Recombinant collagen composite nanoliposome, preparation method and application thereof

Abstract

This patent application discloses recombinant collagen composite nanoliposomes, their preparation method, and applications. The composite nanoliposomes of this invention comprise: liposomes having a closed lipid bilayer and contents encapsulated within the closed lipid bilayer; and a polyquaternium-51 layer encapsulating the liposomes. This invention utilizes a layered encapsulation technology to form supramolecular assembly, phospholipid encapsulation, and a polyquaternium-51 protective layer on the surface of the protein. This multi-layered technology significantly improves the stability of the protein and liposomes, as well as their transdermal absorption efficiency.

Description

Technical Field

[0001] This invention relates to the field of active substance delivery systems, and in particular to a recombinant collagen composite nanoliposome with highly efficient intradermal deep delivery, its preparation method and application. Background Technology

[0002] Recombinant collagen is a humanized protein produced using genetic engineering technology and is an important anti-aging and repairing ingredient in the cosmetics industry. Compared with animal-derived collagen, its advantages lie in its higher biocompatibility, greater safety, and avoidance of animal-derived disease risks. In skincare products, recombinant collagen (especially type I and type III) not only has excellent moisturizing properties but also promotes the synthesis of the skin's own collagen and inhibits its degradation through signal transduction, thereby exerting firming, anti-wrinkle, and barrier-repairing effects.

[0003] As a biological macromolecule, recombinant collagen faces fundamental challenges in transdermal absorption. The core issue lies in its enormous molecular weight (typically tens of thousands of Daltons) and strong hydrophilicity, which directly clashes with the strongly hydrophobic physical barrier of the stratum corneum, the outermost layer of the skin. Therefore, recombinant collagen struggles to efficiently diffuse through this dense structure; the vast majority remains on the skin's surface, providing moisturizing effects, but fails to effectively penetrate the dermis to directly achieve its crucial anti-aging and repairing effects of promoting collagen regeneration. This is the main technical bottleneck restricting its ability to maximize its skincare efficacy.

[0004] Liposomes are artificially prepared tiny spherical vesicles. In water, the hydrophilic head of the phospholipid molecule is inserted into the water, while the hydrophobic tail of the liposome extends into the air. After stirring, a spherical liposome with a bilayer of lipid molecules is formed, with a diameter ranging from 25 to 1000 nm.

[0005] Because human skin has a natural barrier function, the transdermal penetration and active absorption of components are significantly affected and restricted, thus greatly reducing or weakening the biological effects and therapeutic efficacy of active substances. Liposomes, similar in structure to biological membranes, can increase the permeability of active substances into the stratum corneum, preventing the active ingredients from losing their efficacy due to insufficient penetration. Simultaneously, liposomes can remain in the stratum corneum, slowly releasing active substances into the epidermis, enhancing their sustained efficacy. Furthermore, liposomes themselves are non-toxic, can biodegrade autonomously in vivo, and do not cause skin irritation, making them an ideal carrier for skin care and cosmetic applications.

[0006] Supramolecular technology studies the spontaneous assembly of molecules into ordered, functional structures through non-covalent interactions (such as hydrogen bonds, van der Waals forces, and hydrophobic interactions). By adding appropriate "eutectic components" and "ionic salts," the self-assembly of protein molecules can be achieved, thereby forming ordered nanoscale supramolecular particles that enhance their ability to penetrate the skin barrier.

[0007] While existing technologies, such as liposome encapsulation and supramolecular co-crystallization, have precedents for improving the permeability of biomolecules like collagen, neither single technology can achieve the desired encapsulation and permeation effects. Furthermore, liposome encapsulation technology suffers from a common problem: the long-term stability of liposomes in water. Liposomes stored in water for extended periods are prone to aggregation, fusion, or premature leakage.

[0008] Therefore, developing liposome-based material delivery systems with better encapsulation, higher penetration efficiency, and greater long-term stability in water is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0009] In view of the above-mentioned technical problems existing in the prior art, the purpose of the present invention is to provide a composite nanoliposome with better encapsulation effect, higher permeation efficiency and more stable long-term preservation in water, its preparation method and application.

[0010] Polyquaternium-51, CAS: 125275-25-4, chemically named 2-methacryloyloxyethyl phosphocholine polymer, has been included in the "Catalogue of Used Cosmetic Raw Materials". Polyquaternium-51 exhibits good hydrophilicity and biocompatibility, and has been widely used in cosmetics. Its applications mainly include: (1) In the skin care category, it is used as a long-lasting moisturizing ingredient in products such as face cream, lotion, and serum to enhance the skin's hydration ability.

[0011] (2) It is used as a hair conditioning agent in hair care products such as conditioners and styling products to reduce frizz and enhance the smoothness of hair.

[0012] (3) It is used as a texture improver in sunscreen and makeup products to improve product texture and give it a silky feel.

[0013] The inventors conducted in-depth research to solve the above-mentioned technical problems and discovered that polyquaternium-51 can form a new encapsulation layer on the surface of liposomes. This encapsulation layer can simultaneously improve the encapsulation effect, permeation efficiency, and long-term storage stability in water of liposome-based material delivery systems, thus completing the present invention.

[0014] That is, the present invention includes: The first aspect of the present invention is a composite nanoliposome comprising: A. A liposome having a closed lipid bilayer and contents encapsulated within the closed lipid bilayer; and B. A polyquaternium-51 layer encapsulating the liposomes.

[0015] In this specification, a liposome is a microvesicle formed by encapsulating contents within a lipid bilayer, and its particle size can be 10–1000 nm.

[0016] There are no special restrictions on the lipid components that form the lipid bilayer, as long as they can form a lipid bilayer. These components may include, but are not limited to, one of lecithin, hydrogenated lecithin, phosphatidylcholine, hydrogenated phosphatidylcholine, or any mass ratio of these components.

[0017] There are no particular restrictions on the contents encapsulated within the closed lipid bilayer. These contents typically contain the active substance to be delivered, which may include, but is not limited to, proteins, such as recombinant collagen. The recombinant collagen may be selected from one or a combination of several common types, such as recombinant type I collagen, recombinant type II collagen, recombinant type III collagen, recombinant type IV collagen, recombinant type V collagen, recombinant type VI collagen, recombinant type VII collagen, and recombinant type XVII collagen, preferably recombinant type I collagen, recombinant type III collagen, recombinant type XVII collagen, or combinations thereof. The recombinant collagen may be a full-length chain of recombinant natural collagen, a truncated or spliced ​​full-length chain of natural collagen, or a repeat of natural collagen fragments. The molecular weight of the recombinant collagen may be between 10,000 Daltons and 200,000 Daltons.

[0018] The proteins in the contents preferably form supramolecular proteins. These supramolecular proteins are self-assembled into stable protein structures with nanoparticle sizes by adding one or more salts or alcohols to alter the surface charge of the protein. The salts can be, for example, betaine, sodium chloride, phosphates, ammonium sulfate, magnesium sulfate, etc.; the alcohols can be, for example, polyols such as propylene glycol, glycerol, pentanediol, etc.

[0019] There are no particular limitations on the method for preparing liposomes by encapsulating the contents with lipid components; both active and passive drug delivery methods can be used. Specific preparation methods include, but are not limited to, thin-film dispersion, reverse evaporation, ultrasonic treatment, injection, and microfluidics. Among these, microfluidics, which precisely controls the mixing process of lipids and the aqueous phase through micron-level channels, can prepare well-dispersed nanoscale liposomes. This technology has a high degree of automation and is suitable for industrial production.

[0020] There are no particular limitations on the method for encapsulating the surface of the liposomes with a polyquaternium-51 layer. For example, a certain concentration of polyquaternium-51 aqueous solution can be added to an aqueous solution containing liposomes in a certain proportion and mixed thoroughly. Here, the concentration of the polyquaternium-51 aqueous solution can be, for example, 0.5 to 15% by weight, preferably 1 to 10% by weight, more preferably 2 to 8% by weight, more preferably 3 to 7% by weight, more preferably 4 to 6% by weight, for example, 5% by weight. Here, the aqueous solution containing liposomes can contain 3 to 30% by weight, preferably 7 to 20% by weight, more preferably 6 to 15% by weight, more preferably 5 to 10% by weight of liposomes. Here, the volume ratio of the polyquaternium-51 aqueous solution to the aqueous solution containing liposomes can be, for example, 1:100 to 1:5, preferably 1:50 to 1:8, more preferably 1:30 to 1:10. Here, there are no particular limitations on the method for thoroughly mixing the polyquaternium-51 and the liposomes, as long as they can be thoroughly mixed. For example, a stirring method can be used, with stirring speeds of 50 rpm to 1000 rpm, preferably 100 rpm to 800 rpm, more preferably 200 rpm to 700 rpm, even more preferably 400 rpm to 600 rpm, such as 500 rpm; and stirring time of 1 min to 120 min, preferably 5 min to 90 min, even more preferably 10 min to 60 min, such as about 30 min.

[0021] A second aspect of the present invention is a cosmetic or topical preparation comprising the composite nanoliposomes described in the first aspect of the present invention. The cosmetic or topical preparation may be, for example, a face mask, lotion, serum, liquid, facial cleanser, shampoo, moisturizer, or sunscreen. The cosmetic or topical preparation may also contain other ingredients, such as preservatives. The preservatives are additives added to ensure a longer shelf life and inhibit microbial growth, selected from polyols, nitrocellulose esters, organic acids and salts, preferably polyols, and more preferably one or a combination of pentylene glycol, hexanediol, and octanediol.

[0022] The third aspect of the present invention is a method for preparing the composite nanoliposomes described in the first aspect of the present invention. This preparation method includes: adding an aqueous solution of polyquaternium-51 to an aqueous solution containing liposomes and mixing them.

[0023] Here, the concentration of the polyquaternium-51 aqueous solution can be, for example, 0.5-15% by weight, preferably 1-10% by weight, more preferably 2-8% by weight, more preferably 3-7% by weight, more preferably 4-6% by weight, for example 5% by weight. The aqueous solution containing liposomes can contain 3-30% by weight, preferably 7-20% by weight, more preferably 6-15% by weight, more preferably 5-10% by weight of liposomes. The volume ratio of the polyquaternium-51 aqueous solution to the aqueous solution containing liposomes can be, for example, 1:100-1:5, preferably 1:50-1:8, more preferably 1:30-1:10. There are no particular limitations on the method of thoroughly mixing the polyquaternium-51 and the liposomes, as long as they can be thoroughly mixed. A stirring method can be used, for example, with stirring speeds of 50 rpm to 1000 rpm, preferably 100 rpm to 800 rpm, more preferably 200 rpm to 700 rpm, even more preferably 400 rpm to 600 rpm, such as 500 rpm; and stirring time of 1 min to 120 min, preferably 5 min to 90 min, even more preferably 10 min to 60 min, such as about 30 min. Whether it is "fully mixed" can be determined, for example, by measuring the particle size of the obtained composite nanoliposomes to be <400 nm and / or the homogeneity to be <0.3.

[0024] The preferred method for preparing the composite nanoliposomes is a method for preparing recombinant collagen composite nanoliposomes, which preferably includes: (1) Add salts and / or alcohols to the recombinant collagen solution to form a supramolecular protein solution, which is the aqueous phase; (2) Dissolve the lipid components in an organic solvent to form a lipid component solution, which is the lipid phase; (3) The aqueous phase and the lipid phase are mixed to form an aqueous solution containing liposomes, the contents of which contain recombinant collagen; and (4) Add the aqueous solution of polyquaternium-51 to the aqueous solution containing liposomes and mix them to obtain the recombinant collagen composite nanoliposomes.

[0025] The salts added in step (1) can be, for example, betaine, sodium chloride, phosphate, ammonium sulfate, magnesium sulfate, etc.; the alcohols added can be, for example, propylene glycol, glycerol, pentanediol, etc., polyols. The recombinant collagen in step (1) can be selected from one or a combination of several common types such as recombinant type I collagen, recombinant type II collagen, recombinant type III collagen, recombinant type IV collagen, recombinant type V collagen, recombinant type VI collagen, recombinant type VII collagen, and recombinant type XVII collagen, preferably recombinant type I collagen, recombinant type III collagen, recombinant type XVII collagen, or combinations thereof. The recombinant collagen can be a full-length chain of recombinant natural collagen, a truncated or spliced ​​full-length chain of natural collagen, or a repeat of natural collagen fragments. The molecular weight of the recombinant collagen can be between 10,000 Daltons and 200,000 Daltons.

[0026] The lipid component in step (2) may include, but is not limited to, one of lecithin, hydrogenated lecithin, phosphatidylcholine, hydrogenated phosphatidylcholine, or a mixture thereof in any mass ratio. The organic solvent in step (2) is soluble in water and may be, for example, methanol, ethanol, propanol, ethyl acetate, etc.

[0027] The mixing method in step (3) can be stirring, high-pressure homogenization, microfluidic mixing, etc., with microfluidic mixing being preferred.

[0028] In step (4), the concentration of the polyquaternium-51 aqueous solution can be, for example, 0.5-15% by weight, preferably 1-10% by weight, more preferably 2-8% by weight, more preferably 3-7% by weight, more preferably 4-6% by weight, for example 5% by weight. The aqueous solution containing liposomes can contain 3-30% by weight, preferably 7-20% by weight, more preferably 6-15% by weight, more preferably 5-10% by weight of liposomes. The volume ratio of the polyquaternium-51 aqueous solution to the aqueous solution containing liposomes can be, for example, 1:100-1:5, preferably 1:50-1:8, more preferably 1:30-1:10. There are no particular limitations on the method for thoroughly mixing the polyquaternium-51 and liposomes, as long as they can be thoroughly mixed. A stirring method can be used, for example, with stirring speeds of 50 rpm to 1000 rpm, preferably 100 rpm to 800 rpm, more preferably 200 rpm to 700 rpm, even more preferably 400 rpm to 600 rpm, such as 500 rpm; and stirring time of 1 min to 120 min, preferably 5 min to 90 min, even more preferably 10 min to 60 min, such as about 30 min. Whether it is "fully mixed" can be determined, for example, by measuring the particle size of the obtained composite nanoliposomes to be <400 nm and / or the homogeneity to be <0.3.

[0029] The above-mentioned method for preparing recombinant collagen composite nanoliposomes provides a method applicable to the preparation of macromolecular recombinant collagen composite nanoliposomes with molecular weights ranging from 10,000 Daltons to 200,000 Daltons. It involves adding salts and alcohols to a protein solution to form supramolecular nanoparticles, thereby achieving ordered assembly of disordered protein molecules through hydrogen bonding to form a supramolecular solution. Then, by thoroughly mixing with a lipid-phase phospholipid solution, liposome vesicles are formed on the supramolecular surface for secondary encapsulation. After secondary encapsulation, the liposomes undergo electrostatic adsorption with positively charged polyquaternium-51 on the molecular chain, forming a hydrophilic polymer protective layer on the liposome surface, thus achieving tertiary encapsulation. By employing a multi-layered encapsulation technique, supramolecular assembly, phospholipid encapsulation, and a polyquaternium-51 protective layer are formed on the surface of the protein. This combination of technologies achieves: 1. Highly efficient penetration, combining the effects of supramolecular, liposome, and polyquaternium-51 delivery systems, significantly improving the penetration efficiency of large protein molecules. Transdermal experiments show that the transdermal absorption rate is nearly 5 times higher than that of protein solutions and nearly 3 times higher than that of single encapsulation, significantly enhancing transdermal absorption. 2. Significantly improved protein stability. In aqueous solutions, large protein molecules are prone to hydrolysis or enzymatic degradation during prolonged storage, leading to fragmentation. The multi-layered encapsulation technique creates a charge-stable environment isolated from the external environment, inhibiting degradation caused by changes in ionic strength, temperature, and enzymes. 3. Compared to single liposome encapsulation, the multi-layered encapsulation of liposomes with a hydrophilic polymer protective layer further increases the stability of the entire nanoparticle, preventing liposome aggregation, fusion, or premature leakage. Attached Figure Description

[0030] Figure 1 A graph showing the particle size measurement results of the samples from Example 1 and Comparative Example 1.

[0031] Figure 2 The diagram illustrates the optical Tyndall effect of the supramolecular protein solution formed after steps 1) and 2) in the embodiment.

[0032] Figure 3 Electron micrographs showing the vesicles formed after liposome encapsulation.

[0033] Figure 4 A graph showing the results of Zata potential measurements of the samples from Example 1 and Comparative Example 4. Detailed Implementation

[0034] The present application is further illustrated below with reference to embodiments. It should be understood that the embodiments are only used to further illustrate and explain the present application and are not intended to limit the present invention.

[0035] Unless otherwise specified, % in the following examples refers to weight.

[0036] Example 1: Preparation of composite nanoliposomes of recombinant type I α1 chain mature peptide collagen 1) Preparation of protein solution: Recombinant collagen was prepared according to the method shown in Chinese Patent Application No. 201310701767.2, and a protein solution with a concentration of 0.2% was prepared.

[0037] 2) Preparation of supramolecular solution: Add 2% glycerol, 2% 1,3-propanediol and 1% betaine to the prepared protein solution and mix well.

[0038] 3) Preparation of the lipid phase: Weigh soybean lecithin, dissolve it in anhydrous ethanol to prepare a concentration of 150 g / L, and sonicate to ensure that the phospholipids are fully dissolved, which is the lipid phase.

[0039] 4) Liposome preparation: The flow rate of the supramolecular solution phase was set to 10 mL / min and the flow rate of the lipid phase was 2 mL / min. The microfluidic device was run and the prepared liposome solution was collected, which contained 8.2% liposomes.

[0040] 5) Cation layer encapsulation: After slowly adding 5% polyquaternium-51 solution at 3% of the protein solution, stir at 500 rpm for 30 min on a magnetic stirrer to obtain composite nanoliposomes.

[0041] Example 2: Preparation of composite nanoliposomes of recombinant type III α1 chain mature peptide collagen 1) Preparation of protein solution: Recombinant collagen was prepared according to the method shown in Chinese Patent Application No. 201310701942.8, and a protein solution with a concentration of 0.2% was prepared.

[0042] 2) Preparation of supramolecular solution: Add 3% glycerol, 3% 1,3-propanediol and 1.5% betaine to the prepared protein solution and mix well.

[0043] 3) Preparation of the lipid phase: Weigh phosphatidylcholine, dissolve it in anhydrous ethanol to prepare a concentration of 150 g / L, and sonicate to ensure that the phosphatidylcholine is fully dissolved, which is the lipid phase.

[0044] 4) Liposome preparation: The flow rate of the supramolecular solution phase was set to 15 mL / min and the flow rate of the lipid phase was 2 mL / min. The microfluidic device was run and the prepared liposome solution was collected, which contained 9.5% liposomes.

[0045] 5) Cation layer encapsulation: After slowly adding 5% polyquaternium-51 solution to 10% of the protein solution, stir at 500 rpm for 30 min on a magnetic stirrer to obtain composite nanoliposomes.

[0046] Example 3: Preparation of recombinant type I repeating fragment collagen composite nanoliposomes 1) Preparation of protein solution: Recombinant collagen was prepared according to the method shown in Chinese Patent 202310171755.7 and a protein solution with a concentration of 1.0% was prepared.

[0047] 2) Preparation of supramolecular solution: Add 5% glycerol, 5% 1,3-propanediol and 2.5% betaine to the prepared protein solution and mix well.

[0048] 3) Preparation of the lipid phase: Weigh phosphatidylcholine, dissolve it in anhydrous ethanol to prepare a concentration of 150 g / L, and sonicate to ensure that the phosphatidylcholine is fully dissolved, which is the lipid phase.

[0049] 4) Liposome preparation: Set the supramolecular solution flow rate to 15 mL / min and the lipid phase flow rate to 2 mL / min, run the microfluidic device, and collect the prepared liposome solution, which contains 10% liposomes.

[0050] 5) Cation layer encapsulation: Add 5% polyquaternium-51 solution slowly at 10% of the protein solution, and stir at 500 rpm for 30 min on a magnetic stirrer to obtain composite nanoliposomes.

[0051] Comparative Example 1: Liposome preparation of recombinant type I α1 chain mature peptide collagen 1) Preparation of protein solution: Recombinant collagen was prepared according to the method shown in Chinese Patent Application No. 201310701767.2, and a protein solution with a concentration of 0.2% was prepared.

[0052] 2) Preparation of the lipid phase: Weigh soybean lecithin, dissolve it in anhydrous ethanol to prepare a concentration of 150 g / L, and sonicate to ensure that the phospholipids are fully dissolved, which is the lipid phase.

[0053] 3) Liposome preparation: Set the supramolecular solution flow rate to 10 mL / min and the lipid phase flow rate to 2 mL / min, run the microfluidic device, and collect the prepared liposome solution.

[0054] This comparative example is equivalent to preparing liposomes directly without protein supramolecularization as in Example 1.

[0055] Comparative Example 2: Liposome preparation of recombinant type III α1 chain mature peptide collagen 1) Preparation of protein solution: Recombinant collagen was prepared according to the method shown in Chinese Patent Application No. 201310701942.8, and a protein solution with a concentration of 0.2% was prepared.

[0056] 2) Preparation of the lipid phase: Weigh phosphatidylcholine, dissolve it in anhydrous ethanol to prepare a concentration of 150 g / L, and sonicate to ensure that the phosphatidylcholine is fully dissolved, which is the lipid phase.

[0057] 3) Liposome preparation: Set the supramolecular solution flow rate to 15 mL / min and the lipid phase flow rate to 2 mL / min, run the microfluidic device, and collect the prepared liposome solution.

[0058] This comparative example is equivalent to preparing liposomes directly without protein supramolecularization as in Example 2.

[0059] Comparative Example 3: Preparation of Recombinant Type I Repeat Fragment Collagen Liposomes 1) Preparation of protein solution: Recombinant collagen was prepared according to the method shown in Chinese Patent 202310171755.7 and a protein solution with a concentration of 1.0% was prepared.

[0060] 2) Preparation of the lipid phase: Weigh phosphatidylcholine, dissolve it in anhydrous ethanol to prepare a concentration of 150 g / L, and sonicate to ensure that the phosphatidylcholine is fully dissolved, which is the lipid phase.

[0061] 3) Liposome preparation: Set the supramolecular solution flow rate to 15 mL / min and the lipid phase flow rate to 2 mL / min, run the microfluidic device, and collect the prepared liposome solution.

[0062] This comparative example is equivalent to preparing liposomes directly without protein supramolecularization as in Example 3.

[0063] Comparative Example 4: Liposome preparation of recombinant type I α1 chain mature peptide collagen Proceed as in Example 1, except that only step 4 is performed, and step 5 is not performed.

[0064] Comparative Example 5: Liposome preparation of recombinant type III α1 chain mature peptide collagen It is performed as in Example 2, except that only step 4 is performed, and step 5 is not performed.

[0065] Comparative Example 6: Preparation of Recombinant Type I Repeat Fragment Collagen Liposomes It is performed as in Example 3, except that only step 4 is performed, and step 5 is not performed.

[0066] Comparative Example 7: Preparation of Recombinant Type I Repeat Fragment Collagen Liposomes The procedure is the same as in Example 3, except that the 5% polyquaternium-51 solution from step 5) is first slowly added to the supramolecular protein solution prepared in step 2) at a ratio of 3%, and then subsequent steps are performed, only up to step 4), without proceeding to step 5).

[0067] Example 4: Determination of liposome characteristic parameters and encapsulation efficiency Nine samples were prepared, namely the composite nanoliposomes prepared in Examples 1-3 and the liposomes prepared in Comparative Examples 1-6, respectively, and were designated as Samples 1-9. The average particle size of each sample was determined using a nanoparticle size and Zeta potential analyzer.

[0068] Take a sample and measure the protein concentration C A Then, the sample was treated with Triton-100 before the protein concentration C was measured. B Encapsulation efficiency (%) = C B / (C A +C B) x 100% Table 1. Particle size and encapsulation efficiency results for each sample.

[0069] Note: Samples 1-3 are composite nanoliposomes prepared in Examples 1-3, respectively; Samples 4-9 are liposomes prepared in Comparative Examples 1-6, respectively.

[0070] As shown in Table 1, the triple-encapsulated liposomes have a slightly larger particle size, but the encapsulation efficiency is significantly improved, with an average protein encapsulation efficiency of over 75%, effectively solving the problem of difficult encapsulation of large molecules. Figure 1 A graph showing the particle size measurement results of the samples from Example 1 and Comparative Example 1. Figure 2 The diagram shows the optical Tyndall effect of the supramolecular protein solution formed after steps 1) and 2) in the example. Figure 3 Electron micrographs showing the vesicles formed after liposome encapsulation. Figure 4 To show the zeta potential measurement results of the samples of Example 1 and Comparative Example 4, after the addition of polyquaternium-51, the liposome membrane potential decreased from -30.2 to -16.1, indicating that polyquaternium-51 formed a coating layer on the surface of the liposome membrane and neutralized part of the charge through electrostatic adsorption.

[0071] Example 5: Results of liposome transdermal assay Healthy mice were euthanized by cervical dislocation, and the abdominal skin was peeled off to remove the abdominal hair, subcutaneous mucosa and fat. After being cleaned with physiological saline, transdermal permeation experiments were performed using a modified Franz diffusion cell.

[0072] The mouse skin was placed between the diffusion chamber and its cover; the diffusion chamber had an area of ​​1.00 cm². 2 The sample container has a volume of 18.0 mL, with the stratum corneum facing the supply tank. 2 mg of each of the nine samples from Example 7 was added to the supply tank, along with physiological saline as the receiving solution. The temperature was kept constant at 37°C, and the magnetic stirring speed was 100 r / min.

[0073] Samples were taken at 0.5h, 1h, 1.5h, 2h, and 4h after the start of the experiment, with 2mL samples taken each time for testing and then returned to the sample. The collected receiving solution was diluted, centrifuged, filtered, treated with Triton-100, and then the protein concentration was determined by BCA assay. The percutaneous penetration percentage was calculated, and the average value was taken for three measurements. The results are shown in Table 2.

[0074] Table 2. Results of transdermal penetration percentage for each sample (unit: %)

[0075] Note: Groups 1-3 are the composite nanoliposomes prepared in Examples 1-3, respectively; Groups 4-9 are the liposomes prepared in Comparative Examples 1-6, respectively; Groups 10-12 are the unencapsulated protein solutions prepared in step 1) of Examples 1-3, respectively.

[0076] As shown in Table 2, the transdermal efficiency of the nanocomposite liposomes of this invention reached over 70%, while the transdermal efficiency of unencapsulated proteins (groups 10-12) was below 20%, representing a nearly 5-fold increase in transdermal efficiency. Compared with traditional liposome encapsulation (groups 4-6), the transdermal efficiency was nearly 3 times higher.

[0077] Example 6: Liposome Stability Verification The liposome samples were placed at 40℃ for accelerated stability testing. The particle size of the liposomes was measured at 2, 4, 6 and 8 weeks to assess their storage stability. The results are shown in Table 3.

[0078] Table 3. Particle size detection results of liposomes in accelerated stability test at 40℃ (unit: nm)

[0079] Note: Groups 1-3 are the composite nanoliposomes prepared in Examples 1-3, respectively; Groups 4-7 are the liposomes prepared in Comparative Examples 4-7, respectively.

[0080] As shown in Table 3, the composite nanoliposomes of the present invention underwent accelerated testing at 40°C for 8 weeks without significant changes in particle size or aggregation, indicating stability. In contrast, the control groups all showed varying degrees of aggregation and enlargement, demonstrating significant instability.

[0081] Example 7: Recombinant Collagen Liposome Cosmetics The nanocomposite liposomes prepared in Example 3 are mixed with other ingredients in cosmetics to obtain the relevant cosmetics. The specific formula of the recombinant collagen liposome essence is shown in Table 4 below. The resulting essence can be used for skin moisturizing, soothing and repair.

[0082] Table 4 Recombinant Collagen Liposome Serum Formula

Claims

1. A composite nanoliposome comprising: Liposomes having a closed lipid bilayer and contents encapsulated within the closed lipid bilayer; and The polyquaternary ammonium salt-51 layer encapsulating the liposomes; The contents contain supramolecular proteins; the supramolecular proteins are formed by adding salts and / or alcohols to a recombinant collagen solution; The lipid components forming the lipid bilayer are selected from one of lecithin, hydrogenated lecithin, phosphatidylcholine, hydrogenated phosphatidylcholine, or any mass ratio of them; The composite nanoliposomes are formed by adding an aqueous solution of polyquaternium-51 to an aqueous solution containing the liposomes and mixing them.

2. The complex nanoliposome according to claim 1, wherein, The recombinant collagen is selected from recombinant type I collagen, recombinant type III collagen, recombinant type XVII collagen, or a combination thereof.

3. The complex nanoliposome according to claim 1, wherein, The liposomes are formed by a method selected from thin film dispersion, reverse evaporation, ultrasonic treatment, injection, and microfluidics.

4. A method for preparing the composite nanoliposomes according to claim 1, comprising adding an aqueous solution of polyquaternium-51 to an aqueous solution containing the liposomes and mixing them to form the composite nanoliposomes; wherein, The concentration of the polyquaternium-51 aqueous solution is 1~10% by weight. The aqueous solution containing liposomes contains 6-15% by weight of liposomes; The volume ratio of the polyquaternium-51 aqueous solution to the liposome-containing aqueous solution is 1:50 to 1:

8.

5. The method of making according to claim 4, wherein, The mixing was carried out by stirring; the stirring speed was 50 rpm to 1000 rpm; the stirring time was 1 min to 120 min.

6. A method for preparing recombinant collagen complex nanoliposomes, comprising: (1) Add salts and / or alcohols to the recombinant collagen solution to form a supramolecular protein solution, which is the aqueous phase; (2) Dissolve the lipid components in an organic solvent to form a lipid component solution, which is the lipid phase; (3) The aqueous phase and the lipid phase are mixed to form an aqueous solution containing liposomes, the contents of which contain recombinant collagen; and (4) Add the aqueous solution of polyquaternium-51 to the aqueous solution containing liposomes and mix them to obtain the recombinant collagen composite nanoliposomes; The concentration of the polyquaternium-51 aqueous solution is 1~10% by weight. The aqueous solution containing liposomes contains 6-15% by weight of liposomes; The volume ratio of the polyquaternium-51 aqueous solution to the liposome-containing aqueous solution is 1:50 to 1:

8.

7. A cosmetic or topical preparation comprising any one of the composite nanoliposomes according to claims 1 to 3.

Images