A photodynamic antibacterial hydrogel without chemical crosslinking agent, its preparation method and application

Abstract

This invention discloses a photodynamic antibacterial hydrogel without chemical crosslinking agents, its preparation method, and its application, belonging to the field of biomedical antibacterial materials technology. The method involves mixing an aqueous solution containing polyvinyl alcohol and an aqueous solution containing a sodium salt of a cyclic metallized iridium(III) complex photosensitizer to obtain a mixed precursor solution. This mixed precursor solution is then subjected to freeze-thaw cycle molding to obtain the photodynamic antibacterial hydrogel. This hydrogel utilizes multiple hydrogen bonds formed between the sodium carboxylate groups in IrONa and the hydroxyl groups on the PVA chain to construct an interaction network. Through these multiple hydrogen bonds and the microporous structure formed during the freeze-thaw process, IrONa achieves nanoscale physical confinement. This confinement effect effectively overcomes the common problems of easy leakage and elution in small molecule photosensitizers within physical crosslinking networks.

Description

Technical Field

[0001] This invention belongs to the field of biomedical antibacterial materials technology, specifically relating to a photodynamic antibacterial hydrogel without chemical crosslinking agents, its preparation method, and its application. Background Technology

[0002] In the clinical treatment of acute and chronic wounds, bacterial infection often leads to stalled tissue repair and slow functional recovery. With the widespread prevalence of multidrug-resistant strains, such as methicillin-resistant Staphylococcus aureus (MRSA), the limitations of traditional antibiotic regimens are becoming increasingly apparent, urgently requiring the development of alternative therapies that combine broad-spectrum efficacy with low resistance. Photodynamic therapy (PDT) stands out as an alternative antimicrobial strategy that does not rely on specific metabolic targets. Its core lies in utilizing photo-triggered reactive oxygen species (ROS) for oxidative killing. This therapy not only causes irreversible structural damage to pathogen components but, more importantly, weakens the integrity of the extracellular matrix, thereby effectively penetrating and eliminating the physical diffusion barriers that restrict antibiotic penetration, significantly improving the control of infections in complex wounds.

[0003] The antibacterial properties of photodynamic therapy (PDT) depend on the physicochemical properties of the photosensitizer used. Traditional organic photosensitizers often limit their therapeutic efficiency due to poor water solubility, aggregation-induced quenching, and insufficient photostability. Furthermore, these photosensitizers are mostly neutral or negatively charged, resulting in weak interactions with the predominantly negatively charged bacterial cell membrane and extracellular polymeric substances (EPS). In contrast, positively charged photosensitizers exhibit enhanced antibacterial activity due to electrostatic interactions with the negatively charged bacterial cell membrane and biofilm matrix, promoting surface localization and facilitating ROS-mediated structural disruption. Therefore, the surface charge of the photosensitizer is widely considered an important molecular-level design consideration in photodynamic therapy for biofilm-associated and drug-resistant infections.

[0004] Cyclic metallized iridium(III) (Ir(III)) complexes have become a promising class of photosensitizers for photodynamic applications due to their efficient intersystem crossing, long-lived triplet excited states, high ROS quantum yield, and excellent photostability. Unlike traditional organic photosensitizers, Ir(III) complexes can be designed to achieve inherent water solubility and a permanent positive charge, thus avoiding molecular modification or the need for carrier design.

[0005] However, current Ir(III)-based PDT systems deliver drugs in solution as free molecules, which are easily lost and difficult to distribute in moist wound environments and dense biofilms. Therefore, effectively eradicating biofilm-associated and drug-resistant infections requires not only powerful photosensitizers but also a stable, tissue-adherent delivery platform that can immobilize the photosensitizer, limit its premature diffusion, and maintain local ROS generation under clinically relevant conditions.

[0006] Hydrogels have attracted widespread attention as wound care materials due to their high water content, soft tissue-like mechanical properties, and ability to maintain a moist microenvironment conducive to re-epithelialization and extracellular matrix remodeling. The three-dimensional polymer network of hydrogels can spatially confine photosensitizers to the wound surface, which is particularly advantageous for photodynamic antimicrobial therapy. However, effectively integrating small-molecule photosensitizers into the hydrogel matrix remains an ongoing challenge.

[0007] The weak and nonspecific interactions between photosensitizers and three-dimensional polymer networks often lead to rapid leaching under physiological or exudative conditions, resulting in reduced ROS output and decreased antibacterial efficacy after repeated irradiation. The inherent high permeability and dynamic water exchange of physically cross-linked hydrogels further exacerbate these challenges, highlighting the need for material strategies that can achieve molecular-level retention while maintaining biocompatibility and mechanical compliance. Summary of the Invention

[0008] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0009] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0010] Therefore, the object of this invention is to overcome the shortcomings of the prior art. 1. Solve the problem of photosensitizers being easily lost and having uncontrollable distribution in the wound environment: Current photodynamic therapy based on iridium(III) complexes as photosensitizers primarily delivers the drug in the form of free molecular solutions, which suffers from poor retention and difficulty in achieving local accumulation in moist wounds and dense biofilms. The purpose of this invention is to construct a stable delivery platform. This platform must effectively immobilize the photosensitizer, limiting its premature diffusion and loss under physiological and exudative conditions, thereby ensuring the continuous and localized generation of reactive oxygen species (ROS).

[0011] 2. Solve the problem of weak bonding between photosensitizer and hydrogel matrix, and easy leakage: To address the problem that small molecule photosensitizers, due to their weak and non-specific binding with the hydrogel matrix, rapidly leach out of the hydrogel, leading to a decrease in ROS production and antibacterial efficacy with repeated irradiation, this invention aims to develop a novel material binding strategy. By constructing multiple hydrogen bonds within the hydrogel network and achieving synergistic effects of nanoscale physical encapsulation, efficient and stable retention of photosensitizers is realized at the molecular level.

[0012] 3. Solve the problem of insufficient targeting and penetration of traditional photosensitizers into biological membranes: Although Ir(III) photosensitizers possess the potential to target bacteria and biofilms due to their positive charge, their dispersion in solution limits the full realization of this advantage. This invention aims to combine the inherent positive charge of photosensitizers with the platform advantage of hydrogels, enhancing the interfacial affinity between the hydrogel and negatively charged biofilms through charge-mediated electrostatic interactions. This overcomes the physical diffusion barrier formed by dense extracellular polymers, achieving efficient penetration and removal of mature biofilms.

[0013] To address the aforementioned technical problems, this invention provides a method for preparing a photodynamic antibacterial hydrogel without chemical crosslinking agents: comprising, An aqueous solution of a polymer with hydroxyl side chains and an aqueous solution of a sodium salt photosensitizer containing cyclic metallized iridium(III) complex are mixed and stirred to obtain a mixed precursor solution. The mixed precursor solution is subjected to freeze-thaw cycle molding treatment to obtain a photodynamic antibacterial hydrogel. The mass concentration of the sodium salt photosensitizer containing cyclic metallized iridium(III) complex in the mixed precursor solution is 0.1~0.5wt%.

[0014] In a preferred embodiment of the preparation method of the photodynamic antibacterial hydrogel without chemical crosslinking agent described in this invention, the mass concentration of the polymer in the aqueous solution containing hydroxyl groups in the side chain is 3~8wt%.

[0015] As a preferred embodiment of the preparation method of the photodynamic antibacterial hydrogel without chemical crosslinking agent according to the present invention, wherein: the polymer containing hydroxyl groups in the side chain includes one of polyvinyl alcohol (PVA), polyethylene glycol (PEG), cellulose and its derivatives (such as hydroxypropyl methylcellulose HPMC), hyaluronic acid (HA), sodium alginate, chitosan, and agarose.

[0016] In a preferred embodiment of the preparation method of the chemical crosslinking agent-free photodynamic antibacterial hydrogel of the present invention, the concentration of the sodium cyclometalated iridium(III) complex photosensitizer in the aqueous solution is 0~15 mg / mL, wherein the concentration of the sodium cyclometalated iridium(III) complex photosensitizer is not 0.

[0017] In a preferred embodiment of the preparation method of the photodynamic antibacterial hydrogel without chemical crosslinking agent according to the present invention, the sodium salt photosensitizer of the cyclometalated iridium(III) complex is selected from IrONa or its derivatives, and the derivatives of IrONa have the general structural formula shown in formula (I). Formula (I); In formula (I), R includes one of methyl, ethyl, methoxy, fluorine, chlorine, trifluoromethyl or an analogue thereof.

[0018] It should be noted that the sodium carboxylate group in the sodium salt photosensitizer of the cyclometalated iridium(III) complex in this application serves to provide water solubility and form non-covalent hydrogen bonds with the hydroxyl groups of the PVA (polyvinyl alcohol) network; while the Ir(III) skeleton is responsible for photosensitizing and generating reactive oxygen species. Therefore, as long as these two core elements are retained, conventional chemical modifications to the benzene or pyridine ring of the complex will not change its core antibacterial function in the hydrogel. Thus, analogs with electron-donating groups, such as methyl (-CH3), ethyl (-C2H5), and methoxy (-OCH3) substituted analogs, can be introduced onto the aromatic ring (such as the benzene ring or pyridine ring) of IrONa; or analogs with electron-withdrawing groups, such as fluorine (-F), chlorine (-Cl), and trifluoromethyl (-CF3) substituted analogs, can be introduced.

[0019] In a preferred embodiment of the preparation method of the chemically crosslinking-free photodynamic antibacterial hydrogel of the present invention, the freeze-thaw cycle molding process includes: The mixed precursor solution is injected into a mold and frozen at -20°C for 5 to 15 hours. Then it is thawed at room temperature for 1 hour to complete one freeze-thaw cycle. The above freeze-thaw process is repeated 2 to 10 times.

[0020] Another object of the present invention is to provide a photodynamic antibacterial hydrogel and its application as a biomedical antibacterial material. The photodynamic antibacterial hydrogel exhibits highly efficient antibacterial activity upon application through irradiation treatment, wherein the irradiation intensity is 0~300 mW cm⁻¹. -2 The irradiation time is 15-60 minutes.

[0021] Beneficial effects of this invention: (1) This invention selects water-soluble sodium salt of cyclometalated iridium(III) complex as a photosensitizer, utilizing its high water solubility, long lifetime triplet excited state, and high singlet oxygen ( 1 The O2 yield was improved, overcoming the technical defects of traditional organic photosensitizers that are prone to aggregation-induced quenching (ACQ). By forming multiple hydrogen bonds between the sodium carboxylate group in the sodium salt of the water-soluble cyclometalated iridium(III) complex and the hydroxyl group in the polymer containing hydroxyl groups in the side chain, an interaction network was constructed.

[0022] (2) This invention overcomes the limitations of traditional hydrogels that rely on toxic chemical crosslinking agents such as glutaraldehyde. By using repeated freeze-thaw cycles, the polymer matrix polymer chains crystallize and rearrange at low temperatures, constructing a stable three-dimensional physical network. This process is completed in a pure aqueous phase under mild conditions, which improves the biosafety of the material while preserving the original photophysical activity of the water-soluble cyclometalated iridium(III) complex sodium salt, avoiding the potential damage to the photosensitizer structure caused by chemical crosslinking reactions.

[0023] (3) This invention enables the water-soluble sodium iridium(III) cyclic metallized complex to be physically confined at the nanoscale through multiple hydrogen bonds and the microporous structure formed during the freeze-thaw process. This confinement effect effectively overcomes the common problems of leakage and elution of small molecule photosensitizers in physical cross-linked networks. The FT-PVA / IrONa hydrogel exhibits excellent loading stability in physiological saline and simulated exudate environments, ensuring the long-term retention of the water-soluble sodium iridium(III) cyclic metallized complex on the wound surface and realizing the continuity and repeatability of photodynamic therapy. Attached Figure Description

[0024] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein: Figure 1 Fourier transform infrared spectroscopy results for IrONa photosensitizer, FT-PVA hydrogel, and FT-PVA / IrONa hydrogel; Figure 2 This is a graph showing the reusability verification results of the FT-PVA / IrONa hydrogel prepared in Example 1 of the present invention; Figure 3 This is a graph showing the biosafety evaluation effect of the FT-PVA / IrONa hydrogel prepared in Example 1 of the present invention; Figure 4 This is a diagram illustrating the effect of the FT-PVA / IrONa hydrogel prepared in Example 1 of this invention on hemolysis of erythrocytes. Figure 5 This is a diagram showing the results of a live / dead cell staining experiment after co-culturing FT-PVA / IrONa hydrogel prepared in Example 1 of this invention with 293T cells for 48 hours.

[0025] Figure 6 The antibacterial effects of FT-PVA hydrogel and FT-PVA / IrONa hydrogel on Escherichia coli, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and engineered bacteria are shown in the figure. Figure 7 A bar chart showing the bactericidal effects of FT-PVA hydrogel and FT-PVA / IrONa hydrogel on Escherichia coli, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and engineered bacteria. Figure 8 Scanning electron microscope images of FT-PVA hydrogel and FT-PVA / IrONa hydrogel after sterilization of Escherichia coli, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus and engineered bacteria; Figure 9 The image shows the inhibitory effect of FT-PVA hydrogel and FT-PVA / IrONa hydrogel on crystal violet staining of biofilms of Escherichia coli and methicillin-resistant Staphylococcus aureus. Figure 10 A bar chart showing the biofilm inhibition effects of FT-PVA hydrogel and FT-PVA / IrONa hydrogel on Escherichia coli; Figure 11 A bar chart showing the statistical effects of FT-PVA hydrogel and FT-PVA / IrONa hydrogel on the biofilm inhibition of methicillin-resistant Staphylococcus aureus; Figure 12 Scanning electron microscope image of the FT-PVA hydrogel prepared in Comparative Example 1. Figure 13 The image shown is a scanning electron microscope (SEM) image of the FT-PVA / IrONa hydrogel prepared in Example 1. Figure 14 The figure shows the results of detecting singlet oxygen production in hydrogels with different PVA / IrONa ratios in Example 2 using a DPBF probe.

[0026] Figure 15 Antibacterial effects of FT-PVA / IrONa hydrogels with different amounts of IrONa photosensitizer on Escherichia coli. Figure 16 A bar chart showing the bactericidal effect of FT-PVA / IrONa hydrogels with different amounts of IrONa photosensitizer on Escherichia coli.

[0027] Figure 17 To investigate the antibacterial effects of irradiation treatment under different conditions on FT-PVA / IrONa hydrogel. Detailed Implementation

[0028] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0029] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0030] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0031] The synthesis method of the water-soluble cyclic metallized iridium(III) complex sodium salt (IrONa) used in this invention is as follows: Dalton Trans., 2020, 49, 11493 Specifically: Weigh 0.1 g of [Ir(ppy)2Cl]2 (dichlorotetra(2-(2-pyridyl)phenyl)diiridium(III)) and 0.049 g of 2,2'-bipyridine-4,4'-dicarboxylic acid, and add them to a mixed solvent of 15 mL methanol and 15 mL dichloromethane. Under nitrogen protection and in the dark, the mixture is heated under reflux for 4 hours to obtain a red solution. After the reaction solution cools to room temperature, 0.184 g of KPF6 (potassium hexafluorophosphate) is added, and the mixture is stirred for another 45 minutes at room temperature. The reaction mixture is filtered to separate the solid and liquid phases, and the resulting solid precipitate is collected. The precipitate is washed with petroleum ether to remove residual organic solvents and impurities soluble in petroleum ether. The washed solid is dried to obtain a crude product. This crude product is further purified by silica gel column chromatography using a mixed solvent of acetone and methanol (volume ratio 10:1) as the eluent. After collecting, concentrating, and drying the target component, 0.151 g of red solid, i.e., the intermediate, was obtained. 0.089 g of the obtained intermediate and 0.008 g of NaOH (sodium hydroxide) were dissolved in 5 mL of ultrapure water, and the mixture was stirred continuously at room temperature for 2 hours. After the reaction was completed, the solvent was removed by rotary evaporation, and 0.090 g of IrONa was finally obtained.

[0032] Example 1 This embodiment provides a method for preparing a photodynamic antibacterial hydrogel without chemical crosslinking agents, specifically: 1) Preparation of polyvinyl alcohol solution: 5g of polyvinyl alcohol (PVA) was placed in a 250mL beaker, and 95mL of ultrapure water was added. The solid was dissolved in a 90℃ oil bath using a magnetic stirrer at a stirring speed of 140rpm for 4 hours until the solution became a clear and transparent homogeneous liquid, yielding a 5% polyvinyl alcohol solution. The solution was then cooled to room temperature for later use.

[0033] 2) Preparation of IrONa solution: Accurately weigh 15 mg of IrONa powder, place it in a centrifuge tube, add 1 mL of ultrapure water, and sonicate it to completely dissolve it, thus obtaining a 15 mg / mL IrONa solution.

[0034] 3) Preparation of FT-PVA / IrONa composite solution: 2.4 g of the polyvinyl alcohol solution from step 1) was mixed with 0.6 mL of the IrONa solution from step 2), and stirred for 4 hours using a magnetic stirrer to ensure that IrONa was fully and uniformly dispersed in the polyvinyl alcohol solution, forming a uniform FT-PVA / IrONa yellow mixed precursor solution with a concentration of 0.3 wt%. 4) Freeze-thaw cycle molding: Pour the yellow mixed precursor solution of FT-PVA / IrONa from step 3) into a polytetrafluoroethylene mold, place the mold in a -20°C freezer for 10 hours to allow the solution to freeze completely, remove the mold from the freezer and let it stand at room temperature for 1 hour to allow it to thaw completely. Repeat the above process of "freezing at -20°C for 10 hours - thawing at room temperature for 1 hour" for a total of 6 cycles. This process induces the formation of a stable physical cross-linking network between PVA molecular chains through the formation and melting of ice crystals, thus obtaining the photodynamic antibacterial hydrogel of this embodiment, denoted as FT-PVA / IrONa hydrogel.

[0035] Comparative Example 1 The difference between this comparative example and Example 1 is that IrONa is not added. FT-PVA hydrogel is prepared. Specifically, a 5% polyvinyl alcohol solution is prepared according to the method of Example 1, poured into a polytetrafluoroethylene mold, and placed in a -20°C refrigerator for 10 hours to allow the solution to freeze completely. The mold is then removed from the refrigerator and left to stand at room temperature for 1 hour to allow it to thaw completely. Repeat the above process of "freezing at -20°C for 10 hours - thawing at room temperature for 1 hour" for a total of 6 cycles to obtain the FT-PVA hydrogel of this comparative example.

[0036] Fourier transform infrared spectroscopy was used to characterize the FT-PVA / IrONa hydrogel of Example 1, the FT-PVA hydrogel of Comparative Example 1, and IrONa. The results are as follows: Figure 1 As shown, compared with FT-PVA hydrogel, the OH stretching vibration peak of the hydrogel loaded with IrONa photosensitizer decreased from 3296 cm⁻¹. -1 A significant redshift to 3282 cm -1This demonstrates that a strong non-covalent interaction exists between the hydroxyl groups of PVA and the sodium carboxylate groups of the IrONa photosensitizer.

[0037] Reusability verification The antibacterial reusability of FT-PVA / IrONa hydrogel was evaluated using Escherichia coli, specifically: With a concentration of 10 6 A CFU / mL Escherichia coli bacterial suspension was inoculated onto the surface of the hydrogel prepared in Example 1. After irradiation with blue light for 30 minutes, the bacterial suspension was collected and serially diluted 10 μL with PBS. 4 The solution was then diluted to a multiple, and 100 μL of the diluted solution was evenly spread onto LB agar plates. Bacterial viability was determined by colony counting. After each round of experiments, the hydrogel was washed with ethanol to remove surface bacteria, then rinsed twice with PBS, and subsequently re-inoculated with fresh bacterial suspension. This procedure was repeated for five cycles, and the results are as follows: Figure 2 As shown.

[0038] from Figure 2 It can be seen that even after five consecutive antibacterial cycles, the FT-PVA / IrONa hydrogel maintained high bactericidal efficiency with only a slight decrease in activity, indicating that the IrONa photosensitizer was stably loaded in the hydrogel and did not undergo rapid inactivation after repeated use. Traditional physically cross-linked hydrogels encapsulating small molecule photosensitizers tend to release or lose them rapidly during washing or exudation. The FT-PVA / IrONa hydrogel maintained extremely high bactericidal efficiency after five cycles, with only a slight decrease in activity, demonstrating that multiple hydrogen bonds and nano-physical confinement prevented premature diffusion and elution of the photosensitizer.

[0039] Biosafety verification The biocompatibility of the FT-PVA / IrONa hydrogel was assessed using a cytotoxicity assay (MTT method). Specifically: 293T cells were seeded in 96-well plates and cultured for 24 hours to allow for complete cell adhesion. The cells were then incubated with different concentrations of hydrogel extract for 48 hours. After incubation, 10% MTT solution was added to each well, and the plates were incubated for another 4 hours. The supernatant was then discarded, and 100 μL of dimethyl sulfoxide (DMSO) was added to each well. Finally, the absorbance was measured using a microplate reader at a detection wavelength of 570 nm and a reference wavelength of 630 nm. The results are shown below. Figure 3 As shown.

[0040] Biocompatibility verification The biocompatibility of FT-PVA / IrONa hydrogel was evaluated using a erythrocyte hemolysis assay. All animal experiments complied with the Animal Ethics Committee Guidelines and were approved by Jilin University. Specifically: Fresh blood was collected from anesthetized SD rats, and the blood was pumped at 1000 rpm. -1 The cells were centrifuged for 10 minutes to separate red blood cells (RBCs). The obtained RBCs were then washed three times with PBS and resuspended in PBS, followed by resuspending again to obtain a final 5% (v / v) RBC suspension. Extracts of FT-PVA and FT-PVA / IrONa hydrogel were mixed with the rat RBC suspension and incubated at 37°C for 1 hour. The mixture was then transferred to centrifuge tubes and centrifuged at 1000 rpm for 1 minute. -1 Centrifuge for 10 minutes. Finally, transfer 200 μL of supernatant from each experimental and control group to a 96-well plate and measure the absorbance at 450 nm using a spectrophotometer. Additionally, 0.1% Triton X-100 solution and PBS were used as positive and negative controls, respectively. Hemolysis rate was calculated using the formula: Hemolysis rate % = [(Absorbance of sample group - Absorbance of PBS group) / (Absorbance of 0.1% Triton X-100 solution - Absorbance of PBS group)] × 100%; 293T cells were seeded in 6-well plates and cultured for 24 hours to allow for complete adhesion. They were then co-cultured with different concentrations of hydrogel extract for 48 hours. After culturing, 1 mL of Calcein AM / PI solution was added, and the cells were incubated at 37°C in the dark for 30 minutes. Following incubation, the cells were washed three times with PBS and observed under a fluorescence microscope for imaging.

[0041] Figure 4 This is a diagram showing the effect of a red blood cell hemolysis experiment. Figure 5 These are the results of a live / dead cell staining experiment.

[0042] from Figures 3-5 As can be seen, the FT-PVA / IrONa hydrogel maintained a survival rate of over 90% for 293T cells, and the hemolysis rate was far below the safety threshold of 5%. Live / dead staining showed that cells exposed to the extract primarily exhibited green fluorescence, with good spreadable spindle morphology and intact cell attachment, while only a small amount of red fluorescence was observed. These morphological and cell viability characteristics indicate that the FT-PVA matrix and IrONa photosensitizer do not produce cytotoxicity. The choice of 0.3 wt% instead of 0.5 wt% is because reducing the photosensitizer load by 40% without sacrificing any antibacterial efficacy minimizes the potential toxicity risks associated with photosensitizer accumulation in wounds, conforming to the design principle of "lowest effective dose" for clinical biomaterials. This makes the hydrogel extremely safe for direct contact with broken skin, exudative wounds, and biomedical applications.

[0043] Antibacterial verification Escherichia coli, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and engineered bacteria were selected as representative pathogens to evaluate the antibacterial properties of FT-PVA / IrONa hydrogel. Specifically: Representative pathogens were inoculated as single colonies onto LB medium and cultured with shaking until the bacterial concentration reached approximately 10⁻⁶. 6 CFU / mL, 200 μL of bacterial suspension was added to a 12-well plate, followed by the placement of FT-PVA and FT-PVA / IrONa hydrogels. The samples were then exposed to blue light for 30 minutes. The blue light group received only light treatment, while the control group received no treatment. The groups were designated FT-PVA-BL, FT-PVA / IrONa-BL, Blue Light, and Control, respectively. To quantitatively analyze bacterial viability, the treated bacterial suspension was diluted 10 μL with PBS. 4 Take 100 μL of the diluted solution and spread it evenly on Luria-Bertani agar plates (LB). After incubating at 37°C for 18 hours, count the colonies and calculate the antibacterial efficiency using the formula: Antibacterial efficiency (%) = [(Control group colony count - Experimental group colony count) / Control group colony count] × 100% Simultaneously, 500 μL of the diluted bacterial suspension was inoculated into 3 mL of LB medium and cultured at 37 °C with shaking for 12 hours. The optical density (OD) of the bacterial suspension at 600 nm was measured. 600 This indirectly reflects the growth of bacteria.

[0044] Take 200 μL of the diluted bacterial suspension, centrifuge at 8000 rpm for 5 minutes, discard the supernatant, and resuspend and wash once with phosphate-buffered saline (PBS). Then, add 2.5 wt% glutaraldehyde solution and fix at 4°C for 12 hours. The fixed bacterial sample is washed three times with ultrapure water and then sequentially dehydrated with 30%, 50%, 70%, 80%, 90%, and 100% (v / v) ethanol in a gradient, with each concentration treatment lasting 15 minutes. Finally, the sample is dried and observed under a scanning electron microscope. The results are as follows: Figures 6-8 As shown.

[0045] Figure 6 The graphs show the antibacterial effects of each group against Escherichia coli, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and engineered bacteria. Figure 7 A bar chart is used to statistically analyze the bactericidal effects of each group against Escherichia coli, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and engineered bacteria. Figure 8 The images show scanning electron microscope (SEM) images of each group after sterilization against Escherichia coli, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and engineered bacteria. Figures 6-8It can be seen that the FT-PVA / IrONa hydrogel exhibits a bactericidal efficiency exceeding 97.5% against various pathogens, including Escherichia coli, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and engineered bacteria. Scanning electron microscopy confirms that the bactericidal mechanism of the FT-PVA / IrONa hydrogel is through the generation of... 1 O2-induced bacterial membrane rupture and structural collapse is a type of non-specific oxidative damage that is less likely to lead to drug resistance.

[0046] Biomembrane inhibition verification The experimental method for anti-biofilm performance is as follows: Escherichia coli or MRSA were cultured in tryptone soybean broth (TSB) for 10 hours. 6 A bacterial suspension was prepared at CFU / mL. Then, 1 mL of this suspension was inoculated into a 12-well plate. Finally, the plate was incubated at 37°C for 24 hours to allow for the formation of a mature biofilm. After incubation, the floating culture medium and old culture medium were carefully aspirated, and the adherent biofilm was gently rinsed 2-3 times with PBS to remove any unattached bacteria. The prepared hydrogel sheet (product of Example 1 or Comparative Example 1) was then applied to the biofilm surface. The experimental group was irradiated with blue light for 30 minutes, while the control group was treated in the dark under the same conditions. After treatment, the hydrogel was removed for analysis.

[0047] Total biofilm biomass was quantified using crystal violet staining. The simplified procedure is as follows: Add 500 µL of 0.01% (w / v) crystal violet staining solution to each well and incubate at room temperature in the dark for 30 minutes. Discard the staining solution and rinse each well thoroughly with PBS until the wash buffer is clear. After air drying, photograph the stained biofilm. To dissolve the dye bound to the biofilm, add 500 µL of 33% (v / v) glacial acetic acid to each well and incubate with shaking at room temperature for 30 minutes. Then, transfer 200 µL of the dissolved dye from each well to a new 96-well plate and measure the absorbance (OD) at 570 nm. 570 ). OD 570 The value can relatively reflect the total biofilm biomass.

[0048] To evaluate the antibacterial effect after treatment, 1 mL of PBS was added to each well, and the biofilm was completely dispersed by sonication for 30 minutes. The resulting bacterial suspension was serially diluted, and 100 µL of the appropriately diluted bacterial solution was plated onto LB agar plates and incubated overnight at 37°C. The results were evaluated as follows: Figures 9-11 As shown.

[0049] Figure 9 The images show the inhibition effects of crystal violet staining on biofilms of *Escherichia coli* and methicillin-resistant *Staphylococcus aureus* in each group. Figure 10 A bar chart showing the statistical effect of each group on the biofilm inhibition of Escherichia coli. Figure 11A bar chart showing the statistical effect of each group on biofilm inhibition of methicillin-resistant Staphylococcus aureus. Figures 9-11 The results showed that the FT-PVA / IrONa hydrogel achieved biofilm inhibition rates of 75.8% and 71.2% for mature E. coli and MRSA biofilms, respectively, overcoming the physical diffusion barrier that hinders the penetration of traditional drugs. Compared with the control group, the present invention exhibited significantly improved biofilm clearance ability under the same light conditions.

[0050] Figure 12 The image shown is a scanning electron microscope (SEM) image of the FT-PVA hydrogel without IrONa photosensitizer in Comparative Example 1. Figure 13 This is a scanning electron microscope (SEM) image of the FT-PVA / IrONa hydrogel loaded with IrONa photosensitizer in Example 1. Figure 12 and Figure 13 As can be seen, the FT-PVA hydrogel exhibits a clear, anisotropic porous structure with interconnected lamellar features, consistent with the phase separation induced by the ice template during freezing. After loading with IrONa photosensitizer, the resulting FT-PVA / IrONa hydrogel maintains a highly porous and continuous three-dimensional network structure at both the micron and submicron scales. The high similarity in microstructural characteristics between FT-PVA and FT-PVA / IrONa indicates that the introduction of IrONa photosensitizer did not interfere with the physical cross-linking process of the PVA matrix.

[0051] Example 2 The difference between this embodiment and Example 1 is that the concentration of IrONa added to the mixed precursor solution was adjusted to 0.1wt%, 0.2wt%, 0.25wt%, 0.3wt%, and 0.5wt%, respectively. The remaining steps and processes were the same as in Example 1, resulting in FT-PVA / IrONa hydrogels with different IrONa addition concentrations in this embodiment.

[0052] Spectrophotometry was used with DPBF (1,3-diphenylisobenzofuran) as a probe to monitor singlet oxygen (…). 1 The generation of O2 was investigated. FT-PVA / IrONa hydrogels with different IrONa loadings (0.1wt%, 0.2wt%, 0.25wt%, 0.3wt%, and 0.5wt%) were placed in 96-well plates, and 200 µL of 1 mg / mL DPBF solution was added to each well. The gels were then subjected to blue light (450 nm, 50 mW cm⁻¹). -2 Irradiate for 120 s. After irradiation, quickly remove the hydrogel from the well plate and immediately measure the absorbance of the remaining solution at 412 nm to quantitatively evaluate hydrogels with different loadings. 1 O2 production efficiency.

[0053] like Figure 14 As shown, the singlet oxygen content of FT-PVA / IrONa hydrogels with different IrONa addition concentrations is... 1 O2 production was verified by the absorbance change of DPBF, confirming the ability of the proposed photodynamic system to produce singlet oxygen. Furthermore, increasing the IrONa content led to a gradual increase in the quenching of DPBF absorbance under blue light irradiation, indicating... 1 O2 production is increased. When the loading is further increased to 0.5 wt%, the ability to generate singlet oxygen tends to saturate. This proves that the physical cross-linking network of the present invention can not only effectively carry the photosensitizer, but also maintain the monodisperse state of the IrONa photosensitizer in the range of 0.1 wt%-0.5 wt%, avoiding the aggregation-induced quenching phenomenon commonly seen at high concentrations.

[0054] Escherichia coli was selected as a representative pathogen to evaluate the in vitro antibacterial activity of the control group, the blue light group, the FT-PVA-BL group, and FT-PVA / IrONa hydrogels with different IrONa loadings (0.1wt%, 0.2wt%, 0.25wt%, 0.3wt%, and 0.5wt%). Single colonies were inoculated into LB medium and cultured with shaking until the bacterial concentration reached approximately 10⁻⁶. 6 CFU / mL, 200 μL of bacterial suspension was added to a 12-well plate, followed by the placement of FT-PVA and FT-PVA / IrONa hydrogels. The samples were then exposed to blue light for 30 minutes. The blue light group received only light treatment, while the control group received no treatment. To quantitatively analyze bacterial viability, the treated bacterial suspension was diluted 10 μL with PBS. 4 Take 100 μL of the diluted solution and spread it evenly on Luria-Bertani agar plates (LB). After incubating at 37°C for 18 hours, count the colonies and calculate the antibacterial efficiency using the formula: Antibacterial efficiency (%) = [(number of colonies in control group - number of colonies in experimental group) / number of colonies in control group] × 100%; The results are as shown in 15 and Figure 16 As shown, Figure 15 The graph shows the antibacterial effect of each group against Escherichia coli. Figure 16 A bar chart was used to statistically analyze the bactericidal effects of each group against Escherichia coli. Figure 15 and Figure 16 As shown, the bactericidal efficiency of the hydrogel significantly increases with increasing IrONa photosensitizer loading. While the bactericidal rate improves at loadings of 0.1 wt% and 0.2 wt%, it does not reach the ideal clinical level. However, at a loading of 0.3 wt%, the bactericidal efficiency against *E. coli* approaches 100%. Further increasing the loading to 0.5 wt% does not show a significant difference in antibacterial effect. This confirms the scientific validity of the 0.1-0.5 wt% loading range in this invention, where 0.3 wt% is the optimal concentration for implementation.

[0055] Example 3 This embodiment compares the antibacterial effects of the FT-PVA / IrONa hydrogel, the product of Example 1, by subjecting it to irradiation treatment under different conditions. Specifically: A: FT-PVA / IrONa hydrogel was subjected to a strength of 10 mW / cm². -2 50mW cm -2 100mW cm -2 Irradiation treatment for 30 minutes was performed, and the corresponding antibacterial efficiency was measured. The results are as follows: Figure 17 As shown in (A); B. Apply a strength of 50 mW / cm² to the FT-PVA / IrONa hydrogel. -2 Irradiation treatments for 15 min, 30 min, and 50 min were performed, and the corresponding antibacterial efficiencies were measured. The results are as follows: Figure 17 As shown in (B); from Figure 17 It can be seen that as the irradiation intensity increases from 10 to 300 mW cm⁻¹ -2 This significantly improved antibacterial efficiency, consistent with the increased number of excited-state particles and ROS production resulting from higher photon flux. A similar dose-dependent effect was observed when the irradiation time was extended from 15 min to 60 min, reflecting the cumulative nature of the photodynamic antibacterial process. Importantly, at 50 mW / cm², [the effect was observed]. -2 Significant bacterial killing effects were achieved under moderate light intensity and irradiation time of 30 minutes, indicating that FT-PVA / IrONa hydrogel can be efficiently activated under relatively mild conditions.

[0056] In summary, this application achieves an organic combination of freeze-thaw physical crosslinking without chemical crosslinking agents, sodium salt photosensitizer of cyclometalated iridium(III) complex, multiple hydrogen bonding, and nano-physical confinement. By utilizing the sodium carboxylate group of the sodium salt of cyclometalated iridium(III) complex to form multiple hydrogen bonds with the hydroxyl groups of polyvinyl alcohol, and combining this with the microporous structure constructed by freeze-thaw, the photosensitizer is physically confined at the nanoscale. This solves the common industry problem of easy leakage and easy elution of small molecule photosensitizers at the molecular level. If neutral / negative photosensitizers or hydrogel matrices without side chain hydroxyl groups are used, it is impossible to achieve the crosslinking agent-free anti-leakage effect of this application.

[0057] This application achieves stable loading of photosensitizers and continuous generation of reactive oxygen species without using any toxic chemical cross-linking agents. It exhibits highly efficient antibacterial / film-inhibiting capabilities against Escherichia coli, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), and mature biofilms, while also possessing high biocompatibility and reusability. It overcomes the inherent defects of traditional photodynamic systems and hydrogel carriers.

[0058] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for preparing a photodynamic antibacterial hydrogel without chemical cross-linking agents, characterized in that: include, A mixed precursor solution was obtained by mixing and stirring a polymer aqueous solution containing hydroxyl groups in the side chain and an aqueous solution of sodium salt of cyclometalated iridium(III) complex photosensitizer. The mixed precursor solution was subjected to freeze-thaw cycle molding treatment to obtain photodynamic antibacterial hydrogel. The mass concentration of the sodium salt of cyclic metallized iridium(III) complex photosensitizer in the mixed precursor solution is 0.1~0.5wt%.

2. The method for preparing the photodynamic antibacterial hydrogel without chemical crosslinking agent as described in claim 1, characterized in that: The mass concentration of the polymer in the aqueous solution containing hydroxyl groups in the side chain is 3-8 wt%.

3. The method for preparing the photodynamic antibacterial hydrogel without chemical crosslinking agent as described in claim 2, characterized in that: The polymers containing hydroxyl groups in their side chains include one of polyvinyl alcohol, polyethylene glycol, cellulose and its derivatives, hyaluronic acid, sodium alginate, chitosan, and agarose.

4. The method for preparing the photodynamic antibacterial hydrogel without chemical crosslinking agent as described in claim 1, characterized in that: The concentration of the sodium cyclometalated iridium(III) complex photosensitizer in the aqueous solution is 0~15 mg / mL, wherein the concentration of the sodium cyclometalated iridium(III) complex photosensitizer is not 0.

5. The method for preparing the photodynamic antibacterial hydrogel without chemical crosslinking agent as described in claim 4, characterized in that: The sodium salt photosensitizer of the cyclic metallized iridium(III) complex is selected from IrONa or its derivatives, and the derivatives of IrONa have the general structural formula shown in formula (I). Formula (I); In formula (I), R includes one of methyl, ethyl, methoxy, fluorine, chlorine, trifluoromethyl or an analogue thereof.

6. The method for preparing the photodynamic antibacterial hydrogel without chemical crosslinking agent as described in claim 1, characterized in that: The freeze-thaw cycle molding process includes, The mixed precursor solution is injected into a mold and frozen at -20°C for 5 to 15 hours. Then it is thawed at room temperature for 1 hour to complete one freeze-thaw cycle. The above freeze-thaw process is repeated 2 to 10 times.

7. The photodynamic antibacterial hydrogel prepared by any one of the preparation methods described in claims 1 to 6.

8. The application of the photodynamic antibacterial hydrogel as described in claim 7 as a biomedical antibacterial material, characterized in that: The photodynamic antibacterial hydrogel exhibits highly efficient antibacterial activity through irradiation treatment during application.

9. The application of the photodynamic antibacterial hydrogel as described in claim 7 as a biomedical antibacterial material, characterized in that: The irradiation intensity of the irradiation treatment is 0~300 mW cm⁻¹ -2 The irradiation time is 15-60 minutes.

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