Non-covalent targeted entrapped Ectoin liposome, liposome preparation, cosmetic and preparation method thereof

Abstract

The invention discloses a non-covalent targeted entrapped Ectoine liposome, a liposome preparation, a cosmetic and a preparation method thereof, and belongs to the technical field of cosmetics. The non-covalent targeted entrapped Ectoine liposome is prepared from the following raw materials: phospholipid, an organic solvent, cholesterol, an emulsifier, polyhydric alcohol, Ectoine, chitosan, an acetic acid aqueous solution, hyaluronic acid and water, the emulsifying agent is prepared from beheneth-25. According to the application, hyaluronic acid is modified to the Ectoine liposome by utilizing the electrostatic adsorption effect of chitosan of cationic polysaccharide on hyaluronic acid of anionic polysaccharide, and beheneth-25 is adopted as an emulsifier to prepare the Ectoine liposome which is homogeneous, small in particle size, narrow in particle size distribution and good in stability. According to the present invention, the targeting effect on fibroblasts can be improved, the transdermal effect can be improved, the binding speed with the fibroblasts can be accelerated, the transdermal absorption efficiency of Ectoine can be significantly improved, the effect of Ectoine can be promoted, and the application requirements of Ectoine in the field of cosmetics can be met.

Description

Technical Field

[0001] This application relates to the technical field of cosmetics, and particularly to non-covalently targeted encapsulation of ectoine liposomes, liposome preparations, cosmetics and their preparation methods. Background Art

[0002] Ectoine is a naturally occurring four-carbon heterocyclic compound with good water solubility, having excellent anti-ultraviolet, antibacterial, antioxidant and moisturizing effects, and can effectively enhance the immune protection ability of skin cells, increase cell repair ability, enabling the skin to effectively resist the invasion of microorganisms and allergens. Therefore, ectoine is widely used as a cosmetic efficacy raw material.

[0003] However, due to the complex barrier function of the skin, especially the barrier function of the stratum corneum of the skin, ectoine is not easily absorbed by the skin, which limits its application in cosmetics. Therefore, it is necessary to find a method that can effectively promote the transdermal absorption of ectoine.

[0004] Liposomes are closed vesicles with a bilayer structure formed by amphiphilic substances such as phospholipids, with an internal aqueous phase and a cell membrane-like structure. Water-soluble efficacy raw materials are encapsulated in the hydrophilic group sandwich of the vesicles, while lipid-soluble efficacy raw materials are dispersed in the hydrophobic group sandwich of the vesicles. Liposomes can improve the solubility and stability of efficacy raw materials, and promote their penetration absorption and action time on the skin, and are widely used in cosmetic efficacy raw materials.

[0005] However, ordinary liposomes still have deficiencies in terms of targeting and absorption efficiency, and cannot effectively exert the moisturizing, anti-aging and other effects of ectoine.

[0006] In the current context of the continuous innovation of cosmetic raw material preparation technologies, targeted liposomes, as a highly potential delivery system, provide a new direction for the precise delivery of cosmetic efficacy components. Among them, non-covalently targeted liposomes mainly rely on non-covalent interactions between liposomes and target cells, such as hydrogen bonds, electrostatic interactions, van der Waals forces and hydrophobic interactions, etc. to achieve targeted transportation. These non-covalent interactions are similar to the specific binding between antibodies and antigens, and multiple weak interactions work together, enabling liposomes to specifically bind to the surface of target cells, and then deliver the encapsulated efficacy components into the interior of target cells. Compared with covalently targeted liposomes, the preparation process of non-covalently targeted liposomes is relatively simple, only requiring the modification of ligands with non-covalent binding ability on the surface of liposomes, and the requirements for reaction conditions are also more relaxed.

[0007] Hyaluronic acid (HA) and chitosan (CS) have unique advantages in the construction of non-covalent targeted liposomes. Negatively charged HA can specifically bind to the CD44 receptor, enhancing the targeting of liposomes; positively charged CS has good biocompatibility, adsorption, and certain antibacterial properties, which can improve the stability of liposomes and their interaction with the skin. HA and negatively charged CS can enhance the stability of HA-targeted liposomes through electrostatic adsorption. Applying them to the preparation of non-covalent targeted liposomes is expected to further improve the performance of liposomes and provide a more effective means for the delivery of active ingredients in cosmetics. Summary of the Invention

[0008] Based on this, the main object of this application is to provide a non-covalent targeted liposome encapsulating ectoine, which can significantly improve the transdermal absorption efficiency of ectoine, promote the exertion of its efficacy, and meet its application requirements in the cosmetics field.

[0009] In the first aspect of this application, a non-covalent targeted liposome encapsulating ectoine is provided, comprising the following raw materials:

[0010] Phospholipid, organic solvent, cholesterol, emulsifier, polyol, ectoine, chitosan, acetic acid aqueous solution, hyaluronic acid, and water;

[0011] The emulsifier includes behenyl alcohol polyether-25.

[0012] In some embodiments, by weight, the non-covalent targeted liposome encapsulating ectoine comprises the following raw materials:

[0013] 1-10 parts of phospholipid, 1-10 parts of organic solvent, 0.01-0.5 parts of cholesterol, 0.1-5 parts of emulsifier, 0.1-20 parts of polyol, 1-10 parts of ectoine, 0.1-1 part of chitosan, 5-50 parts of acetic acid aqueous solution, 0.1-2 parts of hyaluronic acid, and 50-100 parts of water.

[0014] In some embodiments, the non-covalent targeted liposome encapsulating ectoine further comprises an auxiliary agent; the auxiliary agent includes at least one of an antioxidant, a preservative, and a pH regulator.

[0015] In some embodiments, the non-covalent targeted liposome encapsulating ectoine satisfies one or more of the following conditions:

[0016] (1) The phospholipid includes at least one of soybean lecithin, cephalin, phosphatidylinositol, sphingomyelin, and egg yolk lecithin;

[0017] (2) The organic solvent includes ethanol;

[0018] (3) The polyol includes at least one of glycerol, butanediol, and pentanediol; preferably glycerol and / or pentanediol;

[0019] (4) The mass concentration of the aqueous acetic acid solution is 0.5 - 2%;

[0020] (5) The antioxidant includes at least one of pentaerythritol tetrakis (di - tert - butyl hydroxyhydrocinnamate), tocopherol, terbutyl hydroxyanisole, tert - butyl - p - cresol, and propyl gallate;

[0021] (6) The preservative includes at least one of hydroxyacetophenone, methyl p - hydroxybenzoate, ethyl p - hydroxybenzoate, butyl p - hydroxybenzoate, benzoic acid, sodium benzoate, and potassium sorbate;

[0022] (7) The pH regulator includes at least one of sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, and triethanolamine;

[0023] (8) The dosage of the antioxidant is 0.01 - 0.4 parts;

[0024] (9) The dosage of the preservative is 0.05 - 1.05 parts;

[0025] (10) The dosage of the pH regulator is based on adjusting the pH of the non - covalently targeted encapsulating ectoine liposome to 4.5 - 7.5.

[0026] In some embodiments, the non - covalently targeted encapsulating ectoine liposome comprises the following raw materials:

[0027] Phospholipid, organic solvent, cholesterol, emulsifier, antioxidant, polyol, ectoine, chitosan, aqueous acetic acid solution, hyaluronic acid, and water.

[0028] In a second aspect, the present application provides a method for preparing the non - covalently targeted encapsulating ectoine liposome described in the first aspect, comprising the following steps: characterized in that, it comprises the following steps:

[0029] Mix the phospholipid, the cholesterol, the emulsifier, and the organic solvent to prepare an oil phase;

[0030] Mix the polyol and the oil phase to prepare a primary emulsion;

[0031] Remove the organic solvent in the primary emulsion to form blank liposomes;

[0032] Mix the ectoine with 30 - 60% of water to form an ectoine solution; mix the ectoine solution with the blank liposomes to prepare ectoine liposomes;

[0033] Prepare a chitosan solution by mixing chitosan with an aqueous acetic acid solution; mix the chitosan solution with the ectoine liposomes to prepare chitosan - modified ectoine liposomes;

[0034] Dissolve hyaluronic acid in the remaining water to form a hyaluronic acid solution; mix the hyaluronic acid solution with the chitosan-modified ectoine liposomes to prepare the non-covalently targeted ectoine-loaded liposomes.

[0035] In some embodiments, the preparation method satisfies one or more of the following conditions:

[0036] (1) The preparation temperature of the oil phase is 40 - 60 °C;

[0037] (2) The method for removing the organic solvent from the primary emulsion includes rotary evaporation or vacuum concentration;

[0038] (3) After the step of preparing the ectoine liposomes, a step of homogenizing the ectoine liposomes is further included.

[0039] In some embodiments, the conditions for rotary evaporation include: evaporation temperature 40 - 60 °C, rotation speed 20 - 100 rpm;

[0040] The conditions for homogenization include: homogenization pressure 300 bar - 700 bar, and the number of homogenization times is 1 - 5 times.

[0041] In a third aspect, the present application provides a liposome preparation, the raw materials of which include the non-covalently targeted ectoine-loaded liposomes described in the first aspect or the non-covalently targeted ectoine-loaded liposomes prepared by the preparation method described in the second aspect.

[0042] In a fourth aspect, the present application provides a cosmetic, which includes the non-covalently targeted ectoine-loaded liposomes described in the first aspect, the non-covalently targeted ectoine-loaded liposomes prepared by the preparation method described in the second aspect, or the liposome preparation described in the third aspect.

[0043] In some embodiments, the cosmetic includes at least one of a facial mask, eye cream, facial cream, primer, essence, lotion, and skin softener.

[0044] Advantages of the present application:

[0045] 1. This application uses chitosan and hyaluronic acid to non-covalently modify ectoin liposomes to prepare non-covalently targeted ectoin-loaded liposomes. The electrostatic adsorption of hyaluronic acid, an anionic polysaccharide, by chitosan, a cationic polysaccharide, is utilized to modify hyaluronic acid onto ectoin liposomes. Behenyl polyether-25 is used as an emulsifier to prepare homogeneous ectoin liposomes with small particle size, narrow distribution, and good stability. Through the targeting effect of hyaluronic acid on fibroblasts, the transdermal effect of ectoin liposomes is improved, and the binding rate between ectoin liposomes and fibroblasts is accelerated, thereby significantly enhancing the transdermal absorption efficiency of ectoin, promoting the exertion of its efficacy, and meeting the application requirements in the cosmetics field.

[0046] 2. The non-covalently targeted ectoin-loaded liposomes of this application have good stability; have a controlled release effect, with a slower release rate compared to unmodified ectoin liposomes; have an improved transdermal effect, with a significantly increased cumulative release amount and cumulative retention amount compared to unmodified ectoin liposomes; have a targeting effect on fibroblasts, with a significantly increased accumulation on fibroblasts compared to unmodified ectoin liposomes; have an improved functional effect, and compared to unmodified ectoin liposomes, can enhance skin hydration, regulate transepidermal water loss, and improve skin elasticity, providing great potential for the delivery of the outer membrane of the skin and laying a foundation for skin health applications.

[0047] 3. The preparation method of the non-covalently targeted ectoin-loaded liposomes of this application is simple, has high repeatability, short preparation time, no residue of harmful substances, and high encapsulation efficiency. Without complex mechanical processes and equipment, the product quality and process can have good reproducibility and stability, and are easy to industrialize. Description of the Drawings

[0048] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. The drawings are only used for the purpose of showing the preferred embodiments and are not considered as a limitation to this application. Throughout the drawings, the same reference signs are used to represent the same components. In the drawings:

[0049] Figure 1 It is a schematic diagram of the preparation of non-covalently targeted ectoin-loaded liposomes in Example 1;

[0050] Figure 2 It is a TEM electron micrograph of the non-covalently targeted ectoin-loaded liposomes prepared from Sample 2 in Example 1;

[0051] Figure 3 It is a graph showing the changing trends of the particle size, PDI, and potential of the non-covalently targeted ectoin-loaded liposomes prepared from Sample 2 in Example 1 under 7 different environments;

[0052] Figure 4 Results graph of the Turbiscan lab stability analyzer for the non-covalently targeted encapsulation of ectoine liposomes prepared from Sample 2 of Example 1;

[0053] Figure 5 Results graph of the Franz diffusion cell transdermal test for the non-covalently targeted encapsulation of ectoine liposomes prepared from Sample 2 of Example 1, the non-targeted encapsulation of ectoine liposomes of Comparative Example 1, and the free ectoine solution of Comparative Example 2, where A is the cumulative permeation amount and B is the cumulative retention amount;

[0054] Figure 6 Results graph of the sustained release test for the non-covalently targeted encapsulation of ectoine liposomes prepared from Sample 2 of Example 1, the non-targeted encapsulation of ectoine liposomes of Comparative Example 1, and the free ectoine solution of Comparative Example 2;

[0055] Figure 7 Results graph of the cell fluorescence data analysis for the non-covalently targeted encapsulation of FITC liposomes and non-targeted FITC liposomes prepared in Test Example 7;

[0056] Figure 8 Results graph of the human efficacy experiment data for the essence of non-covalently targeted encapsulation of ectoine liposomes prepared from Sample 2 of Example 1, the essence of non-targeted encapsulation of ectoine liposomes of Comparative Example 1, the free ectoine solution of Comparative Example 2, and the blank essence, where A is the skin water content increase rate at 1 week, B is the reduction rate of transepidermal water loss at 1 week, and C is the skin elasticity increase rate at 4 weeks. Detailed implementation manners

[0057] To make the objectives, technical solutions, and advantages of this application clearer and the understanding of the disclosed content of this application more thorough and comprehensive, the technical solutions of this application will be clearly and completely described below in conjunction with specific embodiments of this application and the corresponding drawings. The described embodiments are only a part of the embodiments of this application, rather than all of the embodiments.

[0058] The following will describe the implementation of this application in detail with reference to the drawings. This embodiment is implemented on the premise of the technical solution of this application, and detailed implementation manners and specific operation processes are given, but the protection scope of this application is not limited to the following embodiments.

[0059] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this application belongs. The terms used in the description of this application herein are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0060] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. The terms used in the description of this application herein are for the purpose of describing specific embodiments only and are not intended to limit this application. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0061] The terms "preferably", "more preferably", "more preferably", "even more preferably", etc. in this application refer to embodiments of this application that can provide certain beneficial effects in certain cases. However, in the same or other cases, other embodiments may also be preferred. In addition, the description of one or more preferred embodiments does not imply that other embodiments are not available, nor is it intended to exclude other embodiments from the scope of this application. That is, in this application, "preferably", "more preferably", "more preferably", "even more preferably", etc. are only used to describe embodiments or examples with better effects, but do not constitute a limitation on the protection scope of this application.

[0062] In this application, "further", "even further", "especially", etc. are used for descriptive purposes and represent differences in content, but should not be construed as a limitation on the protection scope of this application.

[0063] In this application, "above" and "below" both include the number itself. For example, "below 1" means ≥1.

[0064] In this application, the meaning of "at least one" is more than one, such as one, one and more than one. The meaning of "multiple" or "several" is at least one, such as one, three, etc. The meaning of "multiple layers" is at least two layers, such as two layers, three layers, etc., unless otherwise specifically defined. In the description of this application, the meaning of "several" is at least one, such as one, two, etc., unless otherwise specifically defined.

[0065] When a numerical range is disclosed in this application, the above range is considered continuous and includes the minimum and maximum values of the range, as well as each value between such minimum and maximum values. Further, when the range refers to integers, it includes each integer between the minimum and maximum values of the range. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all sub-ranges subsumed therein.

[0066] Unless otherwise specified, all steps of this application can be carried out sequentially or randomly. For example, the method includes steps (a) and (b), which means that the method may include steps (a) and (b) carried out sequentially, or may also include steps (b) and (a) carried out sequentially. For example, when it is mentioned that the method may further include step (c), it means that step (c) can be added to the method in any order. For example, the method may include steps (a), (b) and (c) in sequence, or may also include steps (a), (c) and (b), or may include steps (c), (a) and (b), etc.

[0067] In this application, the terms "first" and "second" are only used for descriptive purposes and should not be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, features defined with "first" and "second" may explicitly or implicitly include at least one such feature.

[0068] In this application, "comprising", "including", "containing", "having" or other variants are intended to cover non-closed inclusion, and no distinction is made between these terms. The term "including" means that other steps and components can be added without affecting the final result. The compositions and methods / processes of this application comprise, consist of, and consist essentially of the essential elements and limitations described herein, as well as any additional or optional components, ingredients, steps, or limitations described herein.

[0069] In this application, no distinction is made between the terms "efficacy", "performance", "effect", and "function".

[0070] The weights of the relevant components mentioned in the specification of the embodiments of this application not only can refer to the specific contents of each component, but also can represent the weight ratio relationship between each component. Therefore, as long as the contents of the relevant components in the specification of the embodiments of this application are scaled up or down in proportion, they are within the scope disclosed in the specification of the embodiments of this application. Specifically, the weights described in the specification of the embodiments of this application can be mass units well-known in the chemical industry such as μg, mg, g, kg, etc.

[0071] In this application, temperature parameters, unless otherwise specified, are allowed to be either isothermal treatment or treatment within a certain temperature range. The isothermal treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. The room temperature referred to in this application is 0 - 40 °C, preferably 10 °C - 35 °C, and further preferably 20 °C - 30 °C.

[0072] Unless otherwise mentioned, terms in the singular form may include the plural form and should not be construed as having a quantity of one.

[0073] In addition, the drawings of the present application are not drawn to a scale of 1:1, and the relative sizes of the components are only drawn by way of example in the drawings for the convenience of understanding the present application, but are not necessarily drawn to the true scale, and the scale in the drawings does not constitute a limitation to the present application.

[0074] In the present application, unless otherwise specified, the temperature parameter is allowed to be a constant temperature treatment or to vary within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within ranges such as ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

[0075] In the present application, for the units involving data ranges, if the unit is only attached after the right endpoint, it means that the units of the left endpoint and the right endpoint are the same. For example, 2 - 5h means that the units of the left endpoint "2" and the right endpoint "5" are both h (hours).

[0076] In the first aspect of the present application, a non-covalent targeting-encapsulated ectoin liposome is provided, comprising the following raw materials:

[0077] Phospholipids, organic solvents, cholesterol, emulsifiers, polyols, ectoin, chitosan, acetic acid aqueous solution, hyaluronic acid, and water;

[0078] The emulsifier includes behenyl alcohol polyether-25.

[0079] Due to the complex barrier function of the skin, especially the barrier function of the stratum corneum of the skin, ectoin is not easily absorbed by the skin, which limits its application in cosmetics. Liposomes can improve the solubility and stability of efficacy raw materials, and promote their penetration and absorption and the action time on the skin, and are widely used in cosmetic efficacy raw materials. However, ordinary liposomes still have deficiencies in terms of targeting and absorption efficiency, and cannot effectively exert the moisturizing, anti-aging and other effects of ectoin.

[0080] Hyaluronic acid can modify the structure of the stratum corneum of the skin, recognize receptors (such as CD44) on the surface of fibroblasts, and achieve conduction and retention effects. The CD44 receptor has a high affinity for the binding of hyaluronic acid (HA), and is overexpressed in fibroblasts compared with normal somatic cells, making it an ideal active targeting receptor. Chitosan is a cationic polysaccharide existing in nature, with good adsorption, biocompatibility, permeability and low toxicity. Therefore, it is usually used as a biomaterial, especially in drug delivery systems. In addition, the anionic polysaccharide hyaluronic acid can interact with the cationic polysaccharide chitosan through non-covalent binding such as electrostatic attraction.

[0081] The particle size of liposomes has a great impact on their transdermal effect and the degree of uptake by skin cells. Liposomes with smaller particle sizes (≤300 nm) have better transdermal effects and are more easily taken up by skin cells, while liposomes with larger particle sizes are more difficult to penetrate the skin surface layer and are easily phagocytosed by immune cells such as macrophages in the skin. Therefore, their transdermal effect is poor and it is difficult to be taken up by skin cells.

[0082] In this application, chitosan and hyaluronic acid are used to non-covalently modify ectoine liposomes to prepare non-covalently targeted encapsulated ectoine liposomes. The electrostatic adsorption of hyaluronic acid, an anionic polysaccharide, by chitosan, a cationic polysaccharide, is utilized to modify hyaluronic acid onto ectoine liposomes. Through the targeting effect of hyaluronic acid on fibroblasts, the transdermal effect of ectoine liposomes is improved, and the binding rate between ectoine liposomes and fibroblasts is accelerated, thereby significantly enhancing the transdermal absorption efficiency of ectoine, promoting the exertion of its efficacy, and meeting its application requirements in the cosmetics field.

[0083] In addition, the emulsifier helps to disperse lipid materials in the aqueous phase through emulsification to form a stable liposome structure. The structure of the emulsifier has a great impact on the stability of liposomes, the size of liposomes, etc. In this application, by using behenyl alcohol polyether-25 as the emulsifier, liposomes with a homogeneous phase, small particle size, narrow distribution, and good stability can be prepared, improving the transdermal effect of liposomes and promoting the uptake of liposomes by skin cells, thereby enhancing the transdermal absorption efficiency of ectoine.

[0084] In some embodiments, by weight, the non-covalently targeted encapsulated ectoine liposomes comprise the following raw materials:

[0085] 1 - 10 parts of phospholipid, 1 - 10 parts of organic solvent, 0.01 - 0.5 part of cholesterol, 0.1 - 5 parts of emulsifier, 0.1 - 20 parts of polyol, 1 - 10 parts of ectoine, 0.1 - 1 part of chitosan, 5 - 50 parts of acetic acid aqueous solution, 0.1 - 2 parts of hyaluronic acid, and 50 - 100 parts of water.

[0086] In this application, the role of phospholipid is to form the bilayer structure of liposomes, and there is no special limitation on the specific type. Optionally, the phospholipid includes at least one of soybean lecithin, cephalin, phosphatidylinositol, sphingomyelin, and egg yolk lecithin, preferably soybean lecithin, and more preferably soybean lecithin PC60. Its dosage can specifically be 1.0 part, 2.0 parts, 3.0 parts, 4.0 parts, 5.0 parts, 6.0 parts, 7.0 parts, 8.0 parts, 9.0 parts, or 10.0 parts, preferably 2 parts.

[0087] Specifically, the dosage of chitosan can be 0.1 part, 0.15 part, 0.20 part, 0.25 part, 0.30 part, 0.35 part, 0.40 part, 0.45 part, 0.50 part, 0.55 part, 0.60 part, 0.65 part, 0.7 part, 0.75 part, 0.80 part, 0.85 part, 0.90 part, 0.95 part or 1.0 part, preferably 0.25 part.

[0088] Specifically, the mass concentration of the acetic acid aqueous solution is 0.5-2%, such as 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, etc.; its dosage can specifically be 5 parts, 10 parts, 15 parts, 20 parts, 25 parts, 30 parts, 35 parts, 40 parts, 45 parts, 50 parts, etc.

[0089] Specifically, the dosage of hyaluronic acid can be 0.1 part, 0.15 part, 0.20 part, 0.25 part, 0.30 part, 0.35 part, 0.40 part, 0.45 part, 0.50 part, 0.55 part, 0.60 part, 0.65 part, 0.7 part, 0.75 part, 0.80 part, 0.85 part, 0.90 part, 0.95 part, 1.0 part, 1.25 part, 1.5 part, 1.75 part or 2 parts, preferably 0.50 part. The average molecular weight of hyaluronic acid can be 1-20 kDa, preferably 5-10 kDa, and can specifically be 1 kDa, 2 kDa, 4 kDa, 5 kDa, 6 kDa, 7 kDa, 8 kDa, 9 kDa, 10 kDa, 12 kDa, 14 kDa, 16 kDa, 18 kDa, 20 kDa, etc.

[0090] In the present application, the function of the organic solvent is to dissolve phospholipids, cholesterol, emulsifiers, antioxidants, and prepare the oil phase. The organic solvent includes ethanol. The specific dosage of the organic solvent can be 1.0 part, 2.0 part, 3.0 part, 4.0 part, 5.0 part, 6.0 part, 7.0 part, 8.0 part, 9.0 part or 10.0 part, preferably 2.0 part.

[0091] Specifically, the dosage of cholesterol can be 0.01 part, 0.05 part, 0.7 part, 0.10 part, 0.15 part, 0.15 part, 0.20 part, 0.25 part, 0.30 part, 0.35 part, 0.40 part, 0.45 part or 0.50 part, preferably 0.07 part.

[0092] Furthermore, maintaining the mass ratio of the phospholipid to the cholesterol at (28 - 32):1 is beneficial to enhancing the stability of the vesicles and reducing the permeability of the lipid membrane to solutes. It can be understood that the mass ratio of the lecithin to the cholesterol includes but is not limited to 28:1, 28.5:1, 29:1, 29.5:1, 30:1, 30.5:1, 31:1, 31.5:1, or 32:1, preferably 30:1.

[0093] Specifically, the dosage of the emulsifier can be 0.10 parts, 0.50 parts, 0.82 parts, 1.00 parts, 1.5 parts, 2 parts, 2.5 parts, 3 parts, 3.5 parts, 4 parts, 4.5 parts, or 5 parts, etc., preferably 0.82 parts.

[0094] Specifically, the dosage of ectoine can be 1.00 parts, 1.50 parts, 2.00 parts, 2.50 parts, 3.00 parts, 3.50 parts, 4.00 parts, 4.50 parts, 5.00 parts, 5.50 parts, 6.00 parts, 6.50 parts, 7.00 parts, 7.50 parts, 8.00 parts, 8.50 parts, 9.00 parts, 9.50 parts, or 10.00 parts, preferably 1.56 parts.

[0095] In this application, the role of the polyol is to stabilize and protect the liposomes, and there is no special limitation on the specific type. Optionally, the polyol includes at least one of glycerol, butanediol, and pentanediol, and pentanediol also has an antiseptic effect; preferably glycerol and / or pentanediol, more preferably glycerol and pentanediol. The dosage of the polyol can specifically be 0.1 part, 0.5 part, 1.0 part, 2.0 part, 2.7 parts, 3.0 parts, 4.0 parts, 5.0 parts, 6.0 parts, 7.0 parts, 8.0 parts, 9.0 parts, 10.0 parts, 10.8 parts, 11.0 parts, 12.0 parts, 13.0 parts, 14.0 parts, 15.0 parts, 16.0 parts, 17.0 parts, 18.0 parts, 19.0 parts, or 20.0 parts, etc., preferably 2.7 parts.

[0096] In some embodiments, the non-covalently targeted encapsulating ectoine liposomes further include an auxiliary agent; the auxiliary agent includes at least one of an antioxidant, a preservative, and a pH regulator.

[0097] Among them, the antioxidant is beneficial to improving the antioxidant property and stability of the liposomes, the preservative is beneficial to improving the safety and stability of the liposomes, and the pH regulator can adjust the pH of the liposomes to an appropriate level, adjust the particle size and distribution of the liposomes, thereby improving the performance of the non-covalently targeted encapsulating ectoine liposomes. It can be selected according to actual needs, and there is no special limitation on the specific type. Multiple types can be used, or one type can be used.

[0098] Optionally, the antioxidant includes at least one of pentaerythritol tetra(bis-tert-butylhydroxyhydrocinnamate), tocopherol, terbutylhydroxyanisole, tert-butylhydroxytoluene, and propyl gallate, preferably pentaerythritol tetra(bis-tert-butylhydroxyhydrocinnamate). The dosage can be 0.01-0.4 parts, including but not limited to 0.01 part, 0.02 parts, 0.026 parts, 0.05 parts, 0.1 part, 0.15 parts, 0.2 parts, 0.25 parts, 0.3 parts, 0.35 parts, or 0.4 parts, etc., preferably 0.026 parts.

[0099] Optionally, the preservative includes at least one of hydroxyacetophenone, methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, butyl p-hydroxybenzoate, benzoic acid, sodium benzoate, and potassium sorbate, preferably hydroxyacetophenone. The dosage can be 0.05-1.05 parts, including but not limited to 0.05 part, 0.06 parts, 0.07 parts, 0.075 parts, 0.08 parts, 0.09 parts, 0.1 part, 0.2 parts, 0.3 parts, 0.4 parts, 0.5 parts, 0.6 parts, 0.7 parts, 0.8 parts, 0.9 parts, 1 part, or 1.05 parts, etc.

[0100] Optionally, the pH regulator includes at least one of sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, and triethanolamine, preferably sodium hydroxide. The dosage is based on adjusting the pH of the non-covalently targeted encapsulating ectoine liposome to 4.5-7.5, specifically it can be 0.01-0.5 parts, including but not limited to 0.01 part, 0.05 parts, 0.1 part, 0.15 parts, 0.20 parts, 0.25 parts, 0.30 parts, 0.35 parts, 0.40 parts, 0.45 parts, or 0.5 parts, etc. The concentration of the pH regulator can be 0.5-2 mol / L.

[0101] In some embodiments, the non-covalently targeted encapsulating ectoine liposome includes the following raw materials:

[0102] Phospholipid, organic solvent, cholesterol, emulsifier, antioxidant, polyol, ectoine, chitosan, acetic acid aqueous solution, hyaluronic acid, and water.

[0103] In some embodiments, by weight parts, the non-covalently targeted encapsulating ectoine liposome includes the following raw materials:

[0104] Phospholipid 1-10 parts, organic solvent 1-10 parts, cholesterol 0.01-0.5 parts, emulsifier 0.1-5 parts, antioxidant 0.01-0.4 parts, polyol 0.1-20 parts, ectoine 1-10 parts, chitosan 0.1-1 part, acetic acid aqueous solution 5-50 parts, hyaluronic acid 0.1-2 parts, and water 50-100 parts.

[0105] In some embodiments, by weight parts, the non-covalently targeted ectorin-loaded liposome comprises the following raw materials:

[0106] 1 - 3 parts of phospholipid, 1 - 3 parts of organic solvent, 0.01 - 0.2 parts of cholesterol, 0.3 - 1.5 parts of emulsifier, 0.01 - 0.1 parts of antioxidant, 1 - 5 parts of polyol, 1 - 3 parts of ectorin, 0.1 - 1 part of chitosan, 5 - 20 parts of acetic acid aqueous solution, 0.1 - 2 parts of hyaluronic acid and 50 - 100 parts of water.

[0107] In some embodiments, by weight parts, the non-covalently targeted ectorin-loaded liposome comprises the following raw materials:

[0108] 1 - 10 parts of phospholipid, 1 - 10 parts of organic solvent, 0.01 - 0.5 parts of cholesterol, 0.1 - 5 parts of emulsifier, 0.01 - 0.4 parts of antioxidant, 1 - 10 parts of pentanediol, 1 - 10 parts of glycerol, 1 - 10 parts of ectorin, 0.1 - 1 part of chitosan, 5 - 50 parts of acetic acid aqueous solution, 0.1 - 2 parts of hyaluronic acid and 50 - 100 parts of water.

[0109] In some embodiments, taking the sum of the masses of phospholipid, ethanol, cholesterol, emulsifier and antioxidant as A, and the mass of polyol as B, the mass ratio of A to B is (1.5 - 2.0):1, specifically it can be 1.50:1, 1.55:1, 1.60:1, 1.65:1, 1.70:1, 1.75:1, 1.80:1, 1.85:1, 1.90:1, 1.95:1 or 2.0:1, etc.

[0110] In some embodiments, the particle size of the non-covalently targeted ectorin-loaded liposome is 100 nm - 250 nm, including but not limited to 100 nm, 120 nm, 140 nm, 160 nm, 180 nm, 200 nm, 220 nm, 240 nm, 250 nm, etc.; the PDI is 0.3 - 0.6, including but not limited to 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, etc.; the pH is 4.5 - 7.5, including but not limited to 4.5, 4.9, 5.0, 5.5, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0 or 7.5.

[0111] In a second aspect, the present application provides a preparation method of the non-covalently targeted ectorin-loaded liposome described in the first aspect, comprising the following steps:

[0112] Characterized in that it comprises the following steps:

[0113] Mix the phospholipid, the cholesterol, the emulsifier and the organic solvent to prepare an oil phase;

[0114] Mix the polyol and the oil phase to prepare a primary emulsion;

[0115] Remove the organic solvent from the primary emulsion to form blank liposomes;

[0116] Mix the ectoine with 30 - 60% water to form an ectoine solution; mix the ectoine solution with the blank liposomes to prepare ectoine liposomes;

[0117] Prepare a chitosan solution with chitosan and an aqueous acetic acid solution; mix the chitosan solution with the ectoine liposomes to prepare chitosan - modified ectoine liposomes;

[0118] Dissolve hyaluronic acid in the remaining water to form a hyaluronic acid solution; mix the hyaluronic acid solution with the chitosan - modified ectoine liposomes to prepare the non - covalently targeted ectoine - loaded liposomes.

[0119] In some embodiments, the preparation method comprises the following steps:

[0120] Mix the phospholipid, the cholesterol, the emulsifier, the antioxidant and the organic solvent to prepare an oil phase;

[0121] Mix the polyol and the oil phase to prepare a primary emulsion;

[0122] Remove the organic solvent from the primary emulsion to form blank liposomes;

[0123] Mix the ectoine with 30 - 60% water to form an ectoine solution; mix the ectoine solution with the blank liposomes to prepare ectoine liposomes;

[0124] Prepare a chitosan solution with chitosan and an aqueous acetic acid solution; mix the chitosan solution with the ectoine liposomes to prepare chitosan - modified ectoine liposomes;

[0125] Dissolve hyaluronic acid in the remaining water to form a hyaluronic acid solution; mix the hyaluronic acid solution with the chitosan - modified ectoine liposomes to prepare the non - covalently targeted ectoine - loaded liposomes.

[0126] In this application, blank liposomes are first prepared, then the blank liposomes are mixed with an ectoine solution to prepare ectoine liposomes, and then the ectoine liposomes are successively modified with a chitosan solution and a hyaluronic acid solution to prepare the non - covalently targeted ectoine - loaded liposomes. The preparation method is simple, has high repeatability, short preparation time, no harmful substance residues, and high encapsulation efficiency. Without complex mechanical processes and equipment, the product quality and process can have good reproducibility and stability, and are easy for industrial production.

[0127] Optionally, the preparation temperature of the oil phase is 40 - 60°C, such as 40°C, 45°C, 50°C, 55°C, 60°C, etc.

[0128] Optionally, the method for removing the organic solvent from the primary emulsion includes rotary evaporation or vacuum concentration; specifically, the conditions for rotary evaporation include: evaporation temperature 40 - 60°C, such as 40°C, 45°C, 50°C, 55°C, 60°C, etc.; rotary speed 20 - 100 rpm, such as 20 rpm, 30 rpm, 40 rpm, 50 rpm, 60 rpm, 70 rpm, 80 rpm, 90 rpm, 100 rpm, etc.; the conditions for vacuum concentration include: 40 - 60°C, such as 40°C, 45°C, 50°C, 55°C, 60°C, etc.; the time is 30 - 180 min, such as 30 min, 60 min, 90 min, 120 min, 150 min, 180 min, etc.

[0129] It is understandable that when mixing the ectoine solution with the blank liposomes, the mixing order is not restricted. The ectoine solution can be added to the blank liposomes, or the blank liposomes can be added to the ectoine solution.

[0130] It is understandable that homogenization is beneficial to reducing the particle size of liposomes, improving the stability and encapsulation efficiency of liposomes. After the step of preparing the ectoine liposomes, a step of homogenizing the ectoine liposomes can also be included. Specifically, the conditions for homogenization include: homogenization pressure 300 bar - 700 bar, such as 300 bar, 350 bar, 400 bar, 450 bar, 500 bar, 550 bar, 600 bar, 650 bar or 700 bar, etc.; the number of homogenization times is 1 - 5 times, such as 1 time, 2 times, 3 times, 4 times or 5 times.

[0131] It is understandable that stirring is beneficial to improving the reaction efficiency. In the steps of mixing the chitosan solution with the ectoine liposome and mixing the hyaluronic acid solution with the chitosan-modified ectoine liposome, stirring can be employed. The conditions of stirring may include: temperature 40 - 60°C, such as 40°C, 45°C, 50°C, 55°C, 60°C, etc.; stirring speed 100 - 1000 r / min, such as 100 r / min, 200 r / min, 300 r / min, 400 r / min, 500 r / min, 600 r / min, 700 r / min, 800 r / min, 900 r / min, 1000 r / min, etc.; stirring time 180 min - 240 min, such as 180 min, 190 min, 200 min, 210 min, 220 min, 230 min, 240 min, etc.

[0132] In a third aspect, the present application provides a liposomal preparation, the raw materials of which include the non-covalently targeted encapsulated ectoine liposome described in the first aspect or the non-covalently targeted encapsulated ectoine liposome prepared by the preparation method described in the second aspect.

[0133] In a fourth aspect, the present application provides a cosmetic, which includes the non-covalently targeted encapsulated ectoine liposome described in the first aspect, the non-covalently targeted encapsulated ectoine liposome prepared by the preparation method described in the second aspect, or the liposomal preparation described in the third aspect.

[0134] The non-covalently targeted encapsulated ectoine liposome of the present application has good stability; has a controlled release effect, and the release rate is slower than that of the unmodified ectoine liposome; has an improved transdermal effect, and the cumulative release amount and cumulative retention amount are significantly increased compared with the unmodified ectoine liposome; has the effect of targeting fibroblasts, and the accumulation on fibroblasts is significantly increased compared with the unmodified ectoine liposome; has an improved functional effect, and compared with the unmodified ectoine liposome, it can enhance skin hydration, regulate transepidermal water loss and improve skin elasticity, providing great potential for the delivery of the outer membrane of the skin and providing a basis for skin health applications.

[0135] In some embodiments, the cosmetic includes at least one of a facial mask, eye cream, facial cream, primer, essence, lotion and skin softener.

[0136] Unless otherwise specified, the raw materials used in the following tests can all be conventionally purchased from the market.

[0137] Exemplary descriptions of the raw materials used in the examples and comparative examples are as follows:

[0138] Phospholipid: Soybean lecithin PC60, purchased from Hebei Meiye Siwei Biotechnology Co., Ltd.;

[0139] Behenyl Alcohol Polyether-25: Purchased from Shanghai Longquan Chemical Technology Co., Ltd.;

[0140] Chitosan: Purchased from Shanghai Macklin Biochemical Co., Ltd.;

[0141] Hyaluronic acid: with a molecular weight of 8 kDa, purchased from Shanghai Macklin Biochemical Co., Ltd.;

[0142] Polyglycerol-10 Myristate: Purchased from Shanghai Zhaoheng Industry Co., Ltd.;

[0143] Ammonium Acryloyldimethyltaurate / VP Copolymer (AVC): Purchased from Shanghai Macklin Biochemical Co., Ltd.;

[0144] Antioxidant: Pentaerythrityl Tetrakis (3-(3,5-Di-tert-butyl-4-hydroxyphenyl) Propionate), commercially available;

[0145] All other raw materials are of ordinary commercial grade.

[0146] The following are specific examples.

[0147] Basic Example 1

[0148] Preparation of chitosan-modified ectoine liposomes:

[0149] Weigh the raw materials according to Table 1. Mix anhydrous ethanol, phospholipids, and cholesterol, and mechanically stir at 50 °C until completely dissolved; then add the emulsifier and antioxidant and stir until the solution is clear and transparent to obtain the oil phase;

[0150] Add glycerol and pentylene glycol and mix, mechanically stir at 50 °C until completely dissolved, and then mix with the oil phase to prepare the primary emulsion;

[0151] Remove ethanol from the primary emulsion by rotary evaporation (50 °C, 40 rpm) to obtain blank liposomes;

[0152] Dissolve ectoine in water, preheat at 50 °C, and drop it into the prepared blank liposomes. Keep the temperature at 50 °C during the dropping process, heat and stir to mix evenly to obtain ectoine liposomes;

[0153] Homogenize the ectoine liposomes under high pressure 3 times (500 bar); dissolve chitosan in a 1 wt% acetic acid aqueous solution, drop it into the homogenized ectoine liposomes, stir at 50 °C at a speed of 300 r / min for 180 min to mix evenly. After it cools to room temperature, add the preservative and disperse evenly to obtain chitosan-modified ectoine liposomes.

[0154] Table 1 Formulation and properties of chitosan-modified ectoine liposomes (total amount of each raw material is 100 parts)

[0155]

[0156] The chitosan-modified ectoine liposomes prepared in Basic Example 1 showed no layering, leakage, aggregation, or flocculation. As can be seen from Table 1, chitosan can achieve the modification of ectoine liposomes, and uniformly and stably dispersed liposomes can be prepared. Among them, when the chitosan addition amount was 0.5 parts, the liposome potential basically reached saturation, showing no significant upward trend, and the particle size was smaller and the PDI was more uniform.

[0157] Example 1

[0158] Preparation of non-covalently targeted encapsulated ectoine liposomes:

[0159] Weigh the raw materials according to Table 2. Mix anhydrous ethanol, phospholipids, and cholesterol, and mechanically stir at 50 °C until completely dissolved; then add an emulsifier and an antioxidant and stir until the solution is clear and transparent to obtain the oil phase;

[0160] Mix glycerol and pentylene glycol, and mechanically stir at 50 °C until completely dissolved, and then mix with the oil phase to prepare the primary emulsion;

[0161] Remove the ethanol in the primary emulsion by rotary evaporation (50 °C, 40 rpm) to obtain blank liposomes;

[0162] Dissolve ectoine in the first part of water, preheat at 50 °C, and drop it into the prepared blank liposomes. Keep the temperature at 50 °C during the dropping process, heat and stir to mix evenly to obtain ectoine liposomes;

[0163] Homogenize the ectoine liposomes under high pressure three times (500 bar); dissolve chitosan in 1 wt% acetic acid aqueous solution, drop it into the homogenized ectoine liposomes, and stir at 50 °C at 300 r / min for 180 min to mix evenly to obtain chitosan-modified ectoine liposomes;

[0164] Dissolve hyaluronic acid in the second part of water to form a hyaluronic acid solution; drop the hyaluronic acid solution into the chitosan-modified ectoine liposomes, and stir at 50 °C at 300 r / min for 180 min to mix evenly to obtain the non-covalently targeted encapsulated ectoine liposomes. The preparation schematic diagram is shown in Figure 1 .

[0165] Table 2 Formulation and properties of non-covalently targeted encapsulated ectoine liposomes (total amount of each raw material is 100 parts)

[0166]

[0167] The non-covalent targeted encapsulation ectorin liposomes prepared in Example 1 showed no delamination, leakage, aggregation or flocculation. It can be seen that in this application, hyaluronic acid and chitosan were used to modify the ectorin liposomes, and non-covalent targeted encapsulation ectorin liposomes with uniform and stable properties were prepared. Among them, when the addition amount of hyaluronic acid was 0.5 parts, the liposome potential basically reached saturation, there was no significant change, the particle size was small, the encapsulation rate was the highest, and the PDI was also more uniform.

[0168] Example 2

[0169] It had the same formulation as Sample 2 in Example 1, with the only difference being that during the preparation of the ectorin liposomes, the blank liposomes were dropped into the ectorin aqueous solution.

[0170] The prepared liposomes were a light brown translucent solution, with a particle size of 247 nm, a PDI of 0.42, a Zeta potential of -39 mV, and an encapsulation rate of 87.32%.

[0171] Comparative Example 1

[0172] Preparation of non-targeted encapsulation ectorin liposomes:

[0173] 1) Weigh the raw materials according to Table 3, mix absolute ethanol, phospholipids and cholesterol, and stir mechanically at 50 °C until completely dissolved; then add the emulsifier and antioxidant and stir until the solution is clear and transparent to obtain the oil phase;

[0174] 2) Add glycerol and pentylene glycol and mix, stir mechanically at 50 °C until completely dissolved, and then mix with the oil phase to prepare the primary emulsion;

[0175] 3) Remove the ethanol in the primary emulsion by rotary evaporation (50 °C, 40 rpm) to obtain blank liposomes;

[0176] 4) Dissolve ectorin in water to prepare an ectorin solution; preheat it at 50 °C and drop it into the prepared blank liposomes. Keep the temperature at 50 °C during the dropping process, heat and stir to mix evenly. After it cools to room temperature, add the preservative and disperse evenly to obtain the non-targeted encapsulation ectorin liposomes.

[0177] Table 3 Formulation and properties of non-targeted encapsulation ectorin liposomes (total amount of each raw material is 100 parts)

[0178]

[0179] The liposomes were a light brown translucent solution, with a particle size of 233 nm, a PDI of 0.34, and a Zeta potential of -39 mV.

[0180] Comparative Example 2

[0181] Preparation of free ectoine solution:

[0182] Dissolve ectoine in water to prepare a 6.25 wt% free ectoine solution.

[0183] Comparative Example 3

[0184] Except that the emulsifier behenyl alcohol polyether-25 in Sample 2 of Example 1 was replaced with Span80, the rest was the same as Sample 2 of Example 1.

[0185] Comparative Example 4

[0186] Except that the emulsifier behenyl alcohol polyether-25 in Sample 2 of Example 1 was replaced with monoglyceride, the rest was the same as Sample 2 of Example 1.

[0187] Comparative Example 5

[0188] Except that the emulsifier behenyl alcohol polyether-25 in Sample 2 of Example 1 was replaced with poloxamer 407, the rest was the same as Sample 2 of Example 1.

[0189] Comparative Example 6

[0190] Except that the emulsifier behenyl alcohol polyether-25 in Sample 2 of Example 1 was replaced with behenamidopropyl dimethylamine, the rest was the same as Sample 2 of Example 1.

[0191] The properties of the liposomes in Comparative Examples 3-6 are shown in Table 4.

[0192] Table 4 Properties of the liposomes in Comparative Examples 3-6

[0193]

[0194] As can be seen from Table 4, the type of emulsifier has a great influence on the appearance, stability and particle size of the liposomes. In the system of this application, when Span80, monoglyceride, poloxamer 407 or behenamidopropyl dimethylamine is used as the emulsifier, homogeneous liposomes with small particle size and narrow distribution cannot be prepared, and only when behenyl alcohol polyether-25 is used as the emulsifier can the target liposomes with homogeneous, small particle size and narrow distribution be obtained.

[0195] Application Example

[0196] Preparation of essence:

[0197] Separate the non-covalent targeted encapsulation ectorin liposomes of Sample 2 in Example 1, the non-targeted encapsulation ectorin liposomes of Comparative Example 1, and the free ectorin solution of Comparative Example 2 to prepare essence according to the following formula: liposomes or free ectorin solution 64 wt%, glycerol 3 wt%, propylene glycol 5 wt%, butylene glycol 5 wt%, pentylene glycol 1 wt%, polyglyceryl-10 myristate 1 wt%, EDTA-2Na 0.03 wt%, acryloyldimethyltaurine ammonium / VP copolymer 0.4 wt%, p-hydroxyacetophenone 0.2 wt%, and the balance of water. At the same time, replace the liposomes with pure water to prepare a blank essence.

[0198] The preparation method of the essence is as follows: First, stir the acryloyldimethyltaurine ammonium / VP copolymer evenly in glycerol, propylene glycol, butylene glycol, and pentylene glycol, add polyglyceryl-10 myristate, p-hydroxyacetophenone, EDTA-2Na, and water, heat up to 85 °C, and keep warm for 30 min; cool down to 40 °C, then add the corresponding liposomes, free ectorin solution, or water, and mix evenly to obtain the essence.

[0199] Test Example 1

[0200] Perform TEM detection on the non-covalent targeted encapsulation ectorin liposomes prepared from Sample 2 in Example 1, and the results are shown in Figure 2 , and the specific method is as follows:

[0201] Weigh the sample of non-covalent targeted encapsulation ectorin liposomes and drop it on the copper grid. After a few seconds, gently pick up the copper grid sample with forceps, and suck the excess liquid along one side with filter paper. After it is slightly dry, place the copper grid on a 2% phosphotungstic acid staining solution drop for floating staining for 60 s. After picking it up with forceps, suck the excess liquid along one side with filter paper, place the membrane side up on the filter paper to dry, and observe and take pictures with a transmission electron microscope. It can be seen from Figure 2 that the particles are spherical-like and the distribution is relatively uniform.

[0202] Test Example 2 Liposome particle size, PDI, Zeta potential, and encapsulation efficiency test

[0203] 1) Use a ZETASIZER PRO particle size analyzer to measure the particle size, PDI, and Zeta potential of the examples. The particle size and PDI test methods are carried out according to the methods specified in "GB / T 19077-2016 Particle Size Distribution - Laser Diffraction Method". The Zeta potential is detected according to "GB / Z 42353-2023 Zeta Potential Measurement Operation Guide", and the results are shown in Table 1-2.

[0204] 2) Perform an encapsulation efficiency test on the non-covalent targeted encapsulation ectorin liposomes prepared from Samples 1-8 in Example 1. The test method is as follows:

[0205] The encapsulation efficiency of cell membranes in liposomes was determined by high-speed centrifugation. Using a 3KD ultrafiltration tube (microporous UFC501008, USA), 0.5 mL of liposomes was added to the inner tube, and centrifuged at a centrifugal force of 14000 r / min for 15 min in a centrifuge to separate the liposomes. The filtrate was collected and the volume was recorded. The liposomes and the solvent (methanol) were mixed at a ratio of 1:1, and after standing for half an hour, the membrane was disrupted. An appropriate amount of the disrupted liposomes and the ultrafiltration filtrate were taken, appropriately diluted, and the absorbance or peak area was measured by HPLC to obtain the total concentration of water-soluble substances and the concentration of free water-soluble substances in the liposomes. The encapsulation rate (EE%) was calculated according to formula 1, and the results are shown in Table 2.

[0206] Formula 1

[0207] C1—the amount of drug in the liposome suspension ( );

[0208] V1—0.5 (mL);

[0209] C2—the amount of drug in the filtrate ( );

[0210] V2—the volume of the filtrate (mL).

[0211] Test Example 3 Investigation of Liposome Stability

[0212] The non-covalently targeted ectorin-loaded liposomes prepared from the sample 2 of Example 1 were placed under daily, light, dark, 4°C, 45°C, -15°C, and freeze-thaw cycle (one freeze-thaw cycle was 24 h at -15°C followed by 24 h at 45°C) conditions, and their particle size, PDI, and potential stability were measured. The results are as Figure 3 shown. It can be seen that the non-covalently targeted ectorin-loaded liposomes prepared from the sample 2 of Example 1 have good stability under different conditions.

[0213] Test Example 4 Liposome Stability Test

[0214] The non-covalently targeted ectorin-loaded liposomes prepared from the sample 2 of Example 1 were investigated for stability. The sample was taken into the sample bottle of the Turbiscan Lab stability analyzer to measure the dynamic changes in liposome stability. The parameter settings were to scan once every 30 min for 24 h. The results are shown in Figure 4 . Generally, it is considered that the system is in an absolutely stable state when the average value of the backscattered light intensity is less than 0.2. It can be seen that the non-covalently targeted ectorin-loaded liposomes prepared from the sample 2 of Example 1 have good stability.

[0215] Test Example 5 Liposome Transdermal Permeability Test

[0216] Transdermal experiments were conducted on the non-covalently targeted encapsulating ectoine liposomes prepared from the sample 2 of Example 1, the non-targeted encapsulating ectoine liposomes prepared in Comparative Example 1, and the free ectoine solution of Comparative Example 2.

[0217] The transdermal experiment was carried out in a Franz diffusion cell device. The abdominal skin of guinea pigs was fixed between the receiving cell and the supply cell (the inner layer of the skin faced the receiving cell). The effective permeation area of the skin was 1.77 cm 2 , the volume of the receiving cell was 12 mL, and the magnetic stirring speed in the receiving chamber was 300 r / min. The receiving cell was filled with the release medium of physiological saline to remove air bubbles, the stirring was started, and the temperature was kept constant at (37.0 ± 0.5) °C. Samples (with the same content of the active ingredient in the samples) were evenly applied to the skin surface, and were represented by Sample 1, 2, and 3 respectively (see Table 5). At 24 h, 1 mL of the sample was taken with a sampling needle and placed in an ep tube. Then, the receiving cell was first evacuated of air bubbles by traction with a non-porous puncture needle, and then 1 mL of isothermal receiving solution was added to the receiving cell.

[0218] After 24 h, the skin was removed, and the residual sample liquid on the surface of the mouse skin was washed off with ultrapure water. Then, the mouse skin was cut into pieces and placed in a 10 mL ep tube. 3 mL of methanol was added and ultrasonicated for 30 min. After ultrasonication, it was centrifuged at 5000 rpm for 10 min, and the supernatant was taken to determine the content of the active substance by HPLC method, which was the retention amount (Qs) of the active substance in the skin.

[0219] And the cumulative permeation amount Qn of the active substance at different times was calculated by the following formula:

[0220]

[0221] In the formula, Q n is the cumulative permeation amount (μg / cm 2 ) per unit area at the nth time point, V0 is the volume of the liquid in the receiving cell (mL), V is the sampling volume (mL), C n is the concentration of glabridin in the receiving solution measured at the nth time point (μg / ml), C i-1 is the concentration of the active substance in the receiving solution measured at the (i - 1)th sampling point (μg / mL), and S is the effective area (cm 2 ).

[0222] And the retention amount Q s of the active substance in the skin after 24 h was calculated by the following formula:

[0223]

[0224] In the formula, Q s is the retention amount of the active substance per unit area in the skin (μg / cm 2), V is the total volume of the skin extract (mL), C is the concentration of the active substance in the skin extract (μg / mL), and A is the effective diffusion area (cm 2 ).

[0225] Table 5

[0226]

[0227] The samples 2 of Example 1, Comparative Example 1 and Comparative Example 2 of this application were subjected to transdermal tests. The test results are as Figure 5 shown. The results show that the cumulative release amount and retention amount of Sample 3 are the lowest. This may be because free ectoine cannot break through the barrier through active pathways such as fusion and lipid pathway penetration and enter the appropriate release environment in the skin. Compared with Sample 2, the cumulative release amount of Sample 1 increased by 31% and the cumulative retention amount increased by 10%. In summary, it shows that after the liposome is surface-modified with hyaluronic acid, it can further increase the penetration amount and retention amount of ectoine in the skin so as to exert its efficacy.

[0228] Test Example 6 Liposome Sustained Release Test

[0229] An in vitro release test was carried out on the non-covalently targeted ectoine-loaded liposome prepared from Sample 2 of Example 1, the non-targeted ectoine-loaded liposome prepared from Comparative Example 1, and the free ectoine solution of Comparative Example 2. The ectoine in the release medium was measured by PBS dialysis. The sample was placed in a dialysis bag (molecular weight cut-off of 6000 - 8000), and the dialysis bag was placed in a 250 mL beaker containing 200 mL of the release medium. The beaker was incubated in a 37 °C shaking water bath thermostat and shaken at 100 rpm by a magnetic stirrer. At predetermined time intervals (1, 2, 4, 6, 8, 12, 24, 48, 72 hours after the start of the culture), all the release medium (1 mL) was taken out and replaced with the same volume of fresh release medium (1 mL). The release amount of ectoine was determined by HPLC, and a slow release curve was plotted. The results are shown in Figure 6 .

[0230]

[0231] Qi: Cumulative release amount at the i-th sampling time point, %

[0232] V: Volume of the release medium, mL

[0233] ρ n : Concentration of ectoine in the release medium released during the n-th sampling, μg / mL

[0234] ρ i : Concentration of ectoine in the release medium released during the i-th sampling, μg / mL

[0235] Vi: The volume of the i-th sampling, mL

[0236] m: The mass of ectoin in the sample, μg

[0237] It can be seen that the release of ectoin from non-targeted ectoin-loaded liposomes is 30% within 2 h and approximately 80% after 72 h. This may be because the drug is encapsulated in the lipid membrane and is mainly released through the dissolution and diffusion of the lipid bilayer. Liposomes can act as a rate-limiting membrane barrier, thus providing a controllable system.

[0238] The release amount of ectoin from non-covalently targeted ectoin-loaded liposomes is 10% lower than that from non-targeted ectoin-loaded liposomes at 2 h and reaches 69% after 72 h, with the lowest release rate. It can be seen that modification with hyaluronic acid and chitosan can effectively regulate the in vitro release rate of ectoin and achieve a better controlled release effect compared with the drug solution or non-targeted ectoin-loaded liposomes.

[0239] Test Example 7 Liposome Cellular Uptake Test

[0240] To verify the difference in the targeting of fibroblasts between the non-covalently targeted ectoin-loaded liposomes prepared from Sample 2 of Example 1 and the non-targeted ectoin-loaded liposomes prepared from Comparative Example 1, the ectoin in Sample 2 of Example 1 and Comparative Example 1 was replaced with the fluorescent substance FITC to prepare non-covalently targeted FITC-loaded liposomes and non-targeted FITC liposomes, and the following tests were carried out:

[0241] Cultivate fibroblasts, incubate for the same time after administration, stain the cell nuclei with Hoechst33342, and then observe the green fluorescence difference of non-covalently targeted FITC-loaded liposomes and non-targeted FITC liposomes on fibroblasts under a confocal fluorescence microscope.

[0242] The experimental results are as Figure 7 shown. There was a small amount of aggregation around the cells in the fluorescence of the non-targeted FITC liposome group. In contrast, the non-covalently targeted FITC-loaded liposome group showed obvious fluorescence labeling around and inside the cells. This indicates that fibroblasts have a strong interaction with hyaluronic acid-modified liposomes. The main hyaluronic acid-binding receptor of fibroblasts is CD44. Under the active targeting of hyaluronic acid, the accumulation of non-covalently targeted FITC-loaded liposomes on fibroblasts increased significantly, resulting in a difference in the targeting of non-covalently targeted FITC-loaded liposomes and non-targeted FITC liposomes on fibroblasts.

[0243] Test Example 8 Liposome Human Efficacy Test

[0244] To further prove the targeting of hyaluronic acid, the anti-aging efficacy of the non-covalent targeted encapsulation ectoin liposome essence prepared from Sample 2 of Example 1 prepared in the application example, the non-targeted encapsulation ectoin liposome essence of Comparative Example 1, the essence of the free ectoin solution of Comparative Example 2, and the blank essence was verified. The water content of the skin stratum corneum can indirectly reflect the water content of the stratum corneum. The higher the measured value, the higher the water content of the stratum corneum, indicating better moisturizing effect of the product. One of the main functions of the skin barrier is to prevent the body from losing water to the surrounding environment through the skin, and transepidermal water loss (TEWL) is an important non-invasive skin water loss rate test index for characterizing the skin barrier function. The smaller the measured value, the lower the trans-epidermal water loss per unit time and per unit skin cross-sectional area, and the better the skin barrier function. Skin elasticity is the ability of the skin to return to its original state after deformation, and the elasticity of aging skin decreases. The closer the value of the skin elasticity parameter R2 is to 1, the better the skin elasticity, and vice versa. The MMV, TEWL, and elasticity of the subjects before and after the application of different samples were evaluated, and the results are as Figure 8 shown.

[0245] Compared with the non-covalent targeted encapsulation ectoin liposome essence prepared from Sample 2 of Example 1, the non-targeted encapsulation ectoin liposome essence of Comparative Example 1, and the essence of the free ectoin solution of Comparative Example 2, the skin water content increased to 2.34 times and 1.52 times respectively after 1 h, to 2.01 times and 1.38 times respectively at 4 h, and to 2.56 times and 1.44 times respectively at 7 d. In terms of the TEWL value, the non-covalent targeted encapsulation ectoin liposome essence prepared from Sample 2 of Example 1 caused a decrease in TEWL, as Figure 8As shown by C in []. Compared with the essence of the free ectoin solution in Comparative Example 2, the TEWL values of the non-covalently targeted encapsulated ectoin liposome essence prepared from Sample 2 of Example 1 and the non-targeted encapsulated ectoin liposome essence in Comparative Example 1 decreased by 1.88 times and 1.45 times respectively at 1 h, by 2.14 times and 1.37 times respectively at 4 h, and by 2.40 times and 1.69 times respectively at 7 d. Hyaluronic acid has significant water absorption capacity, sufficient to effectively moisturize the stratum corneum and dermis, resulting in changes in the microstructure of lipid self-assembly and a decline in the barrier function of the stratum corneum, ultimately promoting skin penetration. Ectoin reduces skin water loss by adsorbing water molecules and binding to them through hydrogen bonds. Compared with the essence of the free ectoin solution in Comparative Example 2, the non-covalently targeted encapsulated ectoin liposome essence prepared from Sample 2 of Example 1 and the non-targeted encapsulated ectoin liposome essence in Comparative Example 1 improved skin elasticity by 1.89 times and 1.07 times respectively on the 7th day, by 2.03 times and 1.43 times respectively on the 14th day, and by 2.29 times and 1.37 times respectively on the 30th day. Therefore, the non-covalently targeted encapsulated ectoin liposome essence prepared from Sample 2 of Example 1 has effective skin elasticity enhancing properties. This can be attributed to the good skin penetration of the non-covalently targeted encapsulated ectoin liposome obtained after hyaluronic acid modification, thus increasing the efficacy of ectoin. In addition, the strong skin moisturizing property of hyaluronic acid also contributes to this effect.

[0246] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.

[0247] The above-described embodiments only represent several implementation manners of the present application. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present application, several modifications and improvements can still be made, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the appended claims.

Claims

1. A non-covalently targeted encapsulating ectoine liposome, characterized in that, It includes the following raw materials: Phospholipid, organic solvent, cholesterol, emulsifier, polyol, ectoine, chitosan, acetic acid aqueous solution, hyaluronic acid and water; The emulsifier includes behenyl alcohol polyether-25.

2. The non-covalently targeted encapsulating ectoine liposome according to claim 1, wherein, By weight parts, the non-covalent targeted encapsulation of ectoine liposome includes the following raw materials: 1-10 parts of phospholipid, 1-10 parts of organic solvent, 0.01-0.5 parts of cholesterol, 0.1-5 parts of emulsifier, 0.1-20 parts of polyol, 1-10 parts of ectoine, 0.1-1 part of chitosan, 5-50 parts of acetic acid aqueous solution, 0.1-2 parts of hyaluronic acid and 50-100 parts of water.

3. The non-covalently targeted encapsulating ectoine liposome according to claim 1 or 2, characterized in that, The non-covalent targeted encapsulation of ectoine liposome also includes auxiliary agents; the auxiliary agents include at least one of antioxidant, preservative and pH regulator.

4. The non-covalently targeted encapsulating ectoine liposome according to claim 3, wherein The non-covalent targeted encapsulation of ectoine liposome meets one or more of the following conditions: (1) The phospholipid includes at least one of soybean lecithin, cephalin, phosphatidylinositol, sphingomyelin, egg yolk lecithin; (2) The organic solvent includes ethanol; (3) The polyol includes at least one of glycerol, butanediol and pentanediol; preferably glycerol and / or pentanediol; (4) The mass concentration of the acetic acid aqueous solution is 0.5-2%; (5) The antioxidant includes at least one of pentaerythritol tetra(bis-tert-butylhydroxyhydrocinnamate), tocopherol, terbutylhydroxyanisole, tert-butylhydroxytoluene and propyl gallate; (6) The preservative includes at least one of hydroxyacetophenone, methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, butyl p-hydroxybenzoate, benzoic acid, sodium benzoate and potassium sorbate; (7) The pH regulator includes at least one of sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate and triethanolamine; (8) The dosage of the antioxidant is 0.01-0.4 parts; (9) The dosage of the preservative is 0.05-1.05 parts; (10) The dosage of the pH regulator is based on adjusting the pH of the non-covalent targeted encapsulation of ectoine liposome to 4.5-7.

5.

5. The non-covalently targeted encapsulating ectoine liposome according to claim 3, wherein The non-covalent targeted encapsulation of ectoine liposome includes the following raw materials: Phospholipid, organic solvent, cholesterol, emulsifier, antioxidant, polyol, ectoine, chitosan, acetic acid aqueous solution, hyaluronic acid and water.

6. The preparation method of the non-covalently targeted encapsulating ectoine liposome according to claim 1 or 2, characterized in that, It includes the following steps: Mix the phospholipid, the cholesterol, the emulsifier and the organic solvent to prepare an oil phase; Mix the polyol and the oil phase to prepare a primary emulsion; Remove the organic solvent in the primary emulsion to form blank liposomes; Mix the ectoine with 30-60% of water to form an ectoine solution; mix the ectoine solution with the blank liposomes to prepare ectoine liposomes; Prepare a chitosan solution with chitosan and acetic acid aqueous solution; mix the chitosan solution with the ectoine liposomes to prepare chitosan-modified ectoine liposomes; Dissolve hyaluronic acid in the remaining water to form a hyaluronic acid solution; mix the hyaluronic acid solution with the chitosan-modified ectoine liposomes to prepare the non-covalent targeted encapsulation of ectoine liposomes.

7. The preparation method according to claim 6, characterized in that, Meet one or more of the following conditions: (1) The preparation temperature of the oil phase is 40 - 60°C; (2) The method for removing the organic solvent from the primary emulsion includes rotary evaporation or vacuum concentration; optionally, the conditions for rotary evaporation include: evaporation temperature 40 - 60°C, rotation speed 20 - 100 rpm; (3) After the step of preparing the ectoine liposome, it further includes a step of homogenizing the ectoine liposome; optionally, the conditions for homogenization include: homogenization pressure 300 bar - 700 bar, number of homogenization times 1 - 5 times.

8. A liposomal preparation, characterized in that, The raw material includes the non-covalently targeted encapsulated ectoine liposome according to any one of claims 1 - 5 or the non-covalently targeted encapsulated ectoine liposome prepared by the preparation method according to claim 6 or 7.

9. A cosmetic, characterized in that, Includes the non-covalently targeted encapsulated ectoine liposome according to any one of claims 1 - 5 or the non-covalently targeted encapsulated ectoine liposome prepared by the preparation method according to claim 6 or 7 or the liposome preparation according to claim 8.

10. The cosmetic according to claim 9, characterized in that, The cosmetic includes at least one of a facial mask, eye cream, face cream, primer, essence, lotion and skin softener.

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