Wound repair composition and preparation method thereof

Abstract

The invention relates to a wound repairing composition and a preparation method thereof. The wound repairing composition comprises the following components: modified hyaluronic acid containing RGD peptide and collagen. The wound repairing composition can greatly improve the skin bioavailability of collagen, improve the moisturizing and repairing effects of the composition acting on the skin, and accelerate the wound healing time.

Description

Technical Field

[0001] The present application relates to the field of wound repair products, and specifically to a wound repair composition and a preparation method thereof. Background Art

[0002] As people pursue higher standards for skin care and beauty, the number of people who frequently undergo medical beauty projects such as laser treatment, photorejuvenation, and acid peeling is also increasing. However, different medical aesthetics will cause different degrees of wounds, which will also affect the experience of those seeking beauty. In order to accelerate wound healing and prevent postoperative rebound, in addition to daily medicines, some repair products (such as dressings) should be used in conjunction with wound healing and skin repair. Unlike traditional gauze dressing products, new dressing products such as hydrogel dressings, liquid / emulsion dressings or mask-like wound dressings have a very obvious promoting effect in promoting epithelialization, guiding tissue regeneration, and promoting wound healing. The moisturizers in these dressings can maintain the moisture of the wound surface, which is conducive to the dissolution of necrotic tissue, maintain the low oxygen state of the local microenvironment of the wound surface, provide a good healing environment for the wound surface, and are conducive to cell proliferation, differentiation and migration. It retains the active substances in the exudate and promotes the release of active substances, reduces the chance of infection, and does not form dry scabs, thereby significantly improving the healing effect of the wound.

[0003] Currently, the most widely used dressings are mainly sodium hyaluronate medical dressings and collagen medical dressings. The core ingredients of these two types of dressings are hyaluronic acid and collagen, respectively. There are also many problems in the use of these two types of ingredients.

[0004] Hyaluronic acid (HA) is an important component of the extracellular matrix (ECM). HA has a special water-retaining effect. In theory, HA can bind about 1,000 times its own weight of water, and is known as an ideal natural moisturizing factor. However, with age, the skin is affected by endogenous factors and exogenous factors such as ultraviolet rays, which causes natural aging, and the ability of cells in the body to synthesize HA decreases. In addition, if the skin is damaged, the ability to synthesize HA is even slower. Therefore, it is necessary to supplement and repair the hyaluronic acid lost in the skin in medical products that assist in wound healing. Due to the hydrophilicity of hyaluronic acid, it is not conducive to close binding with skin cells, so that the number of cells it acts on and the effect of promoting cell activity are limited, so that its hydrating and moisturizing properties are limited, and the effect of wound repair is slow.

[0005] Collagen is the functional protein with the highest ECM content. Collagen is widely used in the field of medical device products to improve skin elasticity and moisture content, reduce the appearance of wrinkles and fine lines, and make the skin healthier. However, collagen is generally believed to have the following problems: collagen molecules are as high as tens of thousands of Daltons and are extremely hydrophilic. Collagen cannot act on the skin and cells for specific binding. The number of cells it acts on is limited, and the skin bioavailability is low, which limits the repair and moisturizing effect of wound repair products. Collagen is a protein, and protein allergies are not uncommon in the human body. Although in theory recombinant collagen is safer than animal-derived collagen, if the production process is not well controlled, the possibility of causing allergies is also high.

[0006] The addition of collagen to traditional medical wound repair products cannot fully, stably and long-term exert its function, nor can it work well in synergy with hyaluronic acid and other ingredients to exert a repair effect. Summary of the invention

[0007] Based on this, it is necessary to provide an extracellular matrix-based wound repair composition and a preparation method thereof in one embodiment of the present application. The wound repair composition can greatly improve the skin bioavailability of collagen, enhance the moisturizing and repairing effects of the composition on the skin, and accelerate the wound healing time.

[0008] The technical solution includes:

[0009] A wound repair composition comprises the following components: modified hyaluronic acid containing RGD peptide and collagen.

[0010] In one embodiment, the collagen comprises recombinant human collagen.

[0011] Optionally, the recombinant human collagen includes recombinant human type III collagen.

[0012] Optionally, the recombinant human type III collagen includes the recombinant human collagen shown in SEQ ID NO:1 and / or SEQ ID NO:2.

[0013] In one embodiment, the collagen is a recombinant human collagen comprising the sequence shown in SEQ ID NO: 1, and the wound repair composition has a thermal decomposition inflection point between 230°C and 250°C.

[0014] In one embodiment, a moisturizing agent is further included; preferably, the moisturizing agent includes one or more of glycerol, dextran, sodium alginate, and trehalose.

[0015] In one embodiment, the relative molecular weight of the modified hyaluronic acid containing RGD peptide is 80 Da-500 Da.

[0016] In one embodiment, the grafting rate of the modified hyaluronic acid containing RGD peptide is 1.0%-10.0%.

[0017] In one embodiment, the wound repair composition includes modified hyaluronic acid containing RGD peptide, collagen, glycerol and β-glucan.

[0018] Preferably, the wound repair composition comprises the following raw materials in weight percentage: 0.005%-5% of modified hyaluronic acid containing RGD peptide, 0.005%-5% of collagen, 0.5%-20% of glycerol and 0.01%-10% of β-glucan.

[0019] More preferably, the wound repair composition comprises the following raw materials in weight percentage: 0.01%-0.2% of modified hyaluronic acid containing RGD peptide, 0.01%-1% of collagen, 1%-10% of glycerol and 0.05%-5% of β-glucan.

[0020] In one embodiment, the wound repair composition also includes a preservative.

[0021] Optionally, in the wound repair composition, the weight percentage of the preservative is 0.1%-5%.

[0022] Optionally, in the wound repair composition, the weight percentage of the preservative is 0.1%-2%.

[0023] Optionally, the preservative comprises pentylene glycol.

[0024] The method for preparing the wound repair composition comprises the following steps: mixing the modified hyaluronic acid containing RGD peptide and the collagen to prepare the wound repair composition.

[0025] Optionally, the following steps are included:

[0026] S10: mixing the glycerol and water, stirring and heating after mixing;

[0027] S20: mixing the modified hyaluronic acid containing RGD peptide, the collagen, the β-glucan and water;

[0028] S30: adding the material in step S20 to the solution obtained in step S10, stirring, and discharging the material when the temperature drops to ≤45°C.

[0029] The wound repair composition is used in preparing cosmetics, medicines or medical devices for skin moisturizing and / or repairing.

[0030] Compared with the traditional technology, this application has the following beneficial effects:

[0031] The present application invents a wound repair composition of RGD peptide-modified hyaluronic acid combined with collagen and other raw materials, which has the following beneficial effects:

[0032] (1) The RGD in RGD peptide-modified hyaluronic acid enables functional ingredients such as hyaluronic acid and collagen to act on more cells, making the entire RGD peptide-modified hyaluronic acid composition closer to the ECM and more compatible with the skin, thereby improving the microenvironment of the entire RGD peptide-modified hyaluronic acid composition for cells, providing a good healing environment for the wound and surrounding skin, and effectively shortening the wound healing time.

[0033] (2) RGD peptide-modified hyaluronic acid and a recombinant type III humanized collagen described in the present application can combine to form an association and act on skin cells simultaneously. The RGD peptide-modified hyaluronic acid can improve the bioavailability of collagen, accelerate the connection between collagen and cells, and increase cell adhesion, thereby greatly reducing the inflammatory response and adverse reactions after wound healing. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0035] Figure 1 Comparison of the difference in moisture content of the stratum corneum at each test point between the experimental group-1# and the control group; Note: P < 0.05 indicates that the data have statistical differences, and the significance is *; P < 0.01 indicates that the data have significant statistical differences, and the significance is **; P < 0.001 indicates that the test data have extremely significant statistical differences, and the significance is ***.

[0036] Figure 2 Comparison of the difference in moisture content of the stratum corneum at each test point between the experimental group-2# and the control group; Note: P < 0.05 indicates that the data have statistical differences, and the significance is *; P < 0.01 indicates that the data have significant statistical differences, and the significance is **; P < 0.001 indicates that the test data have extremely significant statistical differences, and the significance is ***.

[0037] Figure 3Comparison of the difference in moisture content of the stratum corneum at each test point between the experimental group-3# and the control group; Note: P < 0.05 indicates that the data have statistical differences, and the significance is *; P < 0.01 indicates that the data have significant statistical differences, and the significance is **; P < 0.001 indicates that the test data have extremely significant statistical differences, and the significance is ***.

[0038] Figure 4 Comparison of the difference in moisture content of the stratum corneum at each test point between the experimental group-4# and the control group; Note: P < 0.05 indicates that the data have statistical differences, and the significance is *; P < 0.01 indicates that the data have significant statistical differences, and the significance is **; P < 0.001 indicates that the test data have extremely significant statistical differences, and the significance is ***.

[0039] Figure 5 Schematic diagram of animal experiment grouping.

[0040] Figure 6 Comparison of rabbit skin repair test results.

[0041] Figure 7 This is a bar graph showing the effective rate of rabbit skin repair at different times.

[0042] Figure 8 These are the pathological HE results of the rabbit skin repair test.

[0043] Fig. 9 The thermal decomposition curve of the sample. DETAILED DESCRIPTION

[0044] In order to make the above-mentioned purposes, features and advantages of the present application more obvious and easy to understand, the specific implementation methods of the present application are described in detail below. In the following description, many specific details are set forth to facilitate a full understanding of the present application. However, the present application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present application, so the present application is not limited by the specific embodiments disclosed below.

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0046] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0047] In this article, the extracellular matrix (ECM) is a complex three-dimensional reticular structure and an important component of the extracellular environment of tissues. It is composed of fiber-forming structural molecules, non-fiber-forming structural molecules and matrix-cell proteins, etc. It contains a variety of collagens, proteoglycans (PGs) and glycosaminoglycans (GAGs), elastin and elastic fibers, laminin, fibronectin and other proteins / glycoproteins, etc. These components have significant division of labor and regulatory effects on the morphology, growth, division, differentiation and apoptosis of tissue cells. ECM has multiple functions and is an important component of the cell microenvironment.

[0048] At present, the addition of collagen to medical wound dressings cannot fully, stably and long-term exert its function, nor can it work well in synergy with hyaluronic acid, mussel mucin, silk fibroin, β-glucan, etc. to exert a repair effect.

[0049] Therefore, one embodiment of the present application provides a wound repair composition comprising modified hyaluronic acid and collagen containing RGD peptide.

[0050] The present application adds modified hyaluronic acid (HA-RGD for short) containing RGD peptide to the wound repair composition. Through the integrin site on HA-RGD, it combines with collagen, mussel mucin or silk fibroin, which can simulate ECM to a large extent, provide a suitable microenvironment for cells, and provide a good healing environment for the wound and surrounding skin. It has great prospects in the application of assisting wound healing.

[0051] As the main structural protein of ECM, collagen can regulate various cell behaviors such as cell development, adhesion and migration. Collagen has a wide range of applications in medicine, health care and beauty. In the medical field, collagen is widely used in tissue engineering and regenerative medicine to repair tissue defects and trauma.

[0052] Cell adhesion occurs in the maintenance of tissue structure, wound healing, immune response, tumor metastasis, and integration of biomaterials and tissues. The interaction between cells and collagen is the fundamental problem of using collagen to solve skin damage. Cells are not connected to collagen by direct contact. Cells have a class of transmembrane proteins called integrins. This structure, together with adaptor proteins that play an auxiliary fixation role, plays a role in connecting cells to the extracellular matrix. Cell adhesion provides an important means to study the molecular mechanism of the interaction between cells, ECM and biomaterials, and cell adhesion has also become one of the important indicators for evaluating the biocompatibility of materials.

[0053] The RGD peptide in HA-RGD is a short peptide sequence mainly composed of arginine (Arg)-glycine (Gly)-aspartic acid (Asp), which is widely present in the body. This small molecule peptide exists on the extracellular matrix (ECM) protein of the human body and is the minimum sequence for ECM protein to recognize each other with the transmembrane receptor integrin expressed on almost every cell. When HA-RGD acts on the wound skin, RGD binds to various integrins (α v β 3 , α 5 β1) specifically recognizes and binds to form the RGD-integrin-cytoskeleton transmembrane information system, which regulates cell migration and adhesion and allows more cells to accumulate.

[0054] In one specific example, the collagen includes recombinant human collagen.

[0055] In a specific example, the recombinant human collagen includes recombinant human type III collagen.

[0056] In a specific example, the recombinant human type III collagen includes the recombinant human collagen shown in SEQ ID NO:1 and / or SEQ ID NO:2.

[0057] In a specific example, the collagen is a recombinant human collagen comprising the sequence shown in SEQ ID NO: 1, and the wound repair composition has a thermal decomposition inflection point between 230°C and 250°C.

[0058] The present application found that the use of recombinant humanized type III collagen with a specific sequence can specifically bind to HA-RGD through intermolecular forces to form an association with a specific structure. HA-RGD makes the recombinant humanized type III collagen (SEQ ID NO: 1) have better compatibility with the skin and skin bioavailability. When used, it is evenly applied to non-chronic wounds to form a thin film on the wound surface, isolating the invasion of harmful substances such as external bacteria and dust, improving the living environment of skin cells, reducing discomfort symptoms such as infection and pain, promoting the metabolism of skin tissue, and restoring the skin to a healthy state. It can quickly and effectively restore obvious skin dysfunction caused by medical aesthetics, such as inflammatory reactions such as erythema and edema.

[0059] In a specific example, the wound repair composition further comprises a moisturizer. Optionally, the moisturizer comprises one or more of glycerol, dextran, sodium alginate, and trehalose. Preferably, the moisturizer is glycerol and dextran. Preferably, the dextran comprises β-glucan.

[0060] In a specific example, the relative molecular weight of the modified hyaluronic acid containing the RGD peptide is 80 Da-500 Da. Preferably, the relative molecular weight of the modified hyaluronic acid containing the RGD peptide is 120 Da-130 Da.

[0061] In a specific example, the grafting rate of the modified hyaluronic acid containing RGD peptide is 1.0%-10.0%. Preferably, the grafting rate of the modified hyaluronic acid containing RGD peptide is 1.0%-5.0%, preferably, the grafting rate of the modified hyaluronic acid containing RGD peptide is 5.0%.

[0062] In a specific example, the wound repair composition includes modified hyaluronic acid containing RGD peptide, collagen, glycerol and β-glucan.

[0063] Preferably, the wound repair composition comprises the following raw materials in weight percentage: 0.005%-5% modified hyaluronic acid containing RGD peptide, 0.005%-5% collagen, 0.5%-20% glycerol and 0.01%-10% β-glucan. More preferably, the wound repair composition comprises the following raw materials in weight percentage: 0.01%-0.2% modified hyaluronic acid containing RGD peptide, 0.01%-1% collagen, 1%-10% glycerol and 0.05%-5% β-glucan. By introducing the modified hyaluronic acid containing RGD peptide and compounding with other components, the moisturizing effect of the composition on the skin is enhanced, the moisturizing time is enhanced, and the repair effect is better and faster.

[0064] In a specific example, the weight percentage of modified hyaluronic acid containing RGD peptide in the wound repair composition can be selected as 0.01%, 0.03%, 0.05%, 0.08%, 0.1%, 0.12%, 0.15%, 0.18%, 0.2%, or a range between any two of the above values.

[0065] In a specific example, the weight percentage of collagen in the wound repair composition can be selected as 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, or a range between any two of the above values.

[0066] In a specific example, the weight percentage of glycerol in the wound repair composition can be selected as 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or a range between any two of the above values.

[0067] In a specific example, the weight percentage of β-glucan in the wound repair composition can be selected as 0.05%, 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, or a range between any two of the above values.

[0068] In a specific example, the wound repair composition includes the following raw materials in weight percentage: 0.01%-0.05% modified hyaluronic acid containing RGD peptide, 0.01%-0.2% collagen, 1%-3% glycerol and 0.05%-0.2% β-glucan.

[0069] In one specific example, the wound repair composition further includes a preservative.

[0070] In a specific example, in the wound repair composition, the weight percentage of the preservative is 0.1%-5%, which can be 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, or a range between any two of the above values. In a specific example, in the wound repair composition, the weight percentage of the preservative is 0.1%-2%. The inventors of this application are aware of the restrictions on the amount of preservatives in the national "Technical Specifications for Safety of Cosmetics" and other relevant laws and regulations. The amount of preservatives used in the specific implementation scheme can control the preservative content of the product within the above-mentioned specified addition range.

[0071] In one specific example, the preservative includes pentylene glycol.

[0072] One embodiment of the present application also provides a method for preparing the wound repair composition, comprising: mixing modified hyaluronic acid containing RGD peptide and collagen to prepare the wound repair composition.

[0073] In a specific example, the preparation method includes the following steps S10 to S30:

[0074] S10: Glycerin and water are mixed, and the mixture is stirred and heated.

[0075] In a specific example, in step S10, the stirring speed is 40 rpm-60 rpm, the heating temperature is 80° C.-90° C., and the heating time is 15 min-25 min.

[0076] In a specific example, glycerol is fully stirred and dissolved with purified water, and after mixing evenly, it is added to a vacuum emulsifying pot and stirred and heated. The speed is set to 40rpm-60rpm, optionally 50rpm, the heating temperature is 85±5℃, and kept warm for 15min-20min, optionally 20min.

[0077] S20: Mixing modified hyaluronic acid containing RGD peptide, collagen, β-glucan and water.

[0078] In a specific example, modified hyaluronic acid containing RGD peptide, collagen, β-glucan and purified water are mixed and stirred to dissolve.

[0079] S30: adding the material in step S20 to the solution obtained in step S10, stirring, and discharging the material when the temperature drops to ≤45°C.

[0080] In a specific example, before adding the material in step S20 to the solution obtained in step S10, the step of cooling the solution in step S10 to 45° C.-50° C. is also included.

[0081] In a specific example, in step S30, after stirring, the step of adding a preservative and stirring is also included.

[0082] In a specific example, in step S30, the stirring time is 15 min-20 min.

[0083] In a specific example, after the insulation of step S10 is completed, the temperature is lowered, and when the temperature drops to 48±2°C, the material in step S20 is added to the vacuum emulsifying pot, and the temperature is continued to be lowered and stirred for 15min-20min, and 15min is optional. Pentanediol is added and stirred thoroughly for 15min-20min, and 20min is optional.

[0084] One embodiment of the present application also provides the use of the wound repair composition in the preparation of cosmetics, medicines or medical devices for skin moisturizing and / or repairing. It is understandable that the wound repair composition of the present application can be mixed with a commercially available film towel to form a patch or a mask. In addition to being prepared as a patch or a mask, the application form of the composition of the present application can also be a suitable dosage form such as an aqueous solution or a spray; in addition, the composition of the present application can be provided to a subject in need through a suitable medical device or drug carrier, and is not limited to the content disclosed in the present application.

[0085] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manual or normal conditions in this area, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0086] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0087] The main materials involved in the following examples are as follows:

[0088] The HA-RGD used in the examples of this application is a chemical synthetic raw material purchased from Shaanxi Baihong Synthetic Biotechnology Co., Ltd.; the effective starting materials used include sodium hyaluronate HA and RGD peptide, and HA-RGD is generated by reacting the carboxyl group on the HA long chain with the amino group on RGD. The HA-RGD grafting rate can be 1.0%-10.0%. The HA-RGD grafting rate in the following examples is 5.0%. The molecular formula of HA-RGD is (C 43 H 62 N 12 O 22 )n, relative molecular weight 120-130 Da, the freeze-dried product is white freeze-dried sponge or powder, has a special smell, and can be dissolved in water.

[0089] Recombinant humanized type III collagen (codenamed A) is biosynthesized by establishing a Pichia pastoris eukaryotic expression system and then purified and sterilized. Purity ≥90%, amino acid sequence: EFGERGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGSPGGPGSDGKPGPPGSQGESGRPGPPGPSGPRG QPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGAPGQNGEPGGKGERGAPGEKGEGGPPGPPGGSGPAGPPGPQGVKGER GSPGGPGARGLPGPPGSNGNPGPPGPSGSPGKDGPPGPAGNTGAPGSPGPKGDAGQPGEKGSPGAQGPPGAPGEPGRDGNPGSDGRDGSPGGKGDRGENGSPGAPGA PGHPGPPGPVGPAGKSGDRGESGPAGPAGAPGPAGSRGAPGPQGPRGDKGETGERGNPGAPGSPGPAGQQGPPGPRGNRGERGSEGSPGHPGQPGPPGPPGAP (SEQ ID NO: 1). The inventor unexpectedly discovered that recombinant humanized type III collagen (codenamed A) with a specific sequence can specifically bind to HA-RGD through intermolecular forces to form an association with a specific structure.

[0090] Recombinant human collagen (codenamed B) is a biosynthetic raw material with a purity of ≥90% and an amino acid sequence of (SEQ ID NO: 2).

[0091] The specific biologically active sequences present in the above-mentioned collagen molecules can bind to the RGD integrin site to form an association body, making it easier to be recognized and bound by cell receptors or other biomolecular targets, thereby enhancing biological activity and increasing the repair effect. HA binds to cells while supplementing the extracellular matrix, plays a role in replenishing the dermis while firmly fixing cells and promoting the enrichment of other functional factors to the wound injury site, forming a microenvironment suitable for cell growth, and forming a three-dimensional water storage mechanism of the dermis, thereby greatly exerting the moisturizing, moisturizing, and improving skin condition performance of hyaluronic acid and other moisturizers. By comparing the moisturizing properties and repair effects of different experimental groups through the examples, the optimal formula composition can be screened out.

[0092] Example 1

[0093] A wound repair composition is composed of the following raw materials by weight percentage: HA-RGD 0.01%-0.2%, collagen 0.01%-1%, glycerol 1%-10%, preservative 0.1%-5%, beta-glucan 0.05%-5%, and the balance is purified water.

[0094] The specific formula of each experimental group is shown in Table 1.

[0095] Table 1

[0096] The wound repair composition is prepared according to the formula in Table 1, and the preparation method comprises the following steps:

[0097] Step 1: Stir glycerol thoroughly with purified water to dissolve it. After mixing evenly, add it into a vacuum emulsifier, start stirring and heating. Set the speed to 50 rpm, the heating temperature to 85±5℃, and keep warm for 20 minutes.

[0098] Step 2: Add moisturizer, collagen and β-glucan into a dry and clean container, add purified water to dissolve while stirring, and mix thoroughly.

[0099] Step 3: After the insulation is completed, start cooling down. When the temperature drops to 48±2℃, add the material in step 2 into the vacuum emulsifier, continue cooling and stirring for 15 minutes, add 2% pentanediol and stir thoroughly for 20 minutes.

[0100] Step 4: When the temperature drops to ≤45°C, discharge the material.

[0101] Experimental Example 1 Moisturizing Test

[0102] Objective: To compare the moisturizing effects of human skin between the experimental group (1#, 2#, 3#, 4#) and the control group;

[0103] Number of people and methods for verification: A total of 32 people were recruited this time, and the water content of the inner side of the left and right forearms was tested at 0h, 1h, 2h, and 4h, respectively. See the experimental plan for details;

[0104] Assessment method: Use moisture probe to detect moisture content;

[0105] Experimental plan: According to the inclusion criteria, 32 people were finally included in the group. The left and right arms were modeled by tape tearing. According to the "QBT4256-2011 Cosmetic Moisturizing Efficacy Evaluation Guide", the left arm was used as the test site and divided into 4 areas with an interval of 1 cm. Each area was 4 cm long and 7 cm wide as the experimental area. The sample to be tested was taken at (2.0±0.1) mg / cm 2Apply evenly in the experimental area; the right arm test area is the same size as the left arm modeling area, but no sample is applied, which serves as the control area. Test the moisture content of the experimental area and the control area before applying the test sample and 1h, 2h, and 4h after applying the test sample. Each test site is tested three times, and the average value is taken as the final detection value of the test site. Calculate the difference between the moisture content of the skin stratum corneum and the initial value at each test time point. The results are shown in Tables 2-9. Note: The verification data obtained by the verification method of experimental groups 5# and 6# in this experimental example are close to those of experimental group 2#, with no significant difference.

[0106] Table 2 Statistical results of skin stratum corneum moisture content test data - Sample 1#

[0107]

[0108] Table 3 Statistical analysis of skin stratum corneum moisture content test results - Sample 1#

[0109] Table 4 Statistical results of skin stratum corneum moisture content test data - Sample 2#

[0110] Table 5 Statistical analysis of skin stratum corneum moisture content test results - Sample 2#

[0111]

[0112] Table 6 Statistical results of skin stratum corneum moisture content test data - Sample 3#

[0113]

[0114] Table 7 Statistical analysis of skin stratum corneum moisture content test results - Sample 3#

[0115]

[0116] Table 8 Statistical results of skin stratum corneum moisture content test data - Sample 4#

[0117]

[0118] Table 9 Statistical analysis of moisture content in the stratum corneum of the skin - Sample 4

[0119]

[0120] By calculating the statistical difference between the moisture content of the skin stratum corneum at each test time point and the initial value, the moisturizing effect of the four samples verified in this test was finally concluded to be: 2#>3#>4#>1#.

[0121] According to the above table and Figure 1-Figure 4 Results: The formula design of experimental group 2 is significantly better than that of other groups. The moisturizing properties of samples 2# and 3# with HA-RGD added are better than those of samples 4# and 1# with HA added, indicating that the synergistic effect of HA-RGD and collagen in the formula of experimental group 2 enhances the moisturizing effect of the composition on the skin and the moisturizing time.

[0122] Experimental Example 2 Repair Test

[0123] Objective: To study the effect of the wound repair composition of the present invention on promoting wound healing in rabbit skin.

[0124] The wound surface skin is rich in subcutaneous blood vessels and nerves, and microneedle puncture can cause obvious pain. The open wound formed after surgery is prone to infection and bleeding, and can cause some skin dysfunction, such as inflammatory reactions such as erythema and edema. The wound repair composition of the present invention can form a protective film on the wound surface to play a physical barrier role, and is suitable for the care of non-chronic wounds (such as small wounds, abrasions, cuts, laser / photon / acid peels / micro-plastic surgery wounds, dermatitis eczema, sensitive skin, acne) and surrounding skin. The research was carried out in accordance with GB / T 16886.12-2017 "Biological Evaluation of Medical Devices Part 12: Sample Preparation and Reference Materials" and GB / T 16886.2-2011 "Biological Evaluation of Medical Devices Part 2: Animal Welfare Requirements".

[0125] Methods: Microneedles were used to create skin wounds on the backs of rabbits, and the products of the experimental groups (1#, 2#, 3#, and 4#) were applied respectively for 7 consecutive days to evaluate the repair effects of the products.

[0126] Ethics Statement: The Laboratory Animal Care and Use Committee reviews this experimental procedure regularly every year to ensure that it complies with the ethical requirements of experimental animal welfare.

[0127] Experimental protocol:

[0128] 1. Test Materials

[0129] 1.1 Test samples

[0130] 1) Test group: Samples 1-4 of the experimental group in Example 1 (1#, 2#, 3#, 4#).

[0131] 2) Negative control group: physiological saline, batch number 20240305, specification 250ml.

[0132] 3) Blank control group: no treatment.

[0133] 1.2 Test equipment

[0134] Microneedles, gauze, bandages, iodine, alcohol cotton balls, 4% paraformaldehyde, paraffin, and anesthetics.

[0135] 1.3 Experimental animals

[0136] 1.3.1 Experimental animals

[0137] Breed: New Zealand white rabbit, animal sex and number: either sex, 30 rabbits, body weight: 2.1kg-2.5kg.

[0138] Purchasing unit: Experimental Animal Center, School of Medicine, Xi'an Jiaotong University.

[0139] Animal selection: Adult healthy experimental animals that have not used topical drugs or products and have no skin damage. Both males and females are acceptable, and females must not be pregnant.

[0140] Animal husbandry: Animals were housed individually in non-attractive steel cages. Each cage was labeled with the trial number, sex, and date of administration.

[0141] 1.3.2. Animal feed

[0142] Ordinary feed (provided by: Experimental Animal Center, School of Medicine, Xi'an Jiaotong University).

[0143] 1.3.3 Animal drinking water

[0144] Tap water that meets the national urban drinking water hygiene standards.

[0145] 1.3.4 Rearing conditions

[0146] Animals were allowed to drink water freely, and the water spouts were checked daily for damage. The room temperature of the animal room was set at 25±2℃, humidity at 40~70%, and light intensity at 12 hours of alternating light and dark. Cages were cleaned 2~3 times a week. The floor and walls were cross-disinfected with 0.1% Sanisol or 0.5% 84 disinfectant once a week.

[0147] 2. Animal Experiment Design

[0148] 2.1 Experimental Grouping

[0149] Remove the hair on the rabbit's back, then clean the area with warm water, dry it with cotton balls and set aside. The depilatory area is divided into six 2cm×2cm areas symmetrically on the left and right: 1#, 2#, 3#, 4#, 5#, and 6#. Among the six areas, the upper left is experimental group 1, code-named 1#; the upper right is experimental group 2, code-named 2#; the lower left is experimental group 4, code-named 4#; the middle left is experimental group 3, code-named 3#; the middle right is the negative control group (normal saline), code-named 5#; the lower right is the blank control group (no treatment), code-named 6#, as shown in the schematic diagram Figure 5 shown.

[0150] 2.2 Experimental steps

[0151] Rabbits were anesthetized with 2.5% sodium pentobarbital at 1 mL / kg along the ear vein. Microneedles were used to create wounds in five areas: 1#, 2#, 3#, 4#, and 5#. The frequency of microneedles was 300 times / min, and each treatment lasted 30 seconds. The treatment lasted 3 to 5 times in total until the rabbits' skin showed severe erythema and formed shallow scars.

[0152] According to the Technical Specifications for External Pharmacological Experiments of Traditional Chinese Medicine (Draft), the sample to be tested was directly applied to the test group area to completely cover the wound surface of the area, and sterile gauze was soaked with sterile saline and covered to the negative control group area. The experimental area was then covered with a bandage for at least 4 hours, the gauze and bandage were removed, the contact area was marked with permanent ink, and the residual test material was washed off with warm water and wiped dry. All areas were given the drug twice a day (once in the morning and once in the evening) for 7 consecutive days, and the repaired skin was photographed and sampled on the 3rd and 7th days after the successful modeling.

[0153] 2.3 Evaluation Method

[0154] ① Skin surface observation: After repair care, observe the rabbit skin surface micro-damage and shallow scar tissue formation with the naked eye.

[0155] Effect judgment criteria: marked effect: the area of ​​erythema and scar disappears by ≥50% as seen by the naked eye; improved: the area of ​​erythema and scar disappears by ≥30% as seen by the naked eye; ineffective: the area of ​​erythema and scar disappears by <30% as seen by the naked eye. The total effective rate is calculated based on marked effect plus improved.

[0156] Statistical method: χ 2 SPSS software was used for statistical analysis.

[0157] ② Take wound tissue for pathological HE examination: randomly select one rabbit for pathological HE examination on the 3rd day; randomly select one rabbit for pathological HE examination on the 7th day.

[0158] 2.4 Statistical methods

[0159] All data were statistically analyzed using SPSS 19.0 software. ±S indicates that the difference was not significant when P≥0.05, the difference was significant when P<0.05, and the difference was extremely significant when P<0.01.

[0160] 2.5 Results

[0161] 2.5.1 Skin surface observation

[0162] The comparative results of the rabbit skin repair test are shown in Table 10 and Figure 6-Figure 7 shown.

[0163] Table 10 Rabbit skin repair efficacy evaluation table

[0164] From the statistical observation of the test results, it was found that 20 cases were significantly effective when using area 1#, 6 cases were improved, and 4 cases were ineffective; 27 cases were significantly effective when using area 2#, 3 cases were improved, and 0 cases were ineffective; 20 cases were significantly effective when using area 3#, 5 cases were improved, and 5 cases were ineffective; 18 cases were significantly effective when using area 4#, 8 cases were improved, and 4 cases were ineffective; in the negative control area (normal saline), 4 cases were significantly effective, 6 cases were improved, and 20 cases were ineffective; there was a significant difference between using the experimental group products and using normal saline (P<0.01).

[0165] Comparison of the effective skin repair rates on the third and seventh days showed that 2# had a better repair effect than the other groups. In particular, after 7 consecutive days of use, 2# significantly reduced the superficial scars, and the wound area basically restored normal skin structure.

[0166] 2.5.2 Histological observation

[0167] The results of the rabbit skin repair test pathology HE are as follows Figure 8 shown.

[0168] According to the HE pathological examination, on the 3rd day, the epidermis of 2# was discontinuous, the structure was clear, and there was inflammatory cell infiltration in the epidermis; the epidermis of the saline group was discontinuous, the structure was unclear, and there were more inflammatory cells infiltrating in the epidermis. On the 7th day, the wound surface of 2# basically returned to normal skin.

[0169] In summary, by observing the repair of the wound, it can be seen that after 7 consecutive days of use, the erythema of the 2# wound was significantly reduced, the shallow scars were significantly reduced, and the normal skin structure of the wound area was basically restored, indicating that 2# has no irritating effect on animal skin. By forming a protective layer on the wound surface and providing a microenvironment for wound healing, it can effectively protect the wound and promote wound healing and recovery. Compared with other groups, 2# has better repair effects.

[0170] The addition of HA-RGD to sample 2# makes recombinant type III humanized collagen (A) have better compatibility with the skin and skin bioavailability, and when used, it can form a thin film on the wound surface, isolating the invasion of harmful substances such as external bacteria and dust, improving the living environment of skin cells, reducing discomfort symptoms such as infection and pain, promoting the metabolism of skin tissue, and restoring the skin to a healthy state. The composition of the present application can effectively restore obvious skin dysfunction caused by medical aesthetics, such as inflammatory reactions such as erythema and edema.

[0171] Experimental Example 3 Structural Identification

[0172] Thermogravimetric analysis was used to identify the structure. A thermogravimetric analyzer was used to analyze the thermal stability of the material and the decomposition temperature of the sample by detecting the loss of mass at elevated temperatures. During the test, appropriate amounts of samples 1# to 4# from the experimental group were placed in a thermogravimetric analyzer. The test temperature range was 30-300°C, and the heating rate was 5°C / min. The mass loss of the sample during the temperature increase was recorded to obtain the thermal gravimetric curve of the sample, i.e., the thermal decomposition curve of the sample. The results are shown in Figure 2. Fig. 9 shown.

[0173] In the TGA image, it was observed that the temperature was below 100°C. The main reason for weight loss in this stage was the evaporation of water in the material. The second weight loss stage appeared at 200-230°C. The weight loss in this stage was mainly caused by the thermal decomposition of HA and HA-RGD. The third weight loss stage appeared at 250-300°C. The weight loss in this stage was mainly caused by the thermal decomposition of recombinant type III humanized collagen (A) and recombinant humanized collagen (B). The 2# sample had an inflection point at 243°C, proving the formation of a new structure. Analysis showed that HA-RGD and recombinant type III humanized collagen (A) may have formed a new structure through electrostatic force.

[0174] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0175] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims, and the description may be used to interpret the content of the claims.

Claims

1. A wound repair composition, characterized in that: The wound repair composition comprises the following components: modified hyaluronic acid containing RGD peptide and collagen.

2. The wound repair composition according to claim 1, characterized in that: The collagen includes recombinant human collagen; Optionally, the recombinant human collagen includes recombinant type III human collagen; Optionally, the recombinant human type III collagen includes the recombinant human collagen shown in SEQ ID NO:1 and / or SEQ ID NO:

2.

3. The wound repair composition according to claim 1, characterized in that: The collagen is a recombinant human collagen containing the sequence shown in SEQ ID NO: 1, and the wound repair composition has a thermal decomposition inflection point between 230° C. and 250° C.

4. The wound repair composition according to any one of claims 1 to 3, characterized in that: It also includes a humectant; preferably, the humectant includes one or more of glycerol, dextran, sodium alginate, and trehalose.

5. The wound repair composition according to any one of claims 1 to 3, characterized in that: The relative molecular weight of the modified hyaluronic acid containing RGD peptide is 80 Da-500 Da.

6. The wound repair composition according to any one of claims 1 to 3, characterized in that: The grafting rate of the modified hyaluronic acid containing the RGD peptide is 1.0%-10.0%.

7. The wound repair composition according to claim 4, characterized in that: The wound repair composition comprises modified hyaluronic acid containing RGD peptide, collagen, glycerol and β-glucan; Preferably, the wound repair composition comprises the following raw materials in weight percentage: 0.005%-5% of modified hyaluronic acid containing RGD peptide, 0.005%-5% of collagen, 0.5%-20% of glycerol and 0.01%-10% of β-glucan; More preferably, the wound repair composition comprises the following raw materials in weight percentage: 0.01%-0.2% of modified hyaluronic acid containing RGD peptide, 0.01%-1% of collagen, 1%-10% of glycerol and 0.05%-5% of β-glucan.

8. The wound repair composition according to any one of claims 1 to 3 and 7, characterized in that: The wound repair composition also includes an antiseptic; Optionally, in the wound repair composition, the weight percentage of the preservative is 0.1%-5%; Optionally, in the wound repair composition, the weight percentage of the preservative is 0.1%-2%; Optionally, the preservative comprises pentylene glycol.

9. The method for preparing the wound repair composition according to any one of claims 1 to 8, characterized in that: The method comprises the following steps: mixing the modified hyaluronic acid containing RGD peptide and the collagen to prepare a wound repair composition; Optionally, the following steps are included: S10: mixing the glycerol and water, stirring and heating after mixing; S20: mixing the modified hyaluronic acid containing RGD peptide, the collagen, the β-glucan and water; S30: adding the material in step S20 to the solution obtained in step S10, stirring, and discharging the material when the temperature drops to ≤45°C.

10. Use of the wound repair composition according to any one of claims 1 to 8 in the preparation of cosmetics, medicines or medical devices for skin moisturizing and / or repairing.

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