Ethosomes, preparation method, use and skin care products

Abstract

The embodiment of the present application discloses an alcohol body and a preparation method, use and skin care product, and the preparation method can be used to enhance the protection of the encapsulated object. The preparation method comprises: mixing glyceryl monooleate and poloxamer 407 at a first temperature, and then cooling to a second temperature to obtain an ester capsule material, wherein the first temperature is greater than the second temperature; obtaining an alcohol mixture containing a short-chain alcohol and an encapsulated object; mixing the alcohol mixture and the ester capsule material to obtain an ester alcohol mixture; adding water to the ester alcohol mixture for hydration treatment to obtain a hydration liquid; homogenizing the hydration liquid to obtain an alcohol body homogenous liquid containing alcohol bodies.

Description

Technical Field

[0001] The invention relates to the technical field of lipid encapsulation, and in particular to ethosomes and preparation methods, uses and skin care products. Background Art

[0002] Lipid encapsulation technology is an important technology that encapsulates active ingredients into lipid vesicles to make them permeable.

[0003] The encapsulation carriers commonly used to load the active ingredients are mainly liposomes, ethosomes, etc. Among them, liposomes are an artificial membrane formed based on phospholipids, and are closed vesicles with a bilayer structure; ethosomes (ES for short) are a new type of flexible nanoliposomes that can be prepared based on phospholipids, water and biocompatible alcohols (such as ethanol, propylene glycol, etc.). It can be seen that traditional liposomes and ethosomes generally need to be prepared from phospholipids.

[0004] In addition, it has been found that in addition to phospholipids, other lipids, such as glycerol monooleate (GMO), can also be used to prepare encapsulation carriers. However, during the preparation process, glycerol monooleate usually needs to be mixed with the encapsulated material at high temperature. This will inevitably produce some difficult-to-overcome defects. For example, when it is necessary to load an encapsulated material that is easily affected by high temperature, the activity of the encapsulated material will be easily destroyed. Summary of the invention

[0005] One purpose of the embodiments of the present application is to solve at least one problem in the above background and provide corresponding beneficial effects.

[0006] Another object of the embodiments of the present application is to provide a method for preparing an ethanosome, which can enhance the protection of the encapsulated object.

[0007] Another object of the embodiments of the present application is to provide an alcohol body prepared according to the aforementioned method, which can enhance the protection of the encapsulated object.

[0008] Another object of the embodiments of the present application is to provide a use of an ethanosome prepared according to the aforementioned preparation method, which can improve the use effect of the encapsulated object.

[0009] Another object of an embodiment of the present application is to provide a skin care product, comprising the aforementioned ethosome.

[0010] The embodiments of the present application mainly achieve the above objectives through the following technical solutions.

[0011] In a first aspect, the present invention provides a method for preparing an ethosome, comprising:

[0012] Mixing monoolein and poloxamer 407 at a first temperature, and then cooling to a second temperature to obtain an ester capsule material, wherein the first temperature is greater than the second temperature;

[0013] obtaining an alcohol mixture containing a short-chain alcohol and an encapsulated substance;

[0014] Mixing the alcohol mixture and the ester capsule material to obtain an ester alcohol mixture;

[0015] adding a water liquid to the ester alcohol mixture to perform a hydration treatment to obtain a hydrated liquid;

[0016] The hydrated liquid is homogenized to obtain an alcoholate homogenous liquid containing alcoholates.

[0017] In some technical solutions, the first temperature is 31-100°C; that is, the first temperature is greater than or equal to 31 degrees and less than or equal to 100 degrees.

[0018] In some technical solutions, the second temperature is 0-30°C, that is, the second temperature is greater than or equal to 0 degrees and less than or equal to 30 degrees.

[0019] In a second aspect, an embodiment of the present application provides an ethosome, which is prepared according to the preparation method of the ethosome described in the first aspect.

[0020] In a third aspect, the embodiments of the present application provide a use of an ethosome in preparing a carrier containing an encapsulated object.

[0021] In a fourth aspect, the embodiments of the present application also provide a use of ethosomes in preparing skin care products.

[0022] In a fifth aspect, the embodiments of the present application further provide a skin care product, comprising the ethosome described in the second aspect.

[0023] The beneficial effects of the embodiments of the present invention include:

[0024] In some embodiments, the preparation method of the present application has an important beneficial effect: on the basis of improving the mixing degree of monoolein and poloxamer 407 in the ester capsule material, the temperature required for hydration can be reduced, thereby reducing or avoiding damage to the encapsulated object. Specifically, on the one hand, on the basis of increasing the mixing degree of monoolein and poloxamer 407 in the ester capsule material by the first temperature, the temperature of the ester capsule material is further reduced through a cooling step; on the other hand, short-chain alcohols such as low-chain alcohols are introduced before hydration to reduce the temperature required for hydration, so that the ester-alcohol mixture composed of the ester capsule material and the alcohol mixture can also hydrate with the water under the condition of not higher than the first temperature; thus, during the preparation process, the damage to the encapsulated object caused by the first temperature can be avoided, thereby solving the defects described in the background. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 A comparison diagram of the appearance and Tyndall effect of an alcoholic homogeneous liquid prepared from different alcohol phases in the examples of the present application;

[0026] Figure 2 A schematic diagram comparing the particle size and PDI of the alcohol bodies prepared from different alcohol phases in the examples of the present application;

[0027] Figure 3 A schematic diagram of drug loading concentration of ethosomes prepared according to different alcohol phases in the embodiments of the present application;

[0028] Figure 4 A schematic diagram comparing the particle size and PDI of the alcohol body prepared according to different homogenization pressures in the embodiment of the present application;

[0029] Figure 5 A schematic diagram comparing the particle size and PDI of the alcohol body at different volume ratios of propylene glycol and dipropylene glycol in the embodiment of the present application;

[0030] Figure 6 A schematic diagram showing a comparison of drug loading concentrations of ethosomes at different volume ratios of propylene glycol to dipropylene glycol in the embodiments of the present application;

[0031] Figure 7 This is a comparison diagram of the appearance and Tyndall effect of an alcoholic homogenate liquid prepared with different stabilizers in the examples of this application;

[0032] Figure 8 A comparative schematic diagram of the particle size and PDI of the alcohol homogenate liquid prepared according to different stabilizers in the embodiment of the present application;

[0033] Fig. 9 A comparative schematic diagram of the particle size and PDI of the homogenous alcoholic liquid prepared with different retinol concentrations in the examples of the present application;

[0034] Fig.10 A schematic diagram comparing drug loading concentrations of ethosome homogenous solutions prepared with different retinol concentrations in the examples of the present application;

[0035] Fig.11 A comparative schematic diagram of the particle size and PDI of the alcohol body homogenate prepared according to different GMO concentrations in the examples of the present application;

[0036] Fig.12 This is a comparative schematic diagram of the retinol retention rates of the ethosome homogenate solutions prepared according to different GMO concentrations at different storage temperatures in the embodiments of the present application;

[0037] Fig.13 A schematic diagram showing the appearance comparison of alcoholic homogenate liquids with different alcohol contents in the embodiments of the present application;

[0038] Fig.14 A transmission electron microscope image (×100000) of an alcoholic homogenate containing 20% ​​alcohol in an embodiment of the present application;

[0039] Fig.15 A schematic diagram showing a comparison of the particle sizes of alcohol homogenous liquids with different alcohol contents at different storage temperatures in the embodiments of the present application;

[0040] Fig.16 A schematic diagram comparing the retinol retention rates of the alcoholic homogenate solutions prepared with different alcohol contents and the retinol solution containing 30% ethanol at different storage temperatures in the embodiments of the present application;

[0041] Fig.17 A columnar comparison diagram of the particle size and PDI of the homogenous alcohol body liquid with different drug-to-lipid ratios at room temperature in the embodiment of the present application;

[0042] Fig.18 This is a comparison chart of the particle sizes of the homogenous alcoholic liquids with different drug-to-lipid ratios after being stored at different storage temperatures for different periods of time in the examples of this application;

[0043] Fig.19 A comparison chart of retinol retention rates of homogenous ethanosomes with different drug-to-lipid ratios after storage at different storage temperatures for different periods of time in the examples of the present application;

[0044] Fig. 20 A comparison chart of the particle sizes of the alcohol homogenate liquid without the antioxidant group after being stored at different storage temperatures for different periods of time in the examples of the present application;

[0045] Fig.21 A comparison chart of the retinol retention rate of the alcohol body homogenate liquid without antioxidant in the embodiment of the present application after being stored at different storage temperatures for different periods of time;

[0046] Fig. 22 A comparison chart of the particle sizes of the alcohol homogenate liquid containing an antioxidant group and the alcohol homogenate liquid without an antioxidant group after being stored at different storage temperatures for different periods of time in the examples of the present application;

[0047] Fig.23 A comparison chart of the retinol retention rates of the alcoholic homogenate solutions containing an antioxidant group and a group without an antioxidant after being stored at different storage temperatures for different periods of time in the examples of the present application;

[0048] Fig.24 This is a typical SAXS characteristic spectrum of lipid cubic liquid crystal;

[0049] Fig.25 This is a SAXS spectrum of an alcohol body in an embodiment of the present application;

[0050] Fig.26 This is a schematic diagram of the homogeneous solution of the alcohol body of Example 25 of the present application under a transmission electron microscope;

[0051] Fig. 27 A comparative schematic diagram of the particle size and PDI of the test liquids of experimental groups 1 to 5 at room temperature in the embodiments of the present application;

[0052] Fig.28 A comparative schematic diagram of the particle size and PDI of the test liquids of experimental groups 1 to 5 at different temperatures in the embodiments of the present application;

[0053] Fig.29 A comparative schematic diagram of the retinol retention rates of the test solutions of experimental groups 1 to 5 at different temperatures in the embodiments of the present application;

[0054] Fig.30 A standard curve diagram formed based on HPLC analysis of retinol;

[0055] Fig.31 A schematic diagram showing the effect of the extraction liquid volume of the sample solution on the extraction recovery rate in the embodiment of the present application;

[0056] Fig.32 A curve diagram of the cumulative permeation amount of a transdermal permeation of each group of sample solutions in the examples of the present application;

[0057] Fig.33 A schematic diagram of the steady-state permeation rate of each group of sample solutions in the examples of the present application;

[0058] Fig.34 A percutaneous permeation evaluation percentage accumulation diagram of each group of sample solutions in the examples of the present application;

[0059] Fig.35This is a schematic diagram of the in vitro cell viability of HaCaT cells to be tested in the examples of this application;

[0060] Fig.36 This is a schematic diagram of the in vitro cell viability of CCC-ESF-1 cells to be tested in the examples of this application;

[0061] Fig.37 A schematic diagram of the left-right comparison method in the embodiment of the present application;

[0062] Fig.38 This is a diagram of the skin state of a guinea pig in a single administration in the examples of this application;

[0063] Fig.39 This is a diagram of the skin state of a guinea pig subjected to multiple dosing in the examples of this application;

[0064] Fig.40 This is a diagram of the microstructural state of guinea pig skin after multiple drug administration in the examples of this application. DETAILED DESCRIPTION

[0065] The present invention is further described in detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

[0066] Reference to "embodiments" herein means that a particular feature, structure, or characteristic described in conjunction with the embodiments may be included in at least one embodiment of the present application. The appearance of the phrase in various locations in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0067] The terms "first", "second", "third", etc. in the embodiments of the present application are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first", "second", etc. may explicitly or implicitly include at least one of the features.

[0068] Furthermore, the terms "comprises," "comprising," "including," "having," and any variations thereof are intended to cover non-exclusive inclusions. For example, a process, method, system, product, or apparatus comprising a series of steps or units is not limited to the listed steps or units, but may optionally include steps or units not listed, or may optionally include other steps or units inherent to the process, method, product, or apparatus.

[0069] When the following terms are involved in the specific implementation methods of this application, unless otherwise stated, their meanings shall be determined according to the corresponding descriptions of the terms:

[0070] The term "PDI" is called Polymer dispersity index in English and polymer dispersion index in Chinese. It is used to describe the molecular weight distribution of polymers. Generally, the smaller the PDI, the more uniform the molecular weight distribution.

[0071] <Method for preparing ethanol body>

[0072] In a first aspect, an embodiment of the present application provides a method for preparing an ethosome, comprising steps S100-500:

[0073] S100, mixing monoolein and poloxamer 407 (F127 for short) at a first temperature, and then cooling to a second temperature to obtain an ester capsule material, wherein the first temperature is greater than the second temperature.

[0074] Monoolein is easier to fully mix with poloxamer 407 at higher temperatures. However, in the preparation process of alcohol bodies, the ester capsule material containing monoolein needs to be mixed with other components later, and too high a temperature will damage components that are not resistant to high temperatures (such as certain encapsulated materials). Through cooling treatment, the temperature of the ester capsule material is adjusted to a second temperature lower than the first temperature, which is beneficial to protect the components that are not resistant to high temperatures.

[0075] At low temperatures and room temperatures, glyceryl monooleate and poloxamer 407 are often solid, which is not conducive to full mixing. In some embodiments, the first temperature is between 31 and 100 degrees, optionally between 31 and 95 degrees, optionally between 50 and 60 degrees, and optionally the first temperature is 60 degrees. This is more conducive to full mixing of glyceryl monooleate and poloxamer 407.

[0076] In some embodiments, the second temperature does not exceed 40 degrees, optionally, the second temperature does not exceed 30 degrees.

[0077] S200, obtaining an alcohol mixture containing short-chain alcohols and encapsulated substances.

[0078] S300, mixing the alcohol mixture containing the short-chain alcohol and the encapsulated material with the ester encapsulating material to obtain an ester alcohol mixture.

[0079] S400, adding water liquid into the ester alcohol mixture to perform hydration treatment to obtain a hydrated liquid.

[0080] In the process of adding water to the ester-alcohol mixture, monooleylglycerol as a capsule material hydrates with water molecules to form a lipid vesicle structure with a lipid bilayer, thereby wrapping the encapsulated object in the alcohol mixture to form a particle structure loaded with the encapsulated object.

[0081] Regarding the aqueous liquid, it should be noted that it refers to a liquid containing water, which can be selected from water or an aqueous solution.

[0082] S500, homogenizing the hydrated liquid to obtain an alcoholic body homogenous liquid containing alcoholic bodies.

[0083] The hydration liquid in S400 is a suspension. In the suspension, the uniformity of the distribution of the particles of the encapsulated material needs to be improved, so it is necessary to improve the uniformity of the distribution of the particles of the encapsulated material through homogenization treatment.

[0084] After the S500 homogenization treatment, the hydration liquid can be converted into an alcohol body homogenous liquid, effectively improving the uniformity of the distribution of the particles of the encapsulated object, thereby obtaining an alcohol body with higher uniformity, and the alcohol body can be used as a carrier for the encapsulated object. As can be seen from the above, the embodiment of the present application provides a method for preparing an alcohol body, and the preparation method can prepare a new type of alcohol body based on monoolein.

[0085] The preparation method of the embodiment of the present application has an important beneficial effect: on the basis of improving the mixing degree of monoolein and poloxamer 407 in the ester capsule material, the temperature required for hydration can be reduced, thereby reducing or avoiding damage to the encapsulated object. Specifically, on the one hand, on the basis of increasing the mixing degree of monoolein and poloxamer 407 in the ester capsule material by the first temperature, the temperature of the ester capsule material is further reduced through a cooling step; on the other hand, short-chain alcohols such as low-chain alcohols are introduced before hydration to reduce the temperature required for hydration, so that the ester-alcohol mixture composed of the ester capsule material and the alcohol mixture can also hydrate with the water liquid under the condition of not higher than the first temperature; thereby, during the preparation process, damage to the encapsulated object caused by the first temperature is avoided.

[0086] In addition, the preparation method of the embodiment of the present application also has the following technical effects:

[0087] 1. The lipid vesicle structure of ethosomes is a multivesicular structure with multiple chambers, which can improve the encapsulation performance of the encapsulated objects.

[0088] 2. Low-chain alcohols can also be inserted between lipid bilayers, thereby changing the structure of lipid nanocubic liquid crystals and adjusting the pore size and pore properties.

[0089] 3. The interaction between low-chain alcohols and lipid molecules (i.e. monoolein molecules) and water molecules can enhance the stability of lipid cubic liquid crystals. This enhanced stability helps prevent the destruction of the lipid cubic liquid crystal structure, thereby extending the effective use time of the ethosome.

[0090] 4. Premixing the alcohol composition with the ester capsule material to form an ester-alcohol mixture can make the encapsulated object disperse better in the ester capsule material, which is helpful to more evenly encapsulate the encapsulated object in the subsequent hydration process (the water is slowly added to the alcohol-ester mixture to form a bilayer vesicle to encapsulate the drug), thereby improving the encapsulation efficiency of the encapsulated object and the stability of the preparation. For example, for the encapsulated object retinol, which has good solubility in alcohol but poor solubility in water, if the water and the alcohol composition are first mixed into an alcohol solution, the following defects may occur: the addition of water will reduce the solubility of retinol in the alcohol solution, so that retinol precipitates during the mixing process, which is not conducive to being encapsulated in the ester capsule material during the hydration process. At the same time, the mixing of the aqueous solution and the alcohol composition into an alcohol solution is not easy to make the encapsulated object fully contact with the ester capsule material, thereby reducing the encapsulation efficiency and the uniformity of the alcohol homogeneous liquid preparation.

[0091] 5. Monoolein has a lipid bilayer and can form a specific lipid vesicle structure through hydration treatment, but it requires a higher hydration temperature. Poloxamer 407 is an amphiphilic block copolymer that can interact with the lipid bilayer of monoolein to enhance stability. Therefore, adding poloxamer 407 and monoolein to mix into a specific ester capsule material, and implementing subsequent hydration through the specific ester capsule material can reduce the dependence of the preparation of ethosomes on monoolein, which is conducive to lowering the hydration temperature.

[0092] 6. The addition of poloxamer, as a surfactant, can interact with the lipid bilayer to form a protective film and enhance the stability of the ethosome.

[0093] 7. Poloxamer has good solubilizing properties, which can help water-soluble encapsulated substances to be better dispersed and dissolved in lipid vesicle structures, thereby improving the stability and bioavailability of the encapsulated substances.

[0094] 8. The lipid vesicle structure is similar to that of human epithelial cells, has greater bioadhesion and good tissue compatibility, increases drug retention in the skin, especially in the dermis, and can form a higher concentration drug reservoir; therefore, the ethosomes of the embodiments of the present application can play a role by better carrying the encapsulated drugs into the skin and forming a drug reservoir in the skin.

[0095] 9. The alcohol bodies prepared according to the method of the embodiments of the present application have a smaller particle size, which is beneficial to improving the transmission efficiency and bioavailability of the encapsulated substances.

[0096] In some embodiments, S100, mixing glyceryl monooleate and poloxamer 407 at a first temperature, comprises:

[0097] Glyceryl monooleate and poloxamer 407 are stirred and mixed at a first temperature and a predetermined rotation speed; wherein the predetermined rotation speed is 300-1000 rpm / min, optionally, 500 rpm / min.

[0098] In some embodiments, heating and melting glyceryl monooleate and poloxamer 407 at a first temperature to a liquid state is more conducive to achieving sufficient mixing. The reason is that after heating and melting, glyceryl monooleate and poloxamer 407 are transformed from an ordered solid structure to a disordered liquid structure, and the molten liquid structure has enhanced fluidity compared to the solid structure (referring to directly mixing GMO and F127). In the mixing process, applying a smaller mechanical action can make the mixing of glyceryl monooleate and poloxamer 407 more uniform.

[0099] It can be understood that the uniform distribution of GMO and F127 in the capsule material after mixing helps to form a stable lipid bilayer in the process of preparing ethosomes, which has both fluidity and flexibility, thereby facilitating efficient encapsulation and protection of the encapsulated molecules; during the storage and use of the ethosomes, it is also conducive to effectively maintaining the integrity of the structure, reducing the degradation and leakage of the encapsulated material, and allowing the encapsulated material to function better and more stably.

[0100] In some embodiments, step S100 may include: heating and melting monoolein and poloxamer 407 independently into liquids and then heating and mixing at a first temperature to obtain a liquid oil ester mixture containing monoolein and poloxamer 407.

[0101] In some other embodiments, step S100 may include: heating and mixing a solid mixture of glyceryl monooleate and poloxamer 407 at a first temperature, and then cooling the mixture to obtain an ester capsule material.

[0102] In some embodiments, S200, obtaining an alcohol mixture containing short-chain alcohols and encapsulated substances comprises:

[0103] The encapsulated material is dissolved in the short-chain alcohol to obtain an alcohol mixture containing the short-chain alcohol and the encapsulated material.

[0104] In some embodiments, S200, obtaining an alcohol mixture containing short-chain alcohols and encapsulated substances, comprises:

[0105] The alcohol mixture and the ester capsule material are stirred together for a first time to obtain an ester alcohol mixture; wherein the first time is 1 to 60 minutes, optionally 5 to 30 minutes, optionally 30 minutes.

[0106] In some embodiments, S300, mixing the alcohol mixture containing the short-chain alcohol and the encapsulated object with the ester capsule material to obtain an ester alcohol mixture, comprises:

[0107] The alcohol mixture and the ester capsule material are stirred together for a second time to obtain an ester alcohol mixture; wherein the second time is 1 to 60 minutes, optionally 5 to 30 minutes, optionally 30 minutes.

[0108] In some embodiments, S400, adding water to the ester alcohol mixture to perform hydration treatment to obtain a hydrated liquid comprises:

[0109] Slowly drop water into the ester alcohol mixture and stir together for a third time to obtain a hydrated liquid; wherein the third time is 1 to 120 minutes, optionally 30 to 90 minutes, optionally 60 minutes.

[0110] In some embodiments, the hydration liquid is homogenized to obtain an alcohol body homogenous liquid containing alcohol bodies, comprising:

[0111] The hydrated liquid is homogenized for a fourth time at a predetermined homogenization pressure to obtain an alcoholic body homogenized liquid containing alcoholic bodies; wherein the fourth time is 1 to 20 minutes, optionally 3 to 10 minutes, optionally 5 minutes.

[0112] Homogenization pressure is also an important factor affecting the properties of the alcohol body, especially the improvement of particle size. In some embodiments, the homogenization pressure is 600-1000 bar; under this condition, the PDI of the alcohol body is basically less than 0.2, and the particle size distribution is uniform. In addition, a homogenization pressure of 1000 bar is conducive to further reducing the particle size of the alcohol body.

[0113] As can be seen from the foregoing, in some embodiments, the solvent system for preparing ethosomes includes at least two solvents, namely, a short-chain alcohol and water in an aqueous solution.

[0114] In some embodiments, the concentration of monoolein, that is, the ratio of the mass of monoolein to the volume of the solvent system is 1-30 mg / mL, which is conducive to the formation of alcohol bodies with better performance, and the particle size and PDI can be maintained in a smaller range.

[0115] In some embodiments, increasing the concentration of monoolein in the ester capsule material can enhance the interaction between molecules in the system, improve the stability of the alcohol body, and prevent the oxidative decomposition of the active ingredient.

[0116] In some embodiments, the mass ratio of glyceryl monooleate to poloxamer 407 is 7:1 to 17:1; alternatively, 7.3:1 to 16.6:1; alternatively, 10:1.

[0117] In some embodiments, changing the amount and type of poloxamer added can adjust the pore size and permeability of the lipid bilayer, affect the structure and pore properties of the lipid vesicle structure, and thus affect the release rate and path of the solute (eg, encapsulated substance).

[0118] In some embodiments, the short-chain alcohol is selected from any one of a monohydric alcohol and a dihydric alcohol. It should be noted that, unless otherwise stated, in the embodiments of the present application, a monohydric alcohol refers to a monomeric alcohol, and a dihydric alcohol refers to a complex alcohol formed by a combination of two monomeric alcohols. Exemplarily, common monohydric alcohols used to prepare alcohol bodies include: propylene glycol (PG), dipropylene glycol (DPG), ethylene glycol (EG), etc.; dihydric alcohols can be selected from any one of ethanol-propylene glycol (EA-PG), ethanol-dipropylene glycol (EA-DPG), and propylene glycol-dipropylene glycol (PG-DPG); wherein PG-DPG is a complex alcohol composed of PG and DPG, EA-DPG is a complex alcohol composed of EA and DPG, and EA-PG is a complex alcohol composed of EA and PG.

[0119] In at least one embodiment, the short-chain alcohol includes one or more of propylene glycol (PG), dipropylene glycol (DPG), and ethylene glycol (EG).

[0120] In some embodiments, when propylene glycol and dipropylene glycol having moisturizing effect are added to the formula of the alcohol body, the dryness of the skin can be reduced to a certain extent when the alcohol body is applied to the skin.

[0121] In some embodiments, the short-chain alcohol is composed of propylene glycol and dipropylene glycol. The type of alcohol phase (i.e., short-chain alcohol) can affect the encapsulation of the encapsulated object to a certain extent. When dipropylene glycol and propylene glycol are used in combination, the viscosity and drug loading concentration of the alcohol body homogeneous liquid can be significantly increased, and the uniformity of the distribution of the alcohol body particles can be improved.

[0122] Optionally, when the short-chain alcohol is composed of propylene glycol and dipropylene glycol, the volume ratio of propylene glycol to dipropylene glycol is (3:7) to (7:3); this is beneficial to reducing the particle size and increasing the drug loading concentration; further optionally, the volume ratio of propylene glycol to dipropylene glycol is 5:5.

[0123] In certain embodiments, the use of short-chain alcohols at an appropriate concentration can appropriately increase the fluidity in the lipid membrane of the ethosome. When the ethosome is used on the skin, a higher concentration of short-chain alcohols can not only increase the solubility of the encapsulated material in the stratum corneum of the skin, but also increase the fluidity in the stratum corneum and the flexibility of the body membrane, and have a strong penetration-promoting effect.

[0124] In some embodiments, the alcohol content in the alcoholic homogenate, that is, the content of short-chain alcohols, is 10-30%.

[0125] In some embodiments, the content of the short-chain alcohol is the volume fraction of the short-chain alcohol in the solvent system, that is, the volume ratio of the short-chain alcohol to the solvent system, which is 10-30%. When the alcohol content is 10-30%, the particle size of the alcohol body is small, the dispersion is uniform, and the drug loading effect is good.

[0126] In some embodiments, the aqueous solution includes water and a stabilizer dissolved in the water, that is, the aqueous solution is a stabilizer aqueous solution.

[0127] In some embodiments, obtaining the aqueous solution includes: dissolving a stabilizer in water, and dissolving by ultrasonication to obtain an aqueous solution.

[0128] In some embodiments, the mass ratio of glyceryl monooleate to the stabilizer is 3:1.

[0129] In some embodiments, the stabilizer includes one or more of ergothioneine (EGT), dipotassium glycyrrhizinate (DG), vitamin E polyethylene glycol succinate (D-α-tocopherylpolyethyleneglycol1000succinate, TPGS) and tocopheryl acetate (TOA).

[0130] In some embodiments, when TPGS is used as a stabilizer, the particle size of the ethosome can be significantly reduced, and the obtained ethosome homogeneous liquid preparation is clear, transparent, and uniformly dispersed. The reason may be that TPGS-modified ethosomes can enhance the stability of the water-soluble components and fat-soluble components in the continuous dual phase and improve the smoothness and transparency of the preparation.

[0131] In some embodiments, when TPGS is used as a stabilizer, not only can the ethosomes have a more uniform and smaller particle size, but also the stability of the ethosomes and the transdermal permeability to the skin can be improved.

[0132] Retinol penetrates the epidermis and is converted into retinoic acid, which has a repairing effect on skin cells. However, it is easy to stimulate the skin to produce hypersensitivity reactions, resulting in dry skin and peeling. In some embodiments, in the ethosomes provided in the embodiments of the present application, when the encapsulated material is selected from retinol and the stabilizer is selected from TPGS, TPGS can assist the dissolution of retinol in the alcohol-water system, which is beneficial to reduce the irritation caused by the large-scale release of retinol drugs at the same time.

[0133] In some embodiments, when tocopherol acetate is used as a stabilizer, the particle size of the prepared ethosomes is larger and the system is more turbid.

[0134] In some embodiments, when any one of ergothioneine and dipotassium glycyrrhizinate is used as a stabilizer, the prepared ethosome system has good dispersion uniformity.

[0135] In some embodiments, the mass ratio of the encapsulated material to glyceryl monooleate (i.e., drug-lipid ratio) is 1:(5-15); optionally, 1:(5-10); the alcohol body has a small particle size, is evenly dispersed, has a good drug loading effect, and has good stability.

[0136] Optionally, the drug-to-lipid ratio is 1:7.5.

[0137] In some embodiments, the concentration of the encapsulated substance, that is, the ratio of the mass of the encapsulated substance to the volume of the solvent system, is 1-4 mg / mL.

[0138] In some embodiments, the encapsulated material includes one or more of retinol, niacinamide, and ferulic acid.

[0139] In some embodiments, the ester capsule material further includes a first antioxidant. Accordingly, step S100 includes: heating and mixing glyceryl monooleate, poloxamer 407, and the first antioxidant at a first temperature, and then cooling to a second temperature to obtain the ester capsule material.

[0140] In some embodiments, the first antioxidant is selected from pentaerythritol tetrakis (di-tert-butylhydroxyoxycinnamate), which is beneficial to improve the storage stability of the encapsulated material.

[0141] In some embodiments, the mass ratio of the first antioxidant to the monoolein is (0.001-0.5):1.

[0142] In some embodiments, the alcohol mixture further includes a second antioxidant, and step S200 includes: dissolving the encapsulated material and the second antioxidant in a short-chain alcohol to obtain an alcohol mixture containing the short-chain alcohol and the encapsulated material.

[0143] In some embodiments, the second antioxidant is selected from tocopherol acetate, which is beneficial to improve the storage stability of the encapsulated material.

[0144] In some embodiments, the mass ratio of the second antioxidant to the encapsulated material is (0.001-2):1.

[0145] Particle size and PDI are very relevant features in the characterization of ethosomes, and are also a key factor affecting transdermal drug delivery. For local skin delivery, the particle size of nanoparticles as ideal drug carriers is usually between 20 and 200 nm. At this particle size, nanoparticles can better enter the living epidermis through the intercellular permeation pathway to play a role. In at least one embodiment, the nanoscale ethosome homogenate prepared in the embodiment of the present application has a particle size of less than 200 nm and a PDI of about 0.3, indicating that in the ethosome homogenate, particles less than 200 nm have a very wide size distribution.

[0146] In some embodiments, the preparation method mainly adopts the injection method to prepare the ethosomes, wherein the capsule material (containing liquid GMO and F127) and the diol solution containing retinol are evenly mixed as the lipid phase, and the aqueous solution containing TPGS is added to the lipid phase at room temperature to obtain a retinol ethosome suspension, and then the particle size of the ethosomes is uniformed by a high-pressure homogenization process to obtain an ethosome homogenous liquid.

[0147] The ethosomes in the embodiments of the present application are formed on the GMO / H2O system. In some embodiments, when the retinol drug is encapsulated in the GMO / H2O system, it has a sustained release effect, which can reduce the severe skin irritation caused by the sudden release of the drug. Compared with the free retinol drug, it shows less irritation to the skin and can be used to help build better skin tolerance.

[0148] <Ethanol body>

[0149] In a second aspect, the embodiments of the present application further provide an alcohol body loaded with an encapsulated object, wherein the alcohol body loaded with an encapsulated object is prepared according to the preparation method of the alcohol body in the above embodiments.

[0150] The ethosomes of the embodiments of the present application are prepared based on the ester capsule material containing GMO and have a multi-capsule structure, which can improve the carrying capacity of water-insoluble encapsulated materials and improve the bioavailability of the encapsulated materials.

[0151] In addition, the alcohol body of the embodiment of the present application can provide a stable hydrophobic packaging environment and improve stability.

[0152] In some embodiments, the ethosomes of the embodiments of the present application can also form a sustained-release system to prevent sudden release of the encapsulated drug.

[0153] In some embodiments, the ethosomes of the embodiments of the present application can increase transdermal absorption and thus enhance the efficacy of the active ingredient.

[0154] In some embodiments, the ethosomes encapsulate retinol and can have an excellent encapsulation effect on retinol.

[0155] Retinol, also known as retinol, retinin, and vitamin A, is one of the manifestations of vitamin A. Retinol has many functions, such as inhibiting the breakage of collagen fibers, stimulating fibroblasts to synthesize collagen, thereby increasing skin elasticity and achieving anti-aging effects. In addition, retinol is also the most commonly used retinoid compound ingredient in cosmetics. Many studies have shown that retinol can continuously improve skin aging, reduce fine lines and wrinkles, and enhance skin firmness and elasticity. However, retinol is very unstable in chemical properties and is easily degraded when exposed to visible light, ultraviolet light, and oxygen, making it difficult to be incorporated into preparations for a long time. The method of incorporating lipids to encapsulate retinol through retinol liposomes has been proven to improve the stability of retinol in the system. However, the liquid phospholipids of retinol liposomes are prone to irreversible oxidative degradation, liposome vesicles are prone to aggregation and sedimentation, and the embedded active substances are prone to leakage, which greatly limits its application in the field of cosmetics. In addition, microencapsulation technology has also been applied to the encapsulation of retinol, but it is necessary to overcome the problems of difficult to control the thickness of the microcapsule wall and high cost. In some embodiments, the ethosomes of the present application can stably encapsulate retinol and are easy to prepare. Therefore, the aforementioned defects can be overcome.

[0156] In some embodiments, when the ethosome acts on the skin, the synergistic effect of lipid vesicles, short-chain alcohols and skin can greatly improve the transdermal performance of retinol, which is more conducive to the retention of retinol in the skin, thereby effectively exerting its efficacy.

[0157] Traditional ethosomes are generally formed by spontaneous formation of phospholipid vesicles by phospholipids in a certain concentration of short-chain alcohols, but they have poor stability, are mainly single-vesicle structures, and the drug loading capacity still needs to be improved, and the cost of the phospholipid materials used is relatively high. The embodiment of the present application is a multi-vesicle structure composed of monoolein and poloxamer, and the lipid membrane is softened by short-chain alcohols, which can improve transdermal permeability.

[0158] <Uses of Ethosomal Forms>

[0159] It can be understood that based on the aforementioned preparation method of the alcohol body, the present embodiment also provides an alcohol body. On this basis, the present embodiment further provides the following uses:

[0160] In some embodiments, the present application also provides a use of an ethosome in preparing a carrier containing an encapsulated object. For example, the ethosome or a solution containing the ethosome can be used as a carrier containing an encapsulated object.

[0161] In some embodiments, the carrier containing the encapsulated object can be used as or for preparing medicines, cosmetics, or skin care products.

[0162] <Skin care products>

[0163] The present application also provides a skin care product, comprising the ethosome described in the above embodiment.

[0164] <Preparation Example of Ethosomal Form>

[0165] The ethosomes of the following examples were prepared.

[0166] Example 1

[0167] The preparation method of Example 1 comprises the following steps:

[0168] Step S101, weighing 600 mg of monoolein and 60 mg of poloxamer 407, placing them on a magnetic stirrer, stirring them at a first temperature (the first temperature is 60° C.) and a predetermined speed (the predetermined speed is 500 rpm / min) until they are fully mixed, and cooling them to a second temperature (the second temperature is 30° C.) to obtain a lipid capsule material;

[0169] Step S102: prepare a short-chain alcohol, wherein the short-chain alcohol is a short-chain alcohol composed of PG and DPG in a volume ratio of 5:5.

[0170] Step S103, accurately weighing 80 mg of an encapsulated substance (the encapsulated substance is selected from retinol) and 6 mL of a short-chain alcohol; dissolving 80 mg of retinol in 6 mL of the short-chain alcohol, and dissolving by ultrasonication to obtain an alcohol mixture;

[0171] Step S104, stirring the alcohol mixture in step S103 and the ester capsule material in step S101 for 30 min to obtain an ester alcohol mixture (the ester alcohol mixture is a retinol suspension);

[0172] Step S105, accurately weigh 200 mg of a stabilizer (the stabilizer is selected from TPGS) and 14 mL of water (the water is selected from ultrapure water), dissolve 200 mg of TPGS in 14 mL of ultrapure water, and dissolve by ultrasonication to obtain an aqueous solution containing the stabilizer (as the aqueous phase);

[0173] Step S106, slowly dropwise adding the aqueous solution of step S105 into the ester alcohol mixture of step S104 and stirring together for 60 minutes to obtain a hydrated solution;

[0174] Step S107, high pressure homogenizing the hydrated liquid in step S106 at a pressure of 1000 bar for 5 min to obtain an alcoholic body homogenous liquid containing alcoholic bodies;

[0175] The volume of the solvent system used to prepare the ethosome, i.e., the sum of the volumes of water and short-chain alcohol, is 20 mL;

[0176] The content (volume fraction) of short-chain alcohol in the solvent system is 30%;

[0177] The concentration of monoolein GMO is 30 mg / mL;

[0178] The mass ratio of monoolein to poloxamer 407 was 10:1;

[0179] The concentration of the stabilizer TPGS was 10 mg / mL;

[0180] The concentration of retinol is 4 mg / mL.

[0181] The preparation conditions of Example 1 can also be further referred to Table 1.

[0182] Table 1. Preparation conditions of Examples 1-12

[0183]

[0184] Embodiment 2-6

[0185] The preparation methods of Examples 2-6 are basically the same as those of Example 1. The same points can be implemented with reference to Example 1. The main difference lies in the different types of short-chain alcohols (the short-chain alcohol in Example 1 is a mixed alcohol composed of PG and DPG). See Table 1 for details.

[0186] Embodiment 7-8

[0187] The preparation methods of Examples 7-8 are basically the same as those of Example 1. The same points can be implemented with reference to Example 1. The main difference is the homogenization pressure (the homogenization pressure of Example 1 is 1000 bar). See Table 1 for details.

[0188] Examples 9-10

[0189] The preparation methods of Examples 9-10 are basically the same as those of Example 1. The same points can be implemented with reference to Example 1. The main difference is that the volume ratio of PG and DPG in the short-chain alcohol is different (the volume ratio of the two alcohols in the alcohol phase of Example 1 is 5:5). See Table 1 for details.

[0190] Examples 11-13

[0191] The preparation methods of Examples 11-13 are basically the same as those of Example 1. The same points can be implemented with reference to Example 1. The main difference lies in the different types of stabilizers (the stabilizer in Example 1 is TPGS), see Table 1-2 for details.

[0192] Table 2. Preparation conditions of Examples 13-21

[0193]

[0194] Examples 14-16

[0195] The preparation methods of Examples 14-16 are basically the same as those of Example 1. The same points can be implemented with reference to Example 1. The main difference lies in the different amounts of retinol (i.e., retinol concentrations) (the amount of retinol in Example 1 is 4 mg / mL). See Table 2 for details.

[0196] Examples 17-19

[0197] The preparation methods of Examples 17-19 are basically the same as those of Example 1. The same points can be implemented with reference to Example 1. The main difference is that the concentration of monoolein is different (the concentration of monoolein in Example 1 is 30 mg / mL). See Table 2 for details.

[0198] Examples 20-21

[0199] The preparation methods of Examples 20-21 are basically the same as those of Example 1. The same points can be implemented with reference to Example 1, as shown in Table 2. The main difference is that the alcohol content (content of short-chain alcohol) of the alcohol homogenate liquid is different (the alcohol content of the alcohol homogenate liquid of Example 1 is 30%). More specifically, it is as follows:

[0200] In Example 20, the volume of the short-chain alcohol is 4 mL, the volume of water is 16 mL, and the alcohol content is 20%;

[0201] In Example 21, the volume of the short-chain alcohol is 2 mL, the volume of water is 18 mL, and the alcohol content is 10%.

[0202] Examples 22-24

[0203] The preparation methods of Examples 22-24 are basically the same as those of Example 1, and the mass of monoolein is 600 mg. The same points can be implemented with reference to Example 1. The main difference is that the mass ratio of retinol to monoolein (i.e., the drug-to-lipid ratio) is different (Example 1 is 1:7.5); specifically, the drug-to-lipid ratio of Example 22 is 1:5, the drug-to-lipid ratio of Example 23 is 1:10, and the drug-to-lipid ratio of Example 24 is 1:15.

[0204] Embodiment 25

[0205] The preparation method of Example 25 comprises the following steps:

[0206] Step S201, accurately weighing 300 mg of solid monoolein, 30 mg of solid poloxamer 407, and 14 mg of a first antioxidant [the first antioxidant is selected from pentaerythritol tetra(di-tert-butylhydroxyoxycinnamate) ester], placing them on a magnetic stirrer, stirring at a first temperature of 95° C. and a speed of 500 rpm / min until fully mixed, and cooling to a second temperature of 30° C. to obtain an ester capsule material;

[0207] Step S202, taking 6 mL of short-chain alcohol, wherein the short-chain alcohol is prepared by mixing PG and DPG in a volume ratio of 5:5;

[0208] Step S203, accurately weighing 20 mg of retinol and 40 mg of a second antioxidant (the second antioxidant is selected from tocopherol acetate), dissolving them in 6 mL of a short-chain alcohol, and ultrasonically dissolving them to obtain an alcohol mixture;

[0209] Step S204, stirring the alcohol mixture in step S203 and the ester capsule material in step S201 for 30 min to obtain an ester alcohol mixture (retinol suspension);

[0210] Step S205, accurately weigh 200 mg of a stabilizer (the stabilizer is selected from TPGS) and dissolve it in 14 mL of ultrapure water, and dissolve it by ultrasonication to obtain an aqueous solution (as the aqueous phase);

[0211] Step S206, slowly dropwise adding the aqueous solution of step S205 into the ester alcohol mixture (retinol suspension) of step S204 and stirring together for 60 minutes to obtain a hydrated solution;

[0212] Step S207, high pressure homogenizing the hydrated liquid in step S206 at a pressure of 1000 bar for 5 min to obtain an alcohol body homogenous liquid containing alcohol bodies;

[0213] The volume of the solvent system used to prepare the ethosome, i.e., the sum of the volumes of water and short-chain alcohol, is 20 mL;

[0214] The concentration of monoolein was 15 mg / mL.

[0215] The concentration of poloxamer 407 is 1.5 mg / mL.

[0216] The concentration of pentaerythritol tetra(di-tert-butylhydroxyoxycinnamate) ester was 0.7 mg / mL;

[0217] The concentration of retinol is 1 mg / mL.

[0218] The concentration of tocopherol acetate was 2 mg / mL.

[0219] The concentration of the stabilizer was 10 mg / mL.

[0220] <Comparative Example of Preparation of Ethobody>

[0221] Comparative Example 1: Preparation of unilamellar retinol liposomes

[0222] Unilamellar retinol liposomes were prepared by thin film dispersion method: 30 mg / mL soybean lecithin, 3 mg / mL cholesterol and 4 mg / mL retinol were weighed and dissolved in chloroform-methanol organic mixed solvent (v:v=2:1), and the mixed solution was rotary evaporated under reduced pressure at 30°C to remove the organic mixed solvent. After a uniform thin film was formed, ultrapure water was added and hydrated for 3 h, and ice bath probe ultrasound was performed for 10 min to obtain the retinol-loaded particle solution of unilamellar retinol liposomes.

[0223] Comparative Example 2: Preparation of retinol liposomes containing poloxamer 407

[0224] Retinol liposomes were prepared by thin film dispersion method: 30 mg / mL soybean lecithin, 3 mg / mL cholesterol and 4 mg / mL retinol were weighed and dissolved in chloroform-methanol organic mixed solvent (v:v=2:1), and the mixed solution was evaporated under reduced pressure at 30°C to remove the organic mixed solvent. After a uniform thin film was formed, an ultrapure aqueous solution containing 3 mg / mL poloxamer 407 (obtained by dissolving poloxamer 407 in ultrapure water) was added, hydrated for 3 h, and subjected to ice bath probe ultrasound for 10 min to obtain a retinol-loaded particle solution containing retinol liposomes.

[0225] Comparative Example 3: Preparation of Ordinary Retinol Ethosomes

[0226] Preparation of ordinary retinol ethosomes by ethanol injection method:

[0227] Weigh 30 mg / mL soybean lecithin, 3 mg / mL cholesterol and 4 mg / mL retinol dissolved in 3 mL of glycol (propylene glycol and dipropylene glycol) solution and mix well in a magnetic stirrer at 30°C as the alcohol phase;

[0228] Pipette 7 mL of ultrapure water into a syringe as the aqueous phase;

[0229] The aqueous phase in the syringe was slowly added dropwise to the alcohol phase, hydrated for 60 min, and subjected to ice bath probe ultrasound for 10 min to obtain a retinol-loaded particle solution containing ordinary retinol ethosomes.

[0230] Comparative Example 4: Preparation of retinol ethosomes containing poloxamer 407

[0231] Preparation of retinol ethosomes by ethanol injection method:

[0232] Weigh 30 mg / mL soybean lecithin, 3 mg / mL cholesterol and 4 mg / mL retinol dissolved in 3 mL of glycol (propylene glycol and dipropylene glycol) solution and mix well in a magnetic stirrer at 30°C as the alcohol phase;

[0233] Dissolve 3 mg / mL poloxamer 407 in 7 mL ultrapure water as the aqueous phase and draw into a syringe;

[0234] The aqueous phase in the syringe was slowly added dropwise to the alcohol phase, hydrated for 60 min, and subjected to ice bath probe ultrasound for 10 min to obtain a retinol-loaded particle solution containing retinol ethosomes.

[0235] <Preparation Results and Analysis>

[0236] According to the results of Examples 1-25, homogenous ethosome liquids containing retinol-carrying particles (ethosomes) can be prepared. In the process of preparing ethosomes, especially in the hydration process, the temperature environment of the encapsulated material (retinol) does not exceed 30 degrees, indicating that the preparation method of the embodiment of the present application can prevent the encapsulated material from being damaged by high temperature. That is, the purpose of preparing ethosomes can be achieved on the basis of low hydration temperature.

[0237] <Experimental example>

[0238] In order to study the further properties of the ethosomes of the examples of the present application, the homogenous solutions of the ethosomes prepared in Examples 1-25 and the retinol-loaded particle solutions prepared in Comparative Examples 1-4 are taken as the test solutions, respectively. The detection index may involve at least one of the following: particle size, PDI, morphology (including one or more of ordinary appearance, Tyndall appearance and structural morphology), retinol encapsulation efficiency, retinol loading concentration and stability of the retinol-loaded particles in the test solution. Specific detection is detailed in the experimental examples below.

[0239] If there is no special statement, when it comes to the detection of the above indicators, it can be implemented according to the following measurement standards:

[0240] 1. Measurement of particle size and PDI

[0241] The measuring method includes: taking the test solution, diluting it, and measuring the particle size and polydispersity index (PDI) of the retinol-carrying particles using a Malvern particle size detector.

[0242] Regarding dilution, specifically, 100 μL of the test solution can be added to 900 μL of deionized water and gently shaken to ensure that the two are fully mixed. Dilution helps reduce particle aggregation, particle interactions and multiple scattering, ensuring that each particle scatters the light beam independently, thereby improving the accuracy of the measurement results.

[0243] 2. Measurement of general appearance

[0244] Directly observe the appearance of the test liquid and take photos for record.

[0245] 3. Measurement of Tyndall's appearance

[0246] After the Tyndall test is performed on the test liquid, the appearance of the test liquid is directly observed and photographed for record.

[0247] 4. Measurement of electron microscope structure morphology

[0248] The measurement method includes: using a transmission electron microscope to observe the structural morphology of retinol-loaded particles in the test liquid. Specifically, 7 μL of the test liquid is dropped on a 400-mesh carbon support film copper grid. After natural drying, 1% phosphotungstic acid solution is added for staining for 3 minutes, and then the morphology is observed under a transmission electron microscope.

[0249] 5. Measurement of retinol encapsulation efficiency and drug loading concentration

[0250] Measurement methods include:

[0251] (1) Obtaining the total mass of retinol in the test solution: dilute the test solution 10 times with methanol, demulsify by ultrasonication for 30 min, filter through a 0.22 μm microporous filter membrane to obtain a filtrate, and detect the total mass of retinol in the filtrate system using an ELISA reader.

[0252] (2) Separate unencapsulated retinol by ultrafiltration centrifugation: Take 0.5 mL of the test solution, dilute it twice with a solution of corresponding alcohol content (an aqueous solution containing the same type and volume of alcohol as the test solution), centrifuge it in an ultrafiltration centrifuge tube (30 KDa) at 3500 r / min and 4°C for 30 min, take the lower filtrate and use an ELISA instrument to detect the mass of retinol not encapsulated in the retinol-loaded particles.

[0253] (3) Calculate the encapsulation efficiency (EE) and drug loading concentration (DC) of the test solution according to the following formula:

[0254] EE (%) = (W total mass - W free) / W total mass × 100% (Formula 1);

[0255] DC (%) = (W total mass - W free) / V total volume × 100% (Formula 2).

[0256] W free is the mass of retinol in the test solution that is not encapsulated in the retinol-loaded particles;

[0257] W total mass is the total mass of retinol in the test solution;

[0258] V total volume is the total volume of the formula when preparing the test solution, which is basically equal to the volume of all solvents (total solvent) in the formula. Therefore, the volume of the total solvent is taken as V total volume.

[0259] 6. Retinol retention rate

[0260] When the test solution contains retinol-loaded particles, the retinol encapsulated in the retinol-loaded particles may be released over time to become free retinol, that is, the retinol retention rate may change over time.

[0261] Therefore, it can be understood that the retinol retention rate represents the ratio of the final remaining retinol content encapsulated in the retinol-carrying particles in the test solution to the initial retinol content over a period of time. Specifically, the calculation formula of the retinol retention rate can be expressed as:

[0262] Retinol retention rate (%) = (final retinol content / initial retinol content) × 100%.

[0263] If not otherwise stated, the initial retinol content represents the drug loading concentration at the initial time of the test, and the final retinol content represents the drug loading concentration at the terminal time of the test.

[0264] For example, when calculating the retinol retention rate of the ethanosome homogenate after storage for 7 days in Experimental Example 6, the initial retinol content may be the drug loading concentration on the 0th day, and the final retinol content may be the drug loading concentration on the 7th day.

[0265] 7. Storage stability testing

[0266] The test solution is stored at different temperatures (e.g., 4±1°C, 20±1°C, 50±1°C, etc.) in the dark, and one or more of its particle size, PDI, retinol encapsulation efficiency, retinol loading concentration and retinol retention rate are measured at predetermined time (e.g., 0th, 2nd, 7th day, etc.) to investigate the stability of the test solution.

[0267] 8. Other instructions

[0268] Unless otherwise stated, the test temperature involved in the following experimental examples is room temperature. Room temperature, unless otherwise stated, is 20±1℃.

[0269] In addition, when it comes to the measurement of indicators such as particle size (unit nm), PDI, drug loading concentration (unit mg / mL), encapsulation efficiency (unit %), and retinol retention rate (unit %), parallel detection is performed n times, that is, in the detection of each test liquid, n parallel experimental groups are set for each indicator to be detected, and n sets of parallel data are recorded. Exemplarily, when detecting the particle size of the ethanol body homogenous liquid of Example 1, if n=3 is set, 3 portions of ethanol body homogenous liquid are prepared as 3 parallel groups of test liquids according to the method of Example 1, and the particle size data of the 3 test liquids are measured respectively as 3 sets of parallel data, and then the corresponding particle size measurement results are obtained according to the 3 sets of parallel data.

[0270] It should also be understood that, unless otherwise stated, when time involves d, it refers to days; Mean refers to the mean value; SD refers to the standard deviation (Standard Deviation), that is, the variance; "Mean size" represents the average size of the particle size; "ns" represents p>0.05, indicating no statistical difference; "*" represents p<0.05, indicating a statistical difference; "**" represents p<0.01, indicating a significant statistical difference; "***" represents p<0.001, indicating a very significant statistical difference; "****" represents p<0.0001, indicating an extremely significant statistical difference.

[0271] <Experimental Example 1: Effect of Alcohol Phase Type on Ethosome>

[0272] It can be seen from the above preparation examples that the main difference between Examples 1-6 is that the types of alcohol phases (i.e., short-chain alcohol types) in the formulations are different. Therefore, the alcohol phases of Examples 1-6 will be compared below to further study the effect of the alcohol phase on the alcohol phase.

[0273] The experimental method includes: taking the homogenous solution of the ethosome prepared in Examples 1-6 as the test solution, detecting the appearance morphology, particle size, PDI, drug loading concentration and encapsulation efficiency corresponding to the ethosome at room temperature (20±1°C), and recording the results; wherein the number of parallel groups n is 3.

[0274] Results of Experimental Example 1:

[0275] For the appearance morphology, the results are shown in Table 3 and Figure 1 .

[0276] Figure 1 The following are comparison pictures of the normal appearance and Tyndall appearance of alcohol homogenate prepared with different alcohol phases. The first row of pictures represents the appearance before the Tyndall test, and the second row of pictures represents the appearance after the Tyndall test.

[0277] Effect diagram, (A) represents the alcoholic body homogeneous liquid whose alcohol phase is EA, (B) represents the alcoholic body homogeneous liquid whose alcohol phase is PG; (C) represents the alcoholic body homogeneous liquid whose alcohol phase is DPG, (D) represents the alcoholic body homogeneous liquid whose alcohol phase is EA-PG, (E) represents the alcoholic body homogeneous liquid whose alcohol phase is EA-DPG, (F) represents the alcoholic body homogeneous liquid whose alcohol phase is PG-DPG.

[0278] Table 3. Morphology, particle size, PDI, encapsulation efficiency and drug loading concentration of alcoholic homogenates of different alcohol phases in Examples 1 to 6

[0279]

[0280] For indicators such as particle size (unit: nm), PDI, encapsulation efficiency (unit: %) and drug loading concentration (unit: mg / mL), the test results (Mean±sd) of each indicator were determined according to the mean (Mean) and standard deviation (SD) of the corresponding three groups of parallel data, as shown in Table 3.

[0281] For indicators such as particle size and PDI, a bar chart is drawn based on the corresponding test results (Mean±sd). For details, see Figure 2 . Figure 2 A schematic diagram comparing the particle size and PDI of alcohol bodies prepared from different alcohol phases.

[0282] For drug loading concentration, a bar chart is also drawn based on the mean of the corresponding three sets of parallel data. Figure 3 . Figure 3 A schematic diagram of the drug loading concentration of ethosomes prepared according to different alcohol phases.

[0283] Result analysis of Experimental Example 1:

[0284] 1. According to Figure 1 As can be seen from Table 3, the homogeneous alcoholic body liquid prepared in Examples 1-6 has a strong Tyndall effect, strong colloidal properties, and contains dispersed particles inside, that is, the alcoholic body is granular.

[0285] 2. According to Figure 2-3 As shown in Table 3, among the three monohydric alcohols: the ethosomes prepared using ethanol (EA) have a smaller particle size (particle size is 69.6±0.2 nm), but the drug loading concentration for retinol (drug loading concentration is 0.095±0.001 mg / mL) is the lowest, which will reduce the encapsulation efficiency (encapsulation efficiency is 50.8±0.7%) and easily cause drug leakage; propylene glycol (PG) has the characteristics of good stability and high viscosity, which can improve the encapsulation efficiency and drug loading concentration of retinol in ethosomes, but the particle size of the obtained ethosomes is larger; dipropylene glycol (DPG) has a strong ability to co-dissolve with fat-soluble components and high viscosity, which can also improve the encapsulation efficiency and drug loading concentration of retinol, and the particle size of the obtained ethosomes can be maintained at a smaller size.

[0286] 3. In summary, the synergistic use of dipropylene glycol and propylene glycol can significantly increase the drug loading concentration of ethosomes, and the particle distribution is more uniform. Therefore, it is more advantageous to use a diol system composed of propylene glycol and dipropylene glycol (PG-DPG) to prepare ethosomes.

[0287] 4. In addition, dipropylene glycol can be used as an ideal solvent in cosmetics. Therefore, when the short-chain alcohol contains dipropylene glycol, the prepared alcohol body is more conducive to the preparation of cosmetics.

[0288] <Experimental Example 2: Effect of Homogenization Pressure on Ethanol Form>

[0289] The main difference between Example 7-8 and Example 1 is the different homogenization pressures (Pressure). Therefore, the influence of different homogenization pressures (unit bar) on the alcohol phase will be analyzed through Experimental Example 2 below.

[0290] The experimental method of Experimental Example 2 includes: taking the homogenous alcohol solution prepared in Examples 1, 7-8 as the test solution, measuring the corresponding appearance morphology, particle size, and PDI at room temperature (20±1° C.), and recording the results.

[0291] Results of Experimental Example 2

[0292] The appearance and morphology of the ethosomes can be seen in Table 4.

[0293] Table 4 Particle size, PDI and morphology of the homogenous alcohol solutions of Examples 1, 7-8

[0294]

[0295] For indicators such as particle size (unit: nm) and PDI, the test results (Mean±SD) of each indicator are determined according to the mean (Mean) and variance (SD) of the corresponding n groups of parallel data, as shown in Table 4; where n=3.

[0296] For indicators such as particle size (unit: nm) and PDI, a bar chart is drawn based on the corresponding test results (Mean±sd). Figure 4 . Figure 4 A comparative diagram of the particle size and PDI of alcohol bodies prepared according to different homogenization pressures, where n=3.

[0297] Result analysis of Experimental Example 2:

[0298] According to the above results, the particle size is maintained in a small range and the PDI is basically less than or equal to 0.204. Therefore, the performance of the alcohol body is good.

[0299] As the homogenization pressure increases, the particle size of the alcohol body gradually decreases, the PDI first decreases and then increases, and the transparency changes from opaque to clear and transparent.

[0300] <Experimental Example 3: Effect of the Volume Ratio of Propylene Glycol to Dipropylene Glycol in Short-Chain Alcohols on Ethanol Form>

[0301] The main difference between Examples 9-10 and Example 1 is that the volume ratio of PG to DPG in the short-chain alcohol is different. Therefore, the effect on the alcohol body will be analyzed by the volume ratio of PG to DPG.

[0302] Method of Experimental Example 3: Take the homogenous alcohol solution prepared in Examples 1 and 9-10 as the test solution, measure the corresponding particle size, PDI and drug loading concentration at room temperature (20±1° C.), and record the results.

[0303] Results of Experiment 3:

[0304] For indicators such as particle size (unit: nm) and PDI, the test results (Mean±SD) of each indicator are determined according to the mean (Mean) and variance (SD) of the corresponding n groups of parallel data, and then a bar chart is drawn according to the test results (Mean±SD). For details, see Figure 5 ; where n=3. Figure 5 It is a comparative schematic diagram of the particle size and PDI of the alcohol body at different volume ratios of propylene glycol to dipropylene glycol in Examples 1, 9-10.

[0305] For drug loading concentration (unit: mg / mL), the test results (Mean±SD) of each indicator were determined according to the mean (Mean) and variance (SD) of the corresponding three groups of parallel data, and then a bar chart was drawn according to the test results (Mean±SD). For details, see Figure 6 ; where n=3. Figure 6 It is a comparative schematic diagram of the drug loading concentration of ethosomes at different volume ratios of propylene glycol to dipropylene glycol in Examples 1, 9-10.

[0306] Result analysis of Experimental Example 3:

[0307] From the above results, it can be seen that in the experimental results of the ethosomes of Examples 1, 9-10, when the volume ratio of propylene glycol to dipropylene glycol is 5:5 (Example 1), the prepared ethosomes have the largest PDI, but the smallest particle size and the highest drug loading concentration.

[0308] <Experimental Example 4: Effect of Stabilizer Type on Ethosome>

[0309] The main difference between Examples 11-13 and Example 1 is the different types of stabilizers. The effect of the type of stabilizer on the alcohol body will be analyzed through Experimental Example 5 below.

[0310] Method of Experimental Example 4: Take the homogenous alcohol solution prepared in Examples 1 and 11-13 as the test solution, and measure the corresponding particle size, PDI, and appearance morphology.

[0311] Results of Experimental Example 4:

[0312] For the appearance morphology, the results are shown in Table 5 and Figure 7 .

[0313] Figure 7 The figures are a comparison of the normal appearance and the Tyndall effect appearance of the ethanol body homogenate prepared with different stabilizers, wherein the first row of pictures represents the normal appearance before the Tyndall test, the second row of pictures represents the Tyndall effect appearance after the Tyndall test, (A) represents the ethanol body homogenate with ergothioneine EGT as the stabilizer, (B) represents the ethanol body homogenate with dipotassium glycyrrhizinate DG as the stabilizer, (C) represents the ethanol body homogenate with vitamin E polyethylene glycol succinate TPGS as the stabilizer, and (D) represents the ethanol body homogenate with tocopherol acetate TOA as the stabilizer.

[0314] Table 5 Particle size, PDI and morphology of the homogenous alcohol solutions of Examples 1, 11-13

[0315]

[0316] For indicators such as particle size (unit: nm) and PDI, the test results (Mean±sd) of each indicator are determined according to the mean (Mean) and variance (SD) of the corresponding n groups of parallel data, as shown in Table 5; n=3.

[0317] For indicators such as particle size (unit: nm) and PDI, a bar chart is drawn based on the corresponding test results (Mean±sd). Figure 8 . Figure 8 This is a comparative diagram of the particle size and PDI of alcohol bodies prepared with different stabilizers.

[0318] Result analysis of Experimental Example 4:

[0319] According to the above results, the particle size and PDI of all alcohol homogenate liquids can be maintained in a small range.

[0320] Except for TOA as a stabilizer, the Tyndall effect of the alcoholic homogeneous liquid is slightly weaker, while other stabilizers can make the alcoholic homogeneous liquid have a stronger Tyndall effect.

[0321] In addition, when TPGS is used as a stabilizer, the particle size of the alcohol body is the smallest and a smaller PDI can be guaranteed.

[0322] <Experimental Example 5: Effect of Retinol Content on Ethosomes>

[0323] The main difference between Examples 14-16 and Example 1 is that the retinol content in the preparation formula is different; the effect of retinol content on ethosomes will be analyzed below by adding retinol concentration (Dosage concentration of retinol, unit: mg / mL) to the formula when preparing ethosome homogenate.

[0324] The method of Experimental Example 5 includes: taking the homogenous alcohol solution prepared in Examples 1 and 14-16 as the test solution, and detecting the corresponding particle size, PDI, Zeta potential and drug loading concentration at room temperature (20±1° C.).

[0325] Results of Experimental Example 5:

[0326] For indicators such as particle size (unit: nm), PDI, and Zeta potential (unit: mv), the detection results (Mean±sd) of each indicator were determined based on the mean (Mean) and variance (SD) of the corresponding n groups of parallel data, as shown in Table 6; n=3.

[0327] Table 6 Particle size, PDI, Zeta potential and drug loading concentration of the alcohol homogenate solutions of Examples 1, 14-16

[0328]

[0329] For indicators such as particle size (unit: nm) and PDI, a bar chart is also drawn based on the test results (Mean±sd). For details, see Fig. 9 ; where n=3. Fig. 9 A schematic diagram comparing the particle size and PDI of ethosomes prepared with different retinol concentrations.

[0330] For drug loading concentration (unit: mg / mL), the test results (Mean±sd) of each indicator were determined according to the mean (Mean) and variance (SD) of the corresponding three groups of parallel data, and then a bar chart was drawn according to the test results (Mean±sd). For details, see Fig.10 ; where n=3. Fig.10 A schematic comparison of the drug loading concentrations of ethosomes prepared with different retinol concentrations.

[0331] Result analysis of Experimental Example 5:

[0332] According to the above results, the particle size and PDI of all alcohol homogenate liquids can be maintained in a small range.

[0333] In addition, with the increase of retinol content, the particle size and drug loading concentration also gradually increased, but the PDI showed a trend of first decreasing and then increasing, and reached the minimum value when the retinol added concentration was 3 mg / mL.

[0334] <Experimental Example 6: Effect of Monoolein Content on Ethosomes>

[0335] The main difference between Examples 17-19 and Example 1 is that the content of monoolein GMO is different. The effect of the content of monoolein on the ethosomes will be analyzed below by the concentration of monoolein added in the formula when preparing the ethosome homogenate.

[0336] The method of ethosome experimental example 6 includes: taking the homogenous solution of the ethosome prepared in Examples 1 and 17-19 as the test solution, and detecting the corresponding particle size, PDI and stability (drug loading concentration and retinol retention rate) at different storage temperatures at room temperature (20±1°C).

[0337] Results of Experimental Example 6

[0338] For indicators such as particle size (unit: nm) and PDI, the mean (Mean) and standard deviation (SD) were obtained based on the corresponding three sets of parallel data, as shown in Table 7.

[0339] Table 7 Particle size and PDI of the homogenous alcohol solutions of Examples 1 and 17-19

[0340]

[0341] For indicators such as particle size (unit: nm) and PDI, the test results (Mean±SD) of each indicator are determined according to their corresponding mean (Mean) and standard deviation (SD), and then a bar chart diagram is drawn based on the test results (Mean±SD). For details, see Fig.11 . Fig.11 A schematic diagram comparing the particle size and PDI of ethosomes prepared according to different GMO concentrations.

[0342] For indicators such as drug loading concentration (unit: mg / mL) and retinol retention rate (unit: %), the mean (Mean) and the corresponding standard deviation (SD) were obtained according to the corresponding 3 sets of parallel data, and the test results (Mean±SD) of each indicator were determined, as shown in Table 8, where n=3. Table 8 records the drug loading concentration and retinol retention rate on day 0 (d) and day 14 (d) during the storage of the alcohol homogenate solutions of Examples 1 and 17-19 at 4°C, 20°C, and 50°C, respectively.

[0343] Table 8 Drug loading concentration and retinol retention rate of the alcohol homogenate solutions of Examples 1 and 17-19 stored at different temperatures

[0344]

[0345] For the retinol retention rate (unit %) indicator, a bar chart is also drawn based on the corresponding test results (Mean±SD). For details, see Fig.12 . Fig.12 This is a comparative schematic diagram of the retinol retention rates of ethanol body homogenates prepared according to different GMO concentrations at different storage temperatures (4°C, 20°C, and 50°C). In the figure, the horizontal axis represents the storage temperature (Storagetemperature), and the vertical axis represents the retinol retention rate (Retention rate of retlnol). Each temperature on the horizontal axis corresponds to a group of bar graphs, and each group of bar graphs includes 4 bar graphs, which correspond to ethanol body homogenates with GMO concentrations of 1 mg / mL, 10 mg / mL, 20 mg / mL, and 30 mg / mL, respectively, from left to right.

[0346] Result analysis of Experimental Example 6:

[0347] From the above results, it can be seen that when the concentration of monoolein GMO is between 1 mg / mL and 30 mg / mL, the particle size does not exceed 100 nanometers and the PDI is less than 0.35, indicating that the ethosomes provided in the examples of the present application have good performance.

[0348] In addition, when the concentration of GMO is between 1 mg / mL and 30 mg / mL, the PDI first increases to 20 mg / mL and then decreases, while the particle size first decreases to 10 mg / mL and then increases.

[0349] In addition, at storage temperatures of 4°C, 20°C, and 50°C, the retinol retention rate tended to increase with the GMO concentration, but at a storage temperature of 50°C, the increasing trend was no longer obvious.

[0350] <Experimental Example 7: Effect of Alcohol Content on Ethanol Body>

[0351] The main difference between Examples 20-21 and Example 1 is the alcohol content, that is, the content of short-chain alcohols (Percentage of PG-DPG volume); therefore, the effect of alcohol content on alcohol bodies can be analyzed by comparing Examples 1 and 20-21.

[0352] The method of Experimental Example 7 includes:

[0353] Take the alcohol homogenate solutions prepared in Examples 1, 20-21 (alcohol content is 30%, 20%, 10% respectively) as the test solution, detect the appearance, particle size, PDI and drug loading concentration at room temperature (20±1°C), and the stability (particle size, retinol retention rate) stored at different temperatures for different time periods, and record the results;

[0354] In addition, a retinol hydroalcoholic solution (i.e., a retinol solution containing 30% ethanol) was taken to measure the retinol retention rate after storage at different temperatures for different times, for comparison with the results of Examples 1 and 22-23; wherein the retinol hydroalcoholic solution was prepared by dissolving the retinol raw material in 6 mL of ethanol solution, then slowly adding 14 mL of deionized water thereto, and gently stirring to ensure that the two were evenly mixed, to obtain a retinol solution containing 30% ethanol.

[0355] It should be noted that the retinol retention rate of a retinol solution containing 30% ethanol is the ratio of the retinol content in the retinol solution at the end of the test to the retinol content in the retinol solution at the beginning of the test within a certain test time.

[0356] Results of Experimental Example 7

[0357] 1. For the appearance, please refer to Fig.13 ; Fig.13 The figure is a schematic diagram for comparing the common appearance of the alcoholic homogeneous liquid prepared in Examples 1 and 20-21; wherein (A) represents an alcoholic homogeneous liquid with an alcohol content of 10%, (B) represents an alcoholic homogeneous liquid with an alcohol content of 20%, and (C) represents an alcoholic homogeneous liquid with an alcohol content of 30%.

[0358] 2. For the structural morphology under the electron lens, please refer to Fig.14 ; Fig.14 This is a transmission electron micrograph (×100,000) of an alcoholic homogeneous solution containing 20% ​​alcohol.

[0359] 3. For indicators such as particle size (unit: nm) and PDI at room temperature, the mean (Mean) and variance (SD) are obtained based on the corresponding 3 sets of parallel data to determine the test results of each indicator, as shown in Table 9.

[0360] Table 9 Particle size and PDI of the homogenous alcohol solutions prepared in Examples 1, 20-21 at room temperature

[0361]

[0362] 4. For the drug loading concentration (mg / mL) at room temperature, the mean (Mean) and variance (SD) were obtained based on the corresponding three sets of parallel data to determine the test results of each indicator, as shown in Table 10.

[0363] Table 10 Drug loading concentration of alcohol homogenate solutions with different alcohol contents at room temperature in Examples 1, 20-21

[0364]

[0365] 5. For the particle size during storage for different time periods at different storage temperatures such as 4°C, 20°C, and 50°C, the mean (Mean) and variance (SD) were obtained based on the corresponding three sets of parallel data, and the test results (Mean±SD) of each indicator were determined. See Table 11 for details.

[0366] Table 11 Particle size and PDI of the homogenous alcohol solutions of Examples 1, 22-23 stored at different temperatures for different times

[0367]

[0368] 6. For the particle sizes after storage at different storage temperatures and for different time periods, a comparison chart is also drawn based on the corresponding test results (Mean±SD). For details, see Fig.15 . Fig.15 This is a comparative diagram of the particle size and PDI of alcohol homogenates prepared according to different alcohol contents at different storage temperatures (4°C, 20°C, and 50°C).

[0369] 7. For the retinol retention rate after storage at different storage temperatures for different periods of time, the corresponding test results (Mean±SD) are determined according to the mean and standard deviation SD of the three corresponding parallel data sets, and then a comparison chart is drawn based on the test results (Mean±SD). For details, see Fig.16 . Fig.16 This is a comparative diagram of the retinol retention rates of alcoholic homogenates prepared with different alcohol contents and retinol hydroalcoholic solutions at different storage temperatures (4°C, 20°C, and 50°C).

[0370] Results Analysis of Experimental Example 7

[0371] 1. If Fig.13 As shown, the alcohol homogenates with alcohol contents of 10%, 20% and 30% are all transparent emulsions with a yellowish luster.

[0372] 2. If Fig.14 As shown, under transmission electron microscopy, there are multiple ethosome particles in the ethosome homogenate of Example 20, and the basic morphology of the ethosome particles is spherical or elliptical, with a particle size of about 40 nm; in addition, the surface of the particles is uneven, slightly concave, and presents a multi-chamber honeycomb structure. It is easy to understand that due to the multi-chamber, the ethosomes provided in the embodiments of the present application have better structural stability and more outstanding drug loading performance compared with single-chamber vesicles.

[0373] 3. When the alcohol content is between 10-30%, the particle size is small, the dispersion is uniform, and the drug loading effect is good; in addition, with the increase of the short-chain alcohol content, the PDI gradually decreases, and the distribution of the alcohol system is more uniform. This may be because the hydroxyl groups contained in the alcohol form hydrogen bonds with water, increasing the intermolecular force. As the alcohol concentration increases, the particles tend to disperse to form a more uniform dispersion system.

[0374] 4. Stability results analysis

[0375] During the storage at 4℃ and 20℃, the particle size of the alcohol body homogeneous liquid with 10% and 20% alcohol content basically remained at about 150nm; after storage at 50℃ for 7 days, the particle size of the alcohol body homogeneous liquid with 10% and 20% alcohol content increased by approximately 35nm and 119nm, respectively; for the alcohol body homogeneous liquid with 30% alcohol content, the particle size increased by approximately 21nm and 41nm, respectively, after storage at 4℃ and 20℃ for 7 days, and the particle size increase was more significant after storage at 50℃ for 7 days.

[0376] Compared with the retinol hydroalcoholic solution (retinol solution containing 30% ethanol), the ethosomes obtained after GMO encapsulation in the examples of the present application have significantly improved retinol retention rate; in particular, when stored at 4°C, the retinol retention rate of the ethosome homogenate containing 20% ​​alcohol can be increased from 3.3% to 73.6%±1.3% relative to the retinol hydroalcoholic solution.

[0377] The above results indicate that the ethosomes provided in the examples of the present application have excellent stability.

[0378] The stability results of particle size and drug loading concentration at different storage temperatures also show that when ethosomes are stored at low temperatures, the particle size is more stable and the decomposition rate of the loaded retinol can be slowed down.

[0379] <Experimental Example 8: Effect of drug-lipid ratio on ethosomes>

[0380] The main difference between the ethosome homogenates of Examples 22-24 is the drug-to-lipid ratio, where the drug-to-lipid ratio specifically refers to the mass ratio of the encapsulated substance (retinol) to monoolein. The effect of the drug-to-lipid ratio on ethosomes is analyzed through Experimental Example 8 below.

[0381] Experimental method: Take the homogenous alcohol solution prepared according to the method of Examples 22-24 as the test solution, detect the particle size, PDI and potential at room temperature (20±1°C), and the stability (particle size, PDI and retinol retention rate) during storage at different temperatures and in the dark for different time periods, and record the results.

[0382] Results of Experimental Example 8:

[0383] 1. For the Zeta potential (unit: mv), particle size (unit: nm), and PDI at room temperature (20±1°C), the mean (Mean) and variance (SD) were obtained according to the corresponding n (n=3) groups of parallel data, and the test results (Mean±SD) of each index were determined. For the alcohol homogenate solutions with drug-lipid ratios of 1:5, 1:10, and 1:15, the Zeta potential test results (Mean±SD) were -15.3±1.3, -17.7±0.3, and -18.7±0.6, respectively, the particle size test results (Mean±SD) were 32.03±0.85, 49.56±3.50, and 52.99±1.31, respectively, and the PDI test results (Mean±SD) were 0.338±0.033, 0.345±0.016, and 0.380±0.022, respectively.

[0384] 2. For the particle size (in nm) and PDI at room temperature (20±1℃), a comparison chart is also drawn based on the corresponding test results (Mean±SD). For details, see Fig.17 . Fig.17 A columnar comparison diagram of the particle size and PDI of alcohol homogenates with different drug-to-lipid ratios at room temperature.

[0385] 3. For the particle size (in nm) and PDI of alcohol homogenates with different drug-to-lipid ratios stored at different storage temperatures (4°C, 20°C, 50°C) for different time periods (0, 2, 7, 14d), the mean (Mean) and standard deviation (SD) were obtained based on the three sets of parallel data corresponding to each indicator, and the test results (Mean±SD) of each indicator were determined, as shown in Table 12 for details.

[0386] Table 12 Particle size and PDI of the homogenous alcohol solutions of Examples 22-24 stored at different temperatures for different periods of time

[0387]

[0388] 4. For the particle size (in nm) of alcohol homogenate with different drug-to-lipid ratios stored at different storage temperatures (4°C, 20°C, 50°C) for different time periods (0, 2, 7, 14 days), a comparison chart is formed based on the test results (Mean±SD) corresponding to each indicator. Please refer to Fig.18 Each temperature on the horizontal axis of the figure corresponds to a group of bar graphs, and each group of bar graphs includes 3 bar graphs, which correspond to the alcohol body homogenate solutions with drug-lipid ratios of 1:5, 1:10, and 1:15, respectively, from left to right.

[0389] 5. For the retinol retention rate (unit: %) of the alcohol homogenate with different drug-to-lipid ratios stored at different storage temperatures (4°C, 20°C, 50°C) for different time periods (0, 2, 7, 14 days), the mean (Mean) and standard deviation (SD) were obtained based on the three sets of parallel data corresponding to each indicator, and the test results (Mean±SD) of each indicator were determined, as shown in Table 13 for details.

[0390] Table 13. Retinol retention rate of the homogenous alcohol solution of Examples 22-24 after storage at different temperatures for different time periods

[0391]

[0392] 6. For the retinol retention rate (unit: %) of the alcohol homogenate with different drug-to-lipid ratios stored at different storage temperatures (4°C, 20°C, 50°C) for different time periods (2, 7, 14 days), a bar graph is formed based on the mean of the three sets of parallel data corresponding to each indicator. Please refer to Fig.19 In the figure, each time (2, 7, 14d) on the horizontal axis corresponds to a group of bar graphs. Each group of bar graphs includes 3 bar graphs, which correspond to the alcohol body homogenate solutions with drug-lipid ratios of 1:5, 1:10, and 1:15 from left to right.

[0393] Result analysis of Experimental Example 8:

[0394] 1. When the drug-lipid ratio was between 1: (5-15), the particle size of the ethosomes prepared at 20±1°C ranged from 32.03±0.85 nm to 52.99±1.31 nm, the PDI was between 0.338±0.033 and 0.380±0.022, and the potential was between -15.3±1.3 and -18.7±0.6, indicating that the ethosomes had good stability. In addition, with the increase of the mass ratio of monoolein to retinol, the particle size and PDI of the ethosomes also increased.

[0395] 2. According to the preparation results, when the drug-lipid ratio is between 1: (5~15), the particle size of the alcohol bodies with different drug-lipid ratios under different temperature treatments always remains small, basically not exceeding 100nm.

[0396] Further analysis showed that after storage at 4°C for 14 days, the particle sizes of the ethosomes with drug-lipid ratios of 1:5, 1:10, and 1:15 were 53.34 nm, 54.93 nm, and 60.01 nm, respectively; after storage at 20°C for 14 days, the particle sizes of the ethosomes with drug-lipid ratios of 1:5, 1:10, and 1:15 were 35.22 nm, 43.01 nm, and 47.01 nm, respectively; after storage at 50°C for 14 days, the particle sizes of the ethosomes with drug-lipid ratios of 1:5, 1:10, and 1:15 were 75.93 nm, 63.29 nm, and 95.08 nm, respectively. It can be seen that the particle size of the ethosomes with different drug-lipid ratios stored at 4°C and 20°C changed little, and they had good stability. In addition, high temperature may accelerate the aggregation of ethosomes. Therefore, when stored at 50°C, the particle size of ethosomes with different drug-to-lipid ratios increased significantly over time.

[0397] 3. With the increase of drug-lipid ratio, the retention rate of retinol was significantly improved. After storage at 4℃, 20℃, and 50℃ for 14 days, the retinol retention rates of the ethosomes with a drug-lipid ratio of 1:5 were 45.2%, 28.2%, and 4.8%, respectively; the retinol retention rates of the ethosomes with a drug-lipid ratio of 1:10 were 73.3%, 56.6%, and 13.4%, respectively; the retinol retention rates of the ethosomes with a drug-lipid ratio of 1:15 were 86.1%, 71.8%, and 23.8%, respectively. It can be seen that the ethosomes with a drug-lipid ratio of 1:15 have more prominent advantages in maintaining retinol activity at different temperatures.

[0398] <Experimental Example 9: Effect of Antioxidants on Ethanol Forms>

[0399] The alcoholic homogenate liquid prepared according to the method of Example 25 [including antioxidants: pentaerythritol tetra(di-tert-butylhydroxyoxycinnamate) ester and tocopherol acetate] was taken as the test liquid of the antioxidant-containing group; the corresponding alcoholic homogenate liquid was prepared according to the method of Example 24 as the test liquid of the antioxidant-free group.

[0400] The stability of the test solution containing the antioxidant group and the test solution containing the antioxidant group was tested to examine the properties of the ethosome. The specific detection method includes: storing the homogenous solution of each group of ethosomes at different temperatures (4±1℃, 20±1℃ and 50±1℃) in the dark, and measuring the particle size, PDI, drug loading concentration (i.e., retinol loading amount), retinol retention rate and other indicators at different storage times.

[0401] Experimental results of Experimental Example 9

[0402] 1. For the particle size (in nm) and PDI of each group of alcohol homogenate liquid after storage at different storage temperatures (4°C, 20°C, 50°C) for different time periods, the mean (Mean) and standard deviation (SD) were obtained according to the three sets of parallel data corresponding to each indicator, and the test results (Mean±SD) of each indicator were determined, as shown in Table 14 for details.

[0403] Table 14. Particle size and PDI of the group without antioxidant and the group with antioxidant at different temperatures over time (d)

[0404]

[0405] 2. For the particle size (in nm) of the homogenized liquid of the alcoholic body without antioxidant group after storage at different storage temperatures (4℃, 20℃, 50℃) for different time periods, a comparison chart is also formed based on the corresponding test results (Mean±SD), which can be seen in Fig. 20 In the figure, each time on the horizontal axis corresponds to a group of bar graphs, and each group of bar graphs includes three bar graphs, which correspond to the retinol retention rates at 4°C, 20°C, and 50°C from left to right.

[0406] 3. For the retinol retention rate (unit %) of the alcohol homogenate solution without antioxidants after storage at different storage temperatures (4℃, 20℃, 50℃) for different time periods, a comparison chart is also formed based on the corresponding test results (Mean±SD), which can be seen in Fig.21 In the figure, each time on the horizontal axis corresponds to a group of bar graphs, and each group of bar graphs includes three bar graphs, which correspond to the retinol retention rates at 4°C, 20°C, and 50°C from left to right.

[0407] 4. For the particle size (in nm) of each group of alcohol homogenate liquid after storage at different storage temperatures (4℃, 20℃, 50℃) for different time periods, a comparison chart is formed based on the test results (Mean±SD) corresponding to each indicator. Please refer to Fig. 22 In the picture corresponding to each temperature in the figure, each time on the horizontal axis corresponds to a group of bar graphs, and each group of bar graphs includes two bar graphs, corresponding to the non-antioxidant group and the antioxidant-containing group from left to right. 5. For the retinol retention rate (unit %) of each group of alcohol homogenate liquid stored at different storage temperatures (4℃, 20℃, 50℃) for different time (d), according to the corresponding 3 groups of parallel data, the mean (Mean) and standard deviation (SD) are obtained to determine the test results of each indicator (Mean±SD), as shown in Table 15.

[0408] Table 15. Retinol retention rate of the group without antioxidants and the group containing antioxidants at different temperatures over time (d)

[0409]

[0410] 6. For the retinol retention rate (unit %) of each group of alcohol homogenate liquid after storage at different storage temperatures (4℃, 20℃, 50℃) for different time periods, a bar chart is formed according to the corresponding test results (Mean±SD), which can be seen in Fig.23 In the picture corresponding to each temperature in the figure, each time on the horizontal axis corresponds to a group of bar graphs, and each group of bar graphs includes two bar graphs, which correspond to the group without antioxidants and the group containing antioxidants from left to right.

[0411] Result analysis of Experimental Example 9

[0412] 1. After storage at 4℃, 20℃, and 50℃ for 45 days, the particle sizes of the antioxidant-free group were 69.47, 59.04, and 97.51 nm, respectively, and the particle sizes were always maintained at a small size. After storage at 4℃, 20℃, and 50℃ for 14 days, the particle sizes of the antioxidant-containing group were 81.3, 59.4, and 166.9 nm, respectively. The particle size stability was good under room temperature storage, and the particle size increased significantly over time at low and high temperatures. In general, both the antioxidant-free group and the antioxidant-containing group had good particle size stability.

[0413] 2. The retinol retention rates of the antioxidant-free group treated at 4°C, 20°C, and 50°C after 14 days of storage were 86.1±0.6, 71.8±1.0, and 23.8±0.5, respectively. After 14 days of storage at 4°C, 20°C, and 50°C, the retinol retention rates of the antioxidant-containing group were 98.4%, 94.5%, and 55.7%, respectively. Compared with the group without added antioxidants, the retinol retention rates of the antioxidant-containing group increased by about 12.3%, 22.7%, and 31.9%, respectively. Therefore, the addition of antioxidants has a significant effect on improving the storage stability of retinol, especially under high temperature conditions.

[0414] <Experimental Example 10: Characterization of the structure of ethanolate by small-angle X-ray scattering>

[0415] In order to further test the properties of the ethosome, the homogenous solution of the ethosome of Example 20 was taken as the test solution, and the structure of the ethosome was detected by small-angle X-ray scattering to analyze its structure.

[0416] Small angle X-ray scattering (SAXS) is often used to identify the phase structure of liquid crystals. The crystal form of lipid cubic liquid crystals can be determined based on the ratio of the scattering vectors corresponding to each scattering peak (i.e., the scattering factor ratio).

[0417] For typical SAXS characteristic spectra of lipid cubic liquid crystals, please refer to Fig.24 From this spectrum, we know that the scattering peaks are 0.4295, 0.6020, and 0.7681 nm respectively.-1 , and its scattering factor ratio q1∶ q2∶ q3=, therefore, it can be determined that the particles corresponding to the spectrum have a body-centered (Im3m) lipid cubic phase, that is, a lipid cubic liquid crystal structure.

[0418] For the alcohol body of Example 20, the scattering intensity (Intensity) is used as the ordinate, and the scattering vector q (nm -1 ) as the horizontal axis, and a SAXS spectrum of an alcohol body is obtained. Fig.25 .from Fig.25 It can be seen that the long-range ordered structure of the ethosome is not significant within the scattering vector range, and compared with the characteristic SAXS spectrum of lipid cubic liquid crystal, no cubic phase characteristic scattering peak is shown, indicating that there is no cubic liquid crystal structure in the ethosome. Therefore, it can be determined that the ethosome of the embodiment of the present application is a spherical multivesicular carrier that is different from the cubic liquid crystal structure in structure, and should be quite different from the cubic liquid crystal carrier in properties.

[0419] <Experimental Example 11: Comparison of Retinol-Carrying Particles Prepared by Different Methods>

[0420] The retinol-loaded particle solutions prepared in Comparative Examples 1-4 and the ethosome homogenate prepared in Example 25 were taken as the test solutions of Experimental Groups 1 to 5, respectively (the retinol-loaded particles contained in the test solutions of Experimental Groups 1 to 5 were, in order: unilamellar retinol liposomes, retinol liposomes containing poloxamer 407, ordinary retinol ethosomes, retinol ethosomes containing poloxamer 407, and ethosomes of Example 25), and the properties of each test solution were detected for comparative analysis.

[0421] The method of Experimental Example 11: 1. At room temperature (20°C), measure the particle size, PDI, and Zeta potential of each group of test liquids; 2. Store each group of test liquids at 4°C and 20°C, respectively, away from light for 7 days, and test the storage stability. Specifically: measure the particle size (in nm), PDI, and retinol retention rate (in %) corresponding to the 0th, 2nd, and 7th days, respectively; 3. Detect the morphology of the alcohol body of Experimental Group 5 (Example 25) under a transmission electron microscope.

[0422] Experimental results of Experimental Example 11:

[0423] 1. The morphology of the ethosomes of Experimental Group 5 (Example 25) under transmission electron microscopy can be seen in Fig.26 .

[0424] 2. For the particle size (in nm), PDI, and Zeta potential (in mv) at room temperature, the mean (Mean) and standard deviation (SD) were obtained based on the corresponding three sets of parallel data, and the test results (Mean±SD) were determined, see Table 16.

[0425] Table 16. Particle size, PDI, and Zeta potential of experimental groups 1 to 5 at room temperature

[0426]

[0427] 2. For the particle size (in nm) and PDI of experimental groups 1 to 5 at room temperature, a comparative diagram is drawn based on the corresponding test results (Mean±SD), see Fig. 27 .

[0428] 3. For the storage stability indicators such as particle size (unit: nm), PDI and retinol retention rate (unit: %) of experimental groups 1 to 5 at different temperatures, the mean (Mean) and standard deviation (SD) were obtained based on the three sets of parallel data corresponding to each indicator, and the test results (Mean±SD) were determined, as shown in Table 17.

[0429] Table 17 Particle size, PDI and retinol retention rate of experimental groups 1 to 5 stored at different temperatures for different times (d)

[0430]

[0431] 4. For the storage stability indicators such as particle size (in nm) and retinol retention rate (in %) of experimental groups 1 to 5 at different temperatures, a comparative diagram is drawn based on the test results (Mean±SD) corresponding to each indicator, see Figure 28-29 .

[0432] exist Figure 28-29 In the figure, (A), (B), (C), (D), and (E) represent experimental groups 1 to 5, respectively. For each temperature corresponding picture, each time on the horizontal axis corresponds to a group of bar graphs, and each group of bar graphs includes five bar graphs, which correspond to (A), (B), (C), (D), and (E) from left to right.

[0433] Result analysis of Experimental Example 11:

[0434] 1. Particle size and PDI analysis at 20°C on day 0

[0435] The particle size and PDI of the conventional unilamellar retinol liposomes (see the results of Comparative Example 1) were 397.3±5.6 nm and 0.632±0.004, respectively; while the particle size and PDI of the retinol liposomes after the introduction of poloxamer 407 (see the results of Comparative Example 2) decreased slightly, to 232.1±5.0 nm and 0.516±0.016, respectively.

[0436] The particle size and PDI of ordinary retinol ethosomes (see the results of Comparative Example 3) are 106.7±3.4 nm and 0.468±0.011, respectively; while after adding poloxamer 407, the particle size and PDI of retinol ethosomes (see the results of Comparative Example 4) increase to 152.6±2.1 nm and 0.788±0.011, respectively.

[0437] Compared with Comparative Examples 1-4, the ethosomes provided in Example 25 of the present application have the smallest particle size and PDI, which are 53.0±1.3 nm and 0.341±0.018, respectively. Due to the small particle size, the ethosomes provided in the examples of the present application can penetrate the stratum corneum of the skin more deeply to exert their effects and achieve uniform release of the drug.

[0438] It can also be known from the above results that: compared with the thin film dispersion method and the injection method, the method for preparing the alcohol body according to the high-pressure homogenization technology in the embodiment of the present application has unique advantages in particle size and uniformity control, and the operation is safe and simple.

[0439] 2. Analysis of particle size and PDI changes with temperature

[0440] The particle sizes of unilamellar retinol liposomes, retinol liposomes containing poloxamer 407, ordinary retinol ethosomes, and retinol ethosomes containing poloxamer 407 after storage at 4°C for 7 days were 220.2±7.3 nm, 147.0±0.3 nm, 101.0±3.2 nm, and 140.4±2.3 nm, respectively. The particle sizes of retinol ethosomes after storage at 20°C for 7 days were 246.7±2.6 nm, 150.4±1.1 nm, 89.1±6.1 nm, and 121.7±1.9 nm, respectively, indicating that they were larger in size.

[0441] The particle sizes of the alcohol body of the present application example stored at 4° C. and 20° C. for 7 days were 67.6±2.1 and 51.2±1 nm, respectively, which obviously had better particle size stability.

[0442] 3. Analysis of the change of retinol retention rate with temperature

[0443] The unilamellar retinol liposomes, retinol liposomes containing poloxamer 407, ordinary retinol ethosomes, and retinol ethosomes containing poloxamer 407 of Comparative Examples 1 to 4, after storage at 4°C for 2 days, lost 35.2%, 33.3%, 42.5%, and 12.1% of their retinol contents, respectively; after storage at 4°C for 7 days, lost 53.8%, 51.1%, 47.2%, and 32.3%, respectively; in contrast, the ethosomes of the embodiments of the present application had relatively good retinol retention rates of 99.9±0.3% and 98.4±1.6% after storage at 4°C for 2 days and 7 days, respectively, showing significant advantages in maintaining the stability of the retinol active ingredient.

[0444] The unilamellar retinol liposomes, retinol liposomes containing poloxamer 407, ordinary retinol ethosomes, and retinol ethosomes containing poloxamer 407 in Comparative Examples 1 to 4 lost 53.8%, 50.1%, 47.2%, and 32.3% of their retinol contents, respectively, after storage at 20°C for 2 days; and lost 83.0%, 84.0%, 66.5%, and 59.9% of their retinol contents, respectively, after storage at 4°C for 7 days; while the ethosomes of the embodiment of the present application had relatively good retinol retention rates after storage at 20°C for 2 days and 7 days, which were 98.5±0.8% and 97.3±0.3%, respectively; it can be seen that at room temperature, the ethosomes of the embodiment of the present application have a more prominent protective effect on retinol.

[0445] 4. Summary

[0446] In summary, compared with the comparative example, the ethosomes provided in the examples of the present application have the smallest particle size and PDI. Under storage conditions of 4°C and 20°C, the changes in particle size and PDI are small, showing good particle uniformity. In addition, during storage, the retinol retention rate of the ethosomes in the examples of the present application is much higher than that of the retinol-loaded particles in the comparative example, and the protective effect on retinol is significantly better than other preparations.

[0447] The addition of poloxamer 407 had an effect on the particle size and PDI of liposomes and ethosomes, but the effects were different. For liposomes, the particle size and PDI decreased slightly after the addition of poloxamer 407; while for ordinary ethosomes, the particle size and PDI increased after the addition of poloxamer 407. The retinol content loss rate of retinol liposomes and retinol ethosomes with the addition of poloxamer 407 was relatively low, indicating that poloxamer 407 may help protect retinol from oxidative decomposition. In retinol ethosomes stored at 4°C, the retinol retention rate in the group with the addition of poloxamer 407 was significantly improved, showing a strong protective effect. Therefore, the addition of poloxamer 407 to the preparation helps to improve the stability of retinol.

[0448] <Experimental Example 12, in vitro skin permeation test>

[0449] 1. Establishment of a method for retinol analysis

[0450] In the transdermal detection experiment of ethosomes, high performance liquid chromatography (HPLC) and retinol extraction technology are required for analysis. Therefore, the first step is to determine the appropriate analysis method, as follows.

[0451] 1. Establishment of HPLC method for retinol analysis

[0452] (1) Determine the chromatographic conditions for HPLC analysis:

[0453] The chromatographic column was PlatisilODS (250 × 4.6 mm, 5 μm);

[0454] The mobile phase was methanol-water solution (volume ratio v:v = 98:2);

[0455] The column temperature was 40°C;

[0456] The detection wavelength is 325nm;

[0457] The injection volume was 10 μL;

[0458] The flow rate was 1.0 mL / min.

[0459] (2) Draw a standard curve of retinol based on HPLC analysis

[0460] Accurately weigh 20 mg of retinol standard, add methanol to dilute and prepare a retinol stock solution with a mass concentration of 1 mg / mL, which will be used as the retinol mother solution.

[0461] Using 1mg / mL retinol stock solution as the base, dilute to a series of retinol standard solutions with concentrations of 0.1, 1, 10, 20, 40, 80, 100μg / mL, and filter through a 0.22μm organic filter. Then use HPLC to measure the peak area of ​​each concentration of retinol standard solution. Next, draw a standard curve with retinol concentration (C) as the horizontal axis and peak area (Area, A) as the vertical axis, as shown in Figure 1. Fig.30 . Fig.30 The figure is a standard curve diagram for retinol analysis based on HPLC.

[0462] (3) Analysis results

[0463] according to Fig.30 It can be seen that the corresponding linear regression equation obtained by taking retinol concentration (C) and peak area (A) is:

[0464] A= 47.808 C + 27.149, linear correlation coefficient r=0.9996.

[0465] According to the results of the linear regression equation above, the linear relationship between retinol content and peak area is good in the concentration range of 0.1-100.0 µg / mL. Therefore, the retinol content can be detected by peak area.

[0466] 2. Analysis and establishment of retinol extraction method

[0467] (1) The retinol extraction method comprises the following steps:

[0468] A retinol sample solution with a concentration of 100 µg / mL was prepared based on a saline solution containing 1% Tween-80;

[0469] The retinol sample solution (retinol concentration is 0.1 mg / mL) and ethyl acetate-acetone extract (volume ratio of ethyl acetate to acetone v:v=4:1) were mixed at different volume ratios such as 1:0.5, 1:1, and 1:2, and ultrasonic extraction was performed for 15 minutes respectively. The layers were allowed to stand and stratified. The upper extract layer was taken, dried with nitrogen, and then re-dissolved with methanol. The retinol content was detected by HPLC. The extraction recovery rate of retinol at different volume ratios (1:0.5, 1:1, and 1:2) was calculated by the following formula. The test results were recorded in triplicate;

[0470] The calculation formula of the extraction recovery rate is: Extraction recovery rate (%) = W after extraction / W before extraction × 100%; wherein W after extraction is the mass of retinol in the solution obtained after extraction, and W before extraction is the mass of retinol in the solution before extraction.

[0471] The test results are shown in Table 18 and Fig.31 ,in Fig.31 This is a schematic diagram showing the effect of the volume ratio of extracting solvent to sample on the extraction recovery.

[0472] Table 18. Extraction recovery of retinol in retinol sample solution (n=3)

[0473]

[0474] (2) Result analysis:

[0475] As can be seen from the above, the average recovery rates of the groups with a volume ratio of 0.1 mg / mL retinol solution to ethyl acetate acetone extract of 1:0.5, 1:1, and 1:2 were 98.28%, 99.21%, and 99.84%, respectively, and the RSD (relative standard deviation) was 2.07%, 2.50%, and 1.12%, respectively. The extraction recovery results were all between 95% and 105%, and the RSD values ​​were all less than 3%, indicating that the extraction method has a good extraction recovery rate.

[0476] Therefore, in subsequent experiments, a volume ratio of 1:0.5 between retinol solution and ethyl acetate / acetone extract can be used to extract and concentrate retinol.

[0477] 2. Experimental study on the cumulative release and transdermal rate of ethosomes

[0478] 1. The detection experiment includes the following steps:

[0479] The abdominal skin of Sprague-Dawley rats was depilated and peeled, the fascia was scraped off with curved forceps, and washed with saline. The excised skin was checked for damage, and the undamaged skin was selected and divided into 5 experimental groups: experimental groups a to e.

[0480] The undamaged skin of each experimental group was fixed between the drug supply pool (also called drug administration pool) and the receiving pool (specifically the Franz receiving pool), with the stratum corneum facing upward and the dermis facing downward.

[0481] 6.5 mL of release medium (the release medium is a physiological saline solution containing 1% Tween) was added to the Franz receiving cell of each experimental group, and the liquid surface was in contact with the dermis of the ex vivo skin and maintained at a constant temperature of 32°C.

[0482] According to Table 19, 30 μL of the corresponding sample solution for test was added to the dosing pool of each experimental group for dosing treatment. Then, the mixture was magnetically stirred at 300 rpm. The receiving medium (physiological saline solution containing 1% Tween) was taken out from the receiving pool at 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, and 24 h, respectively. At the same time, the same volume of fresh release medium (physiological saline solution containing 1% Tween) preheated to 32 °C was added.

[0483] Table 19. Sample solutions corresponding to each experimental group

[0484]

[0485] Regarding Table 19, the preparation method of the C12-15 alcohol benzoate solution containing retinol is as follows: weigh 3 mg of retinol raw material, mix it with 1 mL of C12-15 alcohol benzoate solution, place the mixture in a constant temperature water bath at 60°C and stir until the retinol raw material is completely dissolved in the C12-15 alcohol benzoate solution, so as to obtain the C12-15 alcohol benzoate solution containing retinol; the preparation method of the pentaerythritol tetra(ethylhexanoate) solution containing retinol is as follows: weigh 3 mg of retinol raw material, mix it with 1 mL of C12-15 alcohol benzoate solution, place the mixture in a constant temperature water bath at 60°C and stir until the retinol raw material is completely dissolved in the pentaerythritol tetra(ethylhexanoate) solution, so as to obtain the pentaerythritol tetra(ethylhexanoate) solution containing retinol.

[0486] Next, the sample solution of each experimental group (whether added to the receiving pool or the dosing pool) was extracted with an extract (the aforementioned ethyl acetate acetone extract, with a volume ratio of 1:0.5 between the sample solution and the ethyl acetate acetone extract) and ultrasonicated for 15 minutes, and allowed to stand at 4°C for 12 hours. 1 mL of the upper extract layer was taken, nitrogen blown, re-dissolved with 0.3 mL of methanol, filtered through a 0.22 μL organic filter membrane, and tested with HPLC. After HPLC testing, the cumulative permeation amount of retinol to the skin at different penetration times was calculated using the following formula:

[0487] The calculation formula of the cumulative permeation of the skin is: Qn = (ρ n ×V+V1) / S;

[0488] Where Qn is the cumulative permeability (in mg / cm 2 ); ρn, ρi are the retinol mass concentrations measured at the nth and ith sampling points (in μg / mL); V1 and V are the sampling volume and the receiving pool volume (in mL, the sampling volume and the receiving pool volume of this experiment are both 6.5 mL); S is the penetration area (in cm 2 , the permeability area of ​​this experiment is 2.8cm 2 ). The sampling point refers to taking a certain volume of the receiving medium from the receiving pool at a predetermined time point in the experiment to determine the amount of drug that has penetrated into the receiving medium. Sampling at different time points can help understand the process of drug penetration into the skin and obtain information such as penetration rate and time dependence.

[0489] 2. Experimental results

[0490] For the cumulative permeation amount of each group at 2, 4, 6, 8, 10, 12 and 24 hours, the mean (Mean) and standard deviation (SD) were obtained based on the corresponding 3 groups of parallel data, and the test results (Mean±SD) were determined, see Tables 20~21.

[0491] Table 20. Cumulative permeation of each experimental group at 2, 4 and 6 hours

[0492]

[0493] Table 21. Cumulative permeation of each experimental group at 8, 10, 12 and 24 hours

[0494]

[0495] The permeation curve of retinol is plotted by comparing the cumulative amount (also called Qn) of the drug (i.e., sample solution) in each group to the sampling time t. Fig.32 . Fig.32 The graph is a cumulative permeation amount of the sample solutions in each group (****: p<0.0001, ***: p<0.001; n=3).

[0496] right Fig.32 Linear regression was performed on the straight line part of the curve to obtain the slope of the line, and the steady-state transdermal flux (Js) corresponding to each group was determined, that is, the cumulative permeation rate in vitro (unit: μg•cm -2 •h -1 ), the results are shown in Table 22.

[0497] Table 22 Steady-state transdermal rates of each experimental group

[0498]

[0499] Draw a bar chart for comparison based on the steady-state transdermal rate (Js), see Fig.33 . Fig.33 A schematic diagram of the steady-state permeability (Transdermal flux) of each group of sample solutions (****: p<0.01; n=3).

[0500] On the basis of the above, we further calculated the skin non-penetration amount (the amount of retinol that did not penetrate / enter the skin), skin retention (the amount of retinol retained in the skin), cumulative permeation (the amount of retinol that penetrated the skin, i.e., cumulative permeation) and other permeation parameters of each group of sample solutions over 24 hours to evaluate the percentage of transdermal permeation (relative abundance in English);

[0501] The calculation formula for relative abundance is: relative abundance (%) = (permeation parameter drug amount / total dosage of sample solution) × 100%; wherein the permeation parameter drug amount is selected from any one of the 24-hour non-penetrated amount, skin retention amount, and cumulative permeation amount.

[0502] The specific results are shown in Table 23 and Fig.34 . Fig.34The figure is a percentage accumulation diagram of the transdermal penetration evaluation of each group of sample solutions. In the figure, (A) represents an alcoholic homogeneous solution with an alcohol content of 10%, (B) represents an alcoholic homogeneous solution with an alcohol content of 20%, (C) represents an alcoholic homogeneous solution with an alcohol content of 30%, (D) represents a retinol solution of C12-15 alcohol benzoate, and (E) represents a retinol solution of pentaerythritol tetra(ethylhexanoate).

[0503] Table 23. Percutaneous penetration evaluation results of experimental groups a~e

[0504]

[0505] 3. Analysis of experimental results

[0506] According to the above results, we can know that:

[0507] (1) The cumulative permeation drug amount of each group of sample solutions within 24 hours was in the following order from large to small: 30% alcohol content alcoholic homogenate solution > 20% alcohol content alcoholic homogenate solution > 10% alcohol content alcoholic homogenate solution > retinol C12-15 alcohol benzoate solution > retinol pentaerythritol tetra(ethyl acetate) ester solution.

[0508] (2) The cumulative permeation amount of the homogenized liquid containing 30% alcohol is (5.888±1.380) μg / cm 2 , which are 4.00, 1.20, 14.19 and 9.62 times of the alcohol homogenate solution with 20% alcohol content, the alcohol homogenate solution with 10% alcohol content, the retinol C12-15 alcohol benzoate solution, and the retinol pentaerythritol tetra(ethyl acetate) ester solution, respectively, indicating that with the increase of alcohol content, the cumulative penetration of retinol also increases accordingly.

[0509] (3) Ethanolsomes can increase the steady-state transdermal rate of drugs (retinol). The steady-state permeation rate of the homogenous solution of ethanolsomes containing 30% alcohol is (0.891±0.110) μg / (cm 2 •h), which are 3.94, 1.04, 15.02, and 10.19 times of those of the alcohol homogenate solution with 20% alcohol content, the alcohol homogenate solution with 10% alcohol content, the retinol C12-15 alcohol benzoate solution, and the retinol pentaerythritol tetra(ethyl acetate) ester solution, respectively. This may be because the alcohol in the system increases the flexibility of the alcohol body, making the drug molecules easy to deform and pass through the stratum corneum to be absorbed deeply into the inner layer of the skin, thereby increasing the transdermal rate and the amount of permeation.

[0510] (4) In addition, the amount of drug that did not penetrate the skin (non-permeated amount) of the alcohol homogenate solution containing 10% and 20% alcohol was 0.7±0.0% and 3.5±0.1% of the total drug, respectively. The alcohol homogenate solution containing 30% alcohol allowed 18.3±4.3% of the retinol drug to penetrate the skin and 81.7±4.3% of the retinol drug to remain in the skin to exert its effect.

[0511] Therefore, as the alcohol concentration increases, the fluidity and flexibility of the alcohol body are greatly enhanced, so that the alcohol body can have better transdermal absorption function. In addition, compared with the retinol solution group of C12-15 alcohol benzoate and pentaerythritol tetra(ethylhexanoate), the alcohol body homogenate of the embodiment of the present application has stronger permeability.

[0512] <Experimental Example 13, Evaluation of Ethosomal Cytotoxicity>

[0513] Ethosome cytotoxicity assay was performed according to methods described in 1 to 3 below.

[0514] 1. Culture of cells to be tested

[0515] The human immortalized keratinocyte cell line HaCaT and the human skin fibroblast cell line CCC-ESF-1 were respectively placed in DMEM medium containing 10% fetal bovine serum and double antibodies, and subcultured in an incubator at 37°C and 5% CO2 concentration to keep the cells in the logarithmic growth phase.

[0516] HaCaT in logarithmic growth phase was digested with trypsin and then 5 The cells were inoculated at a density of 100 μL / mL in a 96-well plate, with a volume of 100 μL per well. The cells were incubated in a 37°C, 5% CO2 incubator for 12 h, and after the cells adhered to the wall, they were used as HaCaT test cells.

[0517] HaCaT and CCC-ESF-1 cells in logarithmic growth phase were digested with trypsin and then cultured at 2×10 5 / mL, 1×10 5 The cells were inoculated at a density of 100 μL / mL in a 96-well plate, with a volume of 100 μL per well. Incubated in a 37°C, 5% CO2 incubator for 12 h. After the cells adhered to the wall, they were used as CCC-ESF-1 test cells.

[0518] 2. Cytotoxicity detection of HaCaT cells

[0519] A normal group, a blank control group and multiple HaCaT cell drug intervention groups were set up; the ethosome homogenates containing 10%, 20% and 30% alcohol were diluted with incomplete culture medium, and each ethosome homogenate containing alcohol was diluted into multiple ethosome samples with retinol concentrations of 0.08, 0.1, 0.5, 0.8, 1.0, 1.5 and 2.0 μg / mL, respectively, and added to the corresponding HaCaT cell drug intervention groups; the same volume of incomplete culture medium was added to the HaCaT cells in the normal group, and the blank control group only contained the same volume of incomplete culture medium and incubated for 24 hours.

[0520] 3. Cytotoxicity test of CCC-ESF-1 cells

[0521] A normal group, a blank control group, and multiple CCC-ESF-1 cell drug intervention groups were set up;

[0522] Ethosome homogenates with alcohol contents of 10%, 20%, and 30% were diluted with incomplete culture medium, and each alcohol content of the ethosome homogenate was diluted into multiple ethosome samples with retinol concentrations of 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0, respectively, and added to the corresponding CCC-ESF-1 cell drug intervention groups; the normal group CCC-ESF-1 cells were added with the same volume of incomplete culture medium, and the blank control group only contained the same volume of incomplete culture medium and incubated for 24 hours.

[0523] Results of Experimental Example 14:

[0524] The following formulas were used to calculate the cell viability of HaCaT cells and CCC-ESF-1 cells:

[0525] The cell survival rate was calculated as follows: cell survival rate (%) = (OD experimental group - OD blank group) / (OD control group - OD blank group) × 100;

[0526] In the formula, OD experimental group is the absorbance of cells in each drug intervention group; OD control group is the absorbance of cells in the non-drug intervention group; OD blank group is the absorbance of cells without cells but only with incomplete culture medium.

[0527] Record the experimental results, see Figure 36-37 .

[0528] Fig.35 A schematic diagram of the in vitro cell survival rate of HaCaT cells to be tested;

[0529] Fig.36 A schematic diagram of the in vitro cell survival rate of CCC-ESF-1 test cells.

[0530] Experimental Example 14 Result Analysis:

[0531] The in vitro cellular safety of ethosomes was evaluated using human immortalized epidermal cells (HaCaT cells). The results showed that when the ethosome drug concentration was less than 0.08 μg / mL, the cell survival rate was greater than 80%, and the cytotoxicity and irritation were low.

[0532] For human embryonic skin fibroblasts (CCC-ESF-1), the results showed that when the concentration of ethosome drug was less than 1 μg / mL, the cell survival rate was greater than 90%, the cytotoxicity and irritation were low, and the safety of the preparation was good.

[0533] <Experimental Example 14, Ethosome Skin Irritation Test>

[0534] 1. Single-dose skin irritation test

[0535] The experimental steps include :

[0536] Sixteen healthy male guinea pigs were randomly divided into four experimental groups, namely, experimental groups f, g, h and i, with four pigs in each group;

[0537] The hair on both sides of the spine of the back of each group of guinea pigs was shaved and divided into a left smear area and a right control area, each area was 3×3 cm 2 , equal in size; using the same body left and right comparison method (can be combined with Fig.37 For understanding), 24 hours after hair removal, the left smear area of ​​each group of guinea pigs was smeared with the corresponding test drug preparation, and the right control area was smeared with normal saline; among them, the test drug preparations corresponding to the experimental groups f, g, h and i were the alcohol homogenate solution containing 10% alcohol, the alcohol homogenate solution containing 20% ​​alcohol, the alcohol homogenate solution containing 30% alcohol, and the retinol solution containing 30% ethanol, respectively;

[0538] 2 hours after administration, the test drug preparations of each group were removed with sterile water, and the occurrence of erythema and edema in the test area was observed and recorded at 24, 48 and 72 hours, and scored according to the scoring criteria in Table 24; the skin irritation intensity of each group was determined according to the standards in the "Technical Specifications for Safety of Cosmetics" (Table 25) based on the highest mean score among the three observation time points of 24, 48 and 72 hours;

[0539] At the end of the experiment, the animals were anesthetized by intraperitoneal injection of 2% sodium pentobarbital, and the skin was removed. After fixation with 4% paraformaldehyde, the tissue sections were embedded in paraffin and stained with HE, and the pathological changes of the skin were observed under a microscope.

[0540] Table 24. Skin irritation reaction scores

[0541]

[0542] Table 25. Skin irritation intensity classification

[0543]

[0544] Experimental results and analysis :

[0545] The experimental results are shown in Table 26 and Fig.38 . Fig.38 The figures are a diagram of the skin status of a guinea pig before and after drug removal in a single drug administration treatment, wherein (A) represents the experimental group f which was administered with an alcoholic homogenate solution containing 10% alcohol, (B) represents the experimental group g which was administered with an alcoholic homogenate solution containing 20% ​​alcohol, (C) represents the experimental group h which was administered with an alcoholic homogenate solution containing 30% alcohol, and (D) represents the experimental group i which was administered with an alcoholic homogenate solution containing 30% alcohol.

[0546] Table 26. Results of single-dose skin irritation test in guinea pigs

[0547]

[0548] According to the above results, after a single administration, no erythema or edema reaction was observed on the skin test sites on both sides of the back of guinea pigs in experimental groups f, g, h and i, and the average score of skin irritation reaction in each group at each time point was 0 points, and the average irritation intensity score was <0.5 points, indicating that a single administration of the ethosomes provided in the examples of the present application is non-irritating to the skin.

[0549] 2. Repeated administration skin irritation test

[0550] The experimental method is as follows:

[0551] 16 healthy guinea pigs were divided into 4 experimental groups, namely, experimental groups j, k, l, and m, with 4 animals in each group. After grouping, each group was given medication once every 24 hours for 14 consecutive days. After removing the test substance every day, observe and record the local skin for erythema, edema, etc. and the disappearance. The administration method was the same as the single administration, and the retinol concentration of each group was 0.1 mg / mL. The test drug preparations corresponding to experimental groups j, k, l, and m were, respectively, 10% alcohol homogenous alcohol solution, 20% alcohol homogenous alcohol solution, 30% alcohol homogenous alcohol solution, and 30% ethanol retinol solution.

[0552] After the administration, the animals were anesthetized by intraperitoneal injection of 2% sodium pentobarbital, and the skin was removed. After fixation with 4% paraformaldehyde, the tissue sections were embedded in paraffin and stained with hematoxylin-eosin (HE), and the pathological changes of the skin were observed under a microscope. In addition, the average score of each animal per day was calculated according to the following formula to determine the intensity of stimulation:

[0553] The calculation formula for the average score per animal per day is:

[0554] ;

[0555] Among them, the erythema and edema scores can be calculated according to the skin irritation reaction scoring standard in Table 24.

[0556] Experimental results and analysis:

[0557] 1. Skin irritation results

[0558] Results Fig.39 And Table 27. Fig.39 The figures show the skin status of guinea pigs in experimental groups j, k, l, m before administration, during administration (5th, 10th, 14th day) and after drug withdrawal; (A) represents the group administered with an alcoholic homogenate solution containing 10% alcohol; (B) represents the group administered with an alcoholic homogenate solution containing 10% alcohol; (C) represents the group administered with an alcoholic homogenate solution containing 10% alcohol; (D) represents the group administered with 30% ethanol retinol solution.

[0559] Table 27. Guinea pig multiple administration skin irritation test

[0560]

[0561] In addition, the average values ​​of total irritation response within 14 days in experimental groups j, k, l, and m were 0.16, 0.16, 0.11, and 0.80, respectively, and the stimulation intensity was non-irritating, non-irritating, non-irritating, and mildly irritating, respectively.

[0562] According to the above results, we can know that:

[0563] On the 5th day after multiple administration, no erythema or edema was observed in the skin test sites on both sides of the back of guinea pigs in each group.

[0564] On the 6th day, the free drug group, which was administered with 30% ethanol retinol solution, began to develop mild erythema.

[0565] On the 9th day, the skin irritation reaction of the free drug group was significantly enhanced, and obvious erythema appeared. This symptom lasted until the end of the dosing cycle. Five days after stopping the drug, there was still slight erythema on the skin of the free drug group.

[0566] In the group given the drug in the form of ethosome homogenate, mild erythema began to appear on the 9th day, while the skin irritation reaction of guinea pigs remained at a low level throughout the administration period. After drug withdrawal, the erythema of the preparation group subsided rapidly, indicating that the presence of ethosome carriers may delay the release of retinol and skin irritation reaction, and has potential advantages in reducing skin irritation.

[0567] The average irritation intensity scores of the alcoholic homogenate solutions containing 10%, 20%, and 30% alcohol, and the 30% ethanol retinol solution were 0.16, 0.16, 0.11, and 0.80 points, respectively. The results showed that 30% ethanol retinol solution was mildly irritating, while the retinol-loaded alcoholic bodies with different alcohol contents showed lower skin irritation after multiple administrations.

[0568] 2. Skin pathological changes results The specific experimental results are shown in Fig.40 . Fig.40 This is a HE staining image of the guinea pig skin after multiple drug administration (HE, ×200); in the figure, (A) represents the staining result of the blank control skin (no drug treatment), (B) represents the staining result of the skin treated with an alcohol homogenate solution containing 10% alcohol, (C) represents the staining result of the skin treated with an alcohol homogenate solution containing 20% ​​alcohol, (D) represents the staining result of the skin treated with an alcohol homogenate solution containing 30% alcohol, and (E) represents the staining result of the skin treated with 30% ethanol retinol solution.

[0569] According to the above results, the skin microstructure of the retinol-containing alcohol preparation groups with alcohol content of 10%, 20%, and 30% did not change compared with the blank control group. The structures were intact, the skin appendages were not damaged, and the epithelium had no edema or hyperplasia. Compared with the preparation group, the epithelium of the 30% ethanol retinol solution group was thickened, the stratum corneum was thinned, and the spinous cell layer was hyperplastic.

[0570] 3. Summary

[0571] (1) Sustained-release delivery. The ethosomes of the present application may slow down the diffusion rate of the drug from the lattice due to the lipid layer encapsulating retinol, thereby achieving a sustained-release effect, thereby reducing its immediate irritation reaction to the skin, helping to reduce the skin's sensitivity to retinol, reducing the occurrence of adverse reactions, and providing the skin with more time to adapt.

[0572] (2) Protect the integrity of the skin barrier. While retinol exerts its anti-aging effects, it may thin the stratum corneum of the skin and weaken the skin barrier function. When the stratum corneum becomes thinner, the skin's resistance to external stimuli decreases, and the skin is more susceptible to damage by bacteria and environmental factors. Compared with the free drug group of 30% ethanol retinol solution, the retinol-loaded plasmosome preparation of the embodiment of the present application shows good skin compatibility, can reduce the potential irritation of retinol, and can also protect and maintain the integrity of the skin barrier, which helps to provide users with a more gentle and effective skin care option.

[0573] (3) Moisturizing and reducing dryness. The thinning of the stratum corneum may also affect the skin's ability to retain moisture, leading to dryness and desquamation. The sustained-release properties of the monooleylglycerol-based ethosomes of the present application can reduce the damage of the retinol drug to the stratum corneum structure and help the skin maintain its natural moisturizing state. In addition, the propylene glycol and dipropylene glycol components in the ethosome preparation formula can enhance skin hydration and effectively moisturize the skin, while also helping to alleviate the problem of dry skin caused by the use of retinol.

[0574] The above specific embodiments have been described in detail for the present invention, but these do not constitute limitations on the present invention. The protection scope of the present invention is not limited to the above embodiments, and any equivalent modifications or changes made by ordinary technicians in the field based on the content disclosed by the present invention should be included in the protection scope recorded in the claims.

Claims

1. A method for preparing an ethosome, characterized in that: include: Stirring monoolein and poloxamer 407 at a first temperature and a predetermined rotation speed to mix, and then cooling to a second temperature to obtain an ester capsule material, wherein the predetermined rotation speed is 300-1000 rpm / min, the first temperature is greater than the second temperature, the first temperature is 31-100° C., and the second temperature is less than or equal to 30° C.; Obtaining an alcohol mixture containing a short-chain alcohol and an encapsulated substance; wherein the short-chain alcohol comprises one or more of ethanol, propylene glycol and dipropylene glycol, and the encapsulated substance is selected from retinol; Mixing the alcohol mixture and the ester capsule material to obtain an ester alcohol mixture; Dissolving a stabilizer in water to obtain an aqueous solution containing the stabilizer; wherein the stabilizer comprises one or more of ergothioneine, dipotassium glycyrrhizinate, vitamin E polyethylene glycol succinate and tocopherol acetate; adding the aqueous solution containing the stabilizer to the ester alcohol mixture to perform a hydration treatment to obtain a hydrated liquid; The hydration liquid is homogenized to obtain an alcoholate homogenized liquid containing alcoholate; wherein the homogenization pressure of the homogenization is 600-1000 Pa; Wherein, the solvent system used to prepare the alcohol body includes the short-chain alcohol and the water; The volume fraction of the short-chain alcohol in the solvent system is 10-30%; The ratio of the mass of the monoolein to the volume of the solvent system is 1 to 30 mg / mL; The ratio of the mass of the encapsulated material to the volume of the solvent system is 1-4 mg / mL; The mass ratio of the glyceryl monooleate to the poloxamer 407 is 7:1 to 17:

1.

2. The method for preparing an ethanosome according to claim 1, characterized in that: The second temperature is 0-30°C.

3. The method for preparing an ethanosome according to claim 1, characterized in that: The first temperature is 50-95°C.

4. The method for preparing an ethanosome according to claim 1, characterized in that: The first temperature is 60-95°C.

5. The method for preparing an ethanosome according to claim 1, characterized in that: The step of stirring and mixing glyceryl monooleate and poloxamer 407 at a first temperature and a predetermined rotation speed comprises: Glyceryl monooleate, poloxamer 407 and a first antioxidant are stirred and mixed at a first temperature and a predetermined rotation speed, wherein the first antioxidant is selected from pentaerythritol tetra(di-tert-butylhydroxyoxycinnamate), and the mass ratio of the first antioxidant to the glyceryl monooleate is (0.001-0.5):

1.

6. The method for preparing an ethosome according to claim 1, characterized in that: The method of obtaining an alcohol mixture containing short-chain alcohols and encapsulated substances comprises: The encapsulated material and the second antioxidant are dissolved in a short-chain alcohol to obtain the alcohol mixture; wherein the second antioxidant is selected from tocopherol acetate, and the mass ratio of the second antioxidant to the encapsulated material is (0.001~2):

1.

7. The method for preparing an ethosome according to claim 1, characterized in that: The short-chain alcohol consists of propylene glycol and dipropylene glycol, The volume ratio of the propylene glycol to the dipropylene glycol is (3:7) to (7:3).

8. The method for preparing an ethosome according to claim 7, characterized in that: The volume ratio of the propylene glycol to the dipropylene glycol is 5:

5.

9. The method for preparing an ethosome according to claim 1, characterized in that: The short-chain alcohol consists of ethanol and propylene glycol.

10. The method for preparing an ethosome according to claim 1, characterized in that: The short-chain alcohol consists of ethanol and dipropylene glycol.

11. The method for preparing an ethosome according to claim 1, characterized in that: The mass ratio of the stabilizer to the glyceryl monooleate is 1:

3.

12. The method for preparing an ethosome according to claim 1, characterized in that: The mass ratio of the encapsulated material to the glyceryl monooleate is 1:5-15.

13. A method for preparing an ethosome, characterized in that: include: Glyceryl monooleate and poloxamer 407 were mixed by stirring at a temperature of 60° C. and a rotation speed of 500 rpm / min, and then cooled to 30° C. to obtain an ester capsule material; Dissolving retinol in a short-chain alcohol and dissolving by ultrasonication to obtain an alcohol mixture; wherein the short-chain alcohol is composed of propylene glycol and dipropylene glycol, and the volume ratio of the propylene glycol to the dipropylene glycol is 3:7 to 7:3; Stirring the alcohol mixture and the ester capsule material for 30 min to obtain an ester alcohol mixture; Dissolving the stabilizer in water and dissolving by ultrasonication to obtain an aqueous solution containing the stabilizer; Slowly dropwise adding the aqueous solution into the ester alcohol mixture and stirring together for 60 min to obtain a hydrated solution; The hydrated liquid is homogenized at a homogenizing pressure of 500-1000 bar for 5 min to obtain an alcoholic homogenous liquid containing alcoholic bodies; Wherein, the solvent system for preparing the alcohol body comprises the short-chain alcohol and the water, and the volume fraction of the short-chain alcohol in the solvent system is 10-30%; The concentration of the monoolein is 1-30 mg / mL; The concentration of retinol is 1-4 mg / mL; The mass ratio of the monoolein to the poloxamer 407 is 10:1; The stabilizer is selected from one or more of ergothioneine, dipotassium glycyrrhizinate, vitamin E polyethylene glycol succinate and tocopherol acetate, and the mass ratio of the stabilizer to the monoolein is 1:

3.

14. A method for preparing an ethosome, characterized in that: include: Glyceryl monooleate, poloxamer 407, and pentaerythritol tetra(di-tert-butylhydroxyoxycinnamate) ester were stirred and mixed at a temperature of 95° C. and a rotation speed of 500 rpm / min, and then cooled to 30° C. to obtain an ester capsule material; Retinol and tocopherol acetate are dissolved in short-chain alcohols and ultrasonically dissolved to obtain an alcohol mixture; wherein the short-chain alcohol is prepared from propylene glycol and dipropylene glycol in a volume ratio of 5:5; Stirring the alcohol mixture and the ester capsule material for 30 min to obtain an ester alcohol mixture; Dissolve vitamin E polyethylene glycol succinate in water and dissolve by ultrasonication to obtain an aqueous solution; Slowly dropwise adding the aqueous solution into the ester alcohol mixture and stirring together for 60 min to obtain a hydrated solution; homogenizing the hydrated liquid at a homogenizing pressure of 1000 bar for 5 min to obtain an alcoholate homogenous liquid containing alcoholates; Wherein, the solvent system for preparing the alcohol body comprises the short-chain alcohol and the water, and the volume fraction of the short-chain alcohol in the solvent system is 10-30%; The concentration of the monoolein is 15 mg / mL; The concentration of Poloxamer 407 is 1.5 mg / mL; The concentration of pentaerythritol tetrakis (di-tert-butylhydroxyoxycinnamate) ester is 0.7 mg / mL; The concentration of retinol is 1 mg / mL; The concentration of tocopherol acetate is 2 mg / mL; The concentration of the vitamin E polyethylene glycol succinate is 10 mg / mL.

15. Ethosome prepared according to the preparation method according to any one of claims 1 to 14.

16. Use of the ethosome according to claim 15 in preparing a carrier containing an encapsulated object.

17. Use of the ethosome according to claim 15 in preparing a skin care product.

18. A skin care product, characterized in that Including the alcohol body described in claim 15.

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