A short peptide for improving skin wrinkles and a subcutaneous injection hydrogel containing the short peptide

Abstract

The present invention relates to a short peptide for improving skin wrinkles and a subcutaneous injection hydrogel containing the short peptide. The amino acid sequence of the short peptide is shown in SEQ ID No. 1 and / or SEQ ID No. 2, and the short peptide has the function of promoting the proliferation of skin fibroblasts and skin keratinocytes, so it can be used to improve skin wrinkles.

Description

Technical Field

[0001] The present invention relates to the medical field, and in particular to polypeptides for medical cosmetic injection and applications thereof. Background Art

[0002] Skin aging is caused by many factors, which can be divided into natural aging and exogenous aging. Natural aging refers to programmed aging caused by irresistible factors within the body, while exogenous aging is mainly caused by environmental factors. The main forms of aging include deepening of skin wrinkles, dryness and roughness, vascular dilation, pigmentation, etc.

[0003] The skin is composed of three major parts: the epidermis, dermis, and subcutaneous tissue. The epidermis has a skin barrier function, and most of its cells are keratinocytes. The decrease in the vitality and proliferation of these cells will lead to a decrease in the skin's moisturizing function or elasticity. The fibroblasts in the dermis can synthesize and secrete a variety of active proteins, such as collagen, hyaluronic acid, elastin, and cell growth factors. These active ingredients are important elements that make up the skin and play a vital role in keeping the skin firm and healthy.

[0004] Existing studies have shown that promoting the proliferation of keratinocytes and fibroblasts can restore normal skin metabolism, increase skin moisture content, and enhance the synthesis and secretion of key factors such as collagen, thereby maintaining the microenvironment homeostasis of normal skin renewal and fundamentally improving skin aging problems such as wrinkles caused by various reasons. Summary of the invention

[0005] One aspect of the present invention provides a short peptide, whose amino acid sequence is shown in SEQ ID No. 1 and / or SEQ ID No. 2.

[0006] The second aspect of the present invention provides a composition, which comprises the short peptide as described in the first aspect of the present invention and an acceptable pharmaceutical excipient.

[0007] In one embodiment, the composition further comprises a dermal filler.

[0008] In a specific embodiment, the dermal filler is a first dermal filler and a second dermal filler, wherein the first dermal filler is bacterial cellulose, and the second dermal filler is sodium hyaluronate and / or collagen.

[0009] In a specific embodiment, the molecular weight of the sodium hyaluronate ranges from 5,000 to 2,000,000 Daltons.

[0010] In a specific embodiment, the molecular weight of the sodium hyaluronate is 600,000 Daltons.

[0011] In one embodiment, the pharmaceutical excipients include sodium carboxymethylcellulose and water, wherein the water may be water for injection, for example.

[0012] In one embodiment, the molecular weight of the sodium carboxymethyl cellulose ranges from 5,000 to 100,000 Daltons.

[0013] In a specific embodiment, the molecular weight of the sodium carboxymethyl cellulose is 90,000 Daltons.

[0014] In a specific embodiment, the amount of the short peptide in the composition is 2.5 to 40 μg / ml.

[0015] In a specific embodiment, the amount of the short peptide in the composition is 2.5 to 20 μg / ml.

[0016] In a specific embodiment, the amount of the short peptide in the composition is 5 to 40 μg / ml.

[0017] In a specific embodiment, the amount of the short peptide in the composition is 5 to 20 μg / ml.

[0018] In a specific embodiment, the amount of the short peptide in the composition is 10 to 20 μg / ml.

[0019] In a specific embodiment, the amount of the first dermal filler in the composition is 1 to 10 mg / mL; preferably, the amount of the bacterial cellulose in the composition is 2.5 mg / mL.

[0020] In a specific embodiment, the amount of the second dermal filler in the composition is 10 to 100 mg / mL; preferably, the amount of the second dermal filler in the composition is 10 mg / mL.

[0021] In a specific embodiment, the amount of sodium carboxymethylcellulose in the composition is 10 to 50 mg / mL, preferably, the amount of sodium carboxymethylcellulose in the composition is 10 mg / mL.

[0022] In one embodiment, the composition is a subcutaneous injectable hydrogel.

[0023] In a specific embodiment, the bacterial cellulose is a textured bacterial cellulose microgel, which is prepared by treating a bacterial cellulose gel sheet with hydrogen peroxide, alkali and acid.

[0024] In one embodiment, the method further comprises the step of treating with an alkaline earth metal salt and / or a transition metal salt.

[0025] In a specific embodiment, the alkaline earth metal salt and / or transition metal salt is at least one of magnesium chloride, calcium chloride, strontium chloride, zinc chloride, magnesium nitrate, calcium nitrate, strontium nitrate and zinc nitrate.

[0026] In a specific embodiment, the method for preparing the textured bacterial cellulose microgel further comprises the step of subjecting the bacterial cellulose gel sheet to freeze-thaw treatment.

[0027] In a specific embodiment, the method for preparing the textured bacterial cellulose microgel further comprises the step of stretching the bacterial cellulose gel sheet.

[0028] In one embodiment, the method for preparing the textured bacterial cellulose microgel comprises the following steps:

[0029] 1) treating the bacterial cellulose gel sheet with an aqueous hydrogen peroxide solution to obtain a hydrogen peroxide-treated bacterial cellulose gel sheet;

[0030] 2) treating the hydrogen peroxide-treated bacterial cellulose gel sheet with an aqueous solution of an alkaline earth metal and / or transition metal salt to obtain a salt-treated bacterial cellulose gel sheet;

[0031] 3) subjecting the salt-treated bacterial cellulose gel sheet to freeze-thaw treatment to obtain a freeze-thaw-treated bacterial cellulose gel sheet;

[0032] 4) treating the freeze-thaw treated bacterial cellulose gel sheet with alkali to obtain an alkali treated bacterial cellulose gel sheet;

[0033] 5) stretching the alkali-treated bacterial cellulose gel sheet to obtain a stretched bacterial cellulose gel sheet;

[0034] 6) treating the stretched bacterial cellulose gel sheet with acid to obtain a textured bacterial cellulose gel sheet.

[0035] In a specific embodiment, the method further comprises step 7) of washing the textured bacterial cellulose gel sheet obtained in step 6) to obtain a washed textured bacterial cellulose gel sheet.

[0036] In a specific embodiment, the method further comprises step 8), homogenizing and crushing the cleaned textured bacterial cellulose gel sheet to obtain a homogenized textured bacterial cellulose microgel.

[0037] In a specific embodiment, in step 1), the mass / volume ratio of the bacterial cellulose gel sheet to the aqueous hydrogen peroxide solution is 1 g:(1-10 mL).

[0038] In a specific embodiment, the volume concentration of the aqueous solution of hydrogen peroxide is 2% to 10%.

[0039] In a specific embodiment, the bacterial cellulose gel sheet is immersed in the aqueous hydrogen peroxide solution for 1 to 2 hours, wherein ultrasonication is performed for 10 to 30 minutes.

[0040] In a specific embodiment, in step 2), the mass / volume ratio of the bacterial cellulose gel sheet to the aqueous salt solution is 1 g:(1-10 mL), for example 1 g:(2-10 mL).

[0041] In a specific embodiment, the molar concentration of the aqueous salt solution is 0.01 to 1 mol / L.

[0042] In a specific embodiment, the bacterial cellulose gel sheet treated with hydrogen peroxide is immersed in the aqueous salt solution for 2 to 24 hours, wherein ultrasonication is performed for 0.5 to 1 hour.

[0043] In one specific embodiment, in step 4), the base is sodium hydroxide.

[0044] In a specific embodiment, the mass / volume ratio of the bacterial cellulose gel sheet to the aqueous alkali solution is 1 g: (1-10 mL), for example 1 g: (5-10 mL); the alkali concentration is 0.1 mol / L.

[0045] In a specific embodiment, the freeze-thaw treated bacterial cellulose gel sheet is immersed in the alkaline aqueous solution for 6 to 24 hours.

[0046] In one embodiment, in step 6), the acid is hydrochloric acid.

[0047] In a specific embodiment, the mass / volume ratio of the bacterial cellulose gel sheet to the acid aqueous solution is 1 g:(1-20 mL), for example 1 g:(5-20 mL); the acid concentration is 0.1 mol / L.

[0048] In a specific embodiment, the alkali-treated bacterial cellulose gel sheet is soaked in the acid aqueous solution for 1 to 2 hours.

[0049] In a specific embodiment, in step 3), freeze-thaw is performed 1 to 5 times.

[0050] In a specific embodiment, in step 5), the alkali-treated bacterial cellulose gel sheet is stretched to an elongation of 10% to 60%.

[0051] In one embodiment, in step 7), the textured bacterial cellulose gel sheet is washed with water (eg, deionized water).

[0052] In a specific embodiment, in step 8), the washed textured bacterial cellulose gel sheet is homogenized and crushed in physiological saline or water for injection to obtain a homogenized textured bacterial cellulose microgel dispersion.

[0053] In a specific embodiment, in the textured bacterial cellulose microgel dispersion, the mass content of the physiological saline or water for injection is 10% to 99%.

[0054] In one embodiment, the textured bacterial cellulose microgel is capable of passing through a 22G to 29G syringe needle.

[0055] The third aspect of the present invention provides the use of the short peptide according to the first aspect of the present invention or the composition according to any one of the second aspect of the present invention in improving skin wrinkles.

[0056] In a specific embodiment, the skin wrinkles include at least one of forehead wrinkles, frown lines, crow's feet, tear trough lines, nasolabial folds, vertical lip lines, mouth corner lines, marionette lines, lower eyebrow lines, neck lines and back of hand wrinkles.

[0057] Beneficial effects of the present invention: The present invention finds that the short peptides with amino acid sequences such as SEQ ID No. 1 and / or SEQ ID No. 2 have the function of promoting the proliferation of skin fibroblasts and skin keratinocytes, and based on this, they can be used to improve skin wrinkles. Furthermore, the present invention further develops a subcutaneous injection hydrogel preparation that can be used to improve skin wrinkles. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 : Analysis of the ability of short peptide 1 and short peptide 2 at different concentrations in Example 1 to promote fibroblast proliferation.

[0059] Figure 2 : Analysis of the ability of short peptide 3 at different concentrations in Example 1 to promote fibroblast proliferation.

[0060] Figure 3 : Analysis of the ability of short peptide 1 and short peptide 2 at different concentrations in Example 2 to promote keratinocyte proliferation.

[0061] Figure 4 :Analysis of the ability of short peptide 1 and short peptide 2 to promote migration of skin fibroblasts.

[0062] Figure 5 :The results of radioactivity counting in various tissues of New Zealand rabbits.

[0063] Figure 6 : Scanning electron micrographs of textured bacterial cellulose microgel 1# and control bacterial cellulose microgel 4#.

[0064] Figure 7 : Analysis of skin tissue morphology after subcutaneous injection of hydrogel containing short peptide 1 or short peptide 2 in rabbits.

[0065] Figure 8 : Comparative analysis of facial wrinkles in one subject before and after subcutaneous short peptide 2 hydrogel injection.

[0066] Fig. 9 : Scanning analysis of wrinkle depth after subcutaneous injection of hydrogel containing short peptide 2 on the face of subjects. DETAILED DESCRIPTION

[0067] The above contents of the present invention are further described in detail below in the form of preferred implementation cases, but they do not constitute a limitation to the present invention.

[0068] Unless otherwise specified, the reagents in the embodiments of the present invention can be purchased through commercial channels.

[0069] The amino acid sequence of short peptide 1 is shown in SEQ ID No. 1, the amino acid sequence of short peptide 2 is shown in SEQ ID No. 2, and the amino acid sequence of short peptide 3 is shown in SEQ ID No. 3 (which is a short peptide derived from FGF1). All three short peptides were synthesized by Nanjing GenScript Biotechnology Co., Ltd.

[0070] Example 1: Analysis of the function of short peptide 1 and short peptide 2 in promoting the proliferation of skin fibroblasts.

[0071] Human skin fibroblasts (NIH-3T3, catalog number: CRL-1658, ATCC, USA) were seeded into Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum, cultured at 37°C to the logarithmic growth phase, and then transferred to 96-well cell culture plates (3000 cells / well, 100 μl of DMEM medium containing 10% fetal bovine serum / well). The lyophilized powders of short peptide 1 and short peptide 2 were diluted to 10 mg / mL (mg / mL) with phosphate buffer (137 mmol / L sodium chloride, 2.7 mmol / L potassium chloride, 10 mmol / L sodium dihydrogen phosphate, 2 mmol / L potassium dihydrogen phosphate, 1 mmol / L calcium chloride and 0.5 mmol / L magnesium chloride, pH = 7.2) to prepare short peptide 1 solution and short peptide 2 solution, and then the short peptide 1 solution was added to the culture wells of the 96-well cell culture plate transferred to human skin fibroblasts in proportion, so that the final concentration of short peptide 1 reached 2.5, 5, 10, 20 and 40 μg / mL, and the short peptide 1 treatment groups at each concentration were obtained; the short peptide 2 solution was added to the culture wells of another 96-well cell culture plate transferred to human skin fibroblasts, so that the final concentration of short peptide 2 reached 2.5, 5, 10, 20 and 40 μg / mL, and the short peptide 2 treatment groups at each concentration were obtained. The blank control group was added with an equal volume of phosphate buffer. After culturing at 37°C for 48 hours, cells were counted using a cell counting kit (Cell Counting kit-8, CCK-8) (Wuhan Punosai Life Science Technology Co., Ltd., catalog number: P-CA-001, Hubei). The operation is as follows: According to the instructions of the cell counting kit, 10 μg of reagent was added to each well of the 96-well cell culture plate to culture cells. After continuing to incubate at 37 degrees Celsius for 1 hour, the absorbance of each well was measured using an enzyme reader (Multiskan, MK3 enzyme reader, Shanghai) (detection wavelength 450nm, reference wavelength 630nm). The absorbance value of the blank control group set at each concentration of the short peptide was used as an internal reference to calculate the relative absorbance of the short peptide 1 treatment group and the short peptide 2 treatment group at each concentration (relative absorbance = absorbance value of the short peptide treatment group / absorbance value of the blank control group). The results are shown in Figure 1 The results showed that compared with the blank control group, short peptide 1 and short peptide 2 could significantly promote the proliferation of skin fibroblasts in the concentration range of 2.5 to 20 μg / ml, among which the final concentration with the most significant proliferation-promoting ability was 10 μg / ml.

[0072] Example 2: Analysis of the function of short peptide 3 in promoting the proliferation of skin fibroblasts.

[0073] The short peptide 1 in Example 1 was replaced by short peptide 3, and an equal volume of phosphate buffer was added as a blank control group. The rest was the same as in Example 1. Finally, the relative absorbance of the short peptide 3 treatment group at each concentration was calculated. The results are shown in Figure 2 .

[0074] Figure 2 The results showed that compared with the blank control group, short peptide 3 did not have the ability to significantly promote fibroblast proliferation in the concentration range of 2.5 to 40 μg / ml.

[0075] Example 3: Analysis of the function of short peptide 1 and short peptide 2 in promoting skin keratinocyte proliferation.

[0076] Human skin keratinocytes (HaCaT, catalog number: PCS-200-011, ATCC, USA) were seeded into Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum, cultured at 37°C to the logarithmic growth phase, and then transferred to 96-well cell culture plates (5000 cells / well, 100 μl of DMEM medium containing 10% fetal bovine serum / well). The lyophilized powders of short peptide 1 and short peptide 2 were diluted to 10 mg / mL (mg / mL) with phosphate buffer (137 mmol / L sodium chloride, 2.7 mmol / L potassium chloride, 10 mmol / L sodium dihydrogen phosphate, 2 mmol / L potassium dihydrogen phosphate, 1 mmol / L calcium chloride and 0.5 mmol / L magnesium chloride, pH = 7.2) to prepare short peptide 1 solution and short peptide 2 solution, and then the short peptide 1 solution was added to the culture wells of the 96-well cell culture plate transferred to human skin fibroblasts in proportion, so that the final concentration of short peptide 1 reached 2.5, 5, 10, 20 and 40 μg / mL, and the short peptide 1 treatment groups at each concentration were obtained; the short peptide 2 solution was added to the culture wells of another 96-well cell culture plate transferred to human skin fibroblasts, so that the final concentration of short peptide 2 reached 2.5, 5, 10, 20 and 40 μg / mL, and the short peptide 2 treatment groups at each concentration were obtained. The blank control group was added with an equal volume of phosphate buffer. After culturing at 37°C for 48 hours, cells were counted using a cell counting kit (Cell Counting kit-8, CCK8). The operation was as follows: According to the instructions of the cell counting kit, 10 μg of the reagent was added to each well of the 96-well cell culture plate to culture cells. After continuing to incubate at 37°C for 1 hour, the absorbance of each well was measured using an enzyme reader (Multiskan, MK3 enzyme reader, Shanghai) (detection wavelength 450nm, reference wavelength 630nm). The absorbance value of the blank control group set at each concentration of the short peptide was used as an internal reference to calculate the relative absorbance of the short peptide 1 treatment group and the short peptide 2 treatment group at each concentration (relative absorbance = absorbance value of the short peptide treatment group / absorbance value of the blank control group). The results are shown in Figure 3 The results showed that compared with the blank control group, short peptide 1 and short peptide 2 could significantly promote the proliferation of skin keratinocytes within the concentration range of 5 to 40 μg / ml, among which the final concentration with the most significant proliferation-promoting ability was 20 μg / ml.

[0077] Example 4: Analysis of the function of short peptide 1 and short peptide 2 in promoting migration of skin fibroblasts.

[0078] Primary skin fibroblasts (Cat. No. PCS-201-012, ATCC, USA) were seeded in Dulbecco's Modified Eagle Medium (DMEM), cultured at 37°C until the logarithmic growth phase, and then seeded into 24-well cell culture plates (10,000 cells / well, 0.5 ml DMEM medium / well). After the cells were cultured to fill the surface of the culture wells (coverage of 100%), scratches were made with a pipette tip (range of 1 to 200 μl), and the scratch width was about 3 mm. The lyophilized powders of short peptide 1 and short peptide 2 were diluted to 10 mg / mL (mg / mL) with phosphate buffer (137 mmol / L sodium chloride, 2.7 mmol / L potassium chloride, 10 mmol / L sodium dihydrogen phosphate, 2 mmol / L potassium dihydrogen phosphate, 1 mmol / L calcium chloride and 0.5 mmol / L magnesium chloride, pH = 7.2) to prepare short peptide 1 solution and short peptide 2 solution. The short peptide 1 solution was slowly added to the cell culture wells of the culture plate to make the final concentration of short peptide 1 10 μg / mL; the short peptide 2 solution was slowly added to the cell culture wells of another culture plate to make the final concentration of short peptide 2 10 μg / mL. The blank control group was added with an equal volume of phosphate buffer. After continuous culture for 24 hours, the cell migration was observed under an inverted microscope. The results are shown in Figure 4 The results showed that compared with the blank control group, both the short peptide 1 treatment group and the short peptide 2 treatment group could significantly promote the rapid migration of skin fibroblasts.

[0079] Example 5: Analysis of tissue distribution of short peptide 1 and short peptide 2 after intravenous injection in New Zealand rabbits.

[0080] In order to understand the dynamic changes and tissue distribution of short peptides entering the body through veins, we first used radioactivity 125 I (isotope iodine 125) was used to label short peptide 1 and short peptide 2, respectively, to obtain radioactivity of 39.58 microcuries / mmole for labeled short peptide 1 and 216.4 microcuries / mmole for labeled short peptide 2, respectively.

[0081] The labeled peptide was diluted with physiological saline to prepare labeled peptide 1 and labeled peptide 2 at a concentration of 125 μg / ml. 0.4 ml of labeled peptide 1 solution was injected into the ear vein of New Zealand rabbits; 0.4 ml of labeled peptide 2 solution was injected into the ear vein of another New Zealand rabbit. Three New Zealand rabbits were treated for each treatment. 96 hours after the injection, the animals were killed, and the heart, liver, spleen, lung, kidney, stomach, intestine, brain, skeletal muscle, skin and genital tissues were collected and their radioactive counts (Counts per minute, CPM) were measured. The results are shown in Figure 5 The results showed that in both treatment groups, the radioactivity was mainly concentrated in the skin, followed by the kidney, and least in the brain. These results indicate that both short peptides have a high affinity for the skin.

[0082] Example 6: Preparation of textured bacterial cellulose microgel.

[0083] Bacterial cellulose gel in sheet or paste form was purchased from Shanghai Puzhen Biotechnology Co., Ltd.

[0084] Preparation of textured bacterial cellulose microgel dispersion 1#: 1) Take 5 grams of bacterial cellulose gel sheet, immerse it in 50 mL of 5% hydrogen peroxide aqueous solution for 1 h, and ultrasonically treat it at 50 W for 30 minutes to obtain a hydrogen peroxide-treated bacterial cellulose gel sheet; 2) Take out the hydrogen peroxide-treated bacterial cellulose gel sheet of step 1), immerse it in 10 mL of 1 mol / L magnesium chloride solution for 2 h, and ultrasonically treat it at 50 W for 0.5 h to obtain a salt-treated bacterial cellulose gel sheet; 3) Take out the salt-treated bacterial cellulose gel sheet of step 2), freeze and thaw it at -40 degrees Celsius / room temperature for 5 times to obtain a freeze-thaw-treated bacterial cellulose gel sheet; 4) Immerse the freeze-thaw-treated bacterial cellulose gel sheet in 50 mL of 0.1 mol / L sodium hydroxide solution for 6 h to obtain an alkali-treated bacterial cellulose gel sheet; 5) Take out the alkali-treated bacterial cellulose gel sheet of step 4) and stretch it to an elongation of 60% to obtain a stretched bacterial cellulose gel sheet; 6) Immerse the stretched bacterial cellulose gel sheet in 100 mL of 0.1 mol / L hydrochloric acid 2 h, to obtain a textured bacterial cellulose gel sheet; 7) taking out the textured bacterial cellulose gel sheet of step 6) and washing it thoroughly with deionized water to obtain a washed textured bacterial cellulose gel sheet; 8) then taking 1 gram of the washed bacterial cellulose gel sheet and adding it to 99 mL of normal saline, using a homogenizer to homogenize and break it until it can pass through a 29G syringe needle, to obtain a textured bacterial cellulose microgel dispersion.

[0085] Preparation of textured bacterial cellulose microgel dispersion 2#: 1) Take 10 grams of bacterial cellulose gel sheet, immerse it in 100 mL of 2% hydrogen peroxide aqueous solution for 2 h, and ultrasonically treat it at 100 W for 10 minutes to obtain a hydrogen peroxide-treated bacterial cellulose gel sheet; 2) Take out the hydrogen peroxide-treated bacterial cellulose gel sheet of step 1), immerse it in 100 mL of 0.1 mol / L magnesium nitrate solution for 24 h, and ultrasonically treat it at 100 W for 1 h to obtain a salt-treated bacterial cellulose gel sheet; 3) Take out the salt-treated bacterial cellulose gel sheet of step 2), freeze and thaw it once at -30 degrees Celsius / room temperature to obtain a freeze-thaw-treated bacterial cellulose gel sheet; 4) Immerse the freeze-thaw-treated bacterial cellulose gel sheet in 100 mL of 0.1 mol / L sodium hydroxide solution for 12 h to obtain an alkali-treated bacterial cellulose gel sheet; 5) Take out the alkali-treated bacterial cellulose gel sheet of step 4) and stretch it to an elongation of 30% to obtain a stretched bacterial cellulose gel sheet; 6) Immerse the stretched bacterial cellulose gel sheet in 100 mL of 0.1 mol / L hydrochloric acid for 1 h to obtain a textured bacterial cellulose gel sheet; 7) the textured bacterial cellulose gel sheet of step 6) is taken out and thoroughly washed with deionized water to obtain a washed textured bacterial cellulose gel sheet; 8) then 1 gram of the washed bacterial cellulose gel sheet is added into 99 mL of water for injection, and the washed bacterial cellulose gel sheet is homogenized and crushed with a homogenizer until it can pass through a 26G syringe needle to obtain a textured bacterial cellulose microgel dispersion.

[0086] Preparation of textured bacterial cellulose microgel dispersion 3#: 1) Take 10 grams of bacterial cellulose gel sheet, immerse it in 100 mL of 10% hydrogen peroxide solution for 2 h, and ultrasonically treat it at 100 W for 30 minutes to obtain a hydrogen peroxide-treated bacterial cellulose gel sheet; 2) Take out the hydrogen peroxide-treated bacterial cellulose gel sheet of step 1), immerse it in 50 mL of 0.2 mol / L zinc chloride solution for 2 h, and ultrasonically treat it at 100 W for 0.5 h to obtain a salt-treated bacterial cellulose gel sheet; 3) Take out the salt-treated bacterial cellulose gel sheet of step 2), freeze and thaw it twice at -20 degrees Celsius / room temperature to obtain a freeze-thaw-treated bacterial cellulose gel sheet; 4) Immerse the freeze-thaw-treated bacterial cellulose gel sheet in 50 mL of 0.1 mol / L sodium hydroxide solution for 24 h to obtain an alkali-treated bacterial cellulose gel sheet; 5) Take out the alkali-treated bacterial cellulose gel sheet of step 4) and stretch it to an elongation of 10% to obtain a stretched bacterial cellulose gel sheet; 6) Immerse the stretched bacterial cellulose gel sheet in 50 mL of 0.1 mol / L hydrochloric acid for 1 h to obtain a textured bacterial cellulose gel sheet; 7) the textured bacterial cellulose gel sheet of step 6) is taken out and thoroughly washed with deionized water to obtain a washed textured bacterial cellulose gel sheet; 8) then 9 grams of the washed bacterial cellulose gel is added with 1 mL of water for injection, and the mixture is homogenized and crushed with a homogenizer until it can pass through a 22G syringe needle to obtain a textured bacterial cellulose microgel dispersion.

[0087] Preparation of control bacterial cellulose microgel dispersion 4#: 1 gram of commercially available bacterial cellulose paste was added to 99 mL of normal saline, and the mixture was homogenized and crushed with a homogenizer until the mixture could pass through a 29G syringe needle to obtain a homogenized bacterial cellulose microgel dispersion.

[0088] The bacterial cellulose microgel dispersions 1# to 4# were injected into the mold through a syringe needle and freeze-dried to obtain bacterial cellulose microgels 1# to 4#. The morphology of the freeze-dried cross-section was observed using a scanning electron microscope (SEM). The results showed that an obvious three-dimensional porous scaffold structure could be observed in the textured bacterial cellulose microgels 1# to 3#, indicating that they can provide a three-dimensional mechanical support structure and sufficient cell proliferation space for tissue regeneration, while the aggregation state of the control bacterial cellulose microgel 4# was not ideal. Among them, the scanning electron microscope images of the textured bacterial cellulose microgel 1# and the control bacterial cellulose microgel 4# are shown in Figure 6 .

[0089] Example 7: Preparation of short peptide hydrogel dermal filling composition.

[0090] In a 2-liter wide-mouth bottle, add 500 ml of water for injection at 60°C, then add 5 g of bacterial cellulose, 10 g of 600,000 Dalton sodium hyaluronate, 10 g of 90,000 Dalton sodium carboxymethyl cellulose, and then add normal saline (i.e., water for injection) to 950 ml, adjust the pH value of the mixture to 6.5 to 7.0, and then stir magnetically (800 rpm), treat at room temperature (25°C) for 12 hours, place in an autoclave, sterilize at 121°C high temperature and moist heat conditions for 15 minutes, and let stand to room temperature to obtain a sterilized hydrogel.

[0091] Weigh 20 mg of lyophilized powder of short peptide 1, dissolve it in 50 ml of physiological saline, filter and sterilize it with a 0.22 μm filter to prepare a short peptide 1 injection solution, slowly add the short peptide 1 injection solution to 950 ml of sterilized hydrogel in a sterile environment (such as a clean bench), stir evenly at room temperature (25°C) for 1 hour with a magnetic stirrer at 500 rpm, and prepare a hydrogel injection of short peptide 1 (off-white). It is recorded as short peptide 1 hydrogel injection-1, which contains a final concentration of 20 μg / ml of short peptide 1, 10 mg / ml of sodium hyaluronate, 10 mg / ml of sodium carboxymethyl cellulose, and 5 mg / ml of bacterial cellulose.

[0092] Short peptide 1 hydrogel injection-2, short peptide 1 hydrogel injection-3, short peptide 1 hydrogel injection-4, short peptide 2 hydrogel injection-1, short peptide 2 hydrogel injection-2, short peptide 2 hydrogel injection-3, and short peptide 2 hydrogel injection-4 were prepared using the same operation as short peptide 1 hydrogel injection-1.

[0093] Short peptide 1 hydrogel injection-2 contains short peptide 1 with a final concentration of 40 μg / ml, 100 mg / ml sodium hyaluronate, 50 mg / ml sodium carboxymethyl cellulose, and 10 mg / ml bacterial cellulose.

[0094] The short peptide 1 hydrogel injection-3 contains short peptide 1 with a final concentration of 2.5 μg / ml, 10 mg / ml sodium hyaluronate, 10 mg / ml sodium carboxymethyl cellulose, and 1 mg / ml bacterial cellulose.

[0095] The short peptide 1 hydrogel injection-4 contains short peptide 1 with a final concentration of 10 μg / ml, 100 mg / ml collagen, 50 mg / ml sodium carboxymethyl cellulose, and 2.5 mg / ml bacterial cellulose.

[0096] Short peptide 2 hydrogel injection-1 contains short peptide 2 with a final concentration of 20 μg / ml, 10 mg / ml sodium hyaluronate, 10 mg / ml sodium carboxymethyl cellulose, and 5 mg / ml bacterial cellulose.

[0097] Short peptide 2 hydrogel injection-2 contains short peptide 2 with a final concentration of 40 μg / ml, 100 mg / ml sodium hyaluronate, 50 mg / ml sodium carboxymethyl cellulose, and 10 mg / ml bacterial cellulose.

[0098] Short peptide 2 hydrogel injection-3 contains short peptide 2 with a final concentration of 2.5 μg / ml, 10 mg / ml sodium hyaluronate, 10 mg / ml sodium carboxymethyl cellulose, and 1 mg / ml bacterial cellulose.

[0099] Short peptide 2 hydrogel injection-4 contains short peptide 2 with a final concentration of 10 μg / ml, 100 mg / ml collagen, 50 mg / ml sodium carboxymethyl cellulose, and 2.5 mg / ml bacterial cellulose.

[0100] Samples from each group were taken and divided into syringes (1 ml / syringe). 100 μl of the hydrogel injection was injected into the back skin (between the epidermis and dermis) of each of 3 New Zealand white rabbits. The control group was injected with an equal volume of saline. After 60 days of treatment, tissues were taken for immunohistochemical observation. The results are shown in Figure 7 . Figure 7 The results showed that after 60 days of treatment with the hydrogel injections in all the above groups, there was no obvious inflammatory reaction in the skin tissue, and the skin tissue structure was not significantly different from that in the control group. These results clearly show that filling the gel containing short peptides by subcutaneous injection will not cause pathological changes in the animal skin tissue.

[0101] Example 8: Dermal filling with short peptide hydrogel improves human skin wrinkles.

[0102] The short peptide 2 hydrogel injection-1 prepared in Example 7 was injected into the skin between the epidermis and dermis by syringe at the wrinkle formation sites on the faces of 20 volunteers. The filling sites were forehead wrinkles, tear grooves and glabellar wrinkles. The photos of one of the subjects before and after the filling were as follows: Figure 8 ImageJ software was used to scan and analyze the depth of facial skin wrinkles before filling (0 day), 30 days after filling, and 90 days after filling, as shown in Figure 2. Fig. 9 The results show that hydrogel filling can effectively improve the main wrinkles on the facial skin, including forehead wrinkles (marked as *1), frown lines (marked as *3) and tear grooves (marked as *2), and this improvement effect can last for more than 90 days.

Claims

1. A short peptide, the amino acid sequence of which is shown in SEQ ID No. 1 or SEQ ID No.

2.

2. A composition comprising the short peptide as claimed in claim 1 and acceptable pharmaceutical excipients.

3. The composition according to claim 2, characterized in that The composition also includes a dermal filler.

4. The composition according to claim 3, characterized in that The skin filler is a first skin filler and a second skin filler, wherein the first skin filler is bacterial cellulose, and the second skin filler is sodium hyaluronate and / or collagen.

5. The composition according to claim 4, characterized in that The molecular weight of the sodium hyaluronate ranges from 5,000 to 2,000,000 Daltons.

6. The composition according to claim 5, characterized in that The molecular weight of the sodium hyaluronate is 600,000 Daltons.

7. The composition according to any one of claims 4 to 6, characterized in that The pharmaceutical excipients include sodium carboxymethyl cellulose and water.

8. The composition according to claim 7, characterized in that The molecular weight of the sodium carboxymethylcellulose ranges from 5,000 to 100,000 Daltons.

9. The composition according to claim 8, characterized in that The molecular weight of the sodium carboxymethyl cellulose is 90,000 Daltons.

10. The composition according to claim 7, characterized in that The amount of the short peptide in the composition is 2.5 to 40 μg / ml; and / or The first dermal filler is present in the composition in an amount of 1 to 10 mg / mL; and / or The second dermal filler is present in the composition in an amount of 10 to 100 mg / mL; and / or The amount of sodium carboxymethylcellulose in the composition is 10 to 50 mg / mL.

11. The composition according to claim 4, characterized in that The bacterial cellulose is a textured bacterial cellulose microgel, and the method for preparing the textured bacterial cellulose microgel is to treat a bacterial cellulose gel sheet with hydrogen peroxide, alkali and acid.

12. The composition according to claim 11, characterized in that The method further comprises the step of treating with an alkaline earth metal salt and / or a transition metal salt.

13. The composition according to claim 12, characterized in that The alkaline earth metal salt and / or transition metal salt is at least one of magnesium chloride, calcium chloride, strontium chloride, zinc chloride, magnesium nitrate, calcium nitrate, strontium nitrate and zinc nitrate.

14. The composition according to claim 11, characterized in that The method further comprises the step of subjecting the bacterial cellulose gel sheet to freeze-thaw treatment.

15. The composition according to claim 11, characterized in that The method further comprises the step of stretching the bacterial cellulose gel sheet.

16. The composition according to claim 11, characterized in that The method for preparing the textured bacterial cellulose microgel comprises the following steps: 1) treating the bacterial cellulose gel sheet with an aqueous hydrogen peroxide solution to obtain a hydrogen peroxide-treated bacterial cellulose gel sheet; 2) treating the hydrogen peroxide-treated bacterial cellulose gel sheet with an aqueous solution of an alkaline earth metal salt and / or a transition metal salt to obtain a salt-treated bacterial cellulose gel sheet; 3) subjecting the salt-treated bacterial cellulose gel sheet to freeze-thaw treatment to obtain a freeze-thaw-treated bacterial cellulose gel sheet; 4) treating the freeze-thaw treated bacterial cellulose gel sheet with alkali to obtain an alkali treated bacterial cellulose gel sheet; 5) stretching the alkali-treated bacterial cellulose gel sheet to obtain a stretched bacterial cellulose gel sheet; 6) treating the stretched bacterial cellulose gel sheet with acid to obtain a textured bacterial cellulose gel sheet.

17. The composition according to claim 16, characterized in that The method for preparing the textured bacterial cellulose microgel further comprises step 7), washing the textured bacterial cellulose gel sheet obtained in step 6), to obtain a washed textured bacterial cellulose gel sheet.

18. The composition according to claim 17, characterized in that The method for preparing the textured bacterial cellulose microgel further comprises step 8), homogenizing and crushing the cleaned textured bacterial cellulose gel sheet to obtain a homogenized textured bacterial cellulose microgel.

19. The composition according to claim 16, characterized in that In step 1), the mass / volume ratio of the bacterial cellulose gel sheet to the hydrogen peroxide aqueous solution is 1 g: (1-10 mL); the bacterial cellulose gel sheet is immersed in the hydrogen peroxide aqueous solution for 1 to 2 hours, wherein the solution is ultrasonicated for 10 to 30 minutes; and / or In step 2), the mass / volume ratio of the bacterial cellulose gel sheet to the salt aqueous solution is 1 g: (1-10 mL); the hydrogen peroxide-treated bacterial cellulose gel sheet is immersed in the salt aqueous solution for 2 to 24 hours, wherein the hydrogen peroxide-treated bacterial cellulose gel sheet is subjected to ultrasound for 0.5 to 1 hour; and / or In step 4), the mass / volume ratio of the bacterial cellulose gel sheet to the alkaline aqueous solution is 1 g: (1-10 mL); the freeze-thawed bacterial cellulose gel sheet is immersed in the alkaline aqueous solution for 6 to 24 hours; and / or In step 6), the mass / volume ratio of the bacterial cellulose gel sheet to the acid aqueous solution is 1 g:(1-20 mL), and the alkali-treated bacterial cellulose gel sheet is immersed in the acid aqueous solution for 1 to 2 hours; and / or In step 5), the alkali-treated bacterial cellulose gel sheet is stretched to an elongation of 10% to 60%.

20. Use of the short peptide according to claim 1 or the composition according to any one of claims 2 to 19 for improving skin wrinkles.

21. The use according to claim 20, characterized in that The skin wrinkles include at least one of forehead wrinkles, frown lines, crow's feet, tear trough lines, nasolabial folds, vertical lip lines, corners of the mouth lines, marionette lines, lower eyebrow lines, neck lines and back of hand wrinkles.

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