A method for preparing an injectable self-healing hydrogel dressing, and products and uses thereof

Abstract

The application discloses a preparation method of an injectable self-healing hydrogel dressing, and relates to the technical field of medical materials, and particularly relates to a preparation method of an injectable self-healing hydrogel dressing: (1) mixing CuCl2.2H2O and protocatechuic aldehyde (PA) in deionized water, adjusting the pH to an alkaline environment, and obtaining a first solution containing a copper protocatechuic aldehyde complex (CuPA) through reaction; (2) dissolving methacrylated gelatin (GelMA) and an initiator in a buffer solution to obtain a second solution; (3) adding the first solution into the second solution and mixing uniformly, and then performing photocuring crosslinking to form the injectable self-healing hydrogel dressing. The application further discloses the injectable self-healing hydrogel dressing obtained through the above preparation method and application of the injectable self-healing hydrogel dressing in preparation of a diabetic infected wound dressing. The hydrogel dressing prepared by the application can realize multiple biological activities such as antibacterial activity, anti-inflammatory activity, antioxidant activity and pro-angiogenic activity at the same time, and has good blood compatibility and biological safety, and can be used for preparing a pro-infected diabetic wound healing dressing.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medical dressings, and particularly relates to a preparation method of an injectable self-healing hydrogel dressing, and products and applications thereof. BACKGROUND

[0002] Diabetic wounds are one of the most serious chronic complications of diabetic patients. High glucose and high oxidative stress levels in the microenvironment of the wound surface seriously hinder the healing of diabetic wounds, and are often accompanied by bacterial infection, leading to the formation of a biological membrane, causing diabetic wounds to develop into chronic refractory wounds, greatly prolonging the wound repair process, and intensifying the pain of patients. Therefore, the complex environment of diabetic wounds puts high demands on new dressings for promoting the repair of diabetic wounds.

[0003] As a class of three-dimensional network structure materials with high water content (up to 99%), hydrogels are very similar to natural tissues, and have good tissue exudate absorption, moisturizing and gas exchange capacity. Through appropriate selection of high molecular materials and functional modification, hydrogel materials can be endowed with various excellent properties such as tissue adhesion, hemostasis, antibiosis, injectability, etc. Further, they can be used as delivery carriers for drug molecules, cytokines, nanomaterials, and therapeutic stem cells, to realize the spatiotemporal regulation of the tissue microenvironment through condition-responsive release, and create a more favorable local environment for tissue repair. 2+ Cu can effectively inhibit and kill various pathogenic microorganisms (such as Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, etc.) through mechanisms such as binding with sulfhydryl groups, changing the permeability of bacterial cell membranes, and producing active oxygen, etc. Moreover, Cu 2+ has a close relationship with fibroblast growth factor (FGF) and has a synergistic stimulating effect on angiogenesis. In addition, Cu 2+ can also directly promote the production of vascular endothelial growth factor (VEGF), increase the expression of hypoxia-inducible factor 1 alpha (HIF-1 alpha) and inhibit its degradation, and promote angiogenesis. Polyphenolic molecules (such as tannic acid, protocatechuic aldehyde, etc.) can promote the conversion of macrophages from M1 type to M2 type in the middle and late stages of tissue repair, and shape an anti-inflammatory immune microenvironment. Metal-polyphenol complexes also have various enzyme-like activities, which can scavenge excess reactive oxygen species (ROS) in tissues and improve the tissue microenvironment. GelMA has RGD sequences for cell adhesion and MMP sequences for supporting enzymolysis, and is widely used in tissue repair and regeneration. It can also achieve tissue adhesion on the skin surface through hydrogen bonding, π-π stacking, etc.

[0004] Therefore, how to design and prepare hydrogel dressings using metals, polyphenols and GelMA to simultaneously realize multiple functions and biological activities will have important significance and application value for promoting the repair of complex wounds such as infected diabetic wounds. SUMMARY

[0005] The application aims to provide a preparation method of an injectable self-healing hydrogel dressing, and the prepared hydrogel dressing has good tissue adhesion, hemostatic function and various biological functions, and can effectively promote the healing of a diabetic wound.

[0006] To achieve the above-mentioned purpose, the application adopts the following technical scheme:

[0007] The application provides a preparation method of an injectable self-healing hydrogel dressing, and the preparation method comprises the following steps:

[0008] (1) mixing copper chloride dihydrate CuCl2·2H2O and protocatechuic aldehyde PA in deionized water, adjusting the pH to an alkaline environment, and obtaining a first solution containing a copper-protocatechuic aldehyde complex CuPA through reaction;

[0009] (2) dissolving methacrylated gelatin GelMA and an initiator in a buffer solution to obtain a second solution;

[0010] (3) adding the first solution into the second solution and mixing uniformly, and then performing light curing crosslinking to form the injectable self-healing hydrogel dressing.

[0011] The technical concept of the application is that copper chloride dihydrate and protocatechuic aldehyde form a copper-protocatechuic aldehyde complex CuPA with active oxygen scavenging properties under weak alkaline conditions (pH 8.7). The aldehyde group in the copper-protocatechuic aldehyde complex forms a dynamic Schiff base bond with the amino group on GelMA, and the methacryl bond in GelMA is polymerized and crosslinked under the action of the initiator and ultraviolet light. The double crosslinking network endows the hydrogel with the performance of injectable self-healing, and the dynamic Schiff base bond can realize the slow release of CuPA in response to an acidic microenvironment. The CuPA complex simultaneously has various biological activities such as antibacterial, anti-inflammatory, antioxidant and pro-angiogenic activities, and can promote the repair of an infected diabetic wound.

[0012] In step (1), the pH is less than 9.0.

[0013] Preferably, in step (1), the preparation method of the copper-protocatechuic aldehyde nanocomposite (CuPA) is as follows: copper chloride dihydrate (CuCl2·2H2O) and protocatechuic aldehyde (PA) are dissolved in deionized water, the pH of the mixed solution is adjusted to 8.7 by using a sodium hydroxide solution, and the copper-protocatechuic aldehyde complex (CuPA) can be obtained after stirring and reaction at room temperature for 3 hours.

[0014] Due to the catechol and aldehyde group structure in protocatechuic aldehyde, oxidation occurs in different degrees under alkaline environment, oxidant and ultraviolet light, forming o-benzoquinone structure, therefore, the reaction conditions of CuPA need to be reasonably controlled, the concentration ratio of copper ion to PA should be controlled at 1:2, the alkaline environment pH should not be higher than 9.0, and the whole reaction needs to be strictly treated in dark to prevent PA from being oxidized under light, thereby affecting the formation of CuPA complex and the antioxidant activity of CuPA.

[0015] In step (2), the preparation method of the methacrylated gelatin GelMA is as follows: pigskin gelatin is added into a buffer solution to be stirred and dissolved, then methacrylic anhydride solution is added dropwise to be stirred and reacted, a buffer solution is added to terminate the reaction to obtain a product solution, the product solution is dialyzed in deionized water by using a dialysis bag with a molecular weight cut-off of 8-14KD, the solution is filtered and freeze-dried to obtain the methacrylated gelatin GelMA.

[0016] As preferred, 10% w / v pigskin gelatin (such as Type A, ~300g bloom) is added into a PBS solution, the mixture solution is magnetically stirred at 45-55℃ until the gelatin is fully dissolved, then 8-12mL of methacrylic anhydride solution is added dropwise, and the reaction is stirred at 50℃ for 3 hours; 50℃ PBS solution with the same volume as the above mixture solution is added to terminate the reaction, the obtained product solution is dialyzed in deionized water by using a dialysis bag with a molecular weight cut-off of 8-14KD for 5-7 days, the solution is filtered and freeze-dried to obtain the methacrylated gelatin GelMA.

[0017] As preferred, in step (2), the initiator is lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP).

[0018] In step (3), the concentration of Cu in the first solution is 0.05M, the concentration of methacrylated gelatin GelMA in the second solution is 5-10%, and the concentration of initiator is 0.1-0.5%, wherein the percentages are mass percentages; the volume of the first solution added is 10-20% of the second solution.

[0019] In step (3), the wavelength of the ultraviolet light source used for photocuring is 300-400nm, and the photocuring time is usually 3-5min.

[0020] The application also provides an injectable self-healing hydrogel dressing obtained by the above preparation method.

[0021] The application also provides application of the above injectable self-healing hydrogel in preparation of a diabetic infected wound dressing.

[0022] 1. The injectable self-healing hydrogel dressing prepared by the method has good injectability, self-healing property and tissue adhesion: the injectability can make the dressing adapt to irregular wound surface, and also facilitate the control of the amount of the dressing used, so as to realize on-demand drug delivery; the self-healing property enables the hydrogel dressing to restore to the original state after being damaged by external force; and the tissue adhesion enables the hydrogel dressing to keep good adhesion with the tissue interface, thereby providing a guarantee for the realization of the effect of the hydrogel dressing in promoting the repair of damaged tissue.

[0023] 2. The injectable self-healing hydrogel dressing prepared by the method has excellent hemostatic performance: the porous structure of the hydrogel dressing can rapidly absorb platelets from blood and form a gel-like substance, thereby forming a pressure barrier and promoting blood coagulation; the catechol group in the copper-protocatechuic aldehyde complex CuPA cooperates with the methacrylated gelatin GelMA to recruit calcium ions to vascular smooth muscle cells, thereby promoting vasoconstriction and promoting hemostasis.

[0024] 3. The injectable hydrogel dressing prepared by the method is crosslinked by radical-initiated polymerization of the acryl group in the methacrylated gelatin GelMA and the dynamic Schiff base bond formed by the aldehyde group in the copper-protocatechuic aldehyde complex CuPA and the amino group in the methacrylated gelatin GelMA, and the pH responsiveness of the Schiff base bond realizes the slow release of CuPA, and at the same time realizes the multiple biological functions such as antibacterial, antioxidant, anti-inflammatory and pro-angiogenic functions.

[0025] Therefore, the present application uses metal copper ions, protocatechuic aldehyde and methacrylated gelatin as raw materials, and prepares an injectable self-healing hydrogel dressing with good tissue adhesion and hemostatic function through radical-initiated polymerization reaction and dynamic Schiff base bond, and utilizes the low pH responsiveness of the Schiff base bond to release the copper-protocatechuic aldehyde complex CuPA to exert its multiple functions and biological activities such as antibacterial, antioxidant, anti-inflammatory and pro-angiogenic functions, thereby improving the tissue microenvironment of the infected diabetic wound and promoting the healing of the diabetic wound. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 A-I in the above table are transmission electron microscope images (scale: 200 nm), element distribution maps (scale: 100 nm), Fourier transform infrared spectrograms (FTIR), Raman spectrograms (Raman), X-ray photoelectron spectrograms (XPS) and other material characterization of the copper-protocatechuic aldehyde complex CuPA in Example 1, as well as the determination of the antioxidant performance of CuPA;

[0027] Figure 2A-I in FIG. 1 are the transmission electron microscopy images (scale bar: 5 nm), element distribution maps (scale bar: 100 nm), Fourier transform infrared spectroscopy (FTIR) images, X-ray diffraction (XRD) spectra, X-ray photoelectron spectroscopy (XPS) images and other material characterization of the ceria nanosensor Ce NPs in Example 1, and the antioxidant performance of the ceria nanosensor Ce NPs is determined;

[0028] Figure 3 A-F in FIG. 2 are the scanning electron microscopy images, pore analysis, element distribution maps, Fourier transform infrared spectroscopy (FTIR) images and other material characterization of the three hydrogel dressings of GCP in Example 2, GelMA in Comparative Example 1 and GC in Comparative Example 2, Figure 3 G-K in FIG. 3 are the injectability, self-healing, tissue adhesion and other property determinations of the GCP hydrogel dressing prepared in Example 2;

[0029] Figure 4 A-E in FIG. 4 are the hemostatic, hemolytic, swelling and other property determinations of the GCP hydrogel dressing in Application Example 1, Figure 4 F-K in FIG. 5 are the antioxidant performance determinations of the three hydrogel dressings of GelMA in Comparative Example 1, GC in Comparative Example 2 and GCP in Example 2.

[0030] Figure 5 A-D in FIG. 6 are the cell compatibility determinations of the GCP hydrogel dressing in Application Example 3, Figure 5 E-F in FIG. 7 are the intracellular reactive oxygen species scavenging properties of the GCP hydrogel dressing in Application Example 2;

[0031] Figure 6 A-K in FIG. 8 are the cell migration promoting, anti-inflammatory and pro-angiogenic function determinations of different hydrogel dressings in Application Example 3;

[0032] Figure 7 A-F in FIG. 9 are the antibacterial property determination results of different hydrogel dressings in Application Example 4;

[0033] Figure 8 A-I in FIG. 10 are the efficacy determinations of different hydrogel dressings in promoting the repair of infected diabetic wounds in Application Example 5. DETAILED DESCRIPTION

[0034] The technical solutions of the present application will be described below in conjunction with the accompanying drawings. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application.

[0035] Example 1

[0036] Under nitrogen protection, 0.14 g of PA was completely dissolved in 5 mL of 0.2 M deionized water at 80 °C. Then, 5 mL of 0.1 M CuCl2·2H2O solution was added to the PA solution. The pH of the mixture was then adjusted to 8.7 with sodium hydroxide solution (1 M). After stirring at 600 rpm for 3 h in the dark, the copper-protocatechuic aldehyde complex CuPA was obtained.

[0037] like Figure 1 As shown, TEM and EDS results indicate that CuPA is a typical nanosheet structure, and that Cu elements are uniformly distributed within it. Figure 1 (AB in the text). FTIR spectrum of protocatechuic aldehyde (PA) ( Figure 1 (C) , 3230cm -1 and 1298cm -1 The stretching vibrations are attributed to the hydroxyl (OH) and CO bonds, respectively, at 1597 cm⁻¹. -1 and 1447cm -1 Belongs to the skeletal vibration of the benzene ring, 1872 cm⁻¹ -1 1754cm -1 877cm -1 and 811cm -1 The indication is that 1, 2, 4 trisubstituted have occurred on the benzene ring, 2820 cm⁻¹ -1 and 2749cm -1 The peak splits due to the Fermi resonance of the CH bond in the aldehyde group. The C=O bond in the aldehyde group is conjugated because it is directly connected to the benzene ring, causing the wavenumber to shift to a lower frequency, down to 1652 cm⁻¹. -1 When Cu is introduced, the overall peak intensity decreases and shifts to higher frequencies, which may be related to hydrogen bond formation and changes in molecular structure. The Raman spectrum of PA is in the range of 300-2000 cm⁻¹. -1 The following characteristic peak appears in the spectral range: 813 cm⁻¹ -1 and 1597cm -1 This is a strong stretching vibration of the benzene ring, 1165 cm⁻¹ -1 The main vibrational modes include CH bond vibrations connected to the benzene ring, and partial OH bond shearing vibrations. 1382 cm⁻¹ -1 The main vibrational modes are benzene ring stretching and partial OH bond shearing vibrations. 1441 cm -1 It can be classified as HC=O shear vibration and part of OH shear vibration, 1647cm -1 It belongs to the CC=O stretching vibration associated with the benzene ring. After adding copper chloride, at 432 cm⁻¹... -1A small peak appears, which can be attributed to the typical Raman vibration of copper chloride molecules. In addition, due to the addition of copper chloride, the overall system changes to a certain extent, thereby causing the Raman characteristic peaks on the CuPA Raman spectrum to generally shift to a certain extent, which is manifested by the characteristic peaks belonging to PA moving, for example, the benzene ring vibration peak 813 cm -1 and 1597 cm -1 are shifted by 15 and 22 wave numbers to 798 cm -1 and 1575 cm -1 , respectively. Some peaks disappear, which may be due to the original peak being insufficient in intensity to be covered by the new background (D in Figure 1 ). (E in Figure 1 ), the XPS peak of CuPA shows that the proportion of Cu 2+ is as high as 91.71%, and Cu + accounts for 8.29%, which shows that the valence state of copper ions in CuPA is mainly +2, which also shows that the CuPA complex is successfully synthesized. The antioxidant kit is used to explore the ROS scavenging ability of CuPA, and the results show that CuPA has good superoxide anion scavenging ability (F and H in Figure 1 ) in the concentration range of 5-100 μg mL -1 , and also shows good DPPH scavenging ability (G and I in Figure 1 ) in the concentration range of 1-5 μg mL -1 . Among them, *p<0.05, **p<0.01, ***p<0.001.

[0038] Example 2

[0039] Preparation of injectable self-healing hydrogel adjuvant GCP:

[0040] (1) Preparation of GelMA: 10% w / v pigskin gelatin was added to a PBS solution, and the mixture solution was magnetically stirred at 45-55°C until the gelatin was fully dissolved. Then 8-12 mL of methacrylic anhydride solution was added dropwise, and the mixture was stirred at 50°C for 3 hours. 50°C PBS solution was added to terminate the reaction, and the resulting product solution was dialyzed in a dialysis bag with a molecular weight cutoff of 8-14 KD in deionized water for 5-7 days. The solution was filtered and lyophilized to obtain methacrylated gelatin GelMA;

[0041] (2) According to Example 1, a copper-protocatechuic aldehyde complex CuPA solution was prepared, and the concentration of Cu was 0.05M;

[0042] (3) 0.5 g GelMA and 0.0125 g of a photoinitiator, lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), were dissolved in 5 mL of PBS (i.e., 10% GelMA and 0.25% LAP) and dissolved by heating and stirring. Then the mixture was mixed well with the CuPA solution at a volume ratio of 9:1, and irradiated under 395 nm ultraviolet light for 3 min to form a GCP hydrogel.

[0043] Comparative Example 1

[0044] Preparation of a photocured GelMA hydrogel dressing: 0.5 g GelMA and 0.0125 g of a photoinitiator, lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), were dissolved in 5 mL of PBS (i.e., 10% GelMA and 0.25% LAP). The mixture was mixed well with the PBS solution at a volume ratio of 9:1, and irradiated under 395 nm ultraviolet light for 3 min to form a GelMA hydrogel.

[0045] Comparative Example 2

[0046] Preparation of a photocured GC hydrogel dressing:

[0047] (1) Preparation of ceria nanoszyme Ce NPs: 0.43 g cerium acetate and 3.25 g oleylamine were mixed well with 15 mL of dimethylbenzene, and stirred at room temperature for 24 hours. The solution was heated to 90°C under vacuum, 1 mL of deionized water was added, and the solution was aged at 90°C for 3 hours. Then, the Ce NPs were precipitated with ethanol, centrifuged and washed with acetone, and the obtained Ce NPs were dispersed in chloroform, and mPEG-DSPE was used to transfer the Ce NPs to the aqueous phase to prepare a 5 mg mL 2k aqueous solution. -1

[0048] (2) 0.5 g GelMA, 0.0125 g of a photoinitiator, lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), and 0.3 mL of a Ce NPs solution (5 mg mL -1 ) were dissolved in 4.7 mL of PBS, and irradiated under 395 nm ultraviolet light for 3 min to form a GC hydrogel.

[0049] Figure 2 A-I in FIG. 1 are transmission electron microscopy images (scale bar: 5 nm), element distribution maps (scale bar: 100 nm), Fourier transform infrared spectroscopy (FTIR) spectra, X-ray diffraction (XRD) spectra, X-ray photoelectron spectroscopy (XPS) spectra, and other material characterization of the ceria nanoszyme Ce NPs in Comparative Example 2, as well as antioxidant performance determination of the ceria nanoszyme Ce NPs. Figure 2 ​The AB diagram indicates the successful preparation of cerium dioxide nanozymes (Ce NPs), with a diameter of approximately 3 nm and a lattice spacing of ~0.32 nm. Elemental mapping analysis confirms the uniform distribution of Ce element in the Ce NPs. FTIR spectra (…) Figure 2 (C) Ce-O bonds can be observed in Ce NPs at 472 cm⁻¹. -1 Stretching vibration at the location. In the XRD characterization results ( Figure 2 In the D), Ce NPs exhibit a cubic fluorite structure, with obvious diffraction peaks observed on the (111), (200), (220), (311), (400), and (331) crystal planes. The XPS fitting results show that Ce... 3+ It accounts for 42.85% of CeNPs, Ce 4+ This accounts for 57.15%, indicating that Ce NPs are composed of Ce 4+ and Ce 3+ The mixture of valence states has a valence ratio of approximately 1.3. Figure 2 (E in the text) Figure 2 In the 2F-I fraction, Ce NPs exhibit strong antioxidant capacity by effectively scavenging reactive oxygen species such as superoxide anions and DPPH. Figure 2 F and H in the figure represent Ce NPs in the range of 1-100 μg / mL -1 Superoxide anion scavenging capacity within the concentration range; Figure 2 G and I in the range are Ce NPs in the range of 1-100 μg / mL. -1 Scavenging ability of DPPH within the concentration range. *p<0.05,**p<0.01,***p<0.001.

[0050] Figure 3 The images in AF represent scanning electron microscopy (SEM) images, pore size analysis, elemental distribution maps, and Fourier transform infrared (FTIR) spectra of the three hydrogel dressings: GCP (Example 2), GelMA (Comparative Example 1), and GC (Comparative Example 2). SEM and elemental analysis show that GelMA, GC, and GCP hydrogels all possess porous network structures, with no significant difference in pore size among the groups. Figure 3 (AB in the text). Copper in GCP hydrogel and cerium in GC hydrogel are uniformly distributed throughout the hydrogel network. Figure 3 (CD in the middle). Figure 3 In the image, E represents the transition from liquid to solid state of GelMA, GC, and GCP hydrogels under ultraviolet irradiation. In the infrared spectrum (… Figure 3 In F), gelatin is at 2500-3300cm. -1 and 3200-3700cm -1The wide peak prominent in the range should be attributed to the stretching vibration of O-H associated with carboxyl and hydroxyl, which is the characteristic peak of gelatin. The peaks of gelatin, GelMA and GCP at 3423 and 3077 cm -1 indicate the stretching vibration of N-H bond. In addition, the peak at 1647 cm -1 indicates the stretching vibration of C=0 bond, while the peak at 1540 cm -1 indicates the stretching vibration of C-N bond and the bending vibration of N-H bond. Compared with gelatin, the addition of MA reduces the absorption peak intensity of GelMA, which is mainly due to the acylation reaction between the carbonyl group on MA and the hydroxyl group on gelatin, which significantly weakens the O-H stretching vibration related absorption peak. The absorption peak intensity of GCP hydrogel is lower than that of GelMA, which may be the result of the change in the molecular structure of GelMA caused by the CuPA complex.

[0051] Application Example 1

[0052] Injectable, self-healing, tissue-adhesive, hemostatic properties of injectable self-healing hydrogel dressing GCP were determined:

[0053] Figure 3 G-K in the above table is the injectable, self-healing, tissue-adhesive, etc. properties of the GCP hydrogel dressing prepared in Example 2:

[0054] Figure 3 In G in the above table, the shear thinning experiment shows that the viscosity of the GCP dressing is logarithmically related to the shear rate, and the viscosity gradually decreases with the increase of the shear rate, indicating that the GCP has good shear thinning performance. The modulus test ( Figure 3 H in the above table) and the structure recovery experiment ( Figure 3 I in the above table) were carried out on the GCP using a rheometer to evaluate the self-healing performance of the hydrogel GCP. When the frequency is kept at 1 Hz and the strain reaches 294.7%, the loss modulus (G”) is greater than the storage modulus (G’), and the hydrogel mainly undergoes viscous deformation, changing from a solid state to a liquid state ( Figure 3 H in the above table). When a strain of 5% is given, the storage modulus is greater than the loss modulus, and the hydrogel is in a solid state. When the strain is adjusted to 500%, the loss modulus is greater than the storage modulus, and the hydrogel becomes a liquid state, and the material changes from elastic deformation to viscous deformation. After three cycles, the GCP alternates between solid and liquid states, and the storage modulus and loss modulus remain basically the same, indicating that the GCP hydrogel has good self-healing function ( Figure 3 I in the above table). Figure 3J in the same way as GCP hydrogel shows significant self-healing properties, the whole process mainly involves the breaking and reversible recombination of Schiff base bonds and copper-catechol coordination bonds. Subsequently, biological tissues of C57BL / 6 mice were used to evaluate the tissue adhesion properties of GCP hydrogel. The results show that GCP has good adhesion performance, and GCP can effectively adhere to the tissues of mouse heart, liver, spleen, kidney Figure 4 K in the same way as A-B, a liver injury model was established using 6-week-old SD rats to evaluate the hemostatic effect of different hydrogel dressings within 1 minute

[0055] Figure 4 A-B in the same way as A-B, a liver injury model was established using 6-week-old SD rats to evaluate the hemostatic effect of different hydrogel dressings within 1 minute Figure 4 The results show that GelMA, GC and GCP hydrogels exhibit different degrees of hemostatic effect; notably, GCP hydrogel treatment significantly reduces bleeding to 60.53±10.60 mg Figure 4 Figure 4 D is the comparison of the swelling properties of GelMA, GC and GCP hydrogels. In the hemolysis experiment Figure 4 E, the hemolysis rate of GelMA hydrogel is 0.36%±0.18%, that of GC hydrogel is 0.20%±0.09%, and that of GCP hydrogel is 0.87%±0.09%, all of which are less than the biological safety threshold of 5%.

[0056] Application Example 2

[0057] Measurement of the scavenging reactive oxygen species properties of the injectable self-healing hydrogel dressing GCP:

[0058] Figure 4 F-K in the same way as A-B, the antioxidant properties of GelMA of Comparative Example 1, GC of Comparative Example 2 and GCP of Example 2 were measured, and the antioxidant properties of the composite hydrogel were studied using an antioxidant kit and mouse fibroblasts (L929). The results of the study show that GCP and GC hydrogels have obvious ability to scavenge ·O2 - and DPPH free radicals, and GCP hydrogel also has significant catalase-like activity Figure 4 F-K in the same way as A-B, the antioxidant properties of GelMA of Comparative Example 1, GC of Comparative Example 2 and GCP of Example 2 were measured, and the antioxidant properties of the composite hydrogel were studied using an antioxidant kit and mouse fibroblasts (L929). The results of the study show that GCP and GC hydrogels have obvious ability to scavenge ·O2 Figure 4 F and I are the determination of the superoxide anion scavenging capacity of GelMA, GC and GCP hydrogel dressings; Figure 4 G and J are the determination of the DPPH scavenging capacity of GelMA, GC and GCP hydrogel dressings; Figure 5 H and K are the determination of the catalase-like activity of GelMA, GC and GCP hydrogel dressings; *p<0.05, **p<0.01, ***p<0.001).

[0059] ​GCP and GC hydrogels showed excellent efficacy in intracellular ROS scavenging after L929 cells were treated with 200 mM hydrogen peroxide (H2O2) and different hydrogels Figure 5 E and F in FIG. 6: F is the analysis of the mean fluorescence intensity of ROS in each group in the E graph, *p < 0.05, **p < 0.01, ***p < 0.001), compared with GC, GCP hydrogel showed stronger ROS scavenging ability.

[0060] Example 3

[0061] Biocompatibility, anti-inflammatory, and pro-angiogenic activity determination of injectable self-healing hydrogel dressing GCP:

[0062] The biocompatibility of GCP hydrogel was evaluated using mouse fibroblasts L929 and human immortalized keratinocytes HaCaT. CCK8 detection and Calcein / PI staining showed that Figure 5 A-D in FIG. 5: Figure 5 A and B in FIG. 5 are cell viability and Calcein / PI staining of L929 cells cultured for 12 and 24 hours, respectively (scale bar: 200 pm); Figure 5 C and D in FIG. 5 are cell viability and Calcein / PI staining of HaCaT cells treated with hydrogels for 12 and 24 hours, respectively (scale bar: 200 pm); Figure 6 E in FIG. 5 is the fluorescence image of ROS determination in L929 cells after treatment with different hydrogel dressings (scale bar: 200 pm). After 12 and 24 hours of treatment with different hydrogel dressings, the survival rates of L929 and HaCaT cells were both more than 95%, and no obvious cell death was observed. These results indicate that GelMA, GC, and GCP hydrogels have good cell compatibility.

[0063] Subsequently, the present application further investigated the ability of hydrogels to promote L929 cell migration. Compared with the control group, GelMA group, and GC group, GCP hydrogel can significantly accelerate the migration of L929 cells within 24 hours Figure 6 A and B in FIG. 6: are the cell migration and migration distance analysis of L929 cells treated with different hydrogel dressings for 24 hours (scale bar: 200 pm).), indicating that GCP hydrogel has strong ability to promote fibroblast migration.

[0064] In diabetic patients, high blood glucose levels can trigger a series of inflammatory responses, promoting the body to produce various inflammatory mediators such as iNOS, IL-1β, and TNF-α. The continuous release of these factors can lead to chronic inflammation and immune dysfunction. Therefore, the present application investigated the anti-inflammatory activity of GCP hydrogel by co-culturing hydrogels with 1 pg mL -1LPS and mouse monocyte macrophage (RAW264.7) co-culture to detect the anti-inflammatory properties of hydrogel materials. Real-time fluorescence quantitative PCR (qRT-PCR) and Western blotting data show that GC and GCP hydrogels can significantly reduce the mRNA and protein expression levels of iNOS, IL-1β and IL-6 in RAW264.7 cells Figure 6 C-I in the above table: Figure 6 C-E in the above table is the relative mRNA expression of iNOS, IL-1β, IL-6 in macrophage Raw264.7 cells after treatment with different hydrogel dressings, Figure 6 F in the above table is the Western blotting graph of iNOS, IL-1β and IL-6 expression in each treatment group, Figure 6 G-I in the above table is the relative protein expression level of iNOS, IL-1β, IL-6, Figure 6 J in the above table is the relative mRNA expression of VEGF in HUVECs treated with different hydrogel dressings, Figure 7 K in the above table is the VEGF protein concentration in HUVECs after intervention with different hydrogel dressings determined by ELISA kit; *p<0.05, **p<0.01, ***p<0.001). GCP hydrogel showed the most obvious anti-inflammatory effect, which may be due to the anti-inflammatory properties of polyphenol compounds.

[0065] Long-term hyperglycemia leads to peripheral circulatory dysfunction, and promoting angiogenesis is beneficial to transport immune cells, sufficient nutrients and oxygen to the wound site, promoting tissue remodeling and regeneration. To evaluate the function of hydrogels in promoting angiogenesis, HUVECs were co-cultured with different hydrogels, and the RNA and protein of HUVECs were extracted for detection by qRT-PCR and ELISA kit. The results show that after GCP intervention, the mRNA and protein expression of VEGF increase Figure 7 J and K in the above table), which proves that GCP has the ability to enhance angiogenesis.

[0066] Application Example 4

[0067] Antibacterial performance test of injectable self-healing hydrogel dressing GCP:

[0068] Diabetic wound healing is often associated with bacterial infection, which can trigger an inflammatory response and may cause fluctuations in blood glucose levels in diabetic patients, and even lead to more serious complications such as cellulitis and sepsis. The antibacterial effect of GCP was evaluated by dilution plate coating method, scanning electron microscope (SEM) and SYTO9 / PI staining. Figure 7 A in the above table is the plate coating graph of Staphylococcus aureus and Escherichia coli after treatment with different hydrogel dressings, Figure 7B in FIG. 1 is a scanning electron microscope image of S. aureus and E. coli after treatment with different hydrogel dressings (scale bar: 1 pm), Figure 7 C and D in FIG. 1 are the bacterial viability of S. aureus and E. coli after treatment with different hydrogel dressings, respectively: Figure 7 A, C and D in FIG. 1 show that GCP significantly reduces the bacterial viability of S. aureus and E. coli, and SEM analysis shows that the inherent morphology of the two bacteria has changed significantly, and the original spherical and rod-shaped structure of the bacteria has been destroyed, resulting in reduced survival ability and bacterial death Figure 7 B in FIG. 1, indicating that the GCP hydrogel has strong antibacterial effect on S. aureus and E. coli. SYTO9 / PI staining further confirms this conclusion. SYTO9 labels the bacterial cell membrane with green fluorescence, while PI penetrates the damaged membrane to stain the bacterial nucleus and emits red fluorescence in dead bacteria. SYTO9 can simultaneously identify live and non-live bacteria, while PI can only specifically label dead bacteria. Figure 7 E and F in FIG. 1 are SYTO9 / PI staining images of S. aureus and E. coli after treatment with different hydrogel dressings, respectively (scale bar: 1 mm). The results of fluorescence staining show that the number of live bacteria of S. aureus Figure 7 E in FIG. 1) and E. coli Figure 8 F in FIG. 1) is significantly reduced after treatment with GCP hydrogel. Figure 8 In FIG. 1: *p < 0.05, **p < 0.01, ***p < 0.001.

[0069] Application Example 5

[0070] Injectable self-healing hydrogel dressing GCP promotes the repair of infected diabetic wounds:

[0071] Figure 8 A in FIG. 2 shows the specific animal experiment scheme. A diabetic rat model was constructed, and the diabetic rats were randomly divided into 4 groups: control group, GelMA group, GC group and GCP group. After anesthesia, a 10 mm full-thickness circular wound was made on the back of the rat. At the same time, 10 pL of S. aureus (1.0 x 10 8 CFU mL -1 ) and 100 pL of different solidified hydrogels were used to treat the wound. The day of the operation was designated as day 0. The wound images of the infected diabetic rats in each group were recorded on days 0, 2, 4, 7, 10 and 14, respectively. On days 7 and 14, the wounds of each group were taken for hematoxylin-eosin (H&E) staining and Masson trichrome staining to evaluate the epidermal thickness, wound length and collagen density. In addition, the wound sections obtained on day 7 were subjected to immunofluorescence (IF) staining to evaluate the anti-inflammatory and angiogenic ability of the hydrogel during the wound healing process. The wound images of the rats in each group are shown in Figure 8B, representative images of the wounds of each group of rats on day 0, 2, 4, 7, 10 and 14; wound healing trajectory is shown in Figure 8 C, fitting graph of the wound healing area of rats in different hydrogel dressing treatment groups on day 14 (blue: day 0; yellow: day 2; green: day 4; orange: day 7; pink: day 10; red: day 14). The results show that the wound area of all groups is gradually decreasing over time, among which the GCP hydrogel shows the most significant efficacy in promoting wound healing. From day 2 to the end of the experiment, the wound area of rats in GC and GCP groups was significantly smaller than that in the control group. In addition, compared with the GC group, the wound area of the GCP group was significantly reduced on day 7 and day 10 Figure 8 D, wound area analysis of rats in different hydrogel dressing treatment groups on day 0, 2, 4, 7, 10, 14. H&E staining and Masson's trichrome staining show that the epithelial gap and wound width gradually decrease and the collagen density increases on day 7 and day 14 after GCP treatment Figure 8 E-I: ​ E and F are respectively representative H&E staining and Masson's trichrome staining images of wound tissue of rats in different hydrogel dressing treatment groups on day 7 and day 14 (scale bar: 500 μm and 200 μm); ​ G-I are respectively the analysis of epidermal thickness, wound length, collagen density of the wounds of rats in different hydrogel dressing treatment groups; *p<0.05, **p<0.01, ***p<0.001). It is suggested that GCP hydrogel can effectively promote the re-epithelialization and collagen fiber formation of diabetic infected wounds, thereby accelerating the healing of infected wounds.

Claims

1. A method of preparing an injectable self-healing hydrogel dressing, characterized in that, The preparation method comprises the following steps: (1) mixing CuCl2·2H2O and protocatechuic aldehyde (PA) in deionized water, adjusting the pH to alkaline environment, and obtaining a first solution containing copper protocatechuic aldehyde complex (CuPA) through reaction; (2) dissolving methacrylated gelatin (GelMA) and an initiator in a buffer solution to obtain a second solution; (3) adding the first solution into the second solution and mixing uniformly, and then performing photocuring crosslinking to form an injectable self-healing hydrogel dressing.

2. The method of claim 1, wherein the injectable self-healing hydrogel dressing is prepared by, In step (1), the pH is less than 9.

0.

3. The method of claim 1, wherein the injectable self-healing hydrogel dressing is prepared by, In step (2), the preparation method of the methacrylated gelatin (GelMA) is as follows: porcine skin gelatin is added into a buffer solution and stirred to dissolve, then a methacrylic anhydride solution is added dropwise and stirred to react, a buffer solution is added to terminate the reaction to obtain a product solution, the product solution is dialyzed in deionized water using a dialysis bag with a molecular weight cut-off of 8-14 KD, the solution is filtered and freeze-dried to obtain the methacrylated gelatin (GelMA).

4. The method of claim 1, wherein the injectable self-healing hydrogel dressing is prepared by, In step (3), the concentration of Cu in the first solution is 0.05 M, the concentration of the methacrylated gelatin (GelMA) in the second solution is 5-10%, and the concentration of the initiator is 0.1-0.5%, wherein the percentages are mass percentages; the volume of the first solution added is 10-20% of the second solution.

5. The method of claim 1, wherein the injectable self-healing hydrogel dressing is prepared by, In step (3), the wavelength of the ultraviolet light source used for photocuring is 300-400 nm, and the photocuring time is usually 3-5 min.

6. An injectable self-healing hydrogel dressing obtained by the preparation method according to any one of claims 1-5.

7. Use of the injectable self-healing hydrogel dressing according to claim 6 in the preparation of a diabetic infected wound dressing.

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