Method for the preparation of a polypeptide gel with mechanical contraction properties and scar inhibition and uses thereof

Abstract

The application discloses a preparation method of a polypeptide gel with mechanical contraction characteristics and scar inhibition, and provides the polypeptide gel with mechanical contraction characteristics and scar inhibition, which is prepared by one-pot method, and has the advantages of simple preparation method, no organic solvent residue and mild reaction condition. Infrared light is used as an exogenous auxiliary therapy, which can adjust the temperature of the gel dressing and realize controllable contraction on one hand, and the photo-thermal effect can enhance the antibacterial activity of the polypeptide and ensure the orderly progress of wound repair on the other hand.

Description

Technical Field

[0001] The present invention belongs to the technical field of gel preparation, and in particular relates to a preparation method and application of a polypeptide gel with mechanical contraction properties and scar inhibition. Background Art

[0002] As a unique class of polymer materials, hydrogels are rich in water, have a soft texture, and are highly plastic. Their physical properties are highly similar to those of biological tissues, exhibiting excellent biocompatibility. They can flexibly carry a variety of substances such as drugs, cells, and exosomes. Their hardness can be adjusted according to actual needs, making them high-quality materials with great potential in the biomedical field. Hydrogel dressings have been used in many medical fields, including anti-tumor, bone repair, and wound repair. In the field of skin wound repair, hydrogels are often used to provide a physical barrier, absorb wound exudate, and intelligently release drugs in response to environmental stimuli.

[0003] A wide variety of polymers can be used to construct hydrogels, among which gelatin (GEL), a hydrolysis product of collagen, is one of the most widely used materials. Gelatin possesses the remarkable properties of collagen, such as biodegradability, good biocompatibility, and non-immunogenicity. It also contains an RGD (R: arginine; G: glycine; D: aspartic acid) peptide sequence, making it a versatile platform with applications in diverse fields such as wound repair, tissue engineering, bone repair, and cartilage repair. An ideal hydrogel scaffold should possess appropriate mechanical properties, good water retention, anti-infection properties, injectability, and excellent cellular biocompatibility.

[0004] Poly(N-isopropylacrylamide) (PNIPAAm), a thermosensitive polymer, has attracted considerable attention in the hydrogel field. It possesses a unique lower critical solution temperature (LCST). When the ambient temperature is below the LCST, PNIPAAm hydrogels exhibit hydrophilic properties, absorbing large amounts of water and swelling. When the ambient temperature is above the LCST, the hydrogel undergoes syneresis and shrinks, becoming hydrophobic. This temperature-sensitive property makes PNIPAAm hydrogels potentially valuable for applications in drug controlled release, smart sensors, and tissue engineering.

[0005] Traditional hydrogels mostly only play a role in providing a physical barrier, absorbing wound exudates, and smartly releasing drugs in response to environmental stimuli, and are difficult to effectively adjust and buffer mechanical forces according to different stages of wound healing and changes in mechanical forces borne by the wound. In the actual wound healing process, a wound will go through different stages such as the inflammation stage, proliferation stage, and remodeling stage, each of which has different needs and tolerances for mechanical forces. For example, in the inflammation stage, the tissue around the wound is relatively fragile and needs the hydrogel to provide gentle support and protection; while in the proliferation and remodeling stages, as cells proliferate and tissues repair, the wound needs to bear certain stretching and contraction forces. Existing hydrogels cannot accurately adapt to these complex changes in mechanical forces, limiting their further application in the biomedical field, especially in wound healing and tissue repair scenarios that have higher requirements for mechanical properties.

[0006] Scars are fibrotic skin diseases caused by abnormal wound healing, mainly including hypertrophic scars and keloids. The overall incidence of hypertrophic scars after trauma is as high as 40%-70%, especially in high-tension areas such as joints, neck, and lower abdomen, where local mechanical tension imbalance caused by activities such as flexion, rotation, and breathing can more easily lead to the formation of hypertrophic scars. Scars not only cause disfigurement, but also often accompany symptoms such as unbearable itching, contracture deformity, and dysfunction, which seriously affect the appearance and mental health of patients. In addition, the course of scars is chronic and prolonged, causing long-term psychological burden to patients, and heavy economic burden to families and society. Therefore, the prevention and treatment of scars has become an important research direction in the medical field.

[0007] Mechanical force regulation is of great significance to wound healing. Appropriate mechanical force can promote cell migration, proliferation, and extracellular matrix synthesis, while excessive mechanical tension can lead to abnormal deposition of collagen fibers, excessive formation of scars, and damage to skin appendages. Traditional wound dressings such as alginate hydrogel, nanofiber hydrogel, and pH-responsive hydrogel have played a certain role in providing a physical barrier, absorbing wound exudates, and smartly releasing drugs in response to environmental stimuli. However, these hydrogels cannot effectively adjust and buffer mechanical forces according to different stages of wound healing and changes in mechanical forces borne by the wound. Dressings based on poly(N-isopropylacrylamide) (PNIPAAm) have an ideal lower critical solution temperature (about 32°C), can be stably stored at room temperature, and spontaneously contract at body temperature. This type of biomaterial not only provides the basic functions of traditional dressings, but also dynamically adjusts the mechanical tension of the wound according to different stages of wound healing and changes in mechanical forces. In this way, the negative effects of mechanical tension on wound healing can be effectively reduced, thereby promoting scarless healing.

[0008] Studies have shown that when mechanical tension increases, the activity of Yes-associated protein (YAP) / transcriptional coactivator containing a PDZ binding motif (TAZ) is activated, prompting its translocation from the cytoplasm to the nucleus. Within the nucleus, YAP / TAZ bind to the transcription factor TEAD, activating the expression of genes associated with cell proliferation, matrix synthesis, and tissue repair. This process is crucial for normal healing, but excessive activation may lead to pathological scarring. Although studies have attempted to alleviate scarring by inhibiting the YAP-TEAD interaction, such as by loading the YAP-TEAD inhibitor verteporfin (VP) into hydrogels for slow release, these approaches suffer from low release efficiency, difficult to control kinetics, and limited targeted delivery capabilities. More importantly, existing research has focused on alleviating the consequences of excessive tension while ignoring the active regulation of local tension in wounds.

[0009] Existing gel dressings cannot regulate wound tension: Although traditional wound dressings, such as gelatin dressings and alginate dressings, play a certain role in providing a physical barrier, absorbing wound exudate and releasing drugs, these hydrogels are unable to properly reduce the high tension of the wound, which often leads to the formation of scar tissue after wound healing. Existing gel dressings have insufficient delivery efficiency: For the regulation of mechanical pathways, some studies have loaded YAP inhibitors into hydrogels in an attempt to achieve precise regulation of the YAP signaling pathway to prevent scar formation. However, the release efficiency and release kinetics of YAP inhibitors in hydrogels are difficult to precisely control, and the targeted delivery ability of hydrogels to VP is limited. Summary of the Invention

[0010] The purpose of the present invention is to provide a method for preparing a polypeptide gel with mechanical contraction properties and scar inhibition.

[0011] The second object of the present invention is to provide a polypeptide gel prepared by the method having mechanical contraction properties and scar inhibition.

[0012] The third object of the present invention is to provide an application of the polypeptide gel having mechanical contraction properties and scar inhibition in the preparation of wound repair products.

[0013] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0014] In a first aspect, the present invention provides a polypeptide, the amino acid sequence of which is shown in SEQ ID NO. 1.

[0015] The amino acid sequence of the polypeptide is as follows: TWLQIGGSGGGKKK (SEQ ID NO. 1).

[0016] The second aspect of the present invention provides a method for preparing a polypeptide gel having mechanical contraction properties and scar inhibition, comprising the following steps:

[0017] A gelatin solution with a mass concentration of 5-15% g / mL (preferably 10% g / mL) and a sodium alginate solution with a mass concentration of 0.5-2% g / mL (preferably 1% g / mL) are stirred evenly, and the polydopamine solution is added, and the mixture is stirred and mixed evenly to obtain a mixed solution a;

[0018] Adding a poly(N-isopropylacrylamide) solution having a mass concentration of 0.1-4% g / mL (preferably 1%, 2.5%, 3% g / mL) to the mixed solution a, stirring and mixing uniformly to obtain a mixed solution b;

[0019] Adding a polypeptide nanoparticle solution having a mass concentration of 0.1-1 mg / mL (preferably 0.5 mg / mL) to the mixed solution b, and mixing uniformly to obtain a mixed solution c;

[0020] The mass ratio of gelatin, sodium alginate, polydopamine, poly(N-isopropylacrylamide), and polypeptide nanoparticles is 1-200:1-50:1-10:1:0.00001-0.1 (preferably 16:1.6:2.4:1:0.004, 50:4:4:1:0.01, and 13.33:1:1.33:1:0.0067);

[0021] The mixed solution c was precooled (at 4°C for 5 minutes), CaCl2 solution was added, and the mixture was allowed to stand for 1 minute to obtain a polypeptide gel with mechanical contraction properties and scar inhibition.

[0022] The preparation method of the gelatin solution comprises the following steps:

[0023] Dissolve gelatin in deionized water, heat and stir (preferably for 20 minutes) at a temperature of 40-60°C (preferably 50°C) to completely dissolve it, and obtain a gelatin solution with a mass concentration of 5-15% g / mL (preferably 10% g / mL) (w / v).

[0024] The preparation method of the sodium alginate solution comprises the following steps:

[0025] Sodium alginate is dissolved in deionized water, and heated and stirred at 40-60°C (preferably 50°C) for 10 minutes to completely dissolve the sodium alginate, to obtain a sodium alginate solution with a mass concentration of 0.5-2% g / mL (preferably 1% g / mL) (w / v).

[0026] The preparation method of the poly (N-isopropylacrylamide) solution comprises the following steps:

[0027] Dissolve poly(N-isopropylacrylamide) (PNIPAAm) in deionized water and stir at room temperature to completely dissolve it to obtain a poly(N-isopropylacrylamide) solution with a mass concentration of 0.1-4% g / mL (preferably 1%, 2.5%, 3% g / mL) (w / v).

[0028] The preparation method of the polypeptide nanoparticle solution comprises the following steps:

[0029] The lyophilized polypeptide nanoparticle powder is dissolved in phosphate buffer to obtain a polypeptide nanoparticle solution with a mass concentration of 0.1-1 mg / mL (preferably 0.5 mg / mL).

[0030] The preparation method of the polypeptide nanoparticle freeze-dried powder comprises the following steps:

[0031] Sodium hydroxide is added to the phosphate buffer to adjust the pH to 9-10, and the polypeptide shown in SEQ ID NO. 1 is added to a concentration of 0.1-1 mg / mL (preferably 0.75 mg / mL). The mixture is stirred for 2-8 hours (preferably 6 hours) to allow the polypeptide to self-assemble into nanoparticles, which are then freeze-dried for at least 24 hours to obtain a freeze-dried powder of polypeptide nanoparticles.

[0032] The concentration of the CaCl2 solution is 0.1-0.5 mol / L (preferably 0.1, 0.2 mol / L).

[0033] The mass ratio of CaCl2 to poly(N-isopropylacrylamide) is 0.1-2:1 (preferably 0.89:1, 1.11:1, 0.56:1).

[0034] The preparation method of the polydopamine solution comprises the following steps:

[0035] Dissolve polydopamine in deionized water and stir at room temperature (preferably for 10 minutes) to uniformly disperse the solution to obtain a polydopamine solution.

[0036] The third aspect of the present invention provides a polypeptide gel prepared by the method having mechanical contraction properties and scar inhibition.

[0037] The fourth aspect of the present invention provides a use of the polypeptide gel having mechanical contraction properties and scar inhibition in the preparation of wound repair products.

[0038] The wound repair product is selected from wound dressings, products that promote wound healing, products that prevent wound expansion, products that prevent wound infection, products that inhibit skin proliferation, products that promote skin collagen remodeling, and products that regenerate skin appendages.

[0039] The fifth aspect of the present invention provides a use of the polypeptide gel having mechanical contraction properties and scar inhibition in the preparation of a tissue engineering scaffold.

[0040] Due to the adoption of the above technical solution, the present invention has the following advantages and beneficial effects:

[0041] The polypeptide gel with mechanical contraction properties and scar inhibition provided by the present invention can be used as a biomedical hydrogel dressing. It can be applied to the skin surface of different parts of the body (such as fingers, wrists, etc.), can adapt to the movement of the skin, and has good mechanical strength and relative stability. In addition, it can self-contract according to body temperature or controllably contract under light and heat. The strong adhesion of the gel can transfer the contraction force to the wound edge, causing the wound to contract rapidly. As the gel degrades, the polypeptide is released into the wound, further preventing scar formation.

[0042] The peptide gel provided by the present invention exhibits mechanical contraction properties and scar inhibition, achieving scarless wound healing through the synergistic combination of mechanical regulation and molecular intervention. Super-TDU is an inhibitory peptide that targets the YAP-TEADs interaction. By mimicking the TDU domain of VGLL4, it competes with YAP / TAZ for TEAD binding, thereby inhibiting the formation of the YAP-TEADs complex. Based on the Super-TDU sequence, the present invention screened a short peptide sequence, Peptide8 (TWLQIGGS), using a molecular docking platform. This peptide sequence retains the key amino acid residues required for Super-TDU to bind to TEADs, enabling Peptide8 to effectively mimic Super-TDU's function and effectively block the formation of the YAP-TEAD complex. Furthermore, the C-terminus of Peptide8 was modified with a lysine (TWLQIGGSGGGKKK), which facilitated the peptide's spontaneous assembly into nanoparticles and conferred antimicrobial activity. The peptide was then loaded into a gel dressing composed of gelatin and PNIPAAm.

[0043] The polypeptide gel with mechanical contraction properties and scar inhibition provided by the present invention has the characteristics of spontaneous contraction at body temperature and photothermal controllable contraction. It generates effective wound traction through spontaneous contraction, resists the tension caused by external activities, and achieves rapid closure of the wound. At the same time, as the hydrogel gradually degrades, the released polypeptides can specifically inhibit the YAP-mediated mechanical signaling pathway, further preventing scar formation at the cellular and molecular levels. In addition, the antibacterial activity of the polypeptide can effectively prevent the risk of infection during wound repair, ensure the stability of the wound microenvironment, and thus provide a strong guarantee for scarless healing. The present invention will provide a new comprehensive treatment strategy for scarless wound healing that covers mechanical regulation and molecular intervention.

[0044] The polypeptide gel with mechanical contraction properties and scar inhibition provided by the present invention is in the form of a solid gel, has excellent tissue adhesion properties, has photothermal controllable mechanical contraction properties, and has good biocompatibility.

[0045] The present invention can regulate the mechanical contraction of the polypeptide gel through photothermal control. The strong adhesion of the polypeptide gel to the tissue can transfer the contraction force of the gel to the wound edge, making the wound resist tension and accelerating wound closure.

[0046] The polypeptide of the present invention has the property of competitively binding to TEAD with YAP. After being loaded into the polypeptide gel, it can be slowly released into the wound as the gel degrades. The released polypeptide will play a role in regulating the mechanical pathway and synergistically inhibit the formation of scars.

[0047] The peptide gel provided by the present invention, which exhibits mechanical contraction properties and scar suppression, is prepared via a simple one-pot process, free of residual organic solvents, and under mild reaction conditions. Infrared light, as an exogenous adjuvant therapy, regulates the temperature of the gel dressing, achieving controlled contraction, while also enhancing the peptide's antibacterial activity through photothermal therapy, ensuring orderly wound repair.

[0048] The polypeptide gel with mechanical contraction properties and scar inhibition provided by the present invention has the characteristics of spontaneous contraction at body temperature and photothermal controllable contraction, and can adapt to the needs of various stages of wound healing. In the early stage of wound healing, effective wound traction is generated through spontaneous contraction to resist the tension caused by external activities and achieve rapid closure of the wound surface; in the later stage of healing, photothermal controllable contraction is used to accurately adjust the mechanical force according to the degree of wound healing to avoid excessive traction. At the same time, as the hydrogel gradually degrades, the released polypeptide can specifically inhibit the YAP-mediated mechanical signaling pathway, further preventing scar formation at the cellular and molecular levels. In addition, the antibacterial activity of the polypeptide can effectively prevent the risk of infection during wound repair, ensure the stability of the wound microenvironment, and thus provide a strong guarantee for scar-free healing.

[0049] Transmitting mechanical contraction stress to the wound requires a dressing with good adhesion. Therefore, this invention incorporates polydopamine (PDA) into the gel system. PDA enhances the peptide gel's adhesion to skin tissue, ensuring a tight fit between the hydrogel and the wound surface. Furthermore, PDA's photothermal conversion capability allows it to rapidly heat the gel dressing under light stimulation, triggering rapid and controlled contraction. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 Schematic diagram of sample peaks after HPLC purification.

[0051] Figure 2 Schematic diagram of the MS spectrum of the peptide.

[0052] Figure 3 Schematic diagram of a series of peptides obtained by screening using the autodock platform.

[0053] Figure 4 This is a scanning electron micrograph of the freeze-dried polypeptide nanoparticle powder prepared in Example 1 and a schematic diagram of its particle size distribution results.

[0054] Figure 5 Schematic diagram of the prepared gel.

[0055] Figure 6 Schematic diagram of the mechanical shrinkage results of the prepared gel at different temperatures.

[0056] Figure 7 Schematic diagram of the scanning electron microscopy results of the prepared gel at different temperatures.

[0057] Figure 8 Schematic diagram of the rheological test results of the prepared gel.

[0058] Figure 9 Schematic diagram of the infrared test results of the prepared gel.

[0059] Figure 10 Schematic diagram of the adhesion results of the prepared gel.

[0060] Figure 11 Schematic diagram of the organ tissue adhesion results of the prepared gel.

[0061] Figure 12 Schematic diagram of the lap shear and tensile testing methods for the prepared gel.

[0062] Figure 13 Schematic diagram showing the results of adhesive strength and wound closure strength of the prepared gel.

[0063] Figure 14 Schematic diagram of the toxicity results of the prepared gel on fibroblasts.

[0064] Figure 15 Schematic diagram showing the compatibility of the prepared gel with endothelial cells.

[0065] Figure 16 Schematic diagram of the biological activity results of the prepared gel at the cellular level.

[0066] Figure 17 Schematic diagram of the photothermal conversion characteristics and antibacterial activity results of the prepared gel.

[0067] Figure 18 Schematic diagram of the results of rapid wound closure achieved by the prepared gel.

[0068] Figure 19 Schematic diagram of the test results of mouse wound skin tissue.

[0069] Figure 20 Schematic diagram of rabbit ear wound healing and scar evaluation results.

[0070] Figure 21 Schematic diagram of the histological evaluation results of rabbit ear skin. DETAILED DESCRIPTION

[0071] In order to explain the present invention more clearly, the present invention is further described below in conjunction with preferred embodiments. Those skilled in the art should understand that the following specific description is illustrative rather than restrictive and should not be used to limit the scope of protection of the present invention.

[0072] Example 1

[0073] The method for preparing the polypeptide comprises the following steps:

[0074] The peptide sequence is H-Thr-Trp-Leu-Gln-Ile-Gly-Gly-Ser-Gly-Gly-Gly-Lys-Lys-Lys-OH, and the solid phase synthesis method is adopted. Wang resin is selected as the solid phase synthesis support, and the peptide sequence is synthesized starting from the C-terminal amino group.

[0075] After Wang resin (3 mmol) was swollen, Fmoc-Lys(Boc)-OH (9 mmol), 10 mL DMF, 1-hydroxybenzotriazole (HOBt, 1.216 g, 9 mmol) and N,N'-diisopropylcarbodiimide (DIC, 1.135 g, 9 mmol) were added and reacted at room temperature for 2 hours. The reaction was monitored by ninhydrin. Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, and Fmoc-Thr(tBu)-OH were added and coupled in the presence of DIC (9 mmol), HOBt (9 mmol), and DMF, respectively. The Fmoc protecting groups of all amino acids were cleaved with a 20% Pip / DMF solution. After solid-phase ligation based on the peptide sequence, 30 mL of 95% TFA solution (TFA:H₂O volume ratio 95:5) was added to cleave the resin and side-chain protecting groups. The resin was filtered, and the filtrate was precipitated with anhydrous ether. The precipitate was washed three times with anhydrous ether and dried in a vacuum dryer at room temperature for 24 hours to obtain 4.5 g of crude product. Purification by reverse-phase preparative liquid chromatography (solution A: 0.1% TFA in H₂O; solution B: ACN; solution A: 82%-62%, solution B: 18%-38%, flow rate: 120 mL / min) yielded 2 g of a peptide with a purity exceeding 95%.

[0076] The target peak solution collected after HPLC purification was first frozen with liquid nitrogen (-196 ° C, 0.5 hours), Figure 1 The figure shows the peaks of the sample after HPLC purification. The sample was then dried in a freeze-dryer under vacuum (cold trap temperature: -108°C, 12 hours) to a powder. A 0.5 mg portion of the lyophilized powder was dissolved in 1 mL of 20% acetonitrile / water and re-analyzed by HPLC (Solution A: 0.1% TFA in H₂O; Solution B: CAN; Solution A: 82%-62%, Solution B: 18%-38%, flow rate: 1 mL / min). The MS results showed that the powder had a purity exceeding 95% as determined by HPLC. The molecular weight determined by MS remained unchanged, indicating that the powder was the target product. Figure 2 Schematic diagram of the MS spectrum of the peptide.

[0077] Peptide sequence: TWLQIGGSGGGKKK (SEQ ID NO. 1), molecular weight: 1416.65; Mass Spectrometry: COSMOSIL 5C18-MS-II 4.6 ID x 250 mm; Mobile Phase: A: 100% H2O containing 0.1% trifluoroacetic acid (TFA); B: acetonitrile; HPLC purity: 99.93%. Appearance: White lyophilized powder; Counterion: trifluoroacetate; Storage Temperature: 2°C–8°C.

[0078] The preparation method of the polypeptide gel having mechanical contraction properties and scar inhibition comprises the following steps:

[0079] The autodock molecular docking platform was used to screen the peptide sequences, such as Figure 3 As shown, Figure 3 Figure 1 shows a series of peptides screened using the Autodock platform. (a) shows the docking results of peptides with different sequences onto TEAD. The specific peptide sequences are as follows: Peptide 31: SVDDHFAKSLGDTWLQIGGSGNPKTANVPQT (SEQ ID NO. 2); Peptide 21: GDTWLQIGGSGNPKTANVPQT (SEQ ID NO. 3); Peptide 10: GDTWLQIGGS (SEQ ID NO. 4); Peptide 8: TWLQIGGS (SEQ ID NO. 5). (b) shows the potential targets of Peptide 8 and TEAD. These results demonstrate that all of the peptides described above interact with TEAD. Based on comprehensive considerations, Peptide 8 was selected as a YAP inhibitor. Its relatively simple structure facilitates further optimization through chemical modification. From a biological function perspective, the core sequence of Peptide8 (TWLQIGGS) fully retains the key amino acid residues that bind to Super-TDU and TEAD. This enables Peptide8 to effectively mimic the function of Super-TDU, effectively blocking the formation of the YAP-TEAD complex, inhibiting the related pathological process at the molecular level, and providing a key target for scar prevention and treatment. The peptide was synthesized using solid-phase synthesis, and glycine and lysine were introduced at its termini (TWLQIGGS-GGG-KKK).

[0080] Preparation of gelatin-alginate-polydopamine hydrogel (GAP hydrogel):

[0081] Preparation of GA hydrogel:

[0082] Dissolve 1 g of gelatin in 10 mL of deionized water, heat at 50°C, and stir magnetically for 20 min to completely dissolve it, obtaining a gelatin solution with a mass concentration of 10% g / mL (w / v).

[0083] 0.1 g of sodium alginate was dissolved in 10 mL of deionized water, heated at 50°C, and magnetically stirred for 10 min to completely dissolve it, obtaining a sodium alginate solution with a mass concentration of 1% g / mL (w / v).

[0084] Mix 2 mL of 10% g / mL (w / v) gelatin solution and 2 mL of 1% g / mL (w / v) sodium alginate solution to obtain mixed solution a. Place 600 μL of mixed solution a in a 24-well plate and pre-cool it in a 4°C refrigerator for 5 minutes. Remove the plate and add 0.5 mL of 0.2 mol / L CaCl₂ solution. Let it stand for 1 minute to obtain a gelatin / sodium alginate hydrogel, referred to as GA hydrogel. Remove the gel from the plate and store it in a 4°C refrigerator until ready for use.

[0085] Preparation of GAP hydrogel:

[0086] Dissolve 0.5 g of polydopamine in 5 mL of deionized water, stir at room temperature for 10 min to disperse it evenly, and store it in a refrigerator at 4°C away from light until use, to obtain a polydopamine (PDA) solution.

[0087] Mix 2 mL of 10% g / mL (w / v) gelatin solution and 2 mL of 1% g / mL (w / v) sodium alginate solution until uniformly mixed. Add 300 μL of the aforementioned polydopamine solution and mix thoroughly to obtain mixed solution a. Place 600 μL of mixed solution a into a 24-well plate and pre-cool in a 4°C refrigerator for 5 minutes. Remove the plate and add 0.5 mL of 0.2 mol / L CaCl₂ solution. Let stand for 1 minute to obtain a gelatin / sodium alginate / polydopamine hydrogel, referred to as a GAP hydrogel. Remove the gel from the plate and store in a 4°C refrigerator until ready for use.

[0088] Preparation of GAPN hydrogel:

[0089] 0.25 g of poly (N-isopropylacrylamide) (PNIPAAm) was dissolved in 10 mL of deionized water and stirred at room temperature to completely dissolve the poly (N-isopropylacrylamide) to obtain a poly (N-isopropylacrylamide) solution with a mass concentration of 2.5% g / mL (w / v).

[0090] Dissolve 0.5 g of polydopamine in 5 mL of deionized water, stir at room temperature for 10 min to disperse it evenly, and store it in a refrigerator at 4°C away from light until use, to obtain a polydopamine solution.

[0091] Mix 2 mL of 10% g / mL (w / v) gelatin solution and 2 mL of 1% g / mL (w / v) sodium alginate solution. Add 300 μL of the aforementioned polydopamine solution and mix thoroughly to obtain mixed solution a. Add 0.5 mL of 2.5% g / mL (w / v) poly(N-isopropylacrylamide) solution to mixed solution a and mix thoroughly to obtain mixed solution b. Place 600 μL of mixed solution b in a 24-well plate and pre-cool in a 4°C refrigerator for 5 minutes. Remove the plate and add 0.5 mL of 0.2 mol / L CaCl₂ solution. Let stand for 1 minute to obtain GAPN hydrogel. Remove the gel from the plate and store in a 4°C refrigerator until ready for use.

[0092] Preparation of polypeptide gel with mechanical contraction properties and scar inhibition (abbreviated as GAPNP hydrogel):

[0093] To 5 mL of phosphate buffer, adjust the pH to 9-10 with sodium hydroxide. Add 3.75 mg of the peptide to a concentration of 0.75 mg / mL. Stir for 6 hours to allow the peptide to self-assemble into nanoparticles. Freeze-dry for 24 hours to obtain a freeze-dried powder of the peptide nanoparticles. Store at -20°C until use. Dissolve 0.5 mg of the freeze-dried powder of the peptide nanoparticles in 1 mL of phosphate buffer to obtain a peptide nanoparticle solution with a mass concentration of 0.5 mg / mL.

[0094] Dissolve 0.5 g of polydopamine in 5 mL of deionized water, stir at room temperature for 10 min to disperse it evenly, and store it in a refrigerator at 4°C away from light until use to obtain a polydopamine solution.

[0095] 2 mL of 10% g / mL (w / v) gelatin solution and 2 mL of 1% g / mL (w / v) sodium alginate solution were stirred uniformly. 300 μL of the aforementioned polydopamine solution was added and mixed uniformly to obtain mixed solution a. 0.5 mL of 2.5% g / mL (w / v) poly(N-isopropylacrylamide) solution was added to mixed solution a and mixed uniformly to obtain mixed solution b. 0.1 mL of 0.5 mg / mL peptide nanoparticle solution was added to mixed solution b and mixed uniformly to obtain mixed solution c, designated GAPNP pregel solution. 600 μL of mixed solution c was placed in a 24-well plate and precooled in a 4°C refrigerator for 5 minutes. The plate was removed and 0.5 mL of 0.2 mol / L CaCl₂ solution was added. The plate was allowed to stand for 1 minute to obtain a peptide gel (abbreviated as GAPNP hydrogel) with mechanical contractile properties and scar inhibition. Remove the gel from the plate and store it in a 4°C refrigerator until use.

[0096] The prepared gel was subjected to scanning electron microscopy, infrared and rheological tests and characterization evaluations:

[0097] Scanning electron microscopy (SEM) results are as follows Figure 4 As shown, Figure 4 The following is a scanning electron micrograph of the freeze-dried polypeptide nanoparticle powder prepared in Example 1, along with a schematic diagram of its particle size distribution. The image clearly shows the uniform size of the polypeptide nanoparticles. Dynamic light scattering (DLS) testing further demonstrated that the hydrodynamic diameter (Dh) of the polypeptide nanoparticles was 69.29 nm, and the particle dispersion (PD) index was 0.26, consistent with the SEM observations. These data demonstrate the successful preparation of the polypeptide nanoparticles.

[0098] Figure 5 Schematic diagram of the prepared gel. It can be seen from the figure that each pre-gel solution is fluid, but does not flow after being made into hydrogel.

[0099] Study on controllable contraction of hydrogels: The prepared hydrogels were placed in a constant temperature environment of 30°C, 35°C, 40°C, and 45°C, and their dimensional changes were measured using a laser rangefinder. The volume changes were calculated and the regulatory effect of the traction force generated by the spontaneous contraction of the hydrogel on the contractile behavior of fibroblasts was evaluated.

[0100] Traditional gel dressings mainly focus on providing physical barriers and creating a moist environment, but ignore the effective regulation and precise management of wound mechanical forces. The hydrogel in the present invention uses PNIPAAm as a thermosensitive component, and further studies the mechanical contraction properties of different hydrogels under different temperature responses. GA hydrogel, GAP hydrogel, GAPN hydrogel, and GAPNP hydrogel were placed in a constant temperature environment of 30°C, 35°C, 40°C, and 45°C, respectively, and allowed to stand for 10 minutes. The appearance of the hydrogel was photographed and recorded, and the gel area was calculated using Image J software. The results are as follows Figure 6 As shown, Figure 6 Schematic diagram of the mechanical shrinkage of the prepared gels at different temperatures. The left figure shows the shrinkage of different hydrogels at different temperatures, and the right figure is a statistical chart of the shrinkage rates of different hydrogels. As can be seen from the left figure, at a temperature of 30°C, all hydrogels showed no significant changes, indicating that the physical properties of the hydrogels are relatively stable at this temperature. When the temperature was raised to 35°C, the edges of the GAPN hydrogel and GAPNP hydrogel began to shrink toward the center, initially demonstrating temperature responsiveness. When the temperature was further increased to 40°C, the shrinkage of the GAPN and GAPNP hydrogels became more significant, with a shrinkage rate of approximately 25%. When the temperature was further increased to 45°C, the shrinkage rates of the GAPN and GAPNP hydrogels increased sharply, reaching approximately 65%.

[0101] In order to observe the microstructural changes of the hydrogel after mechanical shrinkage, the microstructure of the hydrogel was further characterized by SEM. Figure 7 As shown, Figure 7 The following figure shows scanning electron microscopy results of the prepared gel at different temperatures. The results show that under body temperature (37°C) and photothermal stimulation (45°C), the voids in the GAPNP hydrogel gradually decrease and the structure becomes denser, consistent with macroscopic contraction. This demonstrates that the GAPNP hydrogel prepared in this invention exhibits significant contraction properties, which are important for rapid wound closure and preventing wound expansion.

[0102] The mechanical strength of the gel is crucial for wound dressings because the gel is required to provide physical support and resist external mechanical forces during the wound healing process. Figure 8 As shown, Figure 8 Schematic diagram of rheological test results of the prepared gel. No CaCl2 solution was added during the preparation of the hydrogel. The prepared hydrogel in the figure is not CaCl2 2+ Cross-linked hydrogel; GAPNP hydrogel is Ca 2+ Cross-linked hydrogel; GAPNP hydrogel is Ca- 2+Cross-linked hydrogel + infrared irradiation. As can be seen from the figure, the storage modulus (G′) of the GAPNP hydrogel prepared by the present invention is consistently higher than the loss modulus (G″). This phenomenon indicates that the hydrogel's internal cross-linked network structure possesses excellent elastic properties. This property enables the hydrogel to maintain a gel state when subjected to external stress, exhibiting excellent mechanical stability and biocompatibility, providing ideal physical support for wound healing. Under different strain conditions (as the horizontal axis changes), the hydrogel exhibits excellent stability, with its storage modulus (G′) and loss modulus (G″) varying slightly, and G′ consistently exceeding G″. This stability indicates that the hydrogel's internal cross-linked network structure possesses high mechanical strength and deformation resistance, capable of adapting to the complex mechanical environment encountered during wound healing. In contrast, hydrogels that have not undergone calcium ion cross-linking transition from a gel state to a fluid state. Clearly, calcium ion cross-linking can increase the cross-linking density of the hydrogel, significantly enhancing its mechanical strength and stability.

[0103] To further clarify the chemical functional groups that affect the stability of hydrogels, Fourier transform infrared spectroscopy (FTIR) analysis was performed on different gels. The results are shown in Figure 9. Figure 9 The figure shows the infrared test results of the prepared gel. The hydrogel prepared by gelatin is marked as gelatin in the figure; GA hydrogel is marked as gelatin + sodium alginate in the figure without adding CaCl2 solution during the preparation process; GA hydrogel is marked as gelatin + sodium alginate + CaCl2 solution in the figure. 2+ ; During the preparation of GAPNP hydrogel, no CaCl2 solution, polydopamine solution, polypeptide nanoparticle solution, or CaCl2 solution was added, and it is marked as gelatin + sodium alginate + PNIPAAm in the figure; During the preparation of GAPNP hydrogel, no polydopamine solution or polypeptide nanoparticle solution was added, and it is marked as gelatin + sodium alginate + PNIPAAm + CaCl2 solution in the figure 2+ ; As can be seen from the figure, at 3400-3200 cm -1 A broad peak appears in the range of 1410 cm-1, which corresponds to the stretching vibration of hydroxyl (-OH) and amino (-NH). -1 and 1070 cm -1 At the same time, new characteristic peaks appeared, corresponding to the carboxyl group (-COO - ) and CO stretching vibration, which indicates that sodium alginate has been successfully introduced into the system. -1 Near the 400 nm wavelength, new characteristic peaks corresponding to the amide group (C=O stretching vibration) in PNIPAAm appeared, confirming the successful incorporation of PNIPAAm. With the addition of calcium ions, the intensities and shifts of some of these peaks changed. These changes in characteristic peaks reflect the chemical structural changes and interactions of the components in the gel system.

[0104] Figure 10 Schematic diagram showing the adhesion properties of the prepared hydrogels. GAPNP hydrogels prepared without polydopamine (PDA) are labeled as GAPNP hydrogels without PDA; GAPNP hydrogels prepared with PDA are labeled as GAPNP hydrogels with PDA. As can be seen in the figure, the GAPNP hydrogels exhibit excellent adhesion, adhering firmly to glass, plastic, and skin surfaces, and maintaining good mechanical compliance even in areas of high mobility, such as joints.

[0105] The gel was adhered to the organs of mice (heart, liver, spleen, lungs, and kidneys) to observe its adhesion effect. Figure 11 The GAPNP hydrogel prepared in this invention has excellent adhesion to the heart, liver, spleen, lungs, and kidneys, thus expanding its application in wound repair.

[0106] To further evaluate the adhesion strength of the hydrogel dressing to the skin and its wound closure promoting effect, lap shear and wound closure tests were performed on pig skin. The pig skin was fixed on a glass slide with glue, and then the prepared GAPNP hydrogel was adhered to the pig skin. The operation diagram is shown in the figure. Figure 12 As shown, Figure 12 Schematic diagram of the lap shear and tensile test method for the prepared gel. The slide was placed in a universal testing machine for testing. The results are as follows Figure 13 As shown, Figure 13 The following are schematic diagrams showing the adhesion strength and wound closure strength of the prepared gels. The left figure shows the adhesion strength results, while the right figure shows the wound closure strength results. As can be seen from the figures, the addition of PDA significantly improves the adhesion of the GAPNP hydrogel prepared in this invention to skin, achieving an adhesion strength exceeding 15 kPa, enabling the gel to stably adhere to the wound surface. In a porcine skin wound closure test, the GAPNP hydrogel achieved a wound closure force of 2 N, demonstrating that the GAPNP hydrogel prepared in this invention is sufficient to withstand the various mechanical stresses encountered during wound healing.

[0107] In vitro biocompatibility of hydrogels: The biocompatibility of different hydrogels with cells was tested using a CCK-8 assay kit (Biyuntian Biotechnology Co., Ltd.). First, different hydrogels (GA hydrogel, GAP hydrogel, GAPN hydrogel, and GAPNP hydrogel) were diluted in cell culture medium to a series of concentrations (12.5 μg / ml, 25 μg / ml, 50 μg / ml, 100 μg / ml, 200 μg / ml, 400 μg / ml, 600 μg / ml, and 800 μg / ml) and stored in a refrigerator at 4°C. The prepared human dermal fibroblast (HSF) cell suspension was evenly plated in a 96-well plate, with 7,500 cells per well. After the cells adhered, the medium was replaced with fresh medium the next day, and 20 μl of hydrogels of varying concentrations were added to each well. The cells were incubated in a 37°C incubator for 48 hours. Co-culture the cells with serum-free CCK-8 in an incubator at 37°C for 2 hours. Measure the absorbance at 450 nm using a microplate reader and calculate the cell viability according to the following formula:

[0108] Cell viability (%) = [OD 450 (Sample group)-OD 450 (Blank group)] / [OD 450 (Control group) -OD 450 (blank group)] × 100%.

[0109] The cytotoxicity of GAPNP hydrogel to human skin fibroblasts (HSF) was shown in Figure 14 As shown, Figure 14 The figure shows the toxicity of the prepared hydrogels to fibroblasts. As can be seen, after 48 hours of continuous exposure of HSF cells to various concentrations of GAPNP hydrogels, the relative viability of the cells remained high. Even when cells were co-incubated with high-concentration hydrogels (600 μg / ml and 800 μg / ml), cell viability decreased slightly, but the toxicity was minimal.

[0110] To further evaluate the toxicity of the hydrogel on human umbilical vein endothelial cells (HUVEC), live-dead cell double staining was used for detection. HUVEC cells were seeded into 24-well plates and cultured for 24 hours. Different hydrogels (200 μg / ml) were added and cultured for 24 hours. The cytotoxicity of the hydrogel on cells was evaluated using the Calcein AM / PI double staining kit (Maokang Biotechnology Co., Ltd., MX3012). The cells were incubated with the live-dead staining working solution (Calcein-AM / PI) for 30 minutes. The cells were rinsed with PBS and observed under a fluorescence microscope. The results of the compatibility of GAPNP hydrogel with HUVEC cells are shown in Figure 2. Figure 15As shown, Figure 15 The figure shows the compatibility of the prepared hydrogels with endothelial cells. As can be seen, after incubation with HUVECs, the hydrogels with different compositions showed no increase in dead cells compared to the blank control group (PBS alone). These results demonstrate that the GAPNP hydrogels prepared in this invention exhibit good compatibility with human umbilical vein endothelial cells (HUVECs) and do not produce significant cytotoxicity after interaction with these cells.

[0111] In vitro hemocompatibility of the hydrogel: The hemocompatibility of the hydrogel was determined by a hemolysis test. 2 mL of fresh mouse blood was placed in an anticoagulant tube and centrifuged at 3000 rpm for 10 minutes. The supernatant was discarded and the sample was washed three times with 0.9% sodium chloride to obtain purified red blood cells (RBCs). 100 μl of RBCs was placed in a 1.5 ml centrifuge tube, and 100 μl of deionized water, PBS, GAP hydrogel, GAPN hydrogel, and GAPNP hydrogel were added, respectively. 0.9% NaCl was then added to a final volume of 1 mL. The mixture was mixed and incubated at 37°C for 3 hours. Deionized water served as the positive control group, and PBS served as the negative control group. After 3 hours, the sample was centrifuged at 3000 rpm for 10 minutes, and the RBC mixture in the centrifuge tube was photographed. 100 μl of the supernatant was taken, and the absorbance at a wavelength of 545 nm was measured (n = 5). The hemolysis rate (HR%) was calculated according to the following formula:

[0112] Hemolysis rate (%) = [(Ah-An) / (At-An)] × 100%.

[0113] Among them, Ah, At and An represent the absorbance of sample, positive control and negative control at 545nm wavelength respectively. Figure 16 As shown in Figure c, the relative hemolysis rate of each hydrogel is less than 2%, indicating that the GAPNP hydrogel prepared in the present invention does not produce hemolytic side effects on blood and has good compatibility.

[0114] These research results demonstrate that the GAPNP hydrogel prepared in this invention not only exhibits excellent biocompatibility, but also exhibits strong adhesion properties that enable it to closely adhere to skin texture, effectively resisting environmental interference with wounds. Furthermore, at 37°C, the GAPNP hydrogel exhibits both body-temperature autogenous contraction and photothermal controllable contraction. Through strong adhesion, it can be transferred to the wound edge, converting mechanical stress into wound contraction force, providing a stable mechanical microenvironment and precise tension management strategy for wound healing.

[0115] GAPNP hydrogel prepared by the present invention promotes cell proliferation and HUVEC migration test:

[0116] 1. Promoting effect of GAPNP hydrogel prepared by the present invention on cell proliferation

[0117] Cell proliferation was assessed using a CCK-8 assay. Healthy HSF and HUVEC cells were evenly plated onto 96-well plates (5,000 cells / well). After cell attachment, the cell culture medium was removed and 100 μl of DMEM / F12 and ECM culture medium containing GAP hydrogel, GAPN hydrogel, and GAPNP hydrogel (the hydrogel concentration in the culture medium was 50 μg / ml) was added. The cells were incubated at 37°C for 24 hours. Relative cell viability was assessed using CCK-8 assay.

[0118] 2. Evaluation of Hydrogels Promoting HUVEC Migration

[0119] Human umbilical vein endothelial cells (HUVECs) in optimal condition were collected and a cell suspension was prepared. The suspension was then evenly distributed on a 6-well plate and cultured overnight in a constant temperature incubator at 37°C. After the cells formed a uniform monolayer, a controlled scratch was made with a 20μl pipette tip, and then rinsed three times with phosphate-buffered saline (PBS) to gently wash away the scratched cells. Then, 2 mL of extracellular matrix (ECM) medium without fetal bovine serum (FBS) containing GAP hydrogel, GAPN hydrogel, and GAPNP hydrogel at a concentration of 50μg / ml was added to each well and co-cultured with the cells. At 0h and 24h, the wells were observed under a microscope, images were captured, and the width of the wound was measured. Before taking each picture, the floating cells were thoroughly washed with phosphate-buffered saline (PBS) to effectively remove the floating cells. The 24-hour wound closure rate was determined using the following formula:

[0120] Wound closure rate (%) = [(W 0h -W 24h ) / W 0h ]×100%.

[0121] Among them, W 0h Indicates the scratch wound area at 0h, W 24h Indicates the scratch wound area at 24 hours.

[0122] In the clinical application of hydrogels, good biocompatibility is a key requirement. The CCK-8 kit was used to detect the relative viability of cells after incubation with hydrogels. The results are shown in Figure 16. Figure 16Schematic diagram of the bioactivity results of the prepared gels at the cellular level. (a) shows the results of the prepared gels promoting the proliferation of human umbilical vein endothelial cells; (b) shows the results of the prepared gels promoting the proliferation of HSF cells; (c) shows the results of the prepared gels' biocompatibility with blood; (d) shows the results of the prepared gels promoting the migration of endothelial cells; (e) shows the results of the prepared gels' scratch healing rate; (f) shows the results of the prepared gels' inhibition of the contraction of the fibroblast collagen network; (g) shows the results of the prepared gels' relative scratch area; and (h) shows the results of the prepared gels' relative collagen shrinkage rate.

[0123] As shown in Figures a and b, the GAPNP hydrogel prepared in the present invention increased the relative viability of endothelial cells by as much as 180%, and the relative viability of fibroblasts by as much as 150%. This result fully demonstrates that the GAPNP hydrogel prepared in the present invention has an excellent proliferation-promoting effect on both cell types, creating favorable regenerative conditions for wound healing. Furthermore, hemolysis is considered an important parameter for evaluating biomaterial compatibility. Therefore, the present invention conducted a hemolysis test to evaluate the safety of the hydrogels in blood. The results are shown in Figure 16c, where no hemolysis was observed in any of the hydrogel groups.

[0124] The results of the effect of hydrogel on cell migration are as follows Figure 16 As shown in Figure d, compared with the HSF cells in the PBS-treated group, the HSF cells co-treated with the hydrogel showed a more significant wound closure effect. After 24 hours of co-culture of HSF cells with GAPNP hydrogel, the wound healing rate reached about 60% ( Figure 16 The scratch width of other gel groups only remained 45% to 50% ( Figure 16 Compared with the blank control group, the results also showed a good effect in promoting cell migration.

[0125] The GAPNP hydrogel prepared in this invention exhibits excellent biocompatibility with HSF and HUVEC cells, demonstrating high safety. Furthermore, the GAPNP hydrogel significantly enhances the proliferation and migration of HSF and HUVEC cells, two key processes in wound repair. Overall, these properties create a favorable environment for cell growth and subsequent tissue regeneration, demonstrating the potential application of the GAPNP hydrogel in wound healing and other related fields.

[0126] The GAPNP hydrogel prepared by the present invention inhibits collagen contraction in vitro:

[0127] Construction of 3D collagen gel network model of fibroblasts: 200 μl of 5 mg / ml rat tail type I collagen solution was neutralized with 12 μl of 0.1 mol / L NaOH solution, and then 188 μl of DMEM medium was added. 100 μl of HSF cell suspension (1×10 6 ) were mixed thoroughly, and 100 μl of the mixture was added to each well of a 24-well plate. The plates were incubated at 37°C in a 5% CO2 incubator for 30 minutes to allow the collagen to solidify and form a 3D collagen gel network. The plates were then removed, 100 μl of culture medium was added, and the collagen gels were gently peeled from the well walls. 100 μl of culture medium containing GAP hydrogel, GAPN hydrogel, and GAPNP hydrogel (all at a hydrogel concentration of 100 μg / ml) was then added. A control group was treated with 100 μl of pure culture medium. The plates were then incubated in an incubator, and collagen contraction was recorded by photographing at 24, 48, and 72 hours.

[0128] During the wound healing process, fibroblasts can sense mechanical forces and respond accordingly to the surrounding microenvironment in which they are located. When the collagen matrix contracts excessively, this phenomenon will prompt fibroblasts to transform into myofibroblasts. Myofibroblasts have stronger contraction capabilities and secrete a large amount of extracellular matrix (ECM). Excessive and disordered ECM deposition will induce tissue fibrosis, leading to scar formation. The present invention constructs a fibroblast 3D collagen gel network (FPCL) by extracting type I collagen from rat tails. After the FPCL is subjected to different gel treatments, its area changes are observed at different time points. The results are as follows. Figure 16 As shown in Figure f, human skin fibroblasts (HSF) can induce strong contraction of collagen matrix. GAP hydrogel group and GAPN hydrogel group also showed significant collagen contraction. However, the GAPNP hydrogel group containing peptides showed a significant inhibitory effect on collagen contraction. After 3 days of treatment, the collagen area retention rate was 92.49% (see Figure 16 h in the figure).

[0129] The GAPNP hydrogel prepared in this study significantly inhibits the contractile force of fibroblasts, thereby reducing collagen contraction. This inhibitory effect helps reduce excessive and disordered collagen deposition during wound repair, positively impacting the mechanical properties and structure of skin tissue. This effectively reduces the risk of scarring, providing a new theoretical basis and potential strategy for scar prevention and treatment.

[0130] In vitro antibacterial study of the GAPNP hydrogel prepared by the present invention:

[0131] Plate cloning method: the concentrations of 8Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) were diluted 10,000-fold. 500 μL of the bacterial solution was mixed with 200 μL of the hydrogel. The GAPNP+NIR group was exposed to infrared light for 10 minutes. The mixture was then incubated in a 37°C incubator for 60 minutes. 30 μL of the bacterial solution was evenly spread on an agar plate and incubated at 37°C for 24 hours. The resulting colonies were counted.

[0132] Bacterial growth curve method: the concentrations were 10 8 Staphylococcus aureus and Escherichia coli were diluted 10,000-fold, and 20 μl of the diluted culture medium was plated in a 96-well plate. 80 μl of liquid culture medium was added to the control group, while 80 μl of culture medium containing GAP hydrogel, GAPN hydrogel, and GAPNP hydrogel was added to the experimental groups, respectively. The GAPNP+NIR group was exposed to infrared light for 10 minutes. The plates were incubated at 37°C for 48 hours. The optical density (OD) at a wavelength of 600 nm was measured at various time intervals.

[0133] Observation of bacterial morphology: Scanning electron microscopy (SEM) was used to examine bacterial morphology after different treatments. The treated bacteria were fixed with 2.5% glutaraldehyde for 30 minutes and dehydrated using a gradient of ethanol (25%, 50%, 75%, and absolute ethanol). A 5μl sample was evenly applied to a silicon wafer. Prior to SEM analysis, the sample was coated with gold (placed in an automated gold coating machine, which forms a film on the surface to enhance conductivity and facilitate observation).

[0134] In the hydrogel system of this invention, polydopamine (PDA) not only imparts strong adhesive properties but also unique photothermal conversion properties. This property allows precise control of the hydrogel's temperature changes through photothermal action, thereby achieving precise control of its mechanical contraction behavior. More importantly, the photothermal effect significantly enhances the hydrogel's antimicrobial activity, which plays a key role in preventing wound infection and promoting wound healing.

[0135] The results are as follows Figure 17 As shown, Figure 17 Schematic diagram of the photothermal conversion characteristics and antibacterial activity of the prepared hydrogels. (a) Schematic diagram of the photothermal conversion effects of different PDA concentrations; (b) Schematic diagram of the photothermal conversion effects of different illumination times; (c) Schematic diagram of the growth curve of the prepared hydrogels against S. sureus; (d) Schematic diagram of the growth curve of the prepared hydrogels against E. coli; (e) Schematic diagram of the agar plate cloning results of S. sureus and E. coli treated with different hydrogels; (f) Schematic diagram of scanning electron micrographs of S. sureus and E. coli after treatment with different hydrogels.

[0136] from Figure 17 As shown in Figures a and b, the photothermal activity of the hydrogel exhibits a clear positive correlation with the PDA concentration and infrared illumination duration. As the illumination duration gradually increases, photothermal imaging technology allows for precise monitoring of temperature changes, ensuring on-demand contraction of the hydrogel through photothermal control, providing strong support for precise manipulation in applications such as wound repair. Figure 17 The results in Figures c and d show that the GAPNP hydrogel containing PDA exhibited extremely significant antibacterial activity under near-infrared light irradiation. Compared with the control group without hydrogel treatment, the optical density (OD) value at 600nm was significantly reduced, which fully demonstrated that the growth of bacteria was effectively inhibited. In addition, Figure 17 The results of plate colony counting in the middle e further confirmed that compared with the control group without hydrogel treatment, GAPNP hydrogel had the most prominent inhibitory effect on the two bacteria under near-infrared light irradiation. To explore the antibacterial mechanism of the hydrogel, S. sureus and E. coli were tested by scanning electron microscopy after being treated with different hydrogels. The results are as follows Figure 17 As shown in (f), scanning electron microscopy (SEM) observations revealed rupture of the bacterial membrane in the GAPNP hydrogel group, likely attributable to the positively charged lysine residues at the peptide termini. Lysine residues can interact with negative charges on the bacterial membrane surface, destabilizing it and ultimately causing it to rupture, thereby exerting an antibacterial effect.

[0137] The present invention activates the hydrogel through the photothermal effect, which not only can achieve precise temperature-responsive mechanical contraction and meet the demand for regulating the tissue mechanical environment during wound repair, but also can significantly enhance the antibacterial activity, effectively inhibit wound infection, and create favorable conditions for wound healing, showing great potential in the field of biomedical applications.

[0138] Evaluation of rapid wound healing of the GAPNP hydrogel prepared by the present invention:

[0139] All relevant animal experiments of the present application have been approved by the Experimental Animal Welfare and Ethics Committee of Henan University (HUSOM2025-583). C57BL / 6J mice (6 weeks old, male) were purchased in advance and raised in a constant temperature animal room for one week. The mice were anesthetized with isoflurane anesthetic, the hair on the back of the mouse was removed with a hair clipper, and further depilation was performed with depilatory cream. A full-thickness skin injury model was established on the back skin of the mouse with a punch with a diameter of 6 mm, and the mice were randomly divided into 5 groups, a total of 30, 6 in each group, namely the blank control group, the GAP hydrogel group, the GAPN hydrogel group, the GAPNP hydrogel group and the GAPNP+NIR hydrogel group. The wound was covered with a hydrogel with a thickness of 8 mm prepared in advance, so that it completely covered the wound, and the wound was sealed with sterile film, and the GAPNP+NIR hydrogel group was given 10 minutes of light treatment. Observe the wound healing situation regularly, observe the wound closure every 2 hours within the first 6 hours of the first day. The following formula is used to calculate the degree of wound closure:

[0140] Wound closure (%) = (initial wound area - final wound area) / initial wound area x 100%.

[0141] In order to more systematically evaluate the degree of wound healing, the mice were sacrificed on the 8th day, and the skin tissue was collected for HE staining and Masoon staining.

[0142] To verify the effect of the hydrogel prepared by the present application on promoting rapid wound healing through mechanical contraction, the present application establishes a full-thickness skin defect wound model with a diameter of 6 mm on the back of the mouse according to the experimental design of Figure 18 After completing the wound modeling, the mouse was immediately treated with hydrogel dressing and light, and the changes in the wound were closely observed within the first 6 hours.

[0143] Figure 18 To achieve rapid wound closure of the prepared gel, the results are shown in the schematic diagram. Among them, a is a mouse wound model and drug administration schematic diagram; b is a schematic diagram of the effect of gel with mechanical contraction on wound closure; c is a trajectory simulation diagram of wound closure within the first 6 hours; d is a schematic diagram of quantitative analysis of rapid wound closure; e is a schematic diagram of wound healing effect after different gel treatment; f is a wound healing trajectory simulation diagram; g is a schematic diagram of wound healing quantitative analysis.

[0144] As shown in Figure (b), 2 hours after surgery, the wounds in both the control and GAP hydrogel groups expanded, likely due to postoperative pain-induced physical activity and skin stretching in the mice. However, at 4 hours, wounds treated with GAP hydrogel, GAPN hydrogel, and GAPNP hydrogel began to close to varying degrees, with the wounds in the GAPNP+NIR group showing the most significant closure. In contrast, the wounds in the untreated control group continued to expand (Figure (c)). Further analysis revealed that the wound area retention rate in the GAPNP+NIR group was only 70.51% after 6 hours of treatment (Figure (d)), while the wound area retention rate in the control group was as high as 90.69%. These results demonstrate that the GAPNP+NIR hydrogel group significantly accelerated early wound closure by photothermally induced controlled mechanical contraction of the hydrogel, and the strong adhesion of the gel dressing transferred mechanical stress to the wound edge, driving wound contraction.

[0145] To comprehensively evaluate wound healing efficacy, another group of mice was treated with the same method and administered with the same medication. Wound morphology was recorded on days 0, 2, 4, 6, and 8. Representative wound photographs showed significant wound reduction on day 2 in the GAPNP+NIR hydrogel group, while wound area in the control group remained unchanged. The healing trajectory diagram (shown in Figure f) also shows that the wounds in the untreated control group (control) showed significant expansion early in the wound, consistent with the aforementioned observations. Wound healing rate statistics are shown in Figure g. The control group had a spontaneous healing rate of 58.58% on day 6. In contrast, the GAPNP+NIR hydrogel group had a healing rate of 60.35% on day 2 and achieved near-complete closure on day 8, significantly outperforming the control group, demonstrating that the GAPNP hydrogel prepared in this invention accelerates wound healing. Furthermore, mild signs of infection were observed in the GAP and GAPN hydrogel groups on day 2. However, after treatment with the peptide-containing GAPNP hydrogel and infrared imaging, the wound surface remained clean and showed no signs of infection. This result is consistent with in vitro antibacterial experimental data, further verifying that the peptide-modified lysine does indeed impart antibacterial activity to the hydrogel. This antibacterial property not only reduces the risk of infection but also provides a more optimal healing environment for wounds, thereby synergistically promoting healing efficiency.

[0146] The skin of the mouse wound was subjected to histological examination, and the results were as follows Figure 19 As shown, Figure 19Figure 1 is a schematic diagram of the results of mouse wound skin tissue testing. Figure a is a schematic diagram of the HE staining results of the wound-healed skin tissue; b is a schematic diagram of the Masson staining results of the skin tissue; c is a schematic diagram of the quantitative analysis of skin appendages calculated based on the HE staining; and d is a schematic diagram of the relative collagen content calculated based on the Masson staining. As shown in Figure a, the wound healing effect of the GAPNP hydrogel and GAPNP + NIR hydrogel groups was significantly better than that of the control group. In particular, the GAPNP + NIR hydrogel group showed more regenerated appendages in the wound area on day 8 (as shown in Figure c). Figure b shows that the collagen fibers in the control and GAP hydrogel groups are disordered, while the collagen fibers in the GAPNP + NIR hydrogel group are densely and neatly arranged. Quantitative results also show the highest relative collagen content (as shown in Figure d). This demonstrates that the GAPNP hydrogel prepared by the present invention, combined with photothermal controllable shrinkage, can significantly improve the speed and quality of wound healing, providing a new treatment method for scarless wound repair.

[0147] Study on scar-free healing of rabbit ear wounds using the GAPNP hydrogel prepared by the present invention:

[0148] A rabbit ear wound was used as a scar model. Rabbits were anesthetized with sodium pentobarbital, their ears shaved, and a 6 mm full-thickness skin incision was created on the ventral side of the ears using a scalpel. The subjects were randomly divided into five groups: a blank control group, a GAP hydrogel group, a GAPN hydrogel group, a GAPNP hydrogel group, and a GAPNP+NIR hydrogel group. An 8 mm thick hydrogel was applied to the wound, completely covering it. The wound was then sealed with a sterile film. The control group received no treatment, while the GAPNP+NIR hydrogel group received 10 minutes of light therapy. Digital camera images were taken periodically, and image analysis software was used to calculate wound area and healing rate. After wound healing, the rabbit ear skin was harvested for histological analysis. HE staining was performed to observe tissue repair during wound healing, including epidermal regeneration and granulation tissue formation. Masson staining was also performed to observe the extent of scar tissue formation and assess scar thickness and collagen fiber alignment. Image Pro Plus software was used for quantitative analysis of the staining results.

[0149] The results of GAPNP hydrogel prepared by the present invention in inhibiting scar are as follows Figure 20 As shown, Figure 20Schematic diagram of rabbit ear wound healing and scar evaluation results. (a) Schematic diagram of the rabbit ear model and drug administration; (b) Schematic diagram of representative images of rabbit ear wound healing; (c) Schematic diagram of the simulated rabbit ear wound healing trajectory; and (d) Schematic diagram of the wound healing rate calculated based on the rabbit ear wound area. The experiment began at wound initiation (week 0), with wound status recorded weekly. Histological staining was performed at week 4 to assess healing efficacy. The representative wound images shown in (b) show that in week 1, the wound healing rate in the GAPNP+NIR hydrogel group was significantly faster than that in the other groups. The wounds in the GAP hydrogel and GAPN hydrogel groups remained relatively large. In week 2, wound healing in the GAPNP+NIR hydrogel group accelerated further, with a significant reduction in wound area. The healing rate in the GAPNP hydrogel group was also superior to that in the GAP hydrogel and GAPN hydrogel groups. In week 3, the wound in the GAPNP+NIR hydrogel group was almost completely closed, while the wounds in the other groups still had significant areas of unhealed wounds. In week 4, the wound in the GAPNP+NIR hydrogel group was almost completely healed, while the wounds in the control and GAP hydrogel groups still had significant unhealed areas. Figure c shows a simulated trajectory of rabbit ear wound healing, and the results are consistent with the above. Figure d shows the wound healing rates of the different treatment groups at 7, 14, 21, and 28 days. At 21 days, the healing rate of the GAPNP+NIR hydrogel group was close to 95%, while the healing rates of the control group and the GAP hydrogel group were still below 50%.

[0150] The above results indicate that the GAPNP+NIR hydrogel group exhibited excellent closure speed in the early healing stage and a higher healing rate in the later healing stage. Furthermore, the peptide modification in the present invention imparts antibacterial properties to the hydrogel, reducing the risk of infection and further optimizing the healing environment. This result is consistent with the experimental data mentioned above, indicating that the GAPNP hydrogel prepared in the present invention is an ideal wound dressing that can significantly improve wound healing efficiency.

[0151] HE and Masson staining results are as follows Figure 21 As shown, Figure 21 Schematic diagram of the rabbit ear skin histological evaluation results. (a) Schematic diagram of the rabbit ear skin tissue HE staining results; (b) Schematic diagram of the rabbit ear skin tissue Masson staining results; (c) Schematic diagram of the scar height after wound healing; (d) Schematic diagram of the scar index calculated based on HE staining; and (e) Schematic diagram of the collagen deposition fraction calculated based on Masson staining.

[0152] Figures (a) and (c) show HE staining and average skin thickness of rabbit ear wound-healed skin tissue. HE staining results show that normal skin has a thickness of 398.6 μm and is structurally intact. The skin thickness of the control group increased significantly to 1617.3 μm, indicating pathological features of hyperplasia. However, the skin thickness of the GAPNP hydrogel group decreased significantly to 821.6 μm, and the skin thickness of the GAPNP+NIR hydrogel group further decreased to 637.5 μm, approaching that of normal skin, indicating that the synergistic effect of peptide modification and photothermal effect significantly reduced skin hyperplasia. The scar hyperplasia index (SEI) is shown in Figure (d). The SEI of the control group was approximately 2.7, while the SEI of the GAPNP+NIR hydrogel group was only approximately 1.3, indicating that the combined treatment of GAPNP hydrogel and photothermal therapy significantly inhibited scar hyperplasia. As shown in Figure (b), Masson staining was used to assess the distribution and maturity of collagen fibers. The collagen fibers in normal skin were neatly arranged, while those in the control group were disordered, indicating insufficient collagen remodeling during the healing process. The collagen fibers in the GAP hydrogel and GAPN hydrogel groups were still relatively disordered. However, the collagen fiber arrangement in the GAPNP+NIR hydrogel group was close to that of normal tissue, and the collagen volume fraction (CVF) was closest to that of the normal group (shown in Figure (e)), indicating that the GAPNP+NIR hydrogel group significantly promoted collagen remodeling.

[0153] In summary, the GAPNP hydrogel prepared in this invention significantly improved the skin tissue structure after rabbit ear wound healing through the synergistic effect of mechanical contraction induced by the photothermal effect and peptide molecular intervention. It demonstrated excellent performance in inhibiting skin hyperplasia, promoting collagen remodeling, and regenerating skin appendages, providing a repair effect closer to that of normal skin, further validating the potential of the GAPNP hydrogel prepared in this invention for scar-free wound repair.

[0154] Example 2

[0155] Preparation of peptide gel with mechanical contraction properties and scar inhibition:

[0156] Dissolve 0.5 g of polydopamine in 5 mL of deionized water, stir at room temperature for 10 min to disperse it evenly, and store it in a refrigerator at 4°C away from light until use, to obtain a polydopamine solution.

[0157] 0.1 g of poly (N-isopropylacrylamide) (PNIPAAm) was dissolved in 10 mL of deionized water and stirred at room temperature to completely dissolve the poly (N-isopropylacrylamide) to obtain a poly (N-isopropylacrylamide) solution with a mass concentration of 1% g / mL (w / v).

[0158] 2.5 mL of a 10% g / mL (w / v) gelatin solution and 2 mL of a 1% g / mL (w / v) sodium alginate solution were mixed thoroughly. 200 μL of the aforementioned polydopamine solution was added and mixed thoroughly to obtain mixed solution a. 0.5 mL of a 1% g / mL (w / v) poly(N-isopropylacrylamide) solution was added to mixed solution a and mixed thoroughly to obtain mixed solution b. 0.1 mL of a 0.5 mg / mL peptide nanoparticle solution was added to mixed solution b and mixed thoroughly to obtain mixed solution c, designated GAPNP pregel solution. 600 μL of mixed solution c was placed in a 24-well plate and precooled at 4°C for 5 minutes. The plate was removed and 0.5 mL of a 0.1 mol / L CaCl₂ solution was added. The solution was allowed to stand for 1 minute to obtain a peptide gel with mechanical contractile properties and scar inhibition. The gel was removed from the plate and stored in a 4°C refrigerator until further use.

[0159] Example 3

[0160] Preparation of peptide gel with mechanical contraction properties and scar inhibition:

[0161] Dissolve 0.5 g of polydopamine in 5 mL of deionized water, stir at room temperature for 10 min to disperse it evenly, and store it in a refrigerator at 4°C away from light until use, to obtain a polydopamine solution.

[0162] 0.3 g of poly (N-isopropylacrylamide) (PNIPAAm) was dissolved in 10 mL of deionized water and stirred at room temperature to completely dissolve it to obtain a poly (N-isopropylacrylamide) solution with a mass concentration of 3% g / mL (w / v).

[0163] 2.0 mL of a 10% g / mL (w / v) gelatin solution and 1.5 mL of a 1% g / mL (w / v) sodium alginate solution were stirred uniformly. 200 μL of the aforementioned polydopamine solution was added and mixed uniformly to obtain mixed solution a. 0.5 mL of a 3% g / mL (w / v) poly(N-isopropylacrylamide) solution was added to this mixed solution a and mixed uniformly to obtain mixed solution b. 0.2 mL of a 0.5 mg / mL peptide nanoparticle solution was added to this mixed solution b and mixed uniformly to obtain mixed solution c, designated GAPNP pregel solution. 600 μL of mixed solution c was placed in a 24-well plate and precooled in a refrigerator at 4°C for 5 minutes. The plate was then removed and 0.75 mL of a 0.1 mol / L CaCl₂ solution was added. The solution was allowed to stand for 1 minute to obtain a peptide gel with mechanical contractile properties and scar inhibition. Remove the gel from the plate and store it in a 4°C refrigerator until use.

[0164] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as above in terms of a preferred embodiment, it is not intended to limit the present invention. Any technician familiar with the present invention can make slight changes or modifications to equivalent embodiments using the above-mentioned technical contents without departing from the scope of the technical solution of the present invention. However, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the solution of the present invention.

Claims

1. A polypeptide, characterized in that The amino acid sequence is shown in SEQ ID NO.

1.

2. A method for preparing a polypeptide gel having mechanical contraction properties and scar inhibition, characterized in that: The method comprises the following steps: uniformly stirring a gelatin solution with a mass concentration of 5-15% g / mL and a sodium alginate solution with a mass concentration of 0.5-2% g / mL, adding a polydopamine solution, and stirring and mixing to obtain a mixed solution a; Adding a poly(N-isopropylacrylamide) solution having a mass concentration of 0.1-4% g / mL to the mixed solution a, stirring and mixing uniformly to obtain a mixed solution b; Adding a polypeptide nanoparticle solution with a mass concentration of 0.1-1 mg / mL to the mixed solution b, mixing evenly, to obtain a mixed solution c; The mass ratio of gelatin, sodium alginate, polydopamine, poly(N-isopropylacrylamide), and polypeptide nanoparticles is 1-200:1-50:1-10:1:0.00001-0.1; The mixed solution c was pre-cooled, CaCl2 solution was added, and the mixture was allowed to stand to obtain a polypeptide gel with mechanical contraction properties and scar inhibition; The preparation method of the polypeptide nanoparticle solution comprises the following steps: The freeze-dried polypeptide nanoparticle powder was dissolved in phosphate buffer to obtain a polypeptide nanoparticle solution with a mass concentration of 0.1-1 mg / mL; The preparation method of the polypeptide nanoparticle freeze-dried powder comprises the following steps: Sodium hydroxide is added to the phosphate buffer to adjust the pH to 9-10, and the polypeptide shown in SEQ ID NO. 1 is added to a concentration of 0.1-1 mg / mL. The mixture is stirred for 2-8 hours to allow the polypeptide to self-assemble into nanoparticles, which are then freeze-dried for at least 24 hours to obtain a freeze-dried powder of polypeptide nanoparticles.

3. The method for preparing the polypeptide gel having mechanical contraction properties and scar inhibition according to claim 2, characterized in that: The preparation method of the gelatin solution comprises the following steps: dissolving gelatin in deionized water, heating and stirring at a temperature of 40-60° C. to completely dissolve the gelatin, and obtaining a gelatin solution with a mass concentration of 5-15% g / mL.

4. The method for preparing the polypeptide gel having mechanical contraction properties and scar inhibition according to claim 2, characterized in that: The preparation method of the sodium alginate solution comprises the following steps: dissolving sodium alginate in deionized water, heating and stirring at a temperature of 40-60° C. to completely dissolve the sodium alginate, and obtaining a sodium alginate solution with a mass concentration of 0.5-2% g / mL.

5. The method for preparing the polypeptide gel having mechanical contraction properties and scar inhibition according to claim 2, characterized in that: The preparation method of the poly(N-isopropylacrylamide) solution comprises the following steps: dissolving poly(N-isopropylacrylamide) in deionized water, stirring at room temperature to completely dissolve the poly(N-isopropylacrylamide) solution, and obtaining a poly(N-isopropylacrylamide) solution with a mass concentration of 0.1-4% g / mL.

6. The method for preparing the polypeptide gel having mechanical contraction properties and scar inhibition according to claim 2, characterized in that: The concentration of the CaCl2 solution is 0.1-0.5 mol / L; The mass ratio of CaCl2 to poly(N-isopropylacrylamide) is 0.1-2:

1.

7. A polypeptide gel having mechanical contraction properties and scar inhibition prepared by the method according to any one of claims 2 to 6.

8. Use of the polypeptide gel having mechanical contraction properties and scar inhibition according to claim 7 in the preparation of wound repair medicines.

9. The use according to claim 8, characterized in that The wound repair medicine is selected from wound dressings, medicines for promoting wound healing, medicines for preventing wound expansion, medicines for preventing wound infection, medicines for inhibiting skin proliferation, medicines for promoting skin collagen remodeling, and medicines for regenerating skin appendages.

10. Use of the polypeptide gel having mechanical contraction properties and scar inhibition according to claim 7 in preparing tissue engineering scaffolds.

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