Preparation method of a washing and caring cosmetic biological medicine

Abstract

The application discloses a preparation method of biological medicines for washing, protecting and making up, and relates to the technical fields of biological medicines and washing, protecting and making up. The method comprises the following steps: adding a dynamic covalent bond modification reagent into a protein raw material solution, so that multiple dynamic covalent cross-linking points are formed in the protein raw material molecules, the protein raw material molecules are compressed into a nanoscale compact supramolecular structure, and a conformation compression intermediate is obtained; a lipid organic phase composed of ceramide, cholesterols and free fatty acids is mixed with the conformation compression intermediate solution and is homogenized, so that core-shell composite microcapsules are formed; and the dynamic covalent cross-linking points are broken under the pH gradient and reducing conditions of a skin microenvironment, so that the protein returns to a natural conformation. The application can improve the transdermal penetration efficiency without damaging the spatial conformation of the protein, and can improve the stability of the product under the conditions of normal-temperature circulation and repeated use after opening. The application can be used for the preparation of biologically active ingredients in washing, protecting and making up products.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and personal care cosmetics, specifically a method for preparing a biological drug for personal care cosmetics. Background Technology

[0002] With the development of synthetic biology technology, recombinant collagen, recombinant elastin and functional bioactive peptides and other macromolecular bioactive substances have been widely used in hair care and beauty products to exert anti-wrinkle, repair and barrier reconstruction effects. Their efficacy is highly dependent on the natural three-dimensional spatial conformation. Under normal physiological conditions, the stratum corneum of human skin only allows substances with smaller molecular weights to penetrate effectively. Due to their large molecular weight and large hydrodynamic radius, macromolecular bioactive substances are difficult to cross this barrier through passive diffusion.

[0003] Currently, the industry mainly adopts two technical routes to address the above problems. One is the enzymatic degradation and penetration-enhancing scheme, which uses proteases to degrade macromolecules into oligopeptides or polypeptide fragments to reduce molecular weight. However, this process will destroy the original peptide chain folding and intermolecular hydrogen bond network of the protein, causing the product to lose the biological activity supported by the three-dimensional spatial conformation, making it difficult to balance penetration efficiency and activity retention. The other is the pure physical carrier encapsulation scheme. Existing technologies generally believe that macromolecular protein active substances can only be delivered by surface encapsulation or electrostatic adsorption. This application breaks through this prejudice and for the first time achieves active compression of protein volume through dynamic covalent cross-linking at the molecular level before lipid encapsulation. This fundamentally improves the loading rate and isolates the interference of formulation ingredients. Moreover, under the action of surfactants, ethanol, salts and other ingredients in the washing and care formula, demulsification or desorption is easily caused, resulting in premature release of protein.

[0004] Furthermore, existing freeze-drying processes are mainly designed for injection-grade pure aqueous phase formulations, which are difficult to adapt to the dispersion and stability requirements of complex matrices in personal care and beauty products. Proteins are prone to oxidative degradation or aggregation and inactivation under repeated use after opening at room temperature, leading to a decrease in product efficacy. Therefore, existing technologies cannot simultaneously ensure the penetration efficiency, spatial structural integrity, and room temperature stability of macromolecular bioactive substances. To address this, we propose a method for preparing biopharmaceuticals for personal care and beauty products. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing biological pharmaceuticals for washing, care, and beauty purposes.

[0006] The protein raw materials mentioned in this invention refer to macromolecular bioactive substances such as recombinant proteins or recombinant polypeptides, including but not limited to recombinant collagen, recombinant elastin and functional bioactive polypeptides.

[0007] The technical problem to be solved by this invention is: how to improve the transdermal penetration efficiency of protein raw materials without destroying their three-dimensional spatial conformation, and how to maintain the stability of biological drugs containing such protein raw materials under the conditions of room temperature circulation and repeated use after opening in personal care and beauty products.

[0008] To solve the above-mentioned technical problems, this invention provides a method for preparing a biological pharmaceutical for washing and beauty purposes, comprising: dissolving a protein raw material in a buffer solution to prepare a protein solution; adding a dynamic covalent bond modifying agent to the protein solution, causing the dynamic covalent bond modifying agent to react with the active groups on the surface of the protein raw material molecules, forming multiple dynamic covalent cross-linking points within and between the protein raw material molecules, transforming the protein raw material molecules from a loose extended conformation to a nanoscale dense supramolecular structure, obtaining a conformational compression intermediate; purifying the conformational compression intermediate to remove unreacted free modifying agent; and dissolving ceramide, cholesterol, and free fatty acids in a buffer solution. In an organic solvent, a lipid organic phase is formed and mixed with a purified conformational compression intermediate solution. This allows lipid molecules to self-assemble in an aqueous phase to form vesicles that encapsulate the conformational compression intermediate. The vesicles are then homogenized to form core-shell composite microcapsules with a multilayer lipid liquid crystal shell structure, resulting in a microencapsulated suspension. The microencapsulated suspension is then dried to obtain a biopharmaceutical for washing and beauty purposes. The dynamic covalent crosslinking points remain stable in a weakly acidic environment but break in an environment where the pH transitions from weakly acidic to neutral and the concentration of reducing substances increases. This releases the conformational constraints on the protein raw material molecules, allowing them to restore their natural three-dimensional spatial conformation.

[0009] Compared with the prior art, the beneficial effects of the present invention by adopting the above technical solution are as follows:

[0010] 1. This invention achieves volume compression rather than molecular degradation by reversibly dynamic covalent bonds to crosslink protein raw material molecules at multiple points within and between molecules, thereby significantly reducing the hydrodynamic radius while preserving its primary sequence and potential natural conformation information. This overcomes the problem that it is difficult to achieve both improved permeation efficiency and preservation of bioactivity in existing enzymatic degradation schemes.

[0011] 2. This invention utilizes the naturally existing pH gradient and redox potential difference between the stratum corneum and the active epidermal layer as a signal to trigger the dynamic covalent bond breaking. It can achieve in-situ refolding of protein raw materials at the delivery site without the need for additional activating ingredients, and realize the spatiotemporal separation and automatic conversion of the permeable and active states.

[0012] 3. This invention uses a biomimetic lipid liquid crystal shell constructed from ceramides, cholesterol, and free fatty acids to encapsulate the conformational compression intermediate, forming a core-shell composite microcapsule structure. Compared with existing purely physical encapsulation methods, this can more effectively isolate the influence of surfactants, ethanol, salts, and other components in the washing and care formula, and improve the stability of the product under conditions of normal temperature non-cold chain circulation and repeated use after opening. Attached Figure Description

[0013] Figure 1 This is a schematic diagram of the overall complete preparation process in an embodiment of the present invention;

[0014] Figure 2 This is a schematic diagram of the skin penetration response activation process in an embodiment of the present invention;

[0015] Figure 3 This is a simplified process diagram of two modification branches in an embodiment of the present invention. Detailed Implementation

[0016] The following is in conjunction with the appendix Figure 1-3 The specific embodiments of the present invention will be further described below. It should be noted that the description of these embodiments is for the purpose of helping to understand the present invention, but does not constitute a limitation of the present invention.

[0017] Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0018] Example 1 (using recombinant collagen and phenylboronic acid derivative modified system as an example):

[0019] First, the recombinant collagen was dissolved in phosphate buffer at pH 7.5 to prepare a protein solution with a concentration of 5 mg / mL, and stirred at 25°C until completely dissolved and homogeneous.

[0020] Next, a phenylboronic acid derivative-type dynamic covalent bond modification reagent was added to the above protein solution at a ratio of 1:10 between the molar number of surface active groups of recombinant collagen molecules and the molar number of modification reagents. The phenylboronic acid derivative reacted with the diol structure on the surface of the recombinant collagen to form a borate ester bond. The reaction was carried out at 30°C under light-protected conditions for 2 hours. During the reaction, the average hydrodynamic radius of the particles in the solution was monitored in real time by dynamic light scattering. When the detected value decreased by about 65% compared with that before the reaction and tended to stabilize, the conformational compression reaction was determined to be complete, and a conformational compression intermediate was obtained.

[0021] Subsequently, the reaction products were purified by ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa to remove unreacted free phenylboronic acid derivatives and small molecule byproducts, resulting in a purified conformational compression intermediate solution. The purified conformational compression intermediate was found to have a nanoscale dense structure with a particle size of approximately 20 nm. After pretreatment with a reducing agent, its circular dichroism chromatogram was highly consistent with that of natural recombinant collagen, indicating that the compression process is reversible and does not destroy the potential natural conformational information of the protein.

[0022] During conformational compression reactions, the relationship between the diffusion coefficient of protein molecules and their hydrodynamic radius can be quantitatively characterized using the following equation:

[0023] ;

[0024] in, This represents the diffusion coefficient of protein molecules in solution. Boltzmann's constant, The thermodynamic temperature of the solution. Let be the dynamic viscosity of the solution. The hydrodynamic radius of the protein molecule is determined by measuring the diffusion coefficient through dynamic light scattering. The value of the hydrodynamic radius can be deduced from the above relationship. The significant reduction of this value is a quantitative basis for the effectiveness of the conformational compression reaction.

[0025] The reversible equilibrium of the dynamic covalent bond in phenylboronic acid-diol can be described by the following equilibrium constant expression:

[0026] ;

[0027] in, This is the equilibrium constant for the borate esterification reaction. This indicates the concentration of borate ester bonds that have been formed. This indicates the concentration of unreacted free phenylboronic acid groups. This represents the concentration of unreacted free diol groups. This equilibrium constant is affected by the pH of the system. Under acidic conditions, the equilibrium shifts to the left, meaning that the formed borate ester bonds tend to break. This property is the chemical basis for the subsequent microenvironment response release.

[0028] Then, according to the molar ratio of ceramide, cholesterol, and free fatty acid of 1:1:0.6, the three lipid raw materials were dissolved in ethanol solvent and heated to 65°C to fully dissolve them, forming a lipid organic phase. The purified conformational compression intermediate solution was used as the aqueous phase and mixed with the lipid organic phase at a volume ratio of 1:2. The mixture was kept warm and stirred at 65°C to allow the lipid molecules to self-assemble in the aqueous phase to form initial vesicles that encapsulate the conformational compression intermediate.

[0029] Subsequently, the initial vesicle mixture was circulated five times using a high-pressure microfluidic homogenizer at a pressure of 1000 bar to homogenize the vesicle particle size and form a dense multilayer lipid liquid crystal shell structure, resulting in a core-shell composite microcapsule suspension with a particle size of approximately 220 nm. The microencapsulated suspension was then centrifuged to remove unencapsulated free lipids and free recombinant collagen, and the encapsulation efficiency was measured to be approximately 92%.

[0030] The encapsulation efficiency during the microencapsulation process can be calculated using the following formula:

[0031] ;

[0032] in, Indicates the encapsulation ratio. This indicates the total mass of protein-based raw materials input into the system. This indicates the mass of unencapsulated free protein raw materials detected after centrifugation.

[0033] Finally, the microencapsulated suspension purified by centrifugation is freeze-dried to produce a powdered raw material, which is the biological drug for washing and beauty described in this invention. Alternatively, the microencapsulated suspension can be directly added to cosmetic bases such as serums, creams, or shampoos in suspension form to produce the final product.

[0034] Explanation of microenvironment-responsive in-situ recovery processes:

[0035] When the aforementioned biopharmaceuticals for washing and beauty are added to the final product matrix, they are applied to the skin surface along with the product. Due to their structural integrity and nanoscale size, the microcapsules can penetrate through the lipid channels between keratinocytes in the stratum corneum region and enter the active epidermal layer. As the carrier further penetrates into the intercellular and intracellular environment of the epidermal cells, the environmental pH gradually changes from the weakly acidic environment of the stratum corneum to neutral, and the concentration of local reducing substances increases, which promotes the hydrolytic breakage of borate ester bonds. After the dynamic covalent cross-linking points break, the conformational constraints on the protein raw material molecular chain segments are released, and they spontaneously refold according to the thermodynamic minimum energy state determined by their own amino acid sequence, restoring their natural three-dimensional spatial conformation, and re-exhibiting their original physical support and biological activity functions at the target site.

[0036] The breaking process of dynamic covalent bonds triggered by the above microenvironment can be described by the following first-order reaction kinetic equation:

[0037] ;

[0038] in, This indicates the concentration of dynamic covalent crosslinking points in the system that have not yet broken. Indicates time, The observed apparent fracture rate constant increases with decreasing pH or increasing concentration of reducing substances in the microenvironment. The above equation shows that the fracture rate of dynamic covalent bonds is proportional to their instantaneous concentration. The fracture process continues under environmental triggering conditions until the crosslinking points are basically eliminated, allowing the conformation of protein raw material molecules to gradually unfold and recover.

[0039] Example 2 (using a functional bioactive peptide and disulfide bond modified system as an example):

[0040] Functional bioactive peptides were dissolved in phosphate buffer at pH 7.0 to prepare a peptide solution with a concentration of 8 mg / mL. The solution was stirred and dissolved evenly at 4°C. A bifunctional reagent containing a disulfide bond crosslinking structure was added to the peptide solution at a ratio of 1:15 between the molar number of active groups on the surface of the peptide molecule and the molar number of the modifying reagent. The bifunctional reagent reacted with the thiol groups on the surface of the peptide molecule after reduction and exposure to disulfide bonds. The reaction was carried out at 20°C under light-protected conditions for 4 hours. Dynamic light scattering monitoring confirmed that the average hydrodynamic radius of the particles decreased by about 70% compared with that before the reaction, resulting in a conformational compression intermediate. The subsequent purification, lipid liquid crystal microencapsulation, and drying steps were the same as in Example 1 and will not be repeated. The biopharmaceutical for washing and beauty purposes prepared in this example also showed the performance of dynamic covalent crosslinking point breakage and peptide conformation recovery in a reducing environment, verifying that the technical solution of this invention is applicable to different protein raw materials and different dynamic covalent bond types.

[0041] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make possible changes and modifications without departing from the spirit and scope of the present invention. Therefore, any modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention shall fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing a biological drug for washing and beauty purposes, characterized in that, include: Protein raw materials are dissolved in buffer solution to prepare a protein solution. A dynamic covalent bond modifying agent is added to the protein solution, allowing the agent to react with the active groups on the surface of the protein raw material molecules. This creates multiple dynamic covalent cross-linking points within and between the protein raw material molecules, transforming them from a loose, extended conformation to a nanoscale, dense supramolecular structure, resulting in a conformational compression intermediate. The dynamic covalent bond modifying agent is selected from phenylboronic acid derivatives and bifunctional reagents containing disulfide cross-linking structures. The purified conformational compression intermediate solution is mixed with the lipid organic phase at a volume ratio of 1:1 to 1:3 at 60 to 70°C, allowing lipid molecules to self-assemble into initial vesicles encapsulating the conformational compression intermediate. This method overcomes the technical limitations of existing purely physical encapsulation methods, such as low active ingredient loading rates and susceptibility to degradation by surfactants, ethanol, and salts. See, the conformational compression intermediate is purified to remove unreacted free modifying agents, resulting in a purified conformational compression intermediate. Ceramide, cholesterol, and free fatty acids are dissolved in an organic solvent to form a lipid organic phase. The solution of the purified conformational compression intermediate is used as the aqueous phase and mixed with the lipid organic phase, allowing lipid molecules to self-assemble in the aqueous phase to form vesicles encapsulating the conformational compression intermediate. The vesicles are homogenized to form core-shell composite microcapsules with a multilayer lipid liquid crystal shell structure, resulting in a microencapsulated suspension. The microencapsulated suspension is dried to obtain a biopharmaceutical for washing and beauty purposes. The dynamic covalent crosslinking points remain stable in a weakly acidic environment but break in an environment where the pH transitions from weakly acidic to neutral and the concentration of reducing substances increases, releasing the conformational constraints on the protein raw material molecules and restoring the protein raw material molecules to their natural three-dimensional spatial conformation.

2. The method for preparing a biological drug for washing and beauty purposes according to claim 1, characterized in that: The dynamic covalent bond modifying agent is a phenylboronic acid derivative, which reacts with the diol groups on the surface of the protein raw material molecule to form a borate ester bond.

3. The method for preparing a biological drug for washing and beauty purposes according to claim 1, characterized in that: The dynamic covalent bond modification reagent is a bifunctional reagent containing a disulfide bond cross-linking structure. The bifunctional reagent undergoes a disulfide bond exchange reaction with the thiol groups on the surface of the protein raw material molecules to form disulfide bonds.

4. The method for preparing a biological drug for washing and beauty purposes according to claim 1, characterized in that: The buffer solution has a pH of 7.0 to 8.0, a protein solution concentration of 1 to 10 mg / mL, a molar ratio of 1:5 to 1:20, a reaction temperature of 20 to 37°C, and a reaction time of 1 to 4 hours.

5. The method for preparing a biological drug for washing and beauty purposes according to claim 1, characterized in that: When purifying the conformational compression intermediate, an ultrafiltration membrane with a molecular weight cutoff of 10-30 kDa is used for ultrafiltration, and the particle size of the purified conformational compression intermediate is 5-50 nm.

6. The method for preparing a biological drug for washing and beauty purposes according to claim 1, characterized in that: The molar ratio of the ceramide, the cholesterol, and the free fatty acid is 0.5~1.5:0.3~1.

0.

7. The method for preparing a biological drug for washing and beauty purposes according to claim 1, characterized in that: The volume ratio of the purified conformational compression intermediate solution to the lipid organic phase is 1:1 to 1:3, and the mixing temperature is 60 to 70°C.

8. The method for preparing a biological drug for washing and beauty purposes according to claim 1, characterized in that: When homogenizing the vesicles, the processing pressure is 800~1500 bar, and the number of cycles is 3~8. After homogenization, the microencapsulated suspension is centrifuged to remove unencapsulated free lipids and free proteins.

9. The method for preparing a biological drug for washing and beauty purposes according to claim 1, characterized in that: The protein raw materials are selected from one of recombinant collagen, recombinant elastin, and functional bioactive peptides.

10. A biological medicine for washing, care, and cosmetic purposes, characterized in that, It is prepared by any one of claims 1 to 9.

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