Covalent targeting entrapped Ectoine liposome as well as preparation method and application of covalent targeting entrapped Ectoine liposome

Abstract

The invention discloses covalent targeted entrapped Ectoine liposome as well as a preparation method and application thereof, and belongs to the technical field of cosmetics. The covalent targeted entrapped Ectoine liposome is prepared from the following raw materials: phospholipid, targeted phospholipid, an organic solvent, cholesterol, an emulsifier, polyhydric alcohol, Ectoine, hyaluronic acid and water, the targeted phospholipid comprises an amidation reaction product DSPE-HA (distearoyl phosphatidyl ethanolamine) of distearoyl phosphatidyl ethanolamine and hyaluronic acid; the emulsifying agent is prepared from beheneth-25. The covalent targeted entrapped Ectoine liposome is prepared from targeted phospholipid and phospholipid, and the types of the targeted phospholipid and the emulsifier are optimized, so that the Ectoine liposome which is homogeneous, small in particle size, narrow in particle size distribution, high in entrapment efficiency and good in stability can be prepared, covalent targeted modification of the liposome is realized, and the covalent targeted entrapped Ectoine liposome has good application prospects. The transdermal absorption efficiency of the Ectoine is improved, the exertion of the effect of the Ectoine is promoted, and the skin transfer requirement of the outer membrane is met.

Description

Technical Field

[0001] The present application relates to the field of cosmetic technology, and in particular to covalently targeted ectoine-encapsulated liposomes and their preparation method and application. Background Art

[0002] Ectoin, also known as tetrahydropyrimidine carboxylic acid, is an amino acid derivative that can regulate cell osmotic pressure. It is a naturally formed "protective shield" for halophilic bacteria to survive in extreme environments such as high temperature, high salt, and strong ultraviolet rays. Its structural formula is as follows:

[0003] .

[0004] Ectoin has a unique, highly polar molecular structure, resulting in a strong water-binding capacity. One molecule of ectoin can bind four or five water molecules, resulting in excellent moisturizing properties. Furthermore, ectoin can enhance skin immunity and barrier function, effectively repairing problematic skin conditions such as sunburn, a weakened stratum corneum, sensitive skin, and damaged skin.

[0005] However, due to the complex barrier function of the skin, especially the barrier function of the stratum corneum, ectoine is not easily absorbed by the skin, which limits its application in cosmetics. Therefore, it is urgent to improve the transdermal properties, absorption and stability of ectoine to promote its efficacy.

[0006] As a drug delivery system, liposomes have shown certain application prospects in the field of cosmetics. However, it has the problem of limited encapsulation efficiency when encapsulating water-soluble functional substances, and its formula needs to be specially designed according to the properties of the encapsulated substance, which makes the application of liposomes subject to certain restrictions. Although pre-liposomes have improved this, they still cannot meet the needs in terms of targeting. In the delivery process of cosmetic functional substances, due to the lack of precise targeting, liposomes are difficult to accurately transport water-soluble functional substances to target cells or tissues, reducing the utilization efficiency of functional substances. A method discloses a phloretin-hydrophilic active ingredient dual encapsulation delivery preparation based on molecular inclusion complexes, which contains encapsulated carrier particles and molecular inclusion complexes and hydrophilic active ingredients loaded on the encapsulated carrier particles. However, the inclusion complex cannot achieve targeted delivery of drugs.

[0007] Amidst the ongoing advancements in cosmetic raw material formulation technology, targeted liposomes, as a highly promising delivery system, offer a new avenue for the precise delivery of cosmetic active ingredients. Covalently targeted liposomes, with their unique mechanism of action and significant advantages, have become a hot area of research. Covalently targeted liposomes primarily chemically react with specific molecules on target cells through active groups on the liposome surface or within the liposome, forming stable covalent bonds. This allows for specific recognition of target tissues, organs, or cells, enabling efficient drug delivery.

[0008] Hyaluronic acid (HA), a natural biomacromolecule widely present in organisms, possesses excellent biocompatibility, moisturizing properties, and unique targeting characteristics. HA can specifically recognize and bind to the CD44 receptor on the cell surface, which is highly expressed on cells such as fibroblasts. Based on this, covalently linking HA to liposomes to create HA covalently targeted liposomes has the potential to achieve precise targeted delivery to fibroblasts, which is of great significance for enhancing the effectiveness of effective ingredients in cosmetics. Summary of the Invention

[0009] Based on this, the main purpose of this application is to provide a covalently targeted ectoine-loaded liposome that can significantly improve the transdermal absorption efficiency of ectoine, so as to promote the efficacy of ectoine and meet its application needs in the cosmetics field.

[0010] The first aspect of the present application provides a covalently targeted ectoine-loaded liposome, comprising the following raw materials:

[0011] Phospholipids, targeted phospholipids, organic solvents, cholesterol, emulsifiers, polyols, ectoine, hyaluronic acid and water;

[0012] The targeting phospholipids include DSPE-HA, an amidation reaction product of distearoylphosphatidylethanolamine and hyaluronic acid;

[0013] The emulsifier includes Beheneth-25.

[0014] In some embodiments, the covalently targeted ectoine-loaded liposomes include the following raw materials in parts by weight:

[0015] 5-10 parts of phospholipids, 0.05-0.4 parts of targeted phospholipids, 5-10 parts of organic solvents, 0.1-0.5 parts of cholesterol, 2-5 parts of emulsifiers, 1-20 parts of polyols, 6.25-40 parts of ectoine and 50-100 parts of water.

[0016] In some embodiments, the preparation method of DSPE-HA comprises the following steps:

[0017] DSPE, HA and an activating agent are mixed in a solvent and subjected to an amidation reaction to prepare DSPE-HA.

[0018] In some embodiments, the preparation method of DSPE-HA satisfies one or more of the following conditions:

[0019] (1) The activator includes at least one of DCC, NHS, DIC or HOBt;

[0020] (2) The mass ratio of the DSPE to the HA is 1-4:1;

[0021] (3) The average molecular weight of the HA is 1-10 kDa;

[0022] (4) The mass ratio of the activator to the HA is 1-4:1;

[0023] (5) The solvent includes DMF and / or DMSO;

[0024] (6) The volume mass ratio of the solvent to the HA is 50-500 mL:1 g;

[0025] (7) The conditions of the amidation reaction include: reacting at room temperature for 1-5 hours.

[0026] In some embodiments, the covalently targeted ectoine-loaded liposomes further comprise an auxiliary agent; the auxiliary agent comprises at least one of an antioxidant, a preservative, and a pH regulator.

[0027] In some embodiments, the covalently targeted ectoine liposomes satisfy one or more of the following conditions:

[0028] (1) The phospholipids include at least one of soybean lecithin, cephalin, phosphatidylinositol, sphingomyelin, and egg yolk lecithin;

[0029] (2) The organic solvent includes ethanol;

[0030] (3) The polyol includes at least one of glycerol, butanediol and pentanediol; preferably glycerol and / or pentanediol;

[0031] (4) The antioxidant includes at least one of pentaerythritol tetrakis (di-tert-butyl hydroxyhydrocinnamate), tocopherol, tert-butyl hydroxyanethole, tert-butyl hydroxytoluene and propyl gallate;

[0032] (5) The preservative includes at least one of p-hydroxyacetophenone, pentylene glycol, methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, butyl p-hydroxybenzoate, benzoic acid, sodium benzoate and potassium sorbate;

[0033] (6) The pH adjuster includes at least one of sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate and triethanolamine;

[0034] (7) The amount of the antioxidant is 0.1-0.4 parts;

[0035] (8) The amount of the preservative is 0.5-5 parts;

[0036] (9) The amount of the pH regulator is based on adjusting the pH of the covalently targeted ectoine-loaded liposomes to 4.5-7.5.

[0037] In some embodiments, the covalently targeted ectoine liposomes include the following materials:

[0038] Lecithin, targeted lecithin, organic solvent, cholesterol, emulsifier, antioxidant, polyol, ectoine, preservative and water.

[0039] In a second aspect, the present application provides a method for preparing the covalently targeted ectoine-loaded liposomes described in the first aspect, comprising the following steps:

[0040] mixing the phospholipid, the targeted phospholipid, the cholesterol, the emulsifier and the organic solvent to prepare an oil phase;

[0041] mixing the polyol and the oil phase to prepare a primary emulsion;

[0042] removing the organic solvent from the primary emulsion to form blank targeted liposomes;

[0043] The ectoine is mixed with water to form an ectoine solution; the ectoine solution is mixed with the blank targeted liposome to prepare the covalently targeted ectoine-loaded liposome.

[0044] In some embodiments, the preparation method satisfies one or more of the following conditions:

[0045] (1) The preparation temperature of the oil phase is 40-60°C;

[0046] (2) The method of removing the organic solvent from the primary emulsion includes rotary evaporation or reduced pressure concentration; optionally, the conditions of the rotary evaporation include: evaporation temperature 40-60°C, rotation speed 20-100 rpm;

[0047] (3) After the step of preparing the ectoine liposomes, the method further includes a step of homogenizing the ectoine liposomes.

[0048] In a third aspect, the present application provides the use of the covalently targeted ectoine-entrapped liposomes described in the first aspect or the covalently targeted ectoine-entrapped liposomes prepared by the preparation method described in the second aspect in cosmetics.

[0049] In a fourth aspect, the present application provides a cosmetic comprising the covalently targeted ectoine-entrapped liposomes described in the first aspect, the covalently targeted ectoine-entrapped liposomes prepared by the preparation method described in the second aspect, or the liposome preparation described in the third aspect.

[0050] In some embodiments, the cosmetic comprises at least one of a facial mask, an eye cream, a facial cream, a primer, an essence, an emulsion, and a toner.

[0051] Beneficial effects of this application:

[0052] 1. The application adopts targeted phospholipids and phospholipids to prepare covalently targeted encapsulated ectoine liposomes, and targeted phospholipids and emulsifier species are preferred, and homogeneous phase, particle size are small and narrowly distributed, encapsulation efficiency is high and the ectoine liposomes with good stability can be prepared. Targeted phospholipids, as one of the matrix raw materials of liposome, directly participate in the formation of liposome, both can realize the covalently targeted modification of liposome, by the targeting effect of hyaluronic acid on fibroblasts, improve the transdermal effect of ectoine liposomes, accelerate the binding speed of ectoine liposomes and fibroblasts, improve the transdermal absorption efficiency of ectoine, and there is no need to modify liposomes, covalently targeted encapsulated ectoine liposomes can be directly prepared, simple process, easy to industrial production. And liposomes are covalently modified, binding effect is strong, compared with non-covalent modification, it is conducive to improving the stability of liposomes.

[0053] 2. The covalently targeted ectoine-encapsulated liposomes of the present application have good stability, controlled release, improved transdermal effect, fibroblast targeting and improved efficacy, which can promote the efficacy of ectoine, meet the skin delivery requirements of the outer membrane, and have broad application prospects in the field of cosmetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following briefly introduces the drawings required for the embodiments or the prior art description. The drawings are only used to illustrate the preferred embodiments and are not considered to limit the present application. The same reference symbols are used throughout the drawings to represent the same components. In the drawings:

[0055] Figure 1 Schematic diagram of the preparation of covalently targeted ectoine-loaded liposomes according to Examples 1-3 of the present application;

[0056] Figure 2This is the H NMR spectrum of the targeted phospholipid DSPE-HA;

[0057] Figure 3 is the infrared spectrum of the targeted phospholipid DSPE-HA;

[0058] Figure 4 This is a TEM electron micrograph of the covalently targeted ectoine-loaded liposomes of Example 1;

[0059] Figure 5 The particle size, PDI, and potential change trend diagram of the covalently targeted ectoine-loaded liposomes of Example 1 under seven different environments;

[0060] Figure 6 This is a graph showing the results of a Turbiscan lab stability analyzer for the covalently targeted ectoine-loaded liposomes of Example 1;

[0061] Figure 7 Graph showing Franz diffusion cell transdermal test results of the covalently targeted ectoine-loaded liposomes of Example 1, the non-targeted ectoine-loaded liposomes of Comparative Example 1, and the free ectoine solution prepared in Comparative Example 2;

[0062] Figure 8 The sustained release test results of the covalently targeted ectoine-loaded liposomes of Example 1, the non-targeted ectoine-loaded liposomes of Comparative Example 1, and the free ectoine solution of Comparative Example 2 are shown;

[0063] Figure 9 This is a cell fluorescence data analysis diagram of the covalently targeted FITC-loaded liposomes and the non-targeted FITC liposomes prepared in Test Example 9;

[0064] Figure 10 This is an analysis chart of the human efficacy experimental data of the covalently targeted ectoine liposome essence of Example 1, the non-targeted ectoine liposome essence of Comparative Example 1, the free ectoine solution essence of Comparative Example 2 and the blank essence, wherein A is the increase rate of skin water content in 1 week, B is the reduction rate of skin transepidermal water loss in 1 week, and C is the increase rate of skin elasticity in 4 weeks. DETAILED DESCRIPTION

[0065] To make the objectives, technical solutions, and advantages of this application more clear and to provide a more thorough and comprehensive understanding of the disclosure of this application, the following will provide a clear and complete description of the technical solutions of this application in conjunction with the specific embodiments of this application and the corresponding drawings. The described embodiments are only part of the embodiments of this application, not all of them.

[0066] The following is a detailed description of the implementation of this application in conjunction with the accompanying drawings. This embodiment is implemented based on the technical solution of this application, and provides a detailed implementation method and specific operation process, but the scope of protection of this application is not limited to the following embodiment.

[0067] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are for the purpose of describing specific embodiments only and are not intended to limit this application.

[0068] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are for the purpose of describing specific embodiments only and are not intended to limit this application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0069] The words "preferably", "more preferably", "preferably", "better", etc. in this application refer to embodiments of the present application that may provide certain beneficial effects in certain circumstances. However, other embodiments may also be preferred under the same circumstances or other circumstances. In addition, the statement 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", "preferably", "better", etc. are merely descriptions of implementation methods or examples with better effects, but do not constitute a limitation on the scope of protection of this application.

[0070] In the present application, “further”, “further”, “particularly”, etc. are used for descriptive purposes to indicate differences in content, but should not be understood as limiting the scope of protection of the present application.

[0071] In this application, "above" and "below" both include the number itself, such as 1 or below, which means ≥1.

[0072] In this application, "at least one" means more than one, such as one, one and more than one. "Multiple" or "several" means at least one, such as one, three, etc., and "multi-layer" means at least two layers, such as two layers, three layers, etc., unless otherwise clearly defined. In the description of this application, "several" means at least one, such as one, two, etc., unless otherwise clearly defined.

[0073] When a numerical range is disclosed in this application, the range is considered continuous and includes the minimum and maximum values of the range, as well as every value between such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum values of the range is included. In addition, when multiple ranges are provided to describe a feature or characteristic, the ranges can be combined. In other words, unless otherwise indicated, all ranges disclosed herein are understood to include any and all subranges subsumed therein.

[0074] Unless otherwise specified, all steps of the present application may be performed sequentially or randomly. For example, the method includes steps (a) and (b), which means that the method may include steps (a) and (b) performed sequentially, or may include steps (b) and (a) performed sequentially. For example, the method may further include step (c), which means that step (c) may be added to the method in any order. For example, the method may include steps (a), (b) and (c), or may include steps (a), (c) and (b), or may include steps (c), (a) and (b), etc.

[0075] The terms "first" and "second" in this application are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of technical features indicated. Therefore, features specified as "first" or "second" may explicitly or implicitly include at least one of such features.

[0076] As used herein, the words "comprise," "include," "contain," "have," "have," or other variations thereof are intended to encompass non-exclusive inclusions, and no distinction is made between these terms. The term "comprising" means that other steps and ingredients that do not affect the end result may be added. The compositions and methods / processes of the present application comprise, consist of, and consist essentially of the essential elements and limitations described herein, as well as any additional or optional ingredients, components, steps, or limitations described herein.

[0077] In this application, no distinction is made between the terms "efficacy", "performance", "effect" and "efficacy".

[0078] The weights of the relevant components mentioned in the examples of this application may not only refer to the specific content of each component, but also represent the weight ratio between the components. Therefore, as long as the content of the relevant components is proportionally enlarged or reduced according to the examples of this application, it is within the scope disclosed in the examples of this application. Specifically, the weights described in the examples of this application may be mass units known in the chemical industry, such as μg, mg, g, and kg.

[0079] In this application, temperature parameters, unless otherwise specified, allow for both constant temperature treatment and treatment within a certain temperature range. The constant temperature treatment allows for temperature fluctuations within the precision range of instrument control. The room temperature referred to in this application refers to 0-40°C, preferably 10-35°C, and more preferably 20-30°C.

[0080] Unless mentioned otherwise, terms in the singular may include plural forms and should not be construed as having one number.

[0081] In addition, the drawings of the present application are not drawn in a 1:1 scale, and the relative sizes of the elements are drawn in the drawings only as examples to facilitate understanding of the present application, but are not necessarily drawn in true proportion. The proportions in the drawings do not constitute a limitation to the present application.

[0082] In this application, unless otherwise specified, temperature parameters may be either constant temperature or fluctuating within a certain temperature range. It should be understood that constant temperature processing allows for temperature fluctuations within the precision range of instrument control. Fluctuations within ranges such as ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are permitted.

[0083] In this application, when referring to a range of units, if only the right endpoint is followed by the unit, it means that the units of the left and right endpoints are the same. For example, 2-5h means that the units of the left endpoint "2" and the right endpoint "5" are both hours.

[0084] The first aspect of the present application provides a covalently targeted ectoine-loaded liposome, comprising the following raw materials:

[0085] Phospholipids, targeted phospholipids, organic solvents, cholesterol, emulsifiers, polyols, ectoine, hyaluronic acid and water;

[0086] The targeting phospholipids include DSPE-HA, an amidation reaction product of distearoylphosphatidylethanolamine and hyaluronic acid;

[0087] The emulsifier includes Beheneth-25.

[0088] Hyaluronic acid can modify the structure of the skin's stratum corneum and recognize receptors on the surface of fibroblasts (such as CD44), achieving transduction and retention. The CD44 receptor has a high affinity for hyaluronic acid (HA) and is overexpressed in fibroblasts compared to normal somatic cells, making it an ideal active targeting receptor.

[0089] The particle size of liposomes has a great influence on their transdermal effect and the degree of uptake by skin cells. Liposomes with smaller particle sizes (≤300nm) have better transdermal effect and are more easily taken up by skin cells, while liposomes with large particle sizes are more difficult to penetrate the surface of the skin and are easily phagocytosed by immune cells such as macrophages in the skin. Therefore, their transdermal effect is poor and they are difficult to be taken up by skin cells.

[0090] This application uses targeted phospholipid DSPE-HA and phospholipids to prepare covalently targeted ectoine-encapsulated liposomes, and optimizes the types of targeted phospholipids and emulsifiers to prepare homogeneous ectoine liposomes with small particle size and narrow distribution, high encapsulation rate and good stability.

[0091] Among them, the targeted phospholipid, as one of the matrix materials of the liposome, directly participates in the formation of the liposome, which can achieve covalent targeted modification of the liposome, improve the transdermal effect of the ectoine liposome through the targeting effect of hyaluronic acid on fibroblasts, accelerate the binding speed of the ectoine liposomes with fibroblasts, and improve the transdermal absorption efficiency of ectoine. It also does not require modification of the liposome, and can directly prepare covalently targeted ectoine-loaded liposomes. The process is simple and easy to industrialize. In addition, covalent modification of the liposome has a strong binding effect, which is beneficial to improving the stability of the liposome compared to non-covalent modification.

[0092] The role of an emulsifier is to help disperse the lipid material in the aqueous phase through emulsification, forming a stable liposome structure. The type and properties of the emulsifier have a significant impact on the properties and stability of the liposome. The use of behenyl ether-25 in this application facilitates the preparation of homogeneous liposomes with small and narrow particle size distribution, high encapsulation efficiency, and good stability. This improves the transdermal effect of the liposomes and promotes their uptake by skin cells, thereby increasing the transdermal absorption efficiency of ectoine.

[0093] In some embodiments, the covalently targeted ectoine-loaded liposomes include the following raw materials in parts by weight:

[0094] 5-10 parts of phospholipids, 0.05-0.4 parts of targeted phospholipids, 5-10 parts of organic solvents, 0.1-0.5 parts of cholesterol, 2-5 parts of emulsifiers, 1-20 parts of polyols, 6.25-40 parts of ectoine and 50-100 parts of water.

[0095] In the present application, the role of phospholipids is to form a bilayer structure of liposomes, and the specific type is not particularly limited. Optionally, the phospholipids include at least one of soybean lecithin, cephalin, phosphatidylinositol, sphingomyelin, and egg yolk lecithin, preferably soybean lecithin, and more preferably soybean lecithin PC60. The amount thereof can be specifically 1.0 parts, 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 8.3 parts.

[0096] Specifically, the amount of the targeted phospholipid can be 0.05 parts, 0.10 parts, 0.12 parts, 0.15 parts, 0.18%, 0.2 parts, 0.25 parts, 0.3 parts, 0.35 parts or 0.4 parts, preferably 0.12 parts.

[0097] In the present application, the role of the organic solvent is to dissolve phospholipids, cholesterol, emulsifiers, and antioxidants to prepare the oil phase. The organic solvent includes ethanol. The specific amount of the organic solvent can be 5.0 parts, 5.5 parts, 6.0 parts, 6.5 parts, 7.0 parts, 7.5 parts, 8.0 parts, 8.5 parts, 9.0 parts, 9.5 parts, or 10.0 parts, preferably 8.3 parts.

[0098] Specifically, the amount of cholesterol can be 0.10 part, 0.15 part, 0.15 part, 0.20 part, 0.27 part, 0.30 part, 0.35 part, 0.40 part, 0.45 part or 0.50 part, preferably 0.27 part.

[0099] Furthermore, maintaining a mass ratio of the phospholipid to the cholesterol of (28-32):1 is beneficial for enhancing vesicle stability and reducing lipid membrane permeability to solutes. It is understood that the mass ratio of the phospholipid to 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.

[0100] Specifically, the amount of the emulsifier can be 2 parts, 2.5 parts, 3 parts, 3.28 parts, 3.5 parts, 4 parts, 4.5 parts, 5 parts, etc., preferably 3.28 parts.

[0101] Specifically, the amount of ectoine can be 6.25 parts, 10.00 parts, 12.50 parts, 15.00 parts, 20.00 parts, 22.50 parts, 25.00 parts, 27.50 parts, 30.00 parts, 32.50 parts, 35.00 parts, 37.50 parts or 40 parts, preferably 6.25 parts.

[0102] In the present application, the role of polyol is to stabilize and protect liposomes, and specific types are not particularly limited. Optionally, the polyol includes at least one of glycerol, butylene glycol and pentanediol, wherein pentanediol also has an antiseptic effect; preferably glycerol and / or pentanediol, further preferably glycerol and pentanediol. The amount of polyol can specifically be 1.0 parts, 2.0 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 10.8 parts.

[0103] In some embodiments, the covalently targeted ectoine-loaded liposomes further comprise an auxiliary agent; the auxiliary agent comprises at least one of an antioxidant, a preservative, and a pH regulator.

[0104] Among them, antioxidants are beneficial to improving the antioxidant properties and stability of liposomes, preservatives are beneficial to improving the safety and stability of liposomes, and pH regulators can adjust the pH of liposomes to an appropriate level, adjust the particle size and distribution of liposomes, and thus improve the performance of covalently targeted ectoine-encapsulated liposomes. They can be selected according to actual needs, and their specific types are not particularly limited. Multiple or one type can be used.

[0105] Optionally, the antioxidant includes at least one of pentaerythritol tetrakis(di-tert-butylhydroxyhydrocinnamate), tocopherol, tert-butylhydroxyanethole, tert-butylhydroxytoluene, and propyl gallate, preferably pentaerythritol tetrakis(di-tert-butylhydroxyhydrocinnamate). The amount of the antioxidant may be 0.1-0.4 parts, including but not limited to 0.1 parts, 0.15 parts, 0.2 parts, 0.25 parts, 0.3 parts, 0.35 parts, or 0.4 parts, and preferably 0.1 parts.

[0106] Optionally, the preservative includes at least one of p-hydroxyacetophenone, methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, butyl p-hydroxybenzoate, benzoic acid, sodium benzoate, and potassium sorbate, preferably p-hydroxyacetophenone. The amount of p-hydroxyacetophenone used may be 0.5-5 parts, including but not limited to 0.5 parts, 1 part, 1.5 parts, 2 parts, 2.5 parts, 3 parts, 3.5 parts, 4 parts, 4.5 parts, or 5 parts.

[0107] Optionally, the pH regulator includes at least one of sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, and triethanolamine, preferably sodium hydroxide. The amount of the pH regulator is based on adjusting the pH of the covalently targeted ectoine-loaded liposomes to 4.5-7.5, and the pH can be 4.5, 5, 5.5, 6, 6.5, 7, 7.5, etc. The concentration of the pH regulator can be 0.5-2 mol / L.

[0108] In some embodiments, the covalently targeted ectoine liposomes include the following materials:

[0109] Lecithin, targeted lecithin, organic solvent, cholesterol, emulsifier, antioxidant, polyol, ectoine, preservative and water.

[0110] In some embodiments, the covalently targeted ectoine-loaded liposomes include the following raw materials in parts by weight:

[0111] 5-10 parts of phospholipids, 0.05-0.4 parts of targeted phospholipids, 5-10 parts of organic solvents, 0.1-0.5 parts of cholesterol, 2-5 parts of emulsifiers, 0.1-0.4 parts of antioxidants, 1-20 parts of polyols, 6.25-40 parts of ectoine, 0.5-5 parts of preservatives and 50-100 parts of water.

[0112] In some embodiments, the covalently targeted ectoine-loaded liposomes include the following raw materials in parts by weight:

[0113] 5-10 parts of phospholipids, 0.05-0.4 parts of targeted phospholipids, 5-10 parts of organic solvents, 0.1-0.5 parts of cholesterol, 2-5 parts of emulsifiers, 0.1-0.4 parts of antioxidants, 1-10 parts of pentylene glycol, 1-10 parts of glycerol, 6.25-40 parts of ectoine, 0.5-5 parts of preservatives and 50-100 parts of water.

[0114] In some embodiments, the particle size of the covalently targeted ectoine-loaded liposomes is 100 nm-400 nm, including but not limited to 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 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 Zeta potential is -10 mV to -40 mV, for example, -10 mV, -15 mV, -20 mV, -25 mV, -30 mV, -35 mV, -40 mV, 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.

[0115] In some embodiments, the preparation method of DSPE-HA comprises the following steps:

[0116] DSPE, HA and an activating agent are mixed in a solvent and subjected to an amidation reaction to prepare DSPE-HA.

[0117] In the present application, the role of the activator is to activate HA and improve the amidation reaction efficiency of HA and DSPE, and the specific type is not particularly limited. Optionally, the activator includes at least one of DCC, NHS, DIC or HOBt.

[0118] In the present application, there is no special requirement for the average molecular weight of HA, which can be 1-10 kDa, such as 1 kDa, 2 kDa, 3 kDa, 4 kDa, 5 kDa, 6 kDa, 7 kDa, 8 kDa, 9 kDa, 10 kDa, etc.

[0119] The role of the solvent in the present application is to disperse and dissolve DSPE and HA to facilitate the reaction. The specific type is not particularly limited and may include DMF and / or DMSO.

[0120] In the present application, the mass ratio of the DSPE to the HA is 1-4:1, for example, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, etc.; the mass ratio of the activator to the HA is 1-4:1, for example, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, etc.; the volume mass ratio of the solvent to the HA is 50-500 mL:1 g, for example, 50 mL:1 g, 100 mL:1 g, 150 mL:1 g, 200 mL:1 g, 250 mL:1 g, 300 mL:1 g, 350 mL:1 g, 400 mL:1 g, 450 mL:1 g, 500 mL:1 g, etc.; the conditions of the amidation reaction include: reaction at room temperature for 1-5 h, for example, 1 h, 2 h, 3 h, 4 h, 5 h, etc.

[0121] In a second aspect, the present application provides a method for preparing the covalently targeted ectoine-loaded liposomes described in the first aspect, comprising the following steps:

[0122] mixing the phospholipid, the targeted phospholipid, the cholesterol, the emulsifier and the organic solvent to prepare an oil phase;

[0123] mixing the polyol and the oil phase to prepare a primary emulsion;

[0124] removing the organic solvent from the primary emulsion to form blank targeted liposomes;

[0125] The ectoine is mixed with water to form an ectoine solution; the ectoine solution is mixed with the blank targeted liposome to prepare the covalently targeted ectoine-loaded liposome.

[0126] This application uses targeted phospholipids and phospholipids to prepare covalently targeted ectoine-encapsulated liposomes. The targeted phospholipids, as one of the matrix materials of the liposomes, directly participate in the formation of the liposomes, without the need for modification of the liposomes, and can directly prepare covalently targeted ectoine-encapsulated liposomes. The preparation method is simple, highly repeatable, and has a short preparation time. There is no residual harmful substance, a high encapsulation rate, and no complex mechanical processes and equipment are required. The product quality and process have good reproducibility and stability, and are easy to industrialize.

[0127] In some embodiments, the method for preparing the covalently targeted ectoine-loaded liposomes comprises the following steps:

[0128] mixing the phospholipid, the targeted phospholipid, the cholesterol, the emulsifier, the antioxidant and the organic solvent to prepare an oil phase;

[0129] mixing the polyol and the oil phase to prepare a primary emulsion;

[0130] removing the organic solvent from the primary emulsion to form blank targeted liposomes;

[0131] The ectoine is mixed with water to form an ectoine solution; the ectoine solution is mixed with the blank targeted liposome, and a preservative is added to prepare the covalently targeted ectoine-loaded liposome.

[0132] 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.

[0133] Optionally, the method of removing the organic solvent in the primary emulsion includes rotary evaporation or reduced pressure concentration; specifically, the conditions of the rotary evaporation include: evaporation temperature 40-60°C, for example, 40°C, 45°C, 50°C, 55°C, 60°C, etc.; rotation speed 20-100rpm, for example, 20rpm, 30rpm, 40rpm, 50rpm, 60rpm, 70rpm, 80rpm, 90rpm, 100rpm, etc.; the conditions of the reduced pressure concentration include: 40-60°C, for example, 40°C, 45°C, 50°C, 55°C, 60°C, etc.; time is 30-180min, for example, 30min, 60min, 90min, 120min, 150min, 180min, etc.

[0134] In a third aspect, the present application provides the use of the covalently targeted ectoine-entrapped liposomes described in the first aspect or the covalently targeted ectoine-entrapped liposomes prepared by the preparation method described in the second aspect in cosmetics.

[0135] In a fourth aspect, the present application provides a cosmetic comprising the covalently targeted ectoine-entrapped liposomes described in the first aspect or the covalently targeted ectoine-entrapped liposomes prepared by the preparation method described in the second aspect.

[0136] The covalently targeted ectoine liposomes of the present application have good stability; have a controlled release effect, and the release rate is slower than that of the unmodified ectoine liposomes; have an improved transdermal effect, and the cumulative release amount and cumulative retention amount are significantly improved compared with the unmodified ectoine liposomes; have a fibroblast targeting effect, and the accumulation of ectoine liposomes on fibroblasts is significantly increased compared with the unmodified ectoine liposomes; have improved efficacy, and compared with the unmodified ectoine liposomes, can enhance skin hydration, regulate transepidermal water loss and improve skin elasticity, can promote the efficacy of ectoine, meet the skin delivery requirements of the outer membrane, and have broad application prospects in the field of cosmetics.

[0137] In some embodiments, the cosmetic comprises at least one of a facial mask, an eye cream, a facial cream, a primer, an essence, an emulsion, and a toner.

[0138] Unless otherwise specified, the raw materials used in the following experiments can be purchased from the market.

[0139] The information of the instruments and performance test methods used in the Examples and Comparative Examples are as follows:

[0140] Phospholipids: soybean lecithin PC60, purchased from Hebei Meiye Siwei Biotechnology Co., Ltd.;

[0141] Behenyl ether-25: purchased from Shanghai Longquan Chemical Technology Co., Ltd.

[0142] Hyaluronic acid: average molecular weight 3 kDa, purchased from Shandong Freda Biological Co., Ltd.

[0143] Polyglyceryl-10 myristate: purchased from Shanghai Zhaoheng Industrial Co., Ltd.

[0144] Ammonium acryloyldimethyltaurate / VP copolymer (AVC): purchased from Shanghai Macklin Co., Ltd.

[0145] Antioxidant: pentaerythritol tetrakis-di-tert-butylhydroxyhydrocinnamate, commercially available;

[0146] Other raw materials are commonly available on the market.

[0147] The following are specific examples.

[0148] Preparation Example 1

[0149] Preparation of targeted phospholipid DSPE-HA:

[0150] S1: Dissolve 233 mg of DSPE phospholipid, 200 mg of NHS, and 100 mg of HA (3 kDa) in 10 mL of DMF and stir at room temperature for 2 h to obtain a reaction solution;

[0151] S2: The reaction solution was placed in a 3 kDa dialysis bag and dialyzed in water for 3 days, with the water replaced every 12 hours.

[0152] S3: Transfer the dialyzed reaction solution to a culture dish and freeze it for 24 hours until it is completely frozen;

[0153] S4: After freeze-drying for 48 hours, the sample on the surface of the culture dish was collected, which is the targeted phospholipid DSPE-HA. See the schematic diagram. Figure 1 .

[0154] Examples 1-3

[0155] Preparation of covalently targeted ectoine-loaded liposomes:

[0156] 1) Weigh the raw materials according to Table 1, mix anhydrous ethanol, targeted phospholipid (Preparation Example 1), phospholipid, and cholesterol, and mechanically stir at 50°C until completely dissolved; then add an emulsifier and antioxidant and stir until the solution is clear and transparent to obtain an oil phase;

[0157] 2) Add glycerol and pentanediol, mix, mechanically stir at 50°C until completely dissolved, and then mix with the oil phase to prepare a primary emulsion;

[0158] 3) Remove ethanol from the primary emulsion by rotary evaporation (50°C, 40 rpm) to obtain blank targeted liposomes;

[0159] 4) dissolving ectoine in water to prepare an ectoine solution; preheating to 50°C, and adding dropwise to the prepared blank targeted liposomes, maintaining 50°C during the addition process, heating and stirring to mix, and after cooling to room temperature, adding a preservative and dispersing evenly to obtain the covalently targeted ectoine-loaded liposomes.

[0160] Table 1 Formula and properties of covalently targeted ectoine-loaded liposomes (total amount of each raw material is 100 parts)

[0161]

[0162] The covalently targeted ectoine-encapsulated liposomes prepared in Examples 1-3 had no stratification, leakage, agglomeration, or flocculation. As the amount of DAFE-HA added increased, the particle size and PDI increased, but the encapsulation efficiency was above 90%. It can be seen that uniform and stable covalently targeted ectoine-encapsulated liposomes with good properties were prepared. When the addition amount was 0.12%, the particle size was the smallest and the encapsulation efficiency was the highest.

[0163] Comparative Example 1

[0164] Preparation of non-targeted ectoine-loaded liposomes: The only difference from Example 1 was that the targeting phospholipid DSPE-HA was not used. 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.

[0165] Comparative Example 2

[0166] Preparation of free ectoine aqueous solution:

[0167] Ectoin was dissolved in water to prepare a 6.25 wt% free ectoin aqueous solution.

[0168] Comparative Example 3

[0169] Except that the targeted phospholipid DSPE-HA in Example 1 was replaced by DOPE-HA (the preparation process was basically the same as that in Preparation Example 1, the only difference being that DOPE was used instead of DSPE), the rest was the same as Example 1.

[0170] Comparative Example 4

[0171] Except that the emulsifier Beheneth-25 was replaced by Span 40, the rest was the same as in Example 1.

[0172] Comparative Example 5

[0173] Except that the emulsifier Beheneth-25 was replaced by Span 60, the rest was the same as in Example 1.

[0174] Comparative Example 6

[0175] Except that the emulsifier Beheneth-25 was replaced by Tween 80, the rest was the same as in Example 1.

[0176] Table 2 Summary of the properties of the liposomes of Example 1 and Comparative Examples 3-6

[0177]

[0178] As can be seen from Table 2, the encapsulation efficiency of Comparative Examples 3-6 is significantly inferior to that of Example 1, and there are also large differences in particle size and PDI. It can be seen that the specific type of targeted phospholipid and the type of emulsifier have a great influence on the properties of liposomes. Only by using the targeted phospholipids and emulsifiers defined in this application can we prepare homogeneous ectoine liposomes with small and narrow particle size distribution, high encapsulation efficiency and good stability.

[0179] Application Examples

[0180] Preparation of essence:

[0181] The covalently targeted ectoine liposomes of Example 1, the non-targeted ectoine liposomes of Comparative Example 1, and the free ectoine aqueous solution of Comparative Example 2 were respectively taken to prepare an essence according to the following formula: liposomes or free ectoine aqueous solution 64wt%, glycerol 3wt%, propylene glycol 5wt%, butylene glycol 5wt%, pentylene glycol 1wt%, polyglycerol-10 myristate 1wt%, EDTA-2Na 0.03wt%, ammonium acryloyldimethyltaurate / VP copolymer 0.4wt%, parahydroxyacetophenone 0.2wt% and the balance water. At the same time, the liposomes were replaced with pure water to prepare a blank essence.

[0182] The preparation method of the essence is as follows: first, stir ammonium acryloyldimethyltaurate / VP copolymer in glycerol, propylene glycol, butylene glycol, and pentylene glycol, add polyglyceryl-10 myristate, parahydroxyacetophenone, EDTA-2 Na and water, raise the temperature to 85°C, and keep warm for 30 minutes; cool to 40°C, then add the corresponding liposomes, free ectoine aqueous solution or water, and mix evenly to prepare the essence.

[0183] Test Example 1 Nuclear Magnetic Resonance Proton Spectroscopy Detection of Targeted Phospholipids

[0184] The targeted phospholipid DSPE-HA was tested by nuclear magnetic resonance hydrogen spectrum to prove the formation of covalent bonds between its phospholipid DSPE and the targeting molecule (hyaluronic acid), proving the existence of targeting at a theoretical level.

[0185] Use dimethyl sulfoxide (DMSO) to dissolve the target phospholipid DSPE-PEG-Nonapeptide-1 and phospholipid DSPE-PEG respectively. After the appropriate concentration is completely dissolved, they are placed in the NMR tube. Ensure that the sample solution in the NMR tube is kept at a height of 4-5 cm to meet the detection requirements. Debug the instrument power system and gas supply system, set the detection parameters and lock, tune, and uniform the field. Then, the NMR hydrogen spectrum of the target phospholipid is detected. The results are as follows Figure 2 As shown in Figure 2, it can be seen that the phospholipid DSPE successfully reacted with the targeting molecule (hyaluronic acid) to prepare DSPE-HA.

[0186] Test Example 2 Nuclear infrared spectroscopy detection of targeted phospholipids

[0187] The targeted phospholipid DSPE-HA was tested by infrared spectroscopy to prove the formation of covalent bonds between its phospholipids and the targeting molecules, proving the existence of targeting at a theoretical level.

[0188] The scanning range is usually set to 400-4000cm -1 ; The resolution is generally set to 2-4cmcm -1; The number of scans can be set to 16-64 times to obtain a better signal-to-noise ratio. Before performing a sample scan, perform a background scan to obtain the background signal of the instrument. During the background scan, no sample is placed in the sample pool. After the scan is completed, the instrument will automatically record the background spectrum and deduct the background signal from the subsequent sample spectra to eliminate the influence of the instrument itself and environmental factors on the test results. Place the pressed DSPE-HA on the sample holder of the infrared spectrometer, make sure that the sample is aligned with the light path, and avoid touching the test surface of the sample during the placement process to prevent contamination and damage to the sample. Close the sample pool cover and click the "Start Scan" button. The instrument starts to scan the infrared spectrum of the sample. During the scan, you can observe the real-time spectrum to check the quality and stability of the spectrum. If any abnormality occurs, stop the scan in time and check the cause. The results are as follows Figure 3 As shown, it can be seen that the phospholipid DSPE and the targeting molecule (hyaluronic acid) successfully reacted to prepare DSPE-HA.

[0189] Test Example 3

[0190] The covalently targeted ectoine-loaded liposomes prepared in Example 1 were subjected to TEM testing, and the results were shown in FIG. Figure 4 , the specific method is as follows:

[0191] Weigh the sample of covalently targeted ectoine liposomes and drop it on the copper mesh. After a few seconds, gently pick up the copper mesh sample with tweezers and absorb the excess liquid along one side with filter paper. After it is slightly dry, place the copper mesh on a drop of 2% phosphotungstic acid dye and float it for 60 seconds. Pick it up with tweezers and absorb the excess liquid along one side with filter paper. Place it on the filter paper with the membrane facing up to dry. Observe and take pictures with a transmission electron microscope. Figure 4 It can be seen that the particles are spherical and distributed relatively evenly.

[0192] Test Example 4 Liposome Particle Size, PDI, Zeta Potential and Encapsulation Efficiency Test

[0193] 1) The particle size, PDI, and zeta potential of the examples were measured using a ZETASIZER PRO particle size analyzer. Particle size and PDI were measured in accordance with the methods specified in GB / T 19077-2016 Particle Size Distribution by Laser Diffraction Method. Zeta potential was measured in accordance with GB / Z 42353-2023 Zeta Potential Determination Operating Guide. The results are shown in Tables 1-2.

[0194] 2) The encapsulation efficiency of the covalently targeted ectoine-loaded liposomes prepared in Examples 1-3 was tested using the following method:

[0195] The encapsulation efficiency of cell membranes in liposomes was determined using high-speed centrifugation. Using 3KD ultrafiltration tubes (Millipore UFC501008, USA), 0.5 mL of liposomes was added to the inner tube and centrifuged at 14,000 rpm for 15 minutes to separate the liposomes. The filtrate was collected and the volume recorded. Liposomes and solvent (methanol) were mixed in a 1:1 ratio and allowed to stand for half an hour before membrane rupture. Appropriate amounts of the ruptured liposomes and ultrafiltration filtrate were collected and diluted appropriately. The absorbance or peak area was measured by HPLC to determine the total concentration of water-soluble substances in the liposomes and the concentration of free water-soluble substances. The encapsulation efficiency (EE%) was calculated using the following formula. The results are shown in Tables 1-2.

[0196]

[0197] C1—amount of drug in liposome suspension ( );

[0198] V1—0.5 (mL);

[0199] C2—amount of drug in the filtrate ( );

[0200] V2—filtrate volume (mL).

[0201] Test Example 5 Liposome Stability Investigation

[0202] The covalently targeted ectoine-loaded liposomes prepared in Example 1 and Comparative Example 3 were placed under normal conditions, light, dark, 4°C, 45°C, -15°C, and freeze-thaw cycle conditions (one freeze-thaw cycle is one after another at -15°C for 24 hours and then at 45°C for 24 hours). The particle size, PDI, and potential stability of the liposomes were determined. Figure 5 As shown, it can be seen that the covalently targeted ectoine liposomes prepared in Example 1 have good stability under different conditions. The covalently targeted ectoine liposomes of Comparative Example 3 have good stability only within 7 days under different test conditions. After 7 days, the liposomes are stratified, while the liposomes prepared by DSPE-HA have good stability for 90 days. Figure 5 As shown in the figure, it is proved that the targeted liposomes prepared by DSPE-HA have better stability than DOPE-HA.

[0203] Test Example 6 Liposome Stability Test

[0204] The stability of the covalently targeted ectoine-loaded liposomes prepared in Example 1 was investigated. Samples were taken from the sample bottle of the Turbiscan Lab stability analyzer to measure the dynamic changes in the stability of the liposomes. The parameters were set to scan once every 30 minutes and the scanning time was 24 hours. The results are shown in Figure 2. Figure 6It is generally believed that when the average backscattered light intensity is less than 0.2, the system is considered to be in an absolutely stable state. It can be seen that the covalently targeted ectoine-loaded liposomes prepared in Example 1 will not precipitate or flocculate, and have good stability.

[0205] Test Example 7 Liposome Transdermal Test

[0206] Transdermal experiments were performed on the covalently targeted ectoine-entrapped liposomes prepared in Example 1, the non-targeted ectoine-entrapped liposomes prepared in Comparative Example 1, and the free ectoine aqueous solution of Comparative Example 2.

[0207] The transdermal experiment was conducted in a Franz diffusion cell apparatus, with the guinea pig abdominal skin fixed between the receiving and supply cells (with the inner layer of the skin facing the receiving cell). The effective permeation area of the skin was 1.77 cm 2 The receiving chamber has a volume of 12 mL and a magnetic stirring speed of 300 rpm. Fill the receiving chamber with saline as the release medium, remove bubbles, start stirring, and maintain a constant temperature of (37.0 ± 0.5)°C. Apply each sample (containing the same active ingredient content) evenly to the skin surface (denoted as Samples 1, 2, and 3, respectively (see Table 3). After 24 hours, use a sampling needle to collect 1 mL of sample into an EP tube. Then, use a non-porous puncture needle to pull the receiving chamber to remove bubbles, and then add 1 mL of isothermal receiving solution to the receiving chamber.

[0208] After 24 hours, the skin was removed and the residual sample liquid on the surface of the mouse skin was washed with ultrapure water. The mouse skin was then cut into pieces and placed in a 10mL eppendorf tube. 3mL of methanol was added and ultrasonicated for 30 minutes. After ultrasonication, the tube was centrifuged at 5000rpm for 10 minutes. The supernatant was taken and the content of the effective substance was determined by HPLC, which is the retention amount of the effective substance in the skin (Qs).

[0209] The cumulative permeation amount Qn of the functional substances at different times is calculated by the following formula:

[0210]

[0211] Where Q n is the cumulative permeation per unit area at the nth time point (μg / cm 2 ), V0 is the volume of the liquid in the receiving pool (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 sampling point (μg / mL), S is the effective area (cm 2 ).

[0212] The retention amount of the active substance in the skin after 24 hours, Q, was calculated by the following formula:s :

[0213]

[0214] Where Q s is the retention 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), A is the effective diffusion area (cm 2 ).

[0215] Table 3

[0216]

[0217] The in vitro retention test was conducted on Example 1, Comparative Example 1 and Comparative Example 2 of the present application. The test results are shown in FIG. Figure 7 As shown, the results show that the cumulative release and retention of sample 3 are the lowest. This may be due to the inability of free ectoine to break through the barrier through active pathways such as fusion and lipid penetration to enter the appropriate release environment in the skin. Compared with sample 2, the cumulative release of sample 1 increased by 41% and the cumulative retention increased by 21%. In summary, it can be seen that the surface modification of liposomes with hyaluronic acid can further increase the penetration and retention of ectoine in the skin, so that it can exert its efficacy.

[0218] Test Example 8 Liposome sustained release test

[0219] In vitro release tests were performed on the covalently targeted ectoine-encapsulated liposomes prepared in Example 1, the non-targeted ectoine-encapsulated liposomes prepared in Comparative Example 1, and the free ectoine aqueous solution of Comparative Example 2, and the ectoine in the release medium was measured by dialysis with PBS. The sample was placed in a dialysis bag (molecular weight cutoff of 6000-8000), and the dialysis bag was placed in a 250 mL beaker containing 200 mL of 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 culture), all release media (1 mL) were removed and replaced with fresh release media (1 mL) of the same volume. The release amount of ectoine was determined by HPLC, and a slow release curve was drawn. The results are shown in Table 1. Figure 8 .

[0220]

[0221] Qi: Cumulative release at the i-th sampling time point, %

[0222] V: volume of release medium, mL

[0223] ρ n : Concentration of ectoine in the medium released during the nth sampling process, μg / mL

[0224] ρ i : The concentration of ectoine released in the medium during the i-th sampling process, μg / mL

[0225] Vi: the volume of the i-th sampling, mL

[0226] m: mass of D-ectoine in the sample, μg

[0227] It can be seen that the release of ectoine from non-targeted ectoine liposomes is 30% within 2 hours and about 80% after 72 hours. This may be because the drug is encapsulated in the lipid membrane and is mainly released through dissolution and diffusion of the lipid bilayer. Liposomes can act as a rate-limiting membrane barrier, thus providing a controllable system.

[0228] The release of ectoine from non-targeted ectoine-encapsulated liposomes was 30% within 2 hours and approximately 80% after 72 hours. This is likely due to the drug being encapsulated in a lipid membrane and released primarily through dissolution and diffusion of the lipid bilayer. The liposome can act as a rate-limiting membrane barrier, thus providing a controllable system. After adding DAPE-HA, the release rate of covalently targeted ectoine liposomes was 26% within 2 hours and approximately 77% after 72 hours. Therefore, the addition of DAPE-HA can effectively regulate the in vitro release rate of ectoine, achieving better controlled release compared to drug solutions or simple liposome formulations.

[0229] Test Example 9 Liposome Cell Uptake Test

[0230] In order to verify the difference between the covalently targeted ectoine-loaded liposomes prepared in Example 1 and the non-targeted ectoine-loaded liposomes prepared in Comparative Example 1 in targeting fibroblasts, the ectoine in Example 1 and Comparative Example 1 was replaced with the fluorescent substance FITC to prepare covalently targeted FITC-loaded liposomes and non-targeted FITC liposomes, and the following experiments were performed:

[0231] Fibroblasts were cultured and incubated for the same time after drug administration. The cell nuclei were stained with Hoechst33342, and then the difference in green fluorescence between covalently targeted FITC-loaded liposomes and non-targeted FITC liposomes on the fibroblasts was observed under a confocal fluorescence microscope.

[0232] The experimental results are as follows Figure 9As shown. The fluorescence of the non-targeted FITC liposome group showed a small amount of aggregation around the cells. In contrast, the covalently targeted FITC liposome group showed obvious fluorescent 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 effect of hyaluronic acid, the accumulation of covalently targeted FITC liposomes on fibroblasts increased significantly, resulting in different targeting properties of covalently targeted FITC liposomes and non-targeted FITC liposomes on fibroblasts.

[0233] Test Example 10 Liposome Human Efficacy Test

[0234] In order to further demonstrate the targeting property of hyaluronic acid, the anti-aging efficacy was verified using the covalently targeted ectoine liposome essence prepared in Example 1, the non-targeted ectoine liposome essence prepared in Comparative Example 1, the free ectoine aqueous solution essence in Comparative Example 2, and a blank essence. The moisture content of the skin stratum corneum can indirectly reflect the moisture content of the stratum corneum. The higher the measured value, the higher the moisture content of the stratum corneum, indicating that the product has a better moisturizing effect. One of the main functions of the skin barrier is to prevent the body from discharging water to the surrounding environment through the skin, and transepidermal water loss (TEWL) is an important non-invasive skin water loss rate test indicator to characterize the skin barrier function. The smaller the measured value, the lower the transepidermal 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 shape 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 were evaluated before and after the application of different samples, and the results are as follows: Figure 10 shown.

[0235] Compared with the covalently targeted ectoine liposome essence prepared in Example 1, the non-targeted ectoine liposome essence of Comparative Example 1 and the free ectoine essence of Comparative Example 2, the skin water content increased by 2.38 times and 1.52 times respectively after 1 hour, increased by 1.86 times and 1.38 times respectively at 4 hours, and increased by 2.51 times and 1.44 times respectively at 7 days. In terms of TEWL value, the covalently targeted ectoine liposome essence prepared in Example 1 resulted in a decrease in TEWL. Compared with the covalently targeted ectoine liposome essence prepared in Example 1, the non-targeted ectoine liposome essence of Comparative Example 1 and the free ectoine essence of Comparative Example 2, the TEWL values decreased by 1.90 times and 1.45 times respectively at 1 hour, decreased by 1.78 times and 1.37 times respectively at 4 hours, and decreased by 2.45 times and 1.69 times respectively at 7 days. Hyaluronic acid has significant water absorption capacity, which is enough to effectively moisturize the stratum corneum and dermis, resulting in changes in the lipid self-assembly microstructure and a decline in the barrier function of the stratum corneum, and ultimately promoting skin penetration. Ectoin reduces skin moisture loss by adsorbing water molecules and binding to them through hydrogen bonds. The covalently targeted entrapped ectoin liposome essence prepared in Example 1, the non-targeted entrapped ectoin liposome essence of Comparative Example 1, and the free ectoin essence of Comparative Example 2 improved skin elasticity by 2.10 times and 1.07 times on the 7th day, 1.91 times and 1.43 times on the 14th day, and 2.88 times and 1.37 times on the 30th day. Therefore, the covalently targeted entrapped ectoin liposome essence prepared in Example 1 has effective skin elasticity enhancing properties. This can be attributed to the good skin penetration of hyaluronic acid-modified liposomes, which increases the efficacy of exotin. In addition, the strong skin moisturizing properties of hyaluronic acid also contribute to this effect.

[0236] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0237] The above-described embodiments merely represent several implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art could make various modifications and improvements without departing from the spirit of the present application, all of which fall within the scope of protection of the present application. Therefore, the scope of protection of the present patent application shall be determined by the appended claims.

Claims

1. A covalently targeted ectoine-loaded liposome, characterized in that: Including the following ingredients: Phospholipids, targeted phospholipids, organic solvents, cholesterol, emulsifiers, polyols, ectoine, hyaluronic acid and water; The targeting phospholipids include DSPE-HA, an amidation reaction product of distearoylphosphatidylethanolamine and hyaluronic acid; The emulsifier includes Beheneth-25.

2. The covalently targeted ectoine-loaded liposome according to claim 1, wherein: The covalently targeted ectoine-loaded liposomes include the following raw materials in parts by weight: 5-10 parts of phospholipids, 0.05-0.4 parts of targeted phospholipids, 5-10 parts of organic solvents, 0.1-0.5 parts of cholesterol, 2-5 parts of emulsifiers, 1-20 parts of polyols, 6.25-40 parts of ectoine and 50-100 parts of water.

3. The covalently targeted ectoine-loaded liposome according to claim 1 or 2, wherein: One or more of the following conditions are met: (1) The covalently targeted ectoine-loaded liposome further comprises an adjuvant; the adjuvant comprises at least one of an antioxidant, a preservative, and a pH regulator; (2) The preparation method of DSPE-HA comprises the following steps: DSPE, HA and an activating agent are mixed in a solvent and subjected to an amidation reaction to prepare DSPE-HA.

4. The covalently targeted ectoine-loaded liposome according to claim 3, wherein: One or more of the following conditions are met: (1) The phospholipids include at least one of soybean lecithin, cephalin, phosphatidylinositol, sphingomyelin, egg yolk lecithin, DSPE-HA, and DOPE-HA; (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 antioxidant includes at least one of pentaerythritol tetrakis (di-tert-butyl hydroxyhydrocinnamate), tocopherol, tert-butyl hydroxyanethole, tert-butyl hydroxytoluene and propyl gallate; (5) The preservative includes at least one of p-hydroxyacetophenone, pentylene glycol, methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, butyl p-hydroxybenzoate, benzoic acid, sodium benzoate and potassium sorbate; (6) The pH adjuster includes at least one of sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate and triethanolamine; (7) The amount of the antioxidant is 0.1-0.4 parts; (8) The amount of the preservative is 0.5-5 parts; (9) The amount of the pH regulator is based on adjusting the pH of the covalently targeted ectoine-loaded liposomes to 4.5-7.5; (10) The activator includes at least one of DCC, NHS, DIC or HOBt; (11) The mass ratio of the DSPE to the HA is 1-4:1; (12) The average molecular weight of the HA is 1-10 kDa; (13) The mass ratio of the activator to the HA is 1-4:1; (14) The solvent includes DMF and / or DMSO; (15) The volume mass ratio of the solvent to the HA is 50-500 mL:1 g; (16) The conditions of the amidation reaction include: reacting at room temperature for 1-5 hours.

5. The covalently targeted ectoine-loaded liposome according to any one of claims 1 to 4, characterized in that: The covalently targeted ectoine liposomes include the following raw materials: Lecithin, targeted lecithin, organic solvent, cholesterol, emulsifier, antioxidant, polyol, ectoine, preservative and water.

6. The method for preparing the covalently targeted ectoine-loaded liposomes according to claim 1 or 2, wherein: The steps include: mixing the phospholipid, the targeted phospholipid, the cholesterol, the emulsifier and the organic solvent to prepare an oil phase; mixing the polyol and the oil phase to prepare a primary emulsion; removing the organic solvent from the primary emulsion to form blank targeted liposomes; The ectoine is mixed with water to form an ectoine solution; the ectoine solution is mixed with the blank targeted liposome to prepare the covalently targeted ectoine-loaded liposome.

7. The preparation method according to claim 6, wherein The preparation method satisfies one or more of the following conditions: (1) The preparation temperature of the oil phase is 40-60°C; (2) The method of removing the organic solvent from the primary emulsion includes rotary evaporation or reduced pressure concentration; optionally, the conditions of the rotary evaporation include: evaporation temperature 40-60°C, rotation speed 20-100 rpm; (3) After the step of preparing the ectoine liposomes, the method further includes a step of homogenizing the ectoine liposomes.

8. Use of the covalently targeted ectoine-loaded liposomes according to any one of claims 1 to 5 or the covalently targeted ectoine-loaded liposomes prepared by the preparation method according to claim 6 or 7 in cosmetics.

9. A cosmetic, characterized in that: The invention comprises the covalently targeted ectoine-encapsulated liposomes according to any one of claims 1 to 5 or the covalently targeted ectoine-encapsulated liposomes prepared by the preparation method according to claim 6 or 7.

10. The cosmetic according to claim 9, wherein The cosmetics include at least one of a facial mask, an eye cream, a facial cream, a primer, an essence, an emulsion and a toner.

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