Azelaic acid liposome as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of cosmetics, and particularly relates to azelaic acid liposome as well as a preparation method and application thereof. The invention firstly provides a composition for preparing the azelaic acid liposome. The composition comprises an organic phase composition and a water phase composition which are used for preparing the azelaic acid liposome, the organic phase composition comprises 1, 2-propylene glycol, hydrogenated phosphatidylcholine and cholesterol; the aqueous phase composition includes an aqueous phase medium and azelaic acid. The invention further provides a method for preparing the composition into the azelaic acid liposome by utilizing a 1, 2-propylene glycol injection method, which comprises the following steps: mixing the components in the organic phase composition to obtain an organic phase solution; mixing the components in the water-phase composition to obtain a water-phase solution; and injecting the organic phase solution into the water phase solution for reaction, and finally performing high-pressure homogenization to obtain the azelaic acid liposome. According to the method, organic solvents do not need to be removed in the preparation process, and the obtained product is good in solubility, good in stability and high in safety and has the effects of removing acnes, controlling oil and whitening.

Description

Technical Field

[0001] The invention belongs to the technical field of cosmetics, and specifically relates to azelaic acid liposome and a preparation method and application thereof. Background Art

[0002] Azelaic acid, also known as azelaic acid, is a saturated dicarboxylic acid containing nine carbon atoms that occurs naturally in plants such as cereals, wheat, rye and barley. Existing studies have confirmed that azelaic acid has at least the following effects: 1) Whitening effect: Azelaic acid can directly inhibit melanocytes, inhibit tyrosinase activity, and reduce pigment synthesis. It has obvious effects on the treatment of pigmentation such as chloasma, freckles, and post-inflammatory pigmentation; 2) Anti-keratinization effect: Azelaic acid can reduce the synthesis of filamentous keratin, prevent excessive keratinization of hair follicles, and help improve acne and clogged pores; 3) Antibacterial effect: Azelaic acid can inhibit bacterial protein synthesis and has a killing effect on a variety of skin bacteria, including Propionibacterium acnes, and is not prone to drug resistance after long-term use; 4) Regulating sebaceous gland function: Azelaic acid can inhibit the action of 5-α reductase and reduce the conversion of androgens into dihydrotestosterone, thereby inhibiting the excessive secretion of free fatty acids in the sebaceous glands and reducing sebum secretion; 5) Anti-inflammatory effect: Azelaic acid can inhibit the production of reactive oxygen free radicals and reduce inflammatory reactions. It has a positive effect on the treatment of inflammatory skin diseases such as acne and rosacea.

[0003] The unique chemical properties and biological activity of azelaic acid give it broad application prospects in the cosmetics and pharmaceutical industries. However, the physical and chemical properties of azelaic acid limit its application in cosmetics to a certain extent, including 1) poor solubility: slightly soluble in water, insoluble in oil; 2) poor permeability, 3) poor stability: easy to demulsify and crystallize; 4) high melting point: around 106.5°C, etc. In addition, azelaic acid also has mild to moderate skin irritation. These problems greatly increase the technical difficulty of azelaic acid product development and affect the stability and comfort of the product.

[0004] Liposomes are spherical carriers with a phospholipid bilayer structure, and their composition generally includes phospholipids and cholesterol. As an effective drug delivery system, liposomes are widely used in the medical and cosmetic fields. The use of liposomes to encapsulate and transport various drug molecules can effectively increase the solubility of poorly soluble substances and improve the stability of active ingredients; and promote the skin permeability of active substances and improve bioavailability. At the same time, liposomes can also achieve sustained release of drugs and reduce the skin irritation of products. Common preparation methods for liposomes include ethanol injection method, thin film hydration method, reverse evaporation method, and multiple emulsion method. These methods all involve the use of organic solvents. Common organic solvents for liposome preparation include ethanol, dichloromethane, chloroform, ether, and n-hexane. Among them, ethanol and n-hexane are flammable, ether is volatile and has anesthetic effects, dichloromethane belongs to carcinogenic category 2A, and chloroform belongs to carcinogenic category 2B, both of which are dangerous. In addition, these organic solvents are difficult to remove using common process methods, which is not conducive to industrial production.

[0005] In the prior art, CN114767559A discloses a nonanedioic acid liposome cream, which includes a nonanedioic acid liposome solution and a cream base. The raw materials for preparing the nonanedioic acid liposome solution include phospholipids, cholesterol, DSPE-mPEG2000, nonanedioic acid active ingredients and physiological saline. The nonanedioic acid active ingredients in the nonanedioic acid liposome solution exist in the form of free nonanedioic acid or DSPE-PEG-nonanedioic acid. The invention adopts a thin film hydration method to prepare the nonanedioic acid liposome solution, and the solubility of nonanedioic acid is relatively low; and the method uses a chloroform-methanol solution, and the chloroform-methanol finally needs to be removed by reduced pressure evaporation, which is not conducive to industrial production. Summary of the invention

[0006] In order to overcome the defects of the prior art, the present invention proposes a nonanedioic acid liposome and a preparation method thereof. The present invention prepares nonanedioic acid liposomes by a propylene glycol injection method, which can effectively solve the solubility, stability and irritation problems of nonanedioic acid, and the nonanedioic acid liposomes are prepared by the method without removing the organic solvent, thus overcoming the problem that the organic solvent is difficult to remove in the existing liposome preparation method, and is more suitable for industrial production.

[0007] One of the objectives of the present invention is to provide a composition for preparing azelaic acid liposomes.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] A composition for preparing azelaic acid liposomes, the composition comprising an organic phase composition for preparing azelaic acid liposomes and an aqueous phase composition for preparing azelaic acid liposomes; the organic phase composition comprises 1,2-propylene glycol, hydrogenated phosphatidylcholine and cholesterol, and the mass ratio thereof is 6-20:4-12:1-3; the aqueous phase composition comprises an aqueous phase medium and azelaic acid, and the mass ratio thereof is 40-85:1-10.

[0010] Furthermore, the organic phase composition includes 1,2-propylene glycol, hydrogenated phosphatidylcholine, cholesterol, a stabilizer and an antioxidant, and the mass ratio thereof is 6-20:4-12:1-3; 0.5-2:0.1-1; the stabilizer is Tween 80; and the antioxidant is tocopherol acetate.

[0011] Furthermore, the aqueous phase composition includes an aqueous phase medium, azelaic acid, a pH adjuster, an anionic surfactant, a preservative and a metal ion chelator, and the mass ratio thereof is 40-85: 1-10: 3.5-5: 0.1-0.5: 0.8-2: 0.01-0.05; the aqueous phase medium is water; the pH adjuster is any one or more of disodium hydrogen phosphate dodecahydrate, citric acid, and sodium hydroxide; the anionic surfactant is sodium stearoyl glutamate; the preservative is any one or two of p-hydroxyacetophenone and 1,2-hexanediol; and the metal ion chelator is EDTA-disodium.

[0012] The second purpose of the present invention is to provide a method for preparing azelaic acid liposomes. The present invention adopts 1,2-propylene glycol injection method to prepare azelaic acid liposomes. The preparation process does not need to remove organic solvents, the steps are simpler, and support is provided for the industrial safe production of azelaic acid liposomes.

[0013] To achieve the above object, the present invention adopts the following technical solutions:

[0014] The method for preparing azelaic acid liposomes is to prepare the above composition into azelaic acid liposomes by a 1,2-propylene glycol injection method, wherein the 1,2-propylene glycol injection method comprises the following steps:

[0015] (1) mixing the components in the aforementioned organic phase composition to obtain an organic phase solution;

[0016] (2) mixing the components in the aforementioned aqueous phase composition to obtain an aqueous phase solution;

[0017] (3) injecting the organic phase solution obtained in step (1) into the aqueous phase solution obtained in step (2), reacting to obtain a suspension;

[0018] (4) The suspension obtained in step (3) is subjected to high-pressure homogenization to obtain azelaic acid liposomes.

[0019] Furthermore, in step (1) and step (2), the components are mixed and stirred at 65-80° C. until the solution becomes clear.

[0020] Furthermore, in step (1), the stirring time is not less than 30 min.

[0021] Furthermore, in step (3), the reaction conditions are stirring at 65-80° C. for 60-180 min.

[0022] Furthermore, in step (4), the high-pressure homogenization includes circulating 3 to 6 times at a pressure of 500 to 1000 bar.

[0023] Preferably, the method comprises the following steps:

[0024] (1) 1,2-propylene glycol, hydrogenated phosphatidylcholine, cholesterol, Tween 80 and tocopherol acetate are mixed and stirred at 65-80° C. for at least 30 minutes to obtain a clear organic phase solution.

[0025] (2) Mix water, azelaic acid, disodium hydrogen phosphate dodecahydrate, sodium hydroxide, citric acid, sodium stearoyl glutamate, disodium EDTA, p-hydroxyacetophenone and 1,2-hexanediol, and stir at 65 to 80° C. until the mixture becomes clear, thereby obtaining a clear aqueous phase solution.

[0026] (3) Slowly inject the organic phase solution into the aqueous phase solution and stir at 65-80°C for at least 60 minutes.

[0027] (4) The solution obtained in step (3) is circulated 3 to 6 times under a pressure of 500 to 1000 bar in a high-pressure homogenizer to obtain azelaic acid liposomes.

[0028] Furthermore, the particle size of the azelaic acid liposome prepared by the method is less than 600 nm.

[0029] Furthermore, the encapsulation efficiency of the azelaic acid liposome prepared by the method is greater than 85%.

[0030] The third object of the present invention is to provide azelaic acid liposome prepared by the above-mentioned preparation method.

[0031] A fourth object of the present invention is to provide a preparation composition.

[0032] To achieve the above object, the present invention adopts the following technical solutions:

[0033] A preparation composition, comprising azelaic acid and liposomes; the liposomes comprising hydrogenated phosphatidylcholine, cholesterol and 1,2-propylene glycol; the mass ratio of azelaic acid, hydrogenated phosphatidylcholine, cholesterol and 1,2-propylene glycol is 1-10:4-12:1-3:6-20; the preparation composition does not contain ethanol, dichloromethane, chloroform, ether and n-hexane.

[0034] Furthermore, the liposome includes hydrogenated phosphatidylcholine, cholesterol, 1,2-propylene glycol, a stabilizer, an antioxidant, an anionic surfactant, a preservative, a pH adjuster, a metal ion chelator and an aqueous phase medium, and the mass ratio thereof is 4-12: 1-3: 6-20: 0.5-2: 0.1-1: 0.1-0.5: 0.8-2: 3.5-5: 0.01-0.05: 40-85; the stabilizer is Tween 80; the antioxidant is tocopherol acetate; the anionic surfactant is sodium stearoyl glutamate; the preservative is any one or two of p-hydroxyacetophenone and 1,2-hexanediol; the pH adjuster is any one or more of disodium hydrogen phosphate dodecahydrate, citric acid, and sodium hydroxide; the metal ion chelator is EDTA-disodium; and the aqueous phase medium is water.

[0035] Furthermore, the liposome is composed of the following components in parts by weight: 4 to 12 parts of hydrogenated phosphatidylcholine, 1 to 3 parts of cholesterol, 6 to 20 parts of 1,2-propylene glycol, 0.5 to 2 parts of Tween 80, 0.1 to 0.5 parts of sodium stearoyl glutamate, 0.4 to 1 parts of p-hydroxyacetophenone, 0.4 to 1 parts of 1,2-hexanediol, 0.1 to 1 parts of tocopherol acetate, 3.5 parts of disodium hydrogen phosphate dodecahydrate, 0.315 parts of citric acid, 0.3 parts of sodium hydroxide, 0.02 parts of EDTA-disodium and 45.365 to 82.365 parts of water.

[0036] Furthermore, the preparation composition is composed of the following components in parts by weight: 1 to 10 parts of azelaic acid, 4 to 12 parts of hydrogenated phosphatidylcholine, 1 to 3 parts of cholesterol, 6 to 20 parts of 1,2-propylene glycol, 0.5 to 2 parts of Tween 80, 0.1 to 0.5 parts of sodium stearoyl glutamate, 0.4 to 1 part of p-hydroxyacetophenone, 0.4 to 1 part of 1,2-hexanediol, 0.1 to 1 part of tocopherol acetate, 3.5 parts of disodium hydrogen phosphate dodecahydrate, 0.315 parts of citric acid, 0.3 parts of sodium hydroxide, 0.02 parts of EDTA-disodium, and 45.365 to 82.365 parts of water.

[0037] The fifth object of the present invention is to provide a use of the aforementioned composition, the aforementioned liposome and / or the aforementioned preparation composition in the preparation of cosmetics with acne-removing, oil-controlling and / or whitening effects.

[0038] Furthermore, the cosmetics include lotions, emulsions, and creams.

[0039] The beneficial effects of the present invention are:

[0040] 1. In order to solve the solubility, stability and irritation problems of azelaic acid, the present invention uses liposome technology to encapsulate azelaic acid in lipid globules, and utilizes the dosage form advantages of liposomes to increase the solubility of azelaic acid, reduce skin irritation, and improve its stability, bioavailability and safety. The azelaic acid liposomes provided by the present invention have a solubility that is about 37 times higher than that of azelaic acid; and the quality is stable when stored at a high temperature of 40°C for 180 days.

[0041] 2. The present invention uses 1,2-propylene glycol as an organic solvent and prepares azelaic acid liposomes by a propylene glycol injection method. The operation is simple, the particle size of the obtained product is less than 600 nm, and the encapsulation rate is greater than 85%. In addition, there is no need to remove the organic solvent during the preparation process, the preparation process is simpler, the operation is more convenient, the safety is higher, and it is more suitable for the industrial production of azelaic acid liposomes.

[0042] 3. The azelaic acid liposomes provided by the present invention have acne-removing, oil-controlling and whitening effects at a relatively low concentration, and can be used as a functional raw material in cosmetics such as lotions, emulsions, creams, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 TEM image of the sample prepared in Example 2;

[0044] Figure 2 The particle size diagram of the sample prepared in Example 2 measured by a nano laser particle size analyzer;

[0045] Figure 3 Potential diagram of the sample prepared in Example 2 measured by a nano-laser particle size analyzer;

[0046] Figure 4 Fluorescence micrograph of lipid droplets after staining with Lipi-Blue for blank control sample;

[0047] Figure 5 This is a bright-field micrograph of cells from a blank control sample;

[0048] Figure 6 Fluorescence micrograph of lipid droplets after staining with Lipi-Blue for negative control samples;

[0049] Figure 7 is a bright-field micrograph of cells from the negative control sample;

[0050] Figure 8 This is a fluorescence micrograph of lipid droplets after the sample of Example 4 was diluted to a concentration of 0.05% and stained with Lipi-Blue;

[0051] Fig. 9This is a bright field micrograph of cells obtained by diluting the sample of Example 4 to a concentration of 0.05%;

[0052] Fig.10 This is a fluorescence micrograph of lipid droplets after the sample of Example 4 was diluted to a concentration of 0.1% and stained with Lipi-Blue;

[0053] Fig.11 This is a bright field micrograph of cells obtained by diluting the sample of Example 4 to a concentration of 0.1%;

[0054] Fig.12 This is a fluorescence micrograph of lipid droplets after the sample of Example 4 was diluted to a concentration of 0.2% and stained with Lipi-Blue;

[0055] Fig.13 This is a bright field micrograph of cells obtained by diluting the sample of Example 4 to a concentration of 0.2%. DETAILED DESCRIPTION

[0056] The technical solution of the present invention will be further described clearly and completely in conjunction with specific embodiments below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work belong to the protection scope of the present invention.

[0057] In the present invention, although liposome technology is widely believed to be able to improve the solubility and bioavailability of drugs, it is still unpredictable whether liposomes can improve the solubility of azelaic acid. Because the liposome preparation process and formulation are diverse and complex, the interaction mechanism between each active ingredient and liposome varies due to its chemical properties, molecular structure and other factors. Azelaic acid, as a compound with specific physicochemical properties, cannot be directly inferred from its dissolution behavior in liposomes based on current knowledge. Therefore, although liposome technology is widely used, it is successfully applied to azelaic acid and significantly improves its solubility, while ensuring that its efficacy can be maintained under low azelaic acid content conditions, and requires careful design and optimization for the specific characteristics of azelaic acid. The present invention has concluded through rigorous experimental design and data analysis that, based on the technical solution provided by the present invention, liposomes can maintain their good efficacy while reducing the dosage of azelaic acid. This discovery not only expands the scope of application of liposome technology in the field of drug delivery, but also provides new possibilities for the clinical application of azelaic acid.

[0058] The conventional preparation method of liposomes uses organic solvents such as methanol, chloroform, dichloromethane, and n-hexane that are dangerous to the human body. These solvents cannot be retained in the product, and must be removed through certain process steps, and their solvent residues are controlled. There are also organic solvents such as ethanol, but ethanol has the properties of volatility and flammability, which is difficult to scale up industrially. It is necessary to increase explosion-proof devices and environmental protection treatment devices, and it is also difficult to control the residual amount of ethanol. Based on this, the present invention has studied the preparation process of liposomes. Propylene glycol is widely used in cosmetics, food and medicine, and it is relatively safe for the human body. Our research found that under the appropriate material ratio, the propylene glycol injection method is used to prepare azelaic acid liposomes, which can not only ensure the formation of complete liposomes, but also can be retained in the product without harm to the human body. Since propylene glycol is retained in the product and its amount is within the safe use limit of propylene glycol, it is not necessary to use process steps to remove it later, and it also overcomes the equipment limitations and difficulties of industrial scale-up to remove organic solvents. Therefore, the present invention selects propylene glycol as the organic solvent of this system.

[0059] Previously, no other existing technologies have adopted the propylene glycol injection method to prepare liposomes. The present invention adopts propylene glycol as the organic solvent, which achieves a high encapsulation rate and high stability of azelaic acid, while also reducing the process step of removing the organic solvent by one step, thereby eliminating the risks of insecurity brought about by the reduced pressure evaporation step, incomplete removal of the organic solvent, equipment limitations, environmental hazards, and personnel hazards.

[0060] In the embodiments of the present invention, the raw materials and proportions of the azelaic acid liposomes of Embodiments 1 to 4 are shown in Table 1.

[0061] Table 1. Composition and ratio of azelaic acid liposomes of Examples 1 to 4

[0062]

[0063]

[0064] In the embodiments of the present invention, the raw materials and proportions of the azelaic acid liposomes of Comparative Examples 1 to 6 are shown in Table 2.

[0065] Table 2. Composition and ratio of azelaic acid liposomes of Comparative Examples 1 to 6

[0066]

[0067]

[0068] Example 1

[0069] A nonanedioic acid liposome is composed of the following components in parts by weight: 82.365 parts of purified water, 6 parts of 1,2-propylene glycol, 4 parts of hydrogenated phosphatidylcholine, 3.5 parts of disodium hydrogen phosphate dodecahydrate, 1 part of cholesterol, 0.5 parts of Tween 80, 1 part of nonanedioic acid, 0.4 parts of p-hydroxyacetophenone, 0.4 parts of 1,2-hexanediol, 0.3 parts of sodium hydroxide, 0.315 parts of citric acid, 0.1 parts of tocopherol acetate, 0.1 parts of sodium stearoyl glutamate, and 0.02 parts of disodium EDTA.

[0070] Azelaic acid liposomes were prepared by 1,2-propylene glycol injection method, and the specific steps are as follows:

[0071] (1) Stir the formulated amounts of 1,2-propylene glycol, hydrogenated phosphatidylcholine, cholesterol, Tween 80 and tocopherol acetate at 65-80° C. for at least 30 minutes to obtain a clear organic phase solution.

[0072] (2) Stir the formulated water, azelaic acid, disodium hydrogen phosphate dodecahydrate, sodium hydroxide, citric acid, sodium stearoyl glutamate, disodium EDTA, p-hydroxyacetophenone and 1,2-hexanediol at 65-80° C. until it becomes clear, thereby obtaining a clear aqueous phase solution.

[0073] (3) Slowly inject the organic phase solution into the aqueous phase solution and stir at 65-80°C for at least 60 minutes.

[0074] (4) The solution obtained in step (3) is circulated 3 to 6 times under a pressure of 500 to 1000 bar in a high-pressure homogenizer to obtain azelaic acid liposomes.

[0075] Example 2

[0076] A nonanedioic acid liposome is composed of the following components in parts by weight: 45.365 parts of purified water, 20 parts of 1,2-propylene glycol, 12 parts of hydrogenated phosphatidylcholine, 3.5 parts of disodium hydrogen phosphate dodecahydrate, 3 parts of cholesterol, 2 parts of Tween 80, 10 parts of nonanedioic acid, 1 part of p-hydroxyacetophenone, 1 part of 1,2-hexanediol, 0.3 parts of sodium hydroxide, 0.315 parts of citric acid, 1 part of tocopherol acetate, 0.5 parts of sodium stearoyl glutamate, and 0.02 parts of EDTA-disodium. The preparation method is the same as that in Example 1.

[0077] Example 3

[0078] A nonanedioic acid liposome is composed of the following components in parts by weight: 64.565 parts of purified water, 15 parts of 1,2-propylene glycol, 8 parts of hydrogenated phosphatidylcholine, 3.5 parts of disodium hydrogen phosphate dodecahydrate, 2 parts of cholesterol, 2 parts of Tween 80, 3 parts of nonanedioic acid, 0.5 parts of p-hydroxyacetophenone, 0.5 parts of 1,2-hexanediol, 0.3 parts of sodium hydroxide, 0.315 parts of citric acid, 0.2 parts of tocopherol acetate, 0.1 parts of sodium stearoyl glutamate, and 0.02 parts of EDTA-disodium. The preparation method is the same as that in Example 1.

[0079] Example 4

[0080] A nonanedioic acid liposome is composed of the following components in parts by weight: 75.565 parts of purified water, 8 parts of 1,2-propylene glycol, 6 parts of hydrogenated phosphatidylcholine, 3.5 parts of disodium hydrogen phosphate dodecahydrate, 2 parts of cholesterol, 2 parts of Tween 80, 1 part of nonanedioic acid, 0.5 parts of p-hydroxyacetophenone, 0.5 parts of 1,2-hexanediol, 0.3 parts of sodium hydroxide, 0.315 parts of citric acid, 0.2 parts of tocopherol acetate, 0.1 parts of sodium stearoyl glutamate, and 0.02 parts of EDTA-disodium. The preparation method is the same as that in Example 1.

[0081] Comparative Example 1

[0082] A nonanedioic acid liposome is composed of the following components in parts by weight: 78.365 parts of purified water, 4 parts of hydrogenated phosphatidylcholine, 3.5 parts of disodium hydrogen phosphate dodecahydrate, 1 part of cholesterol, 0.5 parts of Tween 80, 1 part of nonanedioic acid, 0.4 parts of p-hydroxyacetophenone, 0.4 parts of 1,2-hexanediol, 0.3 parts of sodium hydroxide, 0.315 parts of citric acid, 0.1 parts of tocopherol acetate, 0.1 parts of sodium stearoyl glutamate, 0.02 parts of disodium EDTA, and 10 parts of ethanol.

[0083] Azelaic acid liposomes were prepared by ethanol injection method, and the preparation steps are as follows:

[0084] (1) Stir the formulated amounts of ethanol, hydrogenated phosphatidylcholine, cholesterol, Tween 80 and tocopherol acetate at 65-80° C. for 20 minutes to obtain a clear organic phase solution.

[0085] (2) Stir the formulated water, azelaic acid, disodium hydrogen phosphate dodecahydrate, sodium hydroxide, citric acid, sodium stearoyl glutamate, disodium EDTA, p-hydroxyacetophenone and 1,2-hexanediol at 65-80° C. until it becomes clear, thereby obtaining a clear aqueous phase solution.

[0086] (3) Slowly inject the organic phase solution into the aqueous phase solution and stir at 65-80°C for at least 60 min to obtain a white suspension solution.

[0087] (4) The suspension solution obtained in step (3) is transferred to a round-bottom flask, and the mixture is evaporated under reduced pressure at 50-60° C. for about 40 min until no liquid drops in the receiving bottle. During the reduced pressure evaporation process, it is necessary to prevent the liquid in the round-bottom flask from boiling.

[0088] (5) After the sample obtained in step (4) is cooled to room temperature, it is circulated 3 to 6 times at a pressure of 500 to 1000 bar in a high-pressure homogenizer to obtain azelaic acid liposomes.

[0089] Comparative Example 2

[0090] A nonanedioic acid liposome is composed of the following components in parts by weight: 76.565 parts of purified water, 5 parts of 1,2-propylene glycol, 6 parts of hydrogenated phosphatidylcholine, 3.5 parts of disodium hydrogen phosphate dodecahydrate, 2 parts of cholesterol, 2 parts of Tween 80, 1 part of nonanedioic acid, 0.5 parts of p-hydroxyacetophenone, 0.5 parts of 1,2-hexanediol, 0.3 parts of sodium hydroxide, 0.315 parts of citric acid, 0.2 parts of tocopherol acetate, 0.1 parts of sodium stearoyl glutamate, and 0.02 parts of EDTA-disodium. The preparation method is the same as that in Example 1.

[0091] Comparative Example 3

[0092] A nonanedioic acid liposome is composed of the following components in parts by weight: 62.565 parts of purified water, 21 parts of 1,2-propylene glycol, 6 parts of hydrogenated phosphatidylcholine, 3.5 parts of disodium hydrogen phosphate dodecahydrate, 2 parts of cholesterol, 2 parts of Tween 80, 1 part of nonanedioic acid, 0.5 parts of p-hydroxyacetophenone, 0.5 parts of 1,2-hexanediol, 0.3 parts of sodium hydroxide, 0.315 parts of citric acid, 0.2 parts of tocopherol acetate, 0.1 parts of sodium stearoyl glutamate, and 0.02 parts of EDTA-disodium. The preparation method is the same as that in Example 1.

[0093] Comparative Example 4

[0094] A 1% azelaic acid aqueous solution is composed of the following components in parts by weight: 99 parts of purified water and 1 part of azelaic acid.

[0095] Comparative Example 5

[0096] A nonazelaic acid liposome is composed of the following components in parts by weight: 40 parts of hydrogenated phosphatidylcholine, 5 parts of cholesterol, 3 parts of nonazelaic acid, 1 part of DSPE-mPEG2000, 16 parts of chloroform, 4 parts of methanol, and 51 parts of physiological saline.

[0097] Referring to patent CN114767559A, azelaic acid liposomes were prepared by thin film hydration method, and the specific steps are as follows:

[0098] (1) Azelaic acid, hydrogenated phosphatidylcholine, cholesterol and DSPE-mPEG2000 were dissolved in a chloroform-methanol solution (chloroform:methanol=4:1 V / V), stirred at 37°C to dissolve, and then evaporated under reduced pressure at 37°C to form a film to remove the chloroform and methanol.

[0099] (2) adding physiological saline, hydrating at 37°C for 2 h, and sonicating on ice using an ultrasonic disruptor; then centrifuging at 18,000 g for 3 min, and filtering with a 0.8 μm pore size filter membrane to obtain azelaic acid liposomes.

[0100] Comparative Example 6

[0101] A nonanedioic acid solution is composed of the following components in parts by weight: 60.365 parts of purified water, 20 parts of 1,2-propylene glycol, 3.5 parts of disodium hydrogen phosphate dodecahydrate, 2 parts of Tween 80, 10 parts of nonanedioic acid, 1 part of p-hydroxyacetophenone, 1 part of 1,2-hexanediol, 0.3 parts of sodium hydroxide, 0.315 parts of citric acid, 1 part of tocopherol acetate, 0.5 parts of sodium stearoyl glutamate, and 0.02 parts of EDTA-disodium. The preparation method is the same as that in Example 1.

[0102] Example 5. Process Investigation

[0103] The results of the two preparation processes of Example 1 and Comparative Example 1 are shown in Table 3.

[0104] Table 3. Comparison of process advantages and disadvantages between Example 1 and Comparative Example 1

[0105]

[0106]

[0107] The comparison results show that the 1.2-propylene glycol injection method used in this patent is simple, safe, does not require the removal of organic solvents, does not require anti-boiling and environmental protection of equipment, and can be industrialized. In addition, the encapsulation rate of the azelaic acid liposome prepared by the method of the present invention is higher than that of the product prepared by the ethanol injection method.

[0108] Example 6. Micromorphology, particle size and potential evaluation

[0109] The properties of the azelaic acid liposome sample of the present invention were characterized. Theoretically, if azelaic acid with a high content can be encapsulated, then azelaic acid with a low content can also be encapsulated. Therefore, the present invention evaluated the microscopic morphology, particle size and potential of the azelaic acid liposome with a 10% azelaic acid content prepared in Example 2. The test results are shown in Figure 1 to Figure 3The results showed that the multi-layer vesicle structure of liposomes can be observed under transmission electron microscopy, and the membrane layer is clear, which is characteristic of liposomes. The average particle size of the sample is 385.6nm, and the potential value is -36.8mV, which is consistent with the good stability of nanoliposomes, indicating that 10% azelaic acid can be wrapped by the liposome membrane to form liposomes with multi-layer vesicle structure.

[0110] Example 7. Stability test

[0111] The stability of the azelaic acid liposomes prepared in Examples 1 to 4 and Comparative Example 1 (liposome composition prepared by ethanol injection method), Comparative Example 2 (propylene glycol dosage: 5%), and Comparative Example 3 (propylene glycol dosage: 21%) was tested. The stability of the azelaic acid liposomes was investigated under the test conditions of 5°C, 25°C, 40°C, high and low temperature cycles (5°C / 40°C, 48h cycle once, 3 cycles in total), and light (5000Lx), and the pH, particle size, potential, azelaic acid encapsulation rate, azelaic acid content, and microscopic observation of azelaic acid crystal precipitation were used as detection indicators for evaluation.

[0112] The results are shown in Table 4 below. The results show that the azelaic acid liposomes prepared by the 1,2-propylene glycol injection method in Examples 1 to 4 can be stably stored for 180 days at a high temperature of 40°C, and the pH, particle size, potential, and content are basically stable. The encapsulation rate is >85%, and the azelaic acid does not precipitate, and the stability is excellent. The azelaic acid liposomes prepared by the ethanol injection method in Comparative Example 1 can also be stably stored for 180 days at a high temperature of 40°C, and the pH, particle size, potential, content, and encapsulation rate are basically stable. In comparison, the azelaic acid liposomes prepared by the 1,2-propylene glycol injection method have a smaller particle size and a higher encapsulation rate than the liposome composition prepared by the ethanol injection method. The particle size and encapsulation rate advantages of the azelaic acid liposome composition prepared by the 1,2-propylene glycol injection method are more obvious.

[0113] Further comparison of the test results of Examples 1 to 4, Comparative Example 2, and Comparative Example 3 shows that the stability of the azelaic acid liposomes prepared by the 1,2-propylene glycol injection method is good when the amount of 1,2-propylene glycol in the formula is 8% to 20%. When the content of 1,2-propylene glycol is 5% (lower than 6%), due to the low solvent content, the organic phase cannot be completely dissolved and liposomes cannot be formed. When the content of 1,2-propylene glycol is 21% (higher than 20%), due to the high amount of solvent, the encapsulation efficiency of the prepared azelaic acid liposome composition is 55.3%, which is greatly reduced, indicating that when the content of 1,2-propylene glycol is higher than 20%, it may cause leakage of the azelaic acid liposomes, resulting in a decrease in the encapsulation efficiency. Therefore, the stability of the azelaic acid liposome composition can be maintained, and the 1,2-propylene glycol content range with a higher encapsulation efficiency is 6 to 20%.

[0114] Table 4. Results of stability study of azelaic acid liposomes

[0115]

[0116]

[0117] Example 8. Solubility

[0118] The samples prepared in Examples 1 to 4, Comparative Example 4 and Comparative Example 5 were tested for solubility under the same conditions. The test results are shown in Table 5. The results show that there are a large number of undissolved and precipitated azelaic acid particles in the sample of Comparative Example 4, and the solubility of azelaic acid is relatively low. Comparative Example 5 is a milky white suspension solution, and the samples of Examples 1 to 4 are fine and uniform milky white suspension solutions, without insoluble azelaic acid particles and precipitated crystals. The solubility of azelaic acid was determined and it was found that after the present invention (Examples 1 to 4) prepared azelaic acid into liposomes, the solubility of azelaic acid was significantly improved.

[0119] Table 5. Azelaic acid liposome solubility test results

[0120] Test samples Example 1 Example 2 Example 3 Example 4 Comparative Example 4 Comparative Example 5 Amount of azelaic acid added (%) 1％ 10％ 3％ 1％ 1％ 10％ Azelaic acid solubility (%) 0.99％ 8.96％ 3.00％ 0.98％ 0.24％ 6.88％

[0121] Example 9. Skin irritation evaluation

[0122] Refer to the requirements for skin irritation test in the 2015 version of the Cosmetic Safety Technical Specifications. Take Examples 1 to 4 and Comparative Example 6 (which do not contain hydrogenated phosphatidylcholine and cholesterol, do not form liposomes, and fail to encapsulate azelaic acid) as test samples, and use the rabbit left and right side self-comparison method to evaluate whether the rabbits produce skin irritation reactions (edema, erythema) after continuous administration of the samples for 14 days, and evaluate the intensity of skin irritation.

[0123] The test results are shown in Table 6. The results show that the azelaic acid liposomes prepared in Examples 1 to 4 are non-irritating to mildly irritating, and the irritation score is significantly lower than that of Comparative Example 6. In addition, Example 2 and Comparative Example 6 have the same azelaic acid content, and the difference between the two is that Example 2 forms liposomes, while Comparative Example 6 does not add phospholipids and cholesterol, so Comparative Example 6 does not form liposomes and fails to encapsulate azelaic acid. Therefore, at the same azelaic acid content (10%), the irritation of Example 2, which forms liposomes, is much less than that of the sample in Comparative Example 6. This shows that the use of the method of the present invention to prepare azelaic acid into liposomes can significantly reduce skin irritation and improve safety of use.

[0124] Table 6. Scoring results of multiple skin irritation tests on azelaic acid liposomes in rabbits

[0125]

[0126] Example 10. Evaluation of acne-removing efficacy

[0127] Bacterial lipopolysaccharide-induced mouse mononuclear macrophage RAW264.7 is a classic cell model for studying inflammatory factors. The secretion of inflammatory factors TNF-α and IL-6 is inhibited by the test substance after sampling, so as to evaluate the correlation between the sample and the acne-removing efficacy. Example 4 was used as the test sample. After dilution to different concentrations, the mouse mononuclear macrophage RAW264.7 cell model was used to determine the content of inflammatory factors TNF-α and IL-6 in the cells, and the inhibition rate of inflammatory factors TNF-α and IL-6 was calculated to evaluate the acne-removing efficacy of the sample.

[0128] The test results are shown in Tables 7 and 8. The results show that the test sample Example 4 has an inhibitory effect on the secretion of inflammatory factors TNF-α and IL-6 in mouse mononuclear macrophage RAW264.7 at dilution concentrations of 0.00125%, 0.0025%, and 0.005%, and the results have significant differences (P < 0.05). The test results show that the azelaic acid liposome composition has a more significant acne-removing effect at a lower concentration (0.00125%).

[0129] Table 7. Inhibition rate of TNF-α in RAW264.7 by the tested samples

[0130]

[0131] Note: Compared with negative control: *, p<0.05; **, p<0.01.

[0132] Table 8. Inhibition rate of IL-6 in RAW264.7 by the tested samples

[0133]

[0134] Note: Compared with negative control: *, p<0.05; **, p<0.01.

[0135] Example 11. Evaluation of oil control efficacy

[0136] The evaluation was conducted using human immortalized sebaceous gland cells SZ95. An in vitro oil secretion model was established by stimulating hormone substances. After being treated with the test substance, the oil control efficacy of the test substance was evaluated by the synthesis trend of lipid droplets. Under a microscope, it was observed that Lipi-Blue (lipid droplet staining reagent) combined with intracellular lipid droplets to emit blue fluorescence, and the secretion of lipid droplets could be directly observed. Example 4 was used as the test sample, and after being diluted to different concentrations, its oil control efficacy was evaluated.

[0137] The results are shown in Table 9. Figure 4-Figure 13The data and micrographs directly reflect that the sample of Example 4 has an inhibitory effect on the synthesis of lipid droplets in sebaceous gland cells at a dilution concentration of 0.1% and 0.2%, and there is a significant difference (P < 0.05). The test results show that the azelaic acid liposome composition has a more significant oil control effect at a lower concentration (0.1%).

[0138] Table 9. Results of lipid droplet synthesis inhibition rate of the tested samples

[0139]

[0140] Note: Compared with negative control: *, p<0.05; **, p<0.01.

[0141] Example 12. Evaluation of whitening efficacy

[0142] The experiment was conducted with reference to the group standard "T / HPCIA005-2021 Determination of the whitening effect of cosmetics - zebrafish embryo method", and the inhibition rate of melanin production in zebrafish embryos was used as an evaluation index to evaluate the whitening effect of the test sample. Example 4 was used as the test sample, and after dilution to different concentrations, its whitening effect was evaluated.

[0143] The results are shown in Table 10. When the dilution concentration of the test sample in Example 4 was 0.039%, 0.078%, and 0.156%, the zebrafish was treated to 72hpf (72h after fertilization), and compared with the blank control group, the production of zebrafish melanin was significantly inhibited (P < 0.001). The test results show that the azelaic acid liposome composition has a more significant whitening effect (P < 0.001) at a lower concentration (0.039%). Zebrafish is used to simulate the in vivo environment for experiments, which is different from the human body environment.

[0144] Table 10. Effect of the tested samples on the inhibition rate of melanin production in zebrafish embryos

[0145] Test samples Melanin production inhibition rate (%) P-value Blank control 0.00±1.79 —— Positive control (40 μM N-phenylthiourea) 85.75±0.26 ＜0.001*** Example 4 (dilution concentration: 0.039%) 23.19±0.24 ＜0.001*** Example 4 (dilution concentration: 0.078%) 25.88±0.56 ＜0.001*** Example 4 (dilution concentration: 0.156%) 17.33±1.16 ＜0.001***

[0146] Note: Compared with blank control: *, p<0.05; **, p<0.01, ***, p<0.001.

Claims

1. A composition for preparing azelaic acid liposomes, It is characterized in that The composition comprises an organic phase composition for preparing azelaic acid liposomes and an aqueous phase composition for preparing azelaic acid liposomes; the organic phase composition comprises 1,2-propylene glycol, hydrogenated phosphatidylcholine and cholesterol, and the mass ratio thereof is 6-20:4-12:1-3; the aqueous phase composition comprises an aqueous phase medium and azelaic acid, and the mass ratio thereof is 40-85:1-10.

2. The composition according to claim 1, It is characterized in that The organic phase composition includes 1,2-propylene glycol, hydrogenated phosphatidylcholine, cholesterol, a stabilizer and an antioxidant, and the mass ratio thereof is 6-20:4-12:1-3; 0.5-2:0.1-1; the stabilizer is Tween 80; The antioxidant is tocopherol acetate.

3. The composition according to claim 1, It is characterized in that The aqueous phase composition includes an aqueous phase medium, azelaic acid, a pH regulator, an anionic surfactant, a preservative and a metal ion chelating agent, and the mass ratio thereof is 40-85: 1-10: 3.5-5: 0.1-0.5: 0.8-2: 0.01-0.05; the aqueous phase medium is water; the pH regulator is any one or more of disodium hydrogen phosphate dodecahydrate, citric acid, and sodium hydroxide; the anionic surfactant is sodium stearoyl glutamate; the preservative is any one or two of p-hydroxyacetophenone and 1,2-hexanediol; and the metal ion chelating agent is EDTA-disodium.

4. A method for preparing azelaic acid liposomes, It is characterized in that The composition according to any one of claims 1 to 3 is prepared into azelaic acid liposomes by a 1,2-propylene glycol injection method, wherein the 1,2-propylene glycol injection method comprises the following steps: (1) mixing the components of the organic phase composition according to claim 1 to obtain an organic phase solution; (2) mixing the components of the aqueous phase composition described in claim 1 to obtain an aqueous phase solution; (3) injecting the organic phase solution obtained in step (1) into the aqueous phase solution obtained in step (2), reacting to obtain a suspension; (4) The suspension obtained in step (3) is subjected to high-pressure homogenization to obtain azelaic acid liposomes.

5. The preparation method according to claim 4, It is characterized in that In step (3), the reaction conditions are stirring at 65°C to 80°C for 60 min to 180 min.

6. Azelaic acid liposome prepared by the preparation method according to any one of claims 4 to 5.

7. Preparation composition, It is characterized in that The preparation composition comprises azelaic acid and liposomes; the liposomes comprise hydrogenated phosphatidylcholine, cholesterol and 1,2-propylene glycol; the mass ratio of azelaic acid, hydrogenated phosphatidylcholine, cholesterol and 1,2-propylene glycol is 1-10:4-12:1-3:6-20; the preparation composition does not contain ethanol, dichloromethane, chloroform, ether and n-hexane.

8. The preparation composition according to claim 7, It is characterized in that The liposome comprises hydrogenated phosphatidylcholine, cholesterol, 1,2-propylene glycol, a stabilizer, an antioxidant, an anionic surfactant, a preservative, a pH regulator, a metal ion chelating agent and an aqueous phase medium, and the mass ratio thereof is 4-12: 1-3: 6-20: 0.5-2: 0.1-1: 0.1-0.5: 0.8-2: 3.5-5: 0.01-0.05: 40-85; the stabilizer is Tween 80; the antioxidant is tocopherol acetate; the anionic surfactant is sodium stearoyl glutamate; the preservative is any one or two of p-hydroxyacetophenone and 1,2-hexanediol; the pH regulator is any one or more of disodium hydrogen phosphate dodecahydrate, citric acid and sodium hydroxide; the metal ion chelating agent is disodium EDTA; and the aqueous phase medium is water.

9. The preparation composition according to claim 7, It is characterized in that The preparation composition consists of the following components in parts by weight: 1 to 10 parts of azelaic acid, 4 to 12 parts of hydrogenated phosphatidylcholine, 1 to 3 parts of cholesterol, 6 to 20 parts of 1,2-propylene glycol, 0.5 to 2 parts of Tween 80, 0.1 to 0.5 parts of sodium stearoyl glutamate, 0.4 to 1 part of p-hydroxyacetophenone, 0.4 to 1 part of 1,2-hexanediol, 0.1 to 1 part of tocopherol acetate, 3.5 parts of disodium hydrogen phosphate dodecahydrate, 0.315 parts of citric acid, 0.3 parts of sodium hydroxide, 0.02 parts of EDTA-disodium, and 45.365 to 82.365 parts of water.

10. Use of the composition according to any one of claims 1 to 3, the liposome according to claim 6 and / or the preparation composition according to any one of claims 7 to 9 in the preparation of cosmetics with acne-removing, oil-controlling and / or whitening effects.

Images