A mussel-mimic bacterial-responsive injectable hydrogel and preparation method and application thereof

Abstract

The application discloses a mussel-mimic bacterial-responsive injectable hydrogel and a preparation method and application thereof, and belongs to the technical field of biological medicines. The hydrogel is composed of a mussel-mimic viscous hydrogel support and a bacterial response agent; the viscous hydrogel support is QCS-EC, which is formed by non-covalent combination of quaternary ammonium chitosan QCS and epigallocatechin gallate EGCG; and the bacterial response agent is a supramolecular nanometer cross-linking compound TEBox-B 12， which is self-assembled by hydrogen bonds and electrostatic interactions. 12 H 12 2‑ The hydrogel of the application has NIR-II photothermal and photodynamic antibacterial treatment functions, can realize efficient killing of bacteria in deep tissues, has high adhesion, good biocompatibility and injectability, is convenient and applicable, can provide a good healing environment for a wound, promotes fibroblast proliferation and migration, accelerates wound tissue regeneration and healing process, and reduces scar formation.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a mussel-inspired bacterial responsive injectable hydrogel, its preparation method, and its application. Background Technology

[0002] Invasive infected wounds are a severe pathogenic state caused by the colonization of pathogenic microorganisms in deep tissues. They are characterized by rapid disease progression, a tendency to trigger excessive immune responses leading to septic shock and even death, and are accompanied by local organ dysfunction, severe ulceration, and chronic wounds that are difficult to heal. These wounds are often accompanied by strong systemic inflammatory responses, immune dysfunction, impaired angiogenesis, and an increased risk of multidrug-resistant bacteria, significantly hindering the healing process and imposing a heavy burden on patients and society. Therefore, there is an urgent need to develop a multifunctional intelligent dressing that can effectively isolate the wound, monitor infection dynamics in real time, synergistically eliminate resistant microorganisms, and promote tissue regeneration. However, traditional dressings such as gauze or basic hydrogels only provide a passive physical barrier, lacking integrated functions such as active antibacterial activity, real-time infection monitoring, and healing promotion, and cannot meet the treatment needs of complex infected wounds. Furthermore, the increased bacterial resistance due to antibiotic overuse further complicates infection management. Developing an integrated intelligent treatment strategy that can accurately detect the microenvironment of invasive infected wounds, enabling non-invasive removal of deep pathogens and effective regeneration of wound tissue, is of vital clinical significance and urgent practical need. This strategy can achieve real-time monitoring of infection while avoiding systemic side effects, providing a promising technical path for the precision treatment of invasive infected wounds.

[0003] Inspired by the adhesion mechanism of mussels, hydrogel dressings mimicking the high adhesiveness of mussel tentacle filaments have become a research hotspot for enhancing tissue adhesion and accelerating wound healing. Mussel foot proteins are rich in polyphenols, and their catechol groups can achieve strong underwater adhesion to different surfaces through various mechanisms such as hydrogen bonds and coordination bonds, providing an effective approach for constructing biomimetic hydrogels that closely adhere to the skin and maintain a moist healing environment. In existing research, epigallocatechin gallate (EGCG), a natural antibacterial polyphenol with a similar chemical structure to mussels, can form hydrogen bonds with skin proteins and lipids, improving the adhesion and antibacterial effect of dressings. Chitosan (CS) is an ideal base material due to its good solubility under weakly acidic conditions. Furthermore, the positively charged free amino groups on the quaternized chitosan (QCS) molecular chain can bind to polyphenol molecules through electrostatic interactions and hydrogen bonds, further enhancing the adhesion properties of the hydrogel. Despite the advantages of the aforementioned materials in specific properties, existing mussel-inspired hydrogels still face significant technical bottlenecks: their functions are relatively limited, lacking the ability to monitor specific responses to bacteria in the infected microenvironment and the ability to synergistically fight bacteria through multiple mechanisms; they are difficult to achieve efficient penetration and non-invasive removal of stubborn bacteria that have colonized deep within the wound; and in complex infected microenvironments, their adhesion strength and structural integrity are easily affected, lacking an intelligent response mechanism based on supramolecular dynamic regulation, which limits their practical application in invasive infected wounds.

[0004] In recent years, supramolecular self-assembly multifunctional biomimetic nanomaterials have shown broad clinical application prospects due to their dynamic reversibility and environmental responsiveness. Among them, the "liquid-free effect" is an important mechanism driving their self-assembly. This mechanism can significantly affect the solubility, aggregation state, and self-assembly behavior of biomolecules by adjusting the type and concentration of ions, thereby precisely controlling the microstructure and macroscopic properties of the material. However, research on introducing the liquid-free effect-driven mechanism into mussel-inspired quaternized chitosan / polyphenol hydrogel systems to construct integrated smart dressings with precise bacterial response, deep pathogen clearance, and real-time monitoring functions is still insufficient. How to overcome the technical defects of existing mussel-inspired hydrogels, such as unstable adhesion, incomplete antibacterial effect, and lack of real-time infection monitoring, and how to optimize the supramolecular self-assembly process using the liquid-free effect to develop a novel smart hydrogel for precise diagnosis and treatment and tissue repair of invasive infected wounds, remains an urgent problem to be solved in this field.

[0005] To address the problem that traditional dressings in the prior art have limited functions and cannot simultaneously achieve real-time infection monitoring, efficient removal of bacteria from deep tissues, and effective promotion of wound healing, this invention aims to provide an injectable hydrogel that can firmly adhere to wound tissue, enable real-time visual monitoring of infection, and efficiently remove bacteria and promote epithelialization and wound healing through multimodal synergistic effects. Summary of the Invention

[0006] The first objective of this invention is to provide an injectable hydrogel that mimics mussel bacteria responsiveness, and the second objective of this invention is to provide applications of the said injectable hydrogel that mimics mussel bacteria responsiveness.

[0007] The first objective of this invention is achieved as follows: a mussel-inspired bacterial responsive injectable hydrogel, wherein the hydrogel is composed of a mussel-inspired adhesive hydrogel scaffold and a bacterial responsive agent; the adhesive hydrogel scaffold is QCS-EC, formed by the non-covalent bonding of quaternized chitosan (QCS) and epigallocatechin gallate (EGCG); the bacterial responsive agent is a supramolecular nano-crosslinked complex TEBox-B. 12， The TEBox is a box-shaped quaternary ammonium pyridine heterocyclic organic salt molecule containing superionized liquid anion B. 12 H 12 2- Salts are formed through self-assembly via hydrogen bonding and electrostatic interactions.

[0008] The method for preparing the mussel-inspired bacterial responsive injectable hydrogel is to mix a 5% QCS-EC aqueous solution with TEBox-B. 12 The solutions were mixed at a volume ratio of 1:1 and stirred at room temperature until a gel was formed.

[0009] The second objective of this invention is achieved by applying the mussel-inspired bacterial responsive injectable hydrogel to wound infection monitoring.

[0010] The principle of this invention is as follows:

[0011] 1. Inspired by the characteristics of mussels, this invention utilizes hydrogen bonding to non-covalently combine the traditional catechin polyphenol antibacterial agent EGCG with the high molecular weight polysaccharide chain QCS to form a functionalized hydrogel framework QCS-EC, thereby improving the viscosity of the hydrogel and enabling it to adhere tightly to the tissue surface.

[0012] 2. This invention utilizes the liquid ionization effect to dissociate superionized liquid anions B. 12 H 12 2- The TEBox-B supramolecular self-assembled complex is formed by the tight binding of the box-shaped quaternary ammonium pyridine heterocyclic organic salt molecule TEBox. 12 This complex not only possesses the photodynamic therapy (PDT) effect to generate ROS to clear bacteria, but it can also be reduced in situ by bacteria to generate stable organic free radicals for real-time infection monitoring and near-infrared II (NIR-II) photothermal therapy (PTT). The multi-effect synergy not only effectively clears stubborn bacteria from infected wounds, but also promotes rapid wound healing.

[0013] 3. This invention utilizes the supramolecular self-assembled complex TEBox-B 12By combining the QCS-EC nano-crosslinking agent with the functionalized hydrogel framework, a bacterial-responsive multifunctional antibacterial hydrogel, MQCEC-TEB Gel, was constructed. This hydrogel enables local non-invasive treatment, enhanced antibacterial effects, real-time infection monitoring, and photothermal synergistic multimodal bactericidal action, while also promoting rapid wound healing. The quaternized TEBox serves as the core functional unit, through which this invention constructs a dual self-assembly synergistic gelation system.

[0014] The first self-assembly is the self-assembly of quaternized TEBox and boron clusters through the liquid detachment effect to form functionalized nanomaterials, which serve as the basis of the nanoskeleton of the hydrogel.

[0015] The second stage of self-assembly utilizes the inherent cavity structure of the TEBox macroring to drive the system to achieve a sol-gel transition through specific self-assembly with guest molecules via host-guest interactions.

[0016] The two self-assembly layers work together to achieve controllable preparation of hydrogels and endow the gels with superior structural stability and functional tunability.

[0017] The beneficial effects of this invention are as follows:

[0018] 1. The mussel-inspired bacterial responsive hydrogel provided by this invention can realize real-time infection monitoring of invasive wounds. By observing the color change that occurs when the hydrogel comes into contact with bacteria, the infection can be detected in a timely manner, providing a basis for early diagnosis.

[0019] 2. The hydrogel of this invention has both NIR-II photothermal (PTT) and photodynamic (PDT) antibacterial therapeutic functions, which can achieve efficient killing of bacteria in deep tissues and help alleviate the problem of bacterial resistance caused by antibiotic abuse.

[0020] 3. The hydrogel of this invention has high adhesion and good biocompatibility, which can provide a good healing environment for wounds, promote fibroblast proliferation and migration, accelerate wound tissue regeneration and healing process, and reduce scar formation.

[0021] 4. The hydrogel of this invention has excellent injectability and is suitable for wounds of different locations and shapes, making it convenient and highly applicable. Attached Figure Description

[0022] Figure 1 The glycosidic bond structure of QCS in Example 1 and 1 H NMR spectrum (D2O, 600MHz);

[0023] Figure 2 For QCS, EGCG and QCS-EC in Example 1 1 H NMR spectrum;

[0024] Figure 3 TEBox-B in Example 5 12 Schematic diagram of nano-crosslinked structure and self-assembly; A represents TEBox and B 12 H 12 2- A schematic diagram of the molecular structure; B and C are TEBox and B 12 H 12 2- There are two types of hydrogen bond interactions between them: Region I and Region II; D is TEBox-B. 12 Transmission electron microscopy (TEM) image; E for TEBox-B 12 Dynamic light scattering (DLS) particle size distribution map; F is the transmission electron microscope (TEM) image of MQCEC-TEB gel; G is the TEBox and B... 12 H 12 2- and TEBox-B 12 Powder X-ray diffraction (PXRD) pattern; H represents the energy dispersive X-ray (EDX) mapping of the MQCEC-TEB gel;

[0025] Figure 4 Images and mechanical properties of MQCEC-TEB gel from Example 6; A represents QCS solution, QCS-EC solution, and TEBox-B. 12 Image A shows the solution and MQCEC-TEB gel (light yellow) under natural light; Image B shows scanning electron microscope (SEM) images (scale bar = 100 μm) of QCS, QCS-EC solution and MQCEC-TEB gel; Image C shows the strain scan of MQCEC-TEB gel; Image D shows the frequency scan of MQCEC-TEB gel; Image E shows the viscosity measurement results of MQCEC-TEB gel; Image F shows the results of QCS, EGCG, QCS-EC, TEBox, and B solutions. 12 H 12 2- TEBox-B 12 Fourier transform infrared (FTIR) spectrum of MQCEC-TEB gel; G represents the injectability of MQCEC-TEB gel (dye: methylene blue); H represents the bending of MQCEC-TEB hydrogel at different angles.

[0026] Figure 5 The in vitro NIR-II photodynamic therapy of MQCEC-TEB gel was used to detect the antibacterial effect; F represents the detection of ABDA in different treatment groups of Case 4 at 50mW / cm 2 Fluorescence intensity change curve after 10 minutes of NIR-II 1064nm laser irradiation; G is 50mW / cm²2 Electron paramagnetic resonance spectra of MQCEC-TEB gel before and after 10 minutes of NIR-II 1064nm laser irradiation; H is 50mW / cm². 2 Images of Escherichia coli and Staphylococcus aureus colonies after 10 minutes of NIR-II 1064nm laser irradiation; I and J show the bacterial survival rates of Escherichia coli and Staphylococcus aureus, respectively (n=4, ***p<0.001).

[0027] Figure 6 This study aims to monitor the in vitro real-time infection activity and NIR-II PTT antibacterial activity of MQCEC-TEB gel. A shows a schematic diagram of bacterial in-situ reduction electron transport (ET) and a color-changing photograph of the real-time monitoring. B shows the UV-Vis-NIR absorption spectra of QCS, QCS-EC, and MQCEC-TEB gels before and after in-situ reduction by Staphylococcus aureus at different time points. C and D show the absorption spectra of MQCEC-TEB gel after 10 minutes of NIR-II laser irradiation (1064 nm, power density 1.0 W / cm²) with Staphylococcus aureus. 2 E shows the restored infrared thermal image and corresponding photothermal curve; E represents the photothermal stability of the MQCEC-TEB gel; F shows colony images of Escherichia coli and Staphylococcus aureus; G and H show Escherichia coli and Staphylococcus aureus after 10 minutes of 1064nm NIR-II laser irradiation (power density 1.0W / cm²). 2 Bacterial survival rate results under conditions of n=4 and p<0.001;

[0028] Figure 7 The curve showing the change in water retention rate of MQCEC-TEB gel over time at room temperature and atmospheric pressure (n=3). Figure 8 After incubating the Staphylococcus aureus solution for 6 hours, QCS, QCS-EC, TEBox, and TEBox-B were tested. 12 Electron paramagnetic resonance (EPR) spectra of MQCEC-TEB gel;

[0029] Figure 9 The fluorescence emission spectra (ae) of ABDA under irradiation with 1064 nm laser in the near-infrared region II at different irradiation times and different TEBox concentrations (0, 5, 10, 20 and 50 μM) are shown, as well as the normalized fluorescence intensity change curves (f) of different groups during the irradiation process.

[0030] Figure 10 The MQCEC-TEB gel was irradiated with a 1064nm near-infrared laser (power density 1.0W / cm²) at different time points. 2 The electron paramagnetic resonance spectrum after ( );

[0031] Figure 11 To demonstrate the properties of MQCEC-TEB gel under 1064 nm laser irradiation in the near-infrared II region. 1 O2 content change curve with irradiation time;

[0032] Figure 12 Fluorescence imaging of bacteria in Example 4 after blank dark treatment / light treatment (PDT) and MQCEC-TEB gel dark treatment (scale bar: 20 μm).

[0033] Figure 13 SEM images of Escherichia coli and Staphylococcus aureus after different treatments (blank dark treatment / light treatment group (PDT)) in Example 4 (scale bar: 500nm). Detailed Implementation

[0034] The present invention will be further described below with reference to embodiments, but this is not intended to limit the present invention in any way. Any modifications or substitutions made based on the teachings of the present invention shall fall within the protection scope of the present invention.

[0035] This invention discloses a mussel-inspired bacterial responsive injectable hydrogel, comprising a mussel-inspired adhesive hydrogel scaffold and a bacterial responsive agent; the adhesive hydrogel scaffold is QCS-EC, formed by the non-covalent bonding of quaternized chitosan (QCS) and epigallocatechin gallate (EGCG); the bacterial responsive agent is a supramolecular nano-crosslinked complex TEBox-B. 12， The TEBox is a box-shaped quaternary ammonium pyridine heterocyclic organic salt molecule containing superionized liquid anion B. 12 H 12 2- Salts are formed through self-assembly via hydrogen bonding and electrostatic interactions.

[0036] The QCS-EC is formed by QCS and EGCG under pH 6.0-7.0 conditions.

[0037] TEBox and superionized liquid anion B 12 H 12 2- The molar ratio of salts is 1:2.

[0038] This invention further provides a method for preparing the aforementioned mussel-inspired bacterial responsive injectable hydrogel, which involves mixing a 5% QCS-EC aqueous solution with TEBox-B... 12 The solutions were mixed at a volume ratio of 1:1 and stirred at room temperature until a gel was formed.

[0039] The present invention further provides the application of the aforementioned mussel-inspired bacterial responsive injectable hydrogel in wound infection monitoring.

[0040] This invention further provides the application of the aforementioned mussel-inspired bacterial responsive injectable hydrogel in the preparation of near-infrared synergistic anti-infective therapy.

[0041] The present invention further provides the application of the aforementioned mussel-inspired bacterial responsive injectable hydrogel in promoting the repair of infected wounds.

[0042] Example 1: Preparation of QCS-EC hydrogel scaffold

[0043] Preparation method: Weigh a certain amount of QCS powder, add deionized water, stir until completely dissolved, and prepare a QCS solution with a mass concentration of 5%. Weigh an appropriate amount of EGCG powder, add deionized water to dissolve, and prepare an EGCG solution with a concentration of 30 μg / mL.

[0044] QCS solution and EGCG solution were mixed at a volume ratio of 1:1. The pH of the mixed solution was adjusted to 6.5 with hydrochloric acid or sodium hydroxide. The mixture was stirred at room temperature for 2 hours to obtain QCS-EC solution. The QCS-EC solution was centrifuged at 4℃ and 4500rpm for 10 minutes to remove insoluble matter. The supernatant was then lyophilized to obtain white QCS-EC powder.

[0045] 1 H nuclear magnetic resonance (H nuclear magnetic resonance) 1 ¹H NMR analysis: QCS, EGCG, and QCS-EC were analyzed. 1 H NMR characterization, results showed ( Figure 1-2 EGCG shows a characteristic resonance peak at δ≈7.5 to 2.5 ppm, QCS shows a characteristic resonance peak at δ≈5 to 2 ppm, while QCS-EC shows a characteristic resonance peak at δ≈5 to 2 ppm. 1 The H NMR spectrum showed characteristic resonance peaks of EGCG and QCS simultaneously, with some peak positions slightly shifted, indicating that hydrogen bond interaction was formed between EGCG and QCS, and QCS-EC was successfully prepared.

[0046] Rheological property testing: The viscosity of the QCS-EC solution was tested using a rheometer under the following conditions: temperature 25℃, shear rate 1s. -1 The results showed that the viscosity of the QCS-EC solution was 73 Pa·s, which met the requirements for a highly adhesive hydrogel scaffold. It could enhance the adhesion of the hydrogel to the wound and promote cell proliferation and migration.

[0047] Scanning electron microscopy (SEM) observation: Lyophilized QCS-EC powder was redissolved in deionized water to form a thin film, which was then sputter-coated with gold and its microstructure was observed using SEM. The results showed ( Figure 4B) QCS-EC exhibits a distinct sheet-like structure, which is beneficial for increasing the specific surface area of ​​the hydrogel, enhancing its adsorption capacity and contact area with wound tissue, and providing favorable conditions for wound healing.

[0048] Example 2

[0049] The QCS-EC solution was prepared according to the method in Example 1, except that the concentration of EGCG in the EGCG solution was 10 μg / mL, and the other operations were the same as in Example 1.

[0050] Example 3

[0051] The QCS-EC solution was prepared according to the method in Example 1, except that the concentration of EGCG in the EGCG solution was 20 μg / mL, and the other operations were the same as in Example 1.

[0052] Example 4

[0053] The QCS-EC solution was prepared according to the method in Example 1, except that the concentration of EGCG in the EGCG solution was 40 μg / mL, and the other operations were the same as in Example 1.

[0054] Example 5 TEBox-B 12 Preparation of bacterial response agents

[0055] Preparation process: TEBox (TTzExBox·4Cl) was synthesized according to conventional methods; 120.73 μg (137.80 μmol) of TEBox was accurately weighed and dissolved in 1 mL of deionized water to obtain a TEBox solution with a concentration of 137.80 μmol / mL (137.80 mmol / L); simultaneously, a TEBox solution with a concentration of 275.61 μmol / L (using B...) was prepared. 12 H 12 2- (Calculation) Na2B 12 H 12 Aqueous solution, so that B 12 H 12 2- The molar ratio of B to TEBox is 2:1. Then, add 1 mL of the above B to a 50 mL centrifuge tube. 12 H 12 2- The solution was mixed with TEBox solution and gently stirred at room temperature until fully mixed to obtain turbid TEBox-B. 12 Solution (total volume 2 mL), containing TEBox and B 12 H 12 2-The final concentrations were 68.90 μmol / mL and 137.80 μmol / mL, respectively. The resulting solutions were concentrated under reduced pressure to obtain a pale yellow solid powder, TEBox-B. 12 .

[0056] Transmission electron microscopy (TEM) observation: Take a small amount of TEBox-B 12 The solution was dropped onto a copper grid, stained with phosphotungstic acid, and its morphology and size were observed using TEM. The results showed ( Figure 3 D), TEBox-B 12 The nanostructures form regular cubic structures through self-assembly via the liquid-free effect. The uniform nanostructures facilitate dispersion in the hydrogel, ensuring the uniformity of the hydrogel's properties.

[0057] Powder X-ray diffraction (PXRD) analysis: for TEBox, containing B 12 H 12 2- Salt and TEBox-B 12 PXRD analysis was performed on the lyophilized powder. The results are as follows: Figure 3 As shown in G, TEBox and containing B 12 H 12 2- Each salt has its own characteristic diffraction peaks, while TEBox-B 12 New diffraction peaks appeared in the PXRD pattern, and no TEBox or B-containing peaks were observed. 12 H 12 2- The characteristic diffraction peaks of salt indicate that TEBox and B 12 H 12 2- A new TEBox-B was successfully formed through self-assembly via non-covalent interactions. 12 The structure does not undergo a phase transition during self-assembly.

[0058] Example 6: Preparation of MQCEC-TEB Gel Injectable Hydrogel

[0059] Preparation method: The white QCS-EC powder obtained by freeze-drying in Example 1 was dissolved in deionized water to prepare a 5% (w / w) QCS-EC solution. Subsequently, the TEBox-B prepared in Example 5 was... 12 The solution was mixed with the QCS-EC solution at a volume ratio of 1:1, and stirred at room temperature for 1 hour. The sol-gel transition of the solution was observed until an injectable MQCEC-TEB gel hydrogel was formed. This hydrogel was light yellow, uniform, and transparent. Figure 4 A).

[0060] MQCEC-TEB Gel hydrogel performance testing:

[0061] 1. Injectability Test: 5 mL of MQCEC-TEB Gel hydrogel was placed into a 5 mL syringe. At room temperature, the syringe plunger was pushed to release the contents of the syringe. Figure 4 As can be seen from G, the MQCEC-TEB Gel hydrogel can be continuously and smoothly extruded from the syringe needle and retains its gel state after extrusion, indicating that it has good injectability and is easy to apply to wound sites.

[0062] 2. Rheological property testing: Strain scanning, frequency scanning, and viscosity testing of the MQCEC-TEB gel hydrogel were performed using a rheometer. The strain scanning experiment showed that the hydrogel point of the MQCEC-TEB gel was approximately 637%. Figure 4 C). Further dynamic frequency scanning results ( Figure 4 D) shows that as the frequency gradually increases from 0.1 Hz to 10 Hz, G' (≈870.4 Pa) is consistently higher than G" (≈161.4 Pa), indicating that the MQCEC-TEB gel exhibits dominant elastic behavior. Notably, when the shear rate increases from 0.1 s⁻¹... -1 Increase to 100s -1 The decrease in viscosity of the MQCEC-TEB gel at that time confirmed the injectability of the hydrogel. Figure 4 E).

[0063] 3. Swelling Performance Test: The freeze-dried hydrogel sample (W0) was immersed in phosphate-buffered saline (PBS) and incubated at 37°C until swelling equilibrium was reached. During the swelling process, the hydrogel was removed at preset time intervals, and residual liquid on the surface was absorbed with moistened filter paper. Its wet weight (W1) was then measured using an electronic balance (AUW120D, Japan). The swelling rate was calculated using the following formula: Swelling rate % = [(W1 - W0) / W0] × 100%;

[0064] In the formula, W1 is the mass of the hydrogel sample after swelling, and W0 is the mass of the initial sample.

[0065] The results showed that the hydrogel gradually swelled in PBS, reaching equilibrium swelling after 180 min, with an equilibrium swelling rate of approximately 3250% (32.5 g / g).

[0066] 4. Water-holding capacity test: The freeze-dried hydrogel sample was pre-immersed in PBS until swelling equilibrium (denoted as W0). The sample that had reached swelling equilibrium was then transferred to a 37°C oven, and its mass (W0) was measured at different time points. d The water retention rate of the hydrogel is calculated using the following formula: Water retention rate % = [W d / W0]×100%; where W d W0 represents the mass of the hydrogel sample at a specified time point, where W0 is the initial mass of the hydrogel.

[0067] The results are as follows Figure 7 As shown, the MQCEC-TEB Gel has suitable water retention and release characteristics: it can quickly release some water in the early stage to form a moist microenvironment at the application site; in the later stage, it can maintain a stable water retention capacity, providing continuous humidity conditions for wound repair or local treatment, while matching the biodegradation behavior of the material, which meets the application requirements of medical biomaterials.

[0068] 5. Degradation performance testing

[0069] Experimental methods: The lyophilized MQCEC-TEB Gel was prepared into samples of uniform size (consistent in mass and size); the samples were immersed in PBS buffer at pH 7.4 and placed in a constant temperature environment of 37℃ (simulating the human physiological detection cycle: different time nodes were set (focusing on monitoring degradation within 5 days), and the samples were taken out periodically; the degradation process was judged by weighing the remaining mass of the sample, observing the changes in appearance and morphology, and observing the integrity of the internal structure of the gel by SEM.

[0070] Results: Degradation process: Initial stage (1-2 days): The gel surface gradually swells and softens, some network structures begin to disintegrate, and the mass slowly decreases;

[0071] Mid-term (3-4 days): The main structure of the gel disintegrates, forming dispersed tiny particles, and the mass is rapidly lost;

[0072] Later stage (5 days): Most of the gel particles dissolve or degrade, leaving only a small amount of soluble products, which meets the application requirements of "short-term degradation and no accumulation" for biomedical materials.

[0073] In summary, MQCEC-TEB Gel exhibits rapid degradation characteristics, achieving near-complete degradation within 5 days of immersion in PBS buffer with no significant solid residue.

[0074] 6. Fourier Transform Infrared (FTIR) Spectroscopy Test: Verification of QCS-EC and TEBox-B 12 Interaction and network formation with MQCEC-TEB gel. In TEBox-B 12 B was observed in MQCEC-TEB hydrogel 12 H 12 2- (2472cm) -1 ), TEBox (3030cm) -1 ) and QCS (1660cm) -1 ) characteristic peaks ( Figure 4 F). C–H signal of the TEBox pyrazine ring (from 3030 to 3020 cm⁻¹) -1 ) and B 12 H12 2- BH signal (from 2472 to 2445 cm) -1 Significant displacements were observed in all samples, indicating that C–H···H–B dihydrogen bonds were formed through the liquid detachment effect.

[0075] 7. Adhesion performance test

[0076] I. Basic Adhesion Strength Test

[0077] The adhesion strength of MQCEC-TEB Gel hydrogel to pigskin was tested using an overlap shear test. Pigskin was cut into strips of 2cm × 5cm. The hydrogel was evenly applied to one end of one strip (1cm × 1cm area), and then another strip of pigskin was placed on top of the hydrogel. A pressure of 5N was applied and held for 10 minutes. Tensile testing was performed using a universal testing machine at a speed of 5mm / min, and the maximum peel force was recorded. The adhesion strength was calculated (adhesion strength = maximum peel force / adhesion area).

[0078] The results showed that the adhesion strength of MQCEC-TEB Gel hydrogel to pigskin was approximately 15 kPa, higher than that of traditional hydrogel dressings, indicating its excellent adhesion properties. It can closely adhere to wound tissue and prevent detachment during activity. Furthermore, macroscopic adhesion tests revealed that the hydrogel firmly adhered to various substrates, including glass, ceramics, cotton, wood, PTFE, stainless steel, and skin, further demonstrating its superior adhesion ability.

[0079] II. Dynamic Adhesion Performance Test

[0080] (1) Experimental subjects: healthy volunteer fingers (or biocompatible substrates that simulate skin, with human finger joints as the core of the experiment). Before the experiment, the finger joint surface was cleaned with physiological saline to remove grease and impurities and then air-dried.

[0081] (2) Experimental method: TEBox-B 12 Saturated solution was added dropwise to 2 mL of QCS-EC sol (25℃), and stirred rapidly for 30 min. The mixture was then allowed to stand at 25℃ for 1 day to form a homogeneous, injectable hydrogel. The hydrogel was cut into uniformly sized sheets (approximately 2 mm thick and 1 cm × 1 cm in area) for later use. The prepared MQCEC-TEB gel sheets were directly applied to the volunteer's finger joints (selecting the proximal interphalangeal joint of the middle or index finger, ensuring the application area completely covers the joint's movement area). Sterile forceps were used to gently press for 10 seconds to ensure full contact between the hydrogel and the skin surface, removing any air bubbles.

[0082] (3) Angle control and observation:

[0083] Initial state: Keep the fingers naturally straight (0°) and record the adhesion between the hydrogel and the finger joint (no detachment, no displacement);

[0084] Gradient bending: Precisely control the bending angle of your fingers using a protractor, adjusting them sequentially to 90°, 120°, and 135°, holding each angle for 30 seconds;

[0085] Dynamic verification: At each bending angle, gently move the finger joints (small-amplitude flexion and extension 2-3 times) and observe whether the hydrogel detaches, lifts or shifts;

[0086] Recording method: Take front and side photos from every angle using a digital camera to preserve evidence of the adhesion status.

[0087] (4) Repeat and control experiments

[0088] Repeated experiment: The same volunteer and the same finger joint were repeated 3 times, and 2 volunteers were replaced to conduct parallel experiments to ensure the repeatability of the results;

[0089] Control settings: Pure QCS solution and QCS-EC sol were used as control samples. Adhesion experiments were conducted according to the same steps to compare the adhesion advantages of MQCEC-TEB Gel.

[0090] (5) The standard for judging the experimental results is that when the finger is bent to 0°, 90°, 120° and 135°, the hydrogel does not fall off or shift significantly, and only the edges may show slight loosening of the adhesion (which does not affect the overall adhesion).

[0091] Result: From Figure 4 H indicates that MQCEC-TEB Gel hydrogel can firmly adhere to the finger joints, maintaining strong adhesion even when the fingers are bent to 0°, 90°, 120° and 135°.

[0092] Example 1: Real-time infection monitoring performance test of MQCEC-TEB Gel hydrogel

[0093] Experimental Methods: *Escherichia coli* and *Staphylococcus aureus* were inoculated into LB medium and cultured at 37°C with shaking at 200 rpm to obtain bacterial suspensions. 1 mL of the bacterial suspension was mixed with 1 mL of MQCEC-TEB Gel hydrogel and incubated at 37°C. The color changes of the hydrogel were observed at 0 h, 1 h, 2 h, 3 h, 4 h, 5 h, and 6 h. The absorption spectrum of the hydrogel in the wavelength range of 400-1200 nm was measured using a UV-Vis NIR spectrophotometer. Simultaneously, the following groups were set up: QCS solution group (5% QCS solution, 1 mL), EGCG solution group (30 μg / mL EGCG solution, 1 mL), QCS-EC solution group (5% QCS-EC solution prepared in Example 1, 1 mL), and TEBox-B... 12 Solution group (TEBox-B at a concentration of 50 μM in Example 5) 12 A solution (1 mL) was used as a control group, and the same experiment was performed.

[0094] Experimental results: From Figure 6 As shown in Figure A, the MQCEC-TEB Gel hydrogel initially changed from pale yellow to light purple after 1 hour of incubation with the bacterial suspension. The color gradually deepened with increasing incubation time, turning dark purple after 6 hours. The UV-Vis NIR absorption spectrum shows (…). Figure 6 B), two characteristic absorption peaks appeared in the 500-700 nm and 1000-1200 nm wavelength ranges, and the absorption intensity gradually increased with the extension of incubation time, indicating that bacteria induced the generation of organic free radicals in the hydrogel. In the control group, the QCS solution, EGCG solution, and QCS-EC solution did not change color or absorption spectrum throughout the incubation process; TEBox-B 12 After 6 hours of incubation, the solution changed color from light yellow to dark green and sedimentation occurred. The characteristic peaks of its absorption spectrum differed from those of the MQCEC-TEB Gel hydrogel. These results indicate that the MQCEC-TEB Gel hydrogel exhibits good bacterial responsiveness and can enable real-time monitoring of infection through color changes.

[0095] Electron paramagnetic resonance (EPR) analysis: MQCEC-TEB gel hydrogel, QCS solution, QCS-EC solution, TEBox solution, and TEBox-B were incubated with Staphylococcus aureus suspension for 6 h. 12 The solution was subjected to EPR testing.

[0096] The results showed that from Figure 8 It can be seen that the MQCEC-TEB gel hydrogel exhibited a significant EPR signal (g-factor of 2.0028) and the strongest signal intensity, indicating that it generated the most organic free radicals and had the best stability; TEBox-B 12The solution also showed an EPR signal of a certain intensity, but it was weaker than that of the MQCEC-TEB Gel hydrogel. However, no obvious EPR signal was detected in the QCS solution, QCS-EC solution, and TEBox solution, which further confirmed that the MQCEC-TEB Gel hydrogel generated stable organic free radicals under the action of bacteria, providing a basis for real-time infection monitoring.

[0097] Example 2: NIR-II photothermal (PTT) and photodynamic (PDT) antibacterial properties test of MQCEC-TEB Gel hydrogel.

[0098] 1. NIR-II photothermal performance test

[0099] Experimental Methods: 1 mL of MQCEC-TEB Gel hydrogel, after incubation with suspensions of Escherichia coli and Staphylococcus aureus for 6 h respectively, was placed in a petri dish and subjected to a 1064 nm NIR-II laser (power density 1.0 W / cm²). 2 The hydrogel was irradiated, and its temperature change within 0-10 minutes was recorded in real time using an infrared thermal imager. The temperature rise and photothermal conversion efficiency were calculated. Simultaneously, TEBox solution groups and TEBox-B were set up. 12 The solution group served as the control group, and the same experiment was performed.

[0100] Experimental results: From Figure 6 CD indicates that the hydrogel incubated with Staphylococcus aureus for 6 hours, under the same irradiation conditions, increased in temperature to 56°C, with a temperature increase of 31°C. The TEBox solution increased in temperature to 38°C after 10 minutes of irradiation. TEBox-B... 12 The solution temperature was raised to 48°C. According to the formula for calculating photothermal conversion efficiency (η=[hA(T)]),... max -T surround )-Q0] / I(1-10⁻ Aλ Calculations show that the photothermal conversion efficiency of the MQCEC-TEB Gel hydrogel is 37.23%, which is significantly higher than that of the TEBox solution (12.5%) and TEBox-B. 12 The solution (25.8%) indicates that it has excellent NIR-II photothermal properties.

[0101] 2. Photothermal stability test

[0102] The MQCEC-TEB Gel hydrogel prepared in Example 6 was subjected to five NIR-II laser irradiations (1064 nm, 1.0 W / cm²). 2 The experiment included a cooling cycle (10 min) and a cooling cycle. After each irradiation, the temperature was cooled to room temperature before the next irradiation, and the highest temperature after each irradiation was recorded.

[0103] Result: From Figure 6 As can be seen from E, after 5 cycles, the highest temperature of the hydrogel can still reach 54-55°C, which is not significantly lower than the highest temperature of the first irradiation, indicating that it has good photothermal stability and can be repeatedly used for photothermal therapy.

[0104] Test Example 3: NIR-II Photodynamic Performance Test

[0105] Experimental method: The TEBox-B prepared in Example 5 was used... 12 TEBox-B powder was dissolved in deionized water to prepare a 1mM solution. 12 The mother liquor was prepared using a stepwise dilution method. Appropriate amounts of the mother liquor were transferred and diluted with the corresponding solvent to a final volume of 1 mL. After thorough mixing, a series of working solutions with concentrations of 10 μM, 20 μM, 30 μM, 40 μM, and 50 μM were obtained for later use. ABDA was used as... 1 O2 fluorescent probes were used to deliver TEBox-B at different concentrations (10 μM, 20 μM, 30 μM, 40 μM, 50 μM). 12 The solution was mixed with ABDA solution (final concentration 50 μM) and subjected to a 1064 nm NIR-II laser (power density 50 mW / cm²). 2 After irradiation for 10 min, the fluorescence intensity change of ABDA at 420 nm was measured using a fluorescence spectrophotometer, and the fluorescence intensity decrease rate was calculated. Simultaneously, QCS solution group, EGCG solution group, QCS-EC solution group, and MQCEC-TEB Gel hydrogel group (containing 50 μM TEBox-B) were set up. 12 A comparative experiment was conducted.

[0106] Experimental results: such as Figure 9 As shown, with TEBox-B 12 With increasing concentration, the rate of decrease in ABDA fluorescence intensity gradually increases, when TEBox-B 12 At a concentration of 50 μM, after irradiation for 10 min, the fluorescence intensity of ABDA was only 4.0% of the initial value, with a fluorescence intensity decrease rate of 96.0%, indicating that it produced... 1 O2 has the strongest effect. In the comparative experiment, the ABDA fluorescence intensity decrease rate of the MQCEC-TEB Gel hydrogel group also showed a significant PDT effect and produced a large amount of O2. 1 O2; while the ABDA fluorescence intensity of the QCS solution group, EGCG solution group and QCS-EC solution group showed almost no change, and the decrease rate was less than 5%.

[0107] Electron paramagnetic resonance (EPR) experiments were used to determine the specific ROS type produced by MQCEC-TEB gel under 1064 nm NIR-II laser irradiation. Under laser irradiation, specific ROS types were observed in the MQCEC-TEB gel.1 1:1:1 electron cyclone resonance of O2 free radicals ( Figure 10-11 This indicates that MQCEC-TEB gel has significant antibacterial potential in the treatment of wound infections.

[0108] Test Example 4: In vitro antibacterial performance test

[0109] Experimental Methods: The antibacterial activity of MQCEC-TEB Gel hydrogel was determined using the plate count method. *Escherichia coli* and *Staphylococcus aureus* were cultured separately and diluted to the same suitable concentration. 100 μL of bacterial suspension and 100 μL of samples from different treatment groups (MQCEC-TEB Gel hydrogel, QCS solution, EGCG solution, and QCS-EC solution, wherein the MQCEC-TEB Gel hydrogel contained 50 μM TEBox-B) were taken. 12 The samples were mixed and divided into a dark processing group and a light processing group (the light processing group was further divided into a PDT group and a PTT group, which were irradiated with a 1064nm NIR-II laser for 10 minutes at a power density of 50mW / cm²). 2 (PDT) and 1.0 W / cm 2 (PTT), incubated at 37°C for 24 h. Then, 100 μL of the mixture was spread onto LB agar plates and incubated at 37°C for 24 h. The number of colonies was counted, and the antibacterial rate was calculated (antibacterial rate = (number of colonies in the control group - number of colonies in the experimental group) / number of colonies in the control group × 100%). At the same time, a blank control group (bacterial solution only) was set up.

[0110] Experimental results: In the PDT group, TEBox-B containing 50µM was found to be present. 12 The MQCEC-TEB Gel hydrogel showed inhibition rates of 94.47% and 99.17% against Escherichia coli and Staphylococcus aureus, respectively. Figure 5 HJ). In the PTT group, other control groups (blank group, QCS group, EGCG group, and QCS-EC group) all showed poor PTT antibacterial activity. In contrast, the MQCEC-TEB gel phototherapy group showed significant antibacterial activity, with a kill efficiency of over 90% against both Escherichia coli and Staphylococcus aureus. Figure 6 FH).

[0111] Furthermore, the results of the live / dead bacteria test were consistent with those of the plate coating test. Figure 12Bacteria treated with the blank dark / light treatment group (PDT) and the MQCEC-TEB gel dark treatment group showed obvious green fluorescence (live bacteria), while the MQCEC-TEB gel phototherapy group (PDT) showed the strongest red fluorescence (dead bacteria), reflecting its excellent broad-spectrum NIR-II PDT antibacterial effect. Finally, the plating test further confirmed that the MQCEC-TEB gel exhibited antibacterial activity that varied with irradiation time.

[0112] Scanning electron microscopy (SEM) observation: Escherichia coli and Staphylococcus aureus treated with different methods were observed using SEM. The results showed ( Figure 13 In the blank control group, bacterial cell walls were intact, smooth, and regularly shaped; in the QCS solution group, EGCG solution group, and QCS-EC solution group, bacterial morphology showed no significant changes, with only some bacteria showing slight wrinkles on their surface; TEBox-B 12 In the solution-treated group, the bacterial cell walls showed some damage and a small amount of cytoplasm leakage; while in the MQCEC-TEB Gel hydrogel-treated group, the bacterial cell walls were severely ruptured, a large amount of cytoplasm leakage occurred, and the bacterial morphology was significantly shrunken and deformed, further confirming its strong antibacterial effect.

[0113] This multimodal platform offers a promising strategy for the precise management of invasive infected wounds, enabling seamless integration of early diagnosis, non-invasive monitoring, and targeted therapy.

[0114] The above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the protection scope of the claims of the present invention.

Claims

1. A mussel-inspired bacterial responsive injectable hydrogel, characterized in that, The hydrogel is composed of a mussel-inspired adhesive hydrogel scaffold and a bacterial responsive agent; the adhesive hydrogel scaffold is QCS-EC, formed by the non-covalent bonding of quaternized chitosan (QCS) and epigallocatechin gallate (EGCG); the bacterial responsive agent is the supramolecular nano-crosslinked complex TEBox-B. 12 The TEBox is a box-shaped quaternary ammonium pyridine heterocyclic organic salt molecule containing superionized liquid anions B. 12 H 12 2- Salts are formed through self-assembly via hydrogen bonding and electrostatic interactions; The injectable hydrogel is prepared by mixing a 5% QCS-EC aqueous solution with TEBox-B. 12 The solutions were mixed at a volume ratio of 1:1 and stirred at room temperature until a gel was formed.

2. The mussel-inspired bacterial responsive injectable hydrogel according to claim 1, characterized in that, The QCS-EC is formed by QCS and EGCG under pH conditions of 6.0 to 7.

0.

3. The mussel-inspired bacterial responsive injectable hydrogel according to claim 1, characterized in that, TEBox and superionized liquid anion B 12 H 12 2- The molar ratio of salts is 1:

2.

4. The use of the mussel-inspired bacterial responsive injectable hydrogel of claim 1 in the preparation of a wound infection monitoring agent.

5. The use of the mussel-inspired bacterial responsive injectable hydrogel of claim 1 in the preparation of a near-infrared synergistic anti-infective therapeutic agent.

6. The use of the mussel-inspired bacterial responsive injectable hydrogel of claim 1 in the preparation of an agent that promotes the repair of infected wounds.

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