Microstructured supramolecular elastin and preparation method and application thereof

Abstract

The present application relates to a kind of microstructure supermolecular elastin, the microstructure supermolecular elastin includes liposome and supermolecular elastin and hydrolyzed elastin wrapped inside liposome;The membrane material of the liposome includes phospholipid and polyol.The present application creatively constructs an elastin liposome, supermolecular elastin and hydrolyzed elastin are co-encapsulated in the inside of liposome, can significantly improve the skin delivery depth of elastin, realize the efficient, safe transdermal delivery of macromolecular active protein;While supermolecular elastin and hydrolyzed elastin are mutually synergistic, can deeply penetrate skin, realize the effect of moisturizing and anti-wrinkle.

Description

Technical Field

[0001] This invention belongs to the field of cosmetic technology and relates to a microstructured supramolecular elastin, its preparation method, and its application. Background Technology

[0002] Elastin is a key structural protein that maintains skin elasticity and firmness; its degradation and loss are the main causes of skin sagging and wrinkle formation. Exogenous elastin supplementation has become an important strategy for anti-wrinkle skincare. However, elastin is a large molecule protein (molecular weight typically >50 kDa), highly hydrophilic, and has difficulty penetrating the skin's stratum corneum barrier, resulting in low transdermal absorption efficiency and poor bioavailability, which limits its application in cosmetics and pharmaceuticals.

[0003] Liposomes, as nanocarriers with a biomembrane-like structure, can encapsulate both hydrophilic and hydrophobic components, enhancing the skin permeability of active ingredients. Previous studies have attempted to utilize liposomes to deliver macromolecules such as collagen and elastin, but significant limitations remain: traditional liposome preparation often requires the use of organic solvents such as chloroform and methanol, leading to solvent residues and environmental safety issues; the drug loading capacity of a single protein is limited, and the encapsulation efficiency is unstable; furthermore, long-term storage can result in aggregation and leakage, affecting delivery efficiency. In addition, existing liposomes mostly encapsulate only a single protein component, making it difficult to achieve synergistic effects between elastin molecules with different molecular structures.

[0004] Therefore, developing a liposome that can efficiently encapsulate hydrolyzed elastin, is easy to prepare, safe and stable, and can enhance transdermal delivery has become an urgent technical problem to be solved in this field. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a microstructured supramolecular elastin, its preparation method, and its applications.

[0006] To achieve this objective, the present invention adopts the following technical solution: In a first aspect, the present invention provides a microstructured supramolecular elastin, the microstructured supramolecular elastin comprising liposomes and supramolecular elastin and hydrolyzed elastin encapsulated within the liposomes; The membrane material of the liposomes includes phospholipids and polyols.

[0007] This invention creatively constructs an elastin liposome that encapsulates supramolecular elastin and hydrolyzed elastin within the liposome. This significantly enhances the skin delivery depth of elastin, achieving efficient and safe transdermal delivery of large molecular weight active proteins. Simultaneously, the supramolecular elastin and hydrolyzed elastin synergistically penetrate the skin, providing deep moisturizing and anti-wrinkle effects. This liposome system is simple to prepare, structurally stable, and highly safe, offering a new strategy for the efficient delivery of protein-based active ingredients.

[0008] Preferably, the phospholipids include any one or a combination of at least two of soybean lecithin, egg yolk lecithin, and hydrogenated lecithin.

[0009] Preferably, the polyol includes any one or a combination of at least two of 1,2-hexanediol, 1,3-propanediol, 1,2-pentanediol, and 1,3-butanediol.

[0010] Preferably, the polyol is a combination of 1,2-hexanediol and 1,3-propanediol.

[0011] Preferably, the mass ratio of 1,2-hexanediol to 1,3-propanediol is 1:(1-5) (e.g., 1:1, 1:2, 1:3, 1:4, 1:5, etc.).

[0012] Preferably, the raw materials for preparing the microstructured supramolecular elastin include, by weight, 1-3 parts phospholipid (e.g., 1 part, 2 parts, 3 parts, etc.), 3-20 parts polyol (e.g., 3 parts, 5 parts, 7 parts, 10 parts, 13 parts, 15 parts, 20 parts, etc.), 45-70 parts supramolecular elastin (e.g., 45 parts, 50 parts, 55 parts, 60 parts, 65 parts, 70 parts, etc.), and 1-10 parts hydrolyzed elastin (e.g., 1 part, 3 parts, 5 parts, 7 parts, 10 parts, etc.).

[0013] Preferably, the liposome further includes 15-30 parts of glycerol (e.g., 15 parts, 20 parts, 25 parts, 30 parts, etc.).

[0014] Preferably, the raw materials for preparing the supramolecular elastin include: hydrolyzed elastin, surfactant, stabilizer, chelating agent, preservative, eutectic solvent and water.

[0015] Preferably, the raw materials for preparing the supramolecular elastin, by weight, include: 0.01-0.05 parts of hydrolyzed elastin (e.g., 0.01, 0.02, 0.03, 0.04, 0.05, etc.), 0.01-0.1 parts of surfactant (e.g., 0.01, 0.03, 0.05, 0.07, 0.1, etc.), 11-36 parts of stabilizer (e.g., 11, 15, 20, 25, 30, 35, 36, etc.), and 0.01-1 part of chelating agent (e.g., [missing information]). 0.01 parts, 0.05 parts, 0.1 parts, 2 parts, 3 parts, 4 parts, 5 parts, 7 parts, 10 parts, etc.), 0.01-0.2 parts of preservative (e.g., 0.01 parts, 0.03 parts, 0.05 parts, 0.07 parts, 0.1 parts, 0.15 parts, 0.2 parts, etc.), 1-12 parts of eutectic solvent (e.g., 1 part, 2 parts, 5 parts, 7 parts, 9 parts, 10 parts, 12 parts, etc.), and 10-90 parts of water (e.g., 10 parts, 15 parts, 20 parts, 30 parts, 40 parts, 50 parts, 60 parts, 70 parts, 80 parts, 90 parts, etc.).

[0016] Preferably, the eutectic solvent is a combination of betaine and succinic acid.

[0017] Preferably, the mass ratio of betaine to succinic acid is (1-10):(0.1-1) (wherein, the specific values ​​of 1-10 can be 1, 3, 5, 7, 9, 10, etc., and the specific values ​​of 0.1-1 can be 0.1, 0.2, 0.5, 0.7, 0.9, 1, etc.).

[0018] Preferably, the surfactant comprises polysorbate-20.

[0019] Preferably, the stabilizer comprises any one or a combination of at least two of 1,3-propanediol, 1,2-hexanediol, glycerol, and sorbitol.

[0020] Preferably, the stabilizer comprises a combination of 1,3-propanediol, 1,2-hexanediol, glycerol, and sorbitol.

[0021] Preferably, the chelating agent comprises EDTA-disodium.

[0022] Preferably, the preservative includes any one or a combination of at least two of octyl glycol, ethylhexylglycerin, and cycloheptatrienolone.

[0023] Preferably, the preservative comprises a combination of caprylyl glycol, ethylhexylglycerin, and cycloheptatrienolone.

[0024] Preferably, the supramolecular elastin is obtained by a preparation method comprising the following steps: (1) The eutectic solvent is mixed with water and reacted to obtain phase A solution; (2) Hydrolyzed elastin is mixed with phase A solution and reacted. After the reaction, it is mixed with surfactant, stabilizer, chelating agent and preservative and water is added to obtain supramolecular elastin.

[0025] Supramolecular elastin is constructed using a eutectic solvent made from natural succinic acid and betaine, enabling the synergistic application of the eutectic solvent and bioactive components. The eutectic solvent can encapsulate hydrolyzed elastin, achieving better delivery results.

[0026] Preferably, the temperature of the mixing reaction in step (1) is 65-80℃ (e.g., 65℃, 70℃, 75℃, 80℃, etc.), and the time is 1-2 h (e.g., 1 h, 1.2 h, 1.4 h, 1.6 h, 1.8 h, 2 h, etc.).

[0027] Preferably, the mass ratio of the eutectic solvent to water in step (1) is (4-20):1 (for example, it can be 4:1, 8:1, 10:1, 12:1, 14:1, 16:1, 18:1, 20:1, etc.).

[0028] Preferably, the temperature of the reaction between the hydrolyzed elastin and the A-phase solution in step (2) is 40-50℃ (e.g., 40℃, 42℃, 44℃, 46℃, 48℃, 50℃, etc.), and the time is 1-2 h (e.g., 1 h, 1.2 h, 1.4 h, 1.6 h, 1.8 h, 2 h, etc.).

[0029] Preferably, the mixing temperature of polysorbate-20, caprylyl glycol, ethylhexylglycerin, 1,2-hexanediol, cycloheptatrienolone, glycerin, 1,3-propanediol, disodium EDTA, and sorbitol in step (2) is 30-40°C (e.g., 30°C, 32°C, 34°C, 36°C, 38°C, 40°C, etc.), and the reaction time is 1-4 h (e.g., 1 h, 2 h, 3 h, 4 h, etc.).

[0030] In a second aspect, the present invention provides a method for preparing a microstructured supramolecular elastin as described in the first aspect, the method comprising: (1) Phospholipids and polyols are mixed and reacted to obtain phase A solution; Supramolecular elastin, hydrolyzed elastin, and glycerol were mixed and reacted to obtain phase B solution; (2) Mix the A-phase solution and the B-phase solution and homogenize under high pressure to obtain the microstructured supramolecular elastin.

[0031] Preferably, the temperature of the phospholipid and polyol mixture reaction in step (1) is 50-65℃ (e.g., 50℃, 55℃, 60℃, 65℃, etc.), and the time is 20-60 min (e.g., 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, etc.).

[0032] Preferably, the temperature of the mixture reaction of supramolecular elastin, hydrolyzed elastin and glycerol in step (1) is 20-30℃ (e.g., 20℃, 22℃, 24℃, 26℃, 28℃, 30℃, etc.), and the time is 20-60 min (e.g., 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, etc.).

[0033] Preferably, the mixing temperature in step (2) is 20-30℃ (e.g., 20℃, 22℃, 24℃, 26℃, 28℃, 30℃, etc.).

[0034] Preferably, the temperature of the high-pressure homogenization in step (2) is 0-8℃ (e.g., 0℃, 2℃, 4℃, 6℃, 8℃, etc.), and the pressure is 400-450 bar (e.g., 400 bar, 410 bar, 420 bar, 430 bar, 440 bar, 450 bar, etc.).

[0035] Preferably, the high-pressure homogenization cycle in step (2) is repeated 3-8 times (for example, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, etc.).

[0036] All other specific point values ​​not listed above within the numerical ranges mentioned above can be selected and are all within the protection scope of this invention. For the sake of brevity, they will not be described in detail here.

[0037] Thirdly, the present invention provides an application of the microstructured supramolecular elastin as described in the first aspect in the preparation of cosmetics.

[0038] Compared with the prior art, the present invention has the following beneficial effects: This invention creatively constructs an elastin liposome that encapsulates supramolecular elastin and hydrolyzed elastin within the liposome. This significantly enhances the skin delivery depth of elastin, achieving efficient and safe transdermal delivery of large molecular weight active proteins. Simultaneously, the supramolecular elastin and hydrolyzed elastin synergistically penetrate the skin, providing deep moisturizing and anti-wrinkle effects. This liposome system is simple to prepare, structurally stable, and highly safe, offering a new strategy for the efficient delivery of protein-based active ingredients. Attached Figure Description

[0039] Figure 1 The image shows a transmission electron microscope image of the microstructured supramolecular elastin obtained in Example 1. Figure 2 The results of the appearance stability test of the microstructured supramolecular elastin obtained in Example 1; Figure 3 The results of the cytotoxicity test of the microstructured supramolecular elastin obtained in Example 1 are shown. Figure 4 Raman pseudocolor images of hydrolyzed elastin, the microstructured supramolecular elastin obtained in Example 1, and the supramolecular elastin obtained in Preparation Example 1 after permeating pigskin for 15 min; Figure 5 Raman pseudocolor images of hydrolyzed elastin, the microstructured supramolecular elastin obtained in Example 1, and the supramolecular elastin obtained in Preparation Example 1 after permeation in pigskin for 1 h; Figure 6 Raman pseudocolor images of hydrolyzed elastin, the microstructured supramolecular elastin obtained in Example 1, and the supramolecular elastin obtained in Preparation Example 1 after permeation in pigskin for 4 h. Detailed Implementation

[0040] To further illustrate the technical means and effects of the present invention, the following describes the technical solution of the present invention in conjunction with preferred embodiments of the present invention. However, the present invention is not limited to the scope of the embodiments.

[0041] The hydrolyzed elastin of this invention was purchased from IRA ISTITUTO RICERCHE APPLICATE SpA, trade name MARINE ELASTIN PF.

[0042] Preparation Example 1 This preparation example provides a supramolecular elastin, and the preparation method is as follows: (1) At 70℃, 9 parts betaine, 1 part succinic acid and 2 parts water were mixed and reacted for 1.5 h to obtain phase A solution; (2) Cool the A phase solution to 45°C, add 0.05 parts of hydrolyzed elastin and mix and react for 1 h; (3) After the reaction, the system temperature was lowered to 30℃, and then 0.05 parts of polysorbate-20, 0.08 parts of EDTA-disodium, 20 parts of glycerol, 8 parts of 1,3-propanediol, 0.04 parts of 1,2-hexanediol, 3 parts of sorbitol, 0.05 parts of caprylyl glycol, 0.02 parts of ethylhexylglycerol, and 0.0003 parts of cycloheptatrienolone were added. The mixture was reacted for 1 h and homogenized for 30 min. After reacting for another 1 h, water was added to 100 parts to obtain supramolecular elastin.

[0043] Preparation Example 2 This preparation example provides a supramolecular elastin, and the preparation method is as follows: (1) At 75℃, 8 parts betaine, 1 part succinic acid and 1.5 parts water were mixed and reacted for 2 h to obtain phase A solution; (2) Cool the A phase solution to 40°C, add 0.05 parts of hydrolyzed elastin and mix and react for 1 h; (3) After the reaction, the system temperature was lowered to 30℃, and then 0.05 parts of polysorbate-20, 0.08 parts of EDTA-disodium, 18 parts of glycerol, 9 parts of 1,3-propanediol, 0.04 parts of 1,2-hexanediol, 3 parts of sorbitol, 0.05 parts of caprylyl glycol, 0.03 parts of ethylhexylglycerol, and 0.0005 parts of cycloheptatrienolone were added. The mixture was reacted for 1 h and homogenized for 30 min. After reacting for another 1 h, water was added to 100 parts to obtain supramolecular elastin.

[0044] Preparation Example 3 This preparation example provides a supramolecular elastin, and the preparation method is as follows: (1) At 75℃, 9 parts betaine, 1 part succinic acid and 1.5 parts water were mixed and reacted for 2 h to obtain phase A solution; (2) Cool the A phase solution to 45°C, add 0.05 parts of hydrolyzed elastin and mix and react for 1 h; (3) After the reaction, the system temperature was lowered to 35℃, and then 0.05 parts of polysorbate-20, 0.05 parts of EDTA-disodium, 18 parts of glycerol, 7 parts of 1,3-propanediol, 0.05 parts of 1,2-hexanediol, 2 parts of sorbitol, 0.05 parts of caprylyl glycol, 0.03 parts of ethylhexylglycerol, and 0.0005 parts of cycloheptatrienolone were added. The mixture was reacted for 1 h and homogenized for 30 min. After reacting for another 1 h, water was added to 100 parts to obtain supramolecular elastin.

[0045] Preparation Example 4 This preparation example provides a supramolecular elastin, and the preparation method is as follows: (1) At 70℃, 9 parts betaine, 1 part succinic acid and 2 parts water were mixed and reacted for 1.5 h to obtain phase A solution; (2) Cool the A phase solution to 30°C, then add 0.05 parts polysorbate-20, 0.08 parts EDTA-disodium, 20 parts glycerol, 8 parts 1,3-propanediol, 0.04 parts 1,2-hexanediol, 3 parts sorbitol, 0.05 parts caprylyl glycol, 0.02 parts ethylhexylglycerol, and 0.0003 parts cycloheptatrienolone, and mix and react for 1 h; (3) Add 0.05 parts of hydrolyzed elastin to the system, mix and react for 1 h, homogenize for 30 min, react for another 1 h, and then add water to 100 parts to obtain supramolecular elastin.

[0046] Example 1 This embodiment provides a microstructured supramolecular elastin, prepared by the following method: (1) Mix 3 parts soybean lecithin, 3 parts 1,2-hexanediol and 10 parts 1,3-propanediol and heat to 65°C. Stir at 300 rpm for 30 min until completely dissolved. Cool to 25°C to obtain phase A solution. At 25°C, 60 parts of supramolecular elastin obtained in Preparation Example 1, 6 parts of hydrolyzed elastin and 18 parts of glycerol were mixed to obtain phase B solution; (2) Add the A phase solution to the B phase solution at 25℃ with stirring, and stir at 300 rpm for 10 min to obtain a mixed solution; (3) Set the temperature of the cooling circulator to 4℃ and the pressure of the high pressure homogenizer to 450 bar. Homogenize and circulate the mixed solution 5 times at 4℃. After passing the inspection, filter the material with double-layer 200-mesh filter cloth to obtain microstructured supramolecular elastin.

[0047] Example 2 This embodiment provides a microstructured supramolecular elastin, prepared by the following method: (1) Mix 2 parts soybean lecithin, 2 parts 1,2-hexanediol and 8 parts 1,3-propanediol and heat to 65°C. Stir at 300 rpm for 25 min until completely dissolved. Cool to 25°C to obtain phase A solution. At 25°C, 55 parts of supramolecular elastin obtained in Preparation Example 2, 8 parts of hydrolyzed elastin and 20 parts of glycerol were mixed to obtain phase B solution; (2) Add the A phase solution to the B phase solution at 25℃ with stirring, and stir at 300 rpm for 15 min to obtain a mixed solution; (3) Set the temperature of the cooling circulator to 4℃ and the pressure of the high pressure homogenizer to 450 bar. Homogenize and circulate the mixed solution 5 times at 4℃. After passing the inspection, filter the material with double-layer 200-mesh filter cloth to obtain microstructured supramolecular elastin.

[0048] Example 3 This embodiment provides a microstructured supramolecular elastin, prepared by the following method: (1) Mix 2 parts soybean lecithin, 2 parts 1,2-hexanediol and 10 parts 1,3-propanediol and heat to 70°C. Stir at 300 rpm for 25 min until completely dissolved. Cool to 25°C to obtain phase A solution. At 25°C, 58 parts of supramolecular elastin obtained in Preparation Example 3, 8 parts of hydrolyzed elastin and 20 parts of glycerol were mixed to obtain phase B solution; (2) Add the A phase solution to the B phase solution at 25℃ with stirring, and stir at 300 rpm for 15 min to obtain a mixed solution; (3) Set the temperature of the cooling circulator to 4℃ and the pressure of the high pressure homogenizer to 400 bar. Homogenize and circulate the mixed solution 5 times at 4℃. After passing the inspection, filter the material with double-layer 200-mesh filter cloth to obtain microstructured supramolecular elastin.

[0049] Example 4 This embodiment provides a microstructured supramolecular elastin. The only difference between this embodiment and Example 1 is that the supramolecular elastin obtained in Example 1 is replaced with the supramolecular elastin obtained in Example 4, while all other conditions remain unchanged.

[0050] Example 5 This embodiment provides a microstructured supramolecular elastin. The preparation method differs from that in Example 1 only in that glycerol is not added in step (1), while all other conditions remain unchanged.

[0051] Example 6 This embodiment provides a microstructured supramolecular elastin. The preparation method differs from that of Example 1 only in that 1,2-hexanediol is not added in step (1), and the reduced amount of 1,2-hexanediol is allocated to 1,3-propanediol. All other conditions remain unchanged.

[0052] Example 7 This embodiment provides a microstructured supramolecular elastin. The preparation method differs from that of Example 1 only in that 1,3-propanediol is not added in step (1), and the reduced amount of 1,3-propanediol is allocated to 1,2-hexanediol. All other conditions remain unchanged.

[0053] Comparative Example 1 This comparative example provides a supramolecular elastin mixture, prepared by the following method: (1) Mix 60 parts of supramolecular elastin obtained in Preparation Example 1, 6 parts of hydrolyzed elastin and 18 parts of glycerol at 25°C. Set the temperature of the cooling circulator to 4°C and the pressure of the high pressure homogenizer to 450 bar. Homogenize and circulate the mixed solution 5 times at 4°C. After passing the inspection, filter the mixture through a double-layer 200-mesh filter cloth to obtain a mixture of supramolecular elastin.

[0054] Test Example 1 This test example describes the morphology and stability of the microstructured supramolecular elastin obtained in Example 1.

[0055] (1) Morphological characteristics The microstructured supramolecular elastin obtained in Example 1 was observed using transmission electron microscopy, and the results are as follows: Figure 1 As shown in the figure. The results show that the microstructure of supramolecular elastin is a uniformly distributed spherical or near-spherical structure with a size of about 50 nm.

[0056] (2) Stability test The microstructured supramolecular elastin obtained in Example 1 was placed under ultraviolet light, cool room temperature (25°C), -15°C, 4°C, and 45°C for 0, 1, 2, 3, 4, and 8 weeks, respectively, and its appearance, particle size, and pH value were observed.

[0057] The results of the appearance stability test are as follows Figure 2 As shown, the results indicate that the microstructured supramolecular elastin obtained by this invention exhibits excellent appearance stability regardless of whether it is under ultraviolet light, high temperature, or low temperature conditions, and does not show precipitation or stratification.

[0058] The particle size and pH value test results are shown in Table 1. The results show that the microstructured supramolecular elastin obtained by this invention does not show significant changes in particle size and pH value under ultraviolet light irradiation or under room temperature and low temperature conditions, and has good stability. Test Example 2 This test case demonstrates the cytotoxicity of the microstructured supramolecular elastin obtained in Example 1.

[0059] The MTT assay, also known as the MTT colorimetric assay, is a method for detecting cell viability and growth. Its principle is that succinate dehydrogenase in the mitochondria of living cells reduces exogenous MTT to water-insoluble blue-purple formazan crystals, which are then deposited in the cells. Dead cells lack this function. Dimethyl sulfoxide (DMSO) dissolves the formazan in the cells, and the absorbance is measured at 490 nm using an enzyme-linked immunosorbent assay (ELISA) reader, indirectly reflecting the number of living cells. Within a certain cell count range, the amount of MTT crystals formed is directly proportional to the cell count.

[0060] Cells: Human fibroblasts (HXXFB-00001, Cyagen); Reagents: Human fibroblast complete culture medium (HXXFB-90011, Cyagen); MTT (M1020, Solarbio); DMSO (ST038, Beyotime); Fetal bovine serum (FBSST-01033, Cyagen); PBS buffer pH 7.2~7.4 (P1020, Solarbio); Trypsin (T1300, Solarbio).

[0061] Instrument consumables: 96-well plate (Corning); pipette (Eppendorf); centrifuge (OSE-MC8, Tiangen Biotech); CO2 incubator (CLM-170B-8-CN, Taicang Yisigao Medical Device Technology Co., Ltd.); shaker (TS-200, Qilinbell); multi-functional microplate reader (Synergy LX, Yuanxi Biotech).

[0062] Experimental methods: 1) Collect logarithmic-phase cells by trypsin digestion, adjust the cell suspension concentration, and divide into 96-well plates, 180 μL per well, 5.0 × 10⁻⁶ cells / well. 3 Cells / well; 2) Incubate at 37℃ with 5% CO2 to allow cells to adhere to the cell wall for 6-24 hours; 3) Add an appropriate concentration of the test substance and continue culturing for 24 h; the test substance is a microstructural supramolecular elastin sample, and the sample cell culture concentration is diluted to the following concentrations by volume fraction (v / v, %) using serum-free cell culture medium: 0.01%, 0.025%, 0.05%, 0.10%, 0.25%, 0.50%, 1.0%, 2.5%, 5.0%; 4) Carefully aspirate the supernatant, wash the cells once with PBS buffer, add 90 μL of fresh culture medium, then add 10 μL of LTT solution, and continue culturing for 4 h; 5) Remove the supernatant, add 110 μL of Formazan dissolving solution DMSO to each well, place on a shaker and shake at low speed for 10 min to fully dissolve the crystals, and measure the absorbance (OD) of each well at 490 nm using an ELISA reader. 6) Simultaneously set up blank wells (culture medium, MTT, Formazan lysate), negative control wells NC (cells, drug dissolution medium of the same concentration, culture medium, MTT, Formazan lysate), and positive control wells PC (cells, 10% DMSO, culture medium, MTT, Formazan lysate), with 3 replicates for each group.

[0063] 7) Result Calculation: Cell viability (%) = (Drug OD - Blank OD) / (Control OD - Blank OD) × 100% Test results are as follows Figure 3 As shown, the results indicate that the microstructured supramolecular elastin samples have no toxic effect on human fibroblasts in the concentration range of 0.01% to 2.5% (v / v).

[0064] Test Example 3 This test example examines the anti-wrinkle efficacy of the microstructured supramolecular elastin from Examples 1-7 and the mixture of supramolecular elastin from Comparative Example 1, and investigates the effect of microstructured supramolecular elastin on the content of type I and type III collagen in human fibroblasts.

[0065] Method: Enzyme-linked immunosorbent assay (ELISA).

[0066] Cells: Human fibroblasts (HXXFB-00001, Cyagen); Reagents: Human fibroblast complete culture medium (HXXFB-90011, Cyagen); Trypsin (T1300, Solarbio); Transforming growth factor β1 (TGF-β1, CM088-5HP, Chomper); Type I collagen ELISA kit (JM-03329H2, Jingmei Biotechnology); Type III collagen ELISA kit (JM-0961H2, Jingmei Biotechnology); Instrument consumables: 6-well plates (Corning); pipettes (Eppendorf); centrifuges (OSE-MC8, Tiangen Biotech); CO2 incubators (CLM-170B-8-CN, Taicang Yisigao Medical Device Technology Co., Ltd.); multi-functional microplate readers (Synergy LX, Yuanxi Biotech).

[0067] Experimental groups: The microstructured supramolecular elastin of Examples 1-7, the mixture of supramolecular elastin of Comparative Example 1, the supramolecular elastin obtained in Preparation Example 1, and hydrolyzed elastin were used as sample groups. Cells in complete culture medium but without test sample and long-wave ultraviolet (UVA) treatment were used as negative controls (NC), cells treated with UVA only were used as model controls (UVA), and UVA-induced fibroblasts treated with 100 ng / mL TGF-β1 were used as positive controls (PC).

[0068] Experimental steps: 1. Collect logarithmic-phase cells by trypsin digestion, adjust the cell suspension concentration, and aliquot into 6-well plates, 2.0 mL per well, 2.0 × 10⁶ cells / well. 5 Cells / well; 2. Incubate at 37℃ with 5% CO2 to allow cells to adhere. When the cells reach 70% confluence (cell density approximately 70%), add samples and continue incubation. Negative control group (NC): Add complete culture medium containing 0.1% PBS (solvent control), incubate for 2 h, and do not irradiate with UVA.

[0069] Model control group (UVA): Add complete culture medium containing 0.1% PBS, incubate for 2 h, and then irradiate with UVA.

[0070] Positive control group (PC): Add complete culture medium containing 100 ng / mL TGF-β1, incubate for 2 h, and then irradiate with UVA.

[0071] Sample groups: Add complete culture medium containing 0.10%, 0.25%, and 0.50% (v / v) of the sample (the sample was diluted with serum-free DMEM to the corresponding concentration) respectively, and incubate for 2 h.

[0072] After pre-incubation, discard the supernatant from each well and wash each well once with 1 mL of PBS (phenol red-free, pH 7.4). Except for the negative control group, add 1 mL of PBS to each well in all other groups. Place the 6-well plate directly under a UVA light source (wavelength 340–400 nm, peak 365 nm), and calibrate the output intensity using a UV radiometer at 30 J / cm². 2 The control group was irradiated with the same dose; the negative control group was wrapped in aluminum foil and placed in the same environment.

[0073] After irradiation, the PBS was removed, and 2 mL of DMEM medium containing 1% FBS (without any sample or TGF-β1) was added to each well. The cells were then placed back into a 37°C, 5% CO2 incubator and cultured for another 24 h.

[0074] 3. Collect the cell culture medium, centrifuge, take the supernatant and store it at -80℃, and detect the content according to the instructions of the Type I collagen and Type III collagen ELISA kits.

[0075] The test results are shown in Tables 2 and 3, where Table 2 shows the content of type I collagen and Table 3 shows the content of type III collagen; the sample concentration unit is (v / v). The results showed that the microstructured supramolecular elastin of the present invention can significantly increase the content of type I and type III collagen within a concentration range of 0.10% to 0.50% (v / v), indicating excellent anti-wrinkle efficacy. Changing the technical solution of the present invention would lead to a decrease in anti-wrinkle efficacy. If only supramolecular elastin or hydrolyzed elastin is used, the content of type I and type III collagen will be significantly lower compared to the microstructured supramolecular elastin of the present invention, resulting in a weakened anti-wrinkle effect.

[0076] Test Example 4 In this test example, hydrolyzed elastin, the microstructured supramolecular elastin obtained in Example 1, and the supramolecular elastin obtained in Preparation Example 1 were used as test samples for transdermal absorption experiments.

[0077] This test was based on the ex vivo porcine skin-Franz cell system. After the samples were treated for 15 min, 1 h, and 4 h, the residual amount of the samples in the skin was detected by confocal Raman spectroscopy to evaluate the penetration behavior of the three samples in the skin.

[0078] Main equipment: TK-12D transdermal absorption diffusion instrument (Shanghai Kaikai), Franz cell diffusion cell (Shanghai Kaikai), Raman spectrometer (Invia Qontor).

[0079] Transdermal absorption experiment: 1) Fixation of pig skin: Take out the isolated pig skin stored at -20℃, select skin with no hair, no damage, and no wrinkles, cut it into 3.5 cm × 3.5 cm pieces, rinse repeatedly with PBS buffer for 15 s, wipe dry, and fix the pig skin tightly between the supply chamber and the receiving chamber of the Franz diffusion cell. 2) Sample addition: Add 8.0 mL of PBS buffer to the receiving chamber, remove the air, and ensure that the dermal layer of the skin is in close contact with the receiving solution. Add 30 μL of each sample, and distribute the sample radially from the center of the skin to the edge to the surface of the pigskin. Seal the supply chamber with sealing film. 3) Infiltration: Place the Franz diffusion cell in the TK-12D transdermal absorption diffusion instrument, and simultaneously turn on the electromagnetic stirrer at a speed of 300 rpm to maintain a constant temperature water bath of (32 ± 1)℃, and ensure that there are no air bubbles in the water bath jacket. 4) Sample collection: Use a pipette to draw 1 mL of PBS and repeatedly blow and rinse the skin surface, repeating the rinsing process 5 times; 5) Detection: Trim the center of the pigskin to 1 cm × 1 cm, wipe off the moisture, and place it in a centrifuge tube without folding. Place one half of the pigskin on a quartz aluminized glass slide for three-dimensional Raman spectroscopy detection; freeze-embed the other half of the pigskin into sections with a section thickness of 16 μm, and attach them to a quartz aluminized glass slide for two-dimensional Raman spectroscopy detection.

[0080] Raman spectroscopy detection experiment: 1) Before detection, the confocal Raman spectrometer is calibrated using a silicon wafer; 2) Place the quartz aluminized glass slide with frozen sections on the sample stage, and under the microscope select the part where the epidermis is not separated, the skin surface is undamaged, wrinkle-free, and the structure is well-defined, and measure its single spectrum. 3) Select a rectangular range centered on the point where the measured characteristic peak is prominent and the noise is relatively small, and start scanning the sample to obtain the Raman spectrum dataset.

[0081] Statistical Analysis: Witec data analysis software was used to obtain relatively clear spectra through methods such as spectral range selection, cosmic ray removal, polynomial fitting to remove baseline, and Savitzky-Golay spectrum smoothing. The Raman spectroscopy dataset was analyzed using univariate analysis to extract the compositional information of the experimental samples. Witec data analysis software performed univariate imaging, i.e., Raman spectroscopy imaging. A pseudo-color image was reconstructed using the combined intensity of characteristic peaks of a substance in the sample's scanned spectrum. The intensity of the characteristic peak is related to the brightness of the Raman image color. Brightness is directly related to the Raman signal intensity; the higher the signal intensity, the brighter the color; the lower the signal intensity, the darker the color. Red usually indicates a high signal intensity, blue usually indicates a low signal intensity, and color gradients are used for intermediate intensities (color gradients can more subtly reflect changes in signal intensity). Origin software was used to analyze the single-spectrum dataset, plotting multi-point Raman scan spectra of the entire skin layer and reconstructed Raman pseudo-color images after large-scale scanning, as well as performing semi-quantitative analysis of the substance's characteristic peaks. GraphPad Prism was used for plotting, and the results are expressed as Mean ± SD.

[0082] The results are as follows Figures 4-6 As shown, where Figure 4 This represents the amount of residue remaining in the skin after the sample has been exposed for 15 minutes. Figure 5 This represents the residue in the skin after 1 hour of treatment. Figure 6 The values ​​represent the residual amount of the samples in the skin after 4 hours of treatment. The results showed that after 15 minutes, 1 hour, and 4 hours of treatment with hydrolyzed elastin, the supramolecular elastin of Preparation Example 1, and the microstructured supramolecular elastin of Example 1, the residual amount in the skin was 1061 cm⁻¹. -1 Below the peak, the relative penetration depth increased significantly, indicating that elastin, among elastin, supramolecular elastin, and microstructural supramolecular elastin, underwent permeation behavior. Compared to elastin and supramolecular elastin, microstructural supramolecular elastin significantly increased the skin penetration depth.

[0083] The applicant declares that the technical solution of this invention is illustrated by the above embodiments, but this invention is not limited to the above embodiments, that is, it does not mean that this invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the products of this invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of this invention.

[0084] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0085] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A microstructured supramolecular elastin, characterized in that, The microstructured supramolecular elastin includes liposomes and supramolecular elastin and hydrolyzed elastin encapsulated within the liposomes. The membrane material of the liposomes includes phospholipids and polyols.

2. The microstructured supramolecular elastin according to claim 1, characterized in that, The phospholipids include any one or a combination of at least two of soybean lecithin, egg yolk lecithin, and hydrogenated lecithin. Preferably, the polyol includes any one or a combination of at least two of 1,2-hexanediol, 1,3-propanediol, 1,2-pentanediol, and 1,3-butanediol; Preferably, the polyol is a combination of 1,2-hexanediol and 1,3-propanediol; Preferably, the mass ratio of 1,2-hexanediol to 1,3-propanediol is 1:(1-5).

3. The microstructured supramolecular elastin according to claim 1 or 2, characterized in that, The raw materials for preparing the microstructured supramolecular elastin include, by weight, 1-3 parts phospholipids, 3-20 parts polyols, 45-70 parts supramolecular elastin, and 1-10 parts hydrolyzed elastin. Preferably, the liposome also contains 15-30 parts of glycerol.

4. The microstructured supramolecular elastin according to any one of claims 1-3, characterized in that, The raw materials for preparing the supramolecular elastin include: hydrolyzed elastin, surfactant, stabilizer, chelating agent, preservative, eutectic solvent and water.

5. The microstructured supramolecular elastin according to any one of claims 1-4, characterized in that, The raw materials for preparing the supramolecular elastin, by weight, include: 0.01-0.05 parts hydrolyzed elastin, 0.01-0.1 parts surfactant, 11-36 parts stabilizer, 0.01-1 part chelating agent, 0.01-0.2 parts preservative, 1-12 parts eutectic solvent and 10-90 parts water; Preferably, the eutectic solvent is a combination of betaine and succinic acid; Preferably, the mass ratio of betaine to succinic acid is (1-10):(0.1-1).

6. The microstructured supramolecular elastin according to claim 4, characterized in that, The surfactant includes polysorbate-20; Preferably, the stabilizer comprises any one or a combination of at least two of 1,3-propanediol, 1,2-hexanediol, glycerol, and sorbitol; Preferably, the stabilizer comprises a combination of 1,3-propanediol, 1,2-hexanediol, glycerol, and sorbitol; Preferably, the chelating agent comprises EDTA-disodium; Preferably, the preservative includes any one or a combination of at least two of octyl glycol, ethylhexylglycerin, and cycloheptatrienolone; Preferably, the preservative comprises a combination of caprylyl glycol, ethylhexylglycerin, and cycloheptatrienolone.

7. The microstructured supramolecular elastin according to any one of claims 1-6, characterized in that, The supramolecular elastin is obtained by a preparation method comprising the following steps: (1) The eutectic solvent is mixed with water and reacted to obtain phase A solution; (2) Hydrolyzed elastin is mixed with phase A solution and reacted. After the reaction, it is mixed with surfactant, stabilizer, chelating agent and preservative and water is added to obtain supramolecular elastin. Preferably, the temperature of the mixing reaction in step (1) is 65-80℃ and the time is 1-2 h; Preferably, the mass ratio of the eutectic solvent to water in step (1) is (4-20):1; Preferably, the temperature for mixing the hydrolyzed elastin with the A-phase solution in step (2) is 40-50℃, and the time is 1-2h. Preferably, the temperature at which the mixture of polysorbate-20, caprylyl glycol, ethylhexylglycerin, 1,2-hexanediol, cycloheptatrienolone, glycerin, 1,3-propanediol, disodium EDTA, and sorbitol is mixed in step (2) is 30-40°C, and the reaction time is 1-4 h.

8. The method for preparing microstructured supramolecular elastin according to any one of claims 1-7, characterized in that, The preparation method includes: (1) Phospholipids and polyols are mixed and reacted to obtain phase A solution; Supramolecular elastin, hydrolyzed elastin, and glycerol were mixed and reacted to obtain phase B solution; (2) Mix the A-phase solution and the B-phase solution and homogenize under high pressure to obtain the microstructured supramolecular elastin.

9. The preparation method according to claim 8, characterized in that, The temperature for the reaction of the phospholipids and polyols in step (1) is 50-65℃ and the time is 20-60 min. Preferably, the temperature of the reaction of supramolecular elastin, hydrolyzed elastin and glycerol in step (1) is 20-30℃ and the time is 20-60 min; Preferably, the mixing temperature in step (2) is 20-30°C; Preferably, the temperature of the high-pressure homogenization in step (2) is 0-8℃ and the pressure is 400-450 bar; Preferably, the high-pressure homogenization cycle in step (2) is repeated 3-8 times.

10. The use of the microstructured supramolecular elastin according to any one of claims 1-7 in the preparation of cosmetics.

Images