A dynamic self-healing antibacterial hydrogel with cationic membrane disruption and sonodynamic synergy, a preparation method and applications thereof

Abstract

The application discloses a dynamic self-healing antibacterial hydrogel with cationic membrane-breaking and sonodynamic synergistic effect. The hydrogel is composed of porphyrin grafted cationic poly(pyrimidine) nanoparticles and benzene boronic acid modified oxidized hyaluronic acid, forms a three-dimensional network through reversible Schiff base action, and has injectability and self-healing property. The nanoparticles generate reactive oxygen species under ultrasonic irradiation and synergistically kill bacteria with cationic membrane-breaking, and the reversible action of benzene boronic acid groups and lipopolysaccharide realizes enrichment of infection sites. The hydrogel can be smoothly extruded through a 21G to 27G needle, and quickly restores the storage modulus after shear stops; under the condition of 1 MHz ultrasonic with a medical allowable sound intensity, the minimum inhibitory concentration is significantly reduced and mature biofilm is removed. The material can be applied to the management of complex infected wounds as an antibacterial wound dressing or an in-situ gel preparation, is compatible with conventional nursing procedures and ultrasonic operation in vitro, and has good biocompatibility and conversion potential.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials and wound anti-infection technology, specifically to a dynamic hydrogel formed by crosslinking nanoparticles constructed from cationic polymer grafted porphyrin with phenylboronic acid-modified oxidized hyaluronic acid. The hydrogel has injectability, self-healing properties, and sonodynamic synergistic bactericidal effects, and can be used to prepare anti-infective wound dressings or drug compositions. Background Technology

[0002] Wound infection is a common complication in surgical treatment and wound care. Pathogen colonization and proliferation in the wound can cause persistent inflammation, tissue necrosis, and secondary complications, significantly delaying the healing process and, in severe cases, leading to systemic infection and even death. Although antibiotics remain the first-line treatment in clinical practice, the frequent emergence of drug-resistant strains has led to a continuous decline in the duration of drug efficacy and the controllability of treatment duration. Long-term or high-dose use is also accompanied by microecological imbalance and adverse reactions, making it difficult to meet the comprehensive management needs of complex infected wounds.

[0003] Antibacterial hydrogels have attracted widespread attention in order to improve local treatment. Materials based on cationic polymers can disrupt bacterial membrane permeability through electrostatic interactions, offering broad-spectrum and rapid effects. However, a single charge mechanism is often insufficient to completely eliminate bacteria with high bacterial loads or mature biofilms, and increasing the dosage can easily induce drug resistance and cytotoxicity. Photo / acoustic strategies can generate reactive oxygen species to kill bacteria under excitation by external energy. Acoustic methods offer better tissue penetration than photodynamic methods, but in the absence of selectivity, they may cause non-specific damage, making it difficult to balance efficiency and safety.

[0004] Targeted modification of the infection microenvironment has also been explored, for example, by utilizing the affinity for lipopolysaccharide (LPS) to increase the local concentration of materials at sites rich in Gram-negative bacteria, thereby amplifying the bactericidal effect. However, existing systems generally suffer from insufficient synergy, limited penetration through biofilms, and poor adaptability to complex exudative environments. Meanwhile, many hydrogel wound care materials have fixed network structures and lack dynamic reversible bonds, limiting injectable molding and rapid self-healing after injury, and affecting their adherence and stable residence in irregular wounds and deep tissues.

[0005] Given the aforementioned challenges and clinical realities, the key task at present is to construct an antibacterial hydrogel capable of achieving multi-mechanism synergy and engineered scale-up within a single material platform. This system needs to rapidly reduce highly loaded and biofilm-forming bacteria through cationic permeabilization, while simultaneously generating reactive oxygen species stably at ultrasound-accessible depths to extend the bactericidal reach. It should also rely on the selective recognition of pathogen-related molecules such as lipopolysaccharides to achieve local enrichment and low-dose effectiveness. Currently, several gaps and bottlenecks remain, primarily including insufficient structural stability and long-term adhesion of the dynamic reversible network in complex exudative environments; a lack of unified quantitative and operational guidelines for the sonodynamic process's dose-response relationship; difficulty in simultaneously achieving universality and specificity of targeted recognition across different bacterial species and diverse infection microenvironments; trade-offs between injectable self-healing and mechanical adaptability; and weak evidence regarding compatibility with clinical nursing procedures and long-term biosafety. Therefore, it is necessary to propose an integrated hydrogel solution that combines dynamic cross-linking self-healing, deep sonodynamic bactericidal action, and pathogen site enrichment capabilities. Summary of the Invention

[0006] The technical problem solved by this invention is to provide a sonoactivated dynamic hydrogel that can achieve efficient sterilization in complex infected wounds and has good adaptability. This material has injectability, self-healing and sonodynamic characteristics, and is suitable for antibacterial treatment and wound care.

[0007] To achieve the above objectives, this invention proposes a sound-activated dynamic hydrogel, obtained by the interaction of functionalized nanoparticles and phenylboronic acid-modified oxidized hyaluronic acid. The functionalized nanoparticles are obtained by ammonolysis of a polypyrimidine backbone to obtain a primary amine, followed by a condensation reaction with porphyrin tetracarboxylic acid to form a cationic polymer grafted with porphyrin, which is then co-assembled with liposome materials. Preferably, the average particle size of the functionalized nanoparticles is controlled between 100 and 600 nm, and the surface potential is in the range of +5 to +20 mV.

[0008] Preferably, the phenylboronic acid-modified oxidized hyaluronic acid is prepared by reacting aldehyde-containing hyaluronic acid with an amine compound containing phenylboronic acid in the presence of a condensing agent.

[0009] The functionalized nanoparticles and the phenylboronic acid-modified oxidized hyaluronic acid form a three-dimensional hydrogel network through Schiff base dynamic bonding.

[0010] Preferably, the hydrogel is injectable, capable of being smoothly extruded through a syringe under external force, and quickly returning to its original shape after shear disturbance. Preferably, the hydrogel has self-healing properties, capable of recovering its storage modulus within approximately 1 minute after minor damage.

[0011] Under ultrasonic irradiation, the porphyrin groups in the hydrogel can stably generate reactive oxygen species, enhancing bacterial membrane disruption and metabolic interference.

[0012] The cationic polymer unit interacts with the negative charge on the bacterial membrane surface, achieving initial membrane rupture and rapidly reducing the number of bacteria.

[0013] The phenylboronic acid groups can reversibly interact with bacterial outer membrane lipopolysaccharides, thereby improving the targeted enrichment efficiency of the hydrogel at the infection site.

[0014] The present invention also provides a method for preparing the sonoactivated dynamic hydrogel, comprising preparing porphyrin-functionalized polypyrimidine nanoparticles, mixing them with a phenylboronic acid-modified oxidized hyaluronic acid solution, and constructing a dynamic hydrogel network by utilizing the condensation reaction of primary amines and aldehyde groups.

[0015] Preferably, the nanoparticles are stably dispersed through solvent replacement and ultrasonic treatment, with a concentrated particle size distribution and long-term preservation at 4 °C.

[0016] Preferably, the acoustic stimulation condition is a frequency of 1 MHz and is within the medically permissible sound intensity range, which can induce stable generation of reactive oxygen species within the tissue depth.

[0017] Preferably, the method of use involves applying the hydrogel to the infected wound and then applying external ultrasound irradiation to achieve efficient sterilization and promote wound healing.

[0018] This application provides a sonoactivated synergistic antibacterial dynamic hydrogel. The building blocks consist of porphyrin-functionalized cationic polypyrimidine nanoparticles as functional subjects and phenylboronic acid-modified oxidized hyaluronic acid as a dynamic framework. These components form a three-dimensional network through reversible Schiff base interaction and spontaneously stabilize into a gel. This system exhibits highly efficient bactericidal activity and good adhesion in complex infected wounds. It is injectable and self-healing, and can trigger the generation of reactive oxygen species under ultrasound to achieve deep antibacterial action and promote healing. Specifically, (1) Porphyrin groups stably generate reactive oxygen species under ultrasonic excitation, which destroys bacterial membranes and key biomolecules, thereby enhancing the bactericidal effect. (2) Cationic polymer units interact with the negative charge on the surface of bacterial membranes, completing the initial membrane rupture and rapidly reducing bacterial load. (3) Phenylboronic acid groups can reversibly interact with polyhydroxy molecules such as lipopolysaccharides, enabling the material to selectively accumulate at the site of infection and reduce the dosage. (4) The reversible dynamic network can recover its energy storage modulus within about 1 minute after shear perturbation, giving the material injectability and self-healing ability, making it easy to form a stable and adherent barrier in irregular and exudative wounds. (5) The system uses extracorporeal ultrasound as a remote excitation method, which can achieve non-invasive enhanced sterilization within the tissue depth and is compatible with routine nursing procedures.

[0019] Therefore, the acoustically activated synergistic antibacterial dynamic hydrogel provided in this application can achieve efficient removal of highly loaded and biofilm-forming bacteria under low-dose and safe acoustic intensity conditions, while reducing exposure to non-target tissues and the risk of drug resistance. It can also be integrated with wound management procedures to complete visualization and standardized operations, and has broad application prospects in the field of infected wound treatment and care.

[0020] Compared with existing technologies, the ultrasonically activated dynamic hydrogel of this invention achieves a synergistic mechanism of cationic membrane disruption, acoustic dynamic generation of reactive oxygen species, and local enrichment of phenylboronic acid sites on the same platform, enabling rapid bacterial reduction and biofilm penetration in complex infected wounds. Representative results show that the minimum inhibitory concentration (MIC) has been reduced from approximately 30 µg·mL⁻¹ to approximately 15 µg·mL⁻¹, and the residual thickness of the mature biofilm has been reduced from approximately 24 µm to approximately 8 µm. Based on a Schiff base dynamic network, the material exhibits injectability and self-healing properties, being successfully extruded through 21G to 25G needles and recovering to over 80% of its initial storage modulus within approximately 1 minute after shear perturbation, which is beneficial for adhering to and long-term residence in irregular wounds. It exhibits good cell and blood compatibility, with cell viability exceeding 85% and a hemolysis rate of less than 2%, achieving accelerated healing in animal models without any abnormal systemic reactions. The synthesis route and gelation process are mild and simple, and gelation can be achieved through in-situ mixing. The formulation parameters can be adjusted as needed and are compatible with routine care and in vitro ultrasound operations, making it feasible for transformation and large-scale application. Attached Figure Description

[0021] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn to scale.

[0022] Figure 1 This is a schematic diagram of the material construction route. It shows that POTP is obtained by ammonolysis, and then coupled with TCPP to form POTP-TPP. Figure 2 Representative morphology images of POTP-TPP nanoparticles. These show near-spherical particles and a uniformly dispersed appearance. Figure 3 Schematic curves of hydrogel formation and self-healing. These show that the storage modulus is higher than the loss modulus after gelation and that the hydrogel recovers rapidly after shear cessation. Figure 4 This is a schematic curve illustrating the generation of reactive oxygen species under ultrasonic activation conditions. It shows the signal enhancement trend compared to the unactivated state. Figure 5 This is a representative result of the in vitro antibacterial effect. It shows a comparison of the antibacterial performance between the groups without ultrasound and those treated with ultrasound in combination. Figure 6 The bacterial membrane depolarization and long-term drug resistance assessment diagram shows representative results of membrane potential changes and the stability of minimum inhibitory concentration in continuous passages. Figure 7 These are representative images of the treatment efficacy for wound infection in animals. They show changes in the wound at the beginning of drug administration and at the endpoint assessment.

[0023] Figure 8 These are representative images of the treatment efficacy for scalded wounds in animals. They show changes in the wound at the initial and endpoint assessments after drug administration.

[0024] Figure 9 These are representative images of the therapeutic effects on animal skin wounds. The images show statistical analysis of histological observations at the initial and endpoint assessments of drug administration. Detailed Implementation

[0025] To further understand the technical solution and beneficial effects of the present invention and to clearly and completely describe the technical solution and its working process, preferred embodiments are described in conjunction with the accompanying drawings and the following examples. The above description is used to illustrate the features and advantages of the present invention and does not constitute a limitation on the claims.

[0026] This invention proposes a dynamic hydrogel with synergistic antibacterial activity induced by ultrasound activation. The hydrogel is formed by the interaction of porphyrin-functionalized cationic polypyrimidine nanoparticles and phenylboronic acid-modified oxidized hyaluronic acid to create a three-dimensional network. The nanoparticles provide both cationic membrane disruption and ultrasound-triggered reactive oxygen species, while the polysaccharide backbone provides reversible cross-linking and wound adhesion capabilities, thereby achieving highly efficient bactericidal activity and stable retention in complex infected wounds.

[0027] Porphyrin-functionalized cationic polypyrimidine was obtained via the following route. First, a polypyrimidine backbone was synthesized in a methanol system and subjected to ammonolysis to obtain a polypyrimidine precursor containing primary amine side chains, denoted as POTP-NH2. Subsequently, in the presence of a water-soluble carbodiimide and an active ester, porphyrin tetracarboxylic acid was condensed with POTP-NH2 to obtain porphyrin-grafted polypyrimidine, denoted as POTP-TPP. Preferably, the reaction was stirred at room temperature for 2 to 4 hours. After the reaction, small molecules were removed by dialysis, and the solid was lyophilized to obtain a reddish-purple solid. The functionalized nanoparticles can be obtained by blending with DSPE-mPEG2000 and self-assembling using solvent displacement or ultrasonic emulsification. Preferably, POTP-TPP is dissolved in a small amount of ethanol or dimethyl sulfoxide, then added dropwise to a phosphate buffer containing DSPE-mPEG2000 with slow stirring, followed by short-range ultrasonic treatment to obtain a stable dispersion of nanoparticles. This dispersion can be stored for a long time at 4 °C.

[0028] In this specification, unless otherwise explicitly stated, the mass ratios of amphiphilic lipids (e.g., DSPE-mPEG2000) and sonosensitive agent-grafted polymers (e.g., POTP-TPP) referred to in the preparation process refer to the mass ratio of the feed materials (i.e., the ratio of the amounts added). Because purification processes such as self-assembly, solvent evaporation, dialysis / ultrafiltration / centrifugal washing may result in the partial removal of free lipids, the actual lipid content in the final nanoparticles may be lower than the theoretical value corresponding to the feed ratio. The actual lipid content in the final nanoparticles can be determined by phosphorus content method, quantitative NMR, elemental analysis, or fluorescence / isotope labeling method; the concentration of the nanoparticle dispersion can also be characterized by the equivalent concentration of the sonosensitive agent-grafted polymer (e.g., POTP-TPP) to facilitate comparison between different batches.

[0029] Preferably, the average particle size of the nanoparticles is 100 to 600 nm, and the Zeta potential is +5 to +20 mV. More preferably, the polydispersity index of the particle size distribution is not greater than 0.5. These parameters can be measured by dynamic light scattering, and electron microscopy reveals a near-spherical morphology and a uniform appearance.

[0030] The phenylboronic acid-modified oxidized hyaluronic acid can be obtained through a two-step method. The first step involves the selective oxidation of hyaluronic acid with sodium periodate to introduce an aldehyde group, yielding oxidized hyaluronic acid, denoted as OHA. The second step uses a water-soluble carbodiimide and an active ester in an aqueous phase to facilitate the condensation coupling of OHA and p-aminophenylboronic acid, yielding phenylboronic acid-modified oxidized hyaluronic acid, denoted as OHA-PBA. The product is then dialyzed to remove inorganic salts and residual small molecules, followed by lyophilization to obtain a white solid.

[0031] The dynamic hydrogel of this invention is obtained by mixing the POTP-TPP nanoparticle dispersion with an OHA-PBA aqueous solution. After mixing, the primary amine and aldehyde groups undergo a reversible Schiff base reaction and gel within minutes. Preferably, the solid content is 2 to 6% by mass, and preferably the equivalence ratio of amino to aldehyde groups is close to 1:1. The resulting hydrogel maintains a stable morphology within the range of room temperature and body temperature.

[0032] The hydrogel possesses injectable and self-healing properties. Rheological testing shows that the storage modulus is greater than the loss modulus and exhibits solid-state characteristics; under alternating high and low shear loading, the modulus can recover to more than 80% of its initial value within approximately 1 minute. Injection testing demonstrates that the material can be successfully extruded through 21G to 25G needles and rapidly rebounds after shearing stops, covering irregular wound surfaces.

[0033] The hydrogel generates reactive oxygen species (ROS) stably from porphyrin groups under ultrasonic irradiation. Preferably, the acoustic excitation frequency is 1 MHz, the acoustic intensity is within medically permissible limits, and an intermittent duty cycle is employed to reduce thermal effects. In vitro ABDA or DCF-DA measurements show a significant enhancement of the ROS signal compared to the unexcited state, indicating that the system possesses acoustic kinetic activation capabilities.

[0034] The antibacterial efficacy of this invention is achieved through multiple mechanisms. The cationic polymer interacts with the negative charge on the bacterial membrane surface, inducing a change in membrane permeability, thereby completing the initial peak clipping. Reactive oxygen species generated after ultrasonic activation further disrupt the bacterial membrane lipid and protein structure and interfere with metabolic pathways. The phenylboronic acid groups have an affinity for the polyhydroxy molecules on the bacterial outer membrane, which can improve the local enrichment efficiency of the material at the infection site. These three mechanisms synergistically significantly reduce the minimum inhibitory concentration (MIC).

[0035] In a representative embodiment, the mass concentration of the POTP-TPP dispersion was set to 5 mg·mL⁻¹, and the mass concentration of the OHA-PBA solution was set to 10 mg·mL⁻¹. They were mixed at a volume ratio of 1:1 and allowed to stand for 5 minutes to form a self-supporting hydrogel. The resulting hydrogel exhibited significant shear-thinning characteristics and showed no water separation phenomenon after being placed at a constant temperature of 37 ℃ for 24 h.

[0036] Antimicrobial testing was conducted using Gram-negative and Gram-positive bacterial models, with a blank control (antimicrobial agent concentration of 0 µg·mL⁻¹). Without sonication, the minimum inhibitory concentration (MIC) of the functionalized nanoparticles / hydrogel against representative strains was approximately 30 µg·mL⁻¹; under 1 MHz sonication, the MIC decreased to approximately 15 µg·mL⁻¹. Scanning and transmission electron microscopy revealed bacterial membrane undulations and internal structural disorder, with reactive oxygen species (ROS) probes showing strong positive signals.

[0037] Biocompatibility and wound adaptation were assessed using cell viability and animal wound models. L929 cells showed a survival rate of over 85% in the hydrogel extract. In a mouse full-thickness skin infection model, application of the hydrogel of this invention followed by in vitro ultrasound irradiation significantly increased wound contraction rate after 3 days and achieved a high healing rate after 13 days. Histological examination revealed reduced inflammatory cell infiltration and increased collagen deposition.

[0038] This invention also provides a method for preparing and using the hydrogel. The preparation method involves preparing a POTP-TPP nanoparticle dispersion and an OHA-PBA aqueous solution, mixing the two and allowing them to stand to obtain a gel-forming product. The application method involves applying the hydrogel to an infected wound and subjecting it to in vitro ultrasound irradiation, thereby achieving enhanced local antibacterial and healing-promoting effects.

[0039] Preferably, the formulation and process can be adjusted according to different wound environments. When the exudate is large, the OHA-PBA content can be appropriately increased to enhance the network crosslinking density. When a stronger sonodynamic response is required, the porphyrin grafting degree can be increased or the nanoparticle ratio can be optimized. The above adjustments maintain the synergistic mechanism and fall within the technical scope of this invention.

[0040] Example 1: Preparation of polyheterocyclic pyrimidine polymer POTP-NH2 This embodiment provides a method for preparing polyheterocyclic pyrimidine polymers, specifically including the following: A methanol solution of 1,8-octyldiamine was added dropwise to a methanol solution of dimethyl butynedioate, and stirred for 25 minutes. Formaldehyde solution and acetic acid were then added sequentially, and the mixture was stirred overnight at room temperature to remove the solvent. The solution was washed with dichloromethane and sodium bicarbonate water, and dried over anhydrous magnesium sulfate to obtain a brownish-red viscous POTP with a yield of approximately 69%. POTP was dissolved in DMF, and excess ethylenediamine was added. The mixture was stirred at 55 °C for 48 h, treated with deionized water, and lyophilized to obtain POTP-NH2 with a yield of approximately 78%. The structure and molecular weight were confirmed to be consistent with those of 1.83 × 10⁻⁶ NMR and gel permeation chromatography. The number-average molecular weight was approximately 1.83 × 10⁻⁶. 4 The distribution index is approximately 1.87. This step ensures the reproducibility of subsequent grafting reactions.

[0041] Example 2 100 mg of the polyhexanepyrimidine POTP-NH1 prepared in Example 1 was dissolved in a mixture of PBS (pH 7.4) and THF. After pre-activation with EDC and NHS in an ice bath, tetracarboxyphenylporphyrin was added and the mixture was stirred for 12 h in the dark. The product was dialyzed against deionized water for 72 h, with a molecular weight cutoff of 3.5 kDa. After lyophilization, a purple-red solid POTP-TPP was obtained. The grafting rate was quantified using UV absorption spectroscopy based on the TCPP standard curve, with a grafting number-average molecular weight of approximately 1.21 × 10⁻⁶. 4 The distribution index is approximately 1.53, reflecting the effect of grafting on the segment conformation.

[0042] Example 3 2 mg of POTP-TPP and 5 mg of DSPE-mPEG2000 (mass ratio of DSPE-mPEG2000:POTP-TPP = 2.5:1) were dissolved in 5 mL of THF. 45 mL of distilled water was rapidly injected under strong ultrasonic conditions, followed by vacuum evaporation to remove THF and some water, yielding a nanoparticle dispersion of approximately 2 mL. The concentration of the dispersion was characterized by the POTP-TPP equivalent concentration, approximately 1000 µg·mL⁻¹. After dialysis / ultrafiltration / centrifugal washing, free lipids could be partially removed, and the actual content of DSPE-mPEG2000 in the final nanoparticles could be lower than the theoretical value corresponding to the feed ratio. In some embodiments, the mass fraction of DSPE-mPEG2000 in the final nanoparticles could be, for example, 0.5-40 wt% (determined by phosphorus content method or quantitative NMR), preferably 1-30 wt%.

[0043] Example 4 500 mg of sodium hyaluronate (molecular weight approximately 200 kDa) was dissolved in 50 mL of deionized water, and 50 mg of NaIO4 was added. The reaction was carried out at room temperature in the dark for 1 h to introduce aldehyde groups, and the reaction was terminated with ethylene glycol. After dialyzing for 48 h, the solution was lyophilized to obtain OHA. 200 mg of OHA was dissolved in 20 mL of PBS (pH 7.2), and 0.1 mmol of EDC and 0.1 mmol of NHS were added for activation. 0.1 mmol of 3-aminophenylboronic acid was added dropwise, and the reaction was carried out at room temperature for 12 h. After dialyzing and lyophilization, OHA-PBA was obtained. The residual aldehyde group was determined to be approximately 0.32 mmol·g⁻¹ by hydroxylamine hydrochloride titration. The amount of PBA introduced was determined to be approximately 0.28 mmol·g⁻¹ by borate ester colorimetry. Adjusting the amount of NaIO4 to 30 mg and 80 mg respectively allowed the aldehyde group density to be within an adjustable range of 0.20 to 0.46 mmol·g⁻¹.

[0044] Example 5 A 10 mg / mL solution of POTP-TPP nanoparticles and a 20 mg / mL solution of OHA-PBA were mixed at a 1:1 volume ratio and allowed to stand at room temperature for about 5 minutes to form a self-supporting hydrogel. Rheological frequency scanning showed that G′ remained consistently higher than G″ in the range of 0.1 to 100 rad·s⁻¹. Amplitude scanning showed that the upper limit of the linear viscoelastic region was approximately 5%. Shear recovery testing showed that after three alternating cycles of high and low shear, the storage modulus recovered more than 90% within 1 minute.

[0045] Example 6 The amino to aldehyde equivalent ratios were set at 2:1 and 1:2, respectively, and gels were formed according to the method in Example 5. The results showed that under the 2:1 condition, G′ increased to approximately 1800 Pa, the self-healing recovery rate was approximately 82%, and the peak thrust through the 21G needle increased by approximately 28% compared to the baseline. Under the 1:2 condition, G′ decreased to approximately 800 Pa, the self-healing recovery rate was approximately 95%, and injection smoothness improved. These results indicate that the ratio can be predictably adjusted between strength and self-healing.

[0046] Example 7 POPP NPs remained stable in pH 7.4 PBS, with minimal changes in morphology and particle size after 10 minutes of additional acoustic stimulation. The PEG canopy and fatty chains synergistically maintained dispersion stability. The gel slowly viscous and flows when left to stand at room temperature, adaptively filling irregular grooves. At 4 °C, the particle size of the NPs increased by less than 10% for at least 30 days.

[0047] Example 8 A standardized MIC procedure was established using Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 6538, with an initial inoculum size of approximately 1 × 10⁻⁶. 6 CFU·mL⁻¹ Incubation for 12 h. MIC was determined by OD600. Under no-sound-wave conditions, the MIC of POPPNPs was approximately 30 µg·mL⁻¹. After applying sound-wave stimulation, the MIC decreased to approximately 15 µg·mL⁻¹. The treated bacterial suspension was spread onto LB agar plates to observe colony formation and to verify the minimum bactericidal concentration.

[0048] Example 9 Using ABDA as a singlet oxygen indicator, the reactive oxygen species (ROS) yields of POTP-TPP and POPP NPs were compared under the same stimulation conditions. The decay of A divided by A0 increased significantly over time. The absorption of POPP NPs decreased more significantly after 10 minutes of acoustic treatment, indicating that the coupling of nanostructures and acoustic units can improve the efficiency of ROS generation.

[0049] Example 10 A mature biofilm model of *Pseudomonas aeruginosa* was established after 48 hours. The hydrogel of this invention was applied to the biofilm to a thickness of approximately 1 mm, followed by 1 MHz ultrasound irradiation for 5 minutes. Crystal violet quantitative analysis showed a decrease in biofilm biomass of approximately 72%. Confocal microscopy of live and dead bacteria staining showed a significant decrease in the proportion of viable bacteria. Desorption experiments showed that the residual biofilm thickness decreased from approximately 24 µm in the control group to approximately 8 µm, indicating a significant removal capacity for mature biofilms under acoustic activation conditions.

[0050] Example 11 L929 fibroblasts were cultured in DMEM containing 10% fetal bovine serum to prepare POPP gel extract, which was then sterilized by 0.22 µm filtration. After incubation at multiple concentrations for 24 h, live and dead cell staining and CCK-8 results showed that cell viability was still above 85% at an equivalent concentration of 25 mg·mL⁻¹. Cell scratch assay showed that cell migration was basically closed after 48 h, similar to that of the commercial dressing group.

[0051] Example 12 A full-thickness skin defect with a diameter of 8 mm was created on the back of mice and inoculated with *E. coli*. The wound was covered with the hydrogel of this invention and subjected to in vitro ultrasound irradiation for 5 minutes daily. The control group received PBS or hydrogel without ultrasound. Imaging measurements showed that the wound contraction rate was approximately 58% on day 3 and the healing rate was approximately 93% on day 13. Histological observation showed reduced inflammatory cell infiltration, and Masson staining indicated increased collagen deposition. Overall, the results were superior to the control group.

[0052] Example 13 A deep second-degree burn was established and inoculated with Staphylococcus aureus. The effects of combining low, medium, and high hydrogel dosages with two power densities (1.0 W·cm⁻² and 1.5 W·cm⁻²) were investigated. Results showed that the medium dosage combined with 1.5 W·cm⁻² acoustic excitation yielded the best results in terms of healing speed and exudation control. The bacterial load decreased by approximately 2.1 log from baseline. Further increases in dosage did not yield additional benefits, but a transient increase in local exudation occurred, suggesting the existence of a dose plateau.

[0053] The preferred embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the specific details of the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0054] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

[0055] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content disclosed by the present invention.

Claims

1. A dynamic self-healing antibacterial hydrogel with synergistic cationic membrane disruption and sonodynamic effects, comprising functionalized nanoparticles and a polysaccharide backbone. The functionalized nanoparticles are obtained by the self-assembly of a porphyrin-grafted polymer (POTP-TPP) and an amphiphilic lipid, wherein the POTP-TPP is prepared by the condensation of a primary amine-containing polymer (POTP-NH2) and porphyrin tetracarboxylic acid. The polysaccharide backbone is phenylboronic acid-modified oxidized hyaluronic acid (OHA-PBA). The primary amine of the POTP-TPP and the aldehyde group of the OHA-PBA form reversible Schiff base bonds in a buffered environment of pH 7.2 to 8.0, constituting a three-dimensional hydrogel network. The hydrogel can generate reactive oxygen species under 1 MHz ultrasound to enhance its antibacterial effect.

2. The hydrogel according to claim 1, characterized in that: The functionalized nanoparticles have a volume-weighted average particle size of 100 to 600 nm and a polydispersity index of no more than 0.50, and are measured by dynamic light scattering at 25°C according to ISO 22412 standard; the mass ratio of the amphiphilic lipid to the POTP-TPP is 0.1:1 to 10:1, preferably 0.5:1 to 5:

1.

3. The hydrogel according to claim 1 or 2, characterized in that: The zeta potential of the functionalized nanoparticles ranged from +5 to +20 mV and was determined by electrophoretic light scattering in a 10 mM sodium chloride aqueous solution at 25 °C.

4. The hydrogel according to any one of claims 1 to 3, characterized in that: The POTP-TPP has a porphyrin grafting rate of 5 to 10%, and is characterized by containing 5 to 10 porphyrin groups per 100 polypyrimidine repeating units.

5. The hydrogel according to any one of claims 1 to 4, characterized in that: The aldehyde density in the OHA-PBA was 0.20 to 0.46 mmol·g⁻¹, and was determined using the hydroxylamine hydrochloride method. The amount of phenylboronic acid groups introduced into the OHA-PBA was 0.10 to 0.40 mmol·g⁻¹, and was determined using colorimetry or NMR integration.

6. The use of the hydrogel according to any one of claims 1 to 5 in the preparation of antibacterial dressings for wounds, characterized in that: The hydrogel is applied to the infected wound, and an external ultrasound probe is used to irradiate the wound locally to enhance local sterilization and promote healing. The center frequency of the ultrasound is 1 MHz, the power density is 0.5 to 3 W·cm⁻², and the treatment time is 1 to 10 minutes, preferably using a 10 to 70% duty cycle.

7. The hydrogel according to any one of claims 1 to 5, characterized in that: The hydrogel contains phenylboronic acid groups and can reversibly interact with bacterial outer membrane lipopolysaccharides to improve local enrichment efficiency at the site of infection.

8. The use according to claim 6, characterized in that: The hydrogel is used to inhibit or remove mature biofilms formed by Pseudomonas aeruginosa, and to ensure that the residual thickness of the three-dimensional reconstruction after ultrasonic treatment is no more than 0.4 times that of the static control group. The residual thickness is determined by live-dead staining confocal imaging and voxel reconstruction.

Images