Injectable antibacterial hydrogel and preparation method and application thereof

Abstract

The application provides an injectable antibacterial hydrogel and a preparation method and application thereof, and belongs to the technical field of hydrogel preparation.The injectable antibacterial hydrogel is obtained by adding a tannic acid solution drop by drop into a chitosan aqueous solution after heating, stirring, adding a silver nanoparticle solution, and then adding a 3-fluoro-4-formylphenylboronic acid solution drop by drop.The Ag-NPs are coated into the injectable hydrogel formed by CS, TA and 3-F-4-Fm-Ph-B(OH)2 crosslinking, the photo-thermal sterilization performance and the broad-spectrum antibacterial performance of the Ag-NPs are utilized to solve the wound infection caused by bacteria.The hydrogel can adapt to irregular wounds, and has the advantages of simple preparation method, low production cost, mild conditions, and good antibacterial effect on E.coli, S.aureus, S.haemolyticus and S.epidermidis.

Description

Technical Field

[0001] This invention belongs to the field of hydrogel preparation technology, and particularly relates to an injectable antibacterial hydrogel, its preparation method and application. Background Technology

[0002] As the largest organ in the human body, the skin plays a vital role in isolating the body from harmful external substances, maintaining body temperature, and regulating body fluids (Biomaterials, 2017, 139: 229-243; Chemistry of Materials, 2020, 32(23): 9937-9953). Once the integrity of the skin is severely damaged, it becomes susceptible to invasion by pathogenic microorganisms such as Escherichia coli and Staphylococcus aureus, leading to serious wound infections (Biomacromolecules, 2020, 21(5): 1841-1852; Biomaterials Science, 2023, 11(3): 1077-1078; Journal of Materials Chemistry B, 2016, 4(41): 6644-6651), which in turn threatens the patient's life, reduces quality of life, and increases economic burden. Antibiotics have been widely used for a long time, but this has inevitably led to the problem of bacterial resistance. Therefore, it is particularly important to develop a non-antibiotic dressing for wound healing after bacterial infection.

[0003] In recent years, novel wound dressings such as 3D-printed scaffolds, sponges, and hydrogels have emerged. Among them, hydrogels, as a hydrophilic three-dimensional network structure, exhibit good biocompatibility due to their biomimetic structure and similarity to the extracellular matrix of natural living tissue cells. They can not only effectively block the invasion of external pathogens and maintain wound moisture, but also promote collagen deposition and fibroblast proliferation (Frontiers in Bioengineering and Biotechnology, 2023, 11:1180073). In particular, injectable hydrogels, which can be injected non-invasively into targeted areas, are more attractive to patients and clinicians than non-injectable hydrogels due to their greater comfort, less pain, lower cost, and fewer side effects (Materials Science and Engineering: C, 2021, 131: 112489). However, most existing injectable antibacterial hydrogels still face significant bottlenecks: some hydrogels rely on a single antibacterial mechanism, resulting in limited inhibitory effects against multidrug-resistant bacteria; some hydrogels containing silver-based materials suffer from silver nanoparticle aggregation and uncontrolled release rates, which not only reduce antibacterial efficiency but may also induce local tissue toxicity or inflammatory reactions; and some hydrogels have insufficient cross-linking network stability, weak mechanical strength, low photothermal conversion efficiency, and poor cyclic stability, making them difficult to adapt to the complex microenvironment of clinical wounds and the needs of long-term treatment.

[0004] Chitosan (CS), a product of N-deacetylation of chitin, is a natural cationic polysaccharide with antibacterial properties and excellent biocompatibility, often used in the preparation of hydrogels. However, chitosan alone is insufficient to effectively treat bacterial wound infections (Bioactive materials, 2022, 8: 341-354), and pure chitosan hydrogels have weak mechanical strength, rapid degradation rates, and difficulty in maintaining long-term wound protection and antibacterial effects. Furthermore, its antibacterial spectrum is relatively narrow, with limited inhibitory effects on some Gram-positive bacteria.

[0005] Tannic acid (TA) is a natural polyphenol with strong antioxidant properties (ACS Appl. Mater. Interfaces, 2022, 14, 15927−15941; Free Radical Biology and Medicine, 2020,160: 342-355). It is widely found in various plants and is an important raw material for commercial applications. It is commonly used as a tanning agent in the leather, coating, adhesive, surgical, pharmaceutical and food industries (Journal of the American dieteticassociation, 1999, 99(2): 213-218). As a natural building block, tannic acid contains aromatic ring structures of catechol and pyrogallol, possessing a variety of unique chemical properties. It can be used as a crosslinking agent through chemical or physical means to form a multifunctional polymer network (Journal of Polymer Research, 2018, 25: 1-10). However, when used alone as a hydrogel component, its antibacterial activity is weak, and the crosslinked network is easily affected by the pH value of the environment and is unstable, which limits its application alone in wound dressings.

[0006] Silver nanoparticles (Ag-NPs) are materials with diverse antibacterial properties. Under near-infrared light irradiation, they can convert light energy into heat energy to achieve a bactericidal effect, exhibiting excellent photothermal properties (International Journal of Biological Macromolecules, 2023, 226: 870-884). Ag-NPs have broad-spectrum antibacterial activity against Gram-positive bacteria, Gram-negative bacteria, and drug-resistant bacteria. Their antibacterial mechanism mainly involves disrupting cell membranes and cell walls or generating reactive oxygen species during the bactericidal process (International Journal of Biological Macromolecules, 2022, 210:337-349). Studies have reported that Ag-NPs are among the most effective antibacterial materials against bacteria, viruses, and other microorganisms, and are widely used in bioscience, pharmaceuticals, and other fields. Currently, due to their antibacterial properties, they are used as topical ointments in wound management (Microchemical Journal, 2024, 196: 109615). However, Ag-NPs tend to aggregate in hydrogel systems, leading to a decrease in photothermal efficiency and antibacterial activity. Furthermore, the lack of effective encapsulation and controlled release mechanisms makes them prone to burst release, posing a biosafety risk. This greatly limits their large-scale application in injectable hydrogel dressings. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention proposes an injectable antibacterial hydrogel, its preparation method, and its applications. This invention encapsulates Ag-NPs within an injectable hydrogel formed by cross-linking CS, TA, and 3-fluoro-4-aldehyde phenylboronic acid (3-F-4-Fm-Ph-B(OH)2). The photothermal bactericidal and broad-spectrum antibacterial properties of Ag-NPs are utilized to address wound infections caused by bacteria. This hydrogel is adaptable to irregular wound surfaces, and its preparation method is simple, low-cost, and requires mild conditions. It exhibits good antibacterial effects against E. coli, S. aureus, S. haemolyticus, and S. epidermidis.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] This invention proposes a method for preparing an injectable antibacterial hydrogel, comprising the following steps:

[0010] After heating the CS aqueous solution, TA solution was added dropwise, followed by stirring and then Ag-NPs solution was added. Then, 3-F-4-Fm-Ph-B(OH)2 solution was added dropwise to obtain the injectable antibacterial hydrogel.

[0011] Furthermore, the concentration of the tannic acid solution is 2.7 mg / 50 μL, and the volume concentration of the chitosan aqueous solution is 2%.

[0012] Furthermore, the method for preparing the chitosan aqueous solution is as follows:

[0013] Chitosan was dissolved in acetic acid solution and stirred to obtain a chitosan acetic acid solution with a volume concentration of 2%. The chitosan acetic acid solution was then freeze-dried and dissolved in water to obtain the chitosan aqueous solution.

[0014] Furthermore, the 3-fluoro-4-aldehyde phenylboronic acid solution is prepared by dissolving 3-fluoro-4-aldehyde phenylboronic acid in an aqueous ethanol solution.

[0015] Furthermore, the concentration of the 3-fluoro-4-aldehyde phenylboronic acid solution is 4.2 mg / 100 μL.

[0016] Furthermore, the silver nanoparticle solution is synthesized using tannic acid, trisodium citrate, and silver nitrate as raw materials.

[0017] Furthermore, the concentration of the silver nanoparticle solution is 1.21 mg / mL, and the mass ratio of tannic acid, trisodium citrate, and silver nitrate is 8.6:13.7:8.6.

[0018] Furthermore, in the 3-fluoro-4-aldehyde phenylboronic acid, chitosan aqueous solution, and tannic acid solution, the molar ratio of 3-fluoro-4-aldehyde phenylboronic acid, chitosan monomer, and tannic acid is 0.2:1:0.013; wherein the chitosan monomer in the chitosan aqueous solution refers to the basic structural unit constituting the chitosan macromolecule. Chitosan is a linear polysaccharide copolymer composed of two monosaccharide units (D-glucosamine and N-acetylglucosamine) linked by β-1,4-glycosidic bonds. Therefore, the monomer here refers to the basic constituent unit (D-glucosamine and N-acetylglucosamine).

[0019] Furthermore, the volume ratio of the silver nanoparticle solution to the chitosan aqueous solution is 20-60 μL: 1 mL.

[0020] The present invention also proposes an injectable antibacterial hydrogel, which is prepared according to the above preparation method.

[0021] Furthermore, the aldehyde group in the 3-fluoro-4-aldehyde phenylboronic acid undergoes Schiff base crosslinking with the amino group in chitosan, and the boric acid in the 3-fluoro-4-aldehyde phenylboronic acid forms a borate ester bond with the hydroxyl group in the tannic acid.

[0022] Compared with the prior art, the present invention has the following advantages and technical effects:

[0023] This invention encapsulates Ag-NPs within an injectable hydrogel formed by crosslinking CS, TA, and 3-F-4-Fm-Ph-B(OH)2, utilizing the photothermal bactericidal and broad-spectrum antibacterial properties of Ag-NPs to address wound infections caused by bacteria. This hydrogel is adaptable to irregular wound surfaces and is simple to prepare, low in cost, and under mild conditions, exhibiting good antibacterial effects against E. coli, S. aureus, S. haemolyticus, and S. epidermidis. Attached Figure Description

[0024] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0025] Figure 1 Fourier transform infrared analysis results of the hydrogel (CTF / silver) obtained in Example 1 and the raw materials tannic acid, chitosan, and 3-fluoro-4-aldehyde phenylboronic acid;

[0026] Figure 2 The rheological sweep plots of the storage modulus (G') and loss modulus (G") of the hydrogels in Example 1 and Comparative Example 1 as a function of angular frequency are shown.

[0027] Figure 3Shear thinning plots showing the viscosity of the hydrogels (CTF, CTF / Ag) prepared in Example 1 and Comparative Example 1 as a function of shear rate.

[0028] Figure 4 The image shows the UV absorption peak at 410 nm for the Ag-NPs solution obtained in Example 1.

[0029] Figure 5 The particle size analysis results are for the Ag-NPs solution synthesized in Example 1;

[0030] Figure 6 The photothermal performance test results for different Ag-NPs concentrations;

[0031] Figure 7 The photothermal properties of CTF and CTF / Ag hydrogel are shown in the figure.

[0032] Figure 8 The results show the stability of the hydrogel (CTF / Ag) prepared in Example 1 during photothermal cycling.

[0033] Figure 9 The photothermal conversion efficiency test results are for the hydrogel (CTF / Ag) prepared in Example 1;

[0034] Figure 10 The results show the release rate of Ag-NPs in the CTF / Ag hydrogel of Example 1 from 0 to 72 h.

[0035] Figure 11 The results show the scavenging rates of ABTS free radicals by CTF and CTF / Ag hydrogels. In this graph, A represents the absorbance of ABTS at 734 nm after incubation with each hydrogel sample for 20 min; and B is a statistical graph of the free radical scavenging rates of ABTS by the hydrogels.

[0036] Figure 12 The scavenging rates of CTF and CTF / Ag hydrogels against DPPH free radicals are shown in Figure A, where A is the absorbance of DPPH at 518 nm after incubation with each hydrogel sample for 20 min; and B is a statistical graph of the free radical scavenging rates of hydrogels against DPPH.

[0037] Figure 13 The swelling properties of CTF and CTF / Ag hydrogels are shown in Figure A, where A is the swelling volume change of CTF and CTF / Ag from 0 to 24 h, and B is the swelling rate statistics of CTF and CTF / Ag hydrogels from 0 to 48 h.

[0038] Figure 14The results of plate counting experiments on hydrogels under NIR (+NIR) and no NIR (-NIR) conditions are shown. In the figure, A is the plate colony diagram of S. haemolyticus, S. epidermidis, E. coli and S. aureus under NIR and no NIR conditions, and B is the survival rate statistics of S. haemolyticus, S. epidermidis, E. coli and S. aureus.

[0039] Figure 15 Plate inhibition zones of CTF and CTF / Ag hydrogels against S. haemolyticus, S. epidermidis, E. coli and S. aureus. Detailed Implementation

[0040] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0041] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0042] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0043] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0044] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0045] This invention provides a method for preparing an injectable antibacterial hydrogel, comprising the following steps:

[0046] Chitosan aqueous solution was heated and tannic acid solution was added dropwise. After stirring, silver nanoparticle solution was added, followed by dropwise addition of 3-fluoro-4-aldehyde phenylboronic acid solution to obtain injectable antibacterial hydrogel.

[0047] This invention constructs a three-dimensional network structure formed by the cross-linking of chitosan (CS), tannic acid (TA), and 3-F-4-Fm-Ph-B(OH)2 (3-fluoro-4-aldehyde-phenylboronic acid) through the steps of "heating chitosan aqueous solution, adding tannic acid solution dropwise, and then adding silver nanoparticle solution and 3-fluoro-4-aldehyde-phenylboronic acid solution in sequence," achieving uniform coating of Ag-NPs (silver nanoparticles). Simultaneously, it imparts injectability to the hydrogel (suitable for irregular wounds, non-invasive application), and combines the photothermal bactericidal and broad-spectrum antibacterial properties of Ag-NPs to effectively solve wound infections caused by bacteria. Furthermore, the preparation process requires no complex equipment, is simple, has low production cost, and mild reaction conditions, making it suitable for large-scale production. The final hydrogel exhibits excellent antibacterial effects against E. coli, S. aureus, S. haemolyticus, and S. epidermidis.

[0048] In a preferred embodiment of the present invention, the concentration of the tannic acid solution is 2.7 mg / 50 μL, and the volume concentration of the chitosan aqueous solution is 2%. The 2% chitosan aqueous solution provides sufficient and uniform CS monomers, laying a stable framework for the subsequent crosslinking reaction and avoiding insufficient mechanical strength of the hydrogel due to too low a concentration or affecting injectability due to too high a concentration. On the other hand, the tannic acid concentration of 2.7 mg / 50 μL can precisely match the reaction requirements of 3-F-4-Fm-Ph-B(OH)2, ensuring that the hydroxyl groups of TA form stable borate ester bonds with boric acid, while avoiding excessive TA leading to over-crosslinking of the hydrogel (loss of shear-thinning properties, making it uninjectable) or insufficient TA leading to inadequate crosslinking (hydrogel easily disintegrates), ultimately ensuring the mechanical stability and practical performance of the hydrogel.

[0049] In a preferred embodiment of the present invention, the method for preparing the chitosan aqueous solution is as follows:

[0050] Chitosan was dissolved in acetic acid solution and stirred to obtain a chitosan acetic acid solution with a volume concentration of 2%. The chitosan acetic acid solution was then freeze-dried and dissolved in water to obtain a chitosan aqueous solution.

[0051] For example, in this embodiment of the invention, the method for preparing a chitosan (CS) aqueous solution includes the following steps:

[0052] 1) Dissolve CS in 2% (v / v) acetic acid to obtain a CS acetic acid solution;

[0053] 2) The CS acetic acid solution was freeze-dried;

[0054] 3) Dissolve the CS obtained after freeze-drying in step 2) in pure water to obtain a 2% (V / V) CS aqueous solution.

[0055] This invention provides a chitosan aqueous solution prepared by "dissolving chitosan in acetic acid → freeze-drying → dissolving in water". This process removes residual acetic acid, avoids irritation to wounds, and improves the biocompatibility of the hydrogel. Simultaneously, the freeze-drying process improves the aggregation state of chitosan, resulting in more uniform dispersion when dissolved in water. This ensures a more complete and uniform reaction between the CS monomer and the amino group of 3-fluoro-4-aldehyde phenylboronic acid (Schiff base crosslinking) and the hydroxyl group of TA (indirect action), reducing crosslinking defects caused by uneven CS dissolution. This further enhances the structural stability of the hydrogel and the uniformity of the loading of antibacterial components (Ag-NPs).

[0056] In a preferred embodiment of the present invention, the 3-fluoro-4-aldehyde phenylboronic acid solution is prepared by dissolving 3-fluoro-4-aldehyde phenylboronic acid in an aqueous ethanol solution. Dissolving 3-fluoro-4-aldehyde phenylboronic acid in an aqueous ethanol solution utilizes the polarity and solubility of the ethanol solution to ensure complete dissolution of the 3-fluoro-4-aldehyde phenylboronic acid, avoiding solid precipitation and ensuring uniform dispersion in the mixed system during subsequent dropwise addition. This facilitates an efficient and uniform cross-linking reaction with the amino group of CS (forming a Schiff base) and the hydroxyl group of TA (forming a borate ester bond). Using pure water or other solvents can easily lead to insufficient dissolution of the boric acid compound, causing uneven local cross-linking and affecting the mechanical properties of the hydrogel (such as fluctuations in storage modulus G') and injectability. This solvent selection provides a crucial guarantee for stabilizing the cross-linking network.

[0057] In a preferred embodiment of the present invention, the concentration of the 3-fluoro-4-aldehyde phenylboronic acid solution is 4.2 mg / 100 μL. This concentration is the optimized concentration for the cross-linking reaction, providing sufficient cross-linking agent molecules to ensure the formation of a dense and stable three-dimensional network with CS and TA, thus guaranteeing the mechanical strength of the hydrogel and preventing collapse. It also avoids excessive cross-linking agent leading to unreacted monomer residue, reducing potential irritation to the wound. Simultaneously, this concentration matches the concentrations of CS and TA, balancing the degree of cross-linking of the hydrogel, maintaining both shear-thinning properties and swelling performance, achieving a synergistic effect of injectability, stability, and practicality.

[0058] For example, in an embodiment of the present invention, the preparation method of 3-fluoro-4-aldehyde phenylboronic acid (3-F-4-Fm-Ph-B(OH)2) solution includes the following steps:

[0059] 4.2 mg of 3-F-4-Fm-Ph-B(OH)2 was added to 100 μL of a 50% (V / V) aqueous ethanol solution and dissolved completely by vortexing.

[0060] In a preferred embodiment of the present invention, the silver nanoparticle solution is synthesized using tannic acid, trisodium citrate and silver nitrate as raw materials.

[0061] In a preferred embodiment of the present invention, the concentration of the silver nanoparticle solution is 1.21 mg / mL, and the mass ratio of tannic acid, trisodium citrate and silver nitrate is 8.6:13.7:8.6.

[0062] For example, in this embodiment of the invention, the preparation method of the silver nanoparticle (Ag-NPs) solution includes the following steps:

[0063] 1) Prepare 1 mL aqueous solutions of 8.6 mg TA, 13.7 mg CIT, and 8.6 mg AgNO3 respectively;

[0064] 2) Add the above TA aqueous solution and CIT solution to 70 mL of boiling water and stir thoroughly;

[0065] 3) Then slowly add the AgNO3 aqueous solution dropwise to the boiling aqueous solution in step 2). The solution changes from colorless to dark brown. Continue stirring for 5 minutes. After the solution cools, centrifuge at 10,000 rpm for 6 minutes, collect the precipitate and freeze dry to obtain Ag-NPs nanoparticles.

[0066] 4) Weigh 1.21 mg Ag-NPs and disperse them in 1 mL of deionized water to obtain an Ag-NPs solution with a concentration of 1.21 mg / mL.

[0067] This invention uses TA, trisodium citrate (CIT), and silver nitrate (AgNO3) to synthesize Ag-NPs solution. TA and CIT both function as reducing agents and stabilizers; TA can assist in the reduction of Ag. + The formation of Ag-NPs, combined with CIT, inhibits Ag-NP aggregation through adsorption. Compared to a single reducing agent (such as TA or sodium citrate alone), this combination can produce uniformly dispersed Ag-NPs with controllable particle size, ensuring their photothermal conversion efficiency and broad-spectrum antibacterial activity, and avoiding aggregation that leads to decreased photothermal efficiency or reduced antibacterial sites. At the same time, the raw materials are all biocompatible components, meeting the safety requirements of medical materials and avoiding the introduction of toxic impurities.

[0068] In a preferred embodiment of the present invention, the molar ratio of 3-fluoro-4-aldehyde phenylboronic acid, chitosan monomer, and tannic acid in the chitosan aqueous solution and tannic acid solution is 0.2:1:0.013; wherein the chitosan monomer in the chitosan aqueous solution refers to the basic structural unit constituting the chitosan macromolecule. Chitosan is a linear polysaccharide copolymer composed of two monosaccharide units (D-glucosamine and N-acetylglucosamine) linked by β-1,4-glycosidic bonds. Therefore, the monomer here refers to the basic constituent unit (D-glucosamine and N-acetylglucosamine).

[0069] The ratio in this invention is the core optimization parameter for the crosslinking reaction. CS monomer serves as the backbone, and TA and boric acid serve as crosslinking agents. When the molar ratio is within this range, a dual stable network of Schiff base crosslinking (boric acid-CS) and borate ester crosslinking (boric acid-TA) can be formed. This ensures the mechanical strength of the hydrogel while avoiding excessively dense crosslinking that leads to an overly hard hydrogel or excessively sparse crosslinking that leads to easy degradation of the hydrogel. At the same time, the uniform crosslinking network can achieve uniform coating of Ag-NPs, avoiding excessively high or low local Ag-NPs concentrations and ensuring uniform antibacterial effect.

[0070] In a preferred embodiment of the present invention, the volume ratio of silver nanoparticle solution to chitosan aqueous solution is 20-60 μL: 1 mL.

[0071] This invention also proposes an injectable antibacterial hydrogel, prepared according to the above preparation method.

[0072] In the injectable antibacterial hydrogel of the present invention, the aldehyde group in 3-fluoro-4-aldehyde phenylboronic acid undergoes Schiff base crosslinking with the amino group in chitosan, and the boric acid in 3-fluoro-4-aldehyde phenylboronic acid forms a borate ester bond with the hydroxyl group in tannic acid.

[0073] The hydrogel prepared in this invention integrates the advantages of all precursor technologies, possessing injectability (suitable for irregular wounds, non-invasive application), dual antibacterial properties, excellent biocompatibility (no residual irritating components), good swelling properties, antioxidant properties (scavenging ABTS / DPPH free radicals), photothermal cycling stability, and long-lasting antibacterial effect. It exhibits significant antibacterial effects against E. coli, S. aureus, S. haemolyticus, and S. epidermidis. Moreover, it is simple to prepare, low in cost, and mild under mild conditions, making it an ideal dressing for treating wound infections and overcoming the performance deficiencies of existing non-antibiotic wound dressings.

[0074] All raw materials used in the embodiments of the present invention were commercially available, wherein CS has a viscosity of 100 mPa·s and a degree of deacetylation of ≥95%.

[0075] In this embodiment of the invention, "room temperature" refers to "25±2℃".

[0076] The technical solution of the present invention will be further illustrated by the following embodiments.

[0077] Example 1

[0078] A method for preparing an injectable antibacterial hydrogel includes the following steps:

[0079] (1) Take 8.6 mg TA, 13.7 mg CIT and 8.6 mg AgNO3 and add water to prepare 1 mL of aqueous solution respectively; add the obtained TA aqueous solution and CIT aqueous solution to 70 mL of boiling water and stir thoroughly; then slowly add AgNO3 aqueous solution dropwise to the boiling aqueous solution. The solution gradually changes from colorless to dark brown. Continue stirring for 5 min. After the solution cools, centrifuge at 10000 rpm / min for 6 min to enrich Ag-NPs particles. Store at 4℃. Weigh 1.21 mg Ag-NPs particles and disperse them in 1 mL of deionized water to obtain an Ag-NPs solution with a concentration of 1.21 mg / mL.

[0080] (2) Weigh 1g of CS (viscosity 100mPa.s, degree of deacetylation ≥95%, 6×10 -3 1 mol) was dissolved in 2% acetic acid solution (V / V, 49 mL), and stirred thoroughly at 50 °C to obtain a 2% volume concentration chitosan acetate solution. The chitosan acetate solution was then placed in a freeze dryer (-51 °C) for freeze drying until completely dry. The dried CS was weighed and dissolved in pure water to obtain a 2% volume concentration CS aqueous solution.

[0081] (3) Take 2.7 mg of TA (1.6 × 10⁻⁶) -6 Dissolve 1 mol) in 50 μL of pure water to obtain a TA solution, and take 1 mL of the 2% CS aqueous solution obtained in step (2) (1.2 × 10⁻⁶ mol) -4 The solution was heated to 60°C on a magnetic stirrer and stirred at 800 rpm / min. The resulting TA solution was then added dropwise to a heated 2% CS aqueous solution and stirred for 1 h. 60 μL of the Ag-NPs solution obtained in step (1) was then added to the above mixture and stirring continued. Separately, 4.2 mg of 3-F-4-Fm-Ph-B(OH)2 (2.5 × 10⁻⁶ mol) was added. -53-F-4-Fm-Ph-B(OH)2 solution was obtained by dissolving 3-F-4-Fm-Ph-B(OH)2 in 100 μL of 50% ethanol aqueous solution (V / V). The obtained 3-F-4-Fm-Ph-B(OH)2 solution was added dropwise to the above mixed solution at 800 rpm / min (in this step, the molar ratio of 3-F-4-Fm-Ph-B(OH)2, chitosan monomer and tannic acid is 0.2:1:0.013, and the concentration of Ag-NPs is 0.06 mg / mL) until gelation was formed, and an injectable antibacterial hydrogel (denoted as CTF / Ag (0.06 mg / mL)) was obtained.

[0082] Example 2

[0083] Same as Example 1, except that the volume of the Ag-NPs solution obtained in step (1) is 20 μL. The specific preparation method is as follows:

[0084] (1) Take 8.6 mg TA, 13.7 mg CIT and 8.6 mg AgNO3 and add water to prepare 1 mL of aqueous solution respectively; add the obtained TA aqueous solution and CIT aqueous solution to 70 mL of boiling water and stir thoroughly; then slowly add AgNO3 aqueous solution dropwise to the boiling aqueous solution. The solution gradually changes from colorless to dark brown. Continue stirring for 5 min. After the solution cools, centrifuge at 10000 rpm / min for 6 min to enrich Ag-NPs particles. Store at 4℃. Weigh 1.21 mg Ag-NPs particles and disperse them in 1 mL of deionized water to obtain an Ag-NPs solution with a concentration of 1.21 mg / mL.

[0085] (2) Weigh 1g of CS (viscosity 100mPa.s, degree of deacetylation ≥95%, 6×10 -3 1 mol) was dissolved in 2% acetic acid solution (V / V, 49 mL), and stirred thoroughly at 50 °C to obtain a 2% volume concentration chitosan acetate solution. The chitosan acetate solution was then placed in a freeze dryer (-51 °C) for freeze drying until completely dry. The dried CS was weighed and dissolved in pure water to obtain a 2% volume concentration CS aqueous solution.

[0086] (3) Take 2.7 mg of TA (1.6 × 10⁻⁶) -6 Dissolve 1 mol) in 50 μL of pure water to obtain a TA solution, and take 1 mL of the 2% CS aqueous solution obtained in step (2) (1.2 × 10⁻⁶ mol) -4The solution was heated to 60°C on a magnetic stirrer and stirred at 800 rpm / min. The resulting TA solution was then added dropwise to a heated 2% CS aqueous solution and stirred for 1 h. Then, 20 μL of the Ag-NPs solution obtained in step (1) was added to the above mixture and stirring continued. Separately, 4.2 mg of 3-F-4-Fm-Ph-B(OH)2 (2.5 × 10⁻⁶ mol) was added. -5 3-F-4-Fm-Ph-B(OH)2 solution was obtained by dissolving 3-F-4-Fm-Ph-B(OH)2 in 100 μL of 50% ethanol aqueous solution (V / V). The obtained 3-F-4-Fm-Ph-B(OH)2 solution was added dropwise to the above mixed solution at 800 rpm / min (in this step, the molar ratio of 3-F-4-Fm-Ph-B(OH)2, chitosan monomer and tannic acid is 0.2:1:0.013, and the concentration of Ag-NPs is 0.02 mg / mL) until gelation, and an injectable antibacterial hydrogel (denoted as CTF / Ag (0.02 mg / mL)) was obtained.

[0087] Comparative Example 1

[0088] Same as Example 1, except that no Ag-NPs solution was added, and only a hydrogel was obtained. The specific preparation method is as follows:

[0089] (1) Weigh 1g of CS (viscosity 100mPa.s, degree of deacetylation ≥95%, 6×10 -3 1 mol) was dissolved in 2% acetic acid solution (V / V, 49 mL), and stirred thoroughly at 50 °C to obtain a 2% volume concentration chitosan acetate solution. The chitosan acetate solution was then placed in a freeze dryer (-51 °C) for freeze drying until completely dry. The dried CS was weighed and dissolved in pure water to obtain a 2% volume concentration CS aqueous solution.

[0090] (2) Take 2.7 mg of TA (1.6 × 10⁻⁶) -6 Dissolve 1 mol) in 50 μL of pure water to obtain a TA solution, and take 1 mL of the 2% CS aqueous solution (1.2 × 10⁻⁶) obtained in step (1). -4 The solution was heated to 60°C on a magnetic stirrer and stirred at 800 rpm / min. The resulting TA solution was then added dropwise to a heated 2% CS aqueous solution and stirred for 1 h. Separately, 4.2 mg of 3-F-4-Fm-Ph-B(OH)2 (2.5 × 10⁻⁶ mol) was added. -53-F-4-Fm-Ph-B(OH)2 solution was obtained by dissolving 3-F-4-Fm-Ph-B(OH)2 in 100 μL of 50% ethanol aqueous solution (V / V). The obtained 3-F-4-Fm-Ph-B(OH)2 solution was added dropwise to the above mixed solution at 800 rpm / min (in this step, the molar ratio of 3-F-4-Fm-Ph-B(OH)2, chitosan monomer and tannic acid is 0.2:1:0.013) until gelation, and a hydrogel (denoted as CTF) was obtained.

[0091] Performance testing

[0092] 1. Fourier Transform Infrared Analysis

[0093] To demonstrate the successful synthesis of the hydrogel, Fourier transform infrared spectroscopy was used to analyze the changes in chemical bonds in the hydrogel (CTF / Ag) obtained in Example 1 and in the raw materials tannic acid, chitosan, and 3-fluoro-4-aldehyde phenylboronic acid. Figure 1 As shown in the figure. 3345cm -1 The absorption band at 3224 cm⁻¹ originates from the stretching vibration peak of -NH₂ in CS. -1 The absorption peak at 1546 cm⁻¹ is the absorption peak of -CHO of 3-F-4-Fm-Ph-B(OH)₂; the absorption peak of -NH₂ of CS and -CHO of 3-F-4-Fm-Ph-B(OH)₂ is at 1546 cm⁻¹. -1 A new peak of -C=N- appeared at 3288cm. -1 The stretching vibration peak of the band originates from the -OH group in TA, at 3224 cm⁻¹. -1 The absorption peak originates from the -BO group of 3-F-4-Fm-Ph-B(OH)2 at 1408 cm⁻¹. -1 The new peak that appears is the BOC formed by the -OH in TA and the -BO in 3-F-4-Fm-Ph-B(OH)2.

[0094] 2. Rheological characterization

[0095] 1) Rheological sweep frequency test: Using a rheometer, rheological sweep frequency tests were performed on the hydrogels (CTF / Ag, CTF) prepared in Example 1 and Comparative Example 1 to evaluate the response behavior of the hydrogels. The rheological sweep frequency plots of the storage modulus (G') and loss modulus (G") of the hydrogels in Example 1 and Comparative Example 1 as a function of angular frequency are shown in the figure. Figure 2 It can be seen that when the angular frequency (rad / s) is between 10.5 and 135, the storage modulus (G') is greater than the loss modulus (G") in different samples of hydrogel, indicating that the material is solid. When the angular frequency is between 135 and 250, the storage modulus (G') is less than the loss modulus (G"), and the material changes from solid to liquid.

[0096] 2) Viscosity test: The shear-thinning curves of the viscosity of the hydrogels (CTF, CTF / Ag) prepared in Example 1 and Comparative Example 1 as a function of shear rate are shown in the figure. Figure 3 It can be seen that when the shear rate (1 / s) ranges from 0.1 to 100, the viscosity of the hydrogel decreases with the increase of the shear rate, indicating that the hydrogel has shear thinning properties.

[0097] 3. Synthesis and characterization of Ag-NPs

[0098] To demonstrate the successful synthesis of Ag-NPs, the UV absorption peak of the Ag-NPs solution obtained in Example 1 was measured at 410 nm using a UV spectrophotometer. The results are shown in [Figure number missing]. Figure 4 .

[0099] The Ag-NPs solution synthesized in Example 1 was analyzed for particle size using a nanoparticle size and potential analyzer. Figure 5 As shown.

[0100] Depend on Figure 4 As can be seen, the characteristic peak at 410 nm proves the successful synthesis of Ag-NPs. Figure 5 It can be seen that the average particle size of the synthesized Ag-NPs is 40 nm.

[0101] 4. Characterization of the photothermal properties of Ag-NPs

[0102] 1) Photothermal properties of different Ag-NPs concentrations

[0103] Add 1 mL of pure water, 0.25 mg Ag-NPs (Ag-NPs particles obtained in Example 1, the same below) + 1 mL of pure water, 0.5 mg Ag-NPs + 1 mL of pure water, and 0.8 mg Ag-NPs + 1 mL of pure water to four 1.5 mL EP tubes respectively. Then use 808 nm, 1.0 W cm -2 The graph shows the change in solution temperature as the concentration of Ag-NPs increases after 15 minutes of laser irradiation. Figure 6 As can be seen, the temperature of the solution increases significantly with the increase of Ag-NPs concentration.

[0104] 2) Photothermal properties of hydrogels coated with Ag-NPs

[0105] The hydrogels (CTF / Ag and CTF) prepared in Example 1 and Comparative Example 1 were added to 1.5 mL EP tubes, respectively, and analyzed using 808 nm and 1.0 W cm⁻¹. -2 The photothermal properties of CTF and CTF / Ag hydrogel after 15 minutes of laser irradiation are shown in the figure below. Figure 7 As shown, the temperature of the hydrogel coated with Ag-NPs is significantly higher than that of the hydrogel without Ag-NPs.

[0106] 3) Stability of Ag-NPs photothermal cycling

[0107] 1 mL of the hydrogel (CTF / Ag) prepared in Example 1 was added to a 1.5 mL EP tube and incubated at 808 nm and 1.0 W / cm². -2 Under laser irradiation, the temperature was recorded every 50 seconds. After the temperature reached a plateau, the laser was turned off, and the temperature was recorded again every 50 seconds. This process was repeated four times. The stability results of the CTF / Ag hydrogel photothermal cycling are as follows: Figure 8 As shown, the hydrogel still exhibits good photothermal stability after four photothermal cycles.

[0108] The photothermal conversion efficiency test results of the hydrogel (CTF / Ag) prepared in Example 1 are shown in the figure. Figure 9 It can be seen that the hydrogel prepared in Example 1 has excellent photothermal conversion efficiency.

[0109] The aforementioned photothermal conversion efficiency ( ) Calculate according to the following formulas (1)-(3):

[0110] (1)

[0111] (2)

[0112] (3)

[0113] In the above formula, Let M be the thermal time constant, C be the mass of water, S be the specific heat capacity of water, S be the surface area of ​​the container, h be the heat transfer coefficient, and T be the thermal time constant. max T represents the highest temperature in the hydrogel's heating curve. RT Let Q be the temperature at room temperature, I be the heat generated when water is irradiated with near-infrared light, and A be the power of the near-infrared light. 808 It is the absorbance of the sample at 808 nm.

[0114] 5. Ag-NPs release test

[0115] Bacterial infection is a serious problem during wound healing, therefore, it is important to provide a method with long-lasting antibacterial properties to replace antibacterial agents. The accumulation and release behavior of Ag-NPs was evaluated in deionized water. The release rate of Ag-NPs in the CTF / Ag hydrogel of Example 1 from 0-72 h was as follows: Figure 10As shown in the figure, A is the standard curve of release; B is the release rate of AgNPs. It can be seen that the release of Ag-NPs is divided into two stages: a rapid release stage within 0-10 hours, with a release rate of 49%; and a slow release stage after 10-72 hours, with a release rate of 46%. Rapid release can kill bacteria at the wound site, while slow release can prevent bacterial growth.

[0116] 6. Antioxidant properties of hydrogels

[0117] Scavenging rates of ABTS free radicals by CTF and CTF / Ag hydrogels.

[0118] To demonstrate the antioxidant properties of the hydrogel, a commercially available 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) kit and 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonate diammonium salt (ABTS) were used to verify the hydrogel's antioxidant capacity.

[0119] (1) 2 mL of ABTS (7.00 mM) and 2 mL of potassium persulfate (4.95 mM) were reacted at room temperature in the dark for 12 h to obtain stable ABTS free radicals. The above ABTS solution was diluted to a concentration of 5% using PBS (0.1 mM, pH 7.4) buffer to obtain a stable ABTS free radical working solution.

[0120] (2) 5 mg of CTF, CTF / Ag (0.02 mg / mL), and CTF / Ag (0.06 mg / mL) hydrogels were respectively added to 500 μL of ABTS working solution as samples, and 500 μL of ABTS working solution without hydrogel was used as a control. The reaction was carried out in the dark at room temperature for 20 min, and the absorbance was measured at 734 nm using a UV spectrophotometer. The scavenging rates of CTF and CTF / Ag hydrogels against ABTS free radicals are shown in the figure below. Figure 11 As shown, A represents the absorbance of ABTS at 734 nm after incubation with each hydrogel sample for 20 min; B is a statistical graph of the free radical scavenging rate of ABTS by the hydrogel. The scavenging rate of ABTS free radicals by the hydrogel is calculated using formula (4).

[0121] (4)

[0122] In the formula, A control For the control, A sample This is the absorbance value of the sample.

[0123] according to Figure 11It can be seen that the scavenging rates of CTF, CTF / Ag (0.02 mg / mL), and CTF / Ag (0.06 mg / mL) hydrogels for ABTS free radicals are 91%, 97%, and 97%, respectively.

[0124] (3) 5 mg of CTF, CTF / Ag (0.2 mg / mL), and CTF / Ag (0.06 mg / mL) hydrogels were added to 500 μL of a commercially prepared DPPH solution as samples, and 500 μL of DPPH solution without hydrogel was used as a control. The reaction was carried out in the dark at room temperature for 20 min, and the absorbance was measured at 518 nm using a UV spectrophotometer. The scavenging rates of CTF and CTF / Ag hydrogels on DPPH free radicals were as follows: Figure 12 As shown, A is the absorbance of DPPH at 518 nm after incubation with each hydrogel sample for 20 min; B is a statistical graph of the free radical scavenging rate of DPPH by the hydrogel. The scavenging rate of DPPH free radicals by the hydrogel is calculated using formula (5).

[0125] (5)

[0126] In the formula, A control For the control, A sample This is the absorbance value of the sample.

[0127] according to Figure 12 It can be seen that the scavenging rates of CTF, CTF / Ag (0.02 mg / mL), and CTF / Ag (0.06 mg / mL) hydrogels on DPPH free radicals are 85%, 82%, and 74%, respectively.

[0128] 7. Swelling properties of hydrogels

[0129] The swelling properties and volumetric growth rate of hydrogels are important for promoting wound healing because hydrogels can absorb exudate at the wound site, which is beneficial for wound integration. The swelling properties of CTF and CTF / Ag hydrogels are shown in the figure below. Figure 13 As shown in the figure, A represents the swelling volume change of CTF and CTF / Ag hydrogels from 0 to 24 hours, and B represents the swelling rate statistics of CTF and CTF / Ag hydrogels from 0 to 48 hours. It can be seen that within 0-24 hours, the volume of the hydrogel gradually increases with time. The swelling rates of CTF and CTF / Ag are 778.68% and 862.60%, respectively, indicating that the hydrogels possess good swelling properties.

[0130] 8. In vitro antibacterial test of hydrogel

[0131] The bacterial culture was incubated at a volume ratio of 1:100 (V / V) with Luria-Bertani (LB) liquid medium in a shaker at 37°C and 200 rpm / min for 7 hours.

[0132] The incubated bacterial culture was washed three times with sterile PBS and then diluted again with PBS to a concentration of 10. 6 CFU / mL available for use.

[0133] 1 mL of CTF / Ag hydrogel was sterilized by irradiating it under UV light for 30 minutes, and then placed in a sterile EP container.

[0134] 10 6 CFU / mL (3 mL) bacterial suspension was added to EP tubes without hydrogel, EP tubes containing CTF, and EP tubes containing CTF / Ag hydrogel, and then incubated in a 37℃ constant temperature incubator for 4 hours.

[0135] After incubation for 4 hours, take 100 μL of bacterial culture from each EP tube and dilute to 10. 4 CFU / mL was used for plating.

[0136] The petri dishes were incubated in a 37°C incubator for 24 hours, and the results were recorded.

[0137] Figure 14 The results of plate counting experiments on hydrogels under NIR (+NIR) and no NIR (-NIR) conditions are shown. A represents plate colony diagrams of *S. haemolyticus*, *S. epidermidis*, *E. coli*, and *S. aureus* under NIR and no NIR conditions, while B is a statistical graph of the survival rates of *S. haemolyticus*, *S. epidermidis*, *E. coli*, and *S. aureus*. Based on... Figure 14 It can be seen that the number of colonies in the PBS group did not change significantly regardless of whether it was exposed to light. Compared with the PBS group, the number of colonies in the CTF group without AgNPs was slightly reduced, while the number of colonies in the CTF / Ag-NPs group was significantly reduced after light exposure. The antibacterial rates against S. haemolyticus, S. epidermidis, E. coli and S. aureus were 97%, 99%, 99% and 99%, respectively.

[0138] 8. Antibacterial zone test

[0139] The antibacterial properties of the hydrogels were evaluated using *S. haemolyticus*, *S. epidermidis*, *E. coli*, and *S. aureus*.

[0140] The bacterial culture was incubated at a volume ratio of 1:100 (V / V) with Luria-Bertani (LB) liquid medium in a shaker at 37°C and 200 rpm for 7 hours.

[0141] The incubated bacterial culture was washed three times with sterile PBS, and then diluted to 10 with PBS. 6 CFU / mL.

[0142] The CTF and CTF / Ag hydrogels were sterilized by irradiating them under ultraviolet light for 30 minutes.

[0143] Slowly pour 10 ml of LB agar medium into a sterile petri dish and wait for it to cool and solidify.

[0144] 10 6 Add the bacterial culture at CFU / mL (100μL) to LB agar medium and disperse it evenly.

[0145] Use sterile forceps to place CTF and CTF / Ag onto the culture medium containing bacterial solution.

[0146] The petri dishes were placed in a 37°C incubator for 24 hours and the results were recorded.

[0147] Plate inhibition zone diagrams of CTF and CTF / Ag hydrogels against *S. haemolyticus*, *S. epidermidis*, *E. coli*, and *S. aureus* are shown below. Figure 15 It can be seen that due to the inherent antibacterial properties of TA, the CTF group showed an inhibition zone, while the CTF / Ag group containing AgNPs had a larger inhibition zone.

[0148] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for preparing an injectable antibacterial hydrogel, characterized in that, Includes the following steps: Chitosan aqueous solution was heated and tannic acid solution was added dropwise. After stirring, silver nanoparticle solution was added. Then, 3-fluoro-4-aldehyde phenylboronic acid solution was added dropwise to obtain the injectable antibacterial hydrogel. The preparation method of the chitosan aqueous solution is as follows: chitosan is dissolved in acetic acid solution, and after stirring, a chitosan acetic acid solution with a volume concentration of 2% is obtained. Then, the chitosan acetic acid solution is freeze-dried and then dissolved in water to obtain the chitosan aqueous solution. The silver nanoparticle solution was synthesized using tannic acid, trisodium citrate, and silver nitrate as raw materials.

2. The method for preparing the injectable antibacterial hydrogel according to claim 1, characterized in that, The concentration of the tannic acid solution is 1-2.7 mg / 50 μL, and the volume concentration of the chitosan aqueous solution is 2%.

3. The method for preparing the injectable antibacterial hydrogel according to claim 1, characterized in that, The 3-fluoro-4-aldehyde phenylboronic acid solution is prepared by dissolving 3-fluoro-4-aldehyde phenylboronic acid in an aqueous ethanol solution.

4. The method for preparing the injectable antibacterial hydrogel according to claim 3, characterized in that, The concentration of the 3-fluoro-4-aldehyde phenylboronic acid solution is 4.2 mg / 100 μL.

5. The method for preparing the injectable antibacterial hydrogel according to claim 1, characterized in that, The concentration of the silver nanoparticle solution is 1.21 mg / mL, and the mass ratio of tannic acid, trisodium citrate and silver nitrate in the silver nanoparticle solution is 8.6:13.7:8.

6.

6. The method for preparing the injectable antibacterial hydrogel according to claim 1, characterized in that, In the 3-fluoro-4-aldehyde phenylboronic acid, chitosan aqueous solution and tannic acid solution, the molar ratio of 3-fluoro-4-aldehyde phenylboronic acid, chitosan monomer and tannic acid is 0.2:1:0.

013.

7. The method for preparing the injectable antibacterial hydrogel according to claim 1, characterized in that, The volume ratio of the silver nanoparticle solution to the chitosan aqueous solution is 20-60 μL: 1 mL.

8. An injectable antibacterial hydrogel, characterized in that, It is prepared according to any one of claims 1-7.

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