Preparation method of composition for promoting interaction of collagen and hyaluronic acid

Abstract

The invention relates to the technical field of medical biology, and discloses a composition for promoting interaction of collagen and hyaluronic acid, which comprises collagen, hyaluronic acid and a chemical cross-linking agent. According to the preparation method of the composition for promoting interaction of the collagen and the hyaluronic acid and the application of the chemical cross-linking agent N (beta maleimido) propionate, a stable chemical bond is formed between the collagen and the hyaluronic acid, and the molecular structure is optimized, so that the composition is easier to penetrate through the cuticle of the skin, and the interaction of the collagen and the hyaluronic acid is promoted. Physical treatment of ultrasonic waves and a high-voltage pulse electric field accelerates formation of a compound, the skin barrier is reduced through mechanical and electroporation effects, conditions are provided for deep penetration of the compound, particles are refined through the cavitation effect of the ultrasonic waves, and uniformity and fluidity are improved; the electroporation effect of the high-voltage pulsed electric field increases the permeability of cell membranes, an additional permeation path is provided for the compound, and the comprehensive measures effectively improve the transdermal absorption efficiency and the biological activity of the compound.

Description

Technical Field

[0001] The present invention relates to the field of medical biotechnology, and specifically to a method for preparing a composition that promotes the interaction between collagen and hyaluronic acid. Background Art

[0002] Among numerous skin care ingredients in the medical field, collagen and hyaluronic acid have attracted much attention due to their unique biological properties and skin care effects. Collagen is the main structural protein of the skin, responsible for maintaining the elasticity and firmness of the skin. As people age, the synthesis rate of collagen in the body slows down, resulting in skin relaxation and wrinkles. Hyaluronic acid is a high-molecular-weight polysaccharide with strong water retention ability, capable of adsorbing a large amount of water and keeping the skin moist and smooth. However, when using collagen or hyaluronic acid alone, there are often problems such as low absorption rate and poor stability, which limit their application effects in skin care.

[0003] In recent years, research on preparing collagen and hyaluronic acid complexes through chemical and physical methods has become a hot topic, aiming to overcome the above limitations and improve the biocompatibility and functionality of the two. Chemical cross-linking technology, especially the use of specific cross-linking agents, can form stable chemical bonds between collagen and hyaluronic acid molecules, enhancing the structural stability of the complex. At the same time, physical treatments such as the application of ultrasonic waves and high-voltage pulsed electric fields can further optimize the intermolecular interaction, promote the formation of the complex, and reduce possible side effects.

[0004] In the prior art, although there have been cases of using collagen and hyaluronic acid in combination, there are still challenges in improving the transdermal absorption rate, bioavailability, and stability of the complex. Traditional preparation methods often have difficulty achieving these goals simultaneously, resulting in limited effects of the complex in skin care applications. Therefore, we propose a method for preparing a composition that promotes the interaction between collagen and hyaluronic acid. Summary of the Invention

[0005] (I) Technical Problems to be Solved

[0006] Aiming at the deficiencies of the prior art, the present invention provides a method for preparing a composition that promotes the interaction between collagen and hyaluronic acid, which can significantly enhance the interaction between collagen and hyaluronic acid. The formed complex has more excellent moisturizing and skin repair capabilities. Compared with traditional single-component skin care products, it can penetrate deeper into the skin more effectively, improve skin texture, and delay the aging process, solving the problems that although there have been cases of using collagen and hyaluronic acid in combination, there are still challenges in improving the transdermal absorption rate, bioavailability, and stability of the complex, and traditional preparation methods often have difficulty achieving these goals simultaneously, resulting in limited effects of the complex in skin care applications.

[0007] (2) Technical Solution

[0008] To achieve the above object of significantly enhancing the interaction between collagen and hyaluronic acid, the formed complex having more excellent moisturizing and skin repair capabilities, and being able to penetrate more effectively into the deep layer of the skin, improve the skin texture and delay the aging process compared with traditional single-component skin care products, the present invention provides the following technical solutions: A composition for promoting the interaction between collagen and hyaluronic acid, comprising collagen, hyaluronic acid, and a chemical crosslinking agent;

[0009] The collagen is selected from type I collagen derived from fish skin, with a purity ≥ 99%, a molecular weight of about 300 kDa, and is formulated into a 1% (w / v) solution;

[0010] The hyaluronic acid is prepared by microbial fermentation, with a molecular weight range of 1000 - 1500 kDa, and is formulated into a 0.5% (w / v) solution;

[0011] The chemical crosslinking agent is N(β-maleimidopropionate), and the concentration is carefully controlled at 0.5% to 2%.

[0012] A preparation method of a composition for promoting the interaction between collagen and hyaluronic acid, comprising the composition for promoting the interaction between collagen and hyaluronic acid, and further comprising the following steps:

[0013] H1. Raw material preparation: Dissolve collagen and hyaluronic acid in deionized water separately under sterile conditions;

[0014] H2. Pretreatment: Let the collagen and hyaluronic acid solutions stand in a phosphate buffer solution;

[0015] H3. Chemical modification: Chemically modify the collagen using N(β-maleimidopropionate);

[0016] H4. Physical treatment: First, mix the collagen and hyaluronic acid solutions in a volume ratio of 1:1 using an ultrasonic treatment device, and then perform high-voltage pulsed electric field treatment;

[0017] H5. Complex formation and optimization: Let the mixture of collagen and hyaluronic acid continue to stand in a buffer solution, and continuously monitor the particle size distribution of the complex using dynamic light scattering (DLS) technology until the complex is formed;

[0018] H6. Post-treatment and performance testing: After the complex formation is completed, perform necessary post-treatment steps and conduct performance testing.

[0019] Preferably, the complex further comprises a stabilizer and an activator. The stabilizer is selected from one or more of "α-tocopherol", "trehalose", "phosphate buffered saline (PBS)", and "polysorbate 20", and the activator is selected from one or more of "epidermal growth factor (EGF)", "L-proline", "green tea extract", and "vitamin C".

[0020] Preferably, the collagen and hyaluronic acid solution are allowed to stand in a phosphate buffer at a pH value of 6.5 to 7.5 for 12 to 24 hours.

[0021] Preferably, the molar ratio of N-(β-maleimidopropionate) to collagen ranges from 1:5 to 1:20, and chemical modification is carried out for 2 hours at a pH value of 7.4 and a temperature of 25 °C.

[0022] Preferably, the frequency of the ultrasonic treatment device is 15 kHz to 30 kHz, the power is 30 W to 70 W, the treatment time is 5 to 20 minutes, and the mixing mode of the ultrasonic treatment device is an intermittent mode, turning on for 2 seconds and turning off for 2 seconds per second.

[0023] Preferably, the electric field strength of the high-voltage pulsed electric field treatment is 0.5 kV / cm to 2 kV / cm, the pulse width is 5 μs to 20 μs, and the repetition frequency is 0.5 Hz to 5 Hz.

[0024] Preferably, a buffer solution with a pH value of 6.5 to 7.5 can be selected as the buffer solution to meet different bioactivity requirements.

[0025] Preferably, the necessary post-treatment steps include filtration, purification, or sterilization to ensure the safety and quality of the final product. The performance tests include the evaluation of the cytotoxicity of human dermal fibroblasts (HDF) in vitro models, the proliferation stimulation effect, and the ability to promote extracellular matrix synthesis.

[0026] (III) Beneficial effects

[0027] Compared with the prior art, the present invention provides a method for preparing a composition that promotes the interaction between collagen and hyaluronic acid, and has the following beneficial effects:

[0028] 1. The preparation method of the composition that promotes the interaction between collagen and hyaluronic acid effectively enhances the transdermal absorption rate and bioavailability of the collagen and hyaluronic acid complex through finely designed chemical and physical treatment steps. First, the application of the chemical cross-linking agent N-(β-maleimidopropionyl) ester not only establishes stable chemical bonds between collagen and hyaluronic acid molecules but also optimizes the molecular structure of the complex, making it more suitable for penetrating the stratum corneum of the skin. Second, physical treatment, especially the combined application of ultrasonic waves and high-voltage pulsed electric fields, not only accelerates the formation of the complex but also breaks the physical barrier of the skin through mechanical effects and electroporation effects, creating favorable conditions for the deep penetration of the complex. The cavitation effect of ultrasonic waves helps to refine the complex particles, improving their uniformity and fluidity, while the high-voltage pulsed electric field increases the permeability of cell membranes through transient electroporation, providing an additional penetration path for the complex, thus significantly enhancing the transdermal absorption rate and bioavailability of the complex.

[0029] 2. The preparation method of the composition that promotes the interaction between collagen and hyaluronic acid greatly improves the structural stability of the complex through precisely controlled chemical cross-linking processes. This not only extends the shelf life of the complex but also reduces the possibility of its degradation during storage and use. In addition, stabilizers such as α-tocopherol and trehalose, as well as activators such as epidermal growth factor and vitamin C, added to the complex further enhance the antioxidant capacity and biological activity of the complex. The presence of stabilizers helps the complex resist oxidative stress and maintain its biological activity, while activators stimulate the activity of the complex, improving its ability to promote skin cell proliferation and repair. Therefore, the complex of the present invention can not only penetrate deeper into the skin more effectively to improve skin texture but also exert its moisturizing and repair effects more durably, delaying the skin aging process and demonstrating excellent skin care effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is a flow chart of the preparation method of the composition of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0031] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the embodiments and drawings of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0032] Please refer to Figure 1 , a composition that promotes the interaction between collagen and hyaluronic acid, comprising collagen, hyaluronic acid, and a chemical cross-linking agent;

[0033] The collagen is selected from type I collagen derived from fish skin, with a purity of ≥99%, a molecular weight of approximately 300 kDa, and is formulated into a 1% (w / v) solution;

[0034] The hyaluronic acid is prepared by microbial fermentation, with a molecular weight range of 1000 - 1500 kDa, and is formulated into a 0.5% (w / v) solution;

[0035] The chemical cross - linker is N(β - maleimidopropionate), and its concentration is carefully controlled between 0.5% and 2%.

[0036] Specifically, the high purity and appropriate molecular weight of collagen ensure its biocompatibility and skin permeability, the large molecular weight of hyaluronic acid provides excellent water - retaining properties, while the chemical cross - linker establishes stable chemical bonds between the two, enhancing the structural stability and biological activity of the complex.

[0037] A method for preparing a composition that promotes the interaction between collagen and hyaluronic acid, including the composition that promotes the interaction between collagen and hyaluronic acid, and further includes the following steps:

[0038] H1. Raw material preparation: Dissolve collagen and hyaluronic acid in deionized water separately under sterile conditions;

[0039] H2. Pretreatment: Let the collagen and hyaluronic acid solutions stand in phosphate - buffered saline;

[0040] H3. Chemical modification: Chemically modify collagen using N(β - maleimidopropionate);

[0041] H4. Physical treatment: First, mix the collagen and hyaluronic acid solutions in a volume ratio of 1:1 using an ultrasonic treatment device, and then perform high - voltage pulsed electric field treatment;

[0042] H5. Complex formation and optimization: Let the mixture of collagen and hyaluronic acid continue to stand in the buffer solution, and continuously monitor the particle size distribution of the complex using dynamic light scattering (DLS) technology until the complex is formed;

[0043] H6. Post - treatment and performance testing: After the complex is formed, perform necessary post - treatment steps and conduct performance testing.

[0044] Specifically, collagen and hyaluronic acid are separately dissolved in deionized water under sterile conditions, and then stand in phosphate - buffered saline at a specific pH value. This pretreatment step helps to maintain the structural stability and biological activity of the protein, and at the same time promotes the efficiency of subsequent chemical modification and physical treatment.

[0045] Furthermore, the complex also contains a stabilizer and an activator. The stabilizer is selected from one or more of "α-tocopherol", "trehalose", "phosphate buffered saline (PBS)", and "polysorbate 20", and the activator is selected from one or more of "epidermal growth factor (EGF)", "L-proline", "green tea extract", and "vitamin C".

[0046] Specifically, both the stabilizer and the activator can be selected according to the properties of collagen and hyaluronic acid. A stabilizer, such as one or more of α-tocopherol, trehalose, PBS, and polysorbate 20, is added to the complex to improve the antioxidant capacity and structural stability of the complex. At the same time, an activator, such as one or more of epidermal growth factor (EGF), L-proline, green tea extract, and vitamin C, is added to enhance the biological activity and skin repair ability of the complex.

[0047] Furthermore, the collagen and hyaluronic acid solutions are left standing in a phosphate buffer at a pH value of 6.5 to 7.5 for 12 to 24 hours.

[0048] Specifically, both collagen and hyaluronic acid have their optimal pH stability ranges. Collagen is usually more stable in a slightly alkaline environment (around pH 7), while hyaluronic acid also maintains good stability between pH 6.5 and 7.5. Within this pH range, the structures of the two substances do not change significantly, which is beneficial for them to maintain their original biological activities and functional characteristics. At the same time, within this pH range, the charge distribution on the collagen and hyaluronic acid molecules is most conducive to their interaction. Hyaluronic acid is a negatively charged polysaccharide, and collagen may expose more positively charged regions at its specific pH value. This charge complementarity promotes the electrostatic attraction between molecules and is beneficial for the formation of the complex.

[0049] Furthermore, the molar ratio of N-(β-maleimidopropionate) to collagen ranges from 1:5 to 1:20, and chemical modification is carried out continuously for 2 hours under the conditions of a pH value of 7.4 and a temperature of 25°C.

[0050] Specifically, a pH value of 7.4 is an approximation of the human physiological environment, which ensures the structural stability of collagen and avoids conformational changes that may occur under non-physiological pH conditions. This is beneficial for the effective binding of MPA to the active site of collagen. At the same time, 25°C is a mild temperature, which not only avoids protein denaturation caused by high temperature but also ensures the reaction rate, ensuring the mildness and controllability of the chemical modification.

[0051] Furthermore, the frequency of the ultrasonic treatment equipment is from 15 kHz to 30 kHz, the power is from 30 W to 70 W, the treatment time is from 5 to 20 minutes, and the mixing mode of the ultrasonic treatment equipment is the intermittent mode, turning on for 2 seconds and turning off for 2 seconds per second.

[0052] Specifically, "turning on for 2 seconds and turning off for 2 seconds per second" is a specific operation mode. In the ultrasonic treatment equipment, it can make the ultrasonic wave emit for 2 seconds and then stop for 2 seconds every second. The advantage of doing this is that it can avoid the continuous action of the ultrasonic wave on the material and reduce the material damage or thermal effect that may be caused by the long-term high-intensity ultrasonic wave action. Through this intermittent turning on and off, the energy output of the ultrasonic wave can be more precisely controlled, the treatment effect can be optimized, and at the same time, the service life and safety of the equipment can be improved. In the process of preparing the collagen and hyaluronic acid complex, this operation mode helps to achieve a more uniform and gentle mixing and interaction of the materials, thus improving the quality of the final product.

[0053] Furthermore, the electric field strength of the high-voltage pulsed electric field treatment is from 0.5 kV / cm to 2 kV / cm, the pulse width is from 5 μs to 20 μs, and the repetition frequency is from 0.5 Hz to 5 Hz.

[0054] Specifically, the high-voltage pulsed electric field treatment (as one of the key steps to promote the formation of the collagen and hyaluronic acid complex, its parameter setting is carefully considered to maximize the transdermal absorption ability of the complex while ensuring the health and safety of the skin. The electric field strength is set between 0.5 kV / cm and 2 kV / cm. This range is sufficient to generate an instantaneous electroporation effect on the cell membrane, that is, to form transient nanoscale pores on the cell membrane without causing permanent damage. This electroporation effect provides a channel for the deep penetration of the collagen and hyaluronic acid complex, enabling the complex to bypass the natural barrier of the skin and directly reach the deep structure of the skin, thereby improving its bioavailability and therapeutic effect.

[0055] Furthermore, a buffer solution with a pH value of 6.5 to 7.5 can be selected as the buffer solution to adapt to different biological activity requirements.

[0056] Specifically, both collagen and hyaluronic acid are biological macromolecules, and their structural and functional properties highly depend on the pH value of the environment. At a pH value close to neutral, these two molecules can maintain their native conformations, avoiding conformational changes or denaturation caused by too high or too low pH values, which directly affects their biological activities and functions.

[0057] Furthermore, necessary post-treatment steps include filtration, purification or sterilization to ensure the safety and quality of the final product. Performance tests include the evaluation of the cytotoxicity of human dermal fibroblasts (HDF) in vitro models, the proliferation stimulation effect, and the ability to promote extracellular matrix synthesis.

[0058] Specifically, first, the potential harm of the complex to HDF cells is determined by measuring cytotoxicity, which usually involves exposing the cells to different concentrations of the complex and observing the cell viability and morphological changes. If the cytotoxicity test results indicate that the complex is safe for the cells, then its promoting effect on cell proliferation can be further evaluated. The test for the proliferation stimulation effect is usually completed by measuring the cell proliferation rate, which can be achieved by methods such as cell counting, MTT assay, or BrdU incorporation assay. Finally, the evaluation of the ability to promote extracellular matrix synthesis focuses on the effect of the complex on the synthesis of extracellular matrix (such as collagen, elastin, etc.), which can be completed by measuring the concentration of specific proteins in the cell culture medium or by observing the distribution and structure of the extracellular matrix through immunofluorescence staining. Through these comprehensive tests, the potential of the complex in promoting skin repair and regeneration can be comprehensively evaluated to ensure its safety and effectiveness in practical applications.

[0059] In summary, for the preparation method of the composition that promotes the interaction between collagen and hyaluronic acid, through finely designed chemical and physical treatment steps, the transdermal absorption rate and bioavailability of the collagen-hyaluronic acid complex are effectively enhanced. First, the application of the chemical cross-linking agent N-(β-maleimidopropionic acid) not only establishes stable chemical bonds between collagen and hyaluronic acid molecules but also optimizes the molecular structure of the complex, making it more suitable for penetrating the stratum corneum of the skin. Second, physical treatment, especially the combined application of ultrasonic waves and high-voltage pulsed electric fields, not only accelerates the formation of the complex but also breaks the physical barrier of the skin through mechanical effects and electroporation effects, creating favorable conditions for the deep penetration of the complex. The cavitation effect of ultrasonic waves helps to refine the complex particles, improving their uniformity and fluidity, while the high-voltage pulsed electric field increases the cell membrane permeability through short-term electroporation, providing an additional penetration path for the complex, thus significantly increasing the transdermal absorption rate and bioavailability of the complex.

[0060] Moreover, in the preparation method of the composition that promotes the interaction between collagen and hyaluronic acid, through a precisely controlled chemical cross-linking process, the structural stability of the complex has been greatly enhanced. This not only extends the shelf life of the complex but also reduces the possibility of its degradation during storage and use. In addition, stabilizers such as α-tocopherol and trehalose added to the complex, as well as activators such as epidermal growth factor and vitamin C, further enhance the antioxidant capacity and biological activity of the complex. The presence of stabilizers helps the complex resist oxidative stress and maintain its biological activity, while activators stimulate the activity of the complex and improve its ability to promote skin cell proliferation and repair. Therefore, the complex of the present invention can not only penetrate more effectively into the deep layer of the skin to improve the skin texture but also exert its moisturizing and repair effects more persistently, delaying the skin aging process and demonstrating excellent skin care effects, solving the problem that although there have been cases of compound use of collagen and hyaluronic acid, there are still challenges in improving the transdermal absorption rate, bioavailability, and stability of the complex. Traditional preparation methods often struggle to achieve these goals simultaneously, resulting in limited effects of the complex in skin care applications.

[0061] All relevant modules involved in this system are hardware system modules or functional modules that combine computer software programs or protocols in the prior art with hardware. The computer software programs or protocols themselves involved in this functional module are all well-known technologies to those skilled in the art and are not the improvements of this system. The improvement of this system lies in the interaction relationship or connection relationship between each module, that is, the overall structure of the system is improved to solve the corresponding technical problems to be solved by this system.

[0062] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A composition for promoting the interaction between collagen and hyaluronic acid, characterized in that, It includes collagen, hyaluronic acid, and a chemical crosslinking agent; The collagen is selected from type I collagen derived from fish skin, with a purity ≥ 99%, a molecular weight of approximately 300 kDa, and is formulated into a 1% (w / v) solution; The hyaluronic acid is obtained by microbial fermentation, with a molecular weight range of 1000 - 1500 kDa, and is formulated into a 0.5% (w / v) solution; The chemical crosslinking agent is N(β - maleimidopropionate), and its concentration is carefully controlled within 0.5% to 2%.

2. A method for preparing a composition that promotes the interaction between collagen and hyaluronic acid, characterized in that, It includes the composition for promoting the interaction between collagen and hyaluronic acid described in claim 1, and further includes the following steps: H1. Raw material preparation: Dissolve collagen and hyaluronic acid in deionized water separately under sterile conditions; H2. Pretreatment: Let the collagen and hyaluronic acid solutions stand in a phosphate buffer solution; H3. Chemical modification: Chemically modify collagen using N(β - maleimidopropionate); H4. Physical treatment: First, mix the collagen and hyaluronic acid solutions in a volume ratio of 1:1 using an ultrasonic treatment device, and then perform high - voltage pulsed electric field treatment; H5. Complex formation and optimization: Let the mixture of collagen and hyaluronic acid continue to stand in a buffer solution, and continuously monitor the particle size distribution of the complex using dynamic light scattering (DLS) technology until the complex is formed; H6. Post - treatment and performance testing: After the complex is formed, perform necessary post - treatment steps and conduct performance testing.

3. The composition for promoting the interaction between collagen and hyaluronic acid according to claim 1, wherein The complex also contains a stabilizer and an activator. The stabilizer is selected from one or more of "α - tocopherol", "trehalose", "phosphate - buffered saline (PBS)", and "polysorbate 20", and the activator is selected from one or more of "epidermal growth factor (EGF)", "L - proline", "green tea extract", and "vitamin C".

4. According to the method for preparing a composition for promoting the interaction between collagen and hyaluronic acid described in claim 2, in H2, the collagen and hyaluronic acid solutions stand in a phosphate buffer solution with a pH value of 6.5 to 7.5 for 12 to 24 hours.

5. According to the method for preparing a composition for promoting the interaction between collagen and hyaluronic acid described in claim 2, in H3, the molar ratio of N(β - maleimidopropionate) to collagen ranges from 1:5 to 1:20, and the chemical modification is carried out for 2 hours under the conditions of a pH value of 7.4 and a temperature of 25°C.

6. According to the method for preparing a composition for promoting the interaction between collagen and hyaluronic acid described in claim 2, in H4, the frequency of the ultrasonic treatment device is 15 kHz to 30 kHz, the power is 30 W to 70 W, the treatment time is 5 to 20 minutes, and the mixing mode of the ultrasonic treatment device is an intermittent mode, turning on for 2 seconds and turning off for 2 seconds per second.

7. A method for preparing a composition for promoting the interaction between collagen and hyaluronic acid according to claim 2, in H4, the electric field strength of the high-voltage pulsed electric field treatment is 0.5 kV / cm to 2 kV / cm, the pulse width is 5 μs to 20 μs, and the repetition frequency is 0.5 Hz to 5 Hz.

8. A method for preparing a composition for promoting the interaction between collagen and hyaluronic acid according to claim 2, in H5, the buffer solution can be a buffer solution with a pH value of 6.5 to 7.5 to meet different bioactivity requirements.

9. A method for preparing a composition for promoting the interaction between collagen and hyaluronic acid according to claim 2, in H6, the necessary post-treatment steps include filtration, purification or sterilization to ensure the safety and quality of the final product. The performance tests include the evaluation of the cytotoxicity of human dermal fibroblasts (HDF) in vitro models, the proliferation stimulation effect, and the ability to promote extracellular matrix synthesis.

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