Red light-pH dual-response targeted antibacterial and anti-inflammatory nanoparticles as well as preparation method and application thereof

Abstract

The invention discloses a red light-pH dual-response targeted antibacterial and anti-inflammatory nanoparticle as well as a preparation method and application thereof. The nano-particle is prepared from the following raw materials: a porphyrin-zirconium metal organic framework carrier, an antibacterial agent, NO donor molecules and a targeting ligand, the mass ratio of the porphyrin-zirconium metal organic framework carrier to the antibacterial drug to the NO donor molecule to the targeting ligand is 1: (0.8-1.2): (0.3-1): (1-3). The red light-pH dual-response targeted antibacterial and anti-inflammatory nano-particle is of a layered composite structure of'carrier-functional molecule-targeted shell ', and has high efficiency, safety and practicability.

Description

Technical Field

[0001] This invention relates to the field of medical nanomaterials technology, specifically to a red light-pH dual-response targeted antibacterial and anti-inflammatory nanoparticle, its preparation method, and its application. Background Technology

[0002] Since the advent of penicillin in 1928, antibiotics have been the core means of preventing and treating bacterial infections. However, with the continuous emergence of pathogens, the appearance of new mutant strains, and the widespread overuse of antibiotics, drug-resistant strains such as methicillin-resistant Staphylococcus aureus (MRSA) are increasing, leading to numerous bottlenecks in traditional antibiotic treatment. On the one hand, antibiotics struggle to penetrate bacterial biofilm barriers and are unable to effectively eliminate intracellular parasitic bacteria, resulting in poor treatment outcomes. On the other hand, the long-term overuse of antibiotics has accelerated the evolution of bacterial resistance, while the development of new antibiotics is time-consuming, costly, and has a high failure rate; the rate of bacterial resistance evolution far exceeds the rate of antibiotic development. Therefore, developing anti-infective technologies with novel mechanisms of action that can circumvent drug resistance has become a critical issue that urgently needs to be addressed.

[0003] Nanoparticle delivery systems, with their advantages of precise targeting and enhanced tissue penetration, have shown great potential in the treatment of drug-resistant bacterial infections. Since their initial reporting in the 1990s, metal-organic frameworks (MOFs) have demonstrated broad application prospects in various fields, including gas adsorption and separation, catalysis, and drug delivery, thanks to their tunable structure, high specific surface area, and high porosity. Compared to traditional inorganic porous materials, MOFs offer greater flexibility in structural design and functional regulation. Their nanoscale particles can achieve targeted drug delivery, promote cellular uptake, and controllable drug release, particularly helping to improve the solubility and bioavailability of poorly soluble drugs, thereby enabling rapid, localized, and efficient drug therapy.

[0004] Porphyrin compounds, due to their unique conjugated macrocyclic structure and ease of modification, can coordinate with metal ions to form porphyrin metal-organic frameworks (Por-MOFs). Porphyrin-MOFs combine the porosity of MOFs with the excellent photophysical and chemical properties of porphyrins themselves. Among them, Por-MOFs with zirconium (Zr) as the metal node have become a research hotspot due to their good stability and biocompatibility, with the PCN series materials being particularly typical. PCN-224 is synthesized by thermal reaction of zirconium oxychloride octahydrate and tetra-(4-carboxyphenyl)porphyrin in N,N-dimethylformamide solvent, using benzoic acid as an auxiliary ligand. The Zr metal node and the porphyrin carboxyl group are connected by Zr-O bonds. Based on acid-base theory, this type of material exhibits excellent thermodynamic and chemical stability, while its high specific surface area and abundant pore structure make it a promising candidate for drug delivery.

[0005] However, existing porphyrin-based MOF antibacterial systems still have significant drawbacks: First, the ROS generation efficiency is limited by the electron transport characteristics of the material itself, and the bactericidal effect of a single photodynamic mechanism is insufficient to combat highly drug-resistant bacteria and stubborn infections encapsulated by biofilms. Second, the lack of a multi-response synergistic design for the infection microenvironment makes it difficult to achieve integrated treatment of "bactericidal-anti-inflammatory-tissue repair," resulting in persistent inflammation after infection and severely hindering wound healing. Third, insufficient targeting leads to low enrichment efficiency of the material at the infection site, and biocompatibility needs improvement, easily causing off-target toxicity, which limits its in vivo application efficacy. In addition, if the inflammatory response at the infection site is not timely regulated, it will severely hinder wound healing. Existing nano-antibacterial materials mostly focus on bactericidal function, neglecting the synergistic requirement of "bacterial clearance-inflammation suppression," resulting in persistent inflammation after infection and affecting treatment prognosis. At the same time, some materials have problems such as complex synthesis processes, poor batch stability, and limited red light penetration depth, further restricting their clinical translation.

[0006] Therefore, developing a novel nanoplatform that combines efficient synergistic sterilization, precise inflammation regulation, and good biocompatibility, and overcoming existing technological bottlenecks through multi-mechanism synergy, to achieve efficient treatment of drug-resistant bacterial infections and rapid wound repair, has significant clinical value and application prospects. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a red light-pH dual-responsive targeted antibacterial and anti-inflammatory nanoparticle, its preparation method, and its application. The red light-pH dual-responsive targeted antibacterial and anti-inflammatory nanoparticles of this invention have a layered composite structure of "carrier-functional molecule-targeting shell," possessing high efficiency, safety, and practicality.

[0008] To achieve the above objectives, the technical solution designed by the present invention is as follows: This invention provides a red light-pH dual-response targeted antibacterial and anti-inflammatory nanoparticle, wherein the raw materials of the nanoparticle include porphyrin-zirconium metal-organic framework carrier, antibacterial drug, NO donor molecule and targeted ligand; The mass ratio of the porphyrin-zirconium metal-organic framework carrier, the antibacterial drug, the NO donor molecule, and the targeting ligand is 1:0.8~1.2:0.3~1:1~3; The porphyrin-zirconium metal-organic framework support is any one of PCN-224, PCN-225 and MOF-525; The antibacterial drug is any one of ciprofloxacin hydrochloride, levofloxacin, ceftazidime, and vancomycin. The NO donor molecule is any one of arginine, citrulline, and ornithine. The targeting ligand is any one of hyaluronic acid, folic acid, and transferrin.

[0009] Furthermore, the porphyrin-zirconium metal-organic framework carrier is PCN-224; the antibacterial drug is ciprofloxacin hydrochloride; the NO donor molecule is arginine; and the targeting ligand is hyaluronic acid. The mass ratio of the porphyrin-zirconium metal-organic framework carrier, the antibacterial drug, the NO donor molecule, and the targeting ligand is 1:1~1.2:0.5~1:1.5~3. The porphyrin-zirconium metal-organic framework carrier PCN-224 uses Zr 4+ The metal nodes and tetra-(4-carboxyphenyl)porphyrin (TCPP) as the organic ligand coordinate through Zr-O bonds to form a nano-octahedral crystal structure; PCN-224 has a particle size of 90-110 nm, a theoretical pore size of 19.3 Å (1.93 nm), and a specific surface area ≥1000 m². 2 / g; via TCPP with Zr 4＋ The coordination ratio is controlled to form electron-deficient properties, ensuring the charge transfer efficiency in red light response. The porphyrin-zirconium metal-organic framework carrier PCN-224 has the dual functions of "drug loading carrier" and "red light-responsive ROS production unit", and its electronic defect structure provides the basis for subsequent cascade reactions; The antibacterial drug ciprofloxacin hydrochloride is encapsulated in the pores of PCN-224 and its carboxyl groups interact with Zr. 4+ The intermolecular forces are fixed within the carrier pores; ciprofloxacin hydrochloride is subjected to dual regulation by red light-triggered MOF structure dissociation and acidic environment to achieve sustained release. The NO donor molecule arginine interacts with Zr in PCN-224 via the guanidinium and amino groups at its terminal molecule. 4+ In-situ doping is achieved by coordinating unsaturated sites, releasing NO in the acidic microenvironment of the infection site, and destroying the extracellular polymer matrix of bacterial biofilm. The targeted ligand hyaluronic acid forms a hydrated gel shell through ultrasonic dispersion and hydrogen bonding. It is then targeted and enriched at the infection site via CD44 receptor-mediated targeting, while simultaneously improving colloidal stability, reducing non-specific toxicity, and promoting wound healing.

[0010] Furthermore, the mass ratio of PCN-224, ciprofloxacin hydrochloride, arginine, and hyaluronic acid is 1:1:1:3.

[0011] The present invention also provides a method for preparing the aforementioned nanoparticles, comprising the following steps: (1) Weigh the porphyrin-zirconium metal-organic framework carrier, antibacterial drug, NO donor molecule and targeting ligand according to the above mass ratio; (2) Dissolve the antibacterial drug in water, disperse the porphyrin-zirconium metal-organic framework carrier in it, stir in the dark to obtain a PC mixture; (3) Centrifuge the PC mixture to obtain a precipitate, and wash and dry the precipitate in sequence to obtain the PC complex; (4) Disperse the PC complex in water to obtain an aqueous PC solution, dissolve the NO donor molecule in water to obtain an aqueous donor molecule solution, mix the PC aqueous solution and the donor molecule aqueous solution, and stir in the dark to obtain an APC mixture; (5) Centrifuge the APC mixture to obtain a precipitate, and wash and dry the precipitate in sequence to obtain the APC complex; (6) Disperse the APC complex in water, sonicate to obtain an APC aqueous solution, add the targeting ligand, and stir to obtain an HAPC mixture; (7) Centrifuge the HAPC mixture to obtain a precipitate, and wash and dry the precipitate in sequence to obtain red light-pH dual-response targeted antibacterial and anti-inflammatory nanoparticles.

[0012] Furthermore, in steps (2) and (4), the stirring speed in the dark is 200~220 rpm, and the stirring time in the dark is 24~30 hours; In steps (3), (5) and (7), the centrifugation speed is 11000~12000 rpm, the centrifugation time is 10~15 minutes, the drying temperature is 40~50℃, and the drying time is 8~10 hours. In step (6), the ultrasonic dispersion time is 10-15 minutes, the ultrasonic dispersion power is 100-120W, the stirring speed is 200-220rpm, and the stirring time is 24-30 hours.

[0013] Furthermore, in steps (2) and (4), the stirring speed in the dark is 200 rpm, and the stirring time in the dark is 24 hours; In steps (3), (5) and (7), the centrifugation speed is 12000 rpm, the centrifugation time is 10 minutes, the drying temperature is 40℃, and the drying time is 8 hours. In step (6), the ultrasonic dispersion time is 15 minutes, the ultrasonic dispersion power is 100W, the stirring speed is 200rpm, and the stirring time is 24 hours.

[0014] The present invention also provides an application of the nanoparticles described above in inhibiting bacterial growth, wherein the bacteria are any one of Streptococcus suis, Staphylococcus aureus, Glasgow coli, and Escherichia coli.

[0015] The present invention also provides the application of the nanoparticles described herein in the preparation of a drug for treating bacterial infections.

[0016] The present invention also provides a red light-pH dual-response targeted antibacterial and anti-inflammatory drug, the drug comprising the aforementioned nanoparticles and water; The mass-to-volume ratio of the nanoparticles to water is 1~2 mg / mL.

[0017] Furthermore, the mass-to-volume ratio of the nanoparticles to water is 1 mg / mL.

[0018] The principle of this invention: The red light-pH dual-response targeted antibacterial and anti-inflammatory nanoparticles of this invention have a multi-response synergistic working mechanism: 1. Targeted enrichment stage: The hyaluronic acid on the surface of the red light-pH dual-response targeted antibacterial and anti-inflammatory nanoparticles specifically binds to the CD44 receptor overexpressed on the surface of inflammatory cells and bacteria at the site of infection, achieving targeted aggregation and reducing systemic off-target toxicity; 2. Dual-response triggering phase: (1) Red light response: 640-660nm red light irradiation (power density 100mW / cm²) 2 Irradiation time 10-30 minutes) activates the electronic defect properties of PCN-224, generates photogenerated carriers and converts them into reactive oxygen species (ROS, mainly singlet oxygen and hydroxyl radicals), while destroying part of the MOF structure and initiating CIP slow release; (2) pH response: The acidic microenvironment (pH 5.0-5.5) at the site of infection allows Arg and Zr to react. 4+ The coordinate bonds break, releasing NO; 3. Cascade sterilization stage: ROS and NO react rapidly to generate peroxynitrite (ONOO). - ), ONOO - The bacteria are killed by a triple action: oxidizing the bacterial cell membrane, nitrifying the tyrosine residues of proteins, and destroying bacterial DNA. At the same time, CIP enters the bacteria to inhibit DNA synthesis, and Arg degrades the EPS matrix of the biofilm. The three work together to achieve highly efficient sterilization. 4. Anti-inflammatory and repair phase: ONOO - It inhibits the NF-κB inflammatory signaling pathway and significantly reduces the transcription and protein expression of TNF-α, IL-1β, and IL-6; HA promotes fibroblast proliferation and collagen deposition, and accelerates wound tissue remodeling and healing.

[0019] The beneficial effects of this invention are: This invention, through an integrated design of "engineered regulation of electronic defects + red light-pH dual-response cascade + targeted antibacterial-anti-inflammatory-repair," forms red light-pH dual-response targeted antibacterial and anti-inflammatory nanoparticles that combine high efficiency, safety, and practicality. Compared with existing technologies, it exhibits significant technical advantages and beneficial effects, as detailed below: I. Significantly improved sterilization efficiency, breaking through the bottleneck in the treatment of drug-resistant bacteria. 1. Synergistic bactericidal effect through multiple mechanisms, significantly reducing the risk of drug resistance: This invention integrates photodynamic therapy (ROS) and gas therapy (NO / ONOO). - ONOO's triple mechanism of action, including chemotherapy (CIP) and other treatments. - It can simultaneously disrupt bacterial membranes, nitrate proteins, and damage DNA. CIP inhibits bacterial DNA synthesis, while Arg degrades the EPS matrix of biofilms. This multi-target action avoids the activation of single-drug resistance pathways in bacteria. Experimental verification shows that the minimum inhibitory concentration (MIC) of the red light-pH dual-responsive targeted antibacterial and anti-inflammatory nanoparticles against Streptococcus suis SC-19 is only 0.5 μg / mL, which is 0.5 times that of the free antibiotic CIP (1 μg / mL). The red light-pH dual-responsive targeted antibacterial and anti-inflammatory nanoparticles also show highly efficient bactericidal activity against drug-resistant strains such as Streptococcus suis, completely solving the core problems of traditional antibiotics: "easy to induce drug resistance and difficult to penetrate biofilms."

[0020] 2. Optimized response efficiency and improved red light utilization through electronic defect reduction: This is achieved through the Zr... 4＋ Precise coordination with TCPP to construct an electron-deficient structure enhances the quantum yield of red-light (640-660 nm) triggered singlet oxygen. ONOO is generated by the cascade of ROS and NO. - Its bactericidal activity far exceeds that of ROS or NO alone. In vitro colony counting experiments showed that the number of bacterial colonies in the HAPC treatment group under red light irradiation was reduced by more than 99% compared with the control group, achieving rapid elimination of stubborn drug-resistant bacteria.

[0021] II. Dual-response precise regulation to achieve "on-demand treatment" and integrated effects. 1. Dual Red Light-pH Response for Highly Controllable Drug Release: HAPC initiates drug release and cascade reactions only under the dual stimulation of "red light irradiation + acidic microenvironment (pH 5.0-5.5)" at the infection site. It maintains structural stability under normal physiological conditions (pH 7.4, no red light), preventing premature drug leakage. In vitro drug release experiments show that HAPC achieves a cumulative CIP release rate of over 45% after 72 hours at pH 5.5, realizing targeted sustained drug release and solving the problems of "uncontrollable release and high off-target toxicity" associated with traditional nanomaterials.

[0022] 2. Integrated antibacterial, anti-inflammatory, and repair treatment, resulting in significantly improved prognosis: ONOO -Not only does it possess potent bactericidal effects, but it also inhibits the NF-κB inflammatory pathway, reducing TNF-α transcription levels by 10-fold, effectively blocking the vicious cycle of inflammation caused by infection. The HA coating layer can promote fibroblast proliferation and collagen deposition, accelerating wound repair. In vivo experiments verified that the area of ​​infected wounds in mice treated with HAPC shrank to 3% of the initial area within 11 days, significantly better than the CIP group (9.5%). Furthermore, Masson staining showed that collagen deposition in the treatment group was more than 60% higher than that in the control group, achieving a full-cycle treatment of "bactericidal-anti-inflammatory-repair," overcoming the limitations of existing materials that suffer from "functional fragmentation and persistent inflammation."

[0023] III. Excellent targeting and biocompatibility, with significantly improved safety. 1. CD44 receptor-mediated targeting, resulting in high enrichment efficiency at the infection site: The HA on the surface of HAPC specifically binds to the CD44 receptor overexpressed on the surface of inflammatory cells and bacteria at the infection site, significantly increasing the local material concentration. Cell uptake experiments showed that the uptake of HAPC in RAW264.7 cells was more than 40% higher than that of APC without HA modification, effectively reducing off-target toxicity.

[0024] 2. Excellent biocompatibility and no obvious toxic side effects: HA coating changes the Zeta potential of HAPC from -10mV to -22mV, significantly reducing the risk of nonspecific aggregation; CCK-8 assay shows that HAPC treatment of RAW264.7 cells resulted in a survival rate ≥100% with no significant cytotoxicity. In in vivo safety experiments, the weight changes of mice during treatment were consistent with the blank control group, and H&E staining of major organs (heart, liver, spleen, lung, and kidney) showed no significant damage. This solves the problem of "poor biocompatibility and high in vivo toxicity" of traditional nano-antibacterial materials, laying the foundation for clinical translation.

[0025] IV. The preparation process is simple and controllable, with great potential for clinical translation. The preparation process of this invention involves only four conventional steps: PCN-224 synthesis, CIP loading, Arg doping, and HA coating. Each step involves mild reaction conditions (room temperature to 80°C) and easily controllable parameters. Product quality control can be achieved through conventional characterization methods such as EDS, XRD, and FTIR, resulting in excellent batch stability. Compared to existing complex modified nanoplatforms, this invention requires no special equipment or stringent reaction conditions, has low production costs, can be mass-produced, and is applicable to wounds infected with various drug-resistant bacteria related to zoonotic diseases, offering broad clinical applications. It addresses the technical challenges of complex synthesis, batch instability, and difficulty in transformation in some nanomaterials. Attached Figure Description

[0026] Figure 1 Typical SEM images of materials at different synthesis stages; In the diagram, a represents PCN, b represents PC, c represents APC, and d represents HAPC. Figure 2 The distribution of EDS elements in HAPC; Figure 3 Fourier transform infrared spectra of HAPC, APC, PC, and PCN; Figure 4 The result is a graph of the Zata potential. Figure 5 The graph shows the results of CIP drug loading and encapsulation efficiency for HAPC. Figure 6 This is a CIP drug release curve; Figure 7 The results of HAPC treatment of SC-19 are shown in the figure. Figure 8 This is a graph showing the MIC colony count after HAPC light treatment; Figure 9 The results of the cell uptake experiment are shown in the figure. In the figure, A is the control group, B is PCN-224, C is the PC complex, D is the APC complex, and E is HAPC. Figure 10 The graph shows the results of TNF-α transcription level measurement. Figure 11 Figure showing the results of the CCK-8 cytotoxicity assay; Figure 12 The result image generated by ROS for red light response; Figure 13 The graph shows the NO release results of HAPC; Figure 14 This is a comparison of wound healing in mice. Figure 15 Image showing the results of hematoxylin-eosin (HE) staining. Detailed Implementation

[0027] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can understand it.

[0028] Experimental materials description: 1. The Streptococcus suis SC19 of the present invention is the wild-type Streptococcus suis strain SC19 of the invention patent with application number CN201810076108.7.

[0029] 2. The *Glasotherium suis* of this invention is the *Glasotherium suis* of the invention patent with application number CN202411335303.9.

[0030] Example 1 Preparation of electronically defective porphyrin-zirconium MOF support (PCN-224) 1. Weigh 0.9 g of zirconium oxychloride octahydrate (ZrOCl2・8H2O), 8.4 g of benzoic acid (BA, auxiliary ligand), and 0.3 g of tetra-(4-carboxyphenyl)porphyrin (TCPP) according to the proportion, add 300 mL of N,N-dimethylformamide (DMF) solvent to obtain a mixed solution; 2. Add the mixed solution to a three-necked flask, stir magnetically (300 rpm) until completely dissolved, then heat to 80°C and react at a constant temperature for 5 hours. Crystal growth and electronic defect density are controlled by BA. 3. After naturally cooling to room temperature, the mixed solution was centrifuged at 12,000 rpm for 15 minutes and the precipitate was collected. The precipitate was washed three times with DMF (to remove unreacted raw materials) and twice with anhydrous ethanol (to remove residual solvent). The precipitate was then vacuum dried at 60°C for 12 hours to obtain PCN-224 powder.

[0031] Example 2 Preparation of Red Light-pH Dual-Response Targeted Antibacterial and Anti-inflammatory Nanoparticles This invention employs a preparation route of "stepwise synthesis - functional assembly - quality control screening", the specific steps of which are as follows: I. CIP Loading (Preparation of PC Composites) 1. Weigh PCN-224 powder and ciprofloxacin hydrochloride (CIP) in a mass ratio of 1:1. 2. Dissolve CIP in water to prepare a CIP aqueous solution (concentration 5 mg / mL). Disperse the weighed PCN-224 powder in the CIP aqueous solution and stir at room temperature for 24 hours (200 rpm) in the dark to obtain a PC mixture, ensuring that CIP fully penetrates into the PCN-224 pores. 3. Centrifuge the PC mixture at 12000 rpm for 10 minutes, remove the supernatant (containing free CIP), and obtain the precipitate. Wash the precipitate twice with ultrapure water, and dry the precipitate under vacuum at 40℃ for 8 hours to obtain the PC complex (CIP / PCN-224).

[0032] II. Arg doping (preparation of APC complex) 1. Weigh out L-arginine (Arg) according to a mass ratio of PCN-224 to L-arginine of 1:1. 2. Disperse the PC complex obtained above in ultrapure water to obtain a PC aqueous solution (concentration 10 mg / mL). Dissolve the weighed L-arginine in water to obtain an L-arginine aqueous solution (concentration 10 mg / mL). Mix the PC aqueous solution and the L-arginine aqueous solution, and stir continuously for 24 hours (200 rpm) in the dark to obtain an APC mixture. Then, react Arg and Zr...4+ Forming stable coordinate bonds; 3. Centrifuge the APC mixture at 12000 rpm for 10 minutes, collect the precipitate, wash the precipitate three times with ultrapure water (to remove free Arg), and vacuum dry the precipitate at 40℃ for 8 hours to obtain the APC complex (Arg@CIP / PCN-224).

[0033] III. HA Coating 1. Weigh out hyaluronic acid (HA) according to the mass ratio of PCN-224 to hyaluronic acid of 1:3. 2. Disperse the APC complex obtained above in ultrapure water (concentration 1 mg / mL), and sonicate for 15 minutes (power 100 W, frequency 40 kHz) to obtain an APC aqueous solution. Add HA powder and stir at room temperature for 24 hours (speed 200 rpm) to obtain an HAPC mixture. A uniform coating layer is formed on the surface of the APC complex, which serves as a targeted modification layer. 3. Centrifuge the HAPC mixture at 12000 rpm for 10 minutes to obtain a precipitate. Wash the precipitate twice with ultrapure water (to remove free HA). Vacuum dry the precipitate at 40℃ for 8 hours to obtain red light-pH dual-response targeted antibacterial and anti-inflammatory nanoparticles (HAPC).

[0034] In this embodiment, the optional solution is: 1. Antibiotic substitution: CIP can be replaced with antibiotics such as levofloxacin, ceftazidime, and vancomycin. The mass ratio of antibiotic to PCN-224 is 0.8~1.2:1, which is suitable for infections caused by different types of drug-resistant bacteria. 2. Targeted ligand replacement: HA can be replaced with folic acid and transferrin, with the coating amount maintained at 8-12 mg of targeted ligand per 10 mg APC complex, adapting to the receptor expression characteristics of different infection sites. 3. MOF carrier replacement: PCN-224 can be replaced by PCN-225 (Anhui Zesheng Technology Co., Ltd.) and MOF-525 (Anhui Zesheng Technology Co., Ltd.) porphyrin-based Zr-MOF. It is necessary to maintain the electronic defect characteristics and 10-20nm mesoporous structure to ensure drug loading and red light response efficiency. 4. NO donor molecule substitution: Arg can be replaced with citrulline or ornithine as NO donor molecules. The doping amount is maintained at a NO donor to PCN-224 mass ratio of 0.3 to 1:1 to ensure that the NO release amount meets the requirements of the cascade reaction.

[0035] Example 3 Structural analysis of red light-pH dual-responsive targeted antibacterial and anti-inflammatory nanoparticles 1. SEM analysis was performed on the red light-pH dual-response targeted antibacterial and anti-inflammatory nanoparticles HAPC prepared in Example 2. The results are as follows: Figure 1As shown, HAPC has a regular nano-octahedral morphology with a particle size of 90-110 nm.

[0036] 2. Energy dispersive spectroscopy (EDS) was performed on the HAPC, and the results are as follows: Figure 2 As shown, Zr, C, N, and O elements are uniformly distributed in the HAPC structure.

[0037] 3. Fourier transform infrared spectroscopy (FTIR) analysis was performed on HAPC, and the results are as follows: Figure 3 As shown, HAPC at 1710cm -1 (CIP carboxyl group), 1380cm -1 A characteristic peak appears at the (Arg guanidinyl) group.

[0038] 4. The zeta potential of HAPC was tested, and the results are as follows: Figure 4 As shown, HA coating changes the Zeta potential of HAPC from -10mV to -22mV, significantly reducing the risk of nonspecific aggregation.

[0039] 5. The CIP drug loading and encapsulation efficiency of HAPC were determined using a UV-Vis spectrophotometer. A standard solution of ciprofloxacin hydrochloride was prepared by dissolving it in pure water, and the solution was analyzed at 270 nm using a UV-Vis spectrophotometer. The drug loading was determined using an indirect method, and the results are as follows: Figure 5 As shown, the CIP drug loading rate of HAPC is ≥50%, and the encapsulation efficiency of HAPC is ≥95%.

[0040] Example 4 Performance testing of red light-pH dual-response targeted antibacterial and anti-inflammatory nanoparticles I. In vitro drug release experiment 1. Prepare PC solution, APC solution and HAPC solution respectively from PC complex, APC complex and HAPC, with a concentration of 1 mg / ml for all three solutions (calculated based on ciprofloxacin hydrochloride).

[0041] 2. At 37℃, PC, APC, and HAPC solutions were placed in dialysis bags with a molecular weight cutoff of 3500 Da, respectively. The dialysis bags were sealed and immersed in 20 mL of pH 5.5 PBS buffer. They were then irradiated with red light (640-660 nm, 20 min) and incubated again for 2 h. At 0, 1, 2, 4, 6, 8, 10, 12, 24, 36, 48, 60, and 72 hours, 1 mL of release medium was collected and replenished with the corresponding volume of release medium. The absorbance of each sample at 270 nm was measured using a UV spectrophotometer to study the drug release behavior. The following experimental groups were set up: (1) Illumination group (L): ①PC; ②APC; ③HAPC; ④HAPC / JS: When replenishing the corresponding volume of release medium, add an additional 100 μL of *Glassonella suis* (10 μL of ... 8 (CFU / mL).

[0042] (2) Dark Group (D): Not exposed to red light, the four groups are set up the same as the light group.

[0043] The results are as follows Figure 6 As shown, CIP can be released from HAPC in a continuous and controllable manner, which is crucial for achieving long-term therapeutic effects.

[0044] II. Minimum Inhibitory Concentration Determination Experiment The minimum inhibitory concentration (MIC) was determined using the micro-broth dilution method, and the Streptococcus suis SC19 bacterial suspension was adjusted to 1 × 10⁻⁶. 6 After inoculating the drug at a concentration of CFU / mL, it was seeded into 96-well plates. Gradual concentrations of CIP, PC complex, APC complex, and HAPC were added, respectively. The drugs were then irradiated under red light (640-660 nm) for 0, 10, 20, and 30 minutes, followed by incubation at 37°C for 18 to 24 hours. The lowest drug concentration at which no visible bacterial growth was observed was recorded as the MIC value. All experiments were performed in triplicate.

[0045] The results are as follows Figure 7 As shown, the minimum inhibitory concentration (MIC) of the red light-pH dual-response targeted antibacterial and anti-inflammatory nanoparticles against Streptococcus suis SC-19 is only 0.5 μg / mL, which is 0.5 times that of the free antibiotic CIP (1 μg / mL).

[0046] III. In vitro colony counting experiment Streptococcus suis SC19 was cultured to the logarithmic growth phase. It was then diluted to an appropriate concentration with sterile PBS, and 100 μL of the bacterial suspension was added to HAPC or CIP to obtain bacterial suspensions containing different concentrations of HAPC or CIP. These suspensions were then placed in 96-well plates, treated with red light (640-660 nm, 20 min), and incubated at 37°C for 12 hours. Subsequently, each bacterial sample was diluted, spread onto agar plates, and incubated upside down at 37°C for 16 hours before counting. A control group (Dark) was also included, without red light irradiation.

[0047] The results are as follows Figure 8 As shown, the number of bacterial colonies in the HAPC treatment group was reduced by more than 99% compared with the CIP group under red light irradiation, achieving rapid elimination of stubborn drug-resistant bacteria.

[0048] IV. Cell Uptake Experiment In each well of the 24-well plate, at 10 5RAW264.7 cells were added at a concentration of [cells / mL] and incubated at 37°C with 5% CO2 for 24 hours to achieve complete adhesion. The original culture medium was then discarded and replaced with fresh medium containing 8 μg / mL of CIP, PC complex, APC complex, HAPC, or PCN-224. A control group (without any added drugs) was prepared. Incubation was then performed for 4 hours. RAW264.7 cells were subsequently fixed in 4% formaldehyde for 15 minutes, stained with DAPI for 10 minutes, and the cells were collected and observed using a vertical fluorescence microscope.

[0049] The results are as follows Figure 9 As shown, blue represents nuclear dye, and the intensity and distribution of red fluorescence directly reflect the cell's uptake efficiency. HAPC uptake in RAW264.7 cells was more than 40% higher than that of the unmodified HA APC complex.

[0050] V. Measurement of TNF-α transcription levels Mouse mononuclear macrophage leukemia cells (RAW264.7) were seeded into 6-well plates at a concentration of 5 × 10⁶ cells per well. 5 Cells were incubated at 37°C and 5% CO2 for 24 hours to achieve complete cell attachment. Subsequently, the original culture medium was discarded, and a logarithmic growth phase (bacterial liquid density 10⁻⁶) containing an MOI of 100 was added. 8 Streptococcus suis (CFU / mL) was infected for 4 hours, and then the culture medium was replaced with fresh medium containing 8 μg / mL of CIP, PC complex, APC complex, HAPC, or PCN-224. Each group was incubated for 2 hours, followed by red light irradiation (640-660 nm, 20 min), and then incubated again for 2 hours. qPCR was then performed. The 2^-ΔΔCt method was used to calculate the relative expression levels of the target genes in each group. The Positive group was the infection group, containing only Streptococcus suis SC-19 and RAW264.7 cells, while the Control group was the control group, containing only RAW264.7 cells. The CIP, PC, APC, and HAPC groups were each given 8 μg / mL of the corresponding drug.

[0051] The results are as follows Figure 10 As shown, compared with the positive control group, HAPC treatment significantly reduced the expression level of TNF-α, indicating that HAPC has the effect of inhibiting the release of TNF-α inflammatory factors in this experimental system, and may thus play a potential role in alleviating the inflammatory response.

[0052] VI. CCK-8 Experiment RAW264.7 cells were adjusted to 5 × 10⁻⁶ cells. 4After achieving a cell / mL concentration, cells were seeded at a density of 200 μL per well in 96-well plates and cultured at 37°C with 5% CO2 for 24 hours to ensure adequate cell adhesion. The original culture medium was then discarded, and the experimental groups were replaced with fresh medium containing different concentrations of PCN-224 or HAPC. After irradiation with red light for 2 hours, the cells were cultured for another 2 hours. Then, 20 μL of CCK-8 reagent was added to each well, and after incubation for 1 hour, the absorbance at 450 nm was immediately measured to calculate cell viability. A control group (Dark) was also included, without red light irradiation.

[0053] The results are as follows Figure 11 As shown, the survival rate of RAW264.7 cells treated with HAPC light was ≥100%, with no obvious cytotoxicity.

[0054] VII. ROS Generation and Evaluation Intracellular ROS levels were detected using the DCFH-DA fluorescent probe method. Intracellular ROS oxidizes non-fluorescent DCFH into green fluorescent DCF. The intensity of the green fluorescence was observed using a fluorescence microscope or imaging system, reflecting the level of intracellular ROS production.

[0055] Specifically, RAW264.7 cells were seeded into 96-well plates at a concentration of 5 × 10⁶ cells per well. 5 Cells were incubated at 37°C and 5% CO2 for 24 hours to achieve complete cell attachment. Fresh culture medium containing 8 μg / mL of CIP, PC complex, APC complex, HAPC, or PCN-224 was then added. The positive control group received H2O2 treatment. Each group was incubated for 2 hours, followed by red light irradiation (640-660 nm, 20 min), and then incubated again for 2 hours. After incubation, 100 μL of LCFH-DA probe was added, and the cells were incubated in the dark for 30 min. The cells were washed three times with PBS to remove free fluorescent probes, and fluorescence was then observed under an inverted microscope.

[0056] The results are as follows Figure 12 As shown, the fluorescence signal in the HAPC-illuminated group was weaker, indicating that HAPC treatment significantly inhibited the generation of intracellular ROS.

[0057] 8. NO Generation Assessment NO production was detected using the Griess reagent method. HAPC aqueous suspensions with concentration gradients of 128, 64, 32, and 16 μg / mL (based on CIP) were prepared. Griess reagent was added according to the Griess kit (Beyotime). Samples were irradiated with red light at 640-660 nm for 0, 10, 20, 30, 40, 50, and 60 minutes to simulate photoresponse conditions. The absorbance of the supernatant at 540 nm was measured using an enzyme-linked immunosorbent assay (ELISA) instrument. NO release was calculated based on the absorbance values, with three replicates per group to ensure data reliability.

[0058] The results are as follows Figure 13 As shown in the figure, the results indicate that NO release is positively correlated with HAPC concentration, and gradually increases with prolonged light exposure before stabilizing, indicating that HAPC can effectively release NO.

[0059] Example 5 Preparation of red light-pH dual-response targeted antibacterial and anti-inflammatory drugs The HAPC prepared in Example 2 was dispersed in ultrapure water to prepare a 1 mg / mL solution, thus obtaining a red light-pH dual-response targeted antibacterial and anti-inflammatory drug (prepared and used immediately).

[0060] Example 6 Application of red light-pH dual-responsive targeted antibacterial and anti-inflammatory drugs 1. Create a circular incision of a specific diameter on the back of the mouse using a specialized skin puncture device. Apply 50 μL of a 10% concentration of [unspecified substance] to the wound surface. 8 An infection model was established by injecting CFU / mL of Streptococcus suis SC-19 liquid and waiting 24 hours.

[0061] 2. Mice were randomly divided into 10 groups, with 3 mice in each group (n=3). Red light-pH dual-responsive targeted antibacterial and anti-inflammatory drugs or CIP were applied topically or sprayed onto the infected wound surface every 24 hours, with each dose being 50 μL. Immediately after administration, the mice were irradiated with 640-660 nm red light for 20 minutes (power density 100 mW / cm²). 2 Continue treatment for 7-11 days until the wound heals (the wound area shrinks to less than 5% of the initial area).

[0062] The specific groupings are as follows: the light-exposed group received 640-660nm red light irradiation for 20 minutes after drug administration, while the dark-free group did not receive red light treatment after drug administration. L represents the light-exposed group, and D represents the dark-free group. The PBS group used PBS solution instead of the drug, and the control group (Con group) did not receive any drug.

[0063] The results are as follows Figure 14As shown, the area of ​​the infected wound in the HAPC treatment group of mice was reduced to 3% of the initial area within 11 days, which was significantly better than that in the CIP treatment group (9.5%). Furthermore, Masson staining showed that the amount of collagen deposition in the HAPC treatment group was more than 60% higher than that in the control group.

[0064] 3. Collect the major organs of the mice mentioned above and stain the biological samples using hematoxylin-eosin (HE) staining. The results are as follows: Figure 15 As shown, the weight changes of mice during treatment were consistent with those of the control group, and H&E staining of major organs (heart, liver, spleen, lung, and kidney) did not show significant damage.

[0065] All other parts not described in detail are existing technologies. Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A red light-pH dual-responsive targeted antibacterial and anti-inflammatory nanoparticle, characterized in that: The raw materials for the nanoparticles include porphyrin-zirconium metal-organic framework carriers, antibacterial drugs, NO donor molecules, and targeting ligands; The mass ratio of the porphyrin-zirconium metal-organic framework carrier, the antibacterial drug, the NO donor molecule, and the targeting ligand is 1:0.8~1.2:0.3~1:1~3; The porphyrin-zirconium metal-organic framework support is any one of PCN-224, PCN-225 and MOF-525; The antibacterial drug is any one of ciprofloxacin hydrochloride, levofloxacin, ceftazidime, and vancomycin. The NO donor molecule is any one of arginine, citrulline, and ornithine. The targeting ligand is any one of hyaluronic acid, folic acid, and transferrin.

2. The nanoparticles according to claim 1, characterized in that: The porphyrin-zirconium metal-organic framework carrier is PCN-224; the antibacterial drug is ciprofloxacin hydrochloride; the NO donor molecule is arginine; and the targeting ligand is hyaluronic acid. The mass ratio of the porphyrin-zirconium metal-organic framework carrier, the antibacterial drug, the NO donor molecule, and the targeting ligand is 1:1~1.2:0.5~1:1.5~3.

3. The nanoparticles according to claim 2, characterized in that: The mass ratio of PCN-224, ciprofloxacin hydrochloride, arginine, and hyaluronic acid is 1:1:1:

3.

4. A method for preparing nanoparticles according to any one of claims 1 to 3, characterized in that: Includes the following steps: (1) Weigh the porphyrin-zirconium metal-organic framework carrier, antibacterial drug, NO donor molecule and targeting ligand according to the above mass ratio; (2) Dissolve the antibacterial drug in water, disperse the porphyrin-zirconium metal-organic framework carrier in it, stir in the dark to obtain a PC mixture; (3) Centrifuge the PC mixture to obtain a precipitate, and wash and dry the precipitate in sequence to obtain the PC complex; (4) Disperse the PC complex in water to obtain an aqueous PC solution, dissolve the NO donor molecule in water to obtain an aqueous donor molecule solution, mix the PC aqueous solution and the donor molecule aqueous solution, and stir in the dark to obtain an APC mixture; (5) Centrifuge the APC mixture to obtain a precipitate, and wash and dry the precipitate in sequence to obtain the APC complex; (6) Disperse the APC complex in water, sonicate to obtain an APC aqueous solution, add the targeting ligand, and stir to obtain an HAPC mixture; (7) Centrifuge the HAPC mixture to obtain a precipitate, and wash and dry the precipitate in sequence to obtain red light-pH dual-response targeted antibacterial and anti-inflammatory nanoparticles.

5. The preparation method according to claim 4, characterized in that: In steps (2) and (4), the stirring speed in the dark is 200-220 rpm, and the stirring time in the dark is 24-30 hours. In steps (3), (5) and (7), the centrifugation speed is 11000~12000 rpm, the centrifugation time is 10~15 minutes, the drying temperature is 40~50℃, and the drying time is 8~10 hours. In step (6), the ultrasonic dispersion time is 10-15 minutes, the ultrasonic dispersion power is 100-120W, the stirring speed is 200-220rpm, and the stirring time is 24-30 hours.

6. The preparation method according to claim 5, characterized in that: In steps (2) and (4), the stirring speed is 200 rpm and the stirring time is 24 hours. In steps (3), (5) and (7), the centrifugation speed is 12000 rpm, the centrifugation time is 10 minutes, the drying temperature is 40℃, and the drying time is 8 hours. In step (6), the ultrasonic dispersion time is 15 minutes, the ultrasonic dispersion power is 100W, the stirring speed is 200rpm, and the stirring time is 24 hours.

7. The application of the nanoparticles according to any one of claims 1 to 3 in inhibiting bacterial growth, characterized in that: The bacteria are any one of Streptococcus suis, Staphylococcus aureus, Gerasimovicella suis, and Escherichia coli.

8. The use of the nanoparticles according to any one of claims 1 to 3 in the preparation of a medicament for treating bacterial infections.

9. A red light-pH dual-responsive targeted antibacterial and anti-inflammatory drug, characterized in that: The drug comprises the nanoparticles as described in any one of claims 1 to 3 and water; The mass-to-volume ratio of the nanoparticles to water is 1~2 mg / mL.

10. The medicament according to claim 9, characterized in that: The mass-to-volume ratio of the nanoparticles to water is 1 mg / mL.

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