High-permeability heparin sodium liposome as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of liposome preparation, and particularly relates to a high-permeability heparin sodium liposome as well as a preparation method and application thereof. The preparation method of the heparin sodium liposome comprises the following steps: (1) dispersing soybean lecithin in deionized water, and stirring in a water bath to obtain liquid 1; dissolving phytosterol in ethanol, and carrying out ultrasonic treatment to obtain liquid 2; dispersing carboxymethyl chitosan and polyethylene glycol in deionized water, and stirring in a water bath until the solution is clear to obtain a liquid 3; sequentially adding the liquid 1, the liquid 2 and the liquid 3 into a container, and homogenizing to form an oil-in-water microemulsion; and (2) adding heparin sodium into the oil-in-water microemulsion obtained in the step (1), continuing homogenization and ice-bath ultrasonic treatment, adding a natural protective agent into the mixture, uniformly stirring, adjusting the pH value to 5.5-6.5, sterilizing and filtering to obtain the heparin sodium liposome. The heparin sodium liposome has excellent stability and permeability, and when the heparin sodium liposome is applied to cosmetics, the efficacy of the cosmetics can be remarkably improved.

Description

Technical Field

[0001] The invention belongs to the technical field of liposome preparation, and particularly relates to a high-permeability heparin sodium liposome and a preparation method and application thereof. Background Art

[0002] In the cosmetics field, moisturizing, nourishing, and improving various skin problems have always been key areas of research and development, especially given the increasing number of people experiencing skin inflammation in recent years. However, traditional cosmetics have many shortcomings in the delivery and effectiveness of their active ingredients. Ordinary moisturizing ingredients struggle to penetrate deep into the skin's depths, resulting in poor long-term moisturizing effects. Furthermore, ingredients with specific benefits, such as improving skin inflammation and promoting skin repair, often struggle to effectively penetrate the skin's active sites due to their molecular structure or the skin's barrier, significantly reducing the overall effectiveness of the cosmetic.

[0003] Sodium heparin, a substance with multiple biological activities, has been widely used in the medical field for anticoagulation and other applications. If it could be incorporated into cosmetics, leveraging its potential anti-inflammatory and blood circulation-promoting properties, it is expected to play a positive role in improving skin inflammation and enhancing skin vitality. Patent CN118986780A discloses new uses for sodium heparin, a moisturizing composition, its applications, and cosmetics. This invention uses sodium heparin to improve the skin feel of sodium hyaluronate solution. Sodium heparin interacts with sodium hyaluronate via hydrogen bonds, thereby reducing the sticky feel of sodium hyaluronate. When sodium heparin is combined with Dendrobium officinale polysaccharide, the sticky feel of sodium hyaluronate can be further reduced. The invention also provides a moisturizing composition, its applications, and cosmetics. However, sodium heparin itself has problems such as high water solubility and difficulty penetrating the skin's lipid barrier, which greatly limits its effectiveness in cosmetics.

[0004] Liposomes are excellent drug delivery vehicles, offering advantages such as improved drug stability and enhanced drug penetration. However, conventional liposomes, when used in cosmetics, are still not ideal for encapsulating and delivering specialized ingredients like heparin sodium. This prevents the full efficacy of heparin sodium and makes it difficult to meet the cosmetics demand for highly effective, safe, and stable ingredients.

[0005] Therefore, developing a heparin sodium liposome with high permeability and effectively applying it in cosmetics has become an urgent problem to be solved. Summary of the Invention

[0006] The present invention discloses a high-permeability heparin sodium liposome, a preparation method and application thereof. The prepared heparin sodium liposome has excellent stability and permeability, and can be used in cosmetics to significantly improve the efficacy of the cosmetics.

[0007] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] The first aspect of the present invention provides a method for preparing high-permeability heparin sodium liposomes, comprising the following preparation steps:

[0009] (1) Dispersing soybean lecithin in deionized water and stirring in a water bath to obtain liquid 1; dissolving phytosterol in ethanol and ultrasonicating to obtain liquid 2; dispersing carboxymethyl chitosan and polyethylene glycol in deionized water and stirring in a water bath until clear to obtain liquid 3; adding liquid 1, liquid 2, and liquid 3 into a container in sequence and homogenizing to form an oil-in-water microemulsion;

[0010] (2) adding heparin sodium to the oil-in-water microemulsion of step (1), continuing homogenization, ultrasonicating in an ice bath, and then adding a natural protective agent to the mixture, stirring evenly, adjusting the pH, and sterilizing and filtering to obtain heparin sodium liposomes.

[0011] In some embodiments, in step (1), the number average molecular weight of carboxymethyl chitosan is 5,000-50,000, and the number average molecular weight of polyethylene glycol is 2,000-10,000.

[0012] In some embodiments, the mass ratio of soybean lecithin, phytosterol, carboxymethyl chitosan and polyethylene glycol in the oil-in-water microemulsion in step (1) is (4-6):(1-2):(0.5-1):1.

[0013] In some embodiments, the amount of heparin sodium added in step (2) is 1-2.5 wt % of the oil-in-water microemulsion.

[0014] In some embodiments, the natural protective agent in step (2) includes sodium hyaluronate, trehalose and vitamin E.

[0015] In some embodiments, the amount of the natural protective agent in step (2) is 1-5 wt % of the mixture.

[0016] In some embodiments, the pH in step (2) is adjusted to 5.5-6.5.

[0017] In some embodiments, the filter membrane used for filtration in step (2) is of 0.22 μm or 0.45 μm specification.

[0018] The second aspect of the present invention provides high permeability heparin sodium liposomes prepared by the preparation method described in the above scheme.

[0019] In some embodiments, the particle size of the high permeability heparin sodium liposome is ≤250 nm.

[0020] The third aspect of the present invention provides the use of the high permeability heparin sodium liposomes described in the above solution in cosmetics.

[0021] In the preparation process of the present invention, soybean lecithin, as the main film-forming material, is dispersed in deionized water and then initially forms lipid structural units with a certain structure under the action of water bath stirring, thereby obtaining liquid 1; phytosterols are dissolved in ethanol to form liquid 2; when liquid 1 and liquid 2 are mixed, the hydrophobic portion of the phytosterols embeds into the hydrophobic layer of the soybean lecithin, further stabilizing and adjusting the lipid bilayer structure. Carboxymethyl chitosan and polyethylene glycol are dispersed in deionized water to form liquid 3. Carboxymethyl chitosan has good water solubility and certain film-forming properties, while polyethylene glycol plays a role in regulating the viscosity of the system and enhancing dispersibility. After liquid 3 is added to the mixture of the first two, the carboxymethyl chitosan and polyethylene glycol intertwine with the soybean lecithin and phytosterols through intermolecular forces, and during the homogenization process, they jointly construct a stable oil-in-water microemulsion system.

[0022] Sodium heparin is then added to the oil-in-water microemulsion. Due to its water solubility, sodium heparin disperses in the aqueous phase. With continued homogenization and ice-bath ultrasound, the sodium heparin is effectively encapsulated within the aqueous phase of the liposomes. A natural protective agent consisting of sodium hyaluronate, trehalose, and vitamin E is then added. Sodium hyaluronate, with its potent moisturizing properties, forms a hydrated film on the liposome surface, preventing liposome aggregation. The presence of sodium hyaluronate also enhances the liposome's affinity for the skin. When applied to cosmetics, these liposomes can better deliver the encapsulated sodium heparin active ingredient deep into the skin, enhancing overall skincare effectiveness. Trehalose, with its excellent glass transition properties, forms a stable glassy matrix around the liposomes, providing physical protection. Furthermore, trehalose molecules interact with the liposome membrane, filling the spaces between the membrane's phospholipid molecules and enhancing its flexibility and stability. This further protects the encapsulated sodium heparin from leakage and inactivation, ensuring the long-term stability of the liposomes within the cosmetic system. Vitamin E, as an antioxidant, prevents oxidation of the liposome membrane and maintains the stability of the liposome structure, thereby ensuring the liposome's ability to encapsulate and deliver active ingredients such as heparin sodium. It also avoids potential skin irritation from lipid peroxidation products, enhancing the safety and effectiveness of cosmetics containing these liposomes. The pH of the system is adjusted to a suitable pH range of 5.5-6.5, further ensuring the stability of the liposomes. Finally, sterile filtration is performed to obtain highly permeable heparin sodium liposomes.

[0023] The sodium heparin liposomes of the present invention offer the following advantages: 1. Efficient delivery of active ingredients: The highly permeable sodium heparin liposomes prepared in this invention effectively encapsulate sodium heparin, allowing it to penetrate the skin's lipid barrier and penetrate deep into the skin's underlying layers. The anti-inflammatory and blood circulation-boosting properties of sodium heparin are fully realized at its site of action within the skin. Compared to conventional cosmetic ingredients, this significantly improves the effectiveness of cosmetics in alleviating skin inflammation and enhancing skin vitality, significantly enhancing their efficacy. 2. Enhanced cosmetic stability: The liposome structure inherently provides a protective effect, preventing the encapsulated sodium heparin and other active ingredients from being inactivated by environmental factors such as light, temperature, and oxygen. Furthermore, the addition of natural protective agents such as sodium hyaluronate, trehalose, and vitamin E further enhances the stability of the liposomes in various ways, ensuring that the activity and content of the active ingredients remain stable during storage and use, thereby extending the shelf life of the cosmetics. 3. Improved skin compatibility: Ingredients such as soy lecithin and carboxymethyl chitosan exhibit excellent skin compatibility. When present in cosmetics in the form of liposomes, they are more easily absorbed by the skin, reducing skin irritation, making them suitable for all skin types and broadening the range of people for whom cosmetics are intended. 4. Precisely control ingredient release: Liposomes can slowly and continuously release encapsulated heparin sodium and other ingredients according to changes in the skin environment, achieving long-term nourishment and care for the skin. This avoids the waste and adverse effects on the skin caused by the one-time release of active ingredients in traditional cosmetics, and improves the utilization rate of active ingredients. 5. Optimize the texture of cosmetics: The appropriate particle size (≤250nm) allows the highly permeable heparin sodium liposomes to be evenly dispersed in the cosmetic system without affecting the original texture of the cosmetics, such as the fineness of lotions and the smoothness of creams. At the same time, it can also improve the feel of cosmetics applied to the skin, making it more refreshing and comfortable.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1. The present invention discloses a highly permeable heparin sodium liposome, a preparation method thereof, and an application thereof. The raw materials are environmentally friendly and safe, the preparation method is simple, and the prepared heparin sodium liposome has excellent stability and permeability. Its application in cosmetics has many advantages and can significantly improve the efficacy of cosmetics.

[0026] 2. The present invention uses soybean lecithin, phytosterols, carboxymethyl chitosan and polyethylene glycol as liposome raw materials to encapsulate heparin sodium, and adds a natural protective agent composed of sodium hyaluronate, trehalose and vitamin E in a specific proportion to ensure the stability and efficacy of the liposome. DETAILED DESCRIPTION

[0027] Various exemplary embodiments of the present invention are now described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present invention. It should be understood that the terms described in the present invention are only for describing particular embodiments and are not intended to limit the present invention. In addition, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between the intermediate value within any stated value or stated range and any other stated value or intermediate value within the range is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0028] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention. Various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention, as will be apparent to those skilled in the art. Other embodiments obtained from the present invention description will be apparent to those skilled in the art. This application description and examples are exemplary only.

[0029] The molecular weight of the sodium hyaluronate used in the present invention is 3000-10000Da; the molecular weight of the carboxymethyl chitosan (medical grade) used is 9000Da, the degree of carboxylation is ≥80%, and the purity is ≥98%; the sodium heparin used is low molecular weight heparin sodium (4500Da); and the remaining reagents can be obtained through commercial channels.

[0030] Example 1

[0031] A method for preparing high-permeability heparin sodium liposomes comprises the following steps:

[0032] (1) Disperse 5 g of soybean lecithin in 100 mL of deionized water and stir in a water bath at 40°C for 30 min to obtain liquid 1; dissolve 1.5 g of phytosterol in 25 mL of anhydrous ethanol and ultrasonicate at 200 W for 15 min to obtain liquid 2; disperse 0.75 g of carboxymethyl chitosan and 1 g of polyethylene glycol 2000 in 100 mL of deionized water and stir in a water bath at 50°C and 200 rpm until clear to obtain liquid 3; add liquid 1, liquid 2, and liquid 3 into a container in sequence and homogenize at a homogenization pressure of 20 MPa and 12,000 rpm for 10 min to form an oil-in-water microemulsion;

[0033] (2) 5 g of sodium heparin was added to the oil-in-water microemulsion of step (1), and homogenization was continued for 15 min under the same parameters. After ice bath sonication for 15 min, 12 g of natural protective agent was added to the mixture, and the mixture was stirred evenly. The pH was adjusted to 6, and the mixture was filtered through a 0.45 μm sterilizing filter and freeze-dried to obtain sodium heparin liposomes.

[0034] The natural protective agents used include trehalose, sodium hyaluronate and vitamin E in a mass ratio of 60:4:1.

[0035] Example 2

[0036] A method for preparing high-permeability heparin sodium liposomes comprises the following steps:

[0037] (1) Disperse 4 g of soybean lecithin in 100 mL of deionized water and stir in a water bath at 40°C for 30 min to obtain liquid 1; dissolve 1 g of phytosterol in 25 mL of anhydrous ethanol and ultrasonicate at 200 W for 15 min to obtain liquid 2; disperse 0.5 g of carboxymethyl chitosan and 1 g of polyethylene glycol 2000 in 100 mL of deionized water and stir in a water bath at 50°C and 200 rpm until clear to obtain liquid 3; add liquid 1, liquid 2, and liquid 3 into a container in sequence and homogenize at a homogenization pressure of 20 MPa and 10,000 rpm for 15 min to form an oil-in-water microemulsion;

[0038] (2) 2.5 g of heparin sodium was added to the oil-in-water microemulsion of step (1), and homogenization was continued for 15 min under the same parameters. After ice bath ultrasonication for 15 min, 2.5 g of natural protective agent was added to the mixture, and the mixture was stirred evenly. The pH was adjusted to 5.5, and the mixture was filtered through a 0.45 μm sterilizing filter and freeze-dried to obtain heparin sodium liposomes.

[0039] The natural protective agents used include trehalose, sodium hyaluronate and vitamin E in a mass ratio of 55:5:1.

[0040] Example 3

[0041] A method for preparing high-permeability heparin sodium liposomes comprises the following steps:

[0042] (1) Disperse 6 g of soybean lecithin in 100 mL of deionized water and stir in a water bath at 40°C for 30 min to obtain liquid 1; dissolve 2 g of phytosterol in 25 mL of anhydrous ethanol and ultrasonicate at 200 W for 15 min to obtain liquid 2; disperse 1 g of carboxymethyl chitosan and 1 g of polyethylene glycol 2000 in 100 mL of deionized water and stir in a water bath at 50°C and 200 rpm until clear to obtain liquid 3; add liquid 1, liquid 2, and liquid 3 into a container in sequence and homogenize at a homogenization pressure of 20 MPa and 15,000 rpm for 10 min to form an oil-in-water microemulsion;

[0043] (2) 6 g of heparin sodium was added to the oil-in-water microemulsion of step (1), and homogenization was continued for 15 min under the same parameters. After ice bath sonication for 15 min, 12 g of natural protective agent was added to the mixture, and the mixture was stirred evenly. The pH was adjusted to 6.5, and the mixture was filtered through a 0.45 μm sterilizing filter and freeze-dried to obtain heparin sodium liposomes.

[0044] The natural protective agents used include trehalose, sodium hyaluronate and vitamin E in a mass ratio of 65:6:1.

[0045] Example 4

[0046] A method for preparing high-permeability heparin sodium liposomes, the specific implementation method is roughly the same as that of Example 1, except that 2g of carboxymethyl chitosan is used.

[0047] Example 5

[0048] A method for preparing high-permeability heparin sodium liposomes, the specific implementation method is roughly the same as Example 1, except that 7g of heparin sodium is used.

[0049] Example 6

[0050] A method for preparing high-permeability heparin sodium liposomes, the specific implementation method is roughly the same as Example 1, except that 15g of natural protective agent is used.

[0051] Example 7

[0052] A method for preparing highly permeable heparin sodium liposomes, the specific implementation method is substantially the same as that of Example 1, except that sodium hyaluronate is not added to the natural protective agent used.

[0053] Comparative Example 1

[0054] A method for preparing high-permeability heparin sodium liposomes, the specific implementation method is roughly the same as that of Example 1, except that no carboxymethyl chitosan is added.

[0055] Comparative Example 2

[0056] A method for preparing high-permeability heparin sodium liposomes, the specific implementation method is roughly the same as Example 1, except that polyethylene glycol is not added.

[0057] Comparative Example 3

[0058] A method for preparing high-permeability heparin sodium liposomes, the specific implementation method is roughly the same as Example 1, except that no natural protective agent is added.

[0059] Comparative Example 4

[0060] A method for preparing high-permeability heparin sodium liposomes, the specific implementation method is roughly the same as that of Example 1, except that an equal mass of chitosan is used instead of carboxymethyl chitosan.

[0061] Performance Testing

[0062] 1. Particle size: Use dynamic light scattering instrument to test, test 3 times for each group, and take the average value.

[0063] 2. Encapsulation efficiency: 0.1 g of heparin sodium liposomes was dissolved in 100 mL of deionized water to obtain a sample, the concentration of which was marked as V. 总 Centrifuge at 12000 rpm for 30 min, aspirate the supernatant, transfer it to a clean container, measure the volume of the supernatant, and mark it as V 上 , washed with deionized water for three times, and the washing solution was combined with the supernatant; the heparin sodium content in the combined supernatant was determined by high performance liquid chromatography (HPLC), according to the formula: encapsulation efficiency (%) = [(C 总 ×V 总 -C 上 ×V 上 ) / (C 总 ×V 总 )]×100%, where C 总 is the initial heparin sodium concentration when the liposome sample was prepared. The encapsulation efficiency of the heparin sodium liposome was calculated. Each group was tested 3 times and the average value was taken.

[0064] 3. Stability: 0.5 g of heparin sodium liposomes were placed at 45°C and 75% humidity for 30 days. The heparin sodium content was tested by HPLC and the degradation rate was calculated. Each group was tested 3 times and the average value was taken.

[0065] The specific test results are shown in Table 1.

[0066] Table 1

[0067]

[0068]

[0069] As can be seen from Table 1, the heparin sodium liposomes prepared in Examples 1-3 have a high encapsulation efficiency and good stability. Compared with Example 1, the raw material dosage was changed in Examples 5-6, resulting in a decrease in the encapsulation efficiency to varying degrees; in Example 7, the natural protective agent lacked sodium hyaluronate, and the stability of the liposomes decreased; the raw material components in Comparative Examples 1-4 changed, and Comparative Examples 1 and 2 lacked carboxymethyl chitosan and polyethylene glycol, respectively, and had no synergistic effect, which reduced the encapsulation efficiency and stability; Comparative Example 3 lacked a natural protective agent, and the stability of the liposomes was significantly reduced; in Comparative Example 4, chitosan was used instead of carboxymethyl chitosan. Since the molecular structure of carboxymethyl chitosan is modified by carboxymethyl, it can provide suitable steric hindrance during the liposome formation process, which helps to maintain the stability of the liposome structure and encapsulate more drugs. Therefore, the liposome encapsulation efficiency and stability of Comparative Example 4 are also reduced.

[0070] 4. Permeability:

[0071] Experimental groups and sample preparation: control group: 2 wt % heparin sodium aqueous solution; treatment group: the heparin sodium liposomes of Examples 1-3 were prepared into a 2 wt % aqueous solution.

[0072] Assemble and secure the test model: Secure the skin-simulating membrane (Strat-M) between the diffusion chamber and the receiving chamber of a vertical Franz diffusion cell, with the rough surface of the membrane facing the diffusion chamber and the smooth surface facing the receiving chamber. After securing the membrane, add 8 mL of receiving solution (7.4% PBS) to the receiving chamber, ensuring close contact between the membrane and the receiving solution.

[0073] The static vertical transdermal diffusion cell was fixed in the transdermal diffusion instrument, the temperature was set to 32 and the speed of the magnetic stirring rotor was set to 500 rpm.

[0074] Sampling: After the temperature of the diffusion instrument water bath is constant, sample loading is performed. The sample addition amount is 0.1 g. The sample is added to the surface of the skin simulation membrane and spread evenly.

[0075] Receiving pool sample collection: 300 μL of receiving fluid was collected at 2, 6, and 10 hours, and fresh receiving fluid was added. The heparin sodium concentration in the receiving fluid to be tested was quantitatively analyzed by HPLC. Each group was repeated three times, and the test results were averaged.

[0076] The specific test results are shown in Table 2.

[0077] Table 2

[0078]

[0079] As shown in Table 2, the heparin sodium liposomes prepared in Examples 1-3 have good permeability. Starting from the second hour, the cumulative permeation amount is higher than that of the control group.

[0080] The above is only a preferred embodiment of the present invention and does not limit the present application in any form. Although the present application is disclosed as above with a preferred embodiment, it is not intended to limit the present application. Any technician familiar with this profession, without departing from the scope of the technical solution of the present application, makes some changes or modifications using the technical content disclosed above, which are equivalent to equivalent implementation cases. However, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution.

Claims

1. A method for preparing high permeability heparin sodium liposomes, characterized in that: The method comprises the following preparation steps: (1) Dispersing soybean lecithin in deionized water and stirring in a water bath to obtain liquid 1; dissolving phytosterol in ethanol and ultrasonicating to obtain liquid 2; dispersing carboxymethyl chitosan and polyethylene glycol in deionized water and stirring in a water bath until clear to obtain liquid 3; adding liquid 1, liquid 2, and liquid 3 into a container in sequence and homogenizing to form an oil-in-water microemulsion; (2) adding heparin sodium to the oil-in-water microemulsion of step (1), continuing homogenization, ultrasonicating in an ice bath, and then adding a natural protective agent to the mixture, stirring evenly, adjusting the pH, and sterilizing and filtering to obtain heparin sodium liposomes.

2. The method for preparing the highly permeable heparin sodium liposome according to claim 1, wherein: In the step (1), the number average molecular weight of carboxymethyl chitosan is 5000-50000, and the number average molecular weight of polyethylene glycol is 2000-10000.

3. The method for preparing the high permeability heparin sodium liposome according to claim 1, characterized in that: The mass ratio of soybean lecithin, phytosterol, carboxymethyl chitosan and polyethylene glycol in the oil-in-water microemulsion in step (1) is (4-6):(1-2):(0.5-1):

1.

4. The method for preparing the high permeability heparin sodium liposome according to claim 1, characterized in that: The amount of heparin sodium added in step (2) is 1-2.5 wt% of the oil-in-water microemulsion.

5. The method for preparing the high permeability heparin sodium liposome according to claim 1, characterized in that: The natural protective agent in step (2) includes sodium hyaluronate, trehalose and vitamin E.

6. The method for preparing the high permeability heparin sodium liposome according to claim 1, characterized in that: The amount of the natural protective agent in step (2) is 1-5 wt% of the mixture.

7. The method for preparing the high permeability heparin sodium liposome according to claim 1, characterized in that: In the step (2), the pH is adjusted to 5.5-6.

5.

8. A high permeability heparin sodium liposome prepared by the preparation method according to any one of claims 1 to 7.

9. The high permeability heparin sodium liposome according to claim 8, characterized in that The particle size of the heparin sodium liposome is ≤250nm.

10. Use of the high permeability heparin sodium liposome prepared by the preparation method according to any one of claims 1 to 7 or according to claim 8 or 9 in cosmetics.

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