A high-permeability recombinant collagen liposome and its preparation method and application

Abstract

The present invention relates to a kind of high permeability recombinant collagen liposome and its preparation method and application, belong to the technical field of collagen liposome. Including the following components by mass: 5-10 parts of lecithin, 1.5-2.5 parts of recombinant collagen type I, 0.5-1.5 parts of recombinant collagen type III; 0.01-0.02 parts of recombinant collagen type XVII, 4-10 parts of supramolecular solvents, 0.10-5 parts of sodium di(laurylamide glutamine) lysine. The recombinant collagen of the present invention has strong permeability, low bioavailability, and has good stability, does not introduce any toxic and harmful organic solvents in the preparation process, and does not introduce chemical penetration enhancers that cosmetics such as azone are not welcome, and achieves the safe and effective application of collagen.

Description

Technical Field

[0001] The invention relates to the technical field of collagen liposomes, and in particular to a high-permeability recombinant collagen liposome and a preparation method and application thereof. Background Art

[0002] Recombinant humanized collagen refers to the full-length or partial amino acid sequence fragments encoded by the gene of a specific type of human collagen prepared by DNA recombination technology, or a combination of functional fragments of human collagen. Through this method, type I, type III, and type XVII recombinant humanized collagen that is non-immunogenic and has corresponding functions can be prepared according to demand. According to their respective characteristics, they can be compounded in a certain proportion and used in skin care products to achieve anti-aging, skin repair and other effects. It is an emerging research hotspot in the field of skin care products.

[0003] However, there are the following problems for collagen to be widely used in the cosmetics industry:

[0004] ① Permeability problem: Although the recombinant collagen only intercepts part of the active area of ​​the original collagen and its molecular weight is significantly smaller than that of the original collagen, its molecular weight is still as high as tens of thousands of Daltons, and it is extremely hydrophilic, with a hydrophilic-lipophilic balance value LogP < 0. These characteristics make it impossible for it to pass through the hydrophobic channels composed of lipid structures in the stratum corneum of the skin, and therefore it cannot reach the active parts of the skin to play an effective role.

[0005] ②Stability issue: Collagen directly dissolved in water is easily affected by enzymes and heat and degraded, making it difficult to ensure the quality stability of the product during the shelf life of the cosmetics.

[0006] Chinese patent CN116850340A discloses a recombinant collagen liposome excipient and its preparation method, which prepares liposomes by mixing macromolecular recombinant collagen with a penetration enhancer azone, Chinese herbal volatile oil, Tween 80, lecithin, and cholesterol, and uniformly disperses the liposomes in a gelling agent to obtain a collagen liposome excipient with good repair ability. However, since the scheme contains penetration enhancers such as azone, and ethanol may remain in the liposome preparation process, there is a risk of sensitization.

[0007] In summary, recombinant collagen is widely used in the field of skin care products, but it is difficult to take into account the issues of permeability, stability and safety. Summary of the invention

[0008] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a high-permeability recombinant collagen liposome and its preparation method and application, which has the characteristics of high permeability, high bioavailability, good stability, convenient application, etc., and can significantly improve the application effect of recombinant collagen.

[0009] To achieve the above object, the technical solution adopted by the present invention is:

[0010] In a first aspect, the present invention provides a high permeability recombinant collagen liposome, comprising the following components in parts by mass:

[0011] 5-10 parts of lecithin;

[0012] 1.5-2.5 copies of type I recombinant collagen;

[0013] Type III recombinant collagen 0.5-1.5 parts;

[0014] Type XVII recombinant collagen 0.01-0.02 parts;

[0015] 4-10 parts of supramolecular solvent;

[0016] Sodium di(laurylamide glutamine) lysine 0.10-5 parts.

[0017] The high-permeability recombinant collagen liposome of the present invention is prepared by mixing type I recombinant collagen, type III recombinant collagen and type XVII collagen according to the natural ratio in infant skin, which is closer to the collagen ratio of healthy skin, has higher compatibility and affinity with the skin, lower immunogenicity and higher bioavailability. Lecithin (phosphatidylcholine) and sodium di(laurylamide glutamine) lysine are mixed to prepare a lipid membrane to provide protection for the encapsulation of collagen. In addition to improving the stability of collagen, the permeability of collagen can be further improved. Phosphatidylcholine is an important component in human cells, and its fatty acid chain is an unsaturated oleic acid chain, which has a very high skin affinity. The liposome prepared by it has the characteristics of being deformable, and can pass through a gap several dozen times smaller than its diameter by deformation, and has a very high permeability; sodium di(laurylamide glutamine) lysine is a bimolecular surfactant of amino acids, which is composed of three amino acid molecules connected end to end and two molecules of lauric acid connected by amide bonds, and also has a large hydrophilic head and two hydrophobic tails, and its structure is very close to phospholipids, and has the characteristics of low toxicity, high permeability and high skin compatibility. When phosphatidyl and sodium di(laurylamide glutamine) lysine and supramolecular solvent are matched in the ratio described in the present invention, a synergistic effect can be produced. Compared with the traditional penetration aid or emulsification aid system components, the collagen liposome prepared by it can adjust the deformation ability of the lipid membrane, and has higher permeability, and can penetrate into the stratum corneum of the skin to form liquid crystals, improve the layout of the skin liquid crystal fence, introduce collagen deep into the skin, and deeply repair damaged skin. That is, the three work together to significantly improve the penetration-enhancing effect of liposomes.

[0018] Preferably, the high-osmotic recombinant collagen liposome further comprises 20-40 parts of glycerol and 15-25 parts of butylene glycol, which are supplemented with water to make up to 100 parts.

[0019] Preferably, the mass ratio of type I recombinant collagen and type III recombinant collagen is (1-2):1. More preferably, it is 2:1. If the mass ratio of type I recombinant collagen and type III recombinant collagen is lower than 1:1, it will affect the appearance stability and the permeability of the liposomes. This is due to the difference in molecular weight or structure of different types of collagen when dispersed in water. Type I collagen is thick and rod-shaped or bundle-shaped, and type III collagen is sparse and mesh-shaped. Therefore, when preparing liposomes, type III collagen is more likely to form a mesh gel, absorb more water, and its aqueous solution viscosity is also high, so it is more difficult to encapsulate and stabilize. The viscosity of the encapsulated liposomes is also relatively large, the PDI is high, and the stability is low; further, the high viscosity will affect the release rate and thus the permeability.

[0020] Preferably, the supramolecular solvent comprises a mixture of betaine and xylitol; wherein the mass ratio of betaine to xylitol is 1:(1.2-2.0), and more preferably 1:(1.2-1.3). Xylitol acts as a hydrogen bond donor and betaine acts as a hydrogen bond acceptor, and hydrogen bonds interact with each other under certain conditions to form supramolecular aggregates with a stable network structure. The lattice energy of the original single compound can be destroyed or reduced to a certain extent, thereby forming a supramolecular solvent with a lower melting point and the characteristics of solubilization and penetration promotion.

[0021] Preferably, the lecithin is soybean lecithin with a phosphatidylcholine content of ≥90%, and more preferably soybean lecithin with a phosphatidylcholine content of ≥95%. High-purity soybean lecithin can easily prepare double-layer liposomes with soft deformation properties.

[0022] The inventors have found through testing that although the addition of lecithin, sodium di(laurylamide glutamine) lysine and supramolecular solvents has a synergistic effect and can significantly increase the penetration efficiency, the addition of lecithin, sodium di(laurylamide glutamine) lysine and supramolecular solvents will result in stability defects, so it is necessary to adjust the ratio between the three to improve the stability of the liposomes.

[0023] Preferably, the mass ratio of lecithin to sodium di(laurylamide glutamine) lysine is 1:(0.1-1). If the ratio is lower or higher than this range, the stability of the liposome will be reduced.

[0024] Preferably, the mass ratio of the sum of type I recombinant collagen, type III recombinant collagen and type XVII recombinant collagen to the supramolecular solvent is 1:(1-3), and more preferably 1:(1.5-2.0).

[0025] Preferably, the mass ratio of the sum of type I recombinant collagen, type III recombinant collagen and type XVII recombinant collagen to lecithin is 1:(1-2.5), and more preferably 1:(1.6-1.8).

[0026] Preferably, the particle size of the high permeability recombinant collagen liposome is 30 to 100 nm, more preferably 40 to 80 nm; and the particle size of the liposome is mainly affected by the different lecithin contents, because when the lecithin content is too low, the content of collagen is high relative to lecithin, and it is difficult to completely encapsulate the collagen, which has the disadvantages of large particle size and high temperature instability. Therefore, the mass ratio between lecithin and collagen is actually an important parameter affecting the permeability of the product.

[0027] In a second aspect, the present invention provides a method for preparing highly osmotic recombinant collagen liposomes, comprising the following steps:

[0028] (1) adding type I recombinant collagen, type III recombinant collagen, and type XVII recombinant collagen into a supramolecular solvent and continuously stirring for 1 to 2 hours to completely dissolve the collagen and make it transparent, thereby obtaining a first collagen mixed solution;

[0029] (2) freeze-drying the first collagen mixture to obtain a collagen-supramolecular sponge, and then dissolving the collagen-supramolecular sponge in water to obtain a second collagen mixture;

[0030] (3) mixing sodium di(laurylamide glutamine) lysine and lecithin to obtain a first mixed solution;

[0031] (4) adding the second collagen mixed solution to the first mixed solution obtained in step (3) under a water bath and continuous stirring to obtain a second mixed solution;

[0032] (5) homogenizing the second mixed solution to obtain the recombinant collagen liposome.

[0033] Preferably, the method for preparing the supramolecular solvent comprises the following steps: mixing xylitol, betaine and water, then heating to 85-120° C., reacting for 5-8 hours, and then cooling to 30-40° C. to obtain the supramolecular solvent. Under heating conditions and a small amount of water, the crystal lattice of the original single compound changes, promoting the hydrogen bond interaction between the two parties, thereby promoting the formation of the supramolecular solvent.

[0034] Preferably, in step (3), the mixing temperature of the first mixed solution is 45-55°C.

[0035] Preferably, in step (5), the homogenization parameters of the second mixed solution are: pressure of 15000-30000 psi, number of cycles of 1-5 times, and discharge temperature of 10-30°C.

[0036] In a third aspect, the present invention provides the use of the high-permeability recombinant collagen liposome in the preparation of cosmetics, so that the cosmetics can achieve anti-aging and skin repair effects.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] (1) The high permeability recombinant collagen liposome of the present invention is composed of type I recombinant collagen, type III recombinant collagen and type XVII collagen, which is closer to the collagen ratio of healthy skin, has higher compatibility and affinity with the skin, has lower immunogenicity and higher bioavailability, and is combined with high-purity lecithin (phosphatidylcholine) and sodium di(laurylamide glutamine) lysine to prepare a lipid membrane to provide protection for the collagen package. In addition to improving the stability of collagen, it can further improve the permeability of collagen. Therefore, the high permeability recombinant collagen liposome can penetrate into the stratum corneum of the skin to form liquid crystals, improve the layout of the skin liquid crystal fence, introduce collagen deep into the skin, and deeply repair damaged skin.

[0039] (2) The preparation method of the highly permeable recombinant collagen liposome of the present invention is to dissolve collagen using supramolecular solvents, and further increase the interaction between supramolecular molecules containing a large amount of hydrogen bonds and collagen by freeze drying, so that the resistance caused by the interaction with the skin during the penetration process of collagen can be reduced to a certain extent, and the permeability of collagen can be increased; at the same time, liposomes with phosphatidylcholine and sodium di(laurylamide glutamine) lysine as the main membrane materials are wrapped, and homogenized by ultra-high pressure microjet to obtain liposomes with negative surface charge and internal water phase as supramolecular solvent-loaded composite recombinant collagen. In addition, supramolecular solvents including betaine and xylitol are also used as penetration enhancers, which can interfere with lipid arrangement during liposome penetration and release, thereby affecting the permeability of the skin, thereby improving the permeability of collagen. The liposome preparation process of the present invention does not introduce any toxic and harmful organic solvents, and does not introduce chemical penetration enhancers that are not welcome in cosmetics such as azone, thereby achieving safe and effective application of collagen. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is a schematic diagram of the structure of high-osmotic recombinant collagen liposomes in Example 1;

[0041] Figure 2 This is the appearance of the recombinant collagen liposomes of Example 1;

[0042] Figure 3 This is the appearance of the recombinant collagen liposomes of Comparative Example 2;

[0043] Figure 4 The particle size test results of the recombinant collagen liposomes in Example 1;

[0044] Figure 5 The particle size test results of recombinant collagen liposomes in comparative example 2;

[0045] Figure 6 The particle size changes of the recombinant collagen liposomes of Example 1 after storage for one month under four conditions (from left to right 45°C, room temperature, 4°C, -20°C);

[0046] Figure 7 The particle size change results of the recombinant collagen liposomes of Comparative Example 2 after storage for one month under four conditions (from left to right 45°C, room temperature, 4°C, -20°C);

[0047] Figure 8 The results of the permeability test of the high permeability recombinant collagen liposomes of Example 1 and Comparative Examples 1-2 are compared;

[0048] Fig. 9 The results of in vitro permeability fluorescence test of high permeability recombinant collagen liposomes in Example 1;

[0049] Fig.10 This is the in vitro permeability fluorescence test result of high permeability recombinant collagen liposomes in comparative example 1;

[0050] Fig.11 This is the in vitro permeability fluorescence test result of the high permeability recombinant collagen liposomes in comparative example 2. DETAILED DESCRIPTION

[0051] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0052] The parameters of the components used in the following examples are as follows:

[0053] Sodium di(laurylamideglutamine)lysine: Manufacturer: Asahi Kasei Corporation, brand name: Pellicer TM LB-10;

[0054] Lecithin: Lecithin with a phosphatidylcholine content ≥ 95%.

[0055] Unless otherwise specified, other materials, reagents, etc. used in the examples can be obtained from commercial sources.

[0056] Examples 1 to 14

[0057] The proportions of the recombinant collagen liposomes in Examples 1 to 14 are shown in Table 1.

[0058] The method for preparing the above-mentioned high-osmotic recombinant collagen liposomes comprises the following steps:

[0059] (1) xylitol, betaine, and a small amount of water are weighed in the mass ratio and placed in a container, and then heated to 95° C. in a high-pressure container at a pressure of 0.8 MPa, reacted for 8 hours, and then cooled to 35° C. to obtain a supramolecular solvent; wherein the mass ratio of xylitol to betaine is 1:1.25.

[0060] (2) Weighing type I recombinant collagen, type III recombinant collagen, and type XVII recombinant collagen according to the mass ratio, adding them into the supramolecular solvent obtained in step (1), and stirring for 2 hours to completely dissolve the collagen and make it transparent, thereby obtaining a first collagen mixed solution;

[0061] (3) freeze-drying the first collagen mixture obtained in step (2) to obtain a collagen-supramolecular sponge, and then dissolving the collagen-supramolecular sponge in a small amount of water to obtain a uniform transparent liquid, which is the second collagen mixture;

[0062] (4) weighing sodium di(laurylamide glutamine) lysine, butylene glycol, the remaining amount of water, glycerol, and lecithin according to the mass ratio into another clean container, and then heating and stirring in a 50° C. water bath to obtain a first mixed solution;

[0063] (5) In a water bath and under continuous stirring, slowly inject the second collagen mixed solution obtained in step (3) into the first mixed solution obtained in step (4), and continue stirring for 60 minutes to obtain a second mixed solution;

[0064] (6) The second mixed solution obtained in step (5) was homogenized using a microfluidizer, the homogenization pressure was controlled to be 20000 psi, the number of cycles was 2 times, and the discharge temperature was 15° C. to obtain Figure 1 The recombinant collagen liposome of Example 1 has the structure Figure 2 Shown is a transparent bluish appearance.

[0065] Table 1 Components of the recombinant collagen liposomes of Examples 1 to 14 (by weight percentage)

[0066]

[0067] Comparative Example 1

[0068] The difference between Comparative Example 1 and Example 1 is that Comparative Example 1 is a composite collagen solution without any penetration-promoting treatment, that is, sodium di(laurylamide glutamine) lysine, lecithin and supramolecular solvent are not added, and water is used to make up 100 wt%.

[0069] The method for preparing the composite collagen solution comprises the following steps: weighing type I recombinant collagen, type III recombinant collagen and type XVII recombinant collagen according to the mass ratio, and dissolving them to obtain the composite collagen without any penetration-enhancing treatment.

[0070] Comparative Example 2

[0071] The difference between Comparative Example 2 and Example 1 is that: in Comparative Example 2, sodium di(laurylamide glutamine) lysine is not added, and water is used to make up to 100 wt %, to obtain Figure 3 The recombinant collagen liposomes shown have a transparent appearance.

[0072] Comparative Example 3

[0073] The difference between Comparative Example 3 and Example 1 is that sodium di(laurylamide glutamine) lysine is replaced by alkylphenol polyoxyethylene ether-10 (OP-10).

[0074] Comparative Example 4

[0075] The difference between Comparative Example 4 and Example 1 is that the supramolecular solvent is replaced with glycerol, laurocapram, and Tween 80 to form a liposome suspension with phospholipids, wherein Tween 80 is 0.32%, laurocapram 1.5%, and glycerol 38.58%, and the amount of water added is reduced so that the total mass is 100wt%.

[0076] The preparation method of the liposome suspension comprises the following steps:

[0077] (1) weighing type I recombinant collagen, type III recombinant collagen, and type XVII recombinant collagen according to the mass ratio, and then dissolving them in a small amount of water to obtain a collagen solution;

[0078] (2) weighing Tween 80, laurocapram, butylene glycol, the remainder of water, glycerol, and lecithin according to the mass ratio into another clean container, and then heating and stirring in a 50° C. water bath to obtain a first mixed solution;

[0079] (3) In a water bath and under continuous stirring, slowly inject the collagen solution obtained in step (2) into the first mixed solution obtained in step (2), and continue stirring for 60 minutes to obtain a second mixed solution;

[0080] (4) The second mixed solution obtained in step (3) is homogenized using a microfluidizer, the homogenization pressure is controlled to be 20,000 psi, the number of cycles is 2 times, and the discharge temperature is 15° C., to obtain a recombinant collagen liposome suspension containing glycerol, laurocapram, and Tween 80.

[0081] Performance Testing

[0082] 1. Particle size test method: The product was dispersed with laboratory pure water to obtain a 1% pure water dispersion, and then the particle size and PDI were tested using a BeNano 180zeta Pro particle size tester. The test conditions were: 25°C, equilibration time 120s, and test angle 173. See Table 2 for specific data.

[0083] 2. Stability test method: Take the product in a sample bottle and place it under four conditions: room temperature, -20℃, 45℃, and 4℃ for 48 hours. Visually observe the appearance changes of the product at room temperature and without direct sunlight. Specific data are shown in Tables 2-3. Among them, stability means that the color, smell, state, transparency, and apparent viscosity of the sample after returning to room temperature before and after being placed under each condition do not change visible to the naked eye. Solidification means that the state of the sample changes significantly after being placed and returned to room temperature, from a flowing liquid to a non-flowing solid or gel.

[0084] 3. Permeation test: According to the in vitro transdermal test guidelines of OECD / OCDE, the transdermal verification was carried out. The specific operation method is as follows: during the experiment, the Strat-M artificial membrane was first placed in a phosphate buffer solution (pH 7.4) for 30 minutes. After hydration, the surface moisture of the artificial membrane was wiped dry and the artificial membrane was placed between the receiving pool and the supply pool of the Franz diffusion cell, with the smooth side facing the supply pool; the receiving pool was a phosphate buffer solution (pH 7.4); 0.1g of sample was added to the supply pool; the assembled Franz diffusion cell was placed in the transdermal diffusion instrument, and the experimental conditions were set as follows: the temperature was 32°C, the stirring speed was 600rpm, and the penetration time was 12h. The receiving solution after the penetration of each group of samples was taken, and the total collagen in the receiving solution was quantitatively analyzed by UV-visible spectrophotometer. Specific data are shown in Table 2.

[0085] Table 2 Particle size, PDI, stability (48 hours), and cumulative permeation volume in 12 hours for each group of samples

[0086]

[0087] Table 3 Appearance changes of recombinant collagen liposomes in Example 1 and Comparative Example 2 after storage for one month under four conditions (45°C, room temperature, 4°C, -20°C)

[0088]

[0089] Depend on Figure 4 It can be seen that the recombinant collagen liposomes of Example 1 were diluted 100 times with deionized water and tested with a laser particle size analyzer to obtain an average particle size of 57.82 nm and a PDI of 0.177. Figure 6 It can be seen that the recombinant collagen liposomes were stored at 45°C, room temperature, -20°C, and 4°C for one month, and the average particle size and appearance did not change significantly, indicating that the recombinant collagen liposomes have good low-temperature stability, high-temperature stability, and room-temperature stability.

[0090] Depend on Figure 5 It can be seen that the recombinant collagen liposomes of Comparative Example 2 were diluted 100 times with deionized water and tested with a laser particle size analyzer to obtain an average particle size of 57.96 nm and a PDI of 0.495. Figure 7 As shown, the recombinant collagen liposomes of Comparative Example 2 were stored at 45°C, room temperature, -20°C, and 4°C for one month. At high temperature and room temperature, the average particle size and appearance changed significantly, so the high temperature stability and room temperature stability of the product of Comparative Example 2 were lower than those of Example 1. Combined with Table 3, it can be seen that the stability of Example 1 is significantly higher than that of Comparative Examples 1-2.

[0091] Combining Table 2 and Figure 8 It can be seen that the recombinant collagen liposomes prepared by the scheme of Example 1 have the characteristics of high stability and relatively high permeability, and its 12-hour permeability reaches 157.35ug / cm 2 , significantly higher than the comparative example. The permeability of collagen in Example 1 is 5.54 times that of comparative example 1 (untreated collagen), which significantly improves the bioavailability of collagen; Comparative example 2 is a liposome without sodium di(laurylamide glutamine) lysine, and its stability is significantly reduced compared with Example 1, and the permeability is significantly reduced to 76.28ug / cm 2 , indicating that sodium di(laurylamide glutamine) lysine is very important for the stability and permeability of the system. Comparative Example 3 is a liposome prepared with conventional emulsifier OP-10, and its 12-hour permeation amount is only 44.31ug / cm 2 , indicating that conventional liposome formulas can promote collagen penetration to a certain extent, but their penetration enhancement effect is not significant, so the synergistic use of lecithin, sodium di(laurylamide glutamine) lysine and supramolecular solvents in the present technical solution can significantly improve the permeability of collagen; Comparative Example 4 is a collagen liposome prepared by a conventional emulsifier in combination with a chemical penetration enhancer. Compared with Example 1, its particle size is larger and the permeation amount is lower, and there is also an unsafe risk caused by the chemical penetration enhancer.

[0092] As shown in Table 2, compared with Examples 2 to 3, Example 1 has higher stability and permeability within the preferred range, while Example 3 has decreased stability and a certain degree of permeability, indicating that too high type III will affect the stability and permeability of the liposome. Due to the differences in the molecular weight of different types of collagen or the structure when dispersed in water, type I collagen is thick and rod-shaped or bundle-shaped, and type III collagen is sparse and mesh-shaped. Therefore, when preparing liposomes, type III collagen is more likely to form a mesh gel, absorb more water, and its aqueous solution viscosity is also high, so it is more difficult to encapsulate and stabilize. The viscosity of the encapsulated liposomes is also relatively large, the PDI is high, and the stability is low; high viscosity will affect the release rate and thus the permeability.

[0093] Examples 4-7 show the effects of different lecithin contents on the stability and permeability of recombinant collagen liposomes. When the lecithin content is too low, the content of collagen relative to lecithin is high, making it difficult to completely encapsulate the collagen, resulting in the disadvantages of large particle size and high temperature instability. When the lecithin content is too high, the PDI of the system becomes larger (>0.3), indicating the presence of some particles of uneven size, resulting in a relatively large risk of Ostwald ripening, which is not conducive to the long-term stability of the recombinant collagen liposomes.

[0094] Example 1, Example 8-11 explore the effect of supramolecular solvent content on the stability and permeability of recombinant collagen liposomes. When the content of supramolecular solvent relative to the sum of type I recombinant collagen, type III recombinant collagen and type XVII recombinant collagen gradually increases, the permeation effect shows a trend of increasing all the time, but when the content of supramolecular solvent is lower than the sum of collagen, the permeation effect is not obvious, and its permeation amount is not significant compared with the comparative example, and when the content of supramolecular solvent is too low, the proportion of components required to be loaded in the aqueous phase of the liposome is high, and the system is difficult to stabilize, and stratification occurs after 48 hours of high temperature storage. However, the higher the content of supramolecular solvent, the better. When the mass ratio of the sum of type I recombinant collagen, type III recombinant collagen and type XVII recombinant collagen to the supramolecular solvent exceeds 1:3, the stability of the sample is reduced. As shown in Example 11, stratification occurs at 45°C, indicating that too much supramolecular solvent will reduce the high temperature stability of the sample. Therefore, the present invention controls the mass ratio of the sum of type I recombinant collagen, type III recombinant collagen and type XVII recombinant collagen to the supramolecular solvent to be 1:(1-3), and more preferably 1:(1.5-2.0).

[0095] Examples 12-14 show the effect of sodium di(laurylamide glutamine) lysine content on the stability and permeability of recombinant collagen liposomes. Examples 1 and 12 both have high stability and permeability. When the sodium di(laurylamide glutamine) lysine content is reduced, the permeability decreases and the liposomes become unstable. When the sodium di(laurylamide glutamine) lysine content is increased, the permeability does not increase significantly, but the high-temperature stability of the liposomes decreases, and the PDI of the solution increases (>0.3), indicating that there are some particles of uneven size, and there is a relatively large risk of Ostwald ripening, which is not conducive to the long-term stability of the recombinant collagen liposomes.

[0096] 4. Penetration Visualization Test

[0097] Test method: Fix the piglet skin between the diffusion chamber and the receiving chamber of the Franz cell diffusion cell, with the piglet skin stratum corneum facing the diffusion chamber and the dermis facing the receiving chamber. After fixing the piglet skin, add 8mL of receiving solution (7.4PBS) to the receiving chamber to make the piglet skin in close contact with the receiving solution. After the water bath temperature of the diffusion instrument is constant, add 0.1g of samples containing Example 1, Comparative Example 1, and Comparative Example 2 (type III collagen in the three samples is fluorescently labeled with FITC) to the surface of the piglet skin and apply it evenly. Sample collection in the receiving pool: After 12 hours of the penetration experiment, clean the residual samples on the skin surface, and place each group of pig skin in a -80°C refrigerator, followed by OCT embedding and frozen sectioning. The slices are observed under a fluorescence microscope and images are collected.

[0098] Test results: Figures 9-11 The results of fluorescence detection of skin sections show that in the Example 1 group, the recombinant collagen has the strongest fluorescence intensity in the skin and the deepest penetration depth, indicating that the collagen in Example 1 has the strongest penetration ability.

[0099] In summary, the present invention increases its permeability by precisely matching recombinant collagen simulating the ratio of infant skin collagen, dissolving and treating with a supramolecular solvent, and at the same time encapsulating with liposomes using phosphatidylcholine and sodium di(laurylamide glutamine) lysine as main membrane materials, and homogenizing with ultrahigh pressure microjet to obtain liposomes with a positively charged surface and an inner aqueous phase of supramolecular solvent loaded composite recombinant collagen, which has the following beneficial effects:

[0100] Improved stability: Collagen itself is easy to degrade. In addition, liposomes are non-thermodynamically stable systems. During storage, due to Ostwald ripening, particles aggregate, resulting in increased particle size, stratification and other deterioration defects. Conventional collagen liposomes are difficult to stabilize. This solution uses supramolecular solvents to load collagen, and then uses high-purity phosphatidylcholine for secondary encapsulation. More importantly, sodium di(laurylamide glutamine) lysine is used as a membrane stabilizer to further increase the stabilizer of the liposome. The prepared recombinant collagen liposomes are very stable. After being stored for one month under four conditions (45°C, room temperature, 4°C, -20°C), its appearance and particle size remain stable, and PDI remains below 0.3, indicating that the liposome particles are still very uniform and the product has good stability.

[0101] Improved collagen penetration: Recombinant collagen has a large molecular weight and strong hydrophilicity, making it very difficult to use through the skin. In vitro permeation tests have shown that the permeability of collagen that has not been treated to promote permeation is very low, so it is difficult to exert its efficacy when used directly. This solution combines supramolecular technology with liposome technology to obtain recombinant collagen liposomes with a particle size of less than 100nm, which significantly improves its permeability. In vitro permeation tests have shown that its permeability is 5-8 times that of untreated collagen and 2-3 times that of conventional collagen liposomes. Its 12-hour skin flux reaches 157.35ug / cm 2 , and the bioavailability of collagen can be improved. The present invention does not introduce any toxic or harmful organic solvents, and does not introduce chemical penetration enhancers such as azone that are not welcome in cosmetics, thereby achieving safe and effective application of collagen.

[0102] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A highly osmotic recombinant collagen liposome, characterized in that: It includes the following components by mass: 5-10 parts of lecithin; 1.5-2.5 copies of type I recombinant collagen; Type III recombinant collagen 0.5-1.5 parts; Type XVII recombinant collagen 0.01-0.02 parts; 4-10 parts of supramolecular solvent; Sodium di(laurylamide glutamine) lysine 0.50-5 parts; The lecithin is soybean lecithin with a phosphatidylcholine content of ≥90%; The supramolecular solvent comprises a mixture of betaine and xylitol; wherein the mass ratio of betaine to xylitol is 1:(1.2-2.0); The mass ratio of type I recombinant collagen to type III recombinant collagen is (1-2):1; The mass ratio of lecithin to sodium di(laurylamide glutamine) lysine is 1:(0.1-1); The mass ratio of the sum of the type I recombinant collagen, the type III recombinant collagen and the type XVII recombinant collagen to the supramolecular solvent is 1:(1-3); The mass ratio of the sum of the type I recombinant collagen, the type III recombinant collagen and the type XVII recombinant collagen to lecithin is 1:(1-2.5).

2. The highly osmotic recombinant collagen liposome according to claim 1, characterized in that: The particle size of the high-permeability recombinant collagen liposome is 30-100 nm.

3. The method for preparing the highly osmotic recombinant collagen liposome according to any one of claims 1 to 2, characterized in that: The following steps are involved: (1) adding type I recombinant collagen, type III recombinant collagen, and type XVII recombinant collagen into a supramolecular solvent and continuously stirring to obtain a first collagen mixed solution; (2) freeze-drying the first collagen mixture to obtain a collagen-supramolecular sponge, and then dissolving the collagen-supramolecular sponge in water to obtain a second collagen mixture; (3) mixing sodium di(laurylamide glutamine) lysine and lecithin to obtain a first mixed solution; (4) adding the second collagen mixed solution to the first mixed solution obtained in step (3) in a water bath under continuous stirring to obtain a second mixed solution; (5) Homogenizing the second mixed solution to obtain the recombinant collagen liposome.

4. The method for preparing the highly osmotic recombinant collagen liposome according to claim 3, characterized in that: The preparation method of the supramolecular solvent comprises the following steps: mixing xylitol, betaine and water, then heating to 85-120° C., reacting for 5-8 hours, and then cooling to 30-40° C. to obtain the supramolecular solvent.

5. The method for preparing the highly osmotic recombinant collagen liposome according to claim 3, characterized in that: In the step (3), the mixing temperature of the first mixed solution is 45-55°C.

6. The method for preparing the highly osmotic recombinant collagen liposome according to claim 3, characterized in that: In the step (5), the homogenization parameters of the second mixed solution are: pressure of 15000-30000 psi, number of cycles of 1-5 times, and discharge temperature of 10-30°C.

7. Use of the highly osmotic recombinant collagen liposome according to any one of claims 1 to 2 in the preparation of cosmetics.

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