A method for constructing a d-bl biomimetic biofilm

Abstract

The application discloses a kind of D-BL biomimetic membrane construction method, using computer simulation required limited composition environment under receptor (efficacy component) and ligand (modification component) most likely binding mode, calculate binding free energy, and through molecular dynamics method simulation receptor and ligand binding process, finally through the way of computational chemistry screening efficacy component and modification component in the best use ratio of cosmetic formula, preparation into D-BL biomimetic membrane.The D-BL biomimetic membrane has biological safety, targeting, with cell affinity, slow release, enzyme resistance, promote transdermal absorption and other excellent properties, can the efficacy of efficacy component, such as anti-wrinkle, moisturizing, soothing, firming and other effects have targeted application in cosmetic products, achieve efficacy skin care, and solve the instability of peptide, collagen and other macromolecules, low bioavailability, easy enzymolysis, poor permeability, low transmission efficiency and targeting skin care problems.

Description

Technical Field

[0001] This invention belongs to the field of cosmetic technology, specifically relating to the development of D-BL biomimetic membrane technology and its application in cosmetics. It can solve problems such as the instability, low bioavailability, easy enzymatic hydrolysis, poor permeability, and low delivery efficiency and targeting of macromolecules such as peptides and collagen. Background Technology

[0002] Anti-aging ingredients, such as peptides and collagen, share the characteristic of large molecular weight. Some exhibit poor transdermal penetration and retention, forming bypass transdermal pathways that allow them to remain only on the skin surface. A study investigating in vitro skin penetration found that most peptides remained on the skin surface and were subsequently washed away; only 0.22% of the total amount penetrated the skin and remained within the stratum corneum (the outermost layer of skin); 0.01% entered the epidermis. This low skin permeability leads to significant peptide waste, resulting in unnecessarily high production costs and suboptimal products, making skin delivery highly challenging.

[0003] Hexapeptide, also known as acetyl hexapeptide-8 or argirelin, is a neurotransmitter inhibitory anti-wrinkle peptide widely used in anti-wrinkle products in the cosmetics industry. As a water-soluble short peptide, it also has certain drawbacks: 1) It is unstable and inactivated by various complex chemical degradations and physical changes in the in vivo and in vitro environments; 2) It has low bioavailability via non-injection routes; 3) Its poor permeability and small partition coefficient make it difficult to cross biological barriers and lipid membranes, resulting in low delivery efficiency and targeting.

[0004] To address these issues, researchers have found that when applying peptides to cosmetics, methods such as chemical modification, the addition of absorption enhancers, and the application of novel carrier technologies can improve the skin's permeability and resistance to enzymatic degradation. Numerous patents and literature reports have addressed these problems by encapsulating active ingredients in liposomes. This is due to the low toxicity, cell affinity, long-lasting effect, and sustained-release properties of liposomes. However, liposomes obtained using existing methods still suffer from low stability, low encapsulation efficiency, easy leakage of components, bacterial growth and sedimentation during storage, difficulty in controlling particle size, and a wide particle size distribution.

[0005] Therefore, it is particularly important to develop a technology that addresses the problems of unstable properties, low bioavailability, poor permeability, and low delivery efficiency and targeting of macromolecules such as peptides and collagen, as well as the problems of low stability, low encapsulation rate, easy leakage of components, bacterial growth and sedimentation during storage, difficulty in controlling particle size and wide particle size distribution of liposomes. Summary of the Invention

[0006] The purpose of this invention is to provide a method for constructing D-BL biomimetic membranes with properties such as biosafety, stability, sustained release, resistance to enzymatic hydrolysis, promotion of transdermal absorption, and targeting based on digitally controlled artificial biomembrane technology (D-BL biomimetic membrane technology).

[0007] To achieve the above objectives, the method for constructing the D-BL biomimetic membrane provided by the present invention includes the following steps:

[0008] Step 1: Computer Model Construction

[0009] The three-dimensional molecular structure of receptor R was downloaded from the PubChem database. Homologous protein structures of ligand L were searched from the Protein Data Bank (PDB) to find the model backbone of the target docking compound and construct the structural variable region of the target docking compound.

[0010] Step 2: Molecular docking

[0011] Ligand L is placed in the active pocket of receptor R. The interaction between ligand L and receptor R is evaluated according to the complementary matching principles of geometry, electrostatic interaction, hydrogen bonding, and hydrophobic interaction. The optimal binding mode between the two molecules, ligand L and receptor R, is then determined to obtain the initial structural composition. The initial structural composition consists of receptor R, ligand L, and target docking agent in different mass ratios.

[0012] Step 3: Molecular Dynamics Simulation

[0013] The obtained initial structural compositions were subjected to energy minimization and molecular dynamics simulations using Amber software.

[0014] Step 4: Calculate using free energy

[0015] Using the MMPBSA.py module in Amber software, the MM-PBSA method was employed to calculate the system binding free energy of the initial structural composition, obtaining the composition with the lowest energy and greatest stability. The system binding free energy of the composition is expressed by the following formula:

[0016] △G bind =△HT△S=△E MM +△G sol -T△S

[0017] △E MM =△E ele +△E vdw

[0018] △G sol =△G GB+△G surf

[0019] In the formula, △G bind The system binding free energy of the composition is represented by ΔH; ΔE represents the enthalpy change of the composition; MM Represents the molecular mechanical energy in the gas phase; ΔE ele and △E vdw Represent the electrostatic interaction and van der Waals interaction energies of ligand and acceptor formation in the gas phase, respectively; ΔG sol This represents the contribution of the dissolution free energy to ligand-receptor binding; ΔG GB and △G surf Let ΔG and ∠G represent the contributions of polar and nonpolar dissolution free energies to ligand-acceptor binding, respectively. The former is obtained using the finite difference method to solve the molecular mechanics Poisson-Boltzmann equation, while the latter is solved using the following empirical equation: ΔG surf =γSASA + β, where SASA represents the solvent-accessible surface area, and the values ​​of γ and β are set to 0.00542 kcal / (mol). . Å 2 ) and 0.92 kcal / mol; T△S represents the contribution of the entropy change caused by the change in the degrees of freedom of ligand binding to the receptor to the binding free energy, which can be ignored.

[0020] Step 5: Preparation of D-BL biomimetic membrane

[0021] D-BL biomimetic membranes were prepared based on the formulation ratio of the lowest energy and most stable composition, and their performance was verified.

[0022] In step 1 above, the receptor R is a polypeptide, which can be a general oligopeptide (oligopeptide-1, oligopeptide-3, oligopeptide-2, tripeptide-1, hexapeptide-1, oligopeptide-5, hexapeptide-9, oligopeptide-6, pentapeptide-1, hexapeptide-11, pentapeptide-3, hexapeptide-3, etc.), acetyl oligopeptides (acetyl hexapeptide-8, acetyl tetrapeptide-5, acetyl hexapeptide-1, acetyl tetrapeptide-2, acetyl octapeptide-3, acetyl tetrapeptide-9, etc.), palmitoyl oligopeptides (palmitoyl pentapeptide-4, palmitoyl tetrapeptide-7, palmitoyl tripeptide-5, palmitoyl tripeptide-1, palmitoyl oligopeptide, palmitoyl hexapeptide-12, palmitoyl pentapeptide-5, etc.), and other peptides (carnosine, glutathione, etc.).

[0023] In step 1 above, the ligand L is a protein, which may be collagen, elastin, milk protein, deer bone collagen, wild soybean protein, whey protein, serum albumin, keratin, wheat protein, fibronectin, etc.

[0024] In step 1 above, the target docking agent is a bilayer phospholipid composed of lecithin and cholesterol.

[0025] In step 2 above, the GOLD docking procedure is preferably used. A genetic algorithm is employed to search for the binding mode of ligand L in the active site of receptor R. During the docking process, the ligand L is allowed to have a full range of flexible conformations and local flexibility. Based on the GOLD prediction of the binding mode of the composition, the initial structural composition is obtained.

[0026] In step 3 above, the preferred method for minimizing energy is the conjugate gradient method, which eliminates unreasonable contact between atoms in the system. During the slow heating process, the solute molecules in the simulated system are constrained to maintain their conformation. Under constant temperature and pressure conditions, the simulated system reaches thermodynamic equilibrium, and parameters such as conformational changes, temperature, and energy are monitored.

[0027] In step 5 above, the performance includes safety, permeability, resistance to enzymatic hydrolysis, sustained-release properties, and efficacy.

[0028] In step 5 above, the D-BL biomimetic membrane exhibits the characteristics of a small nucleus and a large body, with the core size between 10 and 100 nm and the body size between 100 and 1000 nm.

[0029] This invention utilizes computer simulation technology, leveraging computer algorithms and accumulated bioinformatics data, to simulate the most likely binding modes of receptor R (active ingredient) and ligand L (modifying ingredient) under specific ingredient conditions. (The binding between receptor R and ligand L involves electrostatic interactions, hydrogen bonding, van der Waals interactions, and hydrophobic interactions, and the interaction mode must follow complementary matching principles such as geometry, electrostatic interactions (positive charge to negative charge), hydrogen bonding interactions (hydrogen bond donor to hydrogen bond acceptor), and hydrophobic interactions (hydrophobic region to hydrophobic region).) The binding free energy is calculated, and the receptor-ligand binding process is simulated using molecular dynamics methods. Finally, computational chemistry is used to screen the optimal usage ratio of active ingredients and modifying ingredients in cosmetic formulations, optimizing their proportions to prepare a D-BL biomimetic membrane. The safety, permeability, enzymatic resistance, sustained-release properties, and efficacy of this D-BL biomimetic membrane are then verified using laboratory methods.

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0031] 1. The computer simulation process used in this invention has the advantages of high efficiency, speed and low cost. It is an important auxiliary method for high-throughput screening. It can optimize the best binding mode of receptor R and ligand L, saving a lot of time that would otherwise be required to optimize the ratio of active ingredients through orthogonal experimental methods. It can be used for later interpretation, prediction and guidance of cosmetic formulation design.

[0032] 2. The D-BL biomimetic membrane constructed using the method of this invention has a variety of excellent properties such as safety, targeting, affinity with cells, sustained release, resistance to enzymatic hydrolysis, and promotion of transdermal absorption. It is also a carrier of a class of functional ingredients, which can target modified anti-wrinkle, moisturizing, soothing, and firming functional ingredients to the skin, thereby achieving precise digital skin care and solving the problem of peptide skin care.

[0033] 3. The D-BL biomimetic membrane constructed using the method of this invention is similar to the skin cell membrane, both being bilayer phospholipid structures. They have the characteristic of being like-soluble. The phospholipid bilayer is the basic framework of the cell membrane, so it can play the role of being like-soluble and enhance transdermal absorption.

[0034] 4. The D-BL biomimetic membrane constructed using the method of this invention possesses strong hydration capabilities, allowing it to directly penetrate the skin's brick-and-mortar structure, delivering active ingredients to the skin surface or into the epidermis and dermis, where they accumulate and exert their effects. By regulating different receptor and ligand components, different components can enhance "like dissolves like" and strengthen hydration, and can also act as carriers for active ingredients, thereby achieving a synergistic effect of multiple functions. The derivative significance of this invention lies in the fact that D-BL biomimetic membrane technology can not only be used for the aforementioned polypeptide macromolecules, but can also be derived for other similar peptides, proteins, and other macromolecules, maximizing their effectiveness. Attached Figure Description

[0035] Figure 1 This is the curve of RMSD of DBL-8 over time (molecular dynamics simulation).

[0036] Figure 2 This is a diagram from the DBL-8 chicken embryo chorioallantoic membrane test.

[0037] Figure 3 It represents the cumulative transdermal absorption of DBL-8, LIP-8, and NLIP-8 at different time points.

[0038] Figure 4 This is a graph showing the changes in fluorescence intensity at different time points in the skin permeability test for DBL-8, LIP-8, and NLIP-8.

[0039] Figure 5 These are the enzymatic hydrolysis rate test results for DBL-8, LIP-8, and NLIP-8 at different time points.

[0040] Figure 6 These are the cumulative release rates of acetyl hexapeptide-8 at different time points for DBL-8, LIP-8, and NLIP-8. Detailed Implementation

[0041] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited to these embodiments.

[0042] Example 1

[0043] The receptors R used in this embodiment are acetyl hexapeptide-8, tripeptide-1, and palmitoyl pentapeptide-5, and the ligand L is human recombinant collagen. After treatment with D-BL biomimetic membrane technology, the composition with the lowest energy and the most stable composition was screened out. Finally, this composition was compared with liposomes and non-liposomes to demonstrate their differences in safety, permeability, resistance to enzymatic hydrolysis, sustained-release performance, and efficacy. The specific method is as follows:

[0044] Step 1: Computer Model Construction

[0045] The three-dimensional molecular structure of receptor R was downloaded from the PubChem database, and the homologous protein structure of ligand L was searched from the protein crystal database. The target docking agents were limited to lecithin and cholesterol. A model framework for the target docking agents was found, and the structural variable region of the target docking agents was constructed.

[0046] Step 2: Molecular docking

[0047] Using the GOLD docking procedure, ligand L was placed in the active pocket of acceptor R. Following the principles of complementary matching based on geometry, electrostatic interactions, hydrogen bonding, and hydrophobic interactions, a genetic algorithm was employed to search for binding modes of ligand L within the active site of acceptor R. During docking, the ligand L was allowed to exhibit flexible conformations across its entire range and local flexibility. The binding modes of the compositions were predicted based on GOLD. AutoDock-vina software was used to score all compositions with different binding modes. From a theoretical calculation perspective, the initial structural compositions with higher scores were selected, as shown in Table 1. These initial structural compositions consisted of acceptor R, ligand L, and the target docking agent in different mass ratios.

[0048] Table 1 Scoring results of the initial structural composition

[0049]

[0050] Step 3: Molecular Dynamics Simulation

[0051] The obtained initial structural composition was subjected to energy minimization and molecular dynamics simulations using Amber software. Energy minimization employed the conjugate gradient method to eliminate unreasonable atomic contacts in the system. During slow heating, the solute molecules in the simulated system were constrained to maintain their conformation. Under isothermal and pressure conditions, the simulated system reached thermodynamic equilibrium, and parameters such as conformational changes, temperature, and energy were monitored. Specifically, the composition was placed in water for 50 ns of molecular dynamics simulation, with the conformation during the heating phase used as the reference structure. The root mean square deviation (RMSD) was used to evaluate the stability of the composition. The molecular dynamics simulation results of the composition are shown in Table 2.

[0052] Table 2. Molecular dynamics simulation results of the composition

[0053]

[0054] The molecular dynamics simulation results in Table 2 show that compositions 1, 6, and 12 converged to equilibrium at 30, 32, and 33 ns, respectively, with RMSDs fluctuating around 5.13 Å, 5.47 Å, and 5.61 Å. The RMSDs for different receptors in compositions 1, 6, and 12 were relatively small. Generally, a smaller RMSD value indicates a more stable structure. Figure 1 The RMSD curve of composition 1 (DBL-8) over time also shows that composition 1 gradually stabilizes around 30 ns. Therefore, the molecular dynamics simulation results indicate that the structures of compositions 1, 6, and 12 are relatively stable. Step 4: Calculate the free energy.

[0055] Using the MMPBSA.py module in Amber software, the MM-PBSA method was employed to calculate the system binding free energy of the initial structural composition, obtaining the composition with the lowest energy and greatest stability; the system binding free energy of the composition is expressed by the following formula:

[0056] △G bind =△HT△S=△E MM +△G sol -T△S

[0057] △E MM =△E ele +△E vdw

[0058] △G sol =△G GB +△G surf

[0059] In the formula, △G bind The system binding free energy of the composition is represented by ΔH; ΔE represents the enthalpy change of the composition;MM Represents the molecular mechanical energy in the gas phase; ΔE ele and △E vdw Represent the electrostatic interaction and van der Waals interaction energies of ligand and acceptor formation in the gas phase, respectively; ΔG sol This represents the contribution of the dissolution free energy to ligand-receptor binding; ΔG GB and △G surf Let ΔG and ∠G represent the contributions of polar and nonpolar dissolution free energies to ligand-acceptor binding, respectively. The former is obtained using the finite difference method to solve the molecular mechanics Poisson-Boltzmann equation, while the latter is solved using the following empirical equation: ΔG surf =γSASA + β, where SASA represents the solvent-accessible surface area, and the values ​​of γ and β are set to 0.00542 kcal / (mol). . Å 2 The values ​​are 0.92 kcal / mol; TΔS represents the contribution of the entropy change resulting from the change in the degrees of freedom of ligand binding to the acceptor to the binding free energy, which can be ignored. The calculation results are shown in Table 3.

[0060] Table 3. Calculation results of the system binding free energy of the composition.

[0061]

[0062] As can be seen from the calculation results of the system binding free energy in Table 3, when the ligand L is fixed and different acceptors R are changed, the system binding free energy of composition 1 is the lowest, indicating that the structure of this composition is the most stable.

[0063] Step 5: Preparation of D-BL biomimetic membrane

[0064] Based on the formula (mass ratio) of the lowest energy and most stable composition 1 (DBL-8): lecithin:cholesterol:acetyl hexapeptide-8:human recombinant collagen = 1:0.25:0.75:1, D-BL biomimetic membranes were prepared. The specific preparation method was as follows: lecithin and cholesterol were dissolved in chloroform and added to a flask. The mixture was sonicated in a water bath for 1 min. Then, the flask was placed in a 35°C water bath and evaporated at 140 rpm to form a membrane. After membrane formation, the flask was placed in an ice bath, and ether was added to dissolve the membrane. Then, acetyl hexapeptide-8 and human recombinant collagen, which were completely dissolved in deionized water, were added. The mixture was sonicated using a probe with a power of 40%, with a 3-second sonication followed by a 3-second pause, for 1 min. The mixture was then placed in an ice bath and evaporated at 140 rpm to form a membrane, thus obtaining the DBL-8 biomimetic membrane.

[0065] Using the aforementioned DBL-8 biomimetic membrane (recombinant collagen-modified acetyl hexapeptide-8), LIP-8 (representing acetyl hexapeptide-8 liposomes), and NLIP-8 (representing acetyl hexapeptide-8) as test substances, their performance in terms of safety, permeability, resistance to enzymatic hydrolysis, sustained-release properties, and efficacy was compared. Specific experiments and results are as follows:

[0066] 1. Safety test evaluation

[0067] (1) Chicken embryo chorioallantoic membrane test

[0068] Prepare six fertilized chicken embryos of White Laihang chickens per group, each embryo weighing 50-60g. On day 8 of incubation, examine the eggs using an egg candler, discarding unfertilized and inactive embryos, and selecting those with well-developed blood vessels. Mark the air cell location on the eggshell surface. Weigh 0.3mL of the test substance and apply it to the chorioallantoic membrane (CAM). Use 0.9% sodium chloride solution as a negative control and 0.1mol / L sodium hydroxide solution as a positive control. Begin the experiment on day 9. Carefully peel off the air cell portion of the eggshell using dental tweezers. Apply physiological saline to the surface of the eggshell membrane to thoroughly moisten it, then pour it out. Carefully remove the eggshell membrane with tweezers, ensuring the exposed chorioallantoic membrane remains intact without any damage. Apply 0.3g of the test substance directly to the CAM. Spread it as widely as possible, ensuring coverage of at least 50% of the surface. After 3 minutes of application, gently rinse the CAM surface with double-distilled water, completing the rinsing within 30 seconds. Pour out the liquid and immediately observe the degree of change in each toxic effect under a stereomicroscope, assigning an ES score. Photographs were taken immediately after the 3-minute exposure period. Irritation classification was determined according to the scoring method in SN / T2329-2009 "Eye Irritation / Corrosiveness Test of Chicken Embryo Viral Allantoic Membrane in Cosmetics". The experimental results are shown in Table 4.

[0069] Table 4 Experimental Results

[0070]

[0071] As shown in Table 4, at a test concentration of 1%, the ES scores of DBL-8, LIP-8, and NLIP-8 were 0, 8, and 10, respectively. Compared with the negative and positive controls, they showed no irritation or only slight irritation. Figure 2 As can be seen from the DBL-8 chicken embryo chorioallantoic membrane test, no bleeding, coagulation, or angiolysis was observed in DBL-8. Therefore, this experiment demonstrates that the DBL-8 biomimetic membrane constructed using the method of this invention has a certain degree of safety, providing a guarantee for its safe use in humans.

[0072] (2) Human skin patch test

[0073] Thirty volunteers were selected for a closed patch test, using patches with an area not exceeding 50 mm².2 A qualified patch test apparatus with a depth of approximately 1 mm is used. The test substance is placed in the small chamber of the patch test apparatus at a volume of 0.025 mL. The control well is a blank control (without any substance). The test substance is a diluted sample. The patch test apparatus containing the test substance is applied to the back or flexor side of the forearm of the subject using hypoallergenic adhesive tape. Gently press with the palm of the hand to ensure even application to the skin, and leave for 24 hours. Skin reactions are observed and recorded at 30 minutes (after the indentation disappears), 24 hours, and 48 hours after removing the patch test apparatus, according to the grading standards for skin reactions in occlusive patch tests according to Chapter 7, Section 2, Closure Patch Tests (6.1) of the Cosmetic Technical Specifications (2015 Edition).

[0074] Table 5 Results of patch test

[0075]

[0076] The results of the human skin patch test in Table 5 show that, at a test concentration of 1%, 30 out of 30 people experienced Grade 0 adverse reactions, 0 experienced Grade 1 adverse reactions, 0 experienced Grade 2 adverse reactions, 0 experienced Grade 3 adverse reactions, and 0 experienced Grade 4 adverse reactions. These results indicate that DBL-8, LIP-8, and NLIP-8 are safe for use in humans.

[0077] Therefore, combining the above-mentioned chicken embryo chorioallantoic membrane test and human skin patch test, it is confirmed that acetyl hexapeptide-8 treated by different methods has a certain degree of safety and can be safely used in cosmetic products.

[0078] 2. Skin permeability evaluation

[0079] Transdermal absorption in cosmetics refers to the process by which functional ingredients in cosmetics, according to the product's effectiveness, act on the skin surface or penetrate the epidermis and dermis, accumulating and exerting their effects in that area. The transdermal absorption of cosmetic active ingredients does not require crossing the skin to enter the systemic circulation, which is a key difference between it and drugs. Therefore, the following transdermal assay was used to demonstrate the transdermal absorption of acetyl hexapeptide-8 treated using different methods.

[0080] (1) Transdermal absorption test

[0081] This experiment used piglet skin as a skin model. Before the experiment, the piglet skin was washed with physiological saline and set aside. The treated skin was placed in the center of the diffusion chamber, with the stratum corneum facing the supply chamber. Physiological saline was added to the receiving chamber. The water bath temperature in the diffusion chamber was maintained at (37±0.5)℃, and equilibration was carried out for approximately 30 minutes. Solutions were collected at different time points for analysis. The concentration of acetyl hexapeptide-8 was determined using HPLC, and the cumulative transdermal absorption was calculated.

[0082] Depend on Figure 3The results of the cumulative transdermal absorption determination showed that, under the same active ingredient conditions, the cumulative transdermal absorption of DBL-8, LIP-8, and NLIP-8 within 24 hours were 31.04, 10.47, and 5.06 mg / cm³, respectively. 2 The results demonstrate that, under the same concentration conditions, the transdermal absorption of DBL-8 biomimetic membrane is 3 to 6 times that of liposomes (LIP-8) and non-liposomes (NLIP-8), indicating better transdermal permeability.

[0083] (2) Skin permeability test

[0084] The thiocarbamate bond of fluorescein-5-isocyanate (FITC) can bind to the R-amino group of lysine residues on a peptide chain, forming FITC-peptide conjugates, which exhibit fluorescent properties. In this experiment, the test substance was fluorescently labeled with FITC, and the skin permeability of the FITC-peptide conjugates was tested. Changes in fluorescence intensity were measured using a fluorescent microplate reader at different time points (30, 60, and 120 min). The skin permeability was compared by observing the fluorescence intensity of FITC-DBL-8, FITC-LIP-8, and FITC-NLIP-8 solutions under the same conditions.

[0085] Figure 4 The results showed that, at the same treatment time, the fluorescence intensity of DBL-8 was greater than that of LIP-8 and NLIP-8. For example, under the condition of 60 min, the fluorescence intensities of DBL-8, LIP-8 and NLIP-8 were 75 AU, 50 AU and 35 AU, respectively, indicating that DBL-8 has better skin penetration performance.

[0086] 3. Evaluation of resistance to enzymatic hydrolysis

[0087] This experiment simulated a complex in vitro enzymatic environment using skin extracts. The test substance was mixed with the skin extract and reacted. Free acetyl hexapeptide-8 reacted with and degraded the enzymes in the skin extract. The enzymatic hydrolysis reaction times were 3, 60, 90, 120, 360, and 1440 min. One mL of the sample was then mixed with 10% TRITON X-100 solution, ultrasonically demulsified for 1 min, and brought to a final volume of 5 mL. One mL of the sample was then analyzed by high-performance liquid chromatography (HPLC) to detect the content of acetyl hexapeptide-8 at different time points under this enzyme activity environment, thus comparing the enzymatic resistance of DBL-8, LIP-8, and NLIP-8.

[0088] Figure 5The enzymatic hydrolysis rate test results showed that DBL-8's hydrolysis rate was lower than that of LIP-8 and NLIP-8 at all test time points. The TIC50 of DBL-8 was 355.0 min, LIP-8's was 206.1 min, and NLIP-8's was 100.3 min. This means that the time required for NLIP-8 and LIP-8 to be 50% hydrolyzed was shorter than that of DBL-8. DBL-8's resistance to enzymatic hydrolysis is superior to NLIP-8 and LIP-8, indicating that it can prevent the active ingredients from being destroyed by enzymes in the human body, ultimately ensuring the optimal effect of the active ingredients.

[0089] 4. Evaluation of sustained-release performance

[0090] In this experiment, 10 mL of the test sample was transferred into a dialysis bag, clamped, and placed in 80 mL of physiological saline sustained-release medium. The mixture was stirred at a constant speed under constant temperature conditions. At 2, 4, 6, 8, 12, 24, 30, 36, and 48 hours, 1 mL of the sustained-release medium was collected, and 1 mL of fresh physiological saline was added simultaneously. The sustained-release performance of each sample was compared by detecting the acetyl hexapeptide-8 content in the sample solution using high-performance liquid chromatography (HPLC).

[0091] By fitting the cumulative release of acetyl hexapeptide-8, the in vitro release curves of DBL-8, LIP-8, and NLIP-8 in a physiological saline system all conformed to first-order kinetic equations. Figure 6 It can be seen that after 48 hours, the cumulative release rates of acetyl hexapeptide-8 of DBL-8, LIP-8 and NLIP-8 were 50%, 78% and 95%, respectively. The cumulative release rate of acetyl hexapeptide-8 of DBL-8 was lower than that of LIP-8 and NLIP-8. This result indicates that DBL-8 has a certain sustained-release capacity, and the diffusion rate of the active ingredient in the tissue is reduced, thereby prolonging the time for the active ingredient to exert its effect.

[0092] 5. In vitro efficacy evaluation

[0093] (1) Hyaluronidase activity inhibition test

[0094] Hyaluronidase (HAase) non-selectively hydrolyzes sodium hyaluronate to yield β-N-acetylglucosamine. β-N-acetylglucosamine condenses with borate under alkaline conditions to form a chromogen, which then reacts with p-dimethylaminobenzaldehyde (p-DMAB) to produce a color under acidic conditions. This experiment aims to inhibit hyaluronidase activity by adding a test substance, thus limiting the enzymatic reaction. Given the conjugation effect of the formation system, the wavelength at which maximum absorbance is achieved is used as the optimal selection wavelength. Changes in absorbance are observed to reflect the inhibitory efficiency of the test substance on hyaluronidase.

[0095] The results showed that at a test concentration of 1%, DBL-8, LIP-8 and NLIP-8 inhibited hyaluronidase activity by 67.22%, 42.11% and 32.13% respectively, indicating that DBL-8 has better soothing and repairing effects.

[0096] (2) DPPH free radical scavenging test

[0097] 1,1-Diphenyl-2-trinitrophenylhydrazine (DPPH) is a stable, long-lived free radical with non-shared electrons (N·) within its molecule. Upon reaction with electrons and other free radicals, it transforms into a stable structure (NH). DPPH appears deep purple in alcoholic solution. When the free radical is removed and a covalent bond is formed, the alcoholic solution lightens in color, and the absorbance at the maximum absorption wavelength of 517 nm decreases. The degree of fading of the DPPH alcoholic solution is linearly related to the number of electrons it accepts, thus allowing evaluation of the sample's antioxidant capacity. In this experiment, a blank control and a positive control were used. The test substance was added to a DPPH free radical solution, mixed, and shaken well. The absorbance was measured at 517 nm using a microplate reader, and the color change was observed.

[0098] The results showed that at a test concentration of 1%, the DPPH· free radical scavenging rates of DBL-8, LIP-8 and NLIP-8 were 94.95%, 47.16% and 33.18%, respectively. The antioxidant capacity of DBL-8 was more than twice that of LIP-8, proving that it has strong antioxidant properties and can maximize the effect of the active ingredients.

[0099] 6. Evaluation of human efficacy

[0100] This test recruits healthy subjects and measures the skin stratum corneum content, skin elasticity R2 value, skin firmness F4 value, and skin redness a* value before and after use of DBL-8, LIP-8, and NLIP-8. It also analyzes the number of wrinkles, wrinkle area, and changes in redness to verify the efficacy of the test products in moisturizing, anti-wrinkle, firming, and soothing.

[0101] Qualified volunteers were recruited, signed informed consent forms, and provided with the product and instructions for use. Skin data was tested using a skin analyzer (moisture test probe and elasticity test probe) and VISIA-CR at the beginning, 4 weeks, and 6 weeks after use. The results are as follows:

[0102] Moisturizing efficacy: At a 1% test concentration, the water content of the sample area was tested after 4 and 6 weeks of use. The increase rate of skin stratum corneum moisture content on the DBL-8 side was 32.37% and 42.19%, respectively, while the increase rate of LIP-8 was 20.10% and 28.15%, respectively, and the increase rate of NLIP-8 was 11.16% and 15.10%, respectively. There were significant differences among the samples, indicating that the moisturizing effect of DBL-8 was significantly better than that of LIP-8 and NLIP-8.

[0103] Anti-wrinkle efficacy: After 4 and 6 weeks of use, the wrinkle area ratio in the images of DBL-8 was significantly reduced by 12.03% and 12.78% compared to the initial value. The wrinkle area ratio in the images of LIP-8 after 4 and 6 weeks of use was also significantly reduced compared to the initial value, but the reduction rate was lower, at 7.04% and 8.02% respectively, while NLIP-8 was even less. This result indicates that DBL-8 is significantly more effective than LIP-8 in reducing wrinkles.

[0104] Firming effect: After 4 and 6 weeks of use, the firming effect F4 of DBL-8 changed by 13.16% and 15.14%, respectively, while the firming effect F4 of LIP-8 changed by 7.16% and 9.01%, and the firming effect F4 of NLIP-8 changed by 5.06% and 6.45%. This result indicates that DBL-8 has better firming effect than LIP-8 and NLIP-8.

[0105] Soothing effect: After 4 and 6 weeks of use, the red zone of DBL-8 showed a significant reduction compared to the initial value, and the degree of change was greater than that of LIP-8 and NLIP-8.

[0106] Subject self-assessment: After 6 weeks of continuous use of the test samples, the overall satisfaction rates of subjects with DBL-8, LIP-8 and NLIP-8 products were 89.41%, 61.25% and 40.16%, respectively.

[0107] The above experimental results show that the D-BL biomimetic membrane constructed using the method of the present invention has a variety of excellent properties such as safety, targeting, affinity with cells, sustained release, resistance to enzymatic hydrolysis, and promotion of transdermal absorption. Therefore, the method of the present invention can not only be used for the above-mentioned polypeptide macromolecules, but also be derived to other similar peptides, proteins and other macromolecules to maximize their effects.

Claims

1. A method for constructing a D-BL biomimetic membrane, characterized in that: The method includes the following steps: Step 1: Computer Model Construction The three-dimensional molecular structure of receptor R was downloaded from the PubChem database, and the homologous protein structure of ligand L was searched from the protein crystal database. A model framework for the target docking agent was found, and the structural variable region of the target docking agent was constructed. The receptor R is a polypeptide, the ligand L is a protein, and the target docking agent is a bilayer phospholipid composed of lecithin and cholesterol. Step 2: Molecular docking The ligand L is placed in the active pocket of the receptor R. The interaction between the ligand L and the receptor R is evaluated according to the complementary matching principles of geometry, electrostatic interaction, hydrogen bonding interaction, and hydrophobic interaction. The optimal binding mode between the two molecules, ligand L and receptor R, is found to obtain the initial structural composition. The initial structural composition is composed of receptor R, ligand L, and target docking material in different mass ratios. Step 3: Molecular Dynamics Simulation The obtained initial structural compositions were subjected to energy minimization and molecular dynamics simulations using Amber software. Step 4: Calculate using free energy Using the MMPBSA.py module in Amber software, the MM-PBSA method was employed to calculate the system binding free energy of the initial structural composition, obtaining the composition with the lowest energy and greatest stability. The system binding free energy of the composition is expressed by the following formula: AG bind = AH - TAS = AE MM + AG sol - TAS ΔE MM = ΔE ele + ΔE vdw ΔG sol = ΔG GB + ΔG surf where ΔG bind represents the free energy of mixing of the composition; ΔH represents the enthalpy change of the composition; ΔE MM represents the molecular mechanical energy in the gas phase; ΔE ele and ΔE vdw represent the electrostatic interaction and van der Waals energy of the ligand and acceptor, respectively, generated in the gas phase; AG sol represents the contribution of the free energy of solvation to the binding of the ligand to the receptor; AG GB and AG surf represent the contribution of polar and non-polar solvation free energy, respectively, to the binding of the ligand to the receptor, the former is obtained by solving the Poisson-Boltzmann equation using finite difference method, and the latter is solved using the following empirical equation: AG surf = γSASA + β, where SASA represents the solvent accessible surface area, and the values of γ and β are set to 0.00542 kcal / (mol . Å 2 ) and 0.92 kcal / mol, respectively; TΔS represents the contribution of the entropy change of the ligand upon binding to the receptor to the binding free energy. Step 5: Preparation of D-BL biomimetic membrane D-BL biomimetic membranes were prepared based on the formulation ratio of the lowest energy and most stable composition, and their performance was verified.

2. The method for constructing the D-BL biomimetic membrane according to claim 1, characterized in that: The polypeptide is any one of the following: oligopeptide-1, oligopeptide-3, oligopeptide-2, tripeptide-1, hexapeptide-1, oligopeptide-5, hexapeptide-9, oligopeptide-6, pentapeptide-1, hexapeptide-11, pentapeptide-3, hexapeptide-3, acetyl hexapeptide-8, acetyl tetrapeptide-5, acetyl hexapeptide-1, acetyl tetrapeptide-2, acetyl octapeptide-3, acetyl tetrapeptide-9, palmitoyl pentapeptide-4, palmitoyl tetrapeptide-7, palmitoyl tripeptide-5, palmitoyl tripeptide-1, palmitoyl oligopeptide, palmitoyl hexapeptide-12, palmitoyl pentapeptide-5, carnosine, and glutathione.

3. The method for constructing the D-BL biomimetic membrane according to claim 1, characterized in that: The protein is any one of collagen, elastin, milk protein, wild soybean protein, whey protein, serum albumin, keratin, wheat protein, and fibronectin.

4. The method for constructing the D-BL biomimetic membrane according to claim 1, characterized in that: In step 2, the GOLD docking procedure is used, and a genetic algorithm is employed to search for the binding mode of ligand L in the active site of receptor R. During the docking process, the ligand L is allowed to have a full range of flexible conformations and local flexibility. Based on the GOLD prediction of the binding mode of the composition, the initial structural composition is obtained.

5. The method for constructing the D-BL biomimetic membrane according to claim 1, characterized in that: In step 3, the energy minimization adopts the conjugate gradient method to eliminate unreasonable contact between atoms in the system. During the slow heating process, the solute molecules in the simulated system are constrained to maintain their conformation. Under constant temperature and pressure conditions, the simulated system reaches thermodynamic equilibrium, and the conformational changes, temperature, and energy parameters of the system are monitored.

6. The method for constructing the D-BL biomimetic membrane according to claim 1, characterized in that: In step 5, the performance includes safety, permeability, resistance to enzymatic hydrolysis, sustained-release properties, and efficacy.

7. The method for constructing the D-BL biomimetic membrane according to claim 1, characterized in that: In step 5, the D-BL biomimetic membrane exhibits the characteristics of a small nucleus and a large body, with the core size between 10 and 100 nm and the body size between 100 and 1000 nm.

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