Injection compound liquid and its preparation method and use

Abstract

The present invention relates to an injectable compound solution, its preparation method, and its use, belonging to the field of medical cosmetology. The injectable compound solution comprises the following components, calculated by weight: 0.1 to 35 parts of RGD peptide-modified sodium hyaluronate, 0.1 to 20 parts of collagen, and 0.05 to 25 parts of amino acids. The injectable compound solution exhibits high hydration, anti-inflammatory, repair, and skin quality improvement effects. The preparation method of the injectable compound solution is simple, convenient, and suitable for mass production.

Description

Technical Field

[0001] The present invention belongs to the field of medical cosmetology, and more specifically relates to an injection compound liquid and a preparation method and use thereof. Background Art

[0002] Sodium hyaluronate is widely distributed throughout the human body and is a major component of the epidermis and dermis. Its excellent water-retention properties have earned it the reputation of an ideal natural moisturizing factor. Human skin remains hydrated and smooth thanks to the presence of sodium hyaluronate.

[0003] However, as we age, our skin ages naturally due to endogenous factors and exogenous influences such as ultraviolet rays. This reduces the body's ability to synthesize sodium hyaluronate. Therefore, intradermal injections of sodium hyaluronate are often necessary to supplement the skin with sodium hyaluronate to enhance hydration and elasticity, and improve skin condition. However, unmodified sodium hyaluronate has limited hydrating and moisturizing properties in the human body.

[0004] In view of the problems existing in the existing technology, there is an urgent need to develop an injectable compound liquid that can enhance the hydrating and moisturizing properties, improve the hydrating and moisturizing effects of sodium hyaluronate, and allow sodium hyaluronate to fully play its role to enhance the skin care effect. Summary of the Invention

[0005] In view of the above-mentioned defects and problems of the prior art, the present invention develops an injection compound liquid and its preparation method and use.

[0006] The first aspect of the present invention provides an injection compound solution, the raw materials comprising the following components by mass: 0.1 to 35 parts of RGD peptide-modified sodium hyaluronate (HA-RGD), 0.1 to 20 parts of collagen, and 0.05 to 25 parts of amino acids; preferably, 1 to 10 parts of RGD peptide-modified sodium hyaluronate, 1 to 8 parts of collagen, and 0.3 to 12 parts of amino acids;

[0007] Preferably, the molecular weight of the RGD peptide-modified sodium hyaluronate is 1 to 2 million Da, and the endotoxin content is 0 to 0.05 Eu / mg.

[0008] Optionally, the collagen is recombinant collagen.

[0009] Optionally, the collagen is type I collagen or type III collagen (Col III).

[0010] The inventors of the present invention noticed that the hydrating and moisturizing effect of sodium hyaluronate in the body is closely related to the cell activity (cell migration, adhesion, and proliferation) at the injection site, and the injection compound solution needs to be as skin-friendly as possible. Therefore, the injection compound solution is prepared using raw materials such as collagen, amino acids, and sodium hyaluronate modified with RGD peptide. Among them, RGD peptide is a short peptide sequence containing arginine (Arg)-glycine (Gly)-aspartic acid (Asp). RGD peptide is on the extracellular matrix (ECM) protein in the body, and is the minimum sequence for mutual recognition between ECM proteins and transmembrane receptor integrins on cells.

[0011] First, sodium hyaluronate modified with collagen, amino acids and RGD peptide can be fully absorbed by the skin and shows good compatibility, and is not easy to damage the skin; secondly, each substance acts on the cells in the skin, providing a more suitable environment for the cells, so that the cells can give full play to their metabolism, migration and adhesion properties, and then rely on their own cell activity to play the role of hydrating skin and improving skin condition; in addition, because sodium hyaluronate modified with collagen, amino acids and RGD peptide cooperates with each other, it can play a good anti-inflammatory and repair role while playing a role in hydrating and moisturizing, so that the injection site can be repaired quickly and is not easy to become inflamed.

[0012] Specifically, after the injection compound solution is injected into the dermis, the decomposition time of the RGD peptide-modified sodium hyaluronate in the body is greatly slowed down. The RGD peptide in the RGD peptide-modified sodium hyaluronate specifically recognizes and binds to various integrins (such as αvβ3, α5β1, etc.) on the cell surface to form an RGD-integrin-cytoskeleton transmembrane structure. Through cell signal transduction, it mediates the migration and adhesion of cells containing integrin receptors containing RGD peptides in the body, allowing more cells to be enriched at the injection site, thereby helping sodium hyaluronate to replenish the extracellular matrix to a greater extent while combining with more surrounding cells and firmly forming a microenvironment suitable for cell growth. Sodium hyaluronate and cells, as a functional whole, constitute a three-dimensional water storage mechanism in the dermis, fully exerting the hydrating effect, and helping sodium hyaluronate to maximize its performance in moisturizing, hydrating, and improving skin condition.

[0013] When RGD peptide-modified sodium hyaluronate and collagen coexist in the injection compound, collagen, a major component of the extracellular matrix, not only provides essential nutrients to the dermis through its own properties, but also activates the cell viability of fibroblasts induced to migrate by the RGD peptide-modified sodium hyaluronate, promoting the normal induction of collagen regeneration by the cells. This cycle repeats itself, and the normal metabolism of the cells sustainably promotes the long-term hydration and moisturizing effects of the injection compound. Furthermore, because collagen is a helical, fibrous protein composed of three peptide chains, the entanglement between collagen and RGD peptide-modified sodium hyaluronate creates a larger three-dimensional space that prevents water from being lost, thereby helping to continuously store and slowly release moisture for the skin.

[0014] When preparing injection compound solution, recombinant collagen is more effective. Recombinant collagen is based on the original gene sequence of natural collagen, transferred into engineered cells through recombinant gene technology, fermented and purified. It is lower in cost than animal-derived collagen, has higher safety, lower allergenicity, higher affinity with the human body, and no risk of transmitting animal-derived diseases.

[0015] Recombinant collagen can be either type I or type III collagen. Specifically, when using type III collagen (Col III), the Col III molecule exhibits a sparse, mesh-like structure that surrounds cells, making the skin delicate and more elastic. Furthermore, the numerous hydrophilic groups on the exterior of the Col III molecule, such as amino and carboxyl groups, can also bind to water molecules, thereby enhancing the moisturizing and hydrating properties of the complex solution. Furthermore, Col III and sodium hyaluronate interact and bind to their respective receptors on cells. The mesh-like structure of sodium hyaluronate and collagen forms a three-dimensional cell-hyaluronic acid-collagen scaffold, maintaining the extracellular space of tissue cells, strengthening the cells, and promoting the accumulation of amino acids at the injection site. This further enhances the dermis's three-dimensional water storage mechanism, maintains tissue hydration, and promotes skin metabolism. The normal reproduction and metabolism of cells, along with the production of substances such as sodium hyaluronate and collagen, further enhance the hydration, moisturizing, and skin-improving properties of the entire injection complex solution.

[0016] It should be noted that collagen is present in the dermis of natural skin, accounting for 70% of the subcutaneous dermis. Amino acids are also essential components of the human body and do not pose any harm to the human body.

[0017] HA-RGD has a moderate molecular weight and extremely low endotoxin content, making it suitable for human skin absorption and not prone to skin irritation. Therefore, the compound injection solution prepared in this application is unlikely to have adverse effects on the skin.

[0018] Optionally, the raw material further comprises 1-25 parts by mass of polydeoxyribonucleotides (PDRN), preferably 5-10 parts by mass of the polydeoxyribonucleotides.

[0019] PDRN can expressly reach the dermis, improve the physiological conditions inside the skin at the injection point, restore the internal environment of the skin to normal, and promote rapid repair of the skin in the injection area. In addition, the small oligonucleotides (such as nucleotides, nucleosides, bases, etc.) and water molecules metabolized by PDRN are gradually released in the body, providing nutrients for cell growth, improving the proliferation and growth capacity of fibroblasts, and collaborating with sodium hyaluronate to improve skin condition. After the injection compound solution is injected, PDRN can quickly enter the cell-hyaluronic acid-collagen three-dimensional scaffold and be firmly fixed, thereby further promoting cell activity, allowing the cells and the various components in the injection compound solution to fully play their role, and enhancing the hydration, moisturizing, and skin condition improvement effects of the entire injection compound solution.

[0020] Optionally, the raw material further comprises 0.3 to 11 parts by mass of glutathione, preferably 1 to 5 parts by mass of glutathione.

[0021] Optionally, the raw materials further include 0.1 to 10 parts by mass of water-soluble vitamins. Preferably, the water-soluble vitamin is at least one of vitamin B2, vitamin B3 and vitamin C; more preferably, the water-soluble vitamin is niacinamide; more preferably, the niacinamide is 1 to 5 parts.

[0022] The combined effect of glutathione and niacinamide can improve the stability of the entire injection compound solution, and combined with HA-RGD, collagen and PDRN, it can play an anti-inflammatory role and promote rapid healing of the injection site.

[0023] In summary, the cooperation of HA-RGD, collagen, PDRN, glutathione, niacinamide and amino acids can enhance the hydrating, moisturizing and repairing effects of the injection compound solution, and enhance the hydrating and moisturizing effects of the entire injection compound solution by promoting cell activity.

[0024] Optionally, the amino acid is selected from at least one of glycine, alanine, proline, aspartic acid, leucine and histidine; preferably, the amino acid includes 0.01 to 6 parts of glycine, 0.01 to 6 parts of alanine and 0.01 to 15 parts of proline; more preferably, the amino acid includes 0.1 to 3 parts of glycine, 0.1 to 3 parts of alanine and 0.1 to 6 parts of proline.

[0025] Amino acids, as natural moisturizing factors, bind to water in the skin, regulating and storing it. They are crucial nutrients for maintaining skin hydration. The right amino acid composition can promote cell activation, significantly enhancing the hydrating and repairing effects of the injection compound. Glycine, alanine, and proline, as cellular nutrients, can also indirectly slow the degradation of sodium hyaluronate in the body.

[0026] Another aspect of the present invention provides a method for preparing the above-mentioned injection composite liquid, which is characterized by comprising the following steps:

[0027] (1) Weighing amino acids according to weight, and adding the amino acids to a phosphate buffer solution to obtain a solution A; wherein the pH of the phosphate buffer solution is 5.5 to 7.5;

[0028] (2) Weighing collagen according to weight and dissolving it in solution A to obtain solution B;

[0029] (3) dissolving RGD peptide-modified sodium hyaluronate in solution B to obtain solution C;

[0030] (4) Filtering solution C, sterilizing the filtrate and then aseptically filling it to obtain the injection compound solution.

[0031] During the preparation of the above-mentioned injection compound liquid, amino acids are first added to a phosphate buffer solution to obtain solution A, so that the amino acids are fully dispersed in solution A. Then, in the process of dissolving collagen in solution A, the amino acids can be quickly and evenly wrapped in the network molecular structure of the collagen; when RGD peptide-modified sodium hyaluronate is then added, the collagen wrapped with amino acids is directly entangled with the RGD peptide-modified sodium hyaluronate. In the prepared injection compound liquid, the amino acids are evenly dispersed, which can fully provide nutrients for the cells that migrate into the hyaluronic acid-collagen skeleton. The preparation process is simple and fast, and helps each material to fully exert its function, thereby allowing the entire injection compound liquid to promote cell activity, thereby continuously exerting its hydrating and moisturizing effects.

[0032] Optionally, step (1) further comprises weighing and adding nicotinamide in parts by weight; and step (2) further comprises weighing and adding polydeoxyribonucleotides and glutathione in parts by weight.

[0033] The preparation process of the above-mentioned injection compound liquid is simple and convenient, and the entire production process conditions are easy to achieve, which is conducive to achieving mass production.

[0034] Another aspect of the present invention provides the use of the aforementioned injection compound solution in the preparation of cosmetics, skin care products, pharmaceuticals, or medical devices for skin hydration. The injection compound solution is less likely to cause skin irritation and enhances its hydration and moisturizing properties primarily by promoting cell activity, aiding the skin's self-repair and hydration, thereby significantly enhancing the hydration and moisturizing effects of the entire injection compound solution.

[0035] In summary, this application has the following beneficial effects:

[0036] The injection compound liquid of the present invention uses sodium hyaluronate modified with collagen, amino acids and RGD peptide, and conducts cell signal transduction through the RGD-integrin-cytoskeleton transmembrane structure, thereby achieving cell migration and adhesion, and enrichment in the injection area, thereby enhancing cell activity at the injection site. Collagen further activates the cell activity of nearby cells, effectively inducing collagen regeneration, providing a more suitable environment for cells, and relying on the cells themselves to achieve better hydration. In addition, the collagen and the sodium hyaluronate modified with RGD peptide are entangled with each other, and the larger three-dimensional space can prevent water from being lost, and can also strengthen cells and promote the enrichment of amino acids at the injection site, thereby promoting the metabolism of skin tissue, further enhancing the three-dimensional water storage mechanism of the dermis, and thus helping to continuously play the role of storing and slowly releasing water for the skin, thereby enhancing the hydrating and moisturizing effect of the injection compound liquid. DETAILED DESCRIPTION

[0037] The present invention is further described below with reference to the examples. It should be understood that the examples are only used to further illustrate and explain the present invention and are not intended to limit the present invention.

[0038] Unless otherwise defined, technical and scientific terms used in this specification have the same meanings as commonly understood by those skilled in the art. Although methods and materials similar or identical to those described herein can be used in experiments or applications, the materials and methods are described herein below. In the event of conflict, the present specification, including definitions, will control. The materials, methods, and examples are provided for illustrative purposes only and are not intended to be limiting.

[0039] Unless otherwise specified, the test methods, detection methods and conventional experimental reagent preparation methods used in the embodiments of the present invention are all in accordance with conventional operations in the art.

[0040] Polydeoxyribonucleotides were purchased from Runhui Biotechnology (Weihai) Co., Ltd.

[0041] Type III collagen: purchased from Interfil Bioproducts Research Institute Co., Ltd., batch number 20221214-2.

[0042] RGD peptide-modified sodium hyaluronate was purchased from Shaanxi Future Biomatrix Co., Ltd. The molecular weight of RGD peptide-modified sodium hyaluronate was 800,000 to 1,000,000 Da, and the endotoxin content was 0 to 0.05 Eu / mg.

[0043] Example 1

[0044] The raw materials of the injection compound solution include the following components: collagen, amino acids, polydeoxyribonucleotides (PDRN), nicotinamide, glutathione, and RGD peptide-modified sodium hyaluronate (molecular weight 800,000-1,000,000 Da) in the preparation example. The amount of each component added is shown in Table 1.

[0045] The preparation method of the injection compound solution is as follows:

[0046] (1) Weighing amino acids and nicotinamide, and adding the amino acids and nicotinamide to a 0.9% by mass phosphate buffer solution at room temperature to prepare the final concentrations shown in Table 1, and stirring evenly to obtain solution A; wherein the pH of the phosphate buffer solution is 7;

[0047] (2) Weigh collagen, polydeoxyribonucleotides, and glutathione, dissolve them in solution A, and prepare the final concentrations as shown in Table 1 to obtain solution B;

[0048] (3) Dissolving RGD peptide-modified sodium hyaluronate in solution B to prepare the final concentration in Table 1 to obtain solution C;

[0049] (4) Filtering solution C, sterilizing the filtrate, and aseptically filling it into disposable pre-filled syringes of different specifications to obtain the injection compound solution.

[0050] Comparative Examples 1-7

[0051] The preparation methods of the composite injection solutions in Comparative Examples 1-7 differ from those in Example 1 in that the final concentrations of the components in Comparative Examples 1-7 are different. The final concentrations of the components in Comparative Examples 1-7 are shown in Table 1.

[0052] Table 1. Final concentration of each component in Example 1 and Comparative Examples 1-7

[0053]

[0054] Comparative Example 8

[0055] The difference from Example 1 is that the RGD peptide-modified sodium hyaluronate in Example 1 is replaced with an equal mass of sodium hyaluronate, and the preparation method is the same as that of Example 1.

[0056] Comparative Example 9

[0057] The difference from Example 1 is that the RGD peptide-modified sodium hyaluronate with a molecular mass of 800,000-1,000,000 Da in Example 1 is replaced with an equal amount of RGD peptide-modified sodium hyaluronate with a molecular mass of 2.6-3,000,000 Da, and the preparation method is the same as that of Example 1.

[0058] Comparative Example 10

[0059] The difference from Example 1 is that the preparation method of the injection composite solution is:

[0060] (1) Weighing amino acids and nicotinamide, and adding the amino acids and nicotinamide to a 0.9% by mass phosphate buffer solution at room temperature to prepare a final concentration of the corresponding substances in Example 1 in Table 1, and stirring uniformly to obtain solution A; wherein the pH of the phosphate buffer solution is 7;

[0061] (2) Weighing polydeoxyribonucleotides and glutathione, dissolving them in solution A, and preparing the final concentrations of the corresponding substances in Example 1 in Table 1 to obtain solution B;

[0062] (3) Dissolving collagen and RGD peptide-modified sodium hyaluronate in solution B to prepare the final concentrations of the corresponding substances in Example 1 in Table 1 to obtain solution C;

[0063] (4) Filtering solution C, sterilizing the filtrate, and aseptically filling it into disposable pre-filled syringes of different specifications to obtain the injection compound solution.

[0064] Performance Testing

[0065] Experimental Example 1 Cell activity verification experiment

[0066] Fibroblasts are the primary cells in the dermis of the skin. This test evaluated the effectiveness of the prepared injection compound solutions in improving skin condition by independently injecting the injection compound solutions prepared in Example 1 and Comparative Examples 1-10. The results showed that the prepared injection compound solutions improved skin fibroblast activity. In vitro cell migration, cell adhesion, and cell proliferation experiments can simulate the in vivo processes of cell migration, adhesion, and proliferation, respectively. Therefore, cell migration, cell adhesion, and cell proliferation activity can be used to effectively characterize the cell activity-promoting effects of the injection compound solutions.

[0067] It should be noted that when the injection compound solution promotes faster cell migration, more adherent and proliferating cells, better cell activity, and better skin condition improvement performance. The specific test method is as follows:

[0068] (1) Scratch assay to detect cell migration activity

[0069] Use a marker to evenly draw horizontal lines on the back of a 6-well plate, perpendicular to the centerline of the wells. Count the cells after digestion and seed them into the 6-well plate at a density of 5x105 cells / well. Incubate the plate in an incubator (37°C, 5% CO2) for 24 hours. When the cell plating rate reaches 90%, perform cell streak (using a 1 mL pipette tip to draw a horizontal line perpendicular to the centerline of the 6-well plate).

[0070] After the scratch is made, the supernatant is discarded, 1 mL of PBS is added to each well, and a photo is taken under a 4x microscope. The scratch morphology at 0 h is recorded, and the photo position is marked. After discarding PBS, the injection compound solution prepared in Example 1 and the injection compound solution prepared in Comparative Examples 1-10 are added respectively, with a sample volume of 2 mL per well, and the wells with the above-mentioned sample additions are used as the experimental group, and a well without the sample addition is used as the blank control group. Afterwards, 100 uL of serum-free DMEM culture medium is added to each well of the experimental group and the blank control group, and then the 6-well plate is placed in an incubator (37 ° C, 5% CO2) for incubation and culture, and the cells are observed and photographed under a 4x microscope for 24 h.

[0071] Image J software was used to calculate the migration rate by calculating the scratch area of ​​each sample group at each time and in each photo. Cell migration rate (%) = (scratch area at 0 h - scratch area after 24 h) / scratch area at 0 h × 100%.

[0072] The test results of Example 1 and Comparative Examples 1-10 are shown in Table 3; the cell migration rate of the control group was 11.17%.

[0073] (2) Detection of cell adhesion activity by adherent cell number assay

[0074] The injection composite solution prepared in Example 1 and the injection composite solution prepared in Comparative Examples 1-10 were spread onto the bottom of a 24-well plate (not TC-treated) at 500 μL / well and gently shaken to mix evenly. These wells served as the experimental group, and one well without sample served as the blank control. Subsequently, 100 μL of serum-free DMEM medium was added to each well of the experimental and blank control groups.

[0075] Afterwards, cells were digested and counted, and seeded into pre-treated 24-well plates at a density of 1x10⁴ cells / well, with 300 μL of cell suspension per well. After addition, the 24-well plates were incubated in an incubator (37°C, 5% CO₂). After 24 hours of incubation, cells were observed, photographed, and counted within the field of view. Relative cell adhesion rate (%) was calculated as: number of adherent cells in the test sample / number of adherent cells in the blank control group × 100%.

[0076] The test results of Example 1 and Comparative Examples 1-10 are shown in Table 3; the relative cell adhesion rate of the control group was 103.62%.

[0077] (3) MTT assay to detect cell proliferation

[0078] Human fibroblasts were revived using DMEM medium containing 10% FBS and cultured at 37°C in a humidified atmosphere of 5% CO2. Cells were passaged 2-3 times until they reached the logarithmic growth phase. The cells were trypsinized, harvested, and adjusted to a cell concentration of 1×105 cells / ml for cell experiments.

[0079] The above cells were mixed with serum-free DMEM medium to prepare an experimental group with a concentration of 0.2 g / mL. A serum-free DMEM medium without the above cells was used as a control group. The cell suspension was added to a 96-well plate at 100 μl per well, for a total of 1×104 cells / well. The cells were incubated at 37°C, 5% CO2, and saturated humidity for 24 hours. After the incubation period, the medium was aspirated, and 100 μl of the injection compound prepared in Example 1 and the injection compound prepared in Comparative Examples 1-10 were added to each well of the experimental group. The blank control group was left untreated. The 96-well plate was then placed in a 37°C, 5% CO2, and saturated humidity incubator for 24 hours.

[0080] After 24 hours, the culture medium was aspirated, washed with PBS, and the PBS discarded. The MTT cytotoxicity test was performed. 50 μL of MTT (1 mg / mL) solution was added to each test well and incubated in a 37°C, 5% CO2 saturated humidity incubator for 2 hours. After incubation, the liquid in the culture plate was discarded, and 100 μL of isopropanol solution was added to each well. Mix thoroughly in the dark with shaking for 30 minutes. The plates were then placed in a microplate reader and absorbance was measured at a detection wavelength of 570 nm and a reference wavelength of 650 nm. The relative cell proliferation rate (RCR) was calculated based on the absorbance: average absorbance of the test sample / average absorbance of the blank group × 100%.

[0081] The test results of Example 1 and Comparative Examples 1-10 are shown in Table 3; the relative cell proliferation rate in the control group was 98.16%.

[0082] Table 3. Performance test results of Example 1 and Comparative Examples 1-10

[0083]

[0084]

[0085] Through the above experiments and the data in Table 3, the researchers found that the preparation method within the scope of this application can obtain an injection compound liquid (see Example 1) that can greatly increase cell activity. The injection compound liquid prepared by this application is mainly due to the addition of sodium hyaluronate, collagen, amino acids, glutathione and polydeoxyribonucleotides modified with RGD peptide. The above substances cooperate with each other to improve cell migration rate and cell relative adhesion rate, and also help to effectively improve the relative cell proliferation rate. The injection compound liquid prepared by this application can effectively improve the hydration effect by improving cell activity in the body. In particular, in Example 1, the injection compound liquid can achieve a cell migration rate of 61.42%, a cell relative adhesion rate of 168.52%, and a cell relative proliferation rate of 148.05%. When the addition amount of each component is not appropriate, the cell activity is greatly reduced compared to the cell activity measured in Example 1. It can be seen that the various substances in the formula play a synergistic role. Therefore, when preparing the injection compound liquid, the addition amount of each component needs to be within a suitable range.

[0086] Based on the data in Table 3, the following conclusion can also be drawn: A comparison between Example 1 and Comparative Example 1 shows that when collagen and amino acids are not added during the preparation of the injection composite solution, despite the addition of a sufficient amount of RGD peptide-modified sodium hyaluronate, the cell migration rate, relative cell adhesion rate, and relative cell proliferation rate are generally reduced compared to Example 1, resulting in a very poor effect of enhancing cell activity caused by the injection composite solution. Furthermore, referring to the data between Comparative Example 4 and Example 1, only when collagen, amino acids, and RGD peptide-modified sodium hyaluronate are all added can cell activity be significantly enhanced.

[0087] Similarly, according to Example 1 and Comparative Examples 2-6, collagen, amino acids and RGD peptide-modified sodium hyaluronate play a synergistic role with each other, and none of them can be missing. The lack of any component will lead to a poor effect of enhancing the cell activity caused by it. Even if the amount of a single component added to the collagen, amino acids and RGD peptide-modified sodium hyaluronate is large, the expected effect is not achieved. For example, according to the data of Comparative Example 1 and Example 7, even if the final concentration of RGD peptide-modified sodium hyaluronate is large, it cannot achieve the effect of effectively enhancing cell activity. This may be because when RGD peptide-modified sodium hyaluronate is excessive, it is easy to cause nutrients such as amino acids, or even collagen to be coated therein. In the subsequent preparation of the injection compound solution, it is easy to remain in the filter residue and is not easy to fully play its role; and the various nutrients coated in the RGD peptide-modified sodium hyaluronate cannot effectively activate cells, so after the injection compound solution prepared in Example 7 is injected, cell activity is reduced.

[0088] Furthermore, the data from Comparative Examples 8-9 indicate that when the RGD peptide-modified sodium hyaluronate in Example 1 was replaced with an equal amount of sodium hyaluronate, the resulting injection composite solution failed to demonstrate superior performance in promoting cell activity. Furthermore, the inappropriate molecular weight of the RGD peptide-modified sodium hyaluronate also significantly impacted final cell activity. This is likely due to the structure of the RGD peptide-modified sodium hyaluronate significantly influencing cell migration rate, relative cell adhesion rate, and relative cell proliferation rate.

[0089] According to the data of Example 1 and Comparative Example 10, when the injection compound liquid is not prepared according to the preparation steps provided in this application, the obtained injection compound liquid still cannot maximize the effect of activating cells. This may be because the different addition order affects the interaction mode between the molecular structures of the components, thereby affecting the full play of the advantages of the components in the injection compound liquid, resulting in the injection compound liquid being unable to effectively improve cell activity.

[0090] Experimental Example 2 Moisturizing Performance Test

[0091] Transepidermal water loss (TEWL) represents the repair status of the skin's stratum corneum. A larger value indicates a greater ease of water loss. The TEWL value evaluates the skin's barrier function to moisture by measuring the rate of water loss from the skin's surface, and is currently a commonly used method for evaluating moisturizing efficacy. Therefore, this composition uses the TEWL value to evaluate the moisturizing and hydrating effects of the composition after injection. It should be noted that the injection is generally injected into the face to provide hydration and moisturizing effects. The specific test method for moisturizing performance testing is as follows:

[0092] Eighteen volunteers were randomly divided into six groups, A, B, C, D, E, and F, with three participants in each group. The test area skin was cleaned and disinfected; a topical anesthetic ointment was applied and sealed for 50 minutes, then the topical anesthetic ointment was removed. Group A received the injection compound prepared in Example 1 on the left cheek and the injection compound prepared in Comparative Example 1 on the right cheek; Group B received the injection compound prepared in Comparative Example 1 on the left cheek and the injection compound prepared in Comparative Example 4 on the right cheek; Group C received the injection compound prepared in Example 1 on the left cheek and the injection compound prepared in Comparative Example 4 on the right cheek; Group D received the injection compound prepared in Example 1 on the left cheek and the injection compound prepared in Comparative Example 8 on the right cheek; Group E received the injection compound prepared in Example 1 on the left cheek and the injection compound prepared in Comparative Example 9 on the right cheek; Group F received the injection compound prepared in Example 1 on the left cheek and the injection compound prepared in Comparative Example 10 on the right cheek.

[0093] After the injection, the face was cleaned with saline-soaked gauze. TEWL values ​​at the injection site were observed and recorded at week 0 and week 4 after the injection. The average value was calculated for each group of data. The test results of the above test are shown in Table 4.

[0094] Table 4. Test results of moisturizing performance test

[0095]

[0096] As can be seen from Table 4, based on the data from Groups A to C, when at least one component is missing from the injection compound solution, the moisturizing and hydrating properties of the skin are significantly reduced. It should be noted that the moisturizing and hydrating effects of Comparative Examples 2-3 are similar to those of Comparative Example 1, and the moisturizing and hydrating effects of Comparative Examples 5-6 are similar to those of Comparative Example 4, and therefore, they are not further described here. Combining the data from Tables 3 and 4, it can be seen that when the injection compound solution is injected into the face and cell activity is strong, the injection compound solution has a good moisturizing effect on the face, indicating that the water retention and moisturizing properties of the injection compound solution are positively correlated with cell activity.

[0097] Data from Groups D and E indicate that while both the injection compound containing RGD peptide-modified sodium hyaluronate and the injection compound containing sodium hyaluronate of the same molecular weight can reduce transepidermal water loss (TEWL), overall, the hydrating and moisturizing effects of the injection compound containing sodium hyaluronate of the same molecular weight are not very good. This suggests that RGD peptide-modified sodium hyaluronate plays an important role in the injection compound and cannot be replaced by sodium hyaluronate. Furthermore, RGD peptide-modified sodium hyaluronate of the appropriate molecular weight further contributes to enhancing the hydrating and moisturizing effects of the entire injection compound. Furthermore, data from Group F indicate that the preparation steps of the injection compound also affect its hydrating properties. It should also be noted that when the concentration of RGD peptide-modified sodium hyaluronate is high, the resulting injection compound is not very hydrating and moisturizing.

[0098] It is worth noting that through the above analysis, it can be clearly seen that the moisturizing effect of the injection compound liquid is related to the strength of its promotion of cell activity in the body. The stronger the cell activity of the cells in the body promoted by the injection compound liquid, the better its moisturizing effect and the better the skin improvement effect.

[0099] Summary: When used, the injection compound liquid of the present invention conducts cell signaling through the RGD-integrin-cytoskeleton transmembrane structure, realizes cell migration, adhesion and enrichment to the injection area, and enhances the cell activity at the injection site. Collagen can also activate the cell activity of nearby cells, effectively inducing collagen regeneration, and working in conjunction with amino acids to provide a more suitable environment for cells, relying on their own cells to achieve better hydration. Collagen and RGD peptide-modified sodium hyaluronate are entangled with each other, and the larger three-dimensional space can make it difficult for water to be lost. It can also strengthen cells and promote the continuous enrichment of amino acids to the injection site, promote the metabolism of skin tissue, and further enhance the three-dimensional water storage mechanism of the dermis, thereby helping to continuously play the role of storage-slow release of water for the skin, thereby enhancing the hydrating and moisturizing effect of the injection compound liquid. And after experiments, it was verified that the injection compound liquid prepared by the present application achieves the effect of efficient hydration and moisturizing by enhancing cell activity.

[0100] It should be understood that the present invention disclosed is not limited only to the specific method, scheme and material of description, because these all can change.It should also be understood that the term used herein is only for the purpose of describing specific embodiment scheme, rather than being intended to limit the scope of the present invention, and the scope of the present invention is only limited to the appended claims.

[0101] Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein.Such equivalents are also intended to be encompassed by the appended claims.

Claims

1. An injection compound solution, characterized in that: The raw materials include the following components by mass: 0.1-35 parts of RGD peptide-modified sodium hyaluronate, 0.1-20 parts of collagen, 0.05-25 parts of amino acids, 0.1-10 parts of nicotinamide, 0.3-11 parts of glutathione, and 1-25 parts of polydeoxyribonucleotides; the molecular weight of the RGD peptide-modified sodium hyaluronate is 1 to 2 million Da; the amino acids include glycine, alanine, and proline; The preparation method of the injection composite liquid comprises: (1) Weighing amino acids and nicotinamide according to parts by weight, and adding the amino acids and nicotinamide to a phosphate buffer solution to obtain a solution A; wherein the pH of the phosphate buffer solution is 5.5 to 7.5; (2) Weigh collagen, polydeoxyribonucleotides, and glutathione according to their weight ratios and dissolve them in solution A to obtain solution B; (3) dissolving RGD peptide-modified sodium hyaluronate in solution B to obtain solution C; (4) Filter solution C, sterilize the filtrate, and then aseptically fill it to obtain the injection compound solution.

2. The compound injection solution according to claim 1, characterized in that The collagen is selected from recombinant collagen.

3. The compound injection solution according to claim 2, characterized in that: The collagen is selected from type I collagen or type III collagen.

4. The compound injection solution according to claim 1, characterized in that The polydeoxyribonucleotides are present in an amount of 5-10 parts.

5. The compound injection solution according to claim 1, characterized in that The glutathione is 1 to 5 parts.

6. The compound injection solution according to claim 1, characterized in that The nicotinamide is 1 to 5 parts.

7. The compound injection solution according to claim 1, characterized in that: The amino acids include 0.01 to 6 parts of glycine, 0.01 to 6 parts of alanine, and 0.01 to 15 parts of proline.

8. The compound injection solution according to claim 7, characterized in that: The amino acids include 0.1 to 3 parts of glycine, 0.1 to 3 parts of alanine, and 0.1 to 6 parts of proline.

9. The compound injection solution according to claim 1, characterized in that: The amount of the RGD peptide-modified sodium hyaluronate is 1 to 10 parts, the amount of the collagen is 1 to 8 parts, and the amount of the amino acid is 0.3 to 12 parts.

10. Use of the injection compound solution according to any one of claims 1 to 9 in the preparation of a medicine or medical device product for retaining moisture in the skin.

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