A charge transfer complex hydrogel, its preparation method and application

Abstract

This invention relates to a charge transfer complex hydrogel, its preparation method, and its application. The charge transfer complex hydrogel comprises: a solution of charge transfer complex nanoparticles and a bromothymol blue solution, wherein the charge transfer complex nanoparticles are 3-AP-TQ NPs, Per-TQ NPs, 3-BrP-TQ NPs, or 3-PCA-TQ NPs. The charge transfer complex hydrogel of this invention possesses the following functions: highly efficient photothermal antibacterial properties: utilizing the photothermal effect of NIR-II CTCs, localized high temperatures are generated under laser irradiation to kill drug-resistant bacteria; real-time infection monitoring: the degree of infection is visually indicated by the sensitive color change of BTB on wound pH; ideal dressing properties: relying on a carrageenan base, it provides a suitable moist environment, permeability, and mechanical support.

Description

Technical Field

[0001] This invention relates to the field of hydrogel technology, and more particularly to a charge transfer complex hydrogel, its preparation method, and its application. Background Technology

[0002] Wound infections, especially chronic infections caused by drug-resistant bacteria, pose a significant challenge in clinical treatment. Traditional antibiotic therapy is limited in efficacy due to increasing bacterial resistance and biofilm formation, and is prone to causing systemic side effects. Therefore, developing a novel treatment strategy that can overcome drug resistance, achieve precise local treatment, and promote wound healing is urgently needed. Photothermal therapy (PTT), as an emerging physical antibacterial method, shows great potential in the field of anti-infection due to its advantages such as high spatiotemporal selectivity, low risk of drug resistance, and synergistic therapeutic effects. The core of PTT lies in the ability of PTA to convert absorbed light energy into heat energy, killing bacteria through localized heating. In recent years, various photothermal agents (PTAs), such as precious metals, carbon-based materials, and organic polymers, have been developed, driving the advancement of PTT technology. However, the light absorption of most traditional PTAs is concentrated in the near-infrared I region (NIR-I), limiting their tissue penetration depth and maximum permissible exposure power, thus restricting the treatment effect on deep infections. In contrast, near-infrared II (NIR-II) light has a deeper tissue penetration capability and a higher safety threshold, making the development of efficient NIR-II PTAs a research hotspot.

[0003] Charge-transfer complexes (CTCs) are materials formed by intermolecular charge transfer between electron donors (D) and electron acceptors (A). Their unique electron transfer mechanism enables them to undergo strong nonradiative transitions under photoexcitation, resulting in high photothermal conversion efficiency, making them a highly promising class of phototransferable thermoelectric materials (PTAs). Despite their excellent photothermal properties, most CTC materials struggle to achieve good NIR-II absorption, limiting their application in NIR-II photothermal material transfer (PT) applications. Furthermore, when used directly as therapeutic agents on irregular wound surfaces, they suffer from issues such as easy loss and difficulty in achieving sustained action.

[0004] Therefore, it is necessary to improve one or more of the problems existing in the above-mentioned related technical solutions.

[0005] It should be noted that this section is intended to provide background or context for the technical solutions of the invention as set forth in the claims. The description herein does not imply acceptance of prior art simply because it is included in this section. Summary of the Invention

[0006] The purpose of this invention is to provide a charge transfer complex hydrogel, its preparation method and application, thereby at least partially solving one or more problems caused by the limitations and defects of related technologies.

[0007] The present invention first provides a charge transfer complex hydrogel, the charge transfer complex hydrogel comprising: a charge transfer complex nanoparticle solution and a bromothymol blue solution, wherein the charge transfer complex nanoparticles are 3-AP-TQ NPs, Per-TQ NPs, 3-BrP-TQ NPs or 3-PCA-TQ NPs.

[0008] The present invention further provides a method for preparing a charge transfer complex hydrogel, the method comprising the following steps: S1, dissolve the electron donor material in THF to prepare a donor solution, wherein the electron donor material is 3-AP, Per, 3-BrP or 3-PCA; S2, TCNQ is dissolved in THF to prepare a receptor solution; S3, the donor solution and acceptor solution are mixed, then F127 solution is added, and after mixing evenly, it is added to deionized water, stirred, THF in the mixed solution is removed, and then filtered to obtain charge transfer complex nanoparticle solution; S4. Add bromothymol blue solution and charge transfer complex nanoparticle solution to carrageenan solution, stir evenly to obtain charge transfer complex hydrogel.

[0009] In this invention, in step S1, the concentration of the donor solution is 0.01 mmol·mL. -1 .

[0010] In this invention, in step S2, the concentration of the receptor solution is 0.01 mmol·mL. -1 .

[0011] In this invention, the concentration of the bromothymol blue solution in S4 is 2500 μg·mL. -1 .

[0012] In this invention, in step S4, the concentration of the bromothymol blue solution in the charge transfer complex hydrogel is 100 μg·mL. -1 .

[0013] The present invention also provides an application of a charge transfer complex hydrogel in the preparation of reagents for antibacterial purposes or for monitoring the degree of wound infection.

[0014] The technical solution provided by this invention may include the following beneficial effects: In this invention, the charge transfer complex hydrogel has the following functions: (1) High-efficiency photothermal antibacterial: Utilizing the photothermal effect of NIR-II CTCs, local high temperature is generated under laser irradiation to kill drug-resistant bacteria; (2) Real-time infection monitoring: The degree of infection is visualized by the sensitive color change of wound pH through BTB; (3) Ideal dressing properties: Based on carrageenan base, it provides a suitable moist environment, permeability and absorption capacity and mechanical support. Attached Figure Description

[0015] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with this disclosure and, together with the description, serve to explain the principles of this disclosure. It is obvious that the drawings described below are merely some embodiments of this disclosure, and those skilled in the art can obtain other drawings based on these drawings without any inventive effort.

[0016] Figure 1 This diagram illustrates the preparation and application of the 3-AP-TQ NPs / BTB / carr hydrogel in this invention. Figure 2 This illustrates the analytical content of the present invention; Figure 3 (a) Donor and acceptor molecules used to prepare CTCs NPs; (b) UV-Vis absorption spectra of monomer molecules; (c) Absorption spectra and appearance of CTCs NPs (illustrated). Figure 4 In the middle: (a) is the Ac fitting curve of 3-AP-TQ; (b) is the Ac fitting curve of 3-AP-TQ NPs and the mass extinction coefficient at 1277nm wavelength; Figure 5 (a)-(d) show the nanoparticle diameters and polydispersity index (PDI) of Per-TQ NPs, 3-PCA-TQ NPs, 3-BrP-TQ NPs and 3-AP-TQ NPs in this invention, respectively. Figure 6 Images (a)-(d) show the TEM morphology of 3-AP-TQ NPs, the Zeta potential of 3-AP-TQ NPs, the particle size change of 3-AP-TQ NPs after incubation in PBS, DMEM medium and deionized water for 7 days, respectively; and the absorption wavelength of 3-AP-TQ NPs in PBS buffer at different pH values ​​and the absorbance change curve at the maximum absorption wavelength. Figure 7In the image, (a) shows the images (insets) and absorption spectra of 3-AP-TQ NPs prepared with acceptor to donor (A:D) molar ratios of 1:1, 1:2, 1:3, 2:1, and 3:1; (bf) shows the particle size distribution of 3-AP-TQ NPs nanoparticles prepared with different A:D ratios. Figure 8 In the image, (a) shows different concentrations of 3-AP-TQ NPs (500 µL) under a 1064 nm laser (1 W·cm⁻¹). -2 (a) Temperature change curve under irradiation; (b) Temperature change curve under 1064 nm laser irradiation at different power densities (100 µg·mL⁻¹). -1 (c) shows the temperature response behavior of 3-AP-TQ NPs (100 µg·mL⁻¹); -1 500 µL) in 1064 nm laser (1 W·cm) -2 (d) shows the photothermal cycling stability test results of 3-AP-TQ NPs (100 µg·mL⁻¹); -1 (e) shows the photothermal conversion efficiency of 3-AP-TQ NPs at different concentrations under 1064 nm laser irradiation (500 µL); -2 Infrared thermal image under irradiation of 500 µL; Figure 9 The XRD diffraction patterns of 3-AP-TQ NPs, TCNQ, and 3-AP in this invention are shown. Figure 10 In the figures, (a) and (b) show actual images of hydrogels with different concentrations of 3-AP-TQ NPs in this invention and microstructures of hydrogels with different concentrations taken using SEM. Figure 11 In the figure, (a), (b), (c), and (d) respectively show the in vitro degradation behavior of hydrogels of different concentrations in PBS, the swelling performance statistics of hydrogels of different concentrations, the porosity statistics of hydrogels of different concentrations, and the angular frequency dependence of storage modulus (G′) and loss modulus (G″) of hydrogels of different concentrations in dynamic frequency scanning. Figure 12 In the figures, (a), (b), (c), and (d) show the hydrogels of different concentrations in this invention at 1064 nm (1W cm⁻¹). -2 Temperature changes of hydrogels after 10 minutes of laser irradiation (1 W·cm⁻¹) at different concentrations. -2 Temperature change curves under irradiation, different concentrations of hydrogel at 1064 nm (1 W·cm⁻¹) -2Photothermal cycling stability under laser irradiation, hydrogels of different concentrations at 1064 nm (1 W·cm⁻¹) -2 Photothermal conversion efficiency under laser irradiation; Figure 13 In the figures, (a) and (b) show the effects of different concentrations of 3-AP-TQ NPs on the viability of 293T cells and RAW264.7 cells, respectively; the hemolysis rate and experimental photographs (insets) after co-incubation of mouse whole blood with PBS, hydrogels of different concentrations, and DI Water are also shown, where 1 is the negative control (PBS), and 2-5 are the experimental groups (0, 25, 50, and 100 μg / mL, respectively). -1 Hydrogel), 6 is a positive control (DI Water); Figure 14 In the figure, (a) shows the H&E staining results of the main organs (heart, liver, spleen, lung and kidney) of the mice of the present invention; (b) shows the trend of mouse body weight change within 14 days of treatment in different treatment groups; Figure 15 The images show the in vitro antibacterial effects of the composite hydrogel at different concentrations of the present invention and SEM images of bacteria after treatment with different treatment groups; (a), (b), (c), (d), (e), and (f) respectively show the MRSA agar plate culture image of the present invention; the survival rate of bacteria cultured on MRSA agar plates; SEM images of bacteria after treatment with MRSA in different treatment groups; E. coli agar plate culture image; the survival rate of bacteria cultured on E. coli agar plates; SEM images of bacteria after treatment with E. coli in different treatment groups; Figure 16 The pH responsiveness of the composite hydrogel in this invention is shown in the following figures: (a) color changes of the composite hydrogel after co-incubation with PBS of different pH values; (b) color changes of the composite hydrogel after co-incubation with E. coli bacterial suspensions of different concentrations; and (c) color changes of the composite hydrogel applied to the infected wound surface of a mouse model. Figure 17 (a) and (b) respectively show the temperature change graphs of different treatment groups when treating infected wounds in this invention; and the quantitative temperature analysis graphs of different treatment groups when treating infected wounds. Figure 18 (a) and (b) respectively show the apparent and quantitative analysis of the changes in wound area after 14 days of treatment in the same treatment group in this invention; Figure 19 The following shows the bacterial growth at the wound site of mice after 10 days of treatment in different treatment groups according to the present invention: (a) agar plate culture and (b) colony count; Figure 20 The results of H&E staining of mouse wounds after 14 days of treatment in different treatment groups in this invention are shown. Detailed Implementation

[0017] Exemplary embodiments will now be described more fully with reference to the accompanying drawings. However, these exemplary embodiments can be implemented in many forms and should not be construed as limited to the examples set forth herein; rather, they are provided so that the invention will be more comprehensive and complete, and will fully convey the concept of the exemplary embodiments to those skilled in the art. The described features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0018] Furthermore, the accompanying drawings are merely illustrative diagrams of embodiments of the present invention and are not necessarily drawn to scale. The same reference numerals in the drawings denote the same or similar parts, and therefore repeated descriptions of them will be omitted. Some block diagrams shown in the drawings are functional entities and do not necessarily correspond to physically or logically independent entities.

[0019] In this application, 7,7,8,8-tetracyanodimethyl-p-benzoquinone (TCNQ), as a strong electron acceptor, readily forms stable cell-mediated cytotoxicity (CTCs) with electron-rich donors. Perylene (Per) and its derivatives, with their large conjugated planes and excellent chemical stability, are ideal candidates for electron donors. By introducing functional groups such as bromine, aldehyde, and amino groups onto its backbone, its electronic structure and photophysical properties can be finely tuned, thereby optimizing the light absorption and photothermal conversion capabilities of the formed CTCs. Loading PTAs into hydrogel dressings not only achieves local sustained release and long-term retention of drugs, but its three-dimensional network structure can also absorb exudate, keep the wound moist, and provide a scaffold for cell migration and proliferation, thus synergistically promoting wound healing. Furthermore, the treatment system integrating diagnostic functions (such as pH response) can reflect the wound infection status in real time and intuitively (bacterial metabolism often leads to local microenvironment acidification), achieving integrated "treatment-monitoring."

[0020] Based on the above, this application utilizes a donor molecular engineering strategy to prepare a series of CTCs NPs based on TCNQ and different Per derivatives, and screens those with good NIR-II absorption as PTAs. Furthermore, these are combined with a pH-responsive indicator (BTB) and a biocompatible gel matrix (carr) to construct a novel multifunctional composite hydrogel (such as...). Figure 1 As shown in the figure, this is the charge transfer complex hydrogel described in this application.

[0021] This example embodiment first provides a charge transfer complex hydrogel, which comprises: a charge transfer complex nanoparticle solution and a bromothymol blue solution, wherein the charge transfer complex nanoparticles are 3-AP-TQ NPs, Per-TQ NPs, 3-BrP-TQ NPs, or 3-PCA-TQ NPs. It should be noted that 3-AP is 3-aminoperylene, Per is perylene, 3-BrP is 3-bromoperylene, 3-PCA is 3-perylenecarboxaldehyde, and TQ is TCNQ.

[0022] In this embodiment, the charge transfer complex hydrogel has the following functions: (1) High-efficiency photothermal antibacterial: Utilizing the photothermal effect of NIR-II CTCs, local high temperature is generated under laser irradiation to kill drug-resistant bacteria; (2) Real-time infection monitoring: The degree of infection is visualized by the sensitive color change of wound pH through BTB; (3) Ideal dressing characteristics: Based on carrageenan base, it provides a suitable moist environment, permeability and absorption capacity and mechanical support.

[0023] like Figure 2 As shown, this invention will describe the preparation, characterization, photothermal properties, and biosafety of CTCs NPs, evaluate the physicochemical properties, in vitro antibacterial activity, and pH responsiveness of the composite hydrogel, and finally verify its comprehensive efficacy in promoting wound healing in vivo using a mouse full-thickness skin defect model infected with MRSA, providing a safe, efficient, and intelligent new strategy for the treatment of chronic wounds. Details are as follows: 1. Experimental reagents Table 1. Reagent names and manufacturers.

[0024]

[0025] 2. Experimental equipment Table 2. Equipment Names and Manufacturers.

[0026]

[0027] 3. Preparation of charge-transfer composite nanoparticles (CTCs NPs) In this embodiment, CTCs NPs were prepared using a nanoprecipitation method.

[0028] The following explanation uses the synthesis of 3-AP-TQ NPs as an example to illustrate the preparation method. Figure 1 As shown, the details are as follows: Weigh 2.67 mg pery-3-amine (3-AP, 3-aminoperylene) and dissolve it in 1 mL of THF tetrahydrofuran. Sonicate the solution for 5 minutes to prepare a 0.01 mmol / mL solution.-1 Donor solution (D); Weigh 2.04 mg TCNQ and dissolve it in 1 mL THF. Sonicate the solution using the same method to prepare a 0.01 mmol / mL solution. -1 The receptor solution (A); Mix 1 mL of solution D with 1 mL of solution A and sonicate for 5 minutes to ensure uniform mixing.

[0029] Then add 2 mL of F127 solution (20 mg·mL⁻¹) to the mixture. -1 (dissolved in THF), and sonicated again for 5 minutes to obtain a DA-F127 mixed organic phase; Under ice bath conditions, the mixture was slowly added to 36 mL of deionized water (organic phase: aqueous phase = 1:9, volume ratio) and stirred rapidly overnight. THF was then removed from the system using a rotary evaporator to obtain an aqueous dispersion of 3-AP-TQ NPs. Finally, the resulting solution was filtered through a 0.22 μm filter membrane to remove larger aggregates, yielding a uniformly sized aqueous solution of nanoparticles, i.e., an aqueous solution of 3-AP-TQ NPs.

[0030] Per-TQ NPs, 3-BrP-TQ NPs, and 3-PCA-TQ NPs were all prepared using the same method.

[0031] 4. Physicochemical characterization of CTCs and NPs (1) Absorption spectrum and stability: Monomer absorption test: Per, 3-PCA, 3-BrP, 3-AP, F127, TCNQ and other monomers were dissolved in tetrahydrofuran (THF). Using THF as the reference solvent, the samples were loaded into micro-quartz cuvettes and the absorption spectra of each monomer were measured by UV-Vis spectrophotometer.

[0032] Nanoparticle absorption testing: Using deionized water as the reference solvent, the absorption characteristics of aqueous solutions of three types of nanoparticles—3-AP-TQ NPs, Per-TQ NPs, and 3-BrP-TQ NPs—were tested. Furthermore, the photochemical stability of the target nanoparticles was evaluated by testing their absorption spectra and absorbance under different pH conditions.

[0033] (2) Particle size and stability: An appropriate amount of CTCs NPs aqueous solution was placed in a Malvern cuvette, and its average particle size was measured using dynamic light scattering (DLS). The particle size change was continuously monitored over 0-7 days to evaluate the colloidal stability of the nanoparticles. Simultaneously, the morphology and size distribution were observed using transmission electron microscopy (TEM) to further verify the particle size results. To examine the stability of the nanoparticles in different media, 10 µL of nanoparticle aqueous solution (100 µg·mL⁻¹) was used to measure the particle size. -1 The nanoparticles were mixed with 900 µL PBS (0.1 M) or 900 µL DMEM medium and incubated for 12 hours before their particle size was measured. The particle size changes of the target nanoparticles under co-incubation conditions of PBS and DMEM were further systematically monitored over 0–7 days. In addition, the nanoparticles were added to a dedicated zeta potential cuvette, and their zeta potentials were measured using a DLS system to characterize the surface charge properties of the nanoparticles.

[0034] (3) Optimization of feed ratio: To investigate the effect of the acceptor-donor (A:D) feed ratio on the material properties, 3-AP-TQ NPs were prepared at A:D molar ratios of 1:1, 1:2, 1:3, 2:1, and 3:1, respectively. First, 0.01 mmol·mL⁻¹ of THF was prepared as the solvent. -1 TCNQ and 3-AP solution were mixed in the above proportions, and then an equal volume of F127 solution (20 mg·mL⁻¹) was added. -1 The organic phase was ultrasonically treated for 5 minutes to homogenize it. Under ice bath conditions, the organic phase was rapidly injected into deionized water (organic phase: aqueous phase = 1:9) and continuously stirred rapidly overnight to obtain 3-AP-TQ NPs solutions with different A:D molar ratios. Subsequently, the absorption spectra of 3-AP-TQ NPs solutions with different ratios were measured using a UV-Vis-NIR spectrophotometer, and the nanoparticle diameter was measured using DLS to systematically compare the effects of different A:D ratios on the optical properties and particle size distribution of the materials.

[0035] (4) Concentration Calculation: The actual concentration of 3-AP-TQ NPs was calculated by establishing a standard curve between concentration and absorbance. The specific steps are as follows: First, weigh 500 µg of 3-AP donor molecules and 500 µg of TCNQ acceptor molecules, dissolve them in 1 mL of THF, and prepare a solution with a concentration of 1000 µg·mL⁻¹. -1 3-AP-TQ mixed solution. Subsequently, this mixed solution was sequentially diluted to 20, 10, 5, 2.5, 1.25, and 0.625 µg·mL. -1Standard solutions were prepared. The absorption spectra of each standard solution were scanned in the wavelength range of 300–900 nm using a UV-Vis spectrophotometer, and the absorbance at the maximum absorption wavelength was recorded. A standard curve was plotted with concentration (c) on the x-axis and absorbance (A) on the y-axis, and the linear equation y = ax + b was obtained by fitting the curve. 10 µl of 3-AP-TQ NPs solution was added to 990 µl of THF (diluted 100-fold), thoroughly mixed, and the absorbance value x at the maximum absorption wavelength was measured under the same conditions. This value was substituted into the standard curve equation to calculate the concentration of the diluted solution, and thus the concentration and yield of the original 3-AP-TQ NPs solution were deduced.

[0036] Based on the obtained 3-AP-TQ NPs concentration, prepare 100 µg·mL - ¹3-AP-TQ NPs were used as the stock solution and were successively diluted to obtain 25, 12.5, 6.25, 3.125, 1.5625 and 0.78125 µg·mL⁻¹ -1 Gradient concentration solutions were then calculated. The absorbance of each concentration solution was measured at 1277 nm, and an Ac standard curve was plotted. The corresponding equation was obtained through linear fitting. Based on the slope of the fitted equation, the mass extinction coefficient (ε) of the 3-AP-TQ NPs at that wavelength could be calculated.

[0037] (5) Photothermal performance characterization The photothermal properties of photothermal reagents are a key factor determining the efficacy of PTT (photothermal transluminal therapy). Highly efficient photothermal reagents can effectively convert light energy into heat energy in a short time, thereby improving heat output efficiency and therapeutic effect. In addition, the stability of photothermal reagents under multiple light exposure cycles is also an important indicator for evaluating their performance.

[0038] 3-AP-TQ NPs concentration-dependent temperature variation: Different concentrations (0, 25, 50, 100, 200, 400 µg·mL) were used. -1 500 µL of each of the 3-AP-TQ NPs solution was heated under a 1064 nm laser (1 W·cm⁻¹). -2 Irradiate for 10 minutes. During the irradiation, the real-time temperature of the solution is recorded every minute using a Hikvision H13 Pro infrared thermal imager, and the corresponding thermal images are saved. Based on this, a concentration-dependent temperature change curve is plotted.

[0039] 3-AP-TQ NPs power-dependent temperature change: 100 µg mL -1 3-AP-TQ NPs (500 µL) were used with different power (0.25 W cm⁻¹). -2 0.5 W cm -20.75 W cm -2 1 W cm -2 1.5 W cm -2 The solution was irradiated with a 1064 nm laser for 10 minutes. During the irradiation, the real-time temperature of the solution was captured every minute using a Hikvision H13 Pro infrared thermal imager. Images at different temperatures were saved, and the power-dependent temperature change curve of the 3-AP-TQ NPs solution was plotted.

[0040] Photothermal stability test: 3-AP-TQ NPs (500 µL; 100 µg mL) were tested. -1 Using a 1064 nm laser (1 Wcm) -2 Irradiate for 10 minutes, then stop irradiation until the solution temperature returns to room temperature. During irradiation and recovery, the real-time temperature of the solution is captured using an infrared thermal imager, and the operation is repeated more than 5 times. Finally, the temperature change is analyzed using Origin software to calculate the photothermal conversion efficiency (η). The photothermal conversion efficiency (η) can be obtained by formula (1).

[0041] (1) Where T Max It is the highest steady-state temperature, T Surr It is the ambient temperature. It is the heat transfer time constant. and These are the mass and specific heat capacity of deionized water (4.2 J g), respectively. -1 ), Q Dis This indicates the heat associated with the solvent absorbing the irradiated light. It is the laser power density. It is the absorbance of the 3-AP-TQ NPs aqueous solution at 1064 nm.

[0042] In formula (1) It can be obtained from formula (2). Where, T t t represents the real-time temperature of the 3AP-TQ NPs during the cooling process, and t represents the cooling time.

[0043] (2) In formula (1) It can be calculated using the following formula (3). (3) (6) XRD diffraction spectra: The 3-AP-TQ NPs solution was placed in a 5 mL centrifuge tube, rapidly frozen into a solid state by liquid nitrogen, and then transferred to a freeze dryer for 36 hours to obtain 3-AP-TQ NPs solid powder. The XRD patterns of the 3-AP-TQ NPs solid powder, TCNQ, and 3-AP monomers were determined by X-ray diffraction. By comparing the crystal form differences between the complex and the monomers, the possible structural changes during charge transfer were analyzed to verify the formation of the charge transfer complex.

[0044] 5. Preparation of charge transfer complex hydrogels (also referred to as composite hydrogels in this application) Weigh 2500 μg of bromothymol blue (BTB) solid and add 1 mL of deionized water to prepare a solution with a concentration of 2500 μg·mL. - ¹ Prepare the BTB stock solution. Weigh 625 mg of carrageenan solid, add 24 mL of deionized water, heat to 60 °C and stir continuously until completely dissolved. Adjust the pH of the carrageenan solution to 7.0 using 3.65% dilute hydrochloric acid and 4% sodium hydroxide solution. Then, add 1 mL of the above BTB stock solution and 1 mL of 2500 μg·mL⁻¹ to the solution. -1 The 3-AP-TQ NPs solution was stirred until homogeneous.

[0045] The resulting mixed solution was poured into a mold while still hot to prepare 3-AP-TQ NPs with a concentration of 100 μg·mL⁻¹. -1 BTB concentration of 100 μg·mL -1 Hydrogels were prepared using the same method, with concentrations of 50, 25, and 0 μg·mL⁻¹ of 3-AP-TQ NPs. -1 The concentration gradient of the hydrogels was controlled by adjusting the volume of 3-AP-TQ NPs stock solution added (0.5 mL, 0.25 mL, and 0 mL, respectively). Hydrogels of each concentration were cut into 2 cm × 2 cm square samples, placed in beakers, and rapidly frozen solidified with liquid nitrogen. The frozen samples were then transferred to a freeze dryer and freeze-dried for 36 hours to obtain dehydrated hydrogels. The dehydrated hydrogel samples were then adhered to conductive tape, sputter-coated with gold, and their microstructure was observed using scanning electron microscopy. The effects of different 3-AP-TQ NPs concentrations on the hydrogel micromorphology were systematically compared.

[0046] 6. Characterization of composite hydrogel properties (1) Porosity: The porosity of the lyophilized hydrogel was evaluated using the solution (anhydrous ethanol) displacement method. A small amount of lyophilized 3-AP-TQ NPs / BTB / carr hydrogels of different concentrations were weighed and their dry mass (W1) was measured. Then, the lyophilized hydrogel was added to anhydrous ethanol. Every 10 min, the anhydrous ethanol on the surface of the hydrogel was blotted dry with absorbent paper and the mass change was measured. The mass W2 was measured immediately after the anhydrous ethanol had penetrated into the hydrogel and the hydrogel was saturated. The porosity was calculated using formula (4), where V is the volume of the hydrogel and ρ is the density of anhydrous ethanol.

[0047] (4) (2) Swelling rate: The swelling rate of 3-AP-TQ NPs / BTB / carr hydrogels of different concentrations was determined by gravimetric method. Completely cured lyophilized 3-AP-TQ NPs / BTB / carr hydrogels of different concentrations were cut into pieces of the same size, immersed in PBS, and weighed every hour. The swelling rate of the hydrogel was calculated using formula (5), and a swelling rate-time curve was plotted. Where W d W represents the initial mass of the hydrogel. s The mass of the hydrogel after different soaking times.

[0048] (5) (3) Degradation rate: The degradation rate of 3-AP-TQ NPs / BTB / carr hydrogels of different concentrations was determined by gravimetric method. Hydrogels of different concentrations and the same volume were immersed in 30 mL of PBS (pH = 7.4) and incubated at a constant temperature (37 ℃). Hydrogel samples were removed at predetermined time points, the surface moisture was wiped dry with absorbent paper, and the samples were weighed until the hydrogels were almost completely degraded and could not be weighed. The percentage of weight loss of the hydrogel was calculated according to formula (6), where W0 is the original mass of the solidified hydrogel, and W... t The remaining mass of the hydrogel after co-incubation with PBS.

[0049] (6) (4) Rheological properties: The rheological properties of the hydrogel samples were evaluated using a rheometer. A hydrogel with a diameter of 50 mm and a thickness of 2 mm was placed between 20 mm parallel plates. Under the conditions of constant strain of 1%, temperature of 25 ℃, and frequency range of 0.1 to 100 Hz, a dynamic frequency scan measurement was performed at a height of 1000 µm. The dependence of storage modulus (G') and loss modulus (G'') of hydrogels with different concentrations on angular frequency was determined to characterize the rheological properties of the hydrogel.

[0050] (5) Photothermal performance Hydrogel concentration-dependent temperature variation: 3-AP-TQ NPs / BTB / carr hydrogels of different concentrations were cut into circular shapes with a diameter of 10 mm and a thickness of 3 mm, and subjected to temperature variation using a 1064 nm laser (1 W cm⁻¹). -2 Irradiation was performed for 10 minutes. During the irradiation, the real-time temperature of the hydrogel was captured every minute using a Hikvision H13 Pro infrared thermal imager. Images at different temperatures were saved, and the concentration-dependent temperature change curve of the hydrogel was plotted.

[0051] Photothermal stability and photothermal conversion efficiency of hydrogel: A hydrogel (10 mm in diameter, 3 mm in thickness; 100 µg / mL) was tested. -1 Using a 1064 nm laser (1 W cm⁻¹) -2 Irradiate for 10 minutes, then stop irradiation until the hydrogel temperature returns to room temperature. During irradiation and recovery, the real-time temperature of the hydrogel is captured using an infrared thermal imager, and this process is repeated at least 5 times. Finally, the temperature changes are analyzed using Origin software.

[0052] Calculate the photothermal conversion efficiency (η): The calculation method is the same as above.

[0053] (6) pH response test: In order to evaluate the response performance of the composite hydrogel, this application investigated its response behavior to PBS buffer at different pH, bacterial solutions at different concentrations and the microenvironment of a live wound.

[0054] Take 10 samples of 3-AP-TQ NPs / BTB / carr hydrogel (concentration µg·mL) -1 The items were placed in sterile petri dishes and processed according to the following groups: pH response group: PBS buffer with pH values ​​of 4, 5, 6 and 7 was added to 5 of the samples respectively; Bacterial response group: Add 10% concentration to each of the other 5 samples. 8 10 7 10 6 10 5 10 4 CFU·mL -1 MRSA bacterial solution.

[0055] All in vitro treated samples were incubated at 37 °C for 4 hours, and the color changes of the hydrogel were observed and recorded. Furthermore, in in vivo therapeutic experiments, the color changes of the composite hydrogel after application to the wound were monitored in real time to evaluate its response characteristics in a realistic infection microenvironment.

[0056] 7. Biosafety Characterization (1) Cytotoxicity Before the experiment, the following preparations must be completed: Preheat the water bath to 37 ℃, and prepare and preheat the DMEM complete culture medium according to Table 3. All reagents should be surface sterilized with 75% ethanol and then transferred to a clean bench.

[0057] Preparation of cell culture medium: Under aseptic conditions, mix the above components thoroughly, label the culture medium name, time, and name, and store in a sealed container at 4 ℃.

[0058] Table 3 Preparation of DMEM complete culture medium

[0059] Cell resuscitation, passage and cryopreservation: This experiment used 293T human embryonic kidney cells and Raw 264.7 mouse mononuclear macrophages to evaluate the biotoxicity of the composite hydrogel to human and mouse cells.

[0060] Cell resuscitation: Remove 293T or Raw 264.7 cell cryovials from the liquid nitrogen container, wrap them with sterile gloves, and place them in a water bath with gentle agitation to accelerate thawing. Once the cell suspension is completely dissolved, immediately transfer to a biosafety cabinet, add 2 mL of pre-warmed complete culture medium, and centrifuge at 1000 r / min for 3 minutes at room temperature. Discard the supernatant, retain the cell pellet, gently resuspend it in fresh culture medium, and seed it into 10 cm culture dishes, using a cross-hatching method to ensure even cell distribution. Finally, label the cell line with the seeding date and transfer the culture dishes to a 37 ℃, 5% CO2 incubator for routine culture.

[0061] Cell passage: Cell passage should be performed when cell confluence reaches 80%-90%. First, discard the original culture medium and gently wash the cells twice with 2 mL of sterile PBS buffer. Then, add 2 mL of 0.25% trypsin solution and digest at 37 ℃ for 2-3 minutes. Observe under an inverted microscope; when the intercellular spaces increase, the cells become rounded, and some begin to detach from the cell wall, immediately add an equal volume of complete culture medium containing serum to stop the digestion. Transfer the cell suspension to a 15 mL centrifuge tube and centrifuge at 800-1000 r / min for 3 minutes. Discard the supernatant and retain the cell pellet. Resuspend the cells in fresh culture medium, gently pipette to mix, and seed at a 1:2 ratio into labeled new culture dishes. Incubate at 37 ℃ in a 5% CO2 incubator.

[0062] Cell cryopreservation: Cell cryopreservation should be performed when cell confluence is close to 90%. The cryopreservation solution should be prepared fresh for each use; its standard composition is 90% fetal bovine serum and 10% dimethyl sulfoxide (DMSO), mixed thoroughly under light-protected conditions. Digest and collect cells according to the aforementioned passage method, centrifuge, and resuspend the cell pellet in the pre-prepared cryopreservation solution. Clearly label the cell line name, passage number, cryopreservation date, and operator's name on the outer wall of the cryopreservation tube. Place the cryopreservation tubes in a programmed cooling box, transfer to a -80°C ultra-low temperature freezer for 24 hours, and finally transfer to a liquid nitrogen tank for long-term storage.

[0063] CCK-8 assay for cytotoxicity: Cells were seeded at 5000 cells / well in 96-well plates and cultured in 100 μL of culture medium. After cell attachment, drug treatment was administered as needed. To ensure consistent experimental conditions, the medium was not changed during treatment. After treatment, 10 μL of CCK-8 solution was added to the culture medium, and the plates were incubated in a cell culture incubator. At 0.5 h, 1 h, and 2 h, the OD values ​​at 450 nm were measured using a microplate reader. Data from appropriate time points were then analyzed.

[0064] Group settings and data analysis: Setting blank hole A b (Culture medium only, without CCK-8), Control well A c (No drug treatment), Experimental Group A s (12.5, 25, 50, 100 μg mL -1 Each group has 3 replicates. Calculate cell viability using the following formula: (7) (2) Hemolytic Whole blood preparation: Fresh blood from Kunming mice was collected using the ocular blood collection method and placed in an anticoagulant tube. The stock solution was diluted to 2% (v / v) with PBS and stored at 4 ℃ for later use.

[0065] Experimental groups: A positive control group, a negative control group, and an experimental group were set up, with three replicates for each group. Positive control group: 1 mL deionized water + 20 μL blood; Negative control group: 1 mL PBS + 20 μL blood; Experimental group: 20 mL deionized water + 20 μL blood. -3 Different concentrations (3-AP-TQ NPs concentrations of 0, 12.5, 25, 50, and 100 μg / mL) -1 Hydrogel + 1 mL PBS + 20 μL blood. Incubate all samples in a 37 ℃ water bath for 1 h. After incubation, remove the hydrogel and centrifuge at 2000 rpm / min for 5 min. After centrifugation, collect the supernatant and measure the absorbance (OD) at 545 nm. t This is the OD value of the experimental group, A.n This is the OD value of the negative control group, A. p This is the OD value of the positive control group.

[0066] (8) 8. Characterization of in vitro antibacterial properties (1) Preparation of culture medium: Prepare LB liquid and solid culture medium according to the proportion of each reagent in Table 4. After thorough solvent preparation, sterilize in a high temperature and high pressure autoclave and store the sterilized culture medium in a 4 ℃ refrigerator for later use.

[0067] Table 4 Preparation of LB liquid and solid culture media

[0068] (2) Bacterial recovery, inoculation, and culture: This experiment used methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli (E. coli) to evaluate the in vitro antibacterial properties of the composite hydrogel. MRSA or E. coli bacterial samples were taken from a -80 ℃ freezer. Under aseptic conditions, a small number of colonies were picked up with an inoculation needle and transferred to a shaker tube containing 5 mL of LB liquid medium. The tubes were then incubated overnight at 37 ℃. A small amount of the overnight culture was inoculated onto LB solid medium plates using the streak plate method and incubated again overnight at 37 ℃. After colonies grew, the plates were sealed with sealing film and stored at 4 ℃ for later use.

[0069] Remove the colony plate from the 4 ℃ refrigerator, and under aseptic conditions, use a pipette to aspirate a small amount of colonies, dissolve them in 5 mL of LB liquid medium, and incubate overnight at 37 ℃ to obtain a high-concentration bacterial suspension for subsequent in vitro or in vivo experiments.

[0070] (3) In vitro antibacterial experiment of 3-AP-TQ NPs / BTB / carr hydrogel at different concentrations: The overnight cultured MRSA or E. coli bacterial suspension was aliquoted into centrifuge tubes and centrifuged at 4000 rpm / min for 5 minutes at room temperature. The supernatant was discarded, and the bacterial pellet at the bottom of the tube was retained. Then, an appropriate amount of PBS was added and the pellet was gently resuspended by pipetting. The pellet was centrifuged again under the same conditions for 5 minutes, and the supernatant was discarded. This washing step was repeated three times to ensure the removal of residual culture medium and metabolites. The bacterial suspension was resuspended in PBS, and the OD of the bacterial suspension was measured by an enzyme-linked immunosorbent assay (ELISA) reader. 600 Adjust OD 600 The concentration was approximately 10 at 5-6. 8 CFU·mL -1Prepare the bacterial culture. Cut hydrogels of different concentrations into samples of uniform shape and size, and place them in 24-well plates (three replicates per concentration group). After sterilization by soaking in 75% ethanol for 5 minutes, rinse three times with PBS to thoroughly remove any ethanol residue. Add 50 µL of PBS to each well as a blank control. Add 50 µL of the above bacterial culture (MRSA or E. coli) to the wells containing the hydrogels and PBS blank control, and then incubate the 24-well plates in a 37 ℃ incubator in the dark for 2 hours (dark treatment group). A separate light-treated experimental group was also prepared, where a 1064 nm laser (1 W·cm²) was used after adding the bacterial culture. -2 Irradiate for 10 minutes, then proceed with subsequent treatments. After incubation or irradiation, add 100 µL of PBS to each well to resuspend and collect the bacterial cells. Take 20 µL of the bacterial suspension and spread it evenly on LB agar plates, then incubate overnight at 37 ℃. Calculate the sterilization rate by counting the colony counts of each treatment group according to formula (9).

[0071] (9) (4) Observation of bacterial damage morphology: Scanning electron microscopy (SEM) was used to observe the morphological changes of bacteria after incubation with the modified materials. First, two pieces of 3-AP-TQ NPs / BTB / carr hydrogels with a diameter of 10 mm and a thickness of 2 mm (3-AP-TQ NPs concentration of 100 µg / mL) were placed in the hydrogel. -1 Place the solution into a 24-well plate, and add 1 mL of PBS to the other two wells. Use a pipette to pipette 20 μL of the solution, resulting in a 10% concentration. 8 CFU mL -1 Bacterial suspensions (MRSA or E. coli) were dropped onto two hydrogel surfaces and two portions of PBS, respectively. The well plates were then transferred to a 37 °C bacterial incubator for 1 h to allow for sufficient contact and interaction between the bacteria and the samples. After incubation, the following treatments were performed: 1064 nm laser (1 W cm⁻¹) was used. -2One well containing hydrogel and one well containing PBS were irradiated for 15 min. The other two wells containing hydrogel and PBS were left untreated. After irradiation, the bacteria on the hydrogel were washed and resuspended using PBS. The PBS containing the bacteria was then transferred to a 1 mL centrifuge tube, and the PBS group was also transferred to a 1 mL centrifuge tube. All four centrifuge tubes were centrifuged at 4000 rpm for 5 minutes. The supernatant was discarded, and the bacterial precipitate at the bottom was retained. Then, 1 mL of 4% paraformaldehyde solution was added to the centrifuge tubes, and the samples were fixed at 4°C for 6–8 h. The fixed samples were then subjected to a gradient dehydration process using 20%, 40%, 60%, 80%, 90%, and 99% ethanol solutions. The first three concentrations were dehydrated for 15 min, and the latter three concentrations for 30 min. Finally, the samples were air-dried at room temperature and attached to a SEM sample stage with conductive adhesive. Gold sputtering (10 mA, 90 s) was performed for SEM observation. The photothermal antibacterial properties of hydrogels were observed by comparing the bacterial morphology on the surfaces of different samples.

[0072] 9. Photothermal therapy for wound infection (1) Animal wound infection model construction: 8-week-old Kunming mice (approximately 30 g) were used to construct a wound infection model. Before surgery, mice were anesthetized by inhaling isoflurane. After anesthesia, the hair on the back of the mice was shaved with a razor, and depilatory cream was applied for complete hair removal. After 30 seconds of hair removal, the skin was rinsed with water to prevent burns. Subsequently, a hole was punched in the back of the mouse to create a 10 mm diameter window. Then, 20 μL of 10% concentration of [unspecified ingredient] was dripped into the wound. 8 CFU mL -1 The mouse wound infection model was established by infecting the wound with MRSA suspension and covering the wound with 3M dressing for 24 hours. Once pus appeared at the wound site, the mouse wound infection model was completed.

[0073] (2) The model mice were randomly divided into 4 groups and treated as follows: the wound was cleaned with PBS and then covered with 3M dressing (PBS group); the wound was cleaned with PBS and then treated with a 1064 nm laser (1 W cm⁻¹). -2 Irradiate the wound for 10 minutes, then cover the wound with a 3M dressing (PBS + NIR-II group); apply 3-AP-TQ NPs / BTB / carr hydrogel to the wound, then cover the wound with a 3M dressing (Hydrogel group); apply 3-AP-TQ NPs / BTB / carr hydrogel to the wound, then apply a 1064 nm laser (1 W cm⁻¹). -2The wound was irradiated for 10 minutes, and then covered with a 3M dressing (Hydrogel + NIR-II group). Each of the four treatment groups was performed in triplicate. The treatment was performed twice, on day 1 and day 4.

[0074] The temperature changes at the wound site were recorded using a Hikvision H13 Pro infrared thermal imager when different treatment groups were treating infected wounds.

[0075] Photographs were taken to record the color changes of the composite hydrogel before and after treatment.

[0076] (3) Apparent evaluation of wound healing: The wound healing status of mice was recorded by photographing every 2 days. The wound healing status was represented by the ratio of the exposed wound area visible to the naked eye. In addition, the area of ​​each wound was measured using ImageJ software, and the wound area ratio was calculated to evaluate and statistically analyze the apparent wound healing. The wound area ratio was calculated by formula (10), where A t A0 represents the area of ​​the open wound on the day of photographing after treatment, while A0 represents the initial area of ​​the open wound on the day of modeling.

[0077] (10) (4) Changes in mouse weight: The weight of mice was measured and recorded every 2 days using an electronic balance.

[0078] (5) Evaluation of the in vivo antibacterial properties of the composite hydrogel: Ten days after treatment, pus was collected from the wound using a disposable sterile swab, ensuring that the collection method was consistent for each treatment group. The swab was immersed in 1 mL of sterile saline, and liquid culture medium was added and shaken overnight. The bacterial solution was then diluted 10 mL with sterile saline in a laminar flow hood. 5 The bacterial suspension was spread onto a plate using the plate-spreading method, then the plate was inverted and incubated overnight in a bacterial incubator at 37 °C. Colony counts were recorded using a bacterial counter, and photographs were taken. The results were then analyzed to assess the treatment of bacterial infection at the wound site.

[0079] (6) Tissue hematoxylin-eosin staining (H&E staining) Histological analysis of mouse wound sections: Wound tissue samples were collected from euthanized mice 14 days after treatment in different treatment groups. Intact tissue blocks containing the wound and surrounding normal skin were excised 3-4 mm from the wound edge, rinsed with saline to remove surface blood, and blotted dry with filter paper. The tissue was laid flat on sterile filter paper and divided into two parts along the sagittal plane of the wound center. The tissue samples were then fixed in 4% paraformaldehyde at 4 °C for 24 hours, rinsed with running water for 4 hours, and stored overnight in 75% ethanol. After fixation, the tissues underwent histological processing including paraffin embedding, sectioning, and H&E staining. Finally, the healing status and morphology of the wound tissue were observed and evaluated under a microscope.

[0080] Visceral tissue staining analysis: Mice in different treatment groups were euthanized after 14 days of treatment, and their hearts, livers, spleens, lungs, and kidneys were removed. These tissues were fixed in 4% paraformaldehyde at 4 ℃ for 24 h, rinsed with running water for 4 h, and then placed in 75% ethanol overnight. The fixed viscera were then embedded, sectioned, and stained. Differences in visceral tissue were observed under a microscope.

[0081] 10. Statistical Analysis Significant differences in the data analysis were fitted using analysis of variance. Statistical differences were indicated by "*", where "*" indicates p < 0.05, meaning there is a significant difference between the two groups; "**" indicates p < 0.01, meaning there is a highly significant difference; "***" indicates p < 0.001, meaning there is an extremely significant difference; and "****" indicates p < 0.0001, meaning there is an extremely significant difference.

[0082] Based on the above experiments and tests, the following results were obtained: 1. Physicochemical property analysis of CTCs and NPs (1) Absorption spectroscopy analysis and concentration calculation: The molecular formula and physical appearance of the CTCs NPs monomer molecules used for preparation are as follows: Figure 3 As shown in (a), the monomer molecules used to construct the charge-transfer complex were subjected to absorption spectroscopy, as follows: Figure 3 As shown in (b), the absorption wavelengths of the monomer molecules in THF are all less than 600 nm. Subsequently, Per-TQ NPs, 3-PCA-TQ NPs, 3-BrP-TQ NPs, and 3-AP-TQ NPs were successfully prepared, with the following macroscopic morphologies: Figure 3 (c) As shown in the inset. Absorption spectroscopy analysis shows that, compared to the monomer molecules, the absorption wavelengths of all CTCs NPs exhibit a significant red shift, extending to approximately 1000 nm. Figure 3 (c) Among them, 3-AP-TQ NPs showed the most significant redshift, with a maximum absorption wavelength of 1277 nm, and were therefore selected as photothermal reagents for subsequent experiments.

[0083] By measuring the absorbance of different concentrations of 3-AP-TQ, an absorbance-concentration (Ac) standard curve was plotted. Figure 4 (a) Based on this, the yield of nanoparticles was calculated to be 49.46%. Furthermore, the Ac curve of the 3-AP-TQ NPs aqueous solution was determined. Figure 4 (b) Its mass extinction coefficient was determined to be 2.57 mg. - ¹·mL·cm -1 .

[0084] (2) Particle size characterization and stability analysis: The particle size and stability of nanoparticles in various environments have a significant impact on photothermal conversion efficiency, therapeutic dose control and safety, biocompatibility, and cellular uptake. Therefore, the prepared nanoparticles must have an appropriate particle size. For example... Figure 5 As shown in (ad), the average particle sizes of the four nanoparticles—Per-TQ NPs, 3-PCA-TQ NPs, 3-BrP-TQ NPs, and 3-AP-TQ NPs—are 156.4 nm, 141 nm, 152.9 nm, and 115.2 nm, respectively, with corresponding polydispersity indices (PDI) of 0.264, 0.251, 0.277, and 0.232. All samples have a PDI below 0.3, indicating a relatively uniform particle size distribution.

[0085] The morphology and size of 3-AP-TQ NPs were characterized using transmission electron microscopy (TEM). Figure 6 As shown in (a), the nanoparticles are uniformly dispersed with a particle size distribution ranging from 100 to 200 nm, consistent with the nanoparticle diameter results measured by dynamic light scattering. After co-incubating 3-AP-TQ NPs with PBS and DMEM culture media for 7 days, the particle size did not change significantly. Figure 6 (c) indicates that the nanoparticles possess good colloidal stability. Zeta potential testing shows that its surface potential is -17.9 mV ( Figure 6 (b) The strong negative charge indicates a significant electrostatic repulsion between particles, which helps prevent aggregation and maintain good aqueous phase dispersion stability. Furthermore, the absorbance of 3-AP-TQ NPs remained stable at the maximum absorption wavelength in PBS buffers of different pH values. Figure 6 (d) further proves that it has excellent optical stability.

[0086] (3) Feed ratio optimization analysis: 3-AP-TQ NPs were prepared with acceptor to donor (A:D) molar ratios of 1:1, 1:2, 1:3, 2:1, and 3:1, respectively. Figure 7 As shown in (a), the maximum absorption wavelength (λ) of the prepared 3-AP-TQ NPs at different A:D molar ratios is... max No significant displacement occurred, indicating that the formation and stability of 3-AP-TQ NPs are primarily determined by their chemical structure and are not sensitive to the molar ratio of the reactants. Therefore, the A:D molar ratio is not a sensitive parameter for controlling the light absorption properties of 3-AP-TQ NPs. Figure 7As shown in (bf), 3-AP-TQ NPs were prepared with acceptor-donor (A:D) molar ratios of 1:1, 1:2, 1:3, 2:1, and 3:1, with particle sizes of 115.2 nm, 283.8 nm, 447.4 nm, 248.2 nm, and 245.3 nm, respectively; and PDI values ​​of 0.232, 0.43, 0.345, 0.663, and 0.358, respectively. The results indicate that an A:D ratio of 1:1 yields the smallest particle size and best monodispersity (PDI < 0.3). An excess of donor at A:D ratio of 1:2 leads to a significant increase in particle size and a wider distribution (PDI > 0.4). Further excess of donor at A:D ratio of 1:3 results in a continued increase in particle size to its maximum, but the PDI is improved compared to 1:2. An excess of acceptor at A:D ratio of 2:1 leads to an increase in particle size and extremely poor distribution (PDI > 0.6). When the A:D ratio was 3:1, the acceptor was further in excess, and the particle size was similar to that at 2:1. Comprehensive analysis showed that the 3-AP-TQ NPs prepared at A:D = 1:1 exhibited the optimal particle size distribution and stability. Therefore, subsequent experiments used an A:D ratio of 1:1 to prepare 3-AP-TQ NPs.

[0087] (4) Photothermal performance analysis: The photothermal performance of 3-AP-TQ NPs aqueous solution under 1064 nm laser irradiation was systematically evaluated. Different concentrations (0, 25, 50, 100, 200, 400 µg·mL) were used to evaluate the photothermal performance. -1 1 W·cm -2 ) and different power densities (0.25, 0.5, 0.75, 1, 1.5 W·cm) -2 100 µg·mL -1 Its temperature rise behavior was measured under the following conditions. Figure 8 As shown in (a, e), the temperature rise of the solution increases significantly with increasing nanoparticle concentration, reaching 400 µg·mL⁻¹. - ¹The highest temperature can reach 80 ℃. At 100 µg·mL -1 At this concentration, the temperature rise increases significantly with increasing laser power density, reaching a maximum at 1.5 W·cm⁻¹. -2 The temperature rises to approximately 60°C. Figure 8 (b) Furthermore, the photothermal stability of the nanoparticles was tested (100 µg·mL⁻¹). -1 1 W·cm -2 Five consecutive laser switching cycles were performed. The results showed that the temperature change curves between each cycle were highly consistent, and no significant attenuation was observed, demonstrating its excellent photothermal stability. Figure 8 (c) Finally, the calculated photothermal conversion efficiency of 3-AP-TQ NPs was 36.9% ( Figure 8 (d)).

[0088] (5) XRD Diffraction Spectroscopy Analysis: X-ray single-crystal diffraction (XRD) is a method that uses the diffraction phenomenon produced when X-rays irradiate a single-crystal sample to resolve the three-dimensional atomic structure inside the crystal. For charge-transfer complexes, XRD can reflect the crystal form changes between donor and acceptor molecules, providing the most direct evidence for charge-transfer interactions. For example... Figure 9 As shown, XRD spectral analysis revealed that the main diffraction peaks of 3-AP-TQ NPs shifted to lower angles compared to 3-AP and TCNQ monomers. When 3-AP and TCNQ co-crystallize, the strong charge transfer interaction between them becomes the main driving force for the construction of the dominant crystal structure. The diffraction pattern of 3-AP-TQ NPs is neither a simple superposition of the 3-AP pattern nor a superposition of the TCNQ pattern, but rather a completely new and independent combination of diffraction peaks. This directly proves the formation of a new crystal phase with long-range order.

[0089] 2. Performance analysis of composite hydrogels (1) Analysis of hydrogel morphology and structure: such as Figure 10 As shown in (a), a series of 3-AP-TQ NPs / BTB / carr composite hydrogels were successfully constructed by using different concentrations of 3-AP-TQ NPs as photothermal agents, combined with the pH-responsive indicator bromothymol blue (BTB) and the gel matrix carrageenan. Scanning electron microscopy (SEM) characterization results showed that... Figure 10 (b) All groups of hydrogels exhibited similar porous structures, indicating that the concentration change of 3-AP-TQ NPs did not significantly alter the pore size distribution and water absorption properties of the hydrogels.

[0090] (2) Gel performance analysis: such as Figure 11 As shown in (a), the stability of the hydrogel was evaluated by in vitro degradation experiments. After immersion in PBS for 7 days, the cumulative mass loss rates of samples with different concentrations were 17.4%, 14.8%, 13.3%, and 11.8%, respectively, indicating that the hydrogel could maintain structural stability and did not show significant rapid degradation in a simulated wound exudate environment. Further measurements were made of the swelling behavior and porosity of hydrogels at different concentrations. Figure 11 (bc) The results showed that with the increase of 3-AP-TQ NPs concentration, the swelling ratio and porosity of the hydrogel both increased accordingly, indicating that it has excellent liquid absorption capacity. This property helps to effectively adsorb wound exudate, inhibit the proliferation of bacteria locally, thereby synergistically enhancing the antibacterial effect and promoting the wound healing process. Figure 11As shown in (d), hydrogels containing different concentrations of 3-AP-TQ NPs all exhibited a higher storage modulus (G′) than a loss modulus (G″) in dynamic frequency scanning, and both remained essentially constant within the tested angular frequency range, indicating that all samples exhibited typical solid-like elastic behavior and good structural stability. With increasing 3-AP-TQ NPs content, both G′ and G″ increased significantly, indicating enhanced mechanical strength and viscoelastic response of the hydrogels. This phenomenon can be attributed to the F127 molecules modified on the surface of 3-AP-TQ NPs acting as effective physical cross-linking points, further promoting the interaction of molecular chains in the hydrogel network, thereby strengthening the three-dimensional network structure and improving its overall mechanical properties and elastic response.

[0091] (3) Photothermal performance analysis: such as Figure 12 As shown in (ab), at a 1064 nm laser (1 W·cm⁻¹), -2 Under irradiation, the temperature response of the hydrogel significantly increased with increasing concentration of 3-AP-TQ NPs. After 10 minutes of irradiation, the concentration of 100 µg / mL... - The surface temperature of the ¹NPs hydrogel reached a maximum of 53 °C, after which it tended to stabilize; with continued irradiation for 15 minutes, the temperature further increased to 57 °C, reaching a level that effectively killed microorganisms. (100 µg·mL) - ¹The hydrogel underwent five consecutive laser switching cycle tests. Figure 12 (c) The temperature curves of each cycle were highly consistent, with no significant attenuation, indicating excellent photothermal stability and reusability. Further energy conversion analysis revealed that the photothermal conversion efficiency of this composite hydrogel was 32.7%. Figure 12 (d)).

[0092] 3. Biosafety Analysis (1) Cytotoxicity: Cytotoxicity assessment is the first step in evaluating the biosafety of materials. Its purpose is to detect whether the material releases toxic components during direct contact with cells, thereby inhibiting or damaging key cellular functions related to wound healing. For example Figure 13 As shown in (a), after treatment with different concentrations of nanoparticles, neither type of cell showed obvious toxicity, and the cell viability was not significantly different from that of the blank control group, indicating that the nanoparticles have good in vitro biological safety.

[0093] (2) Hemolytic activity: The hemolysis test is a crucial test for assessing whether the material will cause adverse reactions such as red blood cell rupture and hemoglobin release after contact with blood. The results directly relate to the safety of the material's clinical application. If the material exhibits hemolytic activity, it may lead to serious complications such as anemia and kidney damage. Figure 13As shown in (b), in the positive control group, red blood cells ruptured due to osmotic pressure, and the solution turned bright red; while the color of the solution in the negative control group and each experimental group did not change significantly, indicating that the composite hydrogel did not cause red blood cell lysis. Further quantitative analysis showed that the relative hemolysis rate of the experimental group was significantly lower than that of the positive control (p<0.001), proving that the hydrogel does not cause a hemolytic reaction.

[0094] (3) Organ staining analysis: To evaluate the in vivo biosafety of 3-AP-TQ NPs / BTB / carr hydrogel combined with NIR-II irradiation, H&E staining was performed on the major organs (heart, liver, spleen, lung, and kidney) of mice, such as... Figure 14 As shown in (a), histological analysis revealed that the major organ structures of mice in the Hydrogel + NIR-II treatment group remained intact, with no significant pathological changes in cell morphology compared to the control group, and no significant inflammatory cell infiltration. The results indicate that this treatment strategy did not cause significant systemic toxicity under the experimental conditions and demonstrated good in vivo biocompatibility. Figure 14 As shown in (b), the mice in each treatment group maintained stable body weight within 14 days of treatment, indicating that the hydrogel and NIR-II laser did not pose any health risks to the mice.

[0095] 4. In vitro antibacterial performance analysis The in vitro antibacterial properties of the composite hydrogel were verified through antibacterial experiments against MRSA and E. coli. Figure 15 (ab, de), when the concentration of 3-AP-TQ NPs reaches 50 µg·mL - ¹ At 100 µg·mL⁻¹, it showed significant antibacterial effects against both strains; - At the specified concentration, the bactericidal rates against *E. coli* and MRSA reached 99.7% and 98.5%, respectively. Furthermore, morphological changes in MRSA and *E. coli* were observed using scanning electron microscopy (SEM). Figure 15 As shown in (c, f), in the control groups (PBS, PBS + NIR-II, and Hydrogel), MRSA and E. coli bacteria maintained their typical spherical and rod-shaped morphologies, with intact cell membrane structures and no obvious structural damage. However, in the experimental group (Hydrogel + NIR-II), significant perforation-like damage was observed in the cell membranes of MRSA and E. coli, with some areas exhibiting pore-like structural disruption. These results indicate that the Hydrogel + NIR-II system effectively exerts its bactericidal function by inducing bacterial structural damage through a photothermal effect.

[0096] 5. Analysis of treatment effectiveness (1) pH responsiveness analysis: The pH value of the wound microenvironment can indirectly reflect the bacterial growth at the wound site, providing an apparent analysis of the degree of wound infection and treatment outcomes. For example Figure 16 (a) After co-culturing with PBS at different pH values, the color of the composite hydrogel gradually changed from light green to yellow as the pH decreased. For example... Figure 16 (b) In co-culture experiments with different concentrations of E. coli, the color of the composite hydrogel also transitioned from green to yellow as the bacterial concentration increased. Figure 16 (c) After 12 hours of application in an in vivo wound model, the composite hydrogel also showed a color change from green to yellow. These results indicate that the 3-AP-TQ NPs / BTB / carr hydrogel possesses good pH response characteristics and demonstrates a sensitive ability to monitor wound infection status.

[0097] (2) Photothermal Therapy Analysis: As a non-invasive technique for combating wound infection, the core challenge of photothermal therapy lies in finding the optimal balance between effectively killing bacteria and avoiding damage to normal tissue. This balance largely depends on temperature control. Studies have shown that photothermal therapy can effectively inhibit bacterial growth when the temperature is above 50 ℃. However, when the temperature exceeds 60 ℃, high temperatures can damage healthy skin tissue, hindering wound healing. Therefore, controlling the treatment temperature between 50 and 60 ℃ is the optimal range for photothermal therapy. Figure 17 As shown in (ab), during the treatment of infected wounds, the temperature in the control group (PBS, PBA + NIR-II, Hydrogel) remained below 45 ℃, making it difficult to achieve sterilization. In the experimental group (Hydrogel + NIR-II), the temperature at the wound site reached approximately 50 ℃ after 4 minutes of laser irradiation, and approximately 60 ℃ after 10 minutes of irradiation. Therefore, treating the wound with Hydrogel + NIR-II for 10 minutes was sufficient to kill bacteria.

[0098] (3) Wound healing analysis: such as Figure 18 As shown in (a), on day 4 after treatment of infected wounds, both the control group (PBS, PBS + NIR-II, Hydrogel) and the experimental group (Hydrogel + NIR-II) still had some pus at the wound site. From the second treatment to day 10, all mouse wounds had scabbed over, but the control group recovered slowly with little change in wound area. By day 14, the wound area had decreased, but the control group still had a small amount of wound exudate. In contrast, the wounds in the experimental group almost completely closed, and the apparent wound healing was significantly better than that in the control group. Quantitative analysis of wound area during treatment was performed on the different treatment groups, such as... Figure 18As shown in (b), the wound area ratios in the control group were 82.7% (PBS), 23.8% (Hydrogel), and 35.6% (PBS + NIR-II), while the wound area ratio in the experimental group was 7.8% (Hydrogel + NIR-II). This indicates that Hydrogel + NIR-II has a good therapeutic effect on infected wounds.

[0099] (4) Analysis of antibacterial effect on wounds: Bacterial infection is one of the main reasons why chronic wounds are difficult to heal. To study the antibacterial effect of hydrogels in the wound healing process in vivo, the bacterial count at the wound site of each group of mice was observed using the plate colony counting method. Figure 19 As shown, after 10 days of treatment of infected wounds with different treatments, the bacterial concentration at the wound site in the experimental group was reduced by nearly 80% compared with the control group, indicating that Hydrogel + NIR-II treatment effectively inhibited bacterial growth.

[0100] (5) Staining analysis of mouse wound tissue sections: H&E staining of wound tissue plays an irreplaceable core role in wound healing research, providing a "panoramic" visual information on the quality and dynamics of the healing process. For example... Figure 20 On day 14 post-treatment, H&E staining of mouse wound tissue was used to histologically evaluate the therapeutic effect of the composite hydrogel dressing at the microstructural level. Results showed that all treatment groups exhibited neodermal and dermal formation in the wound area due to epithelial tissue hyperplasia. In the control groups (PBS group, PBS + NIR-II group, and Hydrogel group), a clear structural boundary between the epidermis and dermis was still visible, with only preliminary epithelialization. In contrast, the newly formed epidermal layer in the Hydrogel + NIR-II treatment group was continuous and intact, with visible dermal papillary layer formation, indicating a more mature epidermal structure and higher degree of healing in this group.

[0101] In this invention, four types of CTCs NPs were first prepared using a donor functional group modification strategy. Among them, 3-AP-TQ NPs exhibited excellent NIR-II absorption. Then, using 3-AP-TQ NPs as PTAs to combine with the pH-responsive properties of BTB, a composite hydrogel, 3-AP-TQ NPs / BTB / carr, with both monitoring and therapeutic functions was prepared using carr as the gel substrate. Subsequently, a wound infection model was established using MRSA-infected Kunming mice, and the monitoring and treatment of full-thickness chronic wound healing in mice and its in vivo safety were investigated using the 3-AP-TQ NPs / BTB / carr hydrogel. pH responsiveness results showed that the 3-AP-TQ NPs / BTB / carr hydrogel exhibited good pH responsiveness both in vitro and in vivo, accurately reflecting bacterial proliferation at the infected wound site. In vivo results promoting chronic wound healing showed that the 3-AP-TQ NPs / BTB / carr hydrogel could rapidly kill Staphylococcus aureus in the wound area under NIR-II laser irradiation. Finally, cytotoxicity, hemolysis, H&E staining results of major organs, and mouse body weight changes all demonstrated that 3-AP-TQ NPs and hydrogel exhibit good blood compatibility, no organ toxicity, and high biocompatibility. In conclusion, the 3-AP-TQ NPs / BTB / carr hydrogel not only demonstrates good in vivo safety but also excellent antibacterial activity, providing a novel strategy for promoting the healing and monitoring of chronic wounds.

[0102] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. In addition, those skilled in the art can combine and integrate the different embodiments or examples described in this specification.

[0103] Other embodiments of the invention will readily occur to those skilled in the art upon consideration of the specification and practice of the invention disclosed herein. This application is intended to cover any variations, uses, or adaptations of the invention that follow the general principles of the invention and include common knowledge or customary techniques in the art not disclosed herein. The specification and examples are to be considered exemplary only, and the true scope and spirit of the invention are indicated by the appended claims.

Claims

1. A charge transfer complex hydrogel, characterized in that, The charge transfer complex hydrogel comprises: a charge transfer complex nanoparticle solution and a bromothymol blue solution, wherein the charge transfer complex nanoparticles are 3-AP-TQ NPs, Per-TQ NPs, 3-BrP-TQ NPs or 3-PCA-TQ NPs.

2. The method for preparing the charge transfer complex hydrogel according to claim 1, characterized in that, The preparation method includes the following steps: S1, dissolve the electron donor material in THF to prepare a donor solution, wherein the electron donor material is 3-AP, Per, 3-BrP or 3-PCA; S2, TCNQ is dissolved in THF to prepare a receptor solution; S3, the donor solution and acceptor solution are mixed, then F127 solution is added, and after mixing evenly, it is added to deionized water, stirred, THF in the mixed solution is removed, and then filtered to obtain charge transfer complex nanoparticle solution; S4. Add bromothymol blue solution and charge transfer complex nanoparticle solution to carrageenan solution, stir evenly to obtain charge transfer complex hydrogel.

3. The method for preparing the charge transfer complex hydrogel according to claim 2, characterized in that, In S1, the concentration of the donor solution is 0.01 mmol·mL. -1 .

4. The method for preparing the charge transfer complex hydrogel according to claim 2, characterized in that, In S2, the concentration of the receptor solution is 0.01 mmol·mL. -1 .

5. The method for preparing the charge transfer complex hydrogel according to claim 2, characterized in that, In S4, the concentration of the bromothymol blue solution is 2500 μg·mL. -1 .

6. The method for preparing the charge transfer complex hydrogel according to claim 2, characterized in that, In S4, the concentration of bromothymol blue solution in the charge transfer complex hydrogel is 100 μg·mL⁻¹. -1 .

7. Application of charge transfer complex hydrogels, characterized in that, The use of the charge transfer complex hydrogel of claim 1 in the preparation of reagents for antibacterial purposes or for monitoring the degree of wound infection.

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