Transdermal complexes of albumin encapsulated collagen and methods of making and using the same

Abstract

This application relates to the field of cosmetic technology, specifically disclosing a transdermal complex of albumin-encapsulated collagen, its preparation method, and its application. The preparation process involves sequential pretreatment of serum albumin powder, flexible enzymatic digestion, hydrophobic-driven self-assembly to form polypeptide nanoparticles, directional encapsulation and degradation site shielding of collagen, charge modification, and nanofabrication. The process parameters for each step are precisely defined, resulting in the collagen being encapsulated by albumin. This not only prevents collagen degradation on the skin surface but also enhances its penetration into the skin. Furthermore, the outer layer is modified with a negative charge from hyaluronic acid-metal particles, while the skin interior carries a positive charge, thereby accelerating the overall transdermal absorption efficiency of collagen.

Description

Technical Field

[0001] This application relates to the field of cosmetic technology, and more specifically, to a transdermal complex of albumin-encapsulated collagen, its preparation method, and its application. Background Technology

[0002] In the cosmetics industry, the transdermal absorption of macromolecular ingredients has always been a key challenge. Conventional non-invasive transdermal processes typically involve liposome encapsulation, but this method has several drawbacks. While liposome encapsulation can protect ingredients to some extent, its structure is unstable and prone to rupture under changes in external environmental conditions such as temperature and pH, leading to leakage of the encapsulated ingredients. This results in low transdermal efficiency and difficulty in precise control. Furthermore, the core components of liposomes, phospholipids (such as lecithin and synthetic phospholipids) and cholesterol, can trigger immune responses. For example, impurities in natural phospholipids (such as egg yolk lecithin) (e.g., proteins, fatty acid oxidation products) may act as haptens to activate the immune system, while chemically modified groups in synthetic phospholipids (such as DPPC) (e.g., PEGylated lipids) may induce IgE-mediated allergic reactions.

[0003] In the field of drug delivery, albumin is frequently used as a carrier due to its excellent biocompatibility and bioactivity, primarily administered via injection or oral administration. When injected, albumin rapidly enters the bloodstream, fulfilling its carrier function to precisely deliver the drug to the target site. When administered orally, albumin remains relatively stable in the gastrointestinal environment, protecting the drug from degradation by digestive enzymes and improving bioavailability. The overall stability of albumin carriers is relatively easy to maintain during both injection and oral administration.

[0004] However, in the field of topical skincare, cosmetics need to be applied to the skin's surface. This surface is highly susceptible to external environmental factors such as temperature, pH, and light, as well as degradation enzymes on the skin's surface. This can lead to poor stability or even inactivation of the active ingredients encapsulated in serum albumin. Furthermore, serum albumin itself has a relatively large molecular weight of 66.5 kDa, making it difficult to penetrate the stratum corneum and resulting in extremely low transdermal absorption efficiency. These issues significantly limit the application of cosmetics using serum albumin as a carrier in topical skincare. Summary of the Invention

[0005] To address the issues of improving the transdermal absorption efficiency of cosmetics and enhancing the stability of the encapsulated active ingredients, this application provides a transdermal complex of albumin-encapsulated collagen, its preparation method, and its application in cosmetics.

[0006] Firstly, this application provides the following technical solution: A method for preparing a transdermal complex of albumin-encapsulated collagen includes the following steps: Step 1, Pretreatment of serum albumin powder: Step 1-1: Mix serum albumin powder with 0.01M PBS buffer at 4°C until the volume is adjusted to 100mL to obtain the first mixture. The pH of the first mixture is maintained at 7.1-7.3. Steps 1-2: Add a conformation stabilizer to the first mixture and treat it at 4°C for 30 min to obtain the second mixture; Steps 1-3: Add fatty acids to the second mixture at a final concentration of 0.08-0.12 mM, and treat at 25-30℃ for 20 min to obtain the third mixture; the mass molar ratio of the fatty acids to serum albumin powder is 1:1. Steps 1-4: Add a reducing agent with a concentration of 0.5-1.0 mM to the third mixture and treat it at 25-30℃ for 15 min to obtain the pretreated serum albumin solution; Step 2, flexible enzyme digestion: Step 2-1: Thoroughly mix the hydrolytic enzyme and PBS buffer with a pH of 7.0-7.5 to prepare a hydrolytic enzyme solution with a mass concentration of 1 mg / mL; Step 2-2: Place the hydrolytic enzyme solution on an ice bath and slowly add it dropwise to the pretreated serum albumin solution at an enzyme-substrate ratio of 1:100-1:300 (w / w), mixing gently. Then, perform the enzymatic hydrolysis reaction for 20-40 minutes at 25-35℃, pH 7.0-7.5, and stirring speed of 100 r / min. Add the enzyme inhibitor and incubate at 25℃-30℃ for 10 minutes to gently terminate the reaction. Subsequently, centrifuge at 4℃ and centrifugation force of 8000×g for 10 minutes and collect the enzyme digestion supernatant. Step 3: Hydrophobic-driven self-assembly to form peptide nanoparticles: Step 3-1: The enzyme digestion supernatant is ultrafiltered and centrifuged to concentrate it to a mass concentration of 5-10 mg / mL to obtain concentrated enzyme digestion supernatant; Step 3-2: Let the concentrated enzyme digestion supernatant stand at 25°C for 2-4 hours, and check the turbidity at 600 nm every 30 minutes until the turbidity reaches 0.15±0.02 to obtain the prepolymer solution; Step 3-3: Heat the prepolymer solution to 28-32℃ and maintain it for 30-45 min to obtain a hydrophobic reaction solution; Steps 3-4: The redox pair was thoroughly mixed with 0.01M PBS buffer and added to the hydrophobic reaction solution. The mixture was incubated at 4°C for 4-6 hours and then dialyzed overnight in PBS buffer using a 3.5 kDa MWCO dialysis bag at 4°C to obtain polypeptide nanoparticles with a particle size of 80-120 nm. Step 4, Targeted encapsulation and degradation site shielding of collagen: Step 4-1: Under the temperature conditions of 30℃±2℃, hydrolyzed collagen and polypeptide nanoparticles are incubated at a mass ratio of 1:(5-6) for 58-60 min to obtain albumin nanoparticle system; Step 4-2: Thoroughly mix the transglutaminase solution with the albumin nanoparticle system, and carry out the cross-linking reaction for 2-3 h at a pH of 6.0-6.5 and a temperature of 22-28℃, accompanied by stirring at a speed of 55-60 r / min; then stop the reaction by refrigerating at 4℃ for 30 min, and dialyze to remove unbound transglutaminase to obtain the collagen-encapsulated nanoparticle system; Step 5, Charge Modification and Nanofabrication: Hyaluronic acid solution was added dropwise to the nanoparticle system encapsulating collagen, and incubated at 25℃±2℃ for 25-30 min with stirring at 50 r / min to obtain the charge-modified albumin system. Take 1 mg / mL of gold nanoparticle dispersion and slowly add it dropwise to the charge-modified albumin system at a final concentration of 0.01% (w / v). Stir while adding the mixture at 50 r / min and incubate at 25℃ for 20 min to obtain a transdermal complex of albumin encapsulating collagen.

[0007] The serum albumin powder used in this application can be either recombinant human serum albumin powder or animal serum albumin powder.

[0008] In this application, step 1-1 quantifies the first mixture, which facilitates subsequent operations. Step 1-2 adds a conformational stabilizer to the first mixture, maintaining serum albumin in a tight native conformation, reducing the likelihood of denaturation, aggregation, and precipitation, and minimizing conformational fluctuations in subsequent operations. Step 1-3 ensures a precise ratio between the added fatty acids and serum albumin powder, maximizing the sealing of the IIIA / IIIB domains. Step 1-4, by adding a reducing agent within a reasonable concentration range, only non-critical disulfide bonds on the serum albumin surface are reduced, preserving the disulfide bonds in the core domains (IIIA / IIIB domains) of serum albumin, thus maintaining the protein's cavitary structure and providing favorable conditions for subsequent operations.

[0009] The various operations in step 1 ensure that the pretreated serum albumin solution has a uniform concentration and stable conformation.

[0010] In step 2-1, the hydrolase is fully dissolved in PBS buffer to ensure homogeneity for subsequent operations. In step 2-2, appropriate enzyme-substrate ratios, temperature, pH, stirring speed, and digestion time are used to ensure the enzymatic digestion reaction proceeds fully. Subsequently, an enzyme inhibitor is added to gently terminate the reaction, and undigested protein precipitate is removed by centrifugation. The final enzymatically digested serum albumin is obtained in the enzyme digestion supernatant.

[0011] In step 3-1, the enzyme digestion supernatant is sequentially subjected to ultrafiltration, centrifugation, and concentration to obtain a large number of effective peptide fragments. In step 3-2, the turbidity at 600 nm is detected to determine whether the pre-aggregation is uniform, which is beneficial for obtaining a homogeneous pre-aggregate solution and laying the foundation for subsequent self-assembly.

[0012] In step 3-3, the class III domains (including IIIA / IIIB) retained after enzymatic hydrolysis contain a large number of hydrophobic amino acid residues. During the 4℃ pre-aggregation stage, only a small amount is exposed, and the hydrophobic effect is weak. When the temperature is raised to 28-32℃, the thermal energy breaks the weak intramolecular interactions, causing the protein to gently unfold without destroying the core disulfide bond, thus exposing a large number of hydrophobic groups to provide binding sites.

[0013] The temperature range reached during heating is crucial. If the temperature rises below 28°C, insufficient exposure of hydrophobic groups will result in an inability to form a stable system. If the temperature rises above 32°C, protein denaturation and disulfide bond breakage will occur, leading to precipitation. If the temperature rises to 30°C, both group exposure and conformational integrity can be achieved, driving molecular aggregation to form hydrophobic microregions.

[0014] Meanwhile, the duration of holding the temperature after it has been raised to a suitable range is also extremely important. When the holding time is in the range of 30-45 minutes, the isothermal conditions allow the hydrophobic interactions to fully balance, resulting in ordered molecular arrangement, locking the hydrophobic interface, preventing conformational reversal after cooling, and providing a stable basis for subsequent disulfide bond renaturation.

[0015] In steps 3-4, the three-dimensional structure of the nanoparticles was locked through disulfide bond recombination. After thorough dialysis to remove excess reducing agent, DLS analysis revealed that the particle size of the peptide nanoparticles was 80-120 nm.

[0016] In step 4-1, a relatively mild temperature condition and a reasonable ratio of hydrolyzed collagen to peptide nanoparticles are used. Incubation is performed for an appropriate time to achieve initial encapsulation of collagen peptides through hydrophobic pockets and hydrogen bond networks, avoiding excessive stirring that could damage the hydrophobic pockets. In step 4-2, transglutaminase cross-links with albumin nanoparticles, forming a covalent bond between the N-terminus glutamine (Glu) of the collagen peptide and the lysine (Lys) residue of albumin. The cross-linking reaction is then terminated by refrigeration, and residual trace amounts of transglutaminase are removed by dialysis. This ensures a thorough cross-linking reaction, yielding a larger quantity of encapsulated collagen nanoparticles and minimizing the potential adverse effects of residual transglutaminase on subsequent operations.

[0017] In step 5, a hyaluronic acid solution is added. The hyaluronic acid forms an electrostatic adsorption layer on the surface of albumin nanoparticles by electrostatically adsorbing the amino groups on the surface of the albumin nanoparticles through its own carboxyl groups, thus forming a charge bridging layer.

[0018] Furthermore, the serum albumin powder in step 1-1 has a purity ≥99%, endotoxin <0.125 (EU / mg), is a white to pale yellow powder, has a pH of 6.0-8.0, and an α-helix content ≥60%.

[0019] Furthermore, the conformational stabilizer in steps 1-2 is composed of glycerol and sugars, wherein the final concentration of glycerol is 5-8% (v / v) and the final concentration of sugars is 5-8% (w / v). The fatty acid in steps 1-3 is at least one of palmitic acid and myristic acid; The reducing agent in steps 1-4 is reduced glutathione.

[0020] The conformation stabilizer selected in this application is limited to being composed of glycerol and sugars, and the addition conditions are also specified. The sugars include, but are not limited to, sucrose, and may also include glucose, trehalose, etc., thereby maintaining the cavitary conformation of the protein and reducing the possibility of conformational fluctuations in the protein during subsequent operations.

[0021] In this application, the selection of fatty acids and reducing agents is also limited, so as to achieve the purpose of reducing only the non-critical disulfide bonds on the surface and retaining the disulfide bonds in the core structural domain, so as to stabilize the cavity structure of the protein.

[0022] Furthermore, the hydrolytic enzyme in step 2-1 is porcine pancreatic elastase with an enzyme activity of 250-350 U / mg.

[0023] Furthermore, the enzyme inhibitor in step 2-2 consists of soybean trypsin inhibitor and elastase, wherein the ratio of soybean trypsin inhibitor to elastase is 2:1 (w / w).

[0024] In this application, soybean trypsin inhibitor and elastase compose an enzyme inhibitor to gently terminate the enzymatic hydrolysis reaction.

[0025] Further, in step 3-1, the enzyme digestion supernatant is added to a 30 kDa MWCO centrifugal ultrafiltration tube and centrifuged at 5000×g for 15-20 min at 4°C to concentrate to a mass concentration of 5-10 mg / mL; then it is washed 2-3 times with 0.01 M PBS buffer with a pH of 7.2 to remove impurities and reagent residues with a molecular weight less than 3 kD. In step 3-3, the temperature is increased at a rate of 0.3-0.8℃ / min.

[0026] In this application, a relatively gentle heating rate is used for the heating process, which is beneficial to make the temperature of the prepolymer liquid more uniform and to fully demonstrate the hydrophobic effect.

[0027] Further, in steps 3-4, the redox pair consists of oxidized glutathione and reduced glutathione, wherein the final concentration of oxidized glutathione is 0.2-0.4 mM and the final concentration of reduced glutathione is 0.2-0.4 mM.

[0028] In this application, oxidized glutathione and reduced glutathione are combined in a specific final concentration range to protect the required disulfide bonds, thereby locking the three-dimensional structure of the polypeptide nanoparticles through disulfide bond recombination and stabilizing the conformation of the polypeptide nanoparticles.

[0029] Furthermore, in step 4-1, the mass transfer is promoted by vortexing at 50-100 rpm for the first 20 minutes of incubation, and the remaining time is allowed to stand; the hydrolyzed collagen is obtained by MMP-8 enzymatic hydrolysis of porcine collagen (E / S=1:50 w / w), with a molecular size of 1,500-2,500 Da. The transglutaminase solution in step 4-2 is prepared by transglutaminase and 0.01M PBS buffer, with a mass ratio of transglutaminase to albumin nanoparticles of 1:50.

[0030] During incubation, a low-speed vortex is first applied, followed by a shutdown, to minimize damage to the hydrophobic pockets due to excessive stirring. Initial encapsulation is achieved through the hydrophobic pockets and hydrogen bond network of the serum albumin class III domain.

[0031] Further, in step 5-1, the hyaluronic acid solution is prepared by thoroughly mixing hyaluronic acid with 0.01M PBS buffer, and the mass concentration of the hyaluronic acid solution is 10 mg / mL; the molecular weight of the hyaluronic acid in the hyaluronic acid solution is 50-100 kDa, and the mass ratio of hyaluronic acid to albumin nanoparticles is 1:18.

[0032] Secondly, this application provides the following technical solution: A transdermal complex of albumin-encapsulated collagen was prepared using a method for preparing transdermal complexes of albumin-encapsulated collagen.

[0033] Thirdly, this application provides the following technical solution: Application of a transdermal complex of albumin-encapsulated collagen in cosmetics.

[0034] The albumin-encapsulated collagen transdermal complex prepared in this application can be used in cosmetics, including but not limited to water-based, lotion, and cream-based cosmetics. Furthermore, the amount of the albumin-encapsulated collagen transdermal complex used in cosmetics is 0.1%-1%.

[0035] In summary, this application has the following beneficial effects: The preparation method in this application is quite detailed, taking into account the pretreatment of serum albumin powder, flexible enzymatic digestion, hydrophobic-driven self-assembly to form polypeptide nanoparticles, collagen-directed encapsulation and degradation site shielding, charge modification and nanofabrication in sequence, and limiting the process parameters in each operation step, so that the collagen is encapsulated by albumin, which not only makes the collagen less likely to be degraded on the skin surface, but also helps to improve the penetration effect on the skin. In addition, the outer layer is modified by negative charge of hyaluronic acid-metal particles, while the inside of the skin is positively charged, thereby further accelerating the overall transdermal absorption efficiency of collagen.

[0036] The albumin-encapsulated collagen transdermal complex prepared in this application also has good safety, low skin immune response, and is suitable for a large user group. Attached Figure Description

[0037] Figure 1 These are SDS-PAGE electrophoresis images of the samples before and after enzyme digestion in this application; Figure 2 Raman permeation of control sample 1 in this application; Figure 3 This is a Raman permeation of the test sample 1 in this application. Detailed Implementation Example

[0038] Example 1: A transdermal complex of albumin encapsulating collagen was prepared using the following method: Step 1, Pretreatment of serum albumin powder: Step 1-1: Mix 0.665 g of serum albumin powder with 0.01 M PBS buffer (pH 7.2) at 4°C until the volume is adjusted to 100 mL to obtain the first mixture. Maintain the pH of the first mixture between 7.1 and 7.3. Steps 1-2: Add a conformation stabilizer to the first mixture. The conformation stabilizer consists of 5% glycerol and 5% sucrose at a final concentration. Incubate at 4°C for 30 min to obtain the second mixture. Steps 1-3: Add fatty acids (specifically palmitic acid) to the second mixture to a final concentration of 0.1 mM. The molar ratio of palmitic acid to serum albumin powder is 1:1. Incubate at 25°C for 20 min to obtain the third mixture. Steps 1-4: Add a reducing agent (specifically reduced glutathione) at a concentration of 0.8 mM to the third mixture, and incubate at 25°C for 15 min to obtain the pretreated serum albumin solution. Step 2, flexible enzyme digestion: Step 2-1: Thoroughly mix the hydrolytic enzyme and PBS buffer with a pH of 7.2 to prepare a hydrolytic enzyme solution with a mass concentration of 1 mg / mL; Step 2-2: Place the hydrolytic enzyme solution in an ice bath to avoid enzyme inactivation. Add it slowly dropwise to the pretreated serum albumin solution at an enzyme-substrate ratio of 1:100 (w / w) and stir gently. Then, perform the enzymatic hydrolysis reaction for 30 min at 25°C, pH 7.2, and stirring speed of 100 r / min. After that, add the enzyme inhibitor (soybean trypsin inhibitor: elastase = 2:1 (w / w)) and incubate at 25°C for 10 min to gently terminate the reaction. Then, centrifuge at 4°C and centrifuge force of 8000×g for 10 min and collect the enzyme digestion supernatant. Step 3: Hydrophobic-driven self-assembly to form peptide nanoparticles: Step 3-1: Add the enzyme digestion supernatant to a 30 kDa MWCO ultrafiltration tube, centrifuge at 4℃ and 5000×g for 15-20 min, and concentrate to a mass concentration of 5-10 mg / mL to obtain concentrated enzyme digestion supernatant; then wash twice with 0.01 M PBS buffer (pH=7.2) to remove impurities and reagent residues with a molecular weight lower than 3 kD; Step 3-2: The enzyme digestion supernatant after concentration in step 3-1 is allowed to stand at 25°C for 2-4 hours. The turbidity at 600 nm is measured every 30 minutes using a UV spectrophotometer until the turbidity reaches 0.15±0.02, thus obtaining a homogeneous prepolymer solution. Step 3-3: Heat the prepolymer solution to 30°C at a rate of 0.5°C / min and maintain it for 40 min to improve the overall hydrophobic interaction strength and obtain a hydrophobic reaction solution; Steps 3-4: The redox pair (composed of oxidized glutathione and reduced glutathione at a final concentration of 0.3 mM) was fully dissolved in 0.01 M PBS buffer and added to the hydrophobic reaction solution. The mixture was incubated at 4°C for 4-6 h, and then dialyzed overnight in PBS buffer using a 3.5 kDa MWCO dialysis bag at 4°C to remove excess redox pairs and obtain polypeptide nanoparticles with a particle size of 80-120 nm. Step 4, Targeted encapsulation and degradation site shielding of collagen: Step 4-1: Under the temperature conditions of 30℃±2℃, hydrolyzed collagen and polypeptide nanoparticles were incubated at a mass ratio of 1:6 for 60 min. During the incubation period, the mass transfer was promoted by vortexing at a speed of 50-100 rpm for the first 20 min, and then allowed to stand for the next 40 min to obtain the albumin nanoparticle system. Step 4-2: Thoroughly mix the transglutaminase solution with the albumin nanoparticle system. The transglutaminase solution is prepared by mixing transglutaminase with 0.01M PBS buffer at pH 6.2, with a transglutaminase:albumin nanoparticle ratio of 1:50 (w / w). The cross-linking reaction is carried out for 3 hours at pH 6.2 and 25°C with magnetic stirring at 60 r / min. The reaction is then terminated by refrigerating at 4°C for 30 minutes. Unbound transglutaminase is removed by dialyzing in PBS buffer using a 10 kDa MWCO dialysis bag to obtain the collagen-encapsulated nanoparticle system. Step 5, Charge Modification and Nanofabrication: A 10 mg / mL hyaluronic acid solution was added dropwise to a collagen-encapsulated nanoparticle system. The hyaluronic acid solution was prepared by mixing hyaluronic acid with a molecular weight of 50-100 kDa with 0.01 M PBS buffer at pH 6.2, with a hyaluronic acid:albumin nanoparticle ratio of 1:18 (w / w). The system was incubated at 25℃±2℃ for 30 min with slow stirring at 50 r / min. The hyaluronic acid adsorbed onto the surface of the albumin nanoparticles by electrostatic adsorption through its carboxyl groups, forming a charge bridging layer, thus obtaining a charge-modified albumin system. A 1 mg / mL gold nanoparticle dispersion was slowly added dropwise to the charge-modified albumin system at a final concentration of 0.01% (w / v). The mixture was stirred while being added at 50 r / min and incubated at 25 °C for 20 min to obtain a transdermal complex of albumin encapsulating collagen. The zeta potential was measured using a Zetasizer, with a target value of -25 to -35 mV. DLS analysis showed no significant increase in particle size (≤150 nm).

[0039] The serum albumin powder used in step 1-1 of this embodiment is recombinant human serum albumin with a purity ≥99%, endotoxin <0.125 (EU / mg), white to pale yellow powder, pH 6.0-8.0, and α-helix content ≥60%.

[0040] In step 2-1 of this embodiment, the hydrolytic enzyme used is porcine pancreatic elastase, cosmetic grade, with an enzyme activity of 250-350 U / mg.

[0041] In step 4-1 of this embodiment, the hydrolyzed collagen used is porcine collagen obtained by MMP-8 enzymatic hydrolysis (E / S=1:50 w / w), with a molecular size of 1,500-2,500 Da.

[0042] Example 2: A transdermal complex of albumin encapsulating collagen, which differs from Example 1 in that the conformation stabilizer in steps 1-2 consists of glycerol and sucrose at a final concentration of 8%, while other operation steps and auxiliary agents used are the same.

[0043] Example 3: A transdermal complex of albumin encapsulating collagen, which differs from Example 1 in that the fatty acid in steps 1-3 is myristic acid.

[0044] Example 4: A transdermal complex of albumin encapsulating collagen, which differs from Example 1 in that the final concentration of fatty acids in steps 1-3 is 0.08 mM.

[0045] Example 5: A transdermal complex of albumin encapsulating collagen, which differs from Example 1 in that the final concentration of fatty acids in steps 1-3 is 0.12 mM.

[0046] Example 6: A transdermal complex of albumin encapsulating collagen, which differs from Example 1 in that the concentration of the reducing agent added in steps 1-4 is 0.5 mM.

[0047] Example 7: A transdermal complex of albumin encapsulating collagen, which differs from Example 1 in that the concentration of the reducing agent added in steps 1-4 is 1.0 mM.

[0048] Example 8: A transdermal complex of albumin encapsulating collagen, which differs from Example 1 in that the enzyme-substrate ratio is 1:300 (w / w) when adding the hydrolytic enzyme solution in step 2-2.

[0049] Example 9: A transdermal complex of albumin encapsulating collagen, which differs from Example 1 in that the enzymatic hydrolysis in step 2-2 is performed under the following conditions: temperature 35°C, pH 7.5, and hydrolysis time 20 min.

[0050] Example 10: A transdermal complex of albumin encapsulating collagen, which differs from Example 1 in that: in step 3-3, the prepolymer solution is heated at a rate of 0.8℃ / min and heated to 32℃ and maintained for 30 min.

[0051] Example 11: A transdermal complex of albumin encapsulating collagen, which differs from Example 1 in that: in steps 3-4, the concentration of oxidized glutathione in the redox pair is 0.4 mM, and the final concentration of reduced glutathione is 0.4 mM.

[0052] Comparative Example Comparative Example 1: A type of collagen, specifically YueLiXin® Mic32 small molecule recombinant collagen (SSR), with an average molecular weight of 2000 Da, purchased from Zhejiang Zhuji Juyuan Biotechnology Co., Ltd.

[0053] Comparative Example 2: A collagen complex, which differs from Example 1 in that serum protein powder is not pretreated during the preparation process.

[0054] Comparative Example 3: A collagen complex, which differs from Example 1 in that, in the preparation process, the enzyme-substrate ratio in step 2-1 is 1:350 (w / w).

[0055] Comparative Example 4: A collagen complex, which differs from Comparative Example 3 in that, in step 2-2, the enzyme inhibitor is an equal amount of soybean trypsin inhibitor.

[0056] Comparative Example 5: A collagen complex, which differs from Comparative Example 4 in that the parameters in step 2-2 of the enzymatic hydrolysis reaction are: temperature 37℃, pH 7.6, and stirring speed 110 r / min.

[0057] Comparative Example 6: A collagen complex, which differs from Comparative Example 3 in that, in steps 3-4, the final concentration of oxidized glutathione in the redox pair is 0.1 mM, and the final concentration of reduced glutathione is 0.5 mM.

[0058] test: Experiment 1: Verification experiment of enzyme digestion products after step 2. The experimental sample 1 obtained after step 2 was validated using SDS-PAGE. The separating gel concentration was 12%, and Coomassie Brilliant Blue staining was performed after electrophoresis. The SDS-PAGE electrophoresis image of experimental sample 1 is shown below. Figure 1 As shown, the first lane contains protein molecular weight standards; the second lane is the control group, i.e., undigested albumin with a size of 63 kDa; the third lane is the experimental group, i.e., sample 1, with a target band molecular weight of 30-50 kDa (corresponding to a class III domain fragment), and no obvious 66.5 kDa protein band. This result indicates that the operating steps described in this application ensure complete enzyme digestion, facilitating subsequent operations.

[0059] Experiment 2: pH gradient driven verification after step 5 Simulating skin pH environment: Prepare PBS buffer solutions with pH=5.5 (stratum corneum) and pH=7.4 (deep epidermis); Using Malvern Zetasizer Nano ZS90, in phase analysis light scattering (PALS) mode, with an electric field strength of 15 V / cm, measure 10 times consecutively at each pH point and take the average value to detect the Zeta potential of test sample 1 at the two pH values. The results are detailed in Table 1.

[0060]

[0061] As shown in the table above, the absolute value of the potential decreases at pH=5.5; when pH=7.4, the potential recovers to -25 to -35 mV, verifying the electrostatic attraction mechanism. The reason for these results may be that in the albumin-encapsulated collagen transdermal complex modified with hyaluronic acid and gold, gold can generate significant charge synergy through hyaluronic acid bridging. The albumin-encapsulated collagen transdermal complex generates electrostatic attraction due to the enhanced negative charge, thereby improving the overall penetration and absorption effect of the modified albumin-encapsulated collagen transdermal complex.

[0062] Experiment 3: Raman penetration test Based on the experimental method of T / SHRH 064-2024 "Transdermal Permeation Test of Cosmetic Ingredients - In Vivo Raman Spectroscopy", the permeation of untreated collagen (control sample 1) and the transdermal complex of albumin-encapsulated collagen prepared by the preparation method in this application (test sample 1) were compared.

[0063] When applied to the skin, Raman imaging depth analysis is used to obtain the distribution of the sample at different depths in human skin, such as... Figure 2 and Figure 3As shown, the transdermal complex containing albumin-encapsulated collagen (sample 1) can significantly increase the transdermal absorption of collagen. This permeation-enhancing advantage becomes more pronounced over time. Compared to the control sample 1, the relative permeability was 5.08% and 11.12% after 12 hours of use. Therefore, the permeation absorption efficiency of sample 1 is more than twice that of control sample 1.

[0064] Experiment 4: Efficacy Test Stratum corneum moisture content test: 121 participants aged 20-35 years with healthy skin were selected and randomly divided into 11 groups of 11 participants each. These 11 groups used a blank control sample, experimental samples 1-5, and control samples 1-5, respectively. Each participant performed a simple facial cleansing before using any sample. 0.5g of the sample was applied evenly to the face once in the morning and once in the evening. The trial lasted for 7 days.

[0065] Before and 7 days after using the corresponding samples, the stratum corneum moisture content of the subjects was measured using the German CK Corneometer® CM825 skin analyzer. The data were recorded, and the changes in stratum corneum moisture content were calculated, recorded, and analyzed. Detailed experimental data are shown in Table 2.

[0066] ATP content detection: ATP biofluorescence detection kit method a) Cell seeding: According to 1×10 4 Human keratinocytes (HaCat) were seeded into 96-well plates at a seeding density of cells / well and incubated overnight in an incubator (37°C, 5% CO2).

[0067] b) Cell inoculation and drug administration: After culturing in an incubator (37℃, 5% CO2) for 24 hours, test sample 1 and control sample 1 (untreated collagen) were added.

[0068] c) Sample collection After culturing the cells in an incubator (37℃, 5% CO2) for 24 hours, remove the culture medium, add 20 μL of lysis buffer to each well, lyse the cells, and centrifuge at 12000g for 5 minutes at 4℃. Collect the supernatant for later use.

[0069] d) Fluorescence detection: According to the instructions of the ATP assay kit, fluorescence detection was performed on the supernatant, and the ATP concentration was quantified by fluorescence intensity. The ATP concentration increase rate was calculated by comparing with the blank control sample, and the results were recorded and analyzed. The results are detailed in Table 2.

[0070] ROS clearance rate test According to T / SHRH032-2020 Cosmetic Firming and Anti-wrinkle Efficacy Test - In Vitro Keratinocyte Reactive Oxygen Species (ROS) Inhibition Test Method, the ROS scavenging rate of test samples 1-11 and control samples 1-6 was detected, and the data were recorded and analyzed.

[0071] IL-6 inhibition rate test The inhibition rate of the inflammatory factor IL-6 was detected in test samples 1-11 and control samples 1-6 according to the method of the ELISA (enzyme-linked immunosorbent assay) kit. The specific steps are as follows: a) Establish a keratinocyte (Hacat) inflammation model and induce stimulation using lipopolysaccharide (LPS): b) Add the test samples to the inflammation model respectively; c) Using a relevant enzyme-linked immunosorbent assay (ELISA) kit, the expression concentration of the inflammatory cytokine IL-6 was measured, and the inhibition rate was calculated according to the following formula: Record and analyze the data; the results are detailed in Table 2.

[0072] Type I collagen content detection test According to T / SHRH 031-2020 Cosmetic Firming and Anti-wrinkle Efficacy Test - In Vitro Fibroblast Type I Collagen Content Determination, the collagen content of test samples 1-11 and control samples 1-6 was detected, the data were recorded and analyzed, and the results are detailed in Table 2.

[0073]

[0074] As shown in Table 2, the above experimental results directly indicate that the active ingredients in test samples 1-11 were fully absorbed by the skin and demonstrated good experimental effects.

[0075] Based on the above experimental results, it was found that test samples 1-11 containing 1% of the complex sample showed superior effects compared to control samples 1-6 in many aspects, such as increasing ATP content, inhibiting inflammatory factors, expressing collagen, and repairing the skin barrier.

[0076] The increase in stratum corneum moisture content of test sample 1-11 was greater than that of control sample 1-6, indicating that test sample 1-11 had better moisturizing and water-locking effects.

[0077] The ATP content of test sample 1-11 increased more than that of control sample 1-6, indicating that test sample 1-11 has better effects in enhancing cell energy, anti-dullness, and promoting skin repair.

[0078] The ROS scavenging rate of test sample 1-11 was higher than that of control sample 1-6, indicating that test sample 1-11 had better effects in anti-oxidation, free radical scavenging and anti-photoaging.

[0079] The IL-6 inhibition rate of test sample 1-11 was greater than that of control sample 1-6, indicating that test sample 1-11 was more effective in anti-inflammatory soothing and improving redness and sensitivity.

[0080] The increase rate of type I collagen content in test samples 1-11 was greater than that in control samples 1-6, indicating that test samples 1-11 were more effective in anti-wrinkle firming, promoting collagen regeneration, and improving skin elasticity.

[0081] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A method of preparing a transdermal complex of albumin encapsulated collagen, characterized by, Includes the following steps: Step 1, Pretreatment of serum albumin powder: Step 1-1: Mix serum albumin powder with 0.01M PBS buffer at 4°C until the volume is adjusted to 100mL to obtain the first mixture. The pH of the first mixture is maintained at 7.1-7.

3. Steps 1-2: Add a conformation stabilizer to the first mixture and treat it at 4°C for 30 min to obtain the second mixture; Steps 1-3: Add fatty acids to the second mixture at a final concentration of 0.08-0.12 mM, and treat at 25-30℃ for 20 min to obtain the third mixture; the mass molar ratio of the fatty acids to serum albumin powder is 1:

1. Steps 1-4: Add a reducing agent with a concentration of 0.5-1.0 mM to the third mixture and treat it at 25-30℃ for 15 min to obtain the pretreated serum albumin solution; Step 2, flexible enzyme digestion: Step 2-1: Thoroughly mix the hydrolytic enzyme and PBS buffer with a pH of 7.0-7.5 to prepare a hydrolytic enzyme solution with a mass concentration of 1 mg / mL; Step 2-2: Place the hydrolytic enzyme solution on an ice bath and slowly add it dropwise to the pretreated serum albumin solution at an enzyme-substrate ratio of 1:100-1:300 (w / w), mixing gently. Then, perform the enzymatic hydrolysis reaction for 20-40 min at 25-35℃, pH 7.0-7.5, and stirring speed of 100 r / min. Add the enzyme inhibitor and incubate at 25℃-30℃ for 10 min to gently terminate the reaction. Subsequently, centrifuge at 4℃ and centrifugation force of 8000×g for 10 min and collect the enzyme digestion supernatant. Step 3: Hydrophobic-driven self-assembly to form peptide nanoparticles: Step 3-1: The enzyme digestion supernatant is ultrafiltered and centrifuged to concentrate it to a mass concentration of 5-10 mg / mL to obtain concentrated enzyme digestion supernatant; Step 3-2: Let the concentrated enzyme digestion supernatant stand at 25°C for 2-4 hours, and check the turbidity at 600 nm every 30 minutes until the turbidity reaches 0.15±0.02 to obtain the prepolymer solution; Step 3-3: Heat the prepolymer solution to 28-32℃ and maintain it for 30-45 min to obtain a hydrophobic reaction solution; Steps 3-4: The redox pair was thoroughly mixed with 0.01M PBS buffer and added to the hydrophobic reaction solution. The mixture was incubated at 4°C for 4-6 hours and then dialyzed overnight in PBS buffer using a 3.5 kDa MWCO dialysis bag at 4°C to obtain polypeptide nanoparticles with a particle size of 80-120 nm. Step 4, Targeted encapsulation and degradation site shielding of collagen: Step 4-1: Under the temperature conditions of 30℃±2℃, hydrolyzed collagen and polypeptide nanoparticles are incubated at a mass ratio of 1:(5-6) for 58-60 min to obtain albumin nanoparticle system; Step 4-2: Thoroughly mix the transglutaminase solution with the albumin nanoparticle system, and carry out the cross-linking reaction for 2-3 h at a pH of 6.0-6.5 and a temperature of 22-28℃, accompanied by stirring at a speed of 55-60 r / min. The reaction was then terminated by refrigerating at 4°C for 30 minutes, and unbound transglutaminase was removed by dialysis to obtain a nanoparticle system encapsulating collagen. Step 5, Charge Modification and Nanofabrication: Hyaluronic acid solution was added dropwise to the nanoparticle system encapsulating collagen, and incubated at 25℃±2℃ for 25-30 min with stirring at 50 r / min to obtain the charge-modified albumin system. Take 1 mg / mL of gold nanoparticle dispersion and slowly add it dropwise to the charge-modified albumin system at a final concentration of 0.01% (w / v). Stir while adding the mixture at 50 r / min and incubate at 25℃ for 20 min to obtain a transdermal complex of albumin encapsulating collagen.

2. The method of claim 1, wherein the transdermal collagen-albumin complex is prepared by the steps of: The serum albumin powder in step 1-1 has a purity ≥99%, endotoxin <0.125 (EU / mg), is a white to pale yellow powder, has a pH of 6.0-8.0, and an α-helix content ≥60%.

3. The method of claim 1, wherein the transdermal collagen-albumin complex is prepared by the steps of: The conformation stabilizer in steps 1-2 consists of glycerol and sugars, with the final concentration of glycerol being 5-8% (v / v) and the final concentration of sugars being 5-8% (w / v). The fatty acid in steps 1-3 is at least one of palmitic acid and myristic acid; The reducing agent in steps 1-4 is reduced glutathione.

4. The method of claim 1, wherein the transdermal collagen-albumin complex is prepared by the steps of: The hydrolytic enzyme in step 2-1 is porcine pancreatic elastase with an enzyme activity of 250-350 U / mg; the enzyme inhibitor in step 2-2 consists of soybean trypsin inhibitor and elastase, wherein the ratio of soybean trypsin inhibitor to elastase is 2:1 (w / w).

5. The method for preparing the albumin-encapsulated collagen transdermal complex according to claim 1, characterized in that, In step 3-1, the enzyme digestion supernatant is added to a 30 kDa MWCO centrifugal ultrafiltration tube and centrifuged at 5000×g for 15-20 min at 4°C to concentrate to a mass concentration of 5-10 mg / mL; then it is washed 2-3 times with 0.01 M PBS buffer with a pH of 7.2 to remove impurities and reagent residues with a molecular weight less than 3 kD. In step 3-3, the temperature is increased at a rate of 0.3-0.8℃ / min.

6. The method for preparing the albumin-encapsulated collagen transdermal complex according to claim 1, characterized in that, In steps 3-4, the redox pair consists of oxidized glutathione and reduced glutathione, wherein the final concentration of oxidized glutathione is 0.2-0.4 mM and the final concentration of reduced glutathione is 0.2-0.4 mM.

7. The method for preparing the albumin-encapsulated collagen transdermal complex according to claim 1, characterized in that, In step 4-1, mass transfer is promoted by vortexing at 50-100 rpm for the first 20 minutes of incubation, and the remaining time is allowed to stand; the hydrolyzed collagen is obtained by MMP-8 enzymatic hydrolysis of porcine collagen (E / S=1:50 w / w), with a molecular size of 1,500-2,500 Da. The transglutaminase solution in step 4-2 is prepared by transglutaminase and 0.01M PBS buffer, with a mass ratio of transglutaminase to albumin nanoparticles of 1:

50.

8. The method for preparing the albumin-encapsulated collagen transdermal complex according to claim 1, characterized in that, In step 5-1, the hyaluronic acid solution is prepared by thoroughly mixing hyaluronic acid with 0.01M PBS buffer, and the mass concentration of the hyaluronic acid solution is 10 mg / mL; the molecular weight of the hyaluronic acid in the hyaluronic acid solution is 50-100 kDa, and the mass ratio of hyaluronic acid to albumin nanoparticles is 1:

18.

9. A transdermal complex of albumin-encapsulated collagen, prepared by the method described in any one of claims 1-8.

10. The use of a transdermal complex of albumin-encapsulated collagen as described in claim 9 in cosmetics.

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