greater than 90% can be obtained.

[0077] By changing the concentrations of Liquid A and Liquid B, adjusting the speed of mechanical stirring, and the speed of adding Liquid B to Liquid A, nano raw materials with spherical nanoparticles and an average particle size in the range of 50 - 700 nm can be obtained (such as Figure 1 ). Figure 1 This is the nano particle size distribution and transmission electron microscope (TEM) photograph of the hyaluronic acid ceramide nano sample 1-1 of the present invention measured by a nano particle size and Zeta potential analyzer. Top: average particle size 51.57 nm (PDI = 0.175); Middle: average particle size 111.56 nm (PDI = 0.094); Bottom: average particle size 586.27 nm (PDI = 0.238). TEM observation shows that the nano raw materials of the present invention are spherical nanoparticles in aqueous solution and the outer shell staining is uniform.

[0078] Using microfluidic homogenization instead of mechanical stirring and mixing Liquid A and Liquid B at different speeds, nano raw materials with spherical nanoparticles and an average particle size in the range of 10 - 50 nm can be obtained.

[0079] The method for measuring the particle size of the nano raw materials of the present invention: The nano raw materials are dissolved or diluted with water to an appropriate concentration and measured using a nano particle size and Zeta potential analyzer.

[0080] The electron microscope analysis method of the nano raw materials of the present invention: The nano raw materials are dissolved or diluted with water to an appropriate concentration, then infiltrate a copper mesh, air dry, stain with phosphotungstic acid, and observe and take pictures under a transmission electron microscope.

[0081] Example 4 Stability of the hyaluronic acid ceramide nano raw material

[0082] The samples 1-1 and 1-2 in Example 1 of the present invention were separately packed in sealed aluminum-plastic bags, and the samples 2-1 and 2-2 in Example 2 were separately packed in brown screw-cap glass sample bottles. As the samples for the stability test, they were placed in different temperature environments to investigate the heat resistance stability of the product. Samples were taken at different times to check the properties of the raw material samples and detect the changes in particle size (d.nm / PDI) and drug loading (R / %). The results (Table 4) showed that heating had a certain impact on the nano raw materials of the present invention, and the product was packaged in a light-shielding and sealed manner, and the quality was stable under low-temperature storage conditions.

[0083] Table 4 Results of the stability test of hyaluronic acid ceramide nano raw materials*

[0084]

[0085] * "-" indicates a decrease in drug loading, with the drug loading on day 0 taken as 100%.

[0086] Example 5 Permeation test of hyaluronic acid ceramide nano raw materials

[0087] Experiment 1 Cell phagocytosis test:

[0088] According to the literature method [Reference: Antunes, Patrícia, et al. "Lipid droplets in cancer: from composition and role to imaging and therapeutics." Molecules 27.3 (2022): 991.], human immortalized epidermal cells (HACAT) in the logarithmic growth phase were seeded on a 24-well plate at a density of 50,000 cells per well. After 24 hours, 200 μL of the solution (1 mg / ml) of the hyaluronic acid ceramide nano raw material (Sample 1-1 of Example 1) of the present invention containing Nile red (concentration: 0.625 μg / mL) and 300 μL of culture medium were added to each well, and the cells were incubated in an incubator. After incubation for 0.5, 1, and 2 hours respectively, the culture medium and samples in the well plate were discarded, and the cells were washed 3 times with PBS. After ensuring thorough washing of the samples, 500 μL of paraformaldehyde tissue fixative was added and fixed at room temperature for 15 minutes. After fixation, the cells were washed 3 times with PBS. Finally, DAPI staining solution was added and stained at room temperature for 5 minutes. After staining, the cells were washed 3 times with PBS to ensure thorough washing of DAPI, and the uptake of hyaluronic acid ceramide nanoparticles by the cells was observed using a fluorescence microscope. It can be seen from the figure that the hyaluronic acid ceramide nanoparticles of the present invention began to enter the cells at 0.5 hour, a large number of nanoparticles entered the cells after 1 hour, and the fluorescence intensity in the cells gradually decreased after 2 hours. This indicates that the nano raw material of the present invention can be rapidly phagocytosed and transported by cells, improving the skin penetration of the active ingredients, which is beneficial to exerting various physiological functions of the components of the nano raw material in the skin, such as moisturizing, anti-inflammatory, antioxidant, inhibiting 5A-reductase, and promoting skin barrier recovery, and improving the efficacy of the nano raw material product in terms of moisturizing, anti-aging, whitening, oil control, acne treatment, hair loss prevention, and skin barrier repair.

[0089] Figure 2 This is the result of the cell phagocytosis test of the hyaluronic acid ceramide nano raw material (Sample 1-1 of Example 1) of the present invention. In the photo, B.F is the bright field of the cells, DAPI is the stained cell nucleus, NR is Nile red. The left Merge is the combined image of DAPI and NR, and the right Merge is the combined image of B.F, DAPI, and NR. Human immortalized epidermal cells (HACAT) were incubated in a culture medium containing Nile red (concentration: 0.25 μg / ml) and the solution (concentration: 0.4 mg / ml) of the hyaluronic acid ceramide nano raw material (Sample 1-1 of Example 1) of the present invention. Under a fluorescence microscope, nanoparticles began to enter the cells at 0.5 hour, a large number of nanoparticles entered the cells after 1 hour, and the fluorescence intensity in the cells gradually decreased after 2 hours. This indicates that the nano raw material of the present invention can be rapidly phagocytosed and transported by cells, improving the skin penetration of the active ingredients.

[0090] Experiment 2 Ex vivo skin penetration test

[0091] According to the literature method [Reference: Retinoic acid grafted to hyaluronan for skin delivery: Synthesis, stability studies, and biological evaluation], the in vitro percutaneous experiment on porcine skin was carried out using a vertical Franz diffusion cell method. First, commercially available Panamanian porcine skin was thawed, rinsed, and then dried. Then it was fixed between the receiving chamber and the supply chamber. 0.6 ml of the test sample (Table 5) was respectively injected into the supply chamber. Using PBS (pH 7.4) as the receiving medium, after incubating and permeating at a stirring speed of 600 rpm and 32 °C for 24 h, the skin in the target area was removed, rinsed, dried, and then frozen sections were made. It was observed and photographed under a fluorescence microscope ( Figure 3 ). Figure 3 This is the result of the in vitro skin penetration test of the ceramide nanomaterial of hyaluronic acid of the present invention (Sample 1-1 of Example 1). The test sample in Group I was free nile red at 0.625 μg / ml; the test sample in Group II was the ceramide nanomaterial of hyaluronic acid of the present invention (Sample 1-1 of Example 1) at 1 mg / ml and nile red at 0.625 μg / ml; the test sample in Group III was the ceramide nanomaterial of hyaluronic acid of the present invention (Sample 1-2 of Example 1) at 1 mg / ml and nile red at 0.625 μg / ml. The results showed that after the skin was exposed to the above three test samples for 24 h, a certain amount of nile red was retained and penetrated in the skin. The intensity of the red fluorescence represents the amount of nile red. Among them, free nile red (Group I) mainly stayed on the skin surface layer, and a small amount penetrated into the deep skin tissue, while nile red encapsulated in the ceramide nanomaterial of hyaluronic acid of the present invention (Group II and Group III) entered the deep skin tissue more, indicating that Samples 1-1 and 1-2 of Example 1 of the nanomaterial of the present invention can enhance transport and promote skin penetration.

[0092] Table 5 Samples of the in vitro skin penetration test of the ceramide nanomaterial of hyaluronic acid

[0093]

[0094]

[0095] The results showed that after the skin was exposed to the above three test samples for 24 h, Nile red was retained and penetrated in the skin to a certain extent. The intensity of the red fluorescence represents the amount of Nile red. Among them, free Nile red (Group I) mainly remained in the skin surface layer, and a small amount penetrated into the deep skin tissue, while Nile red encapsulated in the hyaluronic acid ceramide nano-materials of the present invention (Group II and Group III) entered the deep skin tissue more, indicating that the samples 1-1 and 1-2 of Example 1 of the nano-materials of the present invention can enhance transport, promote skin penetration, and are beneficial to exerting various physiological functions of each component of the nano-materials in the skin, such as moisturizing, anti-inflammatory, antioxidant, inhibiting 5α-reductase, and promoting skin barrier recovery, etc., and improving the efficacy of nano-material products in moisturizing, anti-aging, whitening, oil control, acne treatment, hair loss prevention, and skin barrier repair, etc.

[0096] Antioxidant effect of the hyaluronic acid ceramide nano-materials in Example 6

[0097] The antioxidant effect of the hyaluronic acid ceramide nano-materials of the present invention was verified by the test of inhibiting free radicals induced by ultraviolet radiation.

[0098] According to the literature method [Sun He, Wang Lili, Kang Chuanli, et al. Experimental study on ergothioneine inhibiting free radicals induced by ultraviolet radiation, Food and Drug,

[0099] In 2023, Volume 25, Issue 6], using phosphate buffer solution (PBS, pH 7.2, take 2.2 g of Na2HPO4, 0.3 g of NaH2PO4 and 8.5 g of NaCl, dissolve in water to 1000 ml) as the solvent, prepare the test sample solution and 0.001% rhodamine B solution (RhB) according to Table 6 respectively. Take 1.2 ml of RhB solution, 0.4 ml of the sample solution, and 0.4 ml of PBS solution, mix well, and place it under ultraviolet light (wavelength 254 nm, light intensity 5 mW / cm 2 ) for irradiation. At 0 h and 1 h respectively, use an enzyme-labeled instrument to measure the absorbance values (A0 and A1) at a wavelength of 555 nm. Each sample is in parallel for 3 replicates, calculate the average value (ΔA) of the change in absorbance at 0 h and 1 h, and use ΔA 空白 as the baseline, and calculate the antioxidant performance (F) of the active substances in the system according to the following formula.

[0100]

[0101] In the formula,

[0102] ΔA 空白 : the change in the absorbance value of the blank control solution (A 0空白 -A 1空白 );

[0103] ΔA 样品 : the change in the absorbance value of the sample solution (A 0样品 -A1样品 )。

[0104] The results showed that the F values of sodium retinol hyaluronate (Group III) and sodium lauroyl hyaluronate (Group IV), the carriers of the hyaluronic acid ceramide nano raw materials of the present invention, were 2.69% and 0.96% respectively, and the F value of ceramide, with the influence of the cosolvent subtracted from the ceramide plus emulsifier group (Group V) (Group VI), was 1.74%. The F values of retinol hyaluronic acid ceramide and lauroyl hyaluronic acid ceramide nano raw materials (Group I and Group II) of Sample 1-1 and Sample 1-2 in Example 1 of the present invention were 7.33% and 5.79% respectively, both significantly greater than the sum of the F values of their carriers and ceramide (4.43% and 2.7% respectively), showing a synergistic effect. The results indicated that there was a synergistic effect between the carrier and ceramide in the present invention (in Sample 1-1 and Sample 1-2 of Example 1) in terms of antioxidant performance.

[0105] Table 6 Grouping and results of the test on the inhibition of free radicals induced by ultraviolet radiation by hyaluronic acid ceramide nano raw materials

[0106]

[0107] Example 7 Cell protection effect of hyaluronic acid ceramide nano raw materials

[0108] Determination of hemolysis rate and hemolysis inhibition rate of rabbit red blood cells (RBC)

[0109] Prepare rabbit red blood cell suspension (RBC) by referring to the reference method [Reference: Han Jixin, Research on the anti-skin allergy activity of natural Chinese herbal medicine extracts, 2021, Master's thesis of Jiangnan University.]. Use physiological saline to prepare 0.06% SDS (positive control) and sample solutions with different concentrations (Table 7) as test samples. Take 120 μl of the test sample solution and add it to a 5 ml EP tube, then add 60 μl of RBC suspension, and make up the volume with 1.82 ml of physiological saline. After incubating at 150 r / min with shaking at room temperature for 15 min, centrifuge at 10000 rpm for 1 min, measure the absorbance at a wavelength of 530 nm, and calculate the hemolysis rate (H i / % ).

[0110] Another 120 μl of the test sample solution with different concentrations (Table 7) prepared with physiological saline is taken and added to a 5 ml EP tube, add 1.82 ml of physiological saline, mix well, add 60 μL of RBC, mix again, then add 120 μL of 0.06% SDS prepared with physiological saline, incubate with shaking for 15 min, centrifuge at 10000 rpm for 1 min, measure the absorbance at a wavelength of 530 nm, and calculate the hemolysis rate (H j / % ) and hemolysis inhibition rate (I / % ).

[0111] H i / % or H j / % = (A i - A0) / (A t- A0) × 100%

[0112] Where: A i : Absorbance value of the test product tube

[0113] A0: Absorbance value of the blank tube (60 μL RBC added to 1940 μL 0.9% sodium chloride solution)

[0114] A t： Absorbance value of the completely lysed blood tube (60 μL RBC added to 1940 μL ultrapure water).

[0115] I / % = (H0 - H j ) / H0

[0116] In the formula: H j : Hemolysis rate of the test product, %;

[0117] H0: Hemolysis rate of SDS (92.94%).

[0118] The toxicity of the test product to RBC can be judged by the hemolysis rate. The results (Table 7 and Figure 4 ) show that the hemolysis rate of the test product is extremely low (<10%) in the range of 0.2 mg / mL - 5 mg / mL, indicating that the red blood cells are not significantly damaged and stimulated. That is, the hyaluronic acid ceramide nano raw material (Sample 1-1 of Example 1) of the present invention has no obvious irritation to RBC.

[0119] The protective effect of the test product on RBC can be judged by the hemolysis inhibition rate. The results (Table 7 and Figure 5 ) show that the hemolysis inhibition rate of the sample is positively correlated with the concentration. The hemolysis inhibition rate of the sample solution with a concentration above 2 mg / ml is close to 80% or more, indicating that the hyaluronic acid ceramide nano raw material (Sample 1-1 of Example 1) of the present invention can inhibit the stimulation of the surfactant SDS and has an obvious protective effect on red blood cells.

[0120] Table 7 Results of the red blood cell hemolysis test of the hyaluronic acid ceramide nano raw material

[0121]

Claims

1. A hyaluronic acid ceramide nano raw material, characterized in that Ceramide and hyaluronic acid derivatives self-assemble to form nanoparticles with an average particle size of 10 to 1000 nm.

2. The nano raw material according to claim 1, further characterized in that, The drug loading of the ceramide is 2.5 to 50% (mass ratio).

3. The nano raw material according to claim 1, characterized in that, The hyaluronic acid derivative is selected from one or more combinations of sodium retinoil hyaluronate, sodium capryloyl hyaluronate, sodium caprate hyaluronate, sodium lauroyl hyaluronate, sodium myristoyl hyaluronate, sodium palmitoyl hyaluronate, sodium oleoyl hyaluronate, sodium linoleoyl hyaluronate, and / or sodium stearoyl hyaluronate.

4. A method for preparing a hyaluronic acid ceramide nano raw material, characterized in that It includes the following operation steps: a) Dissolve the hyaluronic acid derivative in an aqueous solution to obtain solution A. b) Dissolve the ceramide in an organic solvent to obtain solution B. c) Mix and homogenize solution A and solution B to obtain solution C. d) Purify solution C to remove the organic solvent.

5. The preparation method according to claim 4, further characterized in that Lyophilize the purified solution obtained in step d).

6. The preparation method according to claim 4, further characterized in that Add a preservative to the purified solution obtained in step d).

7. The preparation method according to claim 4, characterized in that The organic solvent described in step b) is selected from one or more combinations of methanol, ethanol, acetone, tetrahydrofuran, dimethyl sulfoxide, and / or dimethylformamide.

8. The preparation method according to claim 4, wherein the hyaluronic acid derivative is selected from one or more combinations of sodium retinoil hyaluronate, sodium capryloyl hyaluronate, sodium caprate hyaluronate, sodium lauroyl hyaluronate, sodium myristoyl hyaluronate, sodium palmitoyl hyaluronate, sodium oleoyl hyaluronate, and / or sodium stearoyl hyaluronate.

9. The nano raw material prepared by the preparation method according to claims 4 to 8.

10. The nano raw material according to claims 1 and 9 is used for preparing cosmetics and medical products for moisturizing, oil control, acne treatment, hair loss prevention, and skin barrier repair.

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