Photosensitizer, dynamic antibacterial hydrogel containing liposome wrapping photosensitizer and preparation method and application of dynamic antibacterial hydrogel

Abstract

The invention discloses a photosensitizer, dynamic antibacterial hydrogel containing lipidosome wrapping the photosensitizer and a preparation method and application of the dynamic antibacterial hydrogel, and belongs to the technical field of biological materials. The invention provides a novel D-A-D type photosensitizer TPT which is simple in structure and simple in synthetic route, and from the angles of safety, practicability and use specificity of hydrogel dressing, modified hyaluronic acid and modified carboxymethyl chitosan are used as raw materials, cationic liposome wrapping the novel photosensitizer TPT is carried, and the hydrogel dressing is prepared. The efficient antibacterial dynamic cross-linked hydrogel material is prepared. The hydrogel disclosed by the invention has two pH response bonds, namely an imine bond and a boric acid ester bond, and has good moisture retention and biocompatibility; meanwhile, the hydrogel is loaded with the cationic liposome wrapping the novel photosensitizer TPT, so that the hydrogel has excellent active oxygen yield. The hydrogel disclosed by the invention aims to quickly resist bacteria, promote wound healing, reduce infection risk and improve the success rate of treatment through the targeted drug release function of the intelligent hydrogel.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological materials, and particularly relates to a photosensitizer, a dynamic antibacterial hydrogel containing liposomes encapsulating the photosensitizer, and a preparation method and application thereof. BACKGROUND

[0002] In daily life, external causes such as collision, scratching, and cutting are prone to cause skin wounds. These wounds destroy the integrity of the skin, making the skin tissue extremely vulnerable to bacterial invasion, leading to infection and inflammation. If the wound infection is not properly treated, the inflammation caused by the infection can develop into an abscess or even more serious complications, such as sepsis, septic shock, multiple organ dysfunction syndrome, etc.

[0003] Photodynamic antibacterial therapy refers to that under specific wavelength irradiation, photosensitizers undergo a series of photophysical and chemical reactions to produce active oxygen with cytotoxicity, thereby destroying the structure of bacteria and exerting antibacterial effect. The key to photodynamic therapy lies in photosensitizers, and the efficacy of photodynamic therapy depends on the yield of active oxygen. The currently developed photosensitizers generally have low active oxygen yield, complex structure, and cumbersome synthesis route, resulting in extremely high preparation cost. Therefore, it is necessary to develop a photosensitizer with high active oxygen yield, simple manufacturing process, and low cost.

[0004] At present, there is still a lack of effective specific drug sustained release system for emergency treatment of susceptible wounds. Under normal conditions, the microenvironment of healthy skin tissue is neutral to slightly alkaline. However, at the site of bacterial infection of the wound, the acidity of the site increases due to the production of malic acid, acetic acid, lactic acid, etc. by bacterial metabolism, and the influence of excessive immune response, presenting an acidic microenvironment (pH = 4.5~6.5). Therefore, pH-responsive drug delivery systems for this situation have attracted widespread attention.

[0005] Hydrogels can simulate the physical and chemical properties of the natural extracellular matrix or tissue, and have good moisturizing properties and biocompatibility. By changing the crosslinking method and chemical composition of the hydrogel, the hydrogel can obtain different properties, such as pH responsiveness. Hyaluronic acid is one of the components of the extracellular matrix, and participates in the hemostasis period, inflammation period, proliferation period, and remodeling period in the skin healing process, and plays a key role in cell differentiation, proliferation, and migration. As a naturally occurring biological macromolecule, hyaluronic acid can maintain a moist environment for the wound and promote the migration of wound tissue cells, helping to promote wound healing, and hyaluronic acid and its derivatives can also form regenerative biomaterials. Carboxymethyl chitosan, as a biological material, has been widely studied in the medical field. Studies have shown that carboxymethyl chitosan has the use of promoting wound healing and repairing skin barrier. At the same time, carboxymethyl chitosan has good antibacterial and anti-inflammatory effects. Since carboxymethyl chitosan contains active amino groups and carboxyl groups, it can be connected with various bioactive substances, and therefore can be used as a good drug controlled release carrier material.

[0006] In summary, this invention provides a novel DAD-type photosensitizer, TPT, with a simple structure and easy synthesis route. From the perspective of the safety, practicality, and specificity of hydrogel dressings, modified hyaluronic acid and modified carboxymethyl chitosan were used as raw materials to prepare a dynamically cross-linked hydrogel material with highly efficient antibacterial activity by encapsulating cationic liposomes containing the novel photosensitizer TPT, using modified hyaluronic acid and modified carboxymethyl chitosan as raw materials. Under light irradiation, the photosensitizer TPT generates a large amount of reactive oxygen species to achieve the antibacterial effect. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a novel DAD-type photosensitizer, TPT, with a simple structure and easy synthesis route. Using modified hyaluronic acid and modified carboxymethyl chitosan as raw materials, and encapsulating cationic liposomes containing the novel photosensitizer TPT, a highly efficient antibacterial dynamically cross-linked hydrogel material is prepared. The hydrogel of this invention possesses both imine and borate ester bonds, exhibiting both good moisturizing properties and biocompatibility. Furthermore, the cationic liposomes encapsulating the novel photosensitizer TPT within the hydrogel demonstrate excellent reactive oxygen species (ROS) yield. This hydrogel is a novel material with potentially broad applications. Through the targeted drug release function of this intelligent hydrogel, it aims to rapidly combat bacteria, promote wound healing, reduce the risk of infection, and improve treatment success rates.

[0008] To achieve the above objectives, the specific technical solution of the present invention is as follows:

[0009] In a first aspect, the present invention provides a DAD-type photosensitizer TPT, comprising a photosensitizing molecule, the chemical structural formula of which is as follows:

[0010] .

[0011] The photosensitizer of this invention exhibits aggregation-induced emission properties and excellent reactive oxygen species (ROS) yield, exceeding that of currently used second-generation photosensitizers such as hematoporphyrin monomethyl ether (HMME) and Rose Bengal. ROS can react with phospholipids on bacterial cell membranes, thereby disrupting membrane structure, increasing bacterial cell membrane permeability, accelerating the outflow of internal bacterial substances, and inhibiting bacterial activity. Furthermore, ROS can interfere with the normal replication and repair processes of intracellular nucleic acids, thus inhibiting bacterial metabolism and growth. In addition, the photosensitizer of this invention has no dark cytotoxicity and is biosafety.

[0012] Secondly, the present invention provides a method for preparing the above-mentioned DAD-type photosensitizer TPT, comprising the following steps:

[0013] S1. Under a nitrogen atmosphere, 4,4'-bis(dimethoxy)benzophenone, zinc powder, and 4-bromobenzophenone were dissolved in an anhydrous solvent, and TiCl4 was added dropwise. The mixture was heated to 60-80°C and refluxed to obtain compound 1. The structural formula of compound 1 is as follows:

[0014] ;

[0015] S2. Under a nitrogen atmosphere, compound 1, pinacol diborate, potassium acetate, and [1,10-bis(diphenylphosphine)ferrocene]palladium(II) dichloride were dissolved in an anhydrous solvent and reacted with stirring at 60-90°C for 8-20 h to obtain compound 2. The structural formula of compound 2 is as follows:

[0016] ;

[0017] S3. Under a nitrogen atmosphere, compound 2,3,8-dibromo-1,10-phenanthroline was dissolved in an anhydrous solvent, and after adding alkali, the mixture was reacted at 80-100°C for 16-24 hours to obtain the DAD-type photosensitizer TPT.

[0018] Preferably, the anhydrous solvent includes at least one of tetrahydrofuran, 1,2-dimethoxyethane, toluene, and xylene.

[0019] Preferably, in step S3, the alkali includes at least one of sodium carbonate, potassium carbonate, cesium carbonate, and triethylamine.

[0020] Thirdly, the present invention provides a cationic liposome encapsulating the above-mentioned DAD-type photosensitizer, which is prepared by the following method: distearate phosphatidylcholine (DSPC), bis(octadecyl dimethyl ammonium bromide) (DDAB), cholesterol and the photosensitizer TPT are mixed and dissolved in chloroform, the solvent is evaporated in a round-bottom flask using a rotary evaporator to form a thin film, deionized water is added and ultrasonically hydrated, and then centrifuged to obtain the liposome TPT@Lip.

[0021] Preferably, the concentration of photosensitizer TPT in the liposome TPT@Lip is 8 wt%~10 wt%.

[0022] Fourthly, the present invention provides a pH-responsive dynamic hydrogel comprising the above-mentioned cationic liposome TPT@Lip, dopamine-modified hyaluronic acid, and phenylboronic acid-modified carboxymethyl chitosan.

[0023] Preferably, the mass ratio of the dopamine-modified hyaluronic acid, the phenylboronic acid-modified carboxymethyl chitosan, and the liposome TPT@Lip is 40:40:(0.2~0.5).

[0024] Preferably, the dopamine-modified hyaluronic acid is prepared by the following method: hyaluronic acid is dissolved in water to obtain a hyaluronic acid solution, then EDC and NHS are added, and the solution is stirred at pH 5-6 for 1-3 hours; then an aqueous solution of dopamine hydrochloride is added, and the solution is stirred for 12-36 hours, the pH is adjusted to 5.0-6.0, and the solution is obtained by dialysis and freeze-drying.

[0025] Preferably, the phenylboronic acid-modified carboxymethyl chitosan is prepared by the following method: phenylboronic acid is dissolved in an aqueous ethanol solution to obtain a phenylboronic acid solution, and then the phenylboronic acid solution is added dropwise to the carboxymethyl chitosan solution. After stirring for 4-12 hours, the solution is dialyzed and freeze-dried to obtain the phenylboronic acid-modified carboxymethyl chitosan.

[0026] Fifthly, the present invention provides a method for preparing the above-mentioned pH-responsive dynamic hydrogel, comprising the following steps: dissolving dopamine-modified hyaluronic acid and phenylboronic acid-modified carboxymethyl chitosan in PBS respectively, then dispersing liposomes TPT@Lip in the phenylboronic acid-modified carboxymethyl chitosan solution, and finally adding dopamine-modified hyaluronic acid solution to obtain the hydrogel.

[0027] In a sixth aspect, the present invention provides the application of the above-mentioned pH-responsive dynamic hydrogel in the preparation of antibacterial dressings or biosensor materials.

[0028] This invention also provides an antibacterial dressing made from a dynamic hydrogel containing TPT liposomes coated with a photosensitizer. This antibacterial dressing is free of antibiotics, metal ions, and antimicrobial peptides, thus offering enhanced safety. Furthermore, testing has shown that this dressing exhibits excellent antibacterial properties.

[0029] Compared with the prior art, the advantages of the present invention are:

[0030] (1) The photosensitizer of the present invention exhibits aggregation-induced emission properties and excellent reactive oxygen species (ROS) yield, which is higher than that of second-generation photosensitizers monomethyl ether hematoporphyrin (HMME) and Rose Bengal. ROS can react with phospholipids on bacterial cell membranes, thereby disrupting the membrane structure, increasing bacterial cell membrane permeability, accelerating the outflow of internal bacterial substances, and inhibiting bacterial activity. In addition, ROS can also interfere with the normal replication and repair processes of intracellular nucleic acids in bacteria, thereby inhibiting bacterial metabolism and growth. Furthermore, the DAD-type photosensitizer TPT of the present invention has a simple structure, a simple preparation route, no cellular dark toxicity, and is biosafety, making it suitable for industrial production.

[0031] (2) The present invention proposes a cationic liposome that encapsulates a photosensitizer, which can promote the fusion of the liposome with the negatively charged bacterial cell membrane through electrostatic interaction, thereby promoting the entry of the photosensitizer into the bacterial cell and enhancing the killing power of the photosensitizer on bacteria.

[0032] (3) The hydrogel matrix in this invention is prepared from modified hyaluronic acid and carboxymethyl chitosan. Hyaluronic acid and carboxymethyl chitosan have advantages such as good biocompatibility and controllable degradation.

[0033] (4) The dynamic hydrogel of the present invention has a rapid response to pH stimulation and possesses the structural basis for drug loading and self-releasing. Carboxymethyl chitosan contains a certain number of amino groups, which can undergo Schiff base reaction crosslinking with the free aldehyde groups in phenylboronic acid to generate acid-responsive Schiff base bonds; hyaluronic acid grafted with dopamine has a catechol structure, which can form dynamic phenylboronic ester bonds with the boric acid bonds in phenylboronic acid-modified carboxymethyl chitosan. Both the boric ester bond structure and the Schiff base structure are pH sensitive and unstable under acidic conditions. Attached Figure Description

[0034] Figure 1 The 1H NMR spectrum of the photosensitizer TPT prepared in Example 1;

[0035] Figure 2 The graph shows a comparison of the reactive oxygen species yields of TPT prepared in Example 1 with those of HMME and Rose Bengal.

[0036] Figure 3 The image shows the results of the cell dark toxicity assay for TPT@Lip prepared in Example 3;

[0037] Figure 4 This is a schematic diagram of the hydrogel preparation process;

[0038] Figure 5 This diagram illustrates the sol-gel transition, self-repair, and injection of hydrogels.

[0039] Figure 6 Images showing the degradation of hydrogels under different pH conditions;

[0040] Figure 7 This is a morphology diagram of the hydrogel;

[0041] Figure 8 This is a schematic diagram illustrating the antibacterial properties of hydrogels. Detailed Implementation

[0042] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0043] This invention provides a DAD-type photosensitizer TPT, comprising a photosensitizing molecule, the chemical structural formula of which is as follows:

[0044] .

[0045] In some embodiments, the preparation method of the photosensitizer TPT includes the following steps:

[0046] S1. Under a nitrogen atmosphere, 4,4'-bis(dimethoxy)benzophenone, zinc powder, and 4-bromobenzophenone were dissolved in anhydrous solvent, and TiCl4 was added dropwise. After the addition was complete, the mixture was heated to 60-80°C and refluxed for 0.5-2 hours. After the reaction was completed, the reaction solution was cooled to room temperature, quenched with Na2CO3 aqueous solution, and extracted three times with dichloromethane. The organic phases were combined and washed three times with saturated brine. The mixture was dried over anhydrous Na2SO4, concentrated under reduced pressure, and purified by column chromatography to obtain compound 1. The structural formula of compound 1 is:

[0047] ;

[0048] S2. Under a nitrogen atmosphere, compound 1, pinacol diborate, potassium acetate, and [1,10-bis(diphenylphosphine)ferrocene]palladium(II) dichloride were dissolved in an anhydrous solvent and reacted with stirring at 60-90°C for 8-20 h. After the reaction was completed, the mixture was cooled to room temperature, quenched with water, extracted three times with dichloromethane, and the combined organic phases were washed three times with saturated brine. The mixture was dried over anhydrous Na2SO4, concentrated under reduced pressure, and purified by column chromatography to obtain compound 2. The structural formula of compound 2 is:

[0049] ;

[0050] S3. Under a nitrogen atmosphere, compound 2,3,8-dibromo-1,10-phenanthroline was dissolved in an anhydrous solvent. After adding alkali, the mixture was reacted at 80-100℃ for 16-24 h. After the reaction was completed, the mixture was cooled to room temperature, quenched with a saturated ammonium chloride aqueous solution, extracted three times with dichloromethane, and the combined organic phases were washed three times with saturated brine. The mixture was dried over anhydrous Na2SO4, concentrated under reduced pressure, and purified by column chromatography to obtain the DAD-type photosensitizer TPT.

[0051] The anhydrous solvent can be any one of tetrahydrofuran, 1,2-dimethoxyethane, toluene, and xylene; the base can be any one of sodium carbonate, potassium carbonate, cesium carbonate, and triethylamine.

[0052] In other embodiments, a cationic liposome (TPT@Lip) encapsulating the photosensitizer TPT is provided, which is prepared by the following method: distearate phosphatidylcholine (DSPC), bis(octadecyl dimethyl ammonium bromide) (DDAB), cholesterol and the photosensitizer TPT are mixed and dissolved in chloroform, the solvent is evaporated in a round-bottom flask using a rotary evaporator to form a thin film, deionized water is added and ultrasonically hydrated, and then centrifuged to obtain the liposome TPT@Lip.

[0053] The concentration of photosensitizer TPT in the liposome TPT@Lip is 8 wt%~10 wt%.

[0054] In other embodiments, a pH-responsive dynamic hydrogel comprising dopamine-modified hyaluronic acid, phenylboronic acid-modified carboxymethyl chitosan, and the above-mentioned TPT@Lip is provided, wherein the mass ratio of the dopamine-modified hyaluronic acid, phenylboronic acid-modified carboxymethyl chitosan, and liposome TPT@Lip is 40:40:(0.2~0.5).

[0055] Example 1

[0056] Synthesis of photosensitizer TPT

[0057] S1. Synthetic intermediate compound 1:

[0058] ;

[0059] Under nitrogen atmosphere, 4,4'-bis(dimethoxy)benzophenone (2.91 g, 9 mmol), zinc powder (6.3 g, 97 mmol), and 4-bromobenzophenone (2.34 g, 9 mmol) were sequentially added to a double-necked round-bottom flask; then anhydrous tetrahydrofuran (50 mL) was added to the reaction mixture while maintaining the temperature between -5°C and 0°C; then, TiCl4 (5.4 mL, 48.6 mmol) was slowly added dropwise with continuous stirring; the reaction mixture was stirred for 30 minutes, then gradually heated to room temperature, and then heated to 75°C under reflux. After the reaction was completed by TLC monitoring, the mixture was cooled to room temperature and quenched with 10% Na2CO3 aqueous solution. The reaction solution was filtered, the filter cake was washed twice with saturated brine, and the filtrates were mixed and extracted three times with dichloromethane (DCM) (100 mL each time). The organic phases were combined and washed three times with saturated brine (50 mL each time). The solution was dried over anhydrous Na2SO4, concentrated under reduced pressure, and purified by column chromatography to obtain compound 1 as a yellow solid (5.52 g, yield 52%).

[0060] S2. Synthetic intermediate compound 2:

[0061] ;

[0062] Under nitrogen atmosphere, compound 1 (1.42 g, 2.8 mmol), pinacol diboronate (1.54 g, 3.0 mmol), potassium acetate (1.12 g, 11.2 mmol), and [1,10-bis(diphenylphosphine)ferrocene]palladium(II) dichloride (80.2 mg, 0.05 mmol) were dissolved in 30 mL of anhydrous dioxane. The reaction mixture was then stirred at 85 °C for 16 h. After the reaction was completed by TLC monitoring, the reaction solution was cooled to room temperature, quenched with 50 mL of water, and extracted three times with dichloromethane (DCM) (100 mL each time). The organic phases were combined and washed with saturated brine (3 × 50 mL), dried over anhydrous Na2SO4, concentrated under reduced pressure, and purified by column chromatography to give compound 2 as a yellow solid (1.02 g, yield 62%).

[0063] S3. Synthesizing TPT:

[0064] ;

[0065] Compound 2 (0.70 g, 2.2 mmol), 3,8-dibromo-1,10-phenanthroline (168 mg, 1.0 mmol), [Pd(PPh3)4] (80.9 mg, 0.2 mmol), and Na2CO3 (286.8 mg, 2.0 mmol) were dissolved in tetrahydrofuran and degassed with nitrogen. The reaction mixture was heated to 90 °C for 24 hours under nitrogen protection. After the reaction was confirmed by TLC, the reaction mixture was cooled to room temperature, quenched with saturated ammonium chloride aqueous solution (50 mL), and extracted with DCM (100 mL × 3). The organic phases were combined and washed with saturated brine (3 × 50 mL), dried over anhydrous Na2SO4, concentrated under reduced pressure, and purified by column chromatography to give a white solid compound TPT (325 mg, yield 63%).

[0066] 1 1H NMR (600 MHz, CDCl3) δ (TMS, ppm): 9.39 (d, J = 2.2 Hz, 2H), 8.34 (d, J = 2.2 Hz, 2H), 7.82 (s, 2H), 7.55 (d, J = 8.3 Hz, 4H), 7.25–6.96 (m, 24H), 6.70 (d, J = 8.8 Hz, 4H), 6.65 (d, J = 8.8 Hz, 4H), 3.75 (s, 12H). The 1H NMR spectrum of compound TPT is shown below. Figure 1 As shown.

[0067] Example 2

[0068] Detection of reactive oxygen species yield

[0069] The generation efficiency of singlet oxygen (one of the types of reactive oxygen species) in solution was investigated using (9,10-anthratridimyl-bis(methylene)dicarboxylic acid) (ABDA) as an indicator. ABDA is a water-soluble anthracene derivative that can react with singlet oxygen to generate the corresponding internal peroxide through photobleaching, as shown in the following reaction formula. This reaction was monitored by spectrophotometric recording of the decrease in optical density at 378 nm.

[0070] ;

[0071] ABDA (50 μM) solution was mixed thoroughly with 20 μM TPT, second-generation photosensitizers monomethyl ether hematoporphyrin (HMME) and rose Bengal (RB) solutions in water, and then irradiated with LEDs for a certain number of times. The irradiation wavelength of TPT was 405 nm, and the irradiation wavelengths of HMME and RB were 535 nm. Simultaneously, 535 nm and 405 nm LED light sources were set up as control groups. The change in absorbance A of ABDA at 378 nm relative to the initial value A0 was recorded at different irradiation times. Figure 2 The graph shows the relationship between the A / A0 ratio of ABDA and irradiation time under LED irradiation. As can be seen from the graph, the decreasing trend of the A / A0 ratio in the ABDA and TPT mixed solution is more pronounced than that in the group mixed with HMME and RB, indicating that the singlet oxygen yield of TPT is higher than that of HMME and RB.

[0072] Example 3

[0073] Preparation of cationic liposomes (TPT@Lip) encapsulating the photosensitizer TPT.

[0074] Cationic liposomes have been reported as local drug delivery carriers to facilitate drug transfer to the skin due to their physical stability and biocompatibility. To prepare TPT cationic liposomes, distearylphosphatidylcholine (DSPC), bis(octadecyldimethylammonium bromide) (DDAB), cholesterol, and TPT were mixed and dissolved in 10 mL of chloroform at a molar ratio of 8:2:1:1. The solvent was evaporated in a round-bottom flask using a rotary evaporator to form a thin film. Then, 5 mL of deionized water was added, and the mixture was sonicated for 30 min to hydrate the lipid film. Finally, the liposome solution was centrifuged at 5000 rpm / min for 10 min, and the supernatant was collected as TPT@Lip.

[0075] The encapsulation efficiency of liposomes for TPT was calculated to be 80.9% and the drug loading rate was 9.2 wt% using ultraviolet-visible spectrophotometry.

[0076] Example 4

[0077] Cytotoxicity assay of TPT@Lip

[0078] Cell compatibility of the materials was evaluated using the MTT assay. After sterilization by filtration through a 0.22 μm filter, TPT@Lip solution was incubated with L929 cells and human umbilical vein endothelial cells (HUVECs) (5000 cells / well) at gradient concentrations in 96-well plates for 24 h. Then, 100 µL of MTT (500 µg / mL) solution was added to each well, and incubation continued at 37 °C for 4 h. Finally, 100 µL of dimethyl sulfoxide was added to dissolve the MTT crystals. The absorbance at 570 nm was measured using a microplate reader, indirectly reflecting the number of viable cells. Within a certain cell number range, the amount of MTT crystals formed was directly proportional to the cell number. The test was repeated three times. Cell viability was calculated using the following formula:

[0079] Cell viability = [(experimental wells - blank wells) / (negative control wells - blank wells)] × 100%

[0080] The results are as follows Figure 3 As shown, at 400 µg / mL, the survival rate of L929 cells and HUVEC cells remained above 85%, indicating that the cells could grow normally in the presence of TPT@Lip, demonstrating that the prepared TPT@Lip has good biocompatibility and safety.

[0081] Example 5

[0082] Preparation of blank hydrogels

[0083] (1) Synthesis of modified hyaluronic acid:

[0084] a) Dissolve 95 mg of dopamine hydrochloride (DA) powder in 5 ml of deionized water and vortex mix until homogeneous to obtain an aqueous solution of dopamine hydrochloride.

[0085] b) Dissolve 201 mg of hyaluronic acid (HA) with a molecular weight of 800-1500 kDa in 50 mL of deionized water and let it stand overnight until it is completely dissolved to obtain an HA solution;

[0086] c) 191 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 115 mg of N-hydroxysuccinimide (NHS) were slowly added to the HA solution, the pH was adjusted to 5.5 ± 0.5, and the mixture was stirred for 2 h to activate the carboxyl group.

[0087] d) Add 5 mL of dopamine hydrochloride aqueous solution to the above solution, stir the mixture at room temperature for 24 h, and use NaOH and HCl aqueous solution to maintain the pH at 5.0~6.0, so that the amino group of dopamine and the carboxyl group of hyaluronic acid are coupled to form an amide bond;

[0088] e) After the reaction, the solution was placed in a dialysis bag (MWCO 8-14 kDa) and dialyzed with deionized water (pH = 5) for 3 days to remove unreacted chemicals and byproducts;

[0089] f) Freeze-dry the solution to obtain the HA-DA grafted product, which is cotton-like and stored at -20°C for later use.

[0090] (2) Synthesis of modified carboxymethyl chitosan:

[0091] a) Dissolve 500 mg of CMCS with a molecular weight of 100-200 KDa in 100 mL of deionized water and let stand overnight until completely dissolved to obtain a CMCS solution.

[0092] b) Dissolve 75 mg of phenylboronic acid (PBA) in a mixed solution of 2 mL ethanol and 4 mL deionized water, and sonicate for 2 h to dissolve, thus obtaining a PBA solution;

[0093] c) At a speed of 500 rpm, the PBA solution from step (b) was slowly added dropwise to the CMCS solution from step (a), and stirred at room temperature for 6 h.

[0094] d) After the reaction, the solution was placed in a dialysis bag (MWCO 8-14 kDa) and dialyzed with deionized water for 3 days to remove unreacted chemicals and byproducts. Then, the solution was freeze-dried to obtain the CMCS-PBA graft product, which was foamy and stored at -20°C for later use.

[0095] (3) Preparation of blank hydrogel:

[0096] Dopamine-modified hyaluronic acid and phenylboronic acid-modified carboxymethyl chitosan were dissolved separately in PBS at a concentration of 40 mg / mL. The two solutions were then mixed in equal volumes, stirred thoroughly, and allowed to stand for 10 minutes to obtain a pH-sensitive dynamically cross-linked hydrogel. The specific technical route is as follows: Figure 4 As shown.

[0097] Example 6

[0098] Sol-gel transition time, self-healing and injectability testing of hydrogels

[0099] (1) Sol-gel transition time: The sol-gel transition time (gelation time) of the hydrogel was tested using the inverted vial method at room temperature. Specifically, the hydrogel precursor solution was placed in a vial, and the vial was tilted every 10 seconds. If the solution did not flow, the sol-gel transition had occurred, and the time at this point was recorded as the gelation time of the hydrogel. Figure 5As shown in Figure a, the sol stopped flowing after the vial was inverted at 80 s, indicating that the gelation time was 80 s.

[0100] (2) Self-healing properties of the hydrogels: Two disc-shaped hydrogels (1 cm in radius) were stained with methyl orange and rhodamine B, respectively, and then cut in half. Subsequently, at room temperature and without any external intervention, the two different colored hemispheres were kept in close contact along the cutting line for 12 h. The self-healing ability of the hydrogels was then determined by direct visual observation. Figure 5 As shown in b, during the healing process, the two dye molecules continuously diffuse on the cut surface and penetrate each other, eventually fusing completely into one. The resulting hydrogel is not significantly different from the hydrogel before repair and can withstand its own gravity in the direction perpendicular to the cut surface without splitting.

[0101] (3) Injectability of the hydrogel: After loading the rhodamine B-stained hydrogel precursor solution into a 1 mL syringe, push the syringe plunger to squeeze it out from the syringe needle. For example... Figure 5 As shown in Figure c, the letters "WHU" can be successfully written on the plate after extrusion, indicating that the hydrogel crosslinked with imine and borate ester bonds has good injectability.

[0102] Example 7

[0103] Acid sensitivity test of hydrogels

[0104] For better observation of the gel-sol transition, methyl orange staining was used. 2 g of methyl orange-stained hydrogel was treated with PBS (pH=7.4) and PBS (pH=4.4) respectively, and observed after standing at room temperature. Figure 6 As shown, the hydrogel almost becomes liquid after the addition of an acidic solution, while the hydrogel in the neutral solution does not change. This is because imine bonds and borate ester bonds are dynamic chemical bonds that are sensitive to pH changes. Under pH stimulation, imine bonds and borate ester bonds break, resulting in the macroscopic disintegration of the cross-linked structure of the hydrogel.

[0105] Example 8

[0106] Morphology detection of hydrogels

[0107] The microstructure of the hydrogel surface was observed using a scanning electron microscope (Tescan VEGA Compact). After freeze-drying the prepared hydrogel, it was slowly submerged in liquid nitrogen, causing it to fracture. The fractured hydrogel was then cut into regularly sized cross-sections (not exceeding the size of the copper stage) using a scalpel. Conductive adhesive was used to fix the samples onto the copper stage. Because the sample is non-conductive, it underwent gold sputtering. The microstructure of the hydrogel was then observed using a low-magnification scanning electron microscope.Figure 7 As shown, the surface of the hydrogel contains a large number of pores that are evenly distributed, with a pore size of about 100 µm. These pores ensure the hydrogel's strong water absorption and retention capacity, making it suitable for use as a wound dressing.

[0108] Example 9

[0109] Preparation of drug-loaded hydrogels

[0110] Dopamine-modified hyaluronic acid and phenylboronic acid-modified carboxymethyl chitosan were dissolved in PBS at a concentration of 40 mg / mL. TPT@Lip was uniformly dispersed in the modified carboxymethyl chitosan solution at a concentration of 300 μg / mL. The two solutions were then mixed in equal volumes, stirred evenly, poured into a mold, and allowed to stand for 10 min to obtain the drug-loaded hydrogel.

[0111] Example 10

[0112] In vitro antibacterial test of hydrogel

[0113] In the in vitro antibacterial experiments, *Escherichia coli* (E. coli) and methicillin-resistant *Staphylococcus aureus* (MRSA) were used. The experiment consisted of three groups: a control group (PBS), the blank gel group from Example 5, and the drug-loaded gel group from Example 9. The hydrogels were prepared under sterile conditions and placed in sterile 24-well plates. 10 μL of bacterial suspension (1×10⁻⁶) was added to each well. 8 CFU / mL, PBS, pH 5.5) was dropped onto the hydrogel surface and co-cultured at 37°C for 3 h. Subsequently, the hydrogel surface was irradiated with an LED at a wavelength of 405 nm for 10 min. After irradiation, the bacterial suspensions of each group were diluted with sterile PBS and inoculated onto LB broth agar plates. After incubation at 37°C for 24 h, the colonies on the plates were photographed. Figure 8 It can be seen that, compared with the PBS control group, the blank gel group has a slight antibacterial effect, while the number of colonies on the plate of the hydrogel group loaded with TPT@Lip is significantly reduced, which proves the photodynamic antibacterial potential of TPT@Lip.

[0114] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A D-A-D type photosensitizer TPT comprising a photosensitizing molecule, characterized in that, The chemical structure of the photosensitive molecule is as follows: 。 2. The method of claim 1 for the preparation of the D-A-D photosensitizer TPT, characterized by, The method comprises the following steps: S1. Under a nitrogen atmosphere, 4, 4'-bis(dimethoxy) benzophenone, zinc powder and 4-bromobenzophenone are dissolved in anhydrous solvent, and TiCl4 is added dropwise, and heated to 60-80℃ to reflux, and after reaction, compound 1 is obtained, and the structural formula of the compound 1 is as follows: ； S2. Under a nitrogen atmosphere, compound 1, pinacol diboronic acid, potassium acetate and [1, 10-bis (diphenylphosphine) ferrocene] dichloropalladium (II) are dissolved in anhydrous solvent, and stirred at 60-90℃ for 8-20h to obtain compound 2, and the structural formula of the compound 2 is as follows: ； S3. Under a nitrogen atmosphere, compound 2 and 3, 8-dibromo-1, 10-phenanthroline are dissolved in anhydrous solvent, and after adding a base, it is reacted at 80-100℃ for 16-24h to obtain the D-A-D type photosensitizer TPT.

3. The preparation method according to claim 2, characterized in that, The anhydrous solvent comprises at least one of tetrahydrofuran, 1, 2-dimethoxyethane, toluene and xylene.

4. The production method according to claim 2, characterized by, In step S3, the base comprises at least one of sodium carbonate, potassium carbonate, cesium carbonate and triethylamine.

5. Liposome TPT@Lip comprising the photosensitizer TPT according to claim 1, characterized in that, The liposome TPT@Lip is prepared by the following method: after mixing distearoylphosphatidylcholine (DSPC), dioctadecyldimethylammonium bromide (DDAB), cholesterol and the photosensitizer TPT, dissolving them in chloroform, using a rotary evaporator to evaporate the solvent in a round-bottom flask to form a thin film, adding deionized water and ultrasonic hydration, and centrifuging to obtain the liposome TPT@Lip.

6. A pH-responsive dynamic hydrogel, characterized in that, The hydrogel comprises dopamine-modified hyaluronic acid, phenylboronic acid-modified carboxymethyl chitosan and the liposome TPT@Lip of claim 5.

7. The pH-responsive dynamic hydrogel according to claim 6, wherein, The mass ratio of the dopamine-modified hyaluronic acid, the phenylboronic acid-modified carboxymethyl chitosan and the liposome TPT@Lip is 40:40: (0.2-0.5).

8. The pH-responsive dynamic hydrogel of claim 6, wherein, The dopamine-modified hyaluronic acid is prepared by the following method: hyaluronic acid is dissolved in water to obtain a hyaluronic acid solution, then EDC and NHS are added, and stirred at pH 5-6 for 1-3h; then dopamine hydrochloride aqueous solution is added, stirred for 12-36h, the pH is adjusted to 5.0-6.0, and after dialysis and freeze-drying, the dopamine-modified hyaluronic acid is obtained; And / or, the phenylboronic acid-modified carboxymethyl chitosan is prepared by the following method: phenylboronic acid is dissolved in an aqueous ethanol solution to obtain a phenylboronic acid solution, then the phenylboronic acid solution is added dropwise into a carboxymethyl chitosan solution, stirred for 4-12h, and after dialysis and freeze-drying, the phenylboronic acid-modified carboxymethyl chitosan is obtained.

9. A method for preparing the pH-responsive dynamic hydrogel according to any one of claims 6 to 8, characterized in that, The hydrogel comprises the following steps: dopamine-modified hyaluronic acid and phenylboronic acid-modified carboxymethyl chitosan are respectively dissolved in PBS, then the liposome TPT@Lip is dispersed in the phenylboronic acid-modified carboxymethyl chitosan solution, and finally the dopamine-modified hyaluronic acid solution is added to obtain the hydrogel.

10. Use of the pH-responsive dynamic hydrogel of any one of claims 6-8 in the preparation of an antibacterial dressing or a biosensor material.

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