Protein hybrid gel dressing of composite drug-loaded fiber and its preparation method and application

Abstract

The present invention provides a protein hybrid gel dressing with a composite drug-loaded fiber, comprising a hybrid hydrogel and esterified hyaluronic acid nanofibers uniformly dispersed within the hybrid hydrogel and loaded with a hydrophobic antibiotic. The hybrid hydrogel is formed by reacting a solution of a biocompatible polymer material with a solution of a protein material having amino and carboxyl groups. The hydrophobic antibiotic is uniformly dispersed within the gel dressing and can be sustained-released from the gel dressing to achieve long-lasting antibacterial properties. The present invention can improve the dispersibility of the hydrophobic antibiotic within the protein hybrid gel, mitigate sudden antibiotic release, and increase the structural stability of the protein hybrid gel within an infected wound environment, thereby enabling application as a wound repair material for bacterial infection.

Description

Technical Field

[0001] The invention belongs to the technical field of biomaterials and relates to a protein hybrid gel dressing of composite drug-loaded fibers and a preparation method and application thereof. Background Art

[0002] The skin is the body's first line of defense. Once a wound occurs, external bacteria immediately migrate to the wound site and rapidly multiply in the moist, warm microenvironment. Localized bacterial infection can lead to suppuration, ulceration, and delayed wound healing, and in severe cases, can even be life-threatening. Traditional dressings such as gauze and bandages lack antimicrobial properties and are prone to secondary damage during replacement. Therefore, hydrogel dressings with an extracellular matrix-like structure and readily imparted antimicrobial properties have attracted widespread attention. Based on their antimicrobial mechanism of action, antimicrobial hydrogels can be categorized as exogenous and endogenous. Exogenous antimicrobial hydrogels primarily utilize external stimuli such as light, heat, ultrasound, and magnetic fields to generate their antimicrobial properties. Endogenous antimicrobial hydrogels primarily utilize inherent physical or chemical properties of the material, such as loaded silver nanoparticles, antimicrobial peptides, and antibiotics. Local delivery of inexpensive, highly effective antibiotics can effectively prevent the development of drug-resistant bacteria. Therefore, compared to other antimicrobial approaches, antibiotic-loaded antimicrobial hydrogels offer significant advantages in their application. However, the healing of infected wounds is a complex process. If a wound gel dressing with both antibacterial and regenerative effects can be developed, it will be very beneficial to better promote the healing of infected wounds.

[0003] Protein materials such as collagen, gelatin, recombinant human protein, elastin, elastin-like peptides, fibrin, and silk fibroin are star materials in the field of skin regeneration, providing a suitable functional protein environment and accelerating wound healing. To meet the needs of infected wound repair, protein gels often need to be compounded with other antimicrobial ingredients. However, most commonly used antibiotics are hydrophobic, making them difficult to effectively disperse in hydrophilic gel systems. There is also the problem of sudden release of antibiotics, which, on the one hand, makes it difficult to ensure antimicrobial efficacy, and on the other hand, can easily lead to the development of drug-resistant bacteria. Furthermore, due to the increased secretion of matrix metalloproteinases in the infected wound environment, protein gels, which are already prone to degradation, also face the problem of excessive degradation and structural instability. Therefore, how to improve the dispersibility of hydrophobic antibiotics in protein gels, alleviate the problem of sudden release of antibiotics, increase the structural stability of protein gels in the infected wound environment, and endow existing protein gel wound dressings with better antimicrobial properties and the ability to promote tissue regeneration and wound healing, remains one of the urgent problems to be solved in this field. Summary of the Invention

[0004] The purpose of the present invention is to overcome the shortcomings of the existing technology and provide a protein hybrid gel dressing with composite drug-loaded fibers and its preparation method and application, so as to improve the dispersibility of hydrophobic antibiotics in the protein hybrid gel, alleviate the sudden release of antibiotics and increase the structural stability of the protein hybrid gel in the infected wound environment, thereby giving the existing protein gel better antibacterial properties and the ability to promote wound healing.

[0005] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is as follows:

[0006] A protein hybrid gel dressing with composite drug-loaded fibers comprises a hybrid hydrogel and esterified hyaluronic acid nanofibers uniformly dispersed in the hybrid hydrogel and loaded with hydrophobic antibiotics. The hybrid hydrogel is formed by reacting a solution of a biocompatible polymer material with a solution of a protein material having amino and carboxyl groups. The biocompatible polymer material is at least one of a thiol-modified polymer material and a catechol functional group-modified polymer material. The hydrophobic antibiotic is uniformly dispersed in the gel dressing and can be sustained-released from the gel dressing to achieve long-lasting antibacterial properties.

[0007] In the technical solution of the above-mentioned composite drug-loaded fiber protein hybrid gel dressing, the mass ratio of the esterified hyaluronic acid nanofibers loaded with hydrophobic antibiotics to the freeze-dried hybrid hydrogel in the protein hybrid gel dressing is determined according to the type of hydrophobic antibiotics actually used, the antibacterial properties and the actual application requirements. Usually, the mass ratio of the esterified hyaluronic acid nanofibers loaded with hydrophobic antibiotics to the freeze-dried hybrid hydrogel is (0.02~0.6):1.

[0008] In the technical solution of the above-mentioned composite drug-loaded fiber protein hybrid gel dressing, when the biocompatible polymer material is a polymer material modified with catechol functional groups, the solution of the biocompatible polymer material and the solution of the protein material having amino and carboxyl groups are subjected to oxidative self-crosslinking of the polymer material modified with catechol functional groups at a pH of 7 to 10, and the polymer material modified with catechol functional groups undergoes Michael addition reaction with the protein material having amino and carboxyl groups to form a hybrid hydrogel; when the biocompatible polymer material is a thiol-modified polymer material, the solution of the biocompatible polymer material and the solution of the protein material having amino and carboxyl groups are subjected to oxidative self-crosslinking of the polymer material modified with thiol functional groups at a pH of 7 to 10, and the acylation reaction occurs with the protein material having amino and carboxyl groups to form a hybrid hydrogel.

[0009] In the technical solution of the above-mentioned composite drug-loaded fiber protein hybrid gel dressing, the hybrid hydrogel is formed by reacting a solution of a biocompatible polymer material with a solution of a protein material having amino and carboxyl groups, with the mass ratio of the biocompatible polymer material to the protein material having amino and carboxyl groups being (0.1 to 1):(0.1 to 1).

[0010] In the technical solution of the above-mentioned composite drug-loaded fiber protein hybrid gel dressing, a feasible catechol functional group-modified polymer material is a dopamine-modified hyaluronic acid having a structural formula as shown in formula (I), wherein the dopamine grafting rate of the dopamine-modified hyaluronic acid is 1% to 70%, and the dopamine grafting rate is preferably 5% to 10%; a feasible thiol-modified polymer material is a thiol-modified hyaluronic acid having a structural formula as shown in formula (II), wherein the thiol grafting rate of the thiol-modified hyaluronic acid is 1% to 70%, and the thiol grafting rate is preferably 5% to 10%;

[0011]

[0012] In the technical solution of the above-mentioned composite drug-loaded fiber protein hybrid gel dressing, the protein material having amino and carboxyl groups includes but is not limited to at least one of collagen, gelatin, recombinant human protein, and silk fibroin. For example, a feasible collagen may be type I collagen.

[0013] In the technical solution of the above-mentioned composite drug-loaded fiber protein hybrid gel dressing, the esterified hyaluronic acid nanofiber is a benzyl-modified hyaluronic acid nanofiber. The structure of the benzyl-modified hyaluronic acid is shown in formula (III). In the benzyl-modified hyaluronic acid, the benzyl grafting rate is 10% to 90%.

[0014]

[0015] In the technical solution of the above-mentioned composite drug-loaded fiber protein hybrid gel dressing, the diameter of the esterified hyaluronic acid nanofibers loaded with hydrophobic antibiotics is at the nanometer level. For example, the diameter of the esterified hyaluronic acid nanofibers loaded with hydrophobic antibiotics can be 10 to 800 nm; the length of the esterified hyaluronic acid nanofibers loaded with hydrophobic antibiotics is usually at the micrometer level.

[0016] In the technical solution of the above-mentioned composite drug-loaded fiber protein hybrid gel dressing, the hydrophobic antibiotic-loaded esterified hyaluronic acid nanofibers are bonded to the esterified hyaluronic acid nanofibers via intermolecular forces. For example, when the loaded hydrophobic antibiotic is a tetracycline antibiotic, the tetracycline antibiotic is bonded to the esterified hyaluronic acid nanofibers via intermolecular forces including π-π stacking.

[0017] In the technical solution of the above-mentioned composite drug-loaded fiber protein hybrid gel dressing, the preparation method of the esterified hyaluronic acid nanofibers loaded with hydrophobic antibiotics is as follows: the esterified hyaluronic acid nanofibers and the hydrophobic antibiotics are dissolved in a hexafluoroisopropanol solution to form an electrospinning solution, and then the nanofiber membrane is obtained by electrospinning, and the nanofiber membrane is dried. The dried nanofiber membrane is placed in water, stirred and dispersed, and dried to obtain the product.

[0018] In the technical solution of the above-mentioned composite drug-loaded fiber protein hybrid gel dressing, the hydrophobic antibiotic is an antibiotic that can be bound to the esterified hyaluronic acid nanofibers through intermolecular forces. For example, it can be rifampicin, roxithromycin, or tetracycline antibiotics. Common tetracycline antibiotics include but are not limited to oxytetracycline, chlortetracycline, tetracycline, doxycycline, minocycline, and methacycline.

[0019] In the technical solution of the above-mentioned composite drug-loaded fiber protein hybrid gel dressing, the porosity of the gel dressing after freeze-drying is in the range of 35% to 75%.

[0020] The present invention also provides a method for preparing the above-mentioned composite drug-loaded fiber protein hybrid gel dressing, comprising the following steps:

[0021] (1) dissolving a biocompatible polymer material in water to form an aqueous solution of the biocompatible polymer material, and uniformly dispersing the esterified hyaluronic acid nanofibers loaded with a hydrophobic antibiotic in the aqueous solution of the biocompatible polymer material to obtain a dispersion; in the dispersion, the concentration of the biocompatible polymer material is 2 wt% to 50 wt%, preferably 2 wt% to 20 wt%, and the concentration of the esterified hyaluronic acid nanofibers loaded with a hydrophobic antibiotic is 0.1 wt% to 50 wt%, preferably 0.1 wt% to 10 wt%;

[0022] (2) dissolving a protein material having amino and carboxyl groups in a 0.2-1 mol / L aqueous acetic acid solution to form a solution of the protein material having amino and carboxyl groups with a concentration of 2 wt% to 50 wt%, preferably 2 wt% to 10 wt%;

[0023] (3) The dispersion obtained in step (1) is fully mixed with the solution of the protein material having amino and carboxyl groups obtained in step (2), the pH value of the resulting mixture is adjusted to 7-10, and the mixture is allowed to stand at 20-40° C. until the mixture is converted into a gel state, thereby obtaining a protein hybrid gel dressing of composite drug-loaded fibers.

[0024] The present invention has demonstrated through a series of experiments that the protein hybrid gel dressing of the composite drug-loaded fiber provided by the present invention has the following properties suitable for use as a dressing for repairing bacterially infected wounds:

[0025] (1) The present invention has experimentally confirmed that by loading hydrophobic antibiotics on esterified hyaluronic acid nanofibers and then integrating the esterified hyaluronic acid nanofibers loaded with hydrophobic antibiotics into hybrid hydrogels, not only can the hydrophobic antibiotics be uniformly and effectively dispersed in the hydrophilic composite hydrogel, but also the hydrophobic antibiotics can be sustained-released from the protein hybrid gel dressing, thereby solving the problems of agglomeration and ineffective dispersion of hydrophobic antibiotics in the prior art of directly dispersing the hydrophobic antibiotics in the hydrogel network, as well as the problem of sudden release of the hydrophobic antibiotics.

[0026] (2) The present invention has been verified through experiments that the protein hybrid gel dressing of the composite drug-loaded fiber provided by the present invention has good water absorption capacity, and the equilibrium swelling percentage is about 532%, which is conducive to the rapid absorption of blood, exudate, etc., and is conducive to maintaining a stable shape at the wound site; at the same time, the protein hybrid gel dressing of the composite drug-loaded fiber has excellent tissue adhesion ability, which is conducive to stable fixation at the wound site; in addition, the protein hybrid gel dressing of the composite drug-loaded fiber also has excellent injectability, which is conducive to easy injection into irregular wounds in actual application.

[0027] (3) The present invention has confirmed through experiments that when the antibiotics loaded by the protein hybrid gel dressing of the composite drug-loaded fiber provided by the present invention are tetracycline antibiotics, the protein hybrid gel dressing has a certain resistance to collagen degradation caused by collagenase, which is beneficial to maintaining the structural stability of the protein hybrid gel and increasing the structural stability of the protein hybrid gel dressing in the infected wound environment, thereby helping to better exert the antibacterial and wound healing effects.

[0028] (4) The present invention has demonstrated through in vitro cell experiments that, when the content of hydrophobic antibiotics is appropriate, the composite drug-loaded fiber protein hybrid gel dressing provided by the present invention has excellent cell compatibility and can promote cell proliferation and migration in vitro; and when the content of hydrophobic antibiotics is appropriate, the composite drug-loaded fiber protein hybrid gel dressing provided by the present invention has good angiogenesis-promoting ability. These are all beneficial for repairing damaged tissues.

[0029] (5) The present invention has confirmed through in vitro antibacterial experiments that when the content of hydrophobic antibiotics is appropriate, the protein hybrid gel dressing of the composite drug-loaded fiber provided by the present invention exhibits good antibacterial properties against Staphylococcus aureus and Escherichia coli.

[0030] (6) The present invention has been verified through animal skin repair experiments: the protein hybrid gel dressing of the composite drug-loaded fiber provided by the present invention can change the wound infection environment, create a sterile and suitable protein environment to accelerate the healing of infected wounds. After 12 days of repair, the protein hybrid gel dressing of the composite drug-loaded fiber of the present invention can achieve complete wound healing. Under the same conditions, 27.83±9.12% and 20.54%±4.64% of the wounds in the HAD / C group and the control group remained open, respectively. After 12 days of repair, the protein hybrid gel dressing of the composite drug-loaded fiber of the present invention can achieve complete wound healing. The experimental group repaired with the gel dressing had more hair follicles and only a small number of neutrophils, and the wound healed the fastest, while the HAD / C group and the control group experienced severe inflammatory reactions; the protein hybrid gel dressing of the composite drug-loaded fiber provided by the present invention can promote the deposition of collagen at the wound site and also has the potential to enhance angiogenesis, which is beneficial to strengthening the repair effect; at the same time, the protein hybrid gel dressing of the composite drug-loaded fiber provided by the present invention can promote the expression of TGF-β and reduce the expression of TNF-α, which is beneficial to reducing the inflammatory period and promoting wound healing.

[0031] Based on the above experimental results, the protein hybrid gel dressing of the composite drug-loaded fiber provided by the present invention can be used as a wound repair material. For example, the above-mentioned protein hybrid gel dressing of the composite drug-loaded fiber can be used in the preparation of anti-bacterial infection wound repair materials. During application, depending on the difference in wound morphology, the formed protein hybrid gel dressing can be directly applied to the wound, or the precursor of the protein hybrid gel dressing before forming (that is, the precursor formed by fully mixing the dispersion with the solution of the protein material having amino and carboxyl groups and adjusting the pH value to 7 to 10) can be injected into the wound for in situ transaction. The former application method is more suitable for skin wounds with regular or relatively regular morphology, and the latter application method is not only suitable for regular skin wounds, but also for irregular wounds.

[0032] Compared with the prior art, the technical solution provided by the present invention produces the following beneficial technical effects:

[0033] 1. The present invention provides a protein hybrid gel dressing of a composite drug-loaded fiber, the protein hybrid gel dressing is composed of a hybrid hydrogel and an esterified hyaluronic acid nanofiber uniformly dispersed in the hybrid hydrogel for loading hydrophobic antibiotics; the hybrid hydrogel is a hydrogel formed by reacting a solution of a biocompatible polymer material with a solution of a protein material having amino and carboxyl groups; the hydrophobic antibiotics are uniformly dispersed in the gel dressing, and the hydrophobic antibiotics can be sustained-released from the gel dressing to achieve long-lasting antibacterial. The present invention loads hydrophobic antibiotics by esterified hyaluronic acid nanofibers, and then integrates the esterified hyaluronic acid nanofibers loaded with hydrophobic antibiotics into the hybrid hydrogel, so that the hydrophobic antibiotics can be uniformly loaded in the hydrophilic hydrogel, and antibiotic sustained-release is achieved to achieve the purpose of long-lasting antibacterial. The problem of agglomeration and ineffective dispersion of the hydrophobic antibiotics present in the hydrogel network directly dispersed in the prior art, as well as the problem of sudden release of hydrophobic antibiotics, is solved.

[0034] 2. Inspired by the structure of the skin, the present invention utilizes cross-linked biocompatible polymer materials and protein materials with amino and carboxyl groups to construct a hybrid hydrogel with a three-dimensional network structure. This stabilizes the structure and slows the degradation of the protein material. Furthermore, when tetracycline is loaded as an antibiotic, the inhibitory effect of tetracycline on collagenase imparts, to a certain extent, the ability of the protein hybrid gel dressing to resist collagen degradation caused by collagenase, which also contributes to the structural stability of the hybrid hydrogel network. These two aspects work together to increase the structural stability of the protein hybrid gel in the infected wound environment, thereby continuously maintaining the protein environment required for wound repair and better promoting wound healing.

[0035] 3. The protein hybrid gel dressing of the composite drug-loaded fiber provided by the present invention can be locally administered through the skin, for example, it can be injected into the wound in situ, that is, the in situ sustained release of the drug can be achieved. Compared with systemic administration, it reduces the toxic and side effects on tissues and organs after systemic administration, and the local high concentration of drugs can better inhibit the production of drug-resistant bacteria.

[0036] 4. The present invention has been confirmed through in vitro cell experiments: when the content of hydrophobic antibiotics is appropriate, the protein hybrid gel dressing provided by the present invention has excellent cell compatibility and can promote cell proliferation and migration in vitro; when the content of hydrophobic antibiotics is appropriate, the protein hybrid gel dressing provided by the present invention has good ability to promote angiogenesis. The present invention has been confirmed through in vitro antibacterial experiments that when the content of hydrophobic antibiotics is appropriate, the protein hybrid gel dressing provided by the present invention exhibits good antibacterial properties against Staphylococcus aureus and Escherichia coli. The present invention has been confirmed through a wound model of full-thickness skin defects in mice infected with Staphylococcus aureus that the protein hybrid gel dressing provided by the present invention can change the wound infection environment, create a sterile and suitable protein environment to accelerate the healing of infected wounds, reduce the inflammatory response during the repair process, and can promote angiogenesis and accelerate the healing of skin wounds faster. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Schematic diagram of the reaction process for preparing HA-Bn in Example 1.

[0038] Figure 2 is the H NMR spectrum of HA-Bn prepared in Example 1 and the raw material hyaluronic acid used, Figure 2 The curves in the figure represent HA-Bn and raw material hyaluronic acid from top to bottom.

[0039] Figure 3 These are SEM images of HA-Bn / T prepared in Example 1 at different magnifications.

[0040] Figure 4 This is the diameter distribution diagram of HA-Bn / T prepared in Example 1.

[0041] Figure 5 These are laser confocal fluorescence images of tetracycline (TC), DAPI-stained HA-Bn / T, and DAPI-stained HAB.

[0042] Figure 6 1 is a Fourier transform infrared image of HA-Bn / T prepared in Example 1 and HAB prepared in Comparative Example 1.

[0043] Figure 7 Figures (a) and (b) are the XPS images of HAB and HA-Bn / T, respectively.

[0044] Figure 8 Schematic diagram of the reaction process for preparing HAD in Example 4.

[0045] Figure 9 is the H NMR spectrum of HAD prepared in Example 4 and the raw material hyaluronic acid used, Figure 9 The curves in the figure represent HAD and raw material hyaluronic acid from top to bottom.

[0046] Figure 10 Fluorescence images of drug distribution in HAD / C / HBT3 and HAD / C / T3 hydrogels.

[0047] Figure 11 These are the drug release curves of HAD / C / HBT3 and HAD / C / T3 hydrogels.

[0048] Figure 12 This is a diagram showing the proliferation of L929 cells in HAD / C / HBT3 and HAD / C / T3 hydrogels.

[0049] Figure 13 These are laser confocal microscopy images of live and dead cells of L929 cells after culture in HAD / C / HBT3 and HAD / C / T3 hydrogels for different time periods.

[0050] Figure 14 are scanning electron micrographs of freeze-dried HAD / C hydrogel, HAD / C / HBT1, HAD / C / HBT3, and HAD / C / HBT5.

[0051] Figure 15 These are the porosity test results of freeze-dried HAD / C hydrogel, HAD / C / HBT1, HAD / C / HBT3, and HAD / C / HBT5.

[0052] Figure 16 The equilibrium swelling histograms of freeze-dried HAD / C hydrogels, HAD / C / HBT1, HAD / C / HBT3, and HAD / C / HBT5 are shown.

[0053] Figure 17 The degradation of HAD / C hydrogel, HAD / C / HBT1, HAD / C / HBT3 and HAD / C / HBT5 in collagenase solution.

[0054] Figure 18 These are photos of the adhesion of HAD / C / HBT3 hydrogel on pig skin.

[0055] Figure 19 These are the viscosity change curves of HAD / C hydrogel, HAD / C / HBT1, HAD / C / HBT3 and HAD / C / HBT5 as a function of shear rate and photos of the injectability performance test.

[0056] Figure 20 These are laser confocal microscopy images of live and dead cells of L929 cells after culture in HAD / C hydrogel, HAD / C / HBT1, HAD / C / HBT3 and HAD / C / HBT5 for different periods of time.

[0057] Figure 21This is a graph showing the cell proliferation of L929 cells in HAD / C / HBT1, HAD / C / HBT3, HAD / C / HBT5, and HAD / C hydrogels.

[0058] Figure 22 These are the results of inverted microscope observation of cell scratch experiments on HAD / C / HBT1, HAD / C / HBT3, HAD / C / HBT5, and HAD / C hydrogels.

[0059] Figure 23 Calculation results of wound healing rates of cell scratch experiments on HAD / C / HBT1, HAD / C / HBT3, HAD / C / HBT5 and HAD / C hydrogels.

[0060] Figure 24 This is a diagram of the angiogenesis of HUVECs cells in HAD / C / HBT1, HAD / C / HBT3, HAD / C / HBT5 and HAD / C hydrogels.

[0061] Figure 25 This is the result graph of the total vascular length of HUVECs cells in HAD / C / HBT1, HAD / C / HBT3, HAD / C / HBT5 and HAD / C hydrogels.

[0062] Figure 26 These are the inhibition zone test results of HAD / C / HBT1, HAD / C / HBT3, HAD / C / HBT5 and HAD / C hydrogels against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). Figure (a) is an optical image of the inhibition zone, and Figures (b) and (c) are the inhibition zone diameter analysis results. The bars in the four groups of histograms in the two figures represent HAD / C / HBT1, HAD / C / HBT3, and HAD / C / HBT5 from left to right, respectively.

[0063] Figure 27 Live-dead staining images of the distribution of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) on HAD / C / HBT1, HAD / C / HBT3, HAD / C / HBT5, and HAD / C hydrogels.

[0064] Figure 28 SEM images of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) cell morphology on HAD / C / HBT1, HAD / C / HBT3, HAD / C / HBT5 and HAD / C hydrogels.

[0065] Figure 29This is the gross view of the wounds in the full-thickness skin defect experiment on mice infected with Staphylococcus aureus in the control group, HAD / C hydrogel group, and HAD / C / HBT3 group.

[0066] Figure 30 This is the wound healing rate in the full-thickness skin defect experiment of mice infected with Staphylococcus aureus in the control group, HAD / C hydrogel group, and HAD / C / HBT3 group. The bars in the three groups of bar graphs in the figure represent the control group, HAD / C hydrogel group, and HAD / C / HBT3 group from left to right, respectively.

[0067] Figure 31 These are H&E-stained images of the control group, HAD / C hydrogel group, and HAD / C / HBT3 group.

[0068] Figure 32 Masson staining images of the control group, HAD / C hydrogel group, and HAD / C / HBT3 group.

[0069] Figure 33 Figures (A) and (B) are the semi-quantitative analysis results of proprotein deposition in wound tissues of the control group, HAD / C hydrogel group, and HAD / C / HBT3 group at 6 days and 12 days, respectively.

[0070] Figure 34 Figures (a) and (b) are the semi-quantitative analysis results of CD31 staining images and the number of blood vessels in the wound tissues of the control group, HAD / C hydrogel group, and HAD / C / HBT3 group. The bars in the two groups of histograms in Figure (b) represent the control group, HAD / C hydrogel group, and HAD / C / HBT3 group from left to right, respectively.

[0071] Figure 35 These are the staining images of TGF-β and TNF-α in wound tissues of the control group, HAD / C hydrogel group, and HAD / C / HBT3 group. DETAILED DESCRIPTION

[0072] The following examples further illustrate the composite drug-loaded fiber protein hybrid gel dressing provided by the present invention, its preparation method, and its application. It is important to note that the following examples are intended only to further illustrate the present invention and are not to be construed as limiting the scope of protection of the present invention. Non-essential improvements and adjustments made by persons skilled in the art based on the above disclosure and implemented in accordance with the present invention remain within the scope of protection of the present invention.

[0073] Example 1

[0074] In this embodiment, controlled-release drug-loaded esterified hyaluronic acid nanofibers were prepared, specifically tetracycline-loaded benzyl-modified hyaluronic acid nanofibers. The reaction process is shown in FIG. Figure 1, the steps are as follows:

[0075] (1) Obtaining activated ion exchange resin

[0076] Weigh the ion exchange resin, disperse it in ultrapure water, and allow it to settle completely. Then, soak the ion exchange resin thoroughly in 2 mol / L hydrochloric acid. Finally, wash the hydrochloric acid-soaked ion exchange resin with ultrapure water to obtain an activated ion exchange resin.

[0077] (2) Preparation of benzyl-modified hyaluronic acid (HA-Bn)

[0078] The reaction process diagram for preparing benzyl-modified hyaluronic acid is shown in FIG. Figure 1 As shown. Hyaluronic acid with an Mn of 960 kDa was weighed and dissolved in ultrapure water to form a 10 mg / mL hyaluronic acid solution. To this hyaluronic acid solution was added an activated ion exchange resin (3 times the mass of the hyaluronic acid) and allowed to react for 12 hours. After the reaction was completed, the mixture was centrifuged and the supernatant was collected. Tetrabutylammonium hydroxide (TBA) (2 times the mass of the hyaluronic acid) was added to the supernatant, allowed to react at room temperature for 2 hours, and then freeze-dried. The freeze-dried product was weighed and dimethyl sulfoxide (DMSO) was added to the freeze-dried product at a ratio of 1 g freeze-dried product to 100 mL DMSO. The reaction was continued at 30°C for 12 hours. After the reaction was completed, benzyl bromide was added to the resulting reaction solution at a mass ratio of 1:0.55 freeze-dried product to benzyl bromide. The reaction was continued at 30°C for 12 hours. The resulting reaction solution was added dropwise to ethyl acetate and allowed to settle to obtain a white precipitate. The white precipitate was washed three times in ethyl acetate and the resulting product was dried in vacuo to obtain HA-Bn.

[0079] HA-Bn was dissolved in deuterated dimethyl sulfoxide and tested at 30°C. 1 H NMR spectra, such as Figure 2 As shown, compared to the raw material hyaluronic acid (HA), HA-Bn exhibits a characteristic benzyl peak at 7.4 ppm, indicating that HA-Bn was successfully prepared. Based on the ratio of the hyaluronic acid carboxyl residue peak at 12.9 ppm to the HA-Bn benzyl peak at 7.4 ppm, the degree of benzyl substitution in HA-Bn can be calculated, which in this example is 71.7%.

[0080] (3) Preparation of benzyl-modified hyaluronic acid nanofibers loaded with tetracycline (HA-Bn / T)

[0081] HA-Bn was dissolved in hexafluoroisopropanol to obtain a 100 mg / mL HA-Bn solution. Tetracycline was added to the HA-Bn solution and stirred in the dark until the tetracycline dissolved to obtain a spinning solution. A nanofiber membrane was electrospun under the following spinning conditions: a needle-to-receiver distance of 10 cm, a flow rate of 2 mL / h, a voltage of 19 kV, and an 18G blunt needle. The resulting nanofiber membrane was vacuum dried, broken into nanofibers, and freeze-dried to obtain HA-Bn / T.

[0082] The scanning electron microscopy image of HA-Bn / T is as follows Figure 3 As shown by Figure 3 It can be seen that HA-Bn / T was successfully prepared. The diameter distribution of HA-Bn / T was analyzed using Image J software, as shown in Figure 2. Figure 4 As shown, the diameter of HA-Bn / T is distributed between 20 and 160 nm.

[0083] Comparative Example 1

[0084] In this comparative example, blank benzyl-modified hyaluronic acid nanofibers (HAB) without tetracycline loading were prepared. The operation of this comparative example was basically the same as that of Example 1, except that the spinning solution in step (3) did not contain tetracycline.

[0085] Example 2

[0086] In this example, the drug loading of HA-Bn / T was verified.

[0087] The HA-Bn / T and HAB prepared in Example 1 and Comparative Example 1 were stained with DAPI dye. The raw material tetracycline (TC), DAPI-stained HA-Bn / T and DAPI-stained HAB were observed using a fluorescence microscope. The results were as follows: Figure 5 As shown. Figure 5 It can be seen that compared with HAB, in HA-Bn / T, tetracycline with fluorescent properties is evenly loaded on the nanofibers.

[0088] Example 3

[0089] In this example, the interaction between tetracycline (TC) and HA-Bn in HA-Bn / T was verified.

[0090] The HA-Bn / T prepared in Example 1 and the HAB prepared in Comparative Example 1 were tested by Fourier transform infrared spectroscopy (FT-IR) and X-ray photoelectron spectroscopy (XPS). The results were as follows: Figure 6 、 7 shown.

[0091] Since both TC and HA-Bn have aromatic structures, their π-π stacking interactions were studied using Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS). Comparing the FTIR spectra of HAB and HA-Bn / T, it can be seen that after the addition of TC, the 1554 cm -1 The characteristic peak at red shifted to 1605 cm -1 , which suggests that π-π stacking interactions may occur between TC and HA-Bn. Figure 7 From the XPS C1s spectrum shown, it can be seen that the binding energy of C=O in HA-Bn / T shifts from 288.91 eV to 289.10 eV compared with that in HAB, which further confirms the existence of π-π stacking interaction between TC and HA-Bn.

[0092] Example 4

[0093] In this example, dopamine-modified hyaluronic acid (HAD) was prepared. The reaction process is shown in FIG. Figure 8 , the steps are as follows:

[0094] Hyaluronic acid with a Mw of 37 kDa was weighed and dissolved in ultrapure water to obtain a hyaluronic acid solution with a concentration of 2 mg / mL. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide were then added, and the reaction was stirred at room temperature for 2 hours. The pH of the resulting reaction solution was adjusted to 4.75-5. Dopamine hydrochloride solution (prepared by dissolving dopamine hydrochloride in ultrapure water) was then added to the pH-adjusted reaction solution, and the reaction was continued for 24 hours. During the reaction, the pH of the reaction solution was maintained at 4.75-5. After completion of the reaction, the resulting reaction solution was dialyzed in ultrapure water for 3 days. The dialyzed product was freeze-dried to obtain dopamine-modified hyaluronic acid (HAD).

[0095] During the above reaction process, the mass ratio of hyaluronic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide and dopamine hydrochloride was controlled to be 400:960:230:56.

[0096] HA-Bn was dissolved in heavy water and tested at 30°C. 1 H NMR spectra, such as Figure 9 Compared with the raw material hyaluronic acid (HA), the three new peaks appearing at 6.8 ppm in HAD indicate that HAD was successfully prepared. Based on the ratio of the peak integrated area of ​​HAD at 6.8 ppm to the methyl proton peak area of ​​HA at 1.9 ppm, the grafting rate of dopamine in HAD was calculated to be 5.3%.

[0097] Example 5

[0098] In this example, a series of protein hybrid gel dressings containing composite drug-loaded fibers were prepared in the following steps:

[0099] (1) HAD prepared in Example 4 was weighed and dissolved in ultrapure water. HA-Bn / T prepared in Example 1 was then added and thoroughly mixed to obtain a reaction solution. Specifically, three reaction solutions were prepared in this step, designated as reaction solutions A1, A3, and A5. The concentration of HAD was 2 wt %, and the concentration of HA-Bn / T was 0.1 wt %, 0.3 wt %, and 0.5 wt %, respectively.

[0100] (2) Weigh type I collagen (COL I), dissolve COL I in 0.5 mol / L acetic acid solution to obtain reaction solution B. The concentration of COL I in reaction solution B is 2 wt%.

[0101] (3) Reaction solutions A1, A3, and A5 were mixed with reaction solution B in equal volumes, the pH value was adjusted to 7.4 with NaOH solution, and the mixture was allowed to stand at 37°C until it gelled, thereby obtaining a series of protein hybrid gel dressings containing composite drug-loaded fibers. The protein hybrid gel dressings obtained by reacting reaction solutions A1, A3, and A5 with reaction solution B were designated as HAD / C / HBT1, HAD / C / HBT3, and HAD / C / HBT5, respectively.

[0102] Comparative Example 2

[0103] In this comparative example, a fiber-free protein hybrid gel dressing was prepared in the following steps:

[0104] (1) The HAD prepared in Example 2 was weighed and dissolved in ultrapure water to obtain a reaction solution C. The concentration of HAD in the reaction solution C was 2 wt %.

[0105] (2) Weigh type I collagen (COL I), dissolve COL I in 0.5 mol / L acetic acid solution to obtain reaction solution B. The concentration of COL I in reaction solution B is 2 wt%.

[0106] (3) Mix equal volumes of reaction solution C and reaction solution B, adjust the pH to 7.4 with NaOH solution, and let it stand at 37°C until it gels to obtain a fiber-free protein hybrid gel dressing, which is recorded as HAD / C hydrogel.

[0107] Comparative Example 3

[0108] In this comparative example, a fiber-free drug-loaded protein hybrid gel dressing was prepared in the following steps:

[0109] (1) The HAD prepared in Example 2 was weighed and dissolved in ultrapure water to obtain a reaction solution C. The concentration of HAD in the reaction solution C was 2 wt %.

[0110] (2) Weigh type I collagen (COL I), dissolve COL I in 0.5 mol / L acetic acid solution to obtain reaction solution B. The concentration of COL I in reaction solution B is 2 wt%.

[0111] (3) Mix equal volumes of reaction solution C and reaction solution B, add tetracycline in an amount equal to that of the HAD / C / HBT3 hydrogel prepared in Example 5, stir and disperse thoroughly, adjust the pH to 7.4 with NaOH solution, and let stand at 37°C until gelled, thereby obtaining a fiber-free drug-loaded protein hybrid gel dressing, designated as HAD / C / HBT3 hydrogel.

[0112] Example 6

[0113] In this example, the drug distribution, drug release, and cytocompatibility of the HAD / C / HBT3 and HAD / C / T3 hydrogels in Example 5 and Comparative Example 3 were characterized to verify that HA-Bn / T can uniformly disperse the drug in the hydrophilic network, has the characteristics of slow drug release, and avoids cytotoxicity.

[0114] (1) The distribution of drugs in HAD / C / HBT3 and HAD / C / T3 hydrogels was observed by fluorescence microscopy. Figure 10 shown.

[0115] (2) The in vitro cytotoxicity of HAD / C / HBT3 and HAD / C / T3 hydrogels was evaluated by cell counting kit-8 (CCK8) assay and fluorescein diacetate (FDA) / propidium iodide (PI) staining. 3 L929 cells were placed in a 24-well plate and 1 mL of complete medium was added for 1, 2, and 3 days. Cell viability was assessed by measuring the absorbance at 450 nm using a CCK counting kit and a microplate reader. The control group was used without the addition of hydrogel. Figure 12 In addition, the cells were stained with FDA / PI staining reagent and then observed under a fluorescence microscope. FDA can mark live cells in green and PI can mark dead cells in red. The results are shown in Figure 13 shown.

[0116] (3) Dissolve collagenase (COLase) in ultrapure water, add activator Cacl2, and stir thoroughly to obtain a collagenase solution. In the collagenase solution, the concentration of COLase is 20U / mL and the concentration of activator is 5mmol / L. Soak HAD / C / HBT3 and HAD / C / T3 hydrogels in equal amounts of 20U / mL collagenase solution. Place in a constant temperature shaker at 37°C and shake at a speed of 80r / min. Samples are taken at regular intervals. The wavelength at 345nm is detected by UV-visible spectrophotometer. The amount of tetracycline released at each sampling time point is compared with the tetracycline standard curve. The drug release curve over time is calculated and plotted.

[0117] Due to the fluorescent properties of tetracycline, the dispersion of the drug can be clearly observed by fluorescence microscopy. Compared with HAD / C / HBT3 hydrogel, the dispersion of tetracycline in HAD / C / T3 hydrogel is poor, and obvious aggregation occurs, such as Figure 10 As shown in Figure 3, this can be attributed to the repulsion between the hydrophobic drug and the hydrophilic matrix. HA-Bn / T can effectively load the drug and distribute it evenly in the HAD / C / HBT3 hydrogel.

[0118] Drug release profiles such as Figure 11 As shown in the figure, compared with HAD / C / HBT3 hydrogel, HAD / C / T3 hydrogel showed the characteristics of sudden drug release, with a drug release rate of about 50% within 10 hours. This is attributed to the free dispersion of small molecule hydrophobic drug tetracycline in the water-filled pores of collagen-based gel, which will lead to rapid drug diffusion. Using HA-Bn to load the drug tetracycline to form HA-Bn / T can achieve sustained release of tetracycline from HAD / C / HBT3. Figure 12 It can be seen that HAD / C / T3 hydrogel inhibits cell proliferation and spread and has certain cytotoxicity. This result shows that the slow release of drugs through HA-Bn / T avoids the cytotoxicity caused by the burst release of drugs. The cell live-death data showed the same trend (see Figure 13 ), compared with the control group, HAD / C / HBT3 hydrogel promoted cell proliferation.

[0119] Example 7

[0120] In this example, the internal structures of HAD / C / HBT1, HAD / C / HBT3, HAD / C / HBT5, and HAD / C hydrogels prepared in Example 5 and Comparative Example 2 were characterized.

[0121] HAD / C hydrogel, HAD / C / HBT1, HAD / C / HBT3 and HAD / C / HBT5 were freeze-dried, and the freeze-dried sponges were gold-sprayed. The internal structure of the sponges was then observed using a scanning electron microscope (SEM), and the porosity of the hydrogels was analyzed using Image J software. The results are shown in Figure 2. Figures 14-15 shown.

[0122] Depend on Figure 14 It can be seen that the interior of the freeze-dried HAD / C hydrogel, HAD / C / HBT1, HAD / C / HBT3 and HAD / C / HBT5 is an interconnected porous structure. This porous structure and pore size are conducive to absorbing and accommodating a large amount of water, maintaining a moist environment at the wound, and providing a good material structure foundation for the delivery of nutrients. The freeze-dried HAD / C hydrogel, HAD / C / HBT1, HAD / C / HBT3 and HAD / C / HBT5 showed similar porosity, about 58%, as shown in Figure 2. Figure 15 .

[0123] Example 8

[0124] In this example, the swelling behaviors of HAD / C / HBT1, HAD / C / HBT3, HAD / C / HBT5 and HAD / C hydrogels prepared in Example 5 and Comparative Example 2 were investigated.

[0125] The same volume of HAD / C / HBT1, HAD / C / HBT3, HAD / C / HBT5 and HAD / C hydrogels were prepared and weighed after freeze-drying, respectively recorded as W1. The freeze-dried hydrogels were then immersed in PBS solution, placed in a constant temperature shaker at 37°C for 24 hours, and weighed, respectively recorded as W2. The equilibrium swelling ratio of the material was calculated using the following formula to reflect the swelling behavior of freeze-dried HAD / C / HBT1, HAD / C / HBT3, HAD / C / HBT5 and HAD / C hydrogels. The results are shown in the figure. Figure 16 shown.

[0126] Equilibrium swelling ratio (%) = (W2-W1) / W1×100%

[0127] Depend on Figure 16 It can be seen that the freeze-dried HAD / C, HAD / C / HBT1, HAD / C / HBT3 and HAD / C / HBT5 hydrogels showed similar solubilization behavior in PBS solution, with an equilibrium swelling percentage of approximately 532%.

[0128] Example 9

[0129] In this example, the in vitro degradation behaviors of HAD / C / HBT1, HAD / C / HBT3, HAD / C / HBT5 and HAD / C hydrogels prepared in Example 5 and Comparative Example 2 were investigated.

[0130] Collagenase (COLase) was dissolved in ultrapure water, and activator CaCl2 was added and stirred thoroughly to obtain a collagenase solution. In the collagenase solution, the concentration of COLase was 50 U / mL and the concentration of activator was 5 mmol / L. HAD / C / HBT1, HAD / C / HBT3, HAD / C / HBT5 and HAD / C hydrogels were freeze-dried and weighed, respectively. i , immersed in collagenase solution, placed in a constant temperature shaker at 37 ° C and shaken at a speed of 80 r / min. After being placed in the constant temperature shaker for 24 hours, the materials were taken out, washed with deionized water, freeze-dried, and weighed, and recorded as W respectively. t The following formula is used to calculate the weight loss percentage of the material to reflect the degradation characteristics of the above samples.

[0131] Weight loss percentage (%) = (W i -W t ) / W i ×100

[0132] The test results of this embodiment are as follows Figure 17 As shown, the degradation behaviors of HAD / C and HAD / C / HBT1 were similar. With increasing HA-Bn / T content, both HAD / C / HBT3 and HAD / C / HBT5 hydrogels exhibited improved resistance to collagenase degradation. This is because TC can disrupt the structure of collagenase and inactivate it, helping to maintain the collagen fiber structure. These results indicate that HAD / C / HBT3 and HAD / C / HBT5 hydrogels can slow collagen degradation in a collagenase-induced environment.

[0133] Example 10

[0134] In this example, the in vitro adhesion behavior of HAD / C / HBT3 hydrogel was investigated.

[0135] The reaction solution A3 and the reaction solution B in Example 5 were mixed in equal volumes, the pH value was adjusted to 7.4 with NaOH solution, and the mixture was injected onto the surface of pigskin to form an in situ gel. The pigskin was twisted and folded continuously to observe the adhesion of the formed hydrogel (HAD / C / HBT3) on the pigskin. The results are shown in FIG. Figure 18As shown in Figure 3, after in situ gelation in porcine epidermis, the hydrogel remained intact and adhered tightly to the tissue when the pig skin was bent and twisted several times. This is primarily due to the high affinity of the catechol groups of HAD for amines, thiols, and imidazoles in tissue peptides and proteins, resulting in the excellent tissue adhesion ability of the HAD / C / HBT3 hydrogel.

[0136] Example 11

[0137] In this example, the injectability of HAD / C / HBT1, HAD / C / HBT3, HAD / C / HBT5 and HAD / C hydrogels prepared in Example 3 and Comparative Example 1 was investigated.

[0138] (1) The shear thinning properties of HAD / C / HBT1, HAD / C / HBT3, HAD / C / HBT5, and HAD / C hydrogels were tested using a rheometer. The viscosity of all hydrogels increased and then decreased with increasing shear rate, as shown in Figure 2. Figure 19 As shown, this indicates that the hydrogel has shear thinning ability, making it easier to inject into irregular wounds in practical applications.

[0139] (2) By injecting HAD / C / HBT1, HAD / C / HBT3, HAD / C / HBT5, and HAD / C hydrogels with a syringe, “SCU” patterns can be drawn, e.g. Figure 19 shown.

[0140] Example 12

[0141] In this example, the effects of HAD / C / HBT1, HAD / C / HBT3, HAD / C / HBT5 and HAD / C hydrogels in promoting the growth of mouse fibroblasts in vitro were investigated.

[0142] The in vitro cytotoxicity of each hydrogel was evaluated by cell counting kit-8 (CCK8) assay and fluorescein diacetate (FDA) / propidium iodide (PI) staining. Specifically, L929 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 1% penicillin-streptomycin and 5% fetal bovine serum as complete culture medium. All cells were routinely cultured in a cell culture incubator containing 5% CO2 at 37°C. Each of the sterilized hydrogels and 5×10 3 L929 cells were placed in a 24-well plate and cultured for 1, 2, and 3 days. The control group was a case without hydrogel. The cells were stained with FDA / PI staining reagent and then observed under a fluorescence microscope. FDA can mark live cells in green, and PI can mark dead cells in red. The results are shown in Figure 2. Figure 20 In addition, the cell viability was evaluated by measuring the absorbance at 450 nm using a CCK counting kit and a microplate reader. Figure 21 shown.

[0143] Depend on Figure 20 It can be seen that in the fluorescent photos of living and dead cells, most cells are marked green, indicating that the cells can survive fairly well. In contrast to the control group and HAD / C / HBT5 group, the cells in the HAD / C, HAD / C / HBT1 and HAD / C / HBT3 groups initially spread rapidly and took on a spindle shape. Over time, the cells in the control group and HAD / C, HAD / C / HBT1 and HAD / C / HBT3 groups proliferated significantly. However, the proliferation rate of HAD / C / HBT5 cells was significantly slower. The results of CCK8 cell activity detection are shown in Figure 2. Figure 21 As shown in the figure, the results showed that HAD / C, HAD / C / HBT1 and HAD / C / HBT3 significantly promoted cell proliferation after 3 days, all faster than the control group. However, the antibiotics in HAD / C / HBT5 had a negative effect on cell proliferation. This phenomenon is consistent with the results of live-dead staining. It is obvious that collagen-based hydrogels can promote cell proliferation, but excessive antibiotics can inhibit cell growth. Therefore, the antibiotic content in HAD / C / HBT1 and HAD / C / HBT3 hydrogels is more appropriate and does not show significant cytotoxicity, while the incorporation of collagen promotes cell proliferation.

[0144] Example 13

[0145] In this example, an in vitro L929 cell migration model was used to investigate the effects of HAD / C / HBT1, HAD / C / HBT3, HAD / C / HBT5, and HAD / C hydrogels in promoting mouse fibroblast migration in vitro.

[0146] L929 cells were cultured in complete medium for 12 hours, and scratches were created using a pipette tip. The hydrogel and cells were then co-cultured in serum-free medium. The control group did not add hydrogel but only serum-free medium. Cell scratches were photographed using an inverted microscope at 0h, 12h, 24h, and 36h of culture. The formula for calculating the scratch healing rate (or area recovery rate) is as follows:

[0147] Area recovery rate (%) = (A0-A1) / A0×100%

[0148] Where A0 is the area of ​​the wound area at 0 h of culture, and A1 is the area of ​​the wound area at a predetermined time point of culture.

[0149] The test results of this embodiment are as follows Figure 22As shown, HAD / C hydrogel, HAD / C / HBT1, and HAD / C / HBT3 significantly enhanced cell migration compared with the control group, and after 36 h, the scratch healing rates of the control group, HAD / C hydrogel, HAD / C / HBT1, HAD / C / HBT3, and HAD / C / HBT5 were 24.14 ± 0.76%, 51 ± 1.44%, 37.85 ± 2.58%, 34.36 ± 2.82%, and 24.3 ± 5.37%, respectively. Figure 23 This indicates that HAD / C hydrogel, HAD / C / HBT1 and HAD / C / HBT3 hydrogels have excellent cell compatibility, promote cell proliferation and migration in vitro, and are beneficial to repair damaged tissues.

[0150] Example 14

[0151] In this example, the ability of HAD / C / HBT1, HAD / C / HBT3, HAD / C / HBT5 and HAD / C hydrogel to promote angiogenesis in vitro was investigated by an in vitro angiogenesis method.

[0152] HUVECs were seeded in a 24-well plate covered with Matrigel. Endothelial cell culture medium extract corresponding to the hydrogel was added and cultured in a 37°C cell culture incubator. After 10 hours, the angiogenesis of the cells was observed under an inverted microscope. Endothelial cell culture medium was added to the control group.

[0153] The test results of this embodiment are as follows Figure 24 As shown. Figure 24 It can be seen that the blood vessel-like structures formed by the HAD / C / HBT1, HAD / C / HBT3, HAD / C / HBT5 and HAD / C hydrogel groups, especially the HAD / C / HBT3 group, were denser than those in the control group. Compared with the control group, the HAD / C / HBT1, HAD / C / HBT3, HAD / C / HBT5 and HAD / C hydrogel groups, especially the HAD / C / HBT3 group, showed longer tube lengths, as shown in Figure 2. Figure 25 This indicates that the hydrogel provided by the present invention has the ability to promote angiogenesis.

[0154] Example 15

[0155] In this example, the antibacterial activities of HAD / C / HBT1, HAD / C / HBT3, HAD / C / HBT5 and HAD / C hydrogel were investigated by agar diffusion method and bacterial co-culture method.

[0156] The antibacterial performance of the hydrogel surface was evaluated by measuring the width of the zone of inhibition (ZOI) using agar diffusion method. 200 μL of bacterial suspension (1×10 6CFUs / mL) were evenly inoculated onto TSA. Afterwards, each sterilized hydrogel was attached to the center of the TSA and statically grown at 37°C. Photos were taken at specific times to observe the outer diameter of the antibacterial zone.

[0157] 100 μL of bacterial suspension (1×10 6 CFUs / mL) and each of the sterilized hydrogels were added to 900 μL of TSB and incubated in a bacterial incubator at 37°C for 12 hours. The resulting bacteria / hydrogel samples were then stained using a live / dead backlit bacterial viability kit and observed using a confocal laser scanning microscope. After dehydration using graded ethanol, the microscopic morphology of bacteria in each hydrogel was observed using a scanning electron microscope.

[0158] The test results of this embodiment are as follows Figures 26-28 As shown. HAD / C hydrogel has no antibacterial effect, while HAD / C / HBT1, HAD / C / HBT3 and HAD / C / HBT5 have good antibacterial effect. Compared with HAD / C / HBT1, HAD / C / HBT3 and HAD / C / HBT5 showed antibacterial effect for up to 24 hours, as shown. Figure 26 . After 24 h, HAD / C / HBT1, HAD / C / HBT3 and HAD / C / HBT5 produced larger inhibition zones against Staphylococcus aureus than those against Escherichia coli, which were 10.56±0.35 mm, 14.43±0.69 mm and 16.07±0.39 mm, respectively. This shows that the antibacterial activity of HAD / C / HBT1, HAD / C / HBT3 and HAD / C / HBT5 against Staphylococcus aureus is better than that against Escherichia coli, which may be due to the ability of tetracycline to penetrate the bacterial cell membrane or the different affinities of the corresponding bacterial receptors. In addition, HAD / C / HBT3 and HAD / C / HBT5 have more lasting antibacterial effects. To further explore the antibacterial effect of the hydrogel, a live / dead cell assay was performed using SYTO9 / PI, and the results are shown in Figure 2. Figure 27 As shown in the figure, all live bacteria were labeled with SYTO9 to emit green fluorescence, while dead cells were labeled with PI to show red fluorescence. Both Staphylococcus aureus and Escherichia coli on HAD / C hydrogels showed clear green fluorescence. In contrast, HAD / C / HBT3 and HAD / C / HBT5 were almost entirely red fluorescence, indicating that most bacteria were killed. SEM was used to observe the morphological changes of Staphylococcus aureus and Escherichia coli after hydrogel treatment to further study the bactericidal effect. The results are shown in Figure 2. Figure 28As shown. The effect of HAD / C hydrogel on bacterial morphology is negligible, and the bacterial membranes of Escherichia coli and Staphylococcus aureus remain smooth and intact. HAD / C / HBT1 can destroy the bacterial membrane of Staphylococcus aureus, causing the leakage of proteins within the bacteria, but the effect on Escherichia coli is significantly reduced, and the surface of some bacteria is no longer smooth and slightly shrunk. In addition, it was observed that HAD / C / HBT3 and HAD / C / HBT5 both exhibited excellent antibacterial activity against Escherichia coli and Staphylococcus aureus; the number of bacteria adhering to the hydrogel surface was significantly reduced, and a large number of bacteria failed to maintain their normal membrane structure and disintegrated, indicating that bacterial adhesion and biofilm formation can be inhibited. In summary, HAD / C / HBT3 and HAD / C / HBT5 exhibited good antibacterial properties against Staphylococcus aureus and Escherichia coli.

[0159] Example 16

[0160] In this example, the full-thickness skin defect wound model of mice infected with Staphylococcus aureus was used to investigate the repair effects of HAD / C / HBT3 and HAD / C hydrogels on infected wounds.

[0161] Male BALB / c mice were randomly divided into a control group, a HAD / C / HBT3 group, and a HAD / C hydrogel group. The control group used Tegaderm TM A full-thickness circular wound with a diameter of 10 mm was formed on the back of each mouse. Afterwards, 20 μL of bacterial (Staphylococcus aureus) suspension was injected into each group of mice at the wound site on the back of the mouse. Finally, Tegaderm was used in the control group. TM The wounds were covered with a transparent dressing. In the HAD / C / HBT3 group, sterilized HAD / C / HBT3 hydrogel was injected into the wound site to cover the wound. In the HAD / C hydrogel group, sterilized HAD / C hydrogel was injected into the wound site to cover the wound. Wound size was measured and imaged on days 0, 4, 8, and 12 after completing the above treatment. Figure 29 The wound area was then measured using Image J software, and the wound healing rate was calculated using the following formula: Figure 30 As shown:

[0162] wound healing rate(%)=(R0-R1) / R0×100%

[0163] In the above formula, R0 is the wound area on day 0 after the completion of treatment, and R1 is the wound area at a predetermined time point after the completion of treatment.

[0164] Depend on Figures 29-30It can be seen that on the 4th day, the repair effects of the HAD / C hydrogel group and the HAD / C / HBT3 group were better than those of the Tegaderm group in the control group. TM The HAD / C / HBT3 group had a wound healing rate exceeding 40% using a transparent dressing. By day 8, the wound healing rates of the HAD / C / HBT3 group were 27.6% and 15.3% higher than those of the control group and the HAD / C hydrogel group, respectively. By day 12, no open wounds appeared in the HAD / C / HBT3 group, and new epidermal tissue had grown. In contrast, 27.83±9.12% and 20.54%±4.64% of wounds in the HAD / C group and the control group, respectively, remained open, with uneven scarring. These experimental results demonstrate that the protein hybrid gel dressing provided by the present invention can modify the wound infection environment, creating a sterile and suitable protein environment to accelerate the healing of infected wounds.

[0165] To assess wound tissue regeneration, mice were euthanized at predetermined time points, and wound tissue was collected and immersed in 4% (vol / vol) paraformaldehyde for 24 hours. The tissues were dehydrated through graded ethanol and embedded in paraffin. 4-μm-thick sections were cut and stained with hematoxylin and eosin (H&E), Masson's trichrome, and immunohistochemical analysis (CD31, TGF-β, and TNF-α). Collagen deposition, vessel number, TGF-β expression, and TNF-α expression were determined using Image J software.

[0166] The results of H&E staining and Masson analysis of the tissues in the control group, HAD / C / HBT3 group, and HAD / C hydrogel group are shown in Figure 2. Figures 31-32 As shown, the control group showed a large number of inflammatory cells on day 6, and this severe symptom persisted until day 12. Although new epidermal tissue appeared in the wound tissue of the HAD / C hydrogel group, the continued inflammation limited the effectiveness of repairing the infected wound. In contrast, the HAD / C / HBT3 group showed more hair follicles, only a small number of neutrophils, and the fastest wound healing.

[0167] like Figure 33 As shown, during wound healing, collagen deposition in the control group was sparse and lighter in color. On day 12, collagen deposition in the HAD / C / HBT3 group (74.07±4.84%) was significantly higher than that in the HAD / C gel group (55.75±4.66%) and the control group (40.11±3.211%). This phenomenon may be attributed to the fact that the protein hybrid gel dressing provided by the present invention provides a beneficial protein environment for wound healing, while tetracycline is effective in antibacterial activities, thereby promoting collagen deposition in the wound.

[0168] The HAD / C / HBT3 group had more blood vessels and hair follicles in the periwound tissue than the HAD / C group and the control group. Figure 34 Figure (a). On day 12, the number of blood vessels in the wound area increased in the control group (24±3) and HAD / C group (35±2), but was still much lower than that in the HAD / C / HBT3 group (65±5). Figure 34 (b) This phenomenon indicates that HAD / C / HBT3 has the potential to enhance angiogenesis.

[0169] The expression of TGF-β and TNF-α in the control group, HAD / C / HBT3 group and HAD / C hydrogel group were as follows: Figure 35 TNF-α is a major pro-inflammatory cytokine that leads to prolonged inflammation. TGF-β is a key mediator required to reduce the inflammatory phase and promote the next stage of healing. Figure 35 It can be seen that the HAD / C / HBT3 group had the highest expression level of TGF-β, followed by the HAD / C group and the control group, while the expression of TNF-α was the opposite of TGF-β. The control group consistently showed a higher expression level of TNF-α, followed by the HAD / C group, and the HAD / C / HBT3 group showed the lowest level of TNF-α expression. This phenomenon can be attributed to the fact that in the infected wound environment, relying solely on the anti-inflammatory effect of catechol in HAD / C is not sufficient. The present invention relies on antibiotics to kill bacteria and change the wound microenvironment, which can enhance the anti-inflammatory effect.

Claims

1. A protein hybrid gel dressing of composite drug-loaded fiber, characterized in that: This protein hybrid gel dressing consists of a hybrid hydrogel and esterified hyaluronic acid nanofibers loaded with hydrophobic antibiotics uniformly dispersed in the hybrid hydrogel. The hybrid hydrogel is formed by reacting a solution of a biocompatible polymer material with a solution of a protein material having amino and carboxyl groups. The biocompatible polymer material is at least one of a thiol-modified polymer material and a catechol-functional group-modified polymer material. The hydrophobic antibiotic is uniformly dispersed in the gel dressing and can be sustained-released from the gel dressing to achieve long-lasting antibacterial effects. The catechol functional group-modified polymer material is dopamine-modified hyaluronic acid, in which the dopamine grafting rate is 1% to 70%; the thiol-modified polymer material is thiol-modified hyaluronic acid, in which the thiol grafting rate is 1% to 70%; the esterified hyaluronic acid nanofiber is benzyl-modified hyaluronic acid nanofiber, in which the benzyl-modified hyaluronic acid has a benzyl grafting rate of 10% to 90%; and the hydrophobic antibiotic is tetracycline.

2. The protein hybrid gel dressing of composite drug-loaded fiber according to claim 1, characterized in that: In the protein hybrid gel dressing, the mass ratio of the hydrophobic antibiotic-loaded esterified hyaluronic acid nanofibers to the freeze-dried hybrid hydrogel is (0.02-0.6):

1.

3. The protein hybrid gel dressing of composite drug-loaded fiber according to claim 1, characterized in that: The hybrid hydrogel is formed by reacting a solution of a biocompatible polymer material with a solution of a protein material having amino and carboxyl groups, with the mass ratio of the biocompatible polymer material to the protein material having amino and carboxyl groups being (0.1-1): (0.1-1).

4. The protein hybrid gel dressing of composite drug-loaded fiber according to claim 1, characterized in that: The protein material having amino and carboxyl groups includes at least one of collagen, gelatin, recombinant human protein, and silk fibroin.

5. The protein hybrid gel dressing of composite drug-loaded fiber according to any one of claims 1 to 4, characterized in that: In the esterified hyaluronic acid nanofibers loaded with the hydrophobic antibiotic, the hydrophobic antibiotic and the esterified hyaluronic acid nanofibers are combined with each other through intermolecular forces.

6. The protein hybrid gel dressing of composite drug-loaded fiber according to any one of claims 1 to 4, characterized in that: The preparation method of esterified hyaluronic acid nanofibers loaded with hydrophobic antibiotics is as follows: esterified hyaluronic acid nanofibers and hydrophobic antibiotics are dissolved in hexafluoroisopropanol to form an electrospinning solution, and then electrospinning is performed to obtain a nanofiber membrane, which is then dried. The dried nanofiber membrane is placed in water, stirred and dispersed, and dried to obtain the nanofiber membrane.

7. The method for preparing the protein hybrid gel dressing of the composite drug-loaded fiber according to any one of claims 1 to 6, characterized in that: The following steps are involved: (1) dissolving a biocompatible polymer material in water to form an aqueous solution of the biocompatible polymer material, and uniformly dispersing esterified hyaluronic acid nanofibers loaded with hydrophobic antibiotics in the aqueous solution of the biocompatible polymer material to obtain a dispersion; in the dispersion, the concentration of the biocompatible polymer material is 2 wt% to 50 wt%, and the concentration of the esterified hyaluronic acid nanofibers loaded with hydrophobic antibiotics is 0.1 wt% to 50 wt%; (2) dissolving the protein material having amino and carboxyl groups in a 0.2-1 mol / L acetic acid aqueous solution to form a solution of the protein material having amino and carboxyl groups having a concentration of 2 wt%-50 wt%; (3) The dispersion obtained in step (1) is fully mixed with the solution of the protein material having amino and carboxyl groups obtained in step (2), the pH value of the resulting mixture is adjusted to 7-10, and the mixture is allowed to stand at 20-40°C until the mixture is converted into a gel state, thereby obtaining a protein hybrid gel dressing of composite drug-loaded fibers.

8. Use of the protein hybrid gel dressing of the composite drug-loaded fiber according to any one of claims 1 to 6 as a wound repair material for resisting bacterial infection.

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