Peptide slow-release temperature-sensitive hydrogel as well as composition, preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to peptide slow-release temperature-sensitive hydrogel as well as a composition, a preparation method and application thereof. The composition consists of a hydrogel monomer polymer and biological peptide, wherein the biological peptide is Leucokinin VIII, and the biological peptide is Leucokinin VIII The hydrogel monomer polymer is prepared by mixing and heating polyethylene glycol, lactide and stannous zincate. The preparation method of the peptide slow-release temperature-sensitive hydrogel comprises the following steps: mixing polyethylene glycol, lactide and stannous zincate, and carrying out heating and stirring reaction to prepare a hydrogel monomer polymer; and then mixing the hydrogel monomer polymer with the Leucokinin VIII, so as to obtain the peptide slow-release temperature-sensitive hydrogel. The peptide sustained-release temperature-sensitive hydrogel has the advantages of excellent temperature-sensitive characteristic, injectability, drug sustained-release performance, biocompatibility and biological safety.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to a peptide sustained-release thermosensitive hydrogel and a composition, a preparation method and an application thereof. Background Art

[0002] Trauma is a global public health problem that seriously threatens human health. From the perspective of epidemiological characteristics, trauma mainly affects young and middle-aged people aged 15-44, mainly traffic accident injuries (about 40%), fall injuries (about 25%) and crush injuries (about 15%). In recent years, the epidemiological trend of trauma has shown the characteristics of younger motor vehicle accidents and increased chronic injuries, which has posed new challenges to trauma prevention and treatment.

[0003] The wound healing process involves three closely related stages: inflammatory response, tissue proliferation and wound remodeling. This complex physiological process requires the precise coordination of multiple cells, growth factors and extracellular matrix components. Among them, keratinocytes regulate the wound microenvironment by secreting cytokines, chemokines and growth factors, promoting epidermal regeneration and wound healing. Especially in the early stages of wound repair, the proliferation and migration ability of keratinocytes directly affects the speed and quality of wound healing. Peptide bioactive molecules have become the focus of wound healing research due to their key regulatory role in cell proliferation, migration and tissue regeneration. These peptides play an important role in promoting wound healing by regulating key signaling pathways such as PI3K / AKT and MAPK.

[0004] Among many bioactive molecules, plant-derived peptides are widely used in wound repair due to their wide sources, mature extraction technology, and good biocompatibility. Although animal-derived peptides have strong biological activity, they are difficult to extract and purify, have poor stability, and are relatively limited in application. Leucokinin VIII is a polypeptide substance that exhibits significant biological activity in tissue repair and regeneration. Studies have shown that Leucokinin VIII exerts its effects by promoting the proliferation and migration of keratinocytes and enhancing tissue regeneration capacity. This polypeptide is homologous to the traditional Chinese medicine Kangfuxinye, and may promote the synthesis and secretion of extracellular matrix and improve the wound microenvironment by regulating key signaling pathways such as JAK / STAT3 and Smad3.

[0005] Hydrogels have shown outstanding advantages in the field of wound healing due to their unique physical and chemical properties and excellent biocompatibility. They can not only maintain a suitable moist environment for wounds, but also serve as an ideal carrier for drug delivery. In particular, injectable, in situ-formed hydrogel systems have the characteristics of controlled drug release, perfect adaptation to irregular wound shapes, and responsiveness to the microenvironment, providing a new research direction for wound treatment. Studies have shown that functional hydrogels can significantly improve wound healing effects by regulating the wound microenvironment, promoting angiogenesis, and accelerating tissue regeneration. Summary of the invention

[0006] In view of this, the present invention combines Leucokinin VIII polypeptide with functional hydrogel materials to develop a PLGA-PEG-PLGA thermosensitive hydrogel containing Leucokinin VIII, referred to as peptide sustained-release thermosensitive hydrogel. The hydrogel has thermosensitive properties, injectability, in-situ molding, good drug sustained-release performance, biocompatibility, and biosafety, and can be used for wound repair and regeneration. It can not only achieve sustained and controlled release of polypeptides, but also protect the wound surface through the physical barrier effect of the hydrogel, thereby improving the wound healing effect.

[0007] One of the purposes of the present invention is to provide a composition for preparing a peptide sustained-release thermosensitive hydrogel.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] The invention discloses a composition for preparing a peptide sustained-release thermosensitive hydrogel, which is composed of a hydrogel monomer (Gel) polymer and a biological peptide; the biological peptide is Leucokinin VIII; the hydrogel monomer polymer is prepared by mixing and heating polyethylene glycol, lactide and stannous zincate; the molar ratio of the lactide to the polyethylene glycol is 20:1 to 40:1, and the molar ratio of the lactide to the stannous zincate is 1000:1 to 2000:1.

[0010] Furthermore, the polyethylene glycol is PEG1000.

[0011] Furthermore, the lactide includes lactide and hexyl lactide; the molar ratio of lactide to hexyl lactide is 2:1 to 4:1.

[0012] Preferably, the molar ratio of lactide to caprylate is 2:1.

[0013] Preferably, the molar ratio of the lactide to the polyethylene glycol is 20:1.

[0014] Preferably, the molar ratio of the lactide to the stannous zincate is 1000:1.

[0015] The second object of the present invention is to provide a method for preparing a peptide sustained-release thermosensitive hydrogel.

[0016] To achieve the above object, the present invention adopts the following technical solutions:

[0017] The preparation method of peptide sustained-release thermosensitive hydrogel comprises the following steps:

[0018] (1) mixing polyethylene glycol, lactide and stannous zincate, and reacting them by heating and stirring to obtain a hydrogel monomer polymer;

[0019] (2) Mixing the hydrogel monomer polymer obtained in step (1) with Leucokinin VIII to prepare the peptide sustained-release thermosensitive hydrogel.

[0020] Furthermore, in step (1), the reaction temperature is 125-140° C., preferably 130° C.; and the reaction time is 8-12 h, preferably 10 h.

[0021] Furthermore, in step (2), the concentration of the hydrogel monomer polymer is 10% to 30%, preferably 15%; the concentration of Leucokinin VIII in the peptide sustained-release thermosensitive hydrogel is 1-10 μg / mL, preferably 5 μg / mL.

[0022] Preferably, in step (1), the molar ratio of the lactide to the polyethylene glycol is 20:1 to 40:1, most preferably 20:1; the molar ratio of the lactide to the stannous zincate is 1000:1 to 2000:1, most preferably 1000:1.

[0023] Furthermore, in step (1), the polyethylene glycol is PEG1000.

[0024] Furthermore, in step (1), the lactide is lactide and hexyl lactide; the molar ratio of lactide to hexyl lactide is 2:1 to 4:1, and most preferably is 2:1.

[0025] Furthermore, the method specifically comprises the following steps:

[0026] 1) mixing polyethylene glycol, lactide and stannous zincate, evacuating the mixture, and heating and stirring the mixture to react to obtain a reaction product A;

[0027] 2) cooling the reaction product A obtained in step 1) to room temperature and then dissolving it, heating it to precipitate a polymer, and freeze-drying it to obtain a hydrogel monomer polymer;

[0028] 3) preparing a solution of the hydrogel monomer polymer obtained in step 2), shaking and mixing, adding Leucokinin VIII; shaking and mixing again to obtain the peptide sustained-release thermosensitive hydrogel.

[0029] Furthermore, in step 3), the first oscillation mixing condition is: oscillation mixing at 180 rpm at 4°C for 72 h; the second oscillation mixing condition is: oscillation mixing at 180 rpm at 4°C for 2 h.

[0030] Preferably, in step 1), the vacuuming time is 20 to 60 minutes, most preferably 30 minutes.

[0031] Preferably, in step 2), the heating temperature is 50-70°C, most preferably 60°C.

[0032] As a preferred solution, step 2) is: after cooling the reaction product A obtained in step 1) to room temperature, adding distilled water, and shaking at low temperature to dissolve. After complete dissolution, the mixture is heated to precipitate the polymer, and the supernatant is removed; after repeating this operation three times, the precipitated polymer is freeze-dried to obtain a hydrogel monomer polymer.

[0033] Preferably, in step 3), the solvent for preparing the hydrogel monomer polymer is double distilled water.

[0034] The third object of the present invention is to provide a peptide sustained-release thermosensitive hydrogel prepared by the aforementioned preparation method.

[0035] A fourth object of the present invention is to provide a use of the aforementioned composition and / or the aforementioned peptide sustained-release thermosensitive hydrogel in the preparation of a product for skin wound repair.

[0036] A fifth object of the present invention is to provide a use of Leucokinin VIII in the preparation of a product for skin wound repair.

[0037] To achieve the above object, the present invention adopts the following technical solutions:

[0038] Application of Leucokinin VIII in preparing products for skin wound repair.

[0039] Furthermore, Leucokinin VIII is used as or in the preparation of a product for promoting the proliferation and migration of keratinocytes.

[0040] The beneficial effects of the present invention are:

[0041] 1. Leucokinin VIII, as a new type of polypeptide substance, exhibits significant biological activity in tissue repair and regeneration. The present invention has found that Leucokinin VIII can promote the proliferation and migration of keratinocytes. The proliferation and migration ability of keratinocytes directly affects the speed and quality of wound healing. That is, the biological peptide Leucokinin VIII has the effect of repairing skin damage.

[0042] 2. The peptide slow-release thermosensitive hydrogel prepared by the present invention is a multifunctional drug-loaded hydrogel, which has excellent thermosensitive properties, injectability, drug slow-release performance, biocompatibility and biosafety, and can be formed in situ. The peptide slow-release thermosensitive hydrogel can not only achieve the slow-release and controlled release of the polypeptide, but also protect the wound surface through the physical barrier effect of the hydrogel, improve the wound healing effect, and is very suitable for wound repair and regeneration. The present invention provides a new treatment strategy for wound repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is the molecular structure diagram of Leucokinin VIII drug;

[0044] Figure 2 is the FTIR image of PLGA-PEG-PLGA;

[0045] Figure 3 For PLGA-PEG-PLGA 1 H NMR spectrum;

[0046] Figure 4 The figure shows the test results of the thermosensitive properties of the drug-loaded hydrogel, where: Figure 4 -A is an image of the drug-loaded hydrogel placed normally at 4°C; Figure 4 -B is an image of the drug-loaded hydrogel placed at an angle at 4°C; Figure 4 -C is an image of the drug-loaded hydrogel placed at an angle at 37°C; Figure 4 -D is an image of the drug-loaded hydrogel placed upside down at 37°C;

[0047] Figure 5 This is a test diagram of the ductility of the drug-loaded hydrogel;

[0048] Figure 6 This is a test diagram of the injectability of the drug-loaded hydrogel;

[0049] Figure 7 This is a test diagram of the adhesion of the drug-loaded hydrogel;

[0050] Figure 8 This is a diagram showing the drug sustained-release performance test results of the drug-loaded hydrogel;

[0051] Fig. 9 The figure is the result of the cell proliferation test of different concentrations of Leucokinin VIII;

[0052] Fig.10 The results of the scratch test are shown in Figure 1, where A is the Control group and B is the Leucokinin VIII group;

[0053] Fig.11 This is the result of Transwell experiment;

[0054] Fig.12 This is the result of the 24-hour skin sensitization test;

[0055] Fig.13 This is the result of the hydrogel cytotoxicity detection experiment;

[0056] Fig.14 The effect diagram of mouse skin wound repair, where A is the Control group and B is the Gel+LeucokininⅧ group;

[0057] Fig.15 This is a graph showing the changes in the area of ​​mouse skin wound repair, where: Fig.15 -A is the change of skin wound repair area in mice of control group, Fig.15 -B is the change of skin wound repair area in mice in Gel+LeucokininⅧ group;

[0058] Fig.16 This is a graph showing the statistical analysis results of the healing rate of mouse skin wound repair. DETAILED DESCRIPTION

[0059] The technical solution of the present invention will be further described clearly and completely in conjunction with specific embodiments below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work belong to the protection scope of the present invention.

[0060] Example 1. Preparation of hydrogel monomer polymer

[0061] The one-step method for preparing the hydrogel monomer polymer specifically comprises the following steps:

[0062] Polyethylene glycol (PEG1000), lactide (LA), hexyl lactide (GA), and stannous zincate (Sn(Oct)2) were added to a single-necked round-bottom flask with a magnetic stirrer, and the mixture was vacuumed for 30 minutes and then sealed. The single-necked round-bottom flask was placed in an oil bath at a preset temperature. After the mixture was completely melted, the reaction system was stirred evenly and the reaction was continued for 10 hours. After the reaction was completed, it was cooled to room temperature, and distilled water was poured into the reaction system, and it was shaken and dissolved at low temperature. After complete dissolution, the mixture was heated to 60°C to precipitate the polymer, and the supernatant was removed; after repeating this operation three times, the precipitated polymer was freeze-dried to obtain a hydrogel monomer polymer (PLGA-PEG-PLGA).

[0063] In the above reaction system, the molar ratio of lactide (LA+GA) to PEG is 20:1, the molar ratio of LA to GA is 2:1, and the molar ratio of lactide to stannous zincate is 1000:1. The reaction temperature is 130°C and the reaction time is 10h.

[0064] Example 2. Preparation of PLGA-PEG-PLGA thermosensitive hydrogel containing Leucokinin VIII

[0065] The PLGA-PEG-PLGA polymer prepared in Example 1 was prepared into a solution with a concentration of 15% using double distilled water, and the solution was shaken and mixed at 180 rpm at 4°C for 72 hours. After Leucokinin VIII was added, the solution was shaken and mixed at 180 rpm at 4°C for 2 hours to obtain a PLGA-PEG-PLGA thermosensitive hydrogel containing Leucokinin VIII, referred to as peptide sustained-release thermosensitive hydrogel. The concentration range of Leucokinin VIII in the hydrogel was 5 μg / mL.

[0066] Example 3. Characterization of hydrogel monomer polymer and peptide sustained-release thermosensitive hydrogel

[0067] (1) Structural characterization - FTIR

[0068] The dried PLGA-PEG-PLGA polymer sample was prepared into a solution with a concentration of 10 mg / mL using dichloromethane. 50 μL of the solution was evenly coated on the surface of a transparent potassium bromide window and dried under an infrared drying lamp. A Fourier transform infrared spectrometer was used to record the test sample at 400-4000 cm -1 Infrared absorption spectrum over a range of wavelengths.

[0069] The infrared spectrum test results of PLGA-PEG-PLGA are as follows Figure 2 As shown, 1749cm -1 The absorption peak at 1084 cm is attributed to the -C=O stretching vibration of the ester carbonyl group. -1 and 1181cm -1 The absorption peaks at 4 and 7 are respectively attributed to the characteristic COC stretching vibration and -COO stretching vibration of the -OCH2CH2 repeating unit of PEG. Therefore, the infrared results show the characteristic peaks of PEG and PLGA segments at the same time, indicating the successful synthesis of PLGA-PEG-PLGA copolymer.

[0070] (2) Structural characterization - NMR

[0071] Using deuterated chloroform as solvent, PLGA-PEG-PLGA polymer was prepared into a sample solution with a concentration of 20 mg / mL. The sample was detected by nuclear magnetic resonance spectrometer at an operating frequency of 500 MHz. 1 H NMR spectrum.

[0072] PLGA-PEG-PLGA 1 H NMR spectrum Figure 3 As shown, δ5.1-5.3ppm (a) belongs to the absorption peak of methylene hydrogen CH2 of lactoyl repeating unit in PLGA chain, δ4.7-4.9ppm (b) belongs to the absorption peak of secondary hydrogen CH of glycolic acid repeating unit in PLGA chain, δ1.52-1.62ppm (d) belongs to the absorption peak of methyl hydrogen CH3 of glycolic acid repeating unit in PLGA chain, δ3.62-3.66ppm (d) belongs to the absorption peak of methylene hydrogen CH2 in PEG chain, indicating that PLGA-PEG-PLGA was successfully synthesized.

[0073] (3) Characterization of drug-loaded hydrogel properties

[0074] The temperature-sensitive properties, extensibility, injectability, adhesion and drug sustained-release performance of the peptide sustained-release thermosensitive hydrogel prepared in Example 2 were further characterized.

[0075] The results of the thermosensitive properties test of peptide sustained-release thermosensitive hydrogel are as follows Figure 4 As shown, the drug-loaded hydrogel is liquid at low temperatures and jelly-like at room temperature or 37°C on the skin. The low-temperature liquid state is conducive to the full mixing of the hydrogel and the drug. At room temperature or 37°C, the drug-loaded hydrogel quickly changes state, which is conducive to fitting the wound.

[0076] The extensibility test results of peptide sustained-release thermosensitive hydrogels are shown in Figure 5 As shown, the drug-loaded hydrogel has good ductility and can cover the wound well.

[0077] The injectability test results of peptide sustained-release thermosensitive hydrogel are as follows Figure 6 As shown, the drug-loaded hydrogel has good injectability, is conducive to better clinical application, and can be used according to different wound shapes and sizes.

[0078] The adhesion test results of peptide sustained-release thermosensitive hydrogel are as follows Figure 7 As shown, the drug-loaded hydrogel has good adhesion and can fit the skin well.

[0079] The drug release performance test results of peptide sustained-release thermosensitive hydrogel are as follows Figure 8 As shown, hydrogel can slowly release drugs, thereby reducing the number of dosing times and achieving a long-lasting effect.

[0080] Example 4. Safety Characterization

[0081] (1) Cell proliferation assay

[0082] Kangfuxinye, a traditional Chinese medicine preparation derived from American cockroaches, shows unique therapeutic value in wound healing. Leucokinin VIII, a homologous peptide drug, has not been effectively explored.

[0083] The steps of the cell proliferation experiment are as follows:

[0084] 1) When the cell growth density reaches 80%, the keratinocytes (HaCaT) cells are digested and centrifuged, and the cells are counted after resuspending;

[0085] 2) The cells were plated in a 96-well plate, with 5000 cells per well (n=6). After the cells adhered to the wall, different concentrations of Leucokinin VIII were added and the plate was placed in a cell culture incubator for 24 hours.

[0086] 3) Prepare sufficient CCK8 solution in a ratio of 90 μL serum-free culture medium and 10 μL CCK8 reagent per well in a dark environment;

[0087] 4) Take out the well plate, discard the supernatant, add 100 μL of CCK8 solution to each well, add only CCK8 solution to the blank control group, and place in an incubator away from light for 1 hour;

[0088] 5) The OD value of each well was detected by an enzyme-labeled instrument at a wavelength of 450 nm, and the mean absorbance of the control group was compared with the mean absorbance of the experimental group, and then statistical analysis was performed.

[0089] Test results such as Fig. 9 As shown, different concentrations of Leucokinin VIII can promote cell proliferation, indicating that the drug can promote the proliferation of keratinocytes near the wound and play a role in promoting wound healing.

[0090] (2) Scratch test

[0091] The scratch test, also known as the wound healing test, artificially creates a blank area on the fused monolayer of cells. The cells at the edge will gradually enter the blank area to heal the "scratch". It can be used to observe the effects of exogenous factors such as drugs and genes on cell migration, repair and interaction. The cell scratch test is currently the most commonly used and simplest method to measure cell migration and repair ability.

[0092] The steps of the scratch test are as follows:

[0093] 1) Digest, centrifuge, resuspend and count the cells; seed each group in a 12-well plate at 1×105 cells per well and culture in a cell culture incubator until the confluence is greater than 90%;

[0094] 2) Use a 200 μL pipette tip to draw a cross at the bottom of the well plate, aspirate the culture medium, wash the cell debris with PBS, replace with serum-free culture medium, and take photos under a microscope;

[0095] 3) Culture in a cell culture incubator for 12-24 hours and take photos under a microscope;

[0096] 4) Compare the healing status of scratch wounds.

[0097] Test results such as Fig.10 As shown, Leucokinin VIII can effectively promote the healing of scratches, indicating that the drug can promote the migration of keratinocytes and promote wound healing.

[0098] (3) Transwell experiment

[0099] The Transwell assay, also known as the Transwell migration or invasion assay, is a laboratory technique used in cell biology and molecular biology to study the movement of cells through porous membranes. It is often used to study cell migration, chemotaxis, and invasion in response to various stimuli, such as growth factors, chemokines, or extracellular matrix components.

[0100] The steps of the Transwell experiment are as follows:

[0101] 1) When the cell density reached 80%, the cells of different groups were digested and centrifuged separately, and the cells were counted after resuspending. The cells were transferred into 1.5 mL EP tubes according to the cell volume of 3×104 cells / well and centrifuged at 800 rpm for 3 min;

[0102] 2) Aspirate the original culture medium and add serum-free culture medium to resuspend the cells;

[0103] 3) Add 600 μL of culture medium containing 10% serum to the lower chamber;

[0104] 4) Use sterile tweezers to gently place the Transwell chamber into a 24-well plate, and then add 200 μL of each group of cell suspensions in step (2) into the upper chamber;

[0105] 5) According to the experimental requirements, place the 24-well plates in an incubator and culture for 12 hours;

[0106] 6) Take out the chamber and place it in 600 μL 4% paraformaldehyde solution and fix it at room temperature for 30 minutes;

[0107] 7) Transfer the chamber to 600 μL crystal violet staining solution and stain at room temperature for 15-30 minutes;

[0108] 8) Soak the cotton swab in pure water until it is soft; take out the chamber with tweezers, soak it in pure water to wash it thoroughly, and then use the cotton swab to gently wipe it from the chamber to ensure that the upper cells are clean;

[0109] 9) Take photos using a microscope.

[0110] Test results such as Fig.11 As shown, Leucokinin VIII can effectively promote the migration of keratinocytes, indicating that the drug can promote wound healing.

[0111] (4) 24h skin sensitization test

[0112] The skin is an important immune organ of the human body. After repeated contact with a substance, the body produces an immune-transmitted skin reaction. In humans, skin allergies may present symptoms such as itching, erythema, papules, blisters, and fused blisters. Therefore, after the hydrogel, drug, and skin come into contact and interact for 24 hours, observe and record the skin changes at the contact site.

[0113] Experimental steps:

[0114] 1) First, a skin irritation test was conducted. The hair on the back of healthy mice was shaved clean and then disinfected with 7.5% povidone-iodine;

[0115] 2) The back area of ​​the mice was divided into left and right parts and then divided into four groups (three mice in each group), namely, control group (PBS), peptide group (Leucokinin VIII), hydrogel group (Gel), and hydrogel plus peptide drug group (Gel+Leucokinin VIII);

[0116] 3) The grouped drugs were covered in symmetrical positions. PBS and Leucokinin VIII were loaded with gauze because they were in liquid form, while the hydrogel was directly covered on the skin. They were fixed with a transparent film to prevent them from falling off when the mice climbed.

[0117] 4) Take photos and record the conditions at 0h and 24h under a large stereo microscope.

[0118] The results are as follows Fig.12 As shown, the hydrogel and the drug have no sensitization to the skin.

[0119] (5) Hydrogel cytotoxicity assay

[0120] Leucokinin VIII, hydrogel and cells were co-cultured to detect cell activity. The experimental steps were the same as (1) cell proliferation experiment. The results are shown in Fig.13 As shown, Leucokinin VIII and hydrogel had no significant effect on cell growth activity and had good biocompatibility.

[0121] Example 5. Skin wound repair application

[0122] This example further investigates the repair effect of the PLGA-PEG-PLGA thermosensitive hydrogel containing Leucokinin VIII prepared in Example 2 on skin wounds. First, a skin wound model was established, and then the healing rate of each group of mice was counted.

[0123] The specific method of establishing the skin wound model is as follows:

[0124] 1) Materials: mice, cages, shavers, depilatory cream, cotton swabs, alcohol, gauze, scissors, tweezers, sterile towels, tribromoethanol, sodium pentobarbital, ruler, punch (ophthalmic trephine), 1mL syringe, weighing scale, lighting, marker, EP tube, paper towel, ear tag, ear tag clamp, etc.

[0125] 2) Numbering of mice: After the mice are taken out, they are fixed and ear-tagged with ear-tagged forceps to mark the order;

[0126] 3) Weighing: weigh the mice using a body weight scale;

[0127] 4) Anesthesia and hair removal: Use 0.2 mL / 10 g of tribromoethanol, and the dosage for mice is about 0.4-0.5 mL for intraperitoneal injection. After the effect takes effect, use a shaver to shave the surface hair; use a cotton swab to dip a small amount of depilatory cream and apply it to the hair; wait for about 10-15 seconds, use gauze dipped in alcohol to wipe off the depilatory cream, and disinfect the skin;

[0128] 5) Modeling: Use an 8 mm ophthalmic trephine to make a mark on the back of the mouse, use a marker to simply mark it, fold the skin in half according to the mark, and use an ophthalmic trephine to make a hole;

[0129] 6) Photography under a large stereo microscope: Place the ruler, number, and mouse under a stereo microscope, record, and take photos.

[0130] 7) Take photos and record the wound conditions on days 0, 1, 3, 5, 7, 9, and 11.

[0131] 8) After wound modeling, the mice were randomly divided into a hydrogel group (Gel) and a hydrogel plus polypeptide drug group (Gel+LeucokininⅧ).

[0132] The healing rate is calculated as follows:

[0133] Healing rate = (day11-day0) / day0*100%

[0134] Test results such as Figure 14 to Figure 16As shown, compared with the control group, the biological peptide Leucokinin VIII sustained-release thermosensitive hydrogel can effectively promote the repair of mouse skin wounds, with an 11-day healing rate of 96.5%, while the 11-day healing rate of the control group was 71.9%.

Claims

1. A composition for preparing a peptide sustained-release thermosensitive hydrogel, characterized in that: The composition consists of a hydrogel monomer polymer and a biological peptide; the biological peptide is Leucokinin VIII; the hydrogel monomer polymer is prepared by mixing and heating polyethylene glycol, lactide and stannous zincate; the molar ratio of the lactide to the polyethylene glycol is 20:1-40:1, and the molar ratio of the lactide to the stannous zincate is 1000:1-2000:

1.

2. The composition according to claim 1, characterized in that The lactide includes lactide and hexyl lactide; the molar ratio of lactide to hexyl lactide is 2:1 to 4:

1.

3. A method for preparing a peptide sustained-release thermosensitive hydrogel, characterized in that: The steps include: (1) mixing polyethylene glycol, lactide and stannous zincate, and reacting them by heating and stirring to obtain a hydrogel monomer polymer; (2) Mixing the hydrogel monomer polymer obtained in step (1) with Leucokinin VIII to prepare the peptide sustained-release thermosensitive hydrogel.

4. The preparation method according to claim 3, characterized in that: In step (1), the reaction temperature is 125-140° C. and the reaction time is 8-12 h.

5. The preparation method according to claim 3, characterized in that: In step (2), the concentration of the hydrogel monomer polymer is 10% to 30%; the concentration of Leucokinin VIII in the peptide sustained-release thermosensitive hydrogel is 1-10 μg / mL.

6. The preparation method according to claim 3, characterized in that: The method specifically comprises the following steps: 1) mixing polyethylene glycol, lactide and stannous zincate, evacuating the mixture, and heating and stirring the mixture to react to obtain a reaction product A; 2) cooling the reaction product A obtained in step 1) to room temperature and then dissolving it, heating it to precipitate a polymer, and freeze-drying it to obtain a hydrogel monomer polymer; 3) preparing a solution of the hydrogel monomer polymer obtained in step 2), shaking and mixing, adding Leucokinin VIII; shaking and mixing again to obtain the peptide sustained-release thermosensitive hydrogel.

7. The preparation method according to claim 6, characterized in that: In step 3), the first oscillation mixing condition is: oscillation mixing at 180 rpm at 4°C for 72 h; the second oscillation mixing condition is: oscillation mixing at 180 rpm at 4°C for 2 h.

8. A peptide sustained-release thermosensitive hydrogel prepared by the preparation method according to any one of claims 3 to 7.

9. Use of the composition according to any one of claims 1 to 2 and / or the peptide sustained-release thermosensitive hydrogel according to claim 8 in the preparation of products for skin wound repair.

10. Use of Leucokinin VIII as or in the preparation of a product for promoting the proliferation and migration of keratinocytes.

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