Transdermal mini collagen for skin light injury repair and dressing product thereof

Abstract

The invention belongs to the field of skin medicine, and particularly relates to a transdermal mini collagen for skin light injury repair and a dressing product of the transdermal mini collagen. The invention provides a transdermal mini collagen for repairing light damage of skin. The transdermal mini collagen has good biocompatibility, and can promote the proliferation and adhesion of fibroblasts. The transdermal mini collagen can effectively penetrate through cells and skin. The invention provides a transdermal mini collagen dressing product which is prepared from the following components: transdermal mini collagen, phenoxyethanol, sodium hyaluronate, methylparaben, glycerol and the like. The transdermal mini collagen dressing product can quickly repair light-damaged skin, and can be widely applied to the fields of cosmetics, dermatology and the like.

Description

Technical Field

[0001] The present invention belongs to the field of dermatology, and in particular relates to a transdermal mini collagen and a dressing product thereof for repairing skin photodamage. Background Art

[0002] As an important organ in direct contact with the external environment, the skin is extremely susceptible to damage from ultraviolet radiation. Short-term intense ultraviolet radiation can cause a variety of skin injuries of different phenotypes, such as erythema, inflammation, papules, blisters, peeling, and pigmentation; long-term ultraviolet radiation may also induce skin cancer, seriously threatening people's life and health. Therefore, it is of great significance to develop effective strategies to promote the repair of skin photodamage.

[0003] The stratum corneum cells of the skin epidermis have a good barrier function, which can effectively prevent the loss of beneficial substances in the body and the invasion of harmful substances outside the body. Studies have found that only some small molecules (<500Da) with moderate lipophilicity (log P value of 1-2) have skin permeability. Many complex hydrophilic macromolecules, peptides, proteins, nucleic acids and other biological molecules are difficult to penetrate the skin barrier into the deep layers of the skin, which seriously limits the application of such molecules in the field of skin damage repair.

[0004] Collagen is the main component of the dermis of the skin and has attracted much attention in the field of skin photodamage repair. Collagen has the properties of moisturizing and film-forming, which can reduce the loss of water through the epidermis of the skin. In addition, collagen can promote the regeneration of collagen in damaged tissues and accelerate wound healing. However, collagen has a large molecular weight and poor water solubility, making it difficult to pass through the barrier function of the skin and reach deep into the skin. Hydrolyzed collagen is a polypeptide fragment produced by the hydrolysis of collagen, which has a low molecular weight and is easily soluble in water. At present, hydrolyzed collagen is mainly prepared by acid, alkali and enzyme methods. However, the hydrolyzed collagen prepared by these methods does not have a triple helix structure, the amino acid sequence is uncontrollable, and it is impossible to mimic the structure and function of natural collagen, which seriously limits its application in skin repair.

[0005] The present invention provides a transdermal mini collagen for the first time, and prepares the transdermal mini collagen into a dressing product for use in skin damage repair. The transdermal mini collagen is a small molecular weight polypeptide with a triple helix structure, a transdermal sequence and a functional sequence. The transdermal mini collagen has good water solubility, biocompatibility and biological activity, and can effectively promote the repair of photodamaged skin. The present invention also provides a transdermal mini collagen dressing product, and evaluates the efficacy of the transdermal mini collagen dressing through cell, mouse and volunteer experiments. The dressing product can effectively help various indicators of damaged skin quickly return to normal levels, promote the repair of photodamaged skin, and has broad application prospects in the fields of medical cosmetology. Summary of the invention

[0006] In order to solve the above technical problems existing in the prior art, the purpose of the present invention is to provide a transdermal mini collagen and its dressing product for skin photodamage repair. Specifically, it includes the following contents:

[0007] In a first aspect, the present invention provides a transdermal mini collagen for repairing skin photodamage, wherein the sequence of the transdermal mini collagen is: N1-GFOGER-N2-(GRR) n ;

[0008] Wherein, the GFOGER is Gly-Phe-Hyp-Gly-Glu-Arg; the GRR is Gly-Arg-Arg;

[0009] The N1 and N2 contain at least 3 repeated Gly-Xaa-Yaa and / or Gly-Xaa-Xaa; the Xaa and Yaa are any different amino acids;

[0010] The n is any integer greater than or equal to 1.

[0011] Preferably, the Xaa is Pro; and the Yaa is Hyp.

[0012] Preferably, the polypeptide sequence of the transdermal mini collagen is: (GPO) m GFOGER(GPO) m (GRR) n ; The GPO is Gly-Pro-Hyp; and m is any integer greater than or equal to 3.

[0013] Preferably, m is any integer greater than or equal to 3 and less than or equal to 5.

[0014] Preferably, the polypeptide sequence of the transdermal mini collagen is: (GPO) 5 GFOGER(GPO) 5 (GRR) n .

[0015] Preferably, n is 1 or 2.

[0016] In a second aspect, the present invention provides the use of the transdermal mini collagen described in the first aspect in the preparation of a skin damage repair product.

[0017] Preferably, the skin damage is skin photodamage.

[0018] Preferably, the light damage is ultraviolet damage.

[0019] Preferably, the products for repairing photodamage of the skin include cosmetics, medical cosmetics and medicines.

[0020] In a third aspect, the present invention provides a dressing for repairing photodamage to the skin, the dressing comprising the following components in percentage by mass: 0.01% of the transdermal mini collagen described in the first aspect above, 0.20% of phenoxyethanol, 0.15% of sodium hyaluronate, 0.01% of methylparaben, 3.00% of glycerin, and the balance being water.

[0021] The beneficial effects of the present invention are:

[0022] (1) The present invention provides a transdermal mini collagen for repairing skin photodamage, wherein the transdermal mini collagen has excellent properties such as good biocompatibility, can promote cell proliferation and cell adhesion, and can effectively penetrate cells and skin;

[0023] (2) The preparation method of the transdermal mini collagen described in the present invention is simple, the conditions are mild, and it has broad application prospects in the fields of tissue regeneration, skin medicine, etc.

[0024] (3) The present invention screened and obtained a transdermal mini collagen dressing product composed of transdermal mini collagen and ingredients such as phenoxyethanol, sodium hyaluronate, methylparaben, glycerin, etc., and the transdermal mini collagen dressing product can repair skin photodamage;

[0025] (4) The transdermal mini collagen dressing product described in the present invention can be widely used in the fields of cosmetics, dermatology, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Circular dichroism spectra of GRR0, GRR1, GRR2 and GRR3 prepared in Example 1 of the present invention; wherein, a1 are respectively the CD full spectrum of GRR0, the thermal variation curve of GRR0, the first derivative of the thermal variation curve of GRR0, the CD full spectrum of GRR1, the thermal variation curve of GRR1, the first derivative of the thermal variation curve of GRR1, the CD full spectrum of GRR2, the thermal variation curve of GRR2, the first derivative of the thermal variation curve of GRR2, the CD full spectrum of GRR3, the thermal variation curve of GRR3, and the first derivative of the thermal variation curve of GRR3;

[0027] Figure 2 Cytotoxicity graph of GRR0, GRR1, GRR2 and GRR3 prepared in Example 1 of the present invention based on HFF-1 cells; wherein, ad are the cytotoxicity of GRR0, GRR1, GRR2 and GRR3, respectively;

[0028] Figure 3Cell adhesion graphs of GRR0, GRR1, GRR2 and GRR3 prepared in Example 1 of the present invention based on HFF-1 cells; wherein af are cell adhesion graphs of PBS, BSA, GRR0, GRR1, GRR2 and GRR3, respectively;

[0029] Figure 4 Cell proliferation diagram of GRR0, GRR1, GRR2 and GRR3 prepared in Example 1 of the present invention based on HFF-1 cells;

[0030] Figure 5 HFF-1 cell penetration diagram of FAM-labeled GRR0, GRR1, GRR2 and GRR3 prepared in Example 3 of the present invention;

[0031] Figure 6 Mouse skin penetration diagram of FAM-labeled GRR0, GRR1, GRR2 and GRR3 prepared in Example 3 of the present invention;

[0032] Figure 7 Ultrasound images of mouse skin tissues in the blank control group, negative control group, commercial product group, and dressing group;

[0033] Figure 8 Chroma test results of mouse skin tissue in blank control group, negative control group, commercial product group and dressing group;

[0034] Fig. 9 The transepidermal water loss (TEWL) test results of mouse skin tissue in the blank control group, negative control group, commercial product group and dressing group;

[0035] Fig.10 HE staining results of mouse skin tissue in blank control group, negative control group, commercial product group and dressing group;

[0036] Fig.11 Masson staining results of mouse skin tissue in the blank control group, negative control group, commercial product group, and dressing group;

[0037] Fig.12 Results of hydroxyproline content in mouse skin tissues in the blank control group, negative control group, commercial product group, and dressing group;

[0038] Fig.13 Adverse reaction feedback results from the subjects;

[0039] Fig.14 Ultrasound images of the subjects’ skin;

[0040] Fig.15 The Combo measures the density, brightness, moisture and transepidermal water loss (TEWL) of the subjects' skin. DETAILED DESCRIPTION

[0041] The technical solution of the present invention is further described below in conjunction with embodiments, but the protection scope of the present invention is not limited to the following.

[0042] definition:

[0043] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those of ordinary skill in the art to which the methods belong. Although any methods and compositions similar or equivalent to those described herein can also be used to achieve self-assembly of collagen polypeptides, they are not limited to the representative exemplary methods and compositions described in the following examples. It should also be understood that the terms used in the present invention are only for the purpose of describing specific embodiments and do not limit the scope of protection of the present invention and the appended claims.

[0044] It must be noted that, as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. For example, reference to "a transdermal mini-collagen" includes a plurality of such transdermal mini-collagens, reference to "the transdermal mini-collagen" is a reference to one or more transdermal mini-collagens and their equivalents known to those skilled in the art, and so on.

[0045] “Comprising” means “including but not limited to”, and is not intended to exclude, for example, other components, integers, etc.

[0046] Where range values ​​are provided, it is understood that each intervening value between the upper and lower limits of the range and any other stated or intermediate values ​​within the stated range are encompassed within the methods and compositions. The upper and lower limits of these smaller ranges may be independently included within the smaller ranges, and are also encompassed within the methods and compositions, subject to any specifically excluded limits in the stated ranges. Where the stated range includes one or both limits, ranges excluding either or both of those included limits are also encompassed within the methods and compositions.

[0047] It should be understood that, for the sake of clarity, certain features described in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, for the sake of brevity, various features of the methods and compositions described in the context of a single embodiment may also be provided separately or in any suitable sub-combination.

[0048] It will be apparent to those skilled in the art upon reading this disclosure that each individual embodiment described and shown herein has individual components and features that may be readily separated or combined with the features of any other embodiment without departing from the scope or spirit of the present method. Any recited method may be performed in the order of events recited or in any other order that is logically possible.

[0049] The term "transdermal mini-collagen" refers to a highly active collagen biomimetic polypeptide with a triple helix structure, a functional sequence and a transdermal sequence.

[0050] The term "collagen" is the main component of animal connective tissue and the most abundant and widely distributed functional protein in mammals. It has a trimer structure composed of three peptide chains, mainly including type I, type II, type III and type IV collagen.

[0051] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to two or more amino acid residues linked to each other by peptide bonds or modified peptide bonds. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers that contain modified residues and non-naturally occurring amino acid polymers.

[0052] The term "thermal transition temperature" refers to the temperature at which half of the triple helical structure of the collagen polypeptide is converted into a random coil. The thermal transition temperature can be determined by known structural analysis methods such as circular dichroism (CD), but is not limited to these and can also be determined by other known methods. It is well known to those skilled in the art that collagen polypeptides with thermal transition temperatures all have a triple helical structure.

[0053] Example 1 Preparation of transdermal mini collagen

[0054] The transdermal mini collagen sequences were synthesized by the classical solid phase synthesis method, which is specifically as follows:

[0055] (1) Add 100 mg of Rink ammonia resin into a reactor with a sieve plate and use 5 mL of dichloromethane to swell the resin;

[0056] (2) removing the N-terminal Fmoc protecting group using a 20% piperidine / N,N-dimethylformamide (DMF) solution, and detecting complete removal of the protecting group using a colorimetric reaction;

[0057] (3) The amino acid (4 eq) with Fmoc protection at the N-terminus, HOBT (4 eq) and HBTU (4 eq) were dissolved in DMF and activated at low temperature for 20 min. DIEA (6 eq) was then added dropwise to the solution. The solution was mixed and added to the reactor for reaction for 3 h.

[0058] (4) After the reaction is completed, the reaction solution is extracted from the reactor, and the resin is washed three times with 5 mL of DMF and DCM, respectively. The color reaction is used to detect that the amino acid condensation is complete. The resin is treated with 20% piperidine / DMF solution three times for 5 min, 5 min and 15 min, respectively. The resin is washed three times with 5 mL of DMF and DCM, respectively, and the color reaction is used to detect that the protecting group is completely removed;

[0059] (5) repeating steps (3) and (4) until the target peptide sequence is completed;

[0060] (6) Block the N-terminus with 25% acetic anhydride (DMF) for 25 min;

[0061] (7) Cleavage: At room temperature, use TFA (trifluoroacetic acid): triisopropylsilane: H 2 O = 95:2.5:2.5, shake the resin for 3 hours to complete the peptide cleavage, precipitate with ice ether, centrifuge and wash to obtain the crude product of transdermal mini collagen.

[0062] (8) The crude transdermal mini collagen product was purified by reverse phase high performance liquid chromatography using a C18 chromatographic column and lyophilized to obtain the target transdermal mini collagen shown in Table 1.

[0063] Table 1 Sequence list of transdermal mini collagen

[0064]

[0065] Example 2 Characterization of transdermal mini collagen

[0066] 1. Circular dichroism

[0067] Circular dichroism (CD) spectra were obtained on a Chirascan CD spectrometer (Appliedphotophysics, England) equipped with a Peltier temperature controller. A transdermal mini-collagen aqueous solution was prepared at a concentration of 2 mg / mL at pH 7.4. The sample was equilibrated at 4°C for at least 24 hours before measurement. A cuvette with a path length of 1 mm was used. The thermal curve was obtained by monitoring the change of the characteristic CD peak at a wavelength of 225 nm with temperature, ranging from 4°C to 90°C, with a heating rate of 0.5°C / min. The first-order derivative of the thermal curve was taken, and the temperature corresponding to the extreme value was the thermal transition temperature (T m ).

[0068] The results are as follows Figure 1As shown: The CD spectra of GRR0, GRR1, GRR2 and GRR3 show the highest absorption peak at 225nm and the lowest absorption peak at 198nm, indicating that they have formed a triple helical structure. The results of the thermal curve and its first-order derivative show that GRR0, GRR1, GRR2 and GRR3 have formed a stable triple helical structure, and the thermal transition temperatures are 66.8℃, 62.9℃, 57.9℃ and 52.9℃ respectively.

[0069] 2. Cell experiments

[0070] 2.1 Cytotoxicity assay

[0071] The cytotoxicity of the above examples to HFF-1 cells was determined by CCK-8 method. HFF-1 cells (DMEM medium containing 10% fetal bovine serum and 1% double antibody) were cultured in 5% CO 2 The cells were cultured in a constant temperature incubator (temperature set at 37°C). A volume of 100 μL of HFF-1 cell suspension (5×10 3 The cells were seeded at a density of 10 cells / mL in 96-well plates and incubated for 24 h (37°C, 5% CO 2 ) to ensure complete adhesion. Then, 100 μL of each sample solution of different concentrations (1 μg / mL, 10 μg / mL, 100 μg / mL, 300 μg / mL and 500 μg / mL) was added to the 96-well plate, and 100 μL of DMEM medium was added to the control wells, and incubation was continued at 37°C for 24 hours. Then, 100 μL of 10% Cell Counting Kit-8 (CCK-8) was added to each well, placed in a cell culture incubator, and the OD value at 450 nm was measured using a Tecan Infinite F200 / M200 multifunctional microplate reader (Tecan, Mannedor, Switzerland). The appropriate time was found, and the culture was stopped when the OD value of the well with only cells and no material reached about 1. The activity of HFF-1 cells was calculated as the average absorbance value of three measurements for each condition.

[0072] The results of cytotoxicity experiments were Figure 2 As shown: GRR0, GRR1 and GRR2 solutions all had 100% cell viability at different concentrations, while when the concentration of GRR3 solution was 100 μg / mL, the viability of HFF-1 cells dropped to 95.98%, and its cell viability decreased significantly with increasing concentration. The above results show that GRR0, GRR1 and GRR2 described in the present application are non-cytotoxic, while GRR3 is non-cytotoxic at low concentrations, but its cytotoxicity increases significantly with increasing concentration.

[0073] 2.2 Cell adhesion assay

[0074] 24 microplates (untreated tissues) were coated with PBS, BSA, GRR0, GRR1, and GRR2 samples. PBS and BSA were used as controls. Before adding HFF-1 cells, the coated wells were washed three times with PBS buffer (10 mM). Then 500 μL (1×10 5 The HFF-1 cell suspension (200 μg / mL, serum-free medium) was inoculated in a 24-well microplate and incubated at 37°C for 5 hours. Finally, the unattached HFF-1 cells were washed with 10 mM PBS buffer. The adhesion effect of HFF-1 cells was observed by inverted microscope.

[0075] The results of cell adhesion experiments were as follows Figure 3 As shown: HFF-1 cells cultured on GRR0, GRR1, GRR2 and GRR3 samples spread into a spindle shape, while HFF-1 cells cultured on PBS and BSA samples remained spherical. This result shows that PBS and BSA have poor adhesion to HFF-1 cells, while GRR0, GRR1, GRR2 and GRR3 described in this application have good cell adhesion ability.

[0076] 2.3 Cell proliferation assay

[0077] The ability of GRR0, GRR1, GRR2 and GRR3 to promote the proliferation of HFF-1 cells was detected by CCK-8 kit at 1, 3, 5 and 7 days. HFF-1 cells (DMEM medium containing 10% fetal bovine serum and 1% double antibody) were cultured in 5% CO 2 The cells were cultured in a constant temperature incubator (temperature set at 37°C). A volume of 100 μL of HFF-1 cell suspension (5×10 3 The cells were seeded at a density of 10 cells / mL in 96-well plates and incubated for 24 h (37°C, 5% CO 2 ) to ensure complete adhesion. Then, 100 μL of 0.1 mg / mL transdermal mini collagen sample solution was added to the 96-well microplate, and 100 μL DMEM culture medium was added to the control wells. The culture solution was replaced every two days. Continue to incubate at 37°C for 1, 3, 5, and 7 days, add 100 μL of 10% CCK-8 to each well, place it in a cell culture incubator, and measure its OD value at 450nm using a Tecan Infinite F200 / M200 multifunctional microplate reader. The activity of HFF-1 cells was calculated as the average absorbance value of three measurements for each condition.

[0078] The results of cell proliferation assays were Figure 4As shown, when co-cultured with HFF-1 cells for 1 day, the cell proliferation rates of the GRR0, GRR1 and GRR2 groups were slightly higher than those of the blank control group, but there was no significant difference, and the cell survival rate of the GRR3 group was lower than that of the other groups; on the 3rd day, the cell proliferation rates of the GRR0, GRR1 and GRR2 groups were significantly higher than those of the blank control group, and the cell survival rate of the GRR3 group continued to decrease compared with the 1st day; as the culture time gradually increased to the 5th and 7th days, the difference in cell proliferation rates between the GRR0, GRR1, GRR 2 groups and the blank control group gradually increased, while the cell survival rate of the GRR3 group significantly decreased with the increase in culture time. This result shows that GRR0, GRR1 and GRR2 described in this application have good ability to promote cell proliferation, while GRR3 will affect cell proliferation and cause cell death.

[0079] Example 3 Preparation of FAM-labeled transdermal mini-collagen

[0080] The FAM-labeled transdermal mini-collagen sequences were synthesized by a classical solid phase synthesis method, which is specifically as follows:

[0081] Steps (1)-(5) are the same as steps (1)-(5) in the solid phase synthesis method of transdermal mini collagen in Example 1;

[0082] (6) 5-fold excess FAM (5 eq), HOBT (4 eq), HBTU (4 eq) and DIEA (6 eq) were dissolved in DMF, the solutions were mixed and added to the reactor, and reacted at 35°C for 16 hours. After the reaction was completed, the reaction solution was withdrawn from the reactor, and the resin was washed three times with 5 mL of DMF and DCM respectively;

[0083] Steps (7)-(8) are the same as steps (7)-(8) in the solid phase synthesis method of transdermal mini collagen in Example 1; finally, the target FAM-labeled transdermal mini collagen shown in Table 2 is obtained.

[0084] Table 2 Sequence list of FAM-labeled transdermal mini collagen

[0085]

[0086] Example 4 Characterization of the properties of FAM-labeled transdermal mini-collagen

[0087] 1. Cell penetration assay

[0088] HFF-1 cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% double antibody in a 5% CO 2 500 μL of HFF-1 cell suspension (5×10 cells per well) was added to the culture medium. 3The cells were seeded at a density of 10 cells / mL in 24-well plates and incubated for 24 h (37°C, 5% CO 2 ) to ensure complete adhesion. Then, 500 μL of each sample solution labeled with 50 μM FAM was added to each 24-well plate. Incubation was continued at 37°C for 5 hours, and the ability of each sample to penetrate HFF-1 cells was observed using a laser confocal microscope. The cell nucleus (blue fluorescence) was observed using 360 nm excitation, and the cytoplasm (green fluorescence) was observed using 488 nm excitation.

[0089] The results of cell penetration experiments are shown in Figure 5 As shown: almost no fluorescence was observed in HFF-1 cells cultured in FAM-GRR0 solution, weak green fluorescence was observed in the cytoplasm of HFF-1 cells cultured in FAM-GRR1 solution, and obvious green fluorescence was observed in the cytoplasm of HFF-1 cells cultured in FAM-GRR2 and FAM-GRR3 solutions. This result shows that the transdermal mini collagen GRR1, GRR2 and GRR3 described in this application all have cell penetration ability.

[0090] 2. Skin penetration test

[0091] Clean-grade male Kunming mice weighing 20±2g were selected, and the back of the mice was shaved with a hair clipper, and then depilatory cream was used to remove hair, so that the back epidermis was completely exposed. The depilatory area was about 2×4cm and stabilized for 24h. 5mL of each sample solution labeled with FAM at a concentration of 0.1mg / mL was applied to the back skin of the mice, respectively, and cultured in the dark. After applying the sample for 2h, 4h and 8h, 3 mice in each group were killed, and the back skin was taken and frozen in the dark. The frozen tissue was embedded with an embedding agent, and the embedded tissue was processed for frozen sectioning. The ability of each sample to penetrate the skin was then observed by laser confocal microscopy, and the skin tissue (green fluorescence) was observed using 488nm excitation.

[0092] Skin penetration test results Figure 6As shown: After 2 hours of culture, green fluorescence was almost invisible in the FAM-GRR0 and FAM-GRR1 groups, weak green fluorescence could be observed in the outer layer of the epidermis in the FAM-GRR2 group, and obvious green fluorescence could be observed in the outer layer of the epidermis in the FAM-GRR3 group; after 4 hours of culture, weak green fluorescence could be observed in the outer layer of the epidermis in the FAM-GRR0 group, obvious green fluorescence could be observed in the outer layer of the epidermis in the FAM-GRR1 and FAM-GRR2 groups, and the fluorescence intensity of the FAM-GRR2 group was higher than that of the FAM-GRR1 group, and the green fluorescence in the FAM-GRR3 group had begun to penetrate deep into the skin; after 8 hours of culture, weak green fluorescence could still only be observed in the outer layer of the epidermis in the FAM-GRR0 group, weak fluorescence began to appear deep in the skin of the FAM-GRR1 group, and the green fluorescence in the FAM-GRR2 and FAM-GRR3 groups penetrated deep into the skin. This result shows that the introduction of the GRR sequence can improve the skin penetration ability of mini collagen.

[0093] Example 5 Preparation of transdermal mini collagen dressing product

[0094] Taking biocompatibility and skin penetration into consideration, we preferred the transdermal mini collagen GRR2 in Example 1 to further prepare a transdermal mini collagen dressing product. The dressing product comprises the following components by mass ratio: transdermal mini collagen 0.01%, phenoxyethanol 0.20%, sodium hyaluronate 0.15%, methylparaben 0.01%, glycerol 3.00%, and the balance water.

[0095] Preparation process: Heat water, glycerin, hyaluronic acid, methylparaben, and phenoxyethanol to 80°C, stir until completely dissolved, homogenize for 3 minutes, and keep warm for 10-15 minutes; cool to 35°C and add transdermal mini collagen, stir until completely dissolved and transparent. Filter with a clean filter, let the material stand to obtain a transdermal mini collagen dressing product solution. Fold the mask cloth and put it into an aluminum strip, place the above solution in the aluminum strip, seal the aluminum strip, and sterilize by irradiation to obtain a transdermal mini collagen dressing product.

[0096] Example 6 UV damage repair ability of transdermal mini collagen dressing product

[0097] 1. UV-damaged skin modeling and treatment

[0098] Clean-grade male Kunming mice weighing 20±2g were selected, and the backs of the mice were shaved with a hair clipper, and then depilatory cream was used to remove hair, so that the back epidermis was completely exposed. The depilatory area was approximately 2×4cm and stabilized for 24 hours. The mice were randomly divided into four groups: blank control group, negative control group, commercially available product group and dressing treatment group. The blank control group did not receive any treatment, and no ultraviolet light irradiation or sample application was used. The back skin of mice in other groups was irradiated with continuous wavelength ultraviolet lamps A (320-440nm) and B (280-320nm). The radiation was quantified using an ultraviolet irradiator to reach 200J / cm 2 , indicating that the model of acute ultraviolet damage to the back skin of mice was successfully established. 5 mL of sample solution was applied to the back skin of mice respectively, and reapplied every 24 hours. The mice in the negative control group were smeared with normal saline, the mice in the commercial product group were smeared with commercially available collagen dressing product solution (Xi'an Juzi Biogene Technology Co., Ltd.), and the mice in the dressing treatment group were smeared with the transdermal mini collagen dressing product solution prepared in Example 5.

[0099] 2. Combo ultrasound images of mouse skin tissue

[0100] Using Combo's ultrasound imaging (ultrasound signal intensity white > yellow > red > blue > green > black), the density and thickness of the skin were detected in vivo, and the ability of mini collagen to promote the repair of UV damage in mouse skin was evaluated.

[0101] Ultrasound images of mouse skin tissue Figure 7 As shown: On the first day after skin UV damage modeling, the density and thickness of the epidermis and dermis of the mice in the blank control group remained at a healthy level. The density and thickness of the epidermis of each group of UV damage increased due to edema, and the density of the dermis decreased. On the third day, the density and thickness of the epidermis and dermis of the mice in the blank control group remained at a healthy level; the density and thickness of the epidermis of the skin of each experimental group were still high due to scab. There was no significant increase in the density of the dermis in the negative control group, and the density of the dermis in the commercial product group and the dressing group began to increase. On the fifth day, the density and thickness of the epidermis and dermis of the mice in the blank control group remained at a healthy level. The epidermal density and thickness of the negative control group were still high, the epidermal thickness of the commercial product group and the dressing group began to decrease, and the epidermal thickness of the dressing group was lower than that of the commercial product group. The dermal density of the negative control group began to increase, and the dermal density of the commercial product group and the dressing group continued to increase. The dressing group was higher than the commercial product group, and both were higher than the negative control group. On the seventh day, the density and thickness of the epidermis and dermis of the mice in the blank control group remained at a healthy level. The epidermal thickness of the negative control group and the commercially available product group decreased, and the density of the dermis increased, but did not return to normal skin levels. The epidermis and dermis of the dressing group returned to normal skin levels. The above results show that the transdermal mini collagen dressing product described in this application helps UV-damaged skin return to a healthy density and thickness.

[0102] 3.Combo evaluation of mouse skin tissue chromaticity and transepidermal water loss (TEWL)

[0103] The skin color probe of Combo was used to detect the chromaticity of the mouse back skin and evaluate the ability of transdermal mini collagen to reduce skin erythema (the erythema index of the skin can reflect the condition of the skin capillaries and directly reflect the degree of inflammation. The lower the value, the better the repair effect). Figure 8 As shown: On the first day of skin UV damage repair, the skin chromaticity of the blank control group remained at a normal level, the skin chromaticity of each UV damage group increased and was significantly higher than that of the blank control group, and there was no significant difference between the commercially available product group and the dressing group. On the third day, the skin chromaticity of the blank control group remained at a normal level, the skin chromaticity of each UV damage group continued to increase, there was no significant difference in skin chromaticity between the commercially available product group and the dressing group, and both were significantly lower than the negative control group. On the fifth day, the skin chromaticity of the blank control group remained at a normal level, the skin chromaticity of the dressing group decreased and was significantly lower than that of the commercially available product group, and both were significantly lower than the negative control group. On the seventh day, the skin chromaticity of the blank control group remained at a normal level, the skin chromaticity of the negative control group and the commercially available product group was still significantly higher than the normal level, and the skin chromaticity of the dressing group returned to normal levels. The above results show that the transdermal mini collagen dressing product described in the present application helps to restore the chromaticity of UV-damaged skin to normal levels.

[0104] The TEWL probe of Combo was used to detect the transepidermal water loss of the mouse back skin, and the ability of mini collagen to promote the rapid recovery of skin TEWL to normal levels was evaluated (TEWL refers to the loss of water in the dermis through evaporation of the epidermis. The higher the value, the more water loss in the skin and the worse the skin barrier function). The TEWL test results of mouse skin tissue are shown in Figure 1. Fig. 9 As shown: On the first day of skin UV damage repair, the skin TEWL of the blank control group remained at a normal level, and the barrier function of the skin in each experimental group was destroyed, resulting in the measured skin TEWL being significantly higher than that of the blank control group, and the skin TEWL of the commercially available product group and the dressing group being significantly lower than that of the negative control group; on the third day, the skin TEWL of the blank control group remained at a normal level, and the skin TEWL of the dressing group decreased and was significantly lower than that of the commercially available product group, both of which were significantly lower than that of the negative control group. On the fifth day, the skin TEWL of the blank control group remained at a normal level, and the skin TEWL of the dressing group continued to decrease and was significantly lower than that of the commercially available product group, both of which were significantly lower than that of the negative control group. On the seventh day, the skin TEWL of the blank control group remained at a normal level, and the skin TEWL of the negative control group and the commercially available product group was still significantly higher than the normal level, and the skin TEWL of the dressing group returned to the normal skin level. The above results show that the transdermal mini collagen dressing product solution described in the present application helps to restore the transepidermal water loss of UV-damaged skin to normal levels.

[0105] 4. HE staining of mouse skin tissue

[0106] HE staining was used to study the ability of transdermal mini collagen to promote the repair of acute UV-damaged skin at the tissue level. Fig.10 As shown: The back skin of mice in the blank control group was in a healthy state throughout the experiment, with no inflammatory reaction, the epidermis was continuous and complete, the cells of each layer of the epidermis were arranged neatly and orderly, the epidermis and the dermis were closely connected, and the junction between the two was wavy. On the first day of skin UV damage repair, the epidermis of the back skin of mice in each UV damage group was damaged and the thickness increased significantly, the basal layer and dermis were edematous, and the dermis was edematous and infiltrated with inflammatory cells. On the third day, the epidermis of the negative control group and the commercial product group began to keratinize, and the dermis was edematous and infiltrated with a large number of inflammatory cells. The epidermis of the dressing group began to keratinize, and the connective tissue close to the epidermis protruded to the epidermis to form dermal papillae, the dermis was edematous, and there was inflammatory cell infiltration. On the fifth day, the epidermis of the negative control group continued to keratinize, and the dermis was edematous and infiltrated with inflammatory cells. The epidermis of the commercial product group continued to keratinize, the connective tissue close to the epidermis protruded to the epidermis to form dermal papillae, the edema of the dermis began to subside, and the number of inflammatory cells decreased slightly. The epidermis of the dressing group began to regenerate, but the keratinized epidermis did not fall off; the dermal papillae close to the epidermis were more obvious, the edema of the dermis basically disappeared, and the number of inflammatory cells decreased. On the 7th day, the epidermis of the negative control group continued to keratinize, and the connective tissue close to the epidermis protruded to the epidermis to form dermal papillae. The keratinized epidermis of the commercial product group was not completely detached, the new epidermis was significantly thicker than that of the blank control group, the epidermal cells had no obvious stratification, the edema of the dermis did not completely subside, and there was still a small amount of inflammatory cell infiltration. The keratinized epidermis of the dressing group was detached, the new epidermis was slightly thicker than that of the blank control group, and the epidermal cells were arranged in an orderly manner; the edema of the dermis completely subsided, and the inflammatory reaction disappeared. The above results indicate that the transdermal mini collagen dressing product described in this application can effectively repair skin damaged by acute ultraviolet light.

[0107] 5. Masson staining of mouse skin tissue

[0108] Masson staining was used to study the ability of transdermal mini-collagen to promote the repair and regeneration of collagen fibers in acute UV-damaged skin. Fig.11As shown: The back skin of mice in the blank control group was in a healthy state throughout the experiment. The fibers in the dermis were arranged continuously and densely, the collagen fibers in the papillary layer were small and slender, and the collagen fibers in the reticular layer were thick. The collagen fibers were roughly arranged parallel to the epidermis. On the first day of skin UV damage repair, the collagen fibers in the dermis of the back skin of mice in each group of UV damage were broken and broken, the overall fiber arrangement was disordered, and some fibers curled and gathered into clumps. On the third day, the collagen fibers in the negative control group and the commercial product group were broken, curled, and gathered into clumps, and the arrangement was disordered. In the dressing group, new collagen fibers began to be generated at the broken parts of the collagen fibers in the dermis, the overall fiber arrangement was disordered, and some fibers curled and gathered into clumps, and inflammatory cells infiltrated in the gaps between the collagen fibers. On the fifth day, the collagen fibers in the negative control group were broken, curled, and gathered into clumps, and the arrangement was disordered. New collagen fibers began to be generated at the broken parts of the collagen fibers in the dermis of the commercial product group, and the collagen fibers were arranged in a disordered manner, and some curled and gathered. The collagen fibers in the dermis of the dressing group continued to regenerate, and the fiber arrangement began to become orderly, but a small number of collagen fibers were still arranged in a disordered manner. On the 7th day, new collagen fibers began to be generated at the broken parts of the collagen fibers in the dermis of the negative control group. The collagen fibers were arranged in a disordered manner, and some were curled and aggregated. The collagen fibers in the commercially available product group continued to increase, and the collagen fibers were arranged in a disordered manner. The collagen fibers in the dermis of the dressing group basically returned to a normal state, and the fibers were arranged in an orderly manner. The above results show that the transdermal mini collagen dressing product solution described in the present application can effectively promote the repair and regeneration of collagen fibers in acute UV-damaged skin.

[0109] 6. Hydroxyproline content in mouse skin tissue

[0110] Take the mouse back skin and dry it in a 60℃ oven, then cut it into pieces, accurately weigh 20mg, add 2mL HCl (6mol / L) and mix well, place it in an 85℃ oven for 12h, take it out and cool it to room temperature, add an appropriate amount of NaOH solution (10mol / L) to adjust the pH to neutral. After filtering, discard the solid impurities, keep the supernatant, dilute it with water to 20.0mL, and get the sample test solution. Take 200μL of the sample test solution in a test tube, add deionized water to 1.0mL; add 2.0mL of chloramine T solution, shake well and place it at room temperature for 20min; add 2.0mL of color developer, mix well and quickly place the test tube in an 85℃ water bath to react for 10min; quickly cool to room temperature; use tube 1 as a blank control, measure the absorbance at a wavelength of 561nm, record the data, and calculate the corresponding hydroxyproline content of the sample according to the standard curve.

[0111] The experimental results are as follows Fig.12As shown: On the 7th day, the hydroxyproline content in the skin of the blank control group was 32.12±2.69μg / mg; the hydroxyproline content in the skin of the negative control group was 19.42±3.05μg / mg; the hydroxyproline content in the skin of the commercial product group was 24.91±0.47μg / mg; the hydroxyproline content in the skin of the dressing group was 31.76±4.23μg / mg, which was significantly higher than that of the negative control group and the commercial product group, and there was no significant difference with the blank control group. The above experimental results show that the transdermal mini collagen dressing product described in this application has the ability to promote the regeneration of collagen in UV-damaged skin.

[0112] Example 7 Human efficacy evaluation of transdermal mini collagen dressing products

[0113] 1. Grouping of subjects

[0114] Based on the principle of voluntariness, 10 subjects who met the inclusion criteria were recruited, including 2 males and 8 females, aged 22-27 years old. The left side of the face of all subjects was the test group (transdermal mini collagen dressing product), and the right side of the face was the commercial product group (commercially available collagen dressing product solution, Xi'an Juzi Biogene Technology Co., Ltd.), with 10 subjects in each group. All subjects used the product after cleaning their face, once every three days, and removed the dressing after 20 minutes of each application and gently massaged the skin to absorb the essence as much as possible (no need to wash), for 28 days. The subjects were required to visit 5 times in total, and the skin condition of the subjects was evaluated at 0, 1, 2, 3, and 4 weeks respectively.

[0115] 2. Adverse reaction feedback from subjects

[0116] The experimenters evaluated the skin adverse reactions of the subjects and measured the parameters according to the skin reaction grading standards for human trial tests in Chapter 7 "Human Safety Test Methods" of the "Technical Specifications for Safety of Cosmetics" (2015 Edition) (no reaction, 0 points; weak erythema, 1 point; erythema, infiltration, papules, 2 points; erythema, edema, papules, blisters, 3 points; erythema, edema, bullae, 4 points). The subjects were asked to report any adverse reactions at any time. Fig.13 As shown: All subjects had no adverse reactions during the entire experiment. One of the subjects reported a tingling sensation when the dressing product was first used, which improved after a few minutes. One subject reported that the tingling sensation was not obvious, and the rest of the subjects reported no discomfort.

[0117] 3. Ultrasound images of the subject’s skin

[0118] Combo ultrasound imaging (ultrasound signal intensity white>yellow>red>blue>green>black) was used to observe the density and thickness of the skin and evaluate the ability of the transdermal mini collagen dressing product to promote the increase of the subject's skin density. Fig.14 As shown: With the increase of experimental days, there was no significant change in the epidermal thickness of the dressing group and the commercial product group, and the dermal skin density increased, but the rate of increase in the dressing group was faster than that in the commercial product group. Except for day 0, the dermal density of the dressing group was higher than that of the commercial product group. The above results show that the transdermal mini collagen dressing described in this application can effectively increase the density of the dermal layer.

[0119] 4. Combo tests the density, brightness, moisture and transepidermal water loss (TEWL) of the subjects’ skin

[0120] The ultrasound probe of the Combo was used to detect the density of the facial skin of the subjects, and the ability of the transdermal mini collagen dressing to promote the increase of skin density was evaluated. The skin color probe of the Combo was used to detect the chromaticity of the facial skin of the subjects, and the ability of the transdermal mini collagen dressing to brighten the skin tone was evaluated (CIE L*a*b* is used to evaluate the color of the skin, where L* is the brightness of the skin color, and the higher the value, the whiter the skin is). The moisture probe of the Combo was used to detect the moisture of the facial skin of the subjects, and the ability of the transdermal mini collagen dressing to increase skin moisture was evaluated. The TEWL probe of the Combo was used to detect the transepidermal water loss of the facial skin of the subjects, and the ability of the dressing to reduce TEWL was evaluated.

[0121] The test results of the subjects' skin density, brightness, moisture, and transepidermal water loss were as follows: Fig.15 As shown: on day 0, there was no significant difference in skin density, brightness, moisture, and transepidermal water loss between the dressing group and the commercial product group; on day 7, the skin density, brightness, and moisture of the dressing group were higher than those of the commercial product group, and TEWL was lower than that of the commercial product group; on day 14, the skin density, brightness, and moisture of the dressing group were higher than those of the commercial product group, and TEWL was significantly lower than that of the commercial product group; on day 21, the skin density, brightness, and moisture of the dressing group were higher than those of the commercial product group, and TEWL was lower than that of the commercial product group; on day 28, the skin density of the dressing group was significantly higher than that of the commercial product group, and the brightness and moisture were higher than those of the commercial product group, and TEWL was significantly lower than that of the commercial product group. The above results show that the transdermal mini collagen dressing described in the present application can effectively improve the skin condition.

[0122] In summary, the transdermal mini-collagen containing two GRR sequences is non-cytotoxic, can promote the adhesion and proliferation of HFF-1 cells, and effectively penetrate cells and skin tissues. The transdermal mini-collagen dressing product prepared therefrom can accelerate the recovery of the density, chromaticity, brightness, moisture and transepidermal water loss of UV-damaged skin to the level of healthy skin, and effectively promote the restoration of the original structure and function of each layer of damaged skin.

[0123] The above description is only the details of individual exemplary implementation cases of the present invention. For those skilled in the art, the present invention may have various modifications and changes according to specific preparation conditions during actual application, and is not intended to limit the present invention. Anything within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A transdermal mini collagen for repairing skin photodamage, characterized in that: The polypeptide sequence of the transdermal mini collagen is: N1-GFOGER-N2-(GRR) n ; Wherein, the GFOGER is Gly-Phe-Hyp-Gly-Glu-Arg; the GRR is Gly-Arg-Arg; The N1 and N2 contain at least 3 repeated Gly-Xaa-Yaa and / or Gly-Xaa-Xaa; the Xaa and Yaa are any different amino acids; The n is any integer greater than or equal to 1.

2. The transdermal mini collagen according to claim 1, characterized in that The Xaa is Pro; the Yaa is Hyp.

3. The transdermal mini collagen according to claim 2, characterized in that The polypeptide sequence of the transdermal mini collagen is: (GPO) m GFOGER(GPO) m (GRR) n ; The GPO is Gly-Pro-Hyp; and m is any integer greater than or equal to 3.

4. The transdermal mini-collagen according to claim 3, characterized in that The m is any integer greater than or equal to 3 and less than or equal to 5.

5. The transdermal mini collagen according to claim 4, characterized in that The polypeptide sequence of the transdermal mini collagen is: (GPO)5GFOGER(GPO)5(GRR) n .

6. The transdermal mini-collagen according to claim 5, characterized in that The n is 1 or 2.

7. Use of the transdermal mini collagen according to any one of claims 1 to 6 in the preparation of skin damage repair products.

8. The use according to claim 7, characterized in that The skin damage is skin photodamage.

9. The use according to claim 8, characterized in that The products for repairing skin photodamage include cosmetics, medical cosmetics and medicines.

10. A dressing for repairing photodamage to the skin, comprising the following components in percentage by mass: 0.01% of the transdermal mini collagen according to any one of claims 1 to 6, 0.20% of phenoxyethanol, 0.15% of sodium hyaluronate, 0.01% of methylparaben, 3.00% of glycerol, and the balance being water.

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