A spiro-engineered cationic iron porphyrin porous organic polymer and a preparation method and application thereof

Abstract

The application discloses a kind of spiro ring engineering cationic iron porphyrin porous organic polymer and its preparation method and application, belong to biological medicine technical field.The octa-formaldehyde porphyrin and double spiro ring cationic diamine bromide that are coordinated with iron ion are used as structural unit, and the non-crystalline organic polymer with hierarchical pore is constructed by forming acetal amine bond between aldehyde group of octa-formaldehyde porphyrin and secondary amine group of spiro ring cationic diamine, i.e.spire ring engineering cationic iron porphyrin porous organic polymer.The application uses TTEP as photoactive unit, DSPZ as double spiro ring unit with two permanent positive charges and two terminal secondary amine reaction sites, and constructs iron porphyrin polymer with high spatial distortion and high cationic charge density.The application reduces porphyrin π-π packing and aggregation quenching, improves photo-thermal conversion and active oxygen generation capacity, and realizes peroxidase-like / peroxide hydrogenase synergistic catalysis by using H2O2 in infected microenvironment, so as to improve antibacterial effect.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a spirocyclic engineered cationic iron porphyrin porous organic polymer, its preparation method, and its applications. Background Technology

[0002] Bacterial resistance and biofilm-associated infections (BAT) pose challenges to traditional antibiotic treatment, including decreased efficacy, high relapse rates, and bacterial imbalance. Photothermal and photodynamic therapies offer advantages such as spatiotemporal control and reduced likelihood of inducing classical resistance; however, single-phototherapy is often limited by factors such as photosensitizer aggregation leading to quenching, short reactive oxygen species (ROS) diffusion distances, and hypoxia in the infection microenvironment. Porphyrins possess visible light absorption and ROS generation capabilities, but their large planar conjugated structures are prone to π-π stacking, leading to aggregation-induced quenching and reduced photodynamic performance. Immobilizing porphyrins in porous covalent networks can limit molecular aggregation to some extent, but most existing porphyrin frameworks still exhibit strong planarity; relying solely on ordinary flexible linkers makes it difficult to simultaneously achieve efficient disruption of porphyrin coplanar stacking, increase cation charge density, and construct stable multifunctional catalytic sites. Furthermore, the infection microenvironment (IME) has several unfavorable characteristics. For example, due to vascular damage and high consumption by immune cells and bacteria, it is typically hypoxic, and bacterial fermentation and inflammatory responses make the IME weakly acidic (pH≈5.5-6.5). In addition, the inflammatory response also produces elevated levels of hydrogen peroxide (H2O2).

[0003] To circumvent these limitations, integrating cationic functional groups into therapeutic platforms has become a highly effective strategy. These groups exert multifaceted synergistic effects: they enhance electrostatic adhesion to negatively charged bacterial surfaces, ensuring close contact while reducing heat and ROS diffusion-mediated losses, and promote biofilm penetration through interaction with anionic extracellular polymeric substances (EPS). Furthermore, cationic groups can promote local enrichment of photothermal agents on bacterial membranes, thereby enhancing photothermal therapy efficiency; and can improve enzyme-like activities (such as peroxidase-like activities) by enriching anionic substrates such as H₂O₂ at catalytic sites. Overall, these advantages highlight the necessity of developing a multifunctional platform that not only overcomes the photophysical defects of conventional photosensitizers but also actively adapts to harsh infectious microenvironments to achieve synergistic therapeutic effects. Therefore, it is necessary to design a cationic porphyrin polymer that can not only overcome aggregation-induced quenching caused by π-π stacking but also enhance enzyme-like activity, integrating photothermal, photodynamic, and enzyme-like activities to improve antibacterial efficacy. Summary of the Invention

[0004] To address the aforementioned prior art, the objective of this invention is to provide a spirocyclic engineered cationic iron porphyrin porous organic polymer, its preparation method, and its applications. This invention employs 5',5''',5'''''',5''''''''''-(21H,23H-porphyrin-5,10,15,20-tetramethyl)tetra([1,1':3',1''-terphenyl]-4,4''-dicarboxaldehyde) as the octaaldehyde-based photoactive building block, and 3,12-diaza-6,9-diazamonium dispiro[5.2.5] 9 .2 6 Hexadecanedibromide, as a bispiral ring building block with two permanent positive charges and two terminal secondary amine reaction sites, is used to construct iron porphyrin polymers with hierarchical channels, exhibiting high spatial distortion and high cationic charge density in an amorphous porous network. This reduces porphyrin π-π stacking and aggregation-induced quenching, enhances photothermal conversion and reactive oxygen species generation, and utilizes H₂O₂ in the infection microenvironment to achieve peroxidase / catalase-like synergistic catalysis, thereby improving antibacterial efficacy.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a spirocyclic engineered cationic iron porphyrin porous organic polymer, which uses an octaaldehyde porphyrin coordinated with iron ions and a bispirocyclic cationic diamine bromide as structural units. The amorphous organic polymer with a porous structure is constructed by forming acetal-amine bonds between the aldehyde group of the octaaldehyde porphyrin and the secondary amine group of the spirocyclic cationic diamine. This is the spirocyclic engineered cationic iron porphyrin porous organic polymer.

[0006] Preferably, the bispirocyclic cationic diamine is a bispirocyclic dicationic diamine dibromide; the octaaldehyde porphyrin is 5',5''',5'''''',5'''''''''''-(21H,23H-porphyrin-5,10,15,20-tetramethyl)tetra([1,1':3',1''-terphenyl]-4,4''-dicarboxaldehyde), with the following structural formula: The acetal-amine bond is an NCN acetal-amine bond.

[0007] Preferably, the bispirocyclic dicationic diamine dibromide is 3,12-diaza-6,9-diaza-onium bispiro[5.2.5]. 9 .2 6 Hexadecane dibromide, its structural formula is: .

[0008] Preferably, its structural formula is: .

[0009] A second aspect of the present invention provides a method for preparing a spirocyclic engineered cationic iron porphyrin porous organic polymer, comprising the following steps: (1) Combine octaaldehyde porphyrin and 3,12-diaza-6,9-diaza-onium dispirol [5.2.5] 9 .2 6 Hexadecane dibromide was added to o-xylene, mixed thoroughly, and then subjected to a solvothermal reaction in a vacuum-sealed environment. After cooling, centrifugation, washing, and drying were performed to obtain a spirocyclic engineered cationic porphyrin porous organic polymer (Por-Aminal-COF-3). (2) The spirocyclic engineered cationic porphyrin porous organic polymer and iron salt were added to a mixed solvent and heated under vacuum. The resulting mixture was added to water to form a brown precipitate. The precipitate was filtered, washed and dried to obtain the spirocyclic engineered cationic iron porphyrin porous organic polymer (Por-Aminal-COF-3@Fe).

[0010] Preferably, in step (1), the octaaldehyde porphyrin and 3,12-diaza-6,9-diaza-onium dispiro[5.2.5] 9 .2 6 The molar ratio of hexadecane dibromide is 1:(4~7); the mixing is homogenized by ultrasonic treatment for 5 min; the temperature of the solvothermal reaction is 110~130℃ and the time is 3 days.

[0011] Preferably, in step (1), the 3,12-diaza-6,9-diaza-onium dispirol [5.2.5] 9 .2 6 Hexadecane dibromide is prepared by the following method: S1. Dissolve triethylenediamine in tetrahydrofuran in an ice bath, add cyanogen bromide, collect the precipitated white solid, wash, and recrystallize in an alcohol-water mixture to obtain white crystals, namely 3,12-dicyano-3,6λ. 5 ,9λ 5 ,12-Tetraazabicyclo[5.2.5] 9 .2 6 Hexadecane-6,9-bis(methylene)dibromide.

[0012] S2. 3,12-dicyano-3,6λ 5 ,9λ 5 ,12-Tetraazabicyclo[5.2.5] 9 .2 6 Hexadecane-6,9-bis(methylene) dibromide was mixed with concentrated hydrochloric acid and water, and the mixture was refluxed. After cooling, it was neutralized with saturated sodium bicarbonate solution, and the solvent was removed under reduced pressure to obtain a white crystalline solid, which is 3,12-diaza-6,9-diaza-onium dispirol [5.2.5]. 9 .26 Hexadecane dibromide.

[0013] Preferably, in step (2), the iron salt is FeCl3·6H2O; the mass ratio of the spirocyclic engineered cationic porphyrin porous organic polymer to the iron salt is 75:19; and the heating treatment temperature is 80℃ and the time is 24h.

[0014] A third aspect of the present invention provides the application of spirocyclic engineered cationic iron porphyrin porous organic polymers in the preparation of antibacterial drugs.

[0015] Preferably, the spirocyclic engineered cationic iron porphyrin porous organic polymer has photothermal activity, photodynamic activity, and enzyme-like activity; the enzyme-like activity includes POD-like activity and CAT-like activity.

[0016] The beneficial effects of this invention are: (1) In this invention, 5',5''',5'''''',5''''''''''-(21H,23H-porphyrin-5,10,15,20-tetramethyl)tetra([1,1':3',1''-terphenyl]-4,4''-dicarboxaldehyde) is used as the octaaldehyde-based photoactive building block, and 3,12-diaza-6,9-diaza-onium dispirol[5.2.5] is used. 9 .2 6 Hexadecanedibromide, as a bispirocyclic building block with two permanent positive charges and two terminal secondary amine reaction sites, was used to construct iron porphyrin polymers with hierarchical channels, exhibiting high steric distortion and high cation charge density in amorphous porous networks. Compared to control polymers using piperazine or monospirocyclic amine linkers, the bispirocyclic building block further reduces skeletal planarity and improves photothermal and photodynamic properties. The bispirocyclic dicationic building block simultaneously provides steric hindrance and permanent positive charges, avoiding the charge instability problems caused by introducing cations solely through post-quaternization or reversible protonation, and improving the contact efficiency between the material and bacterial and biofilm membranes.

[0017] (2) The iron porphyrin polymer with hierarchical channels and high spatial distortion and high cationic charge density in the amorphous porous network constructed by the present invention can reduce porphyrin π-π stacking and aggregation leading to quenching, improve photothermal conversion and reactive oxygen generation capacity, and utilize H2O2 in the infection microenvironment to achieve peroxidase-like / catalase-like synergistic catalysis, thereby improving the antibacterial effect. Attached Figure Description

[0018] Figure 1 Synthetic routes of Por-Aminal-COF-1, Por-Aminal-COF-2, Por-Aminal-COF-3 and Por-Aminal-COF-3@Fe; Figure 2 FTIR spectra of Por-Aminal-COF-3@Fe, Por-Aminal-COF-3, and the corresponding reactive monomers; Figure 3 Solid-state 13C CP / MAS NMR spectrum of Por-Aminal-COF-3@Fe; Figure 4 XRD pattern of Por-Aminal-COF-3@Fe; Figure 5 (a) TGA analysis of Por-Aminal-COF-3@Fe; (b) TGA analysis of Por-Aminal-COF-3; Figure 6 (a) Low-temperature nitrogen adsorption-desorption isotherm of Por-Aminal-COF-3@Fe; (b) Pore size distribution of Por-Aminal-COF-3@Fe obtained from the desorption branch of the isotherm; Figure 7 (a) Scanning electron microscope (SEM) image of Por-Aminal-COF-3@Fe, scale bar 500 nm; (b) Scanning electron microscope (SEM) image of Por-Aminal-COF-3@Fe, scale bar 200 nm; (c) Scanning electron microscope (SEM) image of Por-Aminal-COF-3@Fe, scale bar 100 nm; (d) Transmission electron microscope (TEM) image of Por-Aminal-COF-3@Fe, scale bar 200 nm; (e) Transmission electron microscope (TEM) image of Por-Aminal-COF-3@Fe, scale bar 50 nm; (f) High-resolution transmission electron microscope (HRTEM) image of Por-Aminal-COF-3@Fe at a scale bar of 10 nm; (g) Energy dispersive spectroscopy (EDS) elemental distribution map of Por-Aminal-COF-3@Fe at a scale bar of 200 nm. Figure 8 : EDS of Por-Aminal-COF-3@Fe; Figure 9 (a) XPS full spectrum of Por-Aminal-COF-3@Fe; (b) High-resolution XPS spectrum of C 1s; (c) High-resolution XPS spectrum of N 1s; (d) High-resolution XPS spectrum of Fe 2p; (e) High-resolution XPS spectrum of Br 3d; Figure 10(a) UV-vis absorption spectra of Por-Aminal-COF-3 dispersions at different concentrations (50, 100, 150, and 200 μg / mL) in aqueous solution; (b) UV-vis absorption spectra of Por-Aminal-COF-3@Fe at different concentrations; (c) UV-vis absorption spectra of Por-Aminal-COF-3@Fe after laser irradiation (1.5 W / cm²). 2 (d) Temperature variation with concentration of Por-Aminal-COF-3@Fe at different concentrations (0, 50, 100, 150, 200 μg / mL) under 638 nm laser irradiation (1.5 W / cm²). 2 (e) Infrared thermal imaging at different time points (0-10 min) under different power densities (0.5, 1.0, 1.5 and 2.0 W / cm²). 2 (f) Temperature-time curve under 638 nm laser irradiation; (g) Comparison of temperature changes of Por-Aminal-COF-3@Fe with three other samples under the same conditions; (c) Por-Aminal-COF-3@Fe (200 μg / mL) under repeated "on-off" laser irradiation (1.5 W / cm²). 2 Cyclic photothermal performance in four cycles; (h) Por-Aminal-COF-3@Fe (200 μg / mL) irradiated at 638 nm (1.5 W / cm²) 2 Photothermal conversion kinetics analysis under (i) continuous light irradiation stability in aquatic environment; (j) UV-vis absorption spectrum of Por-Aminal-COF-3@Fe (200 μg / mL) after storage in aqueous solution at room temperature for up to 20 days; Figure 11(a) Time-dependent UV-vis absorption spectra of pure DPBF at 0, 2, 4, 6, 8, and 10 min under 638 nm laser irradiation; (b) Time-dependent UV-vis absorption spectra of DPBF+Por-Aminal-COF-3@Fe at 0, 2, 4, 6, 8, and 10 min; (c) Time-dependent UV-vis absorption spectra of DPBF+Por-Aminal-COF-3 at 0, 2, 4, 6, 8, and 10 min; (d) Time-dependent UV-vis absorption spectrum of pure MB under laser irradiation; (e) Time-dependent UV-vis absorption spectrum of MB+Por-Aminal-COF-3@Fe under laser irradiation; (f) Time-dependent UV-vis absorption spectrum of MB+Por-Aminal-COF-3 under laser irradiation; (g) Time-dependent PL intensity of DHR123 alone under laser irradiation; (h) (i) Time-dependent PL intensity of DHR123+Por-Aminal-COF-3@Fe under laser irradiation; (j) Normalized degradation efficiency (A / A0) of DPBF (pure), DPBF+Por-Aminal-COF-3@Fe and DPBF+Por-Aminal-COF-3 during 0-10 min laser irradiation; (k) Normalized residual absorbance (A / A0) of MB (pure), MB+Por-Aminal-COF-3@Fe and MB+Por-Aminal-COF-3 during 0-12 min laser irradiation; (l) Normalized PL of DHR123 alone, coexisting with Por-Aminal-COF-3@Fe and coexisting with Por-Aminal-COF-3 during 0-10 min laser irradiation. Intensity variation (I / I0); (m) Production under dark conditions (red curve) and laser irradiation (black curve) 1 ESR spectrum of O2; (n) ESR spectrum of ·OH produced under dark state (red curve) and laser irradiation (black curve); (o) O2 produced under dark state (red curve) and laser irradiation (black curve) •- The ESR spectrum; Figure 12(a) UV-vis absorption spectra of TMB, TMB+H2O2, and TMB+H2O2+Por-Aminal-COF-3@Fe; (b) Effect of 638 nm laser irradiation for 1 min on the catalytic activity of Por-Aminal-COF-3@Fe POD-like compounds; (c) UV-vis absorption spectra of Por-Aminal-COF-3@Fe+H2O2+TMB under different pH conditions; (d) UV-vis absorption spectra of Por-Aminal-COF-3@Fe+TMB+H2O2 at different Por-Aminal-COF-3@Fe concentrations; (e) Time-dependent UV-vis absorption of H2O2; (f) Time-dependent UV-vis absorption of Por-Aminal-COF-3@Fe+H2O2; (g) H2O2 (left, colorless) and H2O2+Por-Aminal-COF-3@Fe Visual comparison; (h) Changes in DO of H2O, H2O2 and Por-Aminal-COF-3@Fe+ H2O2 over time; Figure 13 (a) Photographs of *S. aureus* treated with different concentrations of Por-Aminal-COF-1, Por-Aminal-COF-2, Por-Aminal-COF-3, and Por-Aminal-COF-3@Fe; (b) Survival rates of *S. aureus* treated with different concentrations of Por-Aminal-COF-1, Por-Aminal-COF-2, Por-Aminal-COF-3, and Por-Aminal-COF-3@Fe; (c) Photographs of *E. coli* treated with different concentrations of Por-Aminal-COF-1, Por-Aminal-COF-2, Por-Aminal-COF-3, and Por-Aminal-COF-3@Fe; (d) Photographs of *S. aureus* treated with different concentrations of Por-Aminal-COF-1, Por-Aminal-COF-2, Por-Aminal-COF-3, and Por-Aminal-COF-3@Fe. The corresponding survival rate of E. coli after treatment.

[0019] Figure 14(a) Bacterial colony photographs after treatment with *S. aureus* in each group; (b) Bacterial activity after treatment with *S. aureus* in each group; (c) Bacterial colony photographs after treatment with *E. coli* in each group; (d) Bacterial activity after treatment with *E. coli* in each group. Data are expressed as mean ± standard deviation; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; Figure 15 (a) CV quantification of biofilm quality of Staphylococcus aureus; (b) CV quantification of biofilm quality of Escherichia coli; (c) CLSM 3D images of Staphylococcus aureus and Escherichia coli; (d) Quantitative CLSM fluorescence intensity of Staphylococcus aureus; (e) Quantitative CLSM fluorescence intensity of Escherichia coli; Figure 16 (a) Hemolysis assay of Por-Aminal-COF-3@Fe; (b) Cytotoxicity of 3T3 cells; (c) Area of ​​cell migration under 0 and 200 μg / mL Por-Aminal-COF-3@Fe conditions; (d) Representative images of time-dependent non-migrating areas in scratch wound healing assays under different conditions. Figure 17 (a) Representative agar plate images of bacterial colonies from wound swabs in different mouse groups before and after treatment; (b) Quantitative analysis of the reduction in bacterial load in wounds in different mouse groups after treatment; Figure 18 (a) Representative photographs of wound tissue on days 1, 3, 5, 7 and 9, and wound closure marks during treatment; (b) Wound area during treatment; (c) Changes in mouse body weight during treatment; (d) H&E and Masson trichrome staining of wound tissue sections on day 9; (e) H&E staining of heart, liver, spleen, lung and kidney mouse organs collected from each group 9 days after treatment; Figure 19 TTEP 1 H NMR; Figure 20 3,12-diaza-6,9-diaza-onium dispiro[5.2.5] 9 .2 6 hexadecane dibromide 1 H NMR; Figure 21 3,9-diazaspiro[5.5]undecane chloride 1 H NMR. Detailed Implementation

[0020] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0021] As described in the background section, porphyrins possess visible light absorption and reactive oxygen species (ROS) generation capabilities, but their large planar conjugated structures are prone to π-π stacking, leading to aggregation-induced quenching and reduced photodynamic performance. Immobilizing porphyrins within porous covalent networks can limit molecular aggregation to some extent, but most existing porphyrin frameworks still exhibit strong planarity; relying solely on ordinary flexible linkers makes it difficult to simultaneously achieve efficient disruption of porphyrin coplanar stacking, increase cation charge density, and construct stable multifunctional catalytic sites. Furthermore, the infected microenvironment (IME) has several unfavorable characteristics.

[0022] Based on this, the purpose of this invention is to provide a spirocyclic engineered cationic iron porphyrin porous organic polymer, its preparation method, and its applications. This invention utilizes 5',5''',5'''''',5''''''''''-(21H,23H-porphyrin-5,10,15,20-tetramethyl)tetra([1,1':3',1''-terphenyl]-4,4''-dicarboxaldehyde) and 3,12-diaza-6,9-diaza-onium dispiro[5.2.5] 9 .2 6 Using hexadecanedibromide as the structural unit, porous organic polymers were constructed, and spirocyclic engineered cationic iron porphyrin porous organic polymers (Por-Aminal-COF-3@Fe) were obtained through coordination of iron ions with porphyrins. Figure 1As shown, a comparison between porphyrin polymerization and spirocyclic polymerization (from spirocyclic to monospirocyclic to bispirocyclic) reveals that as spirocyclic units are gradually introduced, the planarity of the framework is gradually disrupted, effectively inhibiting aggregation-induced quenching (ACQ). Subsequent post-synthetic iron metallization yields the cationic Por-Aminal-COF-3@Fe. Notably, this cationic framework not only utilizes typical characteristics of the infection microenvironment (IME) (such as excess H2O2 and acidity) but also reshapes the IME by consuming H2O2 and reducing acidity through tertiary amine protonation. Furthermore, the unique cationic framework enhances affinity for bacterial membranes through electrostatic interactions, further improving therapeutic efficiency. Therefore, this platform integrates three synergistic mechanisms: firstly, it enables efficient photothermal conversion for PTT; secondly, it generates various ROS (singlet oxygen, superoxide anion, and hydroxyl radicals) for PDT; and thirdly, it possesses inherent enzyme-like activity. Specifically, its catalase-like activity can convert H2O2 into O2, relieving local hypoxia and enhancing the efficacy of phototherapy (PDT); its peroxidase-like activity can generate cytotoxic hydroxyl radicals (·OH) from H2O2. By bypassing hypoxia dependence, utilizing local acidosis and elevated H2O2, and reducing non-targeted thermal damage through synergistic effects, this multi-action system overcomes the limitations of single phototherapy.

[0023] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0024] Note: Unless otherwise specified, all experiments were repeated three times. Data are expressed as mean ± standard deviation (SD). Statistical analysis was performed using SPSS 26.0 software. For comparisons among multiple groups, one-way ANOVA was performed followed by Tukey's post-hoc test. A p-value less than 0.05 was considered statistically significant. Unless otherwise specified, the pH of the PBS used is 5.5, and the hydrogen peroxide used is prepared from a 30wt% hydrogen peroxide solution.

[0025] The test materials used in the embodiments of this invention are all conventional test materials in the art and can be purchased through commercial channels.

[0026] Example 1: Preparation of Por-Aminal-COF-3@Fe (1) Synthesis of 5',5''',5''''',5''''''''''-(21H,23H-porphyrin-5,10,15,20-tetramethyl)tetra([1,1':3',1''-terphenyl]-4,4''-dicarboxaldehyde)(TTEP) 3,5-Dibromobenzaldehyde (5.05 g, 19.14 mmol) and propionic acid (142 mL) were added to a 400 mL three-necked flask. The mixture was heated to 142 °C with stirring, and the solution began to reflux. Pyrrole (1.38 mL, 19.97 mmol) was then added dropwise to the reflux solution. After complete addition, the reaction was continued under reflux for 4.5 h. The mixture was cooled to room temperature, washed with hot water, and filtered under vacuum. The resulting crude purple product was dried under vacuum to give octabromoporphyrin.

[0027] A mixture of octabromoporphyrin (4.0 g, 3.21 mmol), 4-formylphenylboronic acid (8.04 g, 53.60 mmol), anhydrous K₂CO₃ (12.01 g, 86.91 mmol), and Pd(PPh₃)₄ (120 mg, 0.1 mmol) in THF / H₂O (300 mL, v / v = 3:1) was purged under nitrogen for 15 min. The reaction was refluxed and stirred at 65 °C for 48 h. After cooling, the organic solvent was removed under reduced pressure. The residue was extracted with dichloromethane, dried over anhydrous Na₂SO₄, and concentrated. The crude product was purified by column chromatography (dichloromethane as eluent) to give TTEP as a purple solid (3.23 g, 2.23 mmol, yield: 69.52%), the 1H NMR spectrum of which is shown below. Figure 19 .

[0028] (2) 3,12-diaza-6,9-diaza-onium dispirol [5.2.5] 9 .2 6 Synthesis of hexadecane dibromide (DSPZ) Piperazine (2.4 g, 27.86 mmol) was dissolved in 10 mL of distilled water. Bis(2-chloroethyl)amine hydrochloride (5.44 g, 30.50 mmol) was dissolved in 40 mL of distilled water. The solution of the latter was added to the piperazine solution, followed by the addition of CaCO3 (2.84 g, 28.39 mmol). The mixture was refluxed at 55 °C for 9 hours. The white precipitate was removed by filtration, and the filtrate was concentrated using a rotary evaporator. The product was recrystallized from ethanol, washed three times with ethanol, and dried under vacuum at 50 °C for 8 hours to give triethylenediamine.

[0029] Triethylenediamine (DABCO, 673.2 mg, 6.00 mmol) was dissolved in a 15 mL THF round-bottom flask in an ice bath. BrCN (318 mg, 3.00 mmol) was added to the solution, and a white solid precipitated immediately. The white solid was collected, washed with a small amount of cold ethanol, and recrystallized from a cold water / anhydrous ethanol mixture (1:1, v / v) to give white crystals (600 mg, yield: 93%), namely 3,12-dicyano-3,6λ. 5 ,9λ 5 ,12-Tetraazabicyclo[5.2.5] 9 .2 6 Hexadecane-6,9-bis(methylene)dibromide.

[0030] 3,12-dicyano-3,6λ 5 ,9λ 5 ,12-Tetraazabicyclo[5.2.5] 9 .2 6 Hexadecane-6,9-bis(methylene)dibromide (4.29 g, 9.80 mmol), 25 mL concentrated hydrochloric acid, and 50 mL water were mixed. The mixture was refluxed at 105 °C for 3.5 hours. After cooling, it was neutralized with saturated sodium bicarbonate solution. The solvent was removed under reduced pressure to give a white crystalline solid (1.4 g, 3.60 mmol, yield: 37.0%), which is DSPZ. Its proton NMR spectrum is shown below. Figure 20 .

[0031] (3) Synthesis of Por-Aminal-COF-3 TTEP (600 mg, 0.41 mmol) and DSPZ (634.1 mg, 1.64 mmol) were added to a polymerization tube using o-xylene as solvent and sonicated at 40 kHz and 180 W for 5 minutes to achieve uniform dispersion. The system was degassed by three freeze-vacuum cycles, then sealed under vacuum and heated at 120 °C for 72 hours. After the reaction was complete, the mixture was cooled to room temperature, and the solid precipitate was collected by centrifugation. The obtained solid was washed three times with tetrahydrofuran, followed by Soxhlet extraction with tetrahydrofuran and dichloromethane for 24 h. Finally, a purple powder was collected and dried under vacuum at 100 °C for 8 h to give 721.50 mg (0.10 mmol, yield: 23.5%) of the product.

[0032] (4) Synthesis of Por-Aminal-COF-3@Fe Por-Aminal-COF-3 (300 mg) and FeCl3·6H2O (76 mg, 0.281 mmol) were added to a sealed glass tube containing a mixture of methanol (10 mL), chloroform (45 mL), and DMF (15 mL). The mixture was heated at 80 °C for 24 hours under vacuum, during which the methanol and chloroform were removed. Water (50 mL) was added to the mixture, gradually forming a brown precipitate. The solid was collected by filtration, thoroughly washed with water, and dried under vacuum at 60 °C for 12 h to obtain Por-Aminal-COF-3@Fe (155 mg).

[0033] Comparative Example 1: Synthesis of Por-Aminal-COF-1 TTEP (600 mg, 0.41 mmol) and piperazine (141.1 mg, 1.64 mmol) were added to a polymerization tube using o-xylene as solvent. The mixture was sonicated for 5 minutes to achieve uniform dispersion. The system was degassed by three freeze-vacuum cycles, then sealed under vacuum and heated at 120 °C for 72 hours. After the reaction was complete, the mixture was cooled to room temperature and the solid precipitate was collected by centrifugation. The obtained solid was washed three times with tetrahydrofuran, followed by Soxhlet extraction with tetrahydrofuran and dichloromethane for 24 hours. The purple powder was collected, then washed three times with warm water, N,N-dimethylformamide, ethanol, and diethyl ether, and finally dried under vacuum at 100 °C for 8 hours to give 510.1 mg (0.19 mmol, yield: 46.4%) of the product.

[0034] Comparative Example 2: Synthesis of Por-Aminal-COF-2 The difference from Comparative Example 1 was that piperazine was replaced with 3,9-diazaspiro[5.5]undecane chloride (631.4 mg, 1.64 mmol). The final product was a purple powder, namely Por-Aminal-COF-2 (602.1 mg, yield: 67.12%).

[0035] 3,9-diazaspiro[5.5]undecane chloride was prepared by the following method: piperazine (2.4 g, 27.86 mmol) was dissolved in 10 mL of distilled water. Bis(2-chloroethyl)amine hydrochloride (5.44 g, 30.50 mmol) was dissolved in 40 mL of distilled water. The latter solution was then added to the piperazine solution, along with calcium carbonate (2.96 g, 29.57 mmol). The mixture was refluxed at 55 °C for 9 hours. A white precipitate was removed by filtration, and the filtrate was concentrated using a rotary evaporator. The product was recrystallized from ethanol, washed three times with ethanol, and then dried under vacuum at 50 °C for 8 hours to obtain 3,9-diazaspiro[5.5]undecane chloride, the 1H NMR spectrum of which is shown below. Figure 21 .

[0036] Example 2: Characterization (1) Using FT-IR ( Figure 2 )and 13 CCP / MAS NMR (Figure 3) was used to investigate the chemical transformations during the construction of Por-Aminal-COF-3@Fe. Similar to Por-Aminal-COF-3, the FT-IR spectrum of Por-Aminal-COF-3@Fe also showed the characteristic peak of TTEP (CN bond in the pyrrole ring at 1380 cm⁻¹). -1 C=C stretching vibration at 1603 cm -1 ) and the characteristic peaks of DSPZ (CN) + Stretching absorption of approximately 1455 cm -1 Simultaneously, the characteristic peak of the C=O stretching vibration from the TTEP aldehyde group (approximately 1703 cm⁻¹) is observed. -1 ) and the NH stretching absorption peak from DSPZ (approximately 3432 cm⁻¹) -1 The intensity of the current has significantly decreased. Instead, it has decreased to approximately 1160 cm. -1 A new vibrational band belonging to the acetal-amine linkage appears at [a specific location], confirming the formation of the polymer backbone. In addition to the characteristic peak of Por-Aminal-COF-3, the Por-Aminal-COF-3@Fe spectrum also shows [a specific value] at approximately 999 cm⁻¹. -1 The presence of Fe-N4 peaks confirms the formation of coordinate bonds. 13CCP / MAS NMR spectroscopy further elucidated the carbon skeleton of Por-Aminal-COF-3@Fe. A distinct resonance peak appeared at 89 ppm, a characteristic signal of the acetal amine carbon (NCN), confirming successful linkage between the porphyrin core and the spirocyclic amine linker. Other resonance peaks in the 10–50 ppm range were attributed to saturated methylene (-CH2-) carbons; the peaks at 50 and 53 ppm corresponded to the carbon environment adjacent to nitrogen in the spirocyclic linker. Resonance peaks at 128.3, 136.4, and 139.9 ppm were attributed to the aromatic carbons of the porphyrin macrocycle. Overall, FT-IR and solid-state spectroscopy... 13 The 13C NMR data provide strong evidence for the stepwise transformation of monomers into extended covalent networks and demonstrate that the final Por-Aminal-COF-3@Fe framework was successfully constructed without compromising covalent integrity.

[0037] (2) By powder X-ray diffraction ( Figure 4 The crystallinity of Por-Aminal-COF-3@Fe was characterized. The obtained spectrum showed only broad and diffuse peaks in the range of 2θ = 20~80°, without the sharp Bragg diffraction peaks characteristic of long-range ordered crystal structures, indicating that the material is mainly amorphous.

[0038] (3) The thermal stability of Por-Aminal-COF-3@Fe was evaluated by thermogravimetric analysis (TGA) under N2 atmosphere, with Por-Aminal-COF-3 as a control. Figure 5 (a) As can be seen, the TGA curves of Por-Aminal-COF-3@Fe show three distinct weight loss stages. Specifically, the initial slight weight loss of approximately 2.0% below 100 °C is attributed to the evaporation of physically adsorbed water. More significant weight loss occurs between 100 and 300 °C, with 95% of the residual mass remaining at 300 °C, indicating the beginning of framework decomposition. Subsequently, the mass decreases slowly until 800 °C, with a final residual mass of 70%. In contrast, Figure 5 (b) Under the same conditions, Por-Aminal-COF-3 retains only 59% at 800 °C. This significant difference indicates that Por-Aminal-COF-3@Fe has superior thermal stability, which is a critical property for applications involving thermal stress.

[0039] (4) Figure 6(a) The porous structure of Por-Aminal-COF-3@Fe was characterized using a 77 K nitrogen (N2) adsorption-desorption isotherm. This isotherm exhibits Type IV behavior with a Type H1 hysteresis loop, a typical characteristic of mesoporous materials. Rapid adsorption at low relative pressures (P / P0 < 0.1) indicates the presence of micropores; while a pronounced hysteresis loop between P / P0 = 0.4 and 0.9 reflects uniform mesopores with a narrow size distribution. Further increases at high relative pressures (P / P0 > 0.9) indicate the presence of macropores or intergranular pores in Por-Aminal-COF-3@Fe. The Brunauer-Emmett-Teller (BET) specific surface area of ​​Por-Aminal-COF-3@Fe is 28.9 m². 2 / g, total pore volume is 0.039 cm³ 3 / g. Figure 6 (b) The pore size distribution curve calculated from the desorption branch shows a primary pore size of 3.2 nm, accompanied by secondary peaks at 14.3 nm and 38.8 nm, indicating that the material consists of a hierarchical pore system of micropores, mesopores, and macropores. This hierarchical structure is beneficial for mass transfer and provides significant advantages for antibacterial applications based on photothermal, photodynamic, and enzyme-catalyzed therapies.

[0040] (5) The microstructure of Por-Aminal-COF-3@Fe was characterized by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Figure 7 (a) ~ Figure 7 (c) shows that the material exhibits a porous aggregate morphology at different magnifications, consisting of irregularly shaped subparticles with interconnected porous channels. Importantly, its surface is significantly rough, which facilitates the formation of a hierarchical porous structure. Figure 7 (d)~ Figure 7 (f) TEM and high-resolution transmission electron microscopy (HR-TEM) imaging further revealed nanoscale structural details, showing the material's fine-grained and layered stacking characteristics. Notably, the TEM images clearly show the material contains abundant pores, including mesopores and micropores. Furthermore, the HRTEM images reveal a well-defined microporous structure through variations in light and dark contrast corresponding to the ordered pores within the framework. The coexistence of mesopores and micropores is particularly advantageous for antibacterial applications. Figure 8 Energy-dispersive X-ray spectroscopy (EDS) and Figure 7 Elemental mapping of (g) confirms that nitrogen (N, 11.53%), iron (Fe, 4.98%), and bromine (Br, 0.87%) are uniformly distributed within a carbon-dominated framework (82.52%). This uniform distribution confirms the successful incorporation of iron into the COF framework and verifies the consistency of the material composition.

[0041] (6) X-ray photoelectron spectroscopy (XPS) was used to further confirm the elemental composition within the framework. Figure 9 The XPS full spectrum of (a) provides an overview of the elemental composition, confirming the presence of carbon (C), nitrogen (N), iron (Fe) and bromine (Br) in the material, consistent with Por-Aminal-COF-3@Fe. Figure 9 (b) The high-resolution XPS C 1s spectrum shows two peaks: 284.81 eV and 287.36 eV, corresponding to C=C / CC and C=NC bonds from the porphyrin ring, respectively. Figure 9 In (c), the N 1s spectrum is fitted with two peaks at 399.02 eV and 400.07 eV, which correspond to C=NC and CNH bonds, respectively. Figure 9 In (d), the high-resolution Fe 2p spectrum splits into two peaks with central energies of 711.72 eV and 724.85 eV, respectively, which are attributed to Fe 2p. 3 / 2 and Fe 2p 1 / 2 Energy levels. Furthermore... Figure 9 As shown in (e), the high-resolution Br 3d spectrum exhibits a bimodal distribution at 70.48 eV and 71.43 eV, corresponding to Br 3d... 5 / 2 With Br3d 3 / 2 track.

[0042] Example 3: Effect on photothermal performance The UV-Vis-NIR absorption spectra of Por-Aminal-COF-3 and Por-Aminal-COF-3@Fe were measured at different concentrations (50-200 µg / mL). Figure 10 (a) and Figure 10 (b) It can be seen that both materials exhibit broad and strong absorption in the 400-800 nm range, indicating strong light-trapping capabilities throughout the visible light region and extending into the near-infrared (NIR) window. The absorption intensity gradually increases with concentration. This positive correlation confirms the excellent dispersibility and stable, consistent optical properties of the COF materials in aqueous media. Notably, under the same conditions, Por-Aminal-COF-3@Fe exhibits enhanced absorption compared to Por-Aminal-COF-3, which may be attributed to the introduction of Fe ions, which can introduce additional electronic transitions or alter the local dielectric environment. The broad absorption characteristics indicate that the materials can efficiently trap photons across multiple wavelength ranges, reduce energy loss, and maximize the potential to convert absorbed light into heat energy, thereby achieving efficient PTT (phototransfer).

[0043] To systematically evaluate the photothermal behavior and highlight the structural advantages introduced by iron, water was used as a control. The sample concentration (50-200 μg / mL) or laser power (0.5-2.0 W / cm²) was varied. 2 The Por-Aminal-COF-3@Fe was investigated under 638 nm laser irradiation. Figure 10 As shown in (c), at 1.5 W / cm 2 Under different laser powers, Por-Aminal-COF-3@Fe exhibits a clear concentration-dependent trend. As the concentration of Por-Aminal-COF-3@Fe increases from 0 to 200 μg / mL, the equilibrium temperature steadily increases. Figure 10 Infrared thermography (d) further visually confirmed the temperature increase. Under 638 nm laser irradiation, Por-Aminal-COF-3@Fe solutions of different concentrations (50-200 μg / mL) exhibited a time-dependent heating process, with higher concentrations producing stronger thermal signals. Conversely, Figure 10 (e) At a fixed concentration (200 μg / mL), the laser power was increased from 0.5 to 2.0 W / cm². 2 It can accelerate the heating process and increase the final temperature.

[0044] To compare the differences in photothermal properties of different COF samples, Figure 10 (f) At a fixed laser power of 1.5 W / cm 2 The temperature rise was examined in detail. As shown in the figure, except for PBS, whose temperature change within 10 min was negligible, Por-Aminal-COF-1, Por-Aminal-COF-2, Por-Aminal-COF-3, and Por-Aminal-COF-3@Fe (150 μg / mL) all exhibited significant temperature increases. Among them, Por-Aminal-COF-3@Fe had the highest final temperature (47.6℃), significantly higher than the other COFs: Por-Aminal-COF-3 at 43.0℃, Por-Aminal-COF-2 at 38.4℃, and Por-Aminal-COF-1 at 33.7℃. This superior performance of Por-Aminal-COF-3@Fe stems from the fact that the introduced Fe ions provide additional electronic transition and non-radiative decay pathways, thereby more efficiently converting absorbed light into heat. Therefore, the introduction of Fe not only enhances the photothermal conversion efficiency of the COF framework, but also makes it significantly different from other COF samples, indicating that Por-Aminal-COF-3@Fe is the most effective photothermal agent among the tested materials.

[0045] also, Figure 10 The cyclic stability test (g) further demonstrated the robustness of Por-Aminal-COF-3@Fe. This material (200 μg / mL) underwent repeated 10-min on / off cycles under 638 nm laser irradiation, and consistent temperature profiles were obtained for four consecutive cycles. The photothermal conversion efficiency (η) of Por-Aminal-COF-3@Fe was quantitatively evaluated and calculated according to the formula disclosed in patent application number CN115845086A. Figure 10 (h) Temperature decay curve and heat transfer model, time constant τs is 179.73 s (R2=0.986), under 638 nm laser irradiation (1.5 W / cm²). 2 The η value reaches 69.82%. Combined with its concentration-dependent temperature rise, cycling stability, and photostability, this high η value indicates that Por-Aminal-COF-3@Fe is a highly efficient and durable photothermal agent.

[0046] In addition to photothermal properties, its practicality was evaluated by monitoring the UV-Vis spectral changes of Por-Aminal-COF-3@Fe under continuous laser exposure and long-term aqueous storage conditions. Photostability was compared by 10 min irradiation (638 nm, 1.5 W / cm²). 2 The UV-Vis spectra before and after were evaluated. For example... Figure 10 As shown in (i), the absorption spectrum hardly changes throughout the time period. Figure 10 (j) More significant stability was observed in aqueous media. UV-Vis spectra of the material after immersion in water for 0, 5, 10, 15, and 20 days showed that the absorption curves at all time points almost completely overlapped in the 400-800 nm range, indicating that its optical properties remained largely unchanged. Therefore, Por-Aminal-COF-3@Fe maintains its light absorption capacity and structural integrity in water, confirming its excellent water stability. Overall, these results demonstrate that Por-Aminal-COF-3@Fe maintains its photothermal activity under both light and aqueous conditions. This robust optical behavior supports the material's potential as a high-performance photothermal agent, generating localized high heat under laser irradiation for efficient bacterial removal.

[0047] Example 4: Photodynamic Activity To evaluate the potential of Por-Aminal-COF-3@Fe as a photodynamic therapy (PDT) agent, specific molecular probes were used for detection, including those for detecting singlet oxygen (SO4). 11,3-Diphenylisobenzofuran (DPBF) for detecting O2, methylene blue (MB) for detecting hydroxyl radicals (·OH), and superoxide anion (O2) for detecting O2 •- The dihydrorhodamine 123 (DHR123). Figure 11 (a) It can be seen that, in stark contrast to the pure DPBF probe, there is almost no spectral change within 0-10 min. Figure 11 (b) Por-Aminal-COF-3@Fe and Figure 11 (c) In Por-Aminal-COF-3, the peak at approximately 450 nm in the absorption spectrum is significantly reduced, especially in Por-Aminal-COF-3@Fe. This strong spectral response indicates that Por-Aminal-COF-3@Fe can efficiently generate… 1 O2. Figure 11 (d) shows that the absorption peak of MB at 664 nm remained almost unchanged within 1–10 min in the absence of material, confirming that its self-degradation or aggregation is negligible. However, Figure 11 (e) shows that the peak intensity gradually decreases and broadens in the presence of Por-Aminal-COF-3@Fe. Figure 11 (f) Por-Aminal-COF-3 also showed a similar downward trend, but the adsorption rate and extent were significantly lower in the first 2 min. Figure 11 (g)~ Figure 11 (i) shows the fluorescence intensity of DHR123 (peak at approximately 540 nm) with and without a catalyst. The intensity of DHR123 remained almost constant at all time points, indicating that O2 was present without an active photocatalyst. •- The formation was negligible; however, the addition of Por-Aminal-COF-3@Fe resulted in a significant and time-dependent increase in fluorescence intensity. This behavior is similar to that of DHR123 and O2. •- The reaction and oxidation to the highly fluorescent rhodamine 123 followed the same process. The fluorescence intensity of Por-Aminal-COF-3 also increased over time, but under the same conditions, the increase was significantly smaller than that of Por-Aminal-COF-3@Fe. These results indicate that Por-Aminal-COF-3@Fe is a highly efficient dual-mechanism photosensitizer. Figure 11 (j)~ Figure 11 (l) Further evidence demonstrates that Por-Aminal-COF-3@Fe exhibits advantages in both Type I and Type II PDT. Por-Aminal-COF-3@Fe's... 1The O2 generation rate is approximately 1.2 times that of Por-Aminal-COF-3, while the O2 generation rate of the DPBF control is significantly lower. 1 O2 is negligible. Therefore, iron functionalization significantly enhances type II ROS generation. Compared to the pure probe with almost unchanged absorbance, the addition of Por-Aminal-COF-3@Fe resulted in a loss of approximately 90% of the original intensity of MB after 10 min, similar to Por-Aminal-COF-3. The difference was particularly pronounced in the first 2 min, with Por-Aminal-COF-3@Fe removing approximately 66% of MB, while Por-Aminal-COF-3 removed approximately 33%. Simultaneously, in the Por-Aminal-COF-3@Fe system, the DHR123 fluorescence intensity increased by over 1000% after 10 min (a 3.25-fold increase relative to Por-Aminal-COF-3). Por-Aminal-COF-3@Fe exhibits superior ROS generation capability and overall photodynamic performance.

[0048] In addition, electron paramagnetic resonance (EPR) testing was used to directly identify reactive oxygen species (ROS) generated under laser irradiation. Figure 11 (m)~ Figure 11 (o). Under illumination, Por-Aminal-COF-3@Fe can be clearly detected. 1 O2, ·OH and O2 •- The characteristic signals of the material were observed, while no such signals were observed in the control group (no material). These results further confirm that the material can generate multiple ROS under laser irradiation. The combined EPR and molecular probe results confirm that Por-Aminal-COF-3@Fe exhibits mixed type I / II photodynamic activity.

[0049] Example 5: POD-like and CAT-like activity To evaluate the enzyme-like catalytic performance of Por-Aminal-COF-3@Fe, its peroxidase-like (POD) and catalase-like (CAT) activities were studied using standard colorimetric methods and oxygen monitoring. POD-like activity was evaluated using a two-substrate system consisting of TMB (3,3′,5,5′-tetramethylbenzidine) and H₂O₂, and the reaction progress was tracked using UV-vis absorption spectroscopy. Figure 12As shown in (a), no obvious absorption peak was observed in TMB alone or in the TMB + H2O2 mixture. Conversely, the addition of Por-Aminal-COF-3@Fe resulted in a distinct characteristic absorption band at approximately 652 nm, accompanied by a color change from colorless to blue, confirming that TMB was oxidized to oxTMB. This result indicates that Por-Aminal-COF-3@Fe can efficiently catalyze H2O2 activation and exhibits significant POD-like activity. To further investigate the effect of laser irradiation on this catalytic performance, the activity with and without 638 nm laser irradiation was compared (see...). Figure 12 (b) After 1 min of irradiation, the absorbance at 652 nm was significantly higher than that of the unirradiated group, indicating that irradiation enhanced the POD-like catalytic activity of Por-Aminal-COF-3@Fe. This enhancement may be due to local photothermal effects and improved electron transfer, both of which promote the activation of H2O2 on the iron active sites. Figure 12 (c) shows the pH dependence of the peroxidase-like activity, which exhibits significant pH sensitivity, with the strongest absorption at 652 nm at pH 4.5. Por-Aminal-COF-3@Fe also shows significant catalytic activity in an IME-like acidic environment (pH 5.5). Meanwhile, Figure 12(d) shows that the POD-like activity of Por-Aminal-COF-3@Fe is also concentration-dependent. As the concentration of Por-Aminal-COF-3@Fe increases from 50 μg / mL to 200 μg / mL, the absorbance at 652 nm gradually increases, confirming its good concentration-dependent catalytic behavior.

[0050] Subsequently, the ability of Por-Aminal-COF-3@Fe to decompose H2O2 into water and molecular oxygen (CAT simulated activity) was evaluated. See Figure 12(e) and... Figure 12 As shown in (f), in stark contrast to the pure H2O2 curve, the UV-vis absorption at 240 nm steadily decreased over time after the addition of Por-Aminal-COF-3@Fe, confirming the consumption of H2O2. Furthermore, Figure 12(g) shows that rapid bubble formation was observed after adding Por-Aminal-COF-3@Fe to the H2O2 solution (pH 7.4), indicating O2 production; while under the same conditions, no bubbles appeared in the H2O2 solution without the material. Figure 12(h) Dissolved oxygen (DO) measurements using a portable oxygen meter revealed that in the presence of Por-Aminal-COF-3@Fe, the DO signal rapidly increased within the first 2–5 minutes, subsequently plateauing as H2O2 was consumed. Meanwhile, the DO level in the control group (H2O2 only) remained constant throughout the observation period. These results collectively indicate that Por-Aminal-COF-3@Fe possesses inherent CAT-like activity and can be used in combination with photodynamic and enzymatic therapy to alleviate hypoxia.

[0051] Example 6: In vitro antibacterial effect Staphylococcus aureus and Escherichia coli were cultured in LB medium at 37°C and 110 rpm until mid-log phase. The bacterial concentration was adjusted to 1×10⁻⁶ using PBS. 8 CFU / mL (OD600 ≈ 0.1).

[0052] (1) Bacterial suspension (100 µL, containing a total bacterial count of 1×10⁻⁶) 7 CFU was mixed with different concentrations (0–200 µg / mL) of Por-Aminal-COF-1, Por-Aminal-COF-2, Por-Aminal-COF-3, and Por-Aminal-COF-3@Fe in PBS (total volume 1 mL), and H2O was added. 2， The final H₂O₂ concentration was maintained at 10 mM. The mixture was subjected to a 638 nm laser (1.5 W / cm²). 2 Irradiate for 10 min. Then, spread 50 µL of the mixture (appropriately diluted) onto LB agar plates. After incubation at 37°C for 24 h, count the number of colony-forming units (CFU). Calculate the sterilization efficiency.

[0053] like Figure 13 (a) and Figure 13 As shown in (c), under laser irradiation (1.5 W / cm²), 2In the presence of ) and H₂O₂ (final concentration 10 mM), as the concentration increases (0, 50, 100, 150 and 200 μg / mL), Por-Aminal-COF-1, Por-Aminal-COF-2, Por-Aminal-COF-3, and Por-Aminal-COF-3@Fe all exhibit bactericidal effects. In contrast, the PBS group showed dense, fused colonies at all concentrations, confirming that the antibacterial effect is attributed to COFs. However, under the same conditions, the bacterial survival rates of different treatment groups vary greatly. Among the four COFs, the antibacterial efficacy follows a consistent order: Por-Aminal-COF-1 < Por-Aminal-COF-2 < Por-Aminal-COF-3 < Por-Aminal-COF-3@Fe, and this hierarchical structure is correlated with structural / functional differences. Initially, the gradual increase in photothermal efficiency and ROS production drives the enhanced antibacterial effect. Fe functionalization introduces an additional antibacterial mechanism to amplify ROS production, which synergizes with the photothermal / ROS effect to achieve superior performance. At 150 μg / mL, Por-Aminal-COF-3@Fe only produces a small number of residual colonies, while other COFs still show significant growth. Figure 13 (b) and Figure 13 The quantitative results of (d) confirm that, with the increase of concentration and material efficacy, the colony density decreases, and Por-Aminal-COF-3@Fe produces the least number of colonies under the same conditions. The bacterial survival status is consistent with visual observation: Por-Aminal-COF-3@Fe achieves >99% CFU reduction against both strains at 150 μg / mL. In contrast, even at 200 μg / mL, Por-Aminal-COF-3 still has a survival rate of about 30%, Por-Aminal-COF-2 about 55%, and Por-Aminal-COF-1 about 64%. This assessment indicates that the COF-based photothermal-ROS antibacterial system has concentration dependence, tunability and Fe-enhanced efficacy.

[0054] (2) The experiment was divided into 8 groups: no laser group and laser group. The no laser group included: PBS group (I-no laser), H2O2 group (II-no laser), Por-Aminal-COF-3@Fe group (III-no laser), Por-Aminal-COF-3@Fe+ H2O2 group (IV-no laser); the laser group included: PBS+laser group (I-laser), H2O2+laser group (II-laser), Por-Aminal-COF-3@Fe+laser group (III-laser), Por-Aminal-COF-3@Fe+ H2O2+laser group (IV-laser). In the corresponding groups, the pH of PBS was 5.5, the final concentration of Por-Aminal-COF-3@Fe was 150 µg / mL, and the final concentration of H2O2 was 10 mM. Bacterial suspension (100 µL, 1×10 7 CFU was mixed with the above eight groups (total volume 1 mL). The laser group used a 638 nm laser (1.5 W / cm²). 2 The group was irradiated for 10 min, while the group without laser irradiation was not irradiated. Then, 50 µL of the mixture (appropriately diluted) was spread onto LB agar plates. After incubation at 37°C for 24 h, the number of colony-forming units (CFU) was counted, and the bactericidal efficiency of each group was calculated.

[0055] like Figure 14 (a) and Figure 14 As shown in (c), the bactericidal effect of laser alone on *S. aureus* and *E. coli* was limited, as the PBS group showed virtually no change after irradiation. H2O2 alone had only a slight antibacterial effect, with little improvement after laser irradiation. In contrast, the Por-Aminal-COF-3@Fe group showed a significant reduction in viability after laser irradiation, with the Por-Aminal-COF-3@Fe + H2O2 + laser group showing the most significant decrease, with colonies almost completely eliminated. Figure 14 (b) and Figure 14Quantitative results in (d) showed that the combination of Por-Aminal-COF-3@Fe and laser resulted in a survival rate of 5.01±0.23% for *S. aureus* and 4.89±0.32% for *E. coli*. In contrast, without laser, Por-Aminal-COF-3@Fe+H2O2 produced survival rates of 81.01±1.11% and 76.35±1.57%, respectively, while the survival rates of Por-Aminal-COF-3@Fe+H2O2+laser were 4.31±0.23% and 3.89±0.32%, respectively. Overall, Por-Aminal-COF-3@Fe exhibited broad-spectrum activity against both Gram-positive and Gram-negative bacteria. Its superior performance stems from a synergistic effect of multiple mechanisms: laser-triggered photothermal and ROS effects directly damage bacterial membranes and biomolecules, while Fe promotes the conversion of H2O2 to enhance oxidative stress. Furthermore, the cationic nature of the material enhances electrostatic adsorption to the negatively charged bacterial surface, promoting membrane disruption and improving contact-dependent therapeutic effects. This triple-mode synergistic effect (photothermal / photodynamic / catalytic ROS) combined with cationic electrostatic interactions enables more thorough bacterial inactivation.

[0056] Example 7: Biofilm removal (1) Crystal violet staining Biofilms of *S. aureus* and *E. coli* were synthesized in 96-well plates by adding 150 µL of bacterial suspension (approximately 1 × 10⁻⁶) to each well. 7 CFU / mL was cultured at 37°C for 24 hours to form biofilms. After washing with PBS to remove planktonic cells, the pre-formed biofilms were subjected to laser irradiation (1.5 W / cm²) in the following groups: I) PBS group, II) Por-Aminal-COF-3@Fe group, III) Por-Aminal-COF-3@Fe+H₂O₂ group, IV) Por-Aminal-COF-3@Fe+laser group, and V) Por-Aminal-COF-3@Fe+H₂O₂+laser group. 2 The biofilm was treated with Por-Aminal-COF-3@Fe (150 µg / mL) and H2O2 (final concentration 10 mM). After a further 24 hours of cultivation, the biofilm was washed, fixed in 4% paraformaldehyde, stained with 0.1% CV for 30 min, washed, and dissolved in 33% acetic acid. The absorbance at 590 nm (OD590) was measured to quantify the total biofilm biomass.

[0057] from Figure 15(a) As can be seen, with PBS group (Group I) as 100% baseline, the mass of Staphylococcus aureus biofilm in Group II (Por-Aminal-COF-3@Fe alone) and Group III (Por-Aminal-COF-3@Fe + H2O2) decreased to 78.01 ± 3.56% and 72.22 ± 2.43%, respectively. Similarly, the mass of Escherichia coli biofilm decreased to 79.67 ± 2.60% and 74.71 ± 1.45%, respectively. These small reductions indicate that, without photoactivation, the material itself, or even together with H2O2, can only partially weaken the biofilm, highlighting the necessity of external phototriggering to unleash the full potential of the system. In contrast, under laser irradiation, group IV (Por-Aminal-COF-3@Fe+ laser) resulted in a sharp decrease in biofilm mass to 25.17±5.83% (Staphylococcus aureus) and 30.60±1.74% (Escherichia coli). This significant improvement directly stemmed from the laser-triggered photothermal and photodynamic effects. Notably, the Por-Aminal-COF-3@Fe + H2O2+ laser group further reduced biofilm mass to 21.24±0.92% and 24.66±1.27%, respectively, demonstrating the strongest cleansing effect.

[0058] (2) Confocal laser scanning microscope (CLSM) The biofilm grew on a glass coverslip, treated in the same manner as in Example (1), stained with SYTO 9 and PI, and imaged using a Leica TCS SP8 confocal microscope. Z-stack images were acquired, and biofilm thickness and bacterial viability were analyzed.

[0059] The CLSM results were highly consistent with CV quantification. For example... Figure 15 (c)~ Figure 15 As shown in (e), Group I exhibited a continuous, dense green fluorescent layer, indicating that the biofilm was intact. Groups II and III showed only slight fluorescence decay and localized structural relaxation, confirming that dark-conditioning was insufficient to remove established biofilms. Group IV showed extensive fluorescence decay and significant biofilm collapse, directly demonstrating that photoactivation is the key switch for potent anti-biofilm activity. Finally, Group V showed the weakest green signal, the least residual biofilm, and almost complete loss of three-dimensional structure. The average fluorescence intensity, in the order of I>II>III>IV>V, further demonstrates the superior biofilm ablation ability of the triple treatment.

[0060] Example 8: Biocompatibility (1) Hemolysis test Female BALB / c mice (5 weeks old, purchased from Shandong Pengyue Experimental Animal Technology Co., Ltd.) were used. Fresh whole blood from BALB / c mice was collected and centrifuged to separate red blood cells (RBCs). The RBCs were washed with PBS and resuspended to a 4% (v / v) suspension. Different concentrations of Por-Aminal-COF-3@Fe (0–300 µg / mL) were mixed with the RBC suspension and incubated at 37 °C for 3 hours. After centrifugation, the absorbance of the supernatant was measured at 540 nm. PBS and deionized water were used as negative and positive controls, respectively. Hemolysis rate is calculated using the following formula: Hemolysis rate (%) = (A-An) / (Ap-An) × 100%; where "A" refers to the absorbance obtained from the supernatant after adding Por-Aminal-COF-3@Fe to red blood cells; "An" refers to the absorbance obtained from the supernatant after adding PBS to red blood cells (negative control); and "Ap" represents the absorbance obtained from the supernatant after adding distilled water to red blood cells (positive control).

[0061] like Figure 16 As shown in (a), the supernatant treated with Por-Aminal-COF-3@Fe remained largely clear, with visible erythrocyte sedimentation, while the positive control group exhibited a distinctly red supernatant due to complete hemolysis. Quantitative analysis revealed that the hemolysis rate was well below 2% across a concentration range of 50–300 μg / mL, showing only a slight upward trend, indicating a weak concentration-dependent interaction with the erythrocyte membrane. Importantly, even at the highest concentration (300 μg / mL), the hemolysis induced by Por-Aminal-COF-3@Fe was only slight (1.36 ± 0.079%). Since all measured hemolysis rates were well below the generally accepted biosafety threshold of 5%, the disruptive effect of Por-Aminal-COF-3@Fe on the erythrocyte membrane over a wide concentration range is negligible, demonstrating excellent in vitro blood compatibility.

[0062] (2) Cytotoxicity assay (MTT) Mouse embryonic fibroblasts (NIH / 3T3, purchased from the Shanghai Cell Bank, Chinese Academy of Sciences) were used. Cells were seeded in 96-well plates at a density of 5 × 10⁶ cells / well. 3 Cells were cultured at 100 cells / well for 24 hours. Then, cells were treated with different concentrations of Por-Aminal-COF-3@Fe (0–250 µg / mL) for 24 hours. MTT solution (5 mg / mL, dissolved in PBS) was added to each well, and incubation was performed for 4 hours. Formazan crystals formed were dissolved in DMSO, and absorbance was measured at 490 nm. Cell viability is expressed as a percentage relative to the untreated control group.

[0063] Figure 16(b) shows that the viability remained high at all tested concentrations, indicating that Por-Aminal-COF-3@Fe exhibits weak cytotoxicity. Notably, even at a relatively high concentration of 200 μg / mL, cell viability reached 88.57 ± 9.11%, indicating no significant cytotoxicity. This high viability can be attributed to the material's stable structure and low acute irritation. These properties enable it to maintain good cell tolerance even at higher concentrations.

[0064] (3) Cell migration (scratch wound) experiment NIH / 3T3 cells were seeded in 6-well plates and grown to near confluence. Straight scratches were created using the tip of a sterile 200 µL pipette. Exfoliated cells were washed with PBS and then added to fresh serum-free medium containing Por-Aminal-COF-3@Fe (0 or 200 µg / mL). Scratch images were captured at 0, 12, and 24 hours using an inverted microscope. Wound healing area was quantified using ImageJ software.

[0065] Quantitative analysis in Figures 16(c) and 16(d) showed that at 12 hours, the cell migration rates in the control group (0 µg / mL) and the treatment group (200 µg / mL) were 32.69 ± 8.20% and 31.99 ± 5.33%, respectively, essentially the same. At 24 hours, the migration rates increased to 62.67 ± 4.85% (control group) and 57.32 ± 1.52% (treatment group), respectively. Although the migration rate in the treatment group was slightly lower at 24 hours, the difference between the groups was small, and both groups showed similar time-dependent enhancement of migration. These results confirm that Por-Aminal-COF-3@Fe does not significantly inhibit 3T3 cell migration, indicating no adverse effects on the normal growth and repair function of healthy cells.

[0066] Example 9: Repair of infected wounds Female Kunming mice (5 weeks old) were anesthetized with sodium pentobarbital (40 mg / kg, intraperitoneal injection). Hair was removed from the back of the mice, and the skin was disinfected. A full-thickness circular wound (6 mm in diameter) was created on the back of each mouse using a sterile biopsy needle. The wound was inoculated with 20 µL of Staphylococcus aureus suspension (1 × 10⁻⁶). 8Mice were placed in eight groups (n=5 per group) for 24 hours to establish infection. The groups were randomly assigned to: (I) PBS, (II) H2O2, (III) Por-Aminal-COF-3, (IV) Por-Aminal-COF-3 + laser, (V) Por-Aminal-COF-3@Fe, (VI) Por-Aminal-COF-3@Fe + H2O2, (VII) Por-Aminal-COF-3@Fe + laser, and (VIII) Por-Aminal-COF-3@Fe + H2O2 + laser. 50 µL of each solution (within the corresponding group, the pH of PBS was 5.5, the final concentration of Por-Aminal-COF-3 and Por-Aminal-COF-3@Fe was 150 µg / mL, and the final concentration of H2O2 was 10 mM) was applied topically to the wound at days 1, 3, 5, and 7. For the laser group, the wound was immediately treated with a 638 nm laser (1.5 W / cm²) after application. 2 Irradiation was performed for 10 minutes. Wound area photographs were taken at days 1, 3, 5, 7, and 9, and wound closure rate was calculated using ImageJ software. Mouse body weight was recorded throughout the experiment.

[0067] like Figure 17 As shown, wound swabs collected on days 1 and 9 were used to detect bacterial colony growth in the wound. On day 1, nearly equal numbers of bacterial colonies were observed in all groups, confirming the consistency of the initial infection. By day 9, the bacterial load varied considerably among the treatment groups. The group using Por-Aminal-COF-3@Fe+H2O2+laser showed the most significant reduction in bacterial colonies, indicating strong synergistic antibacterial activity. Quantitative analysis of residual bacterial activity (100% relative to the control group, group I) showed that group VIII (Por-Aminal-COF-3@Fe+H2O2+laser) had the lowest bacterial activity (approximately 3.4%) among the eight treatment groups, indicating that this combination had the strongest antibacterial effect.

[0068] As shown in Figure 18(a), the slowest wound contraction rate was observed in the PBS and H2O2 monotherapy groups, accompanied by persistent eschar formation and residual wound. Treatment with Por-Aminal-COF-3 monotherapy showed some improvement, and its effect was further enhanced by laser irradiation. Better results were observed with Por-Aminal-COF-3@Fe in combination with laser, but group VIII showed the fastest and most significant wound closure, with the wound almost completely closed by day 9. The color-coded wound contour visualization in Figure 18(a) illustrates the progressive wound contraction, with group VIII showing the fastest and most extensive closure. The wound area reduction kinetics, as shown in Figure 18(b), were quantitatively confirmed by monitoring the percentage of residual wound area throughout the treatment period. Group VIII showed rapid wound area reduction, leaving only 8.64% of the original wound area by day 9, corresponding to a closure rate of 91.36%. Figure 18(c) shows that body weight measurements during day 9 indicate that all groups gradually increased their weight and no signs of treatment-related toxicity (such as weight loss or growth retardation) were observed, indicating that these therapies are well biocompatible and tolerable in vivo.

[0069] On day 9, the mice were euthanized. Blood samples were collected for hematological analysis (complete blood cell count). Wound tissue and major organs (heart, liver, spleen, lung, and kidney) were collected, fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) and Masson's trichrome for histological examination.

[0070] Figure 18(d) shows hematoxylin and eosin (H&E) staining of wound tissue. The PBS and H2O2 groups exhibited significant tissue damage, poor epidermal continuity, inflammatory cell infiltration, and residual scabs. The Por-Aminal-COF-3 and Por-Aminal-COF-3+ laser groups showed moderate improvement and partial re-epithelialization, while the Por-Aminal-COF-3@Fe group showed superior tissue repair. The Por-Aminal-COF-3@Fe+H2O2+ laser group achieved the most intact epidermal structure, minimal inflammation, and the closest to normal tissue morphology. Masson trichrome staining further confirmed that this group exhibited the richest and best-arranged collagen deposition, indicating effective promotion of wound reconstruction and collagen regeneration.

[0071] To systematically assess in vivo biocompatibility, major organs were collected on day 9 of treatment and examined using H&E staining. All organs maintained intact structure, with no obvious pathological abnormalities, necrosis, or significant inflammation, indicating that no treatment-induced organ toxicity occurred under experimental conditions (Figure 18(e)).

[0072] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A spirocyclic engineered cationic iron porphyrin porous organic polymer, characterized in that, Using octaaldehyde porphyrin coordinated with iron ions and bispirocyclic cationic diamine bromide as structural units, an amorphous organic polymer with a porous structure is constructed by forming acetal-amine bonds between the aldehyde group of the octaaldehyde porphyrin and the secondary amine group of the spirocyclic cationic diamine. This is the spirocyclic engineered cationic iron porphyrin porous organic polymer.

2. The spirocyclic engineered cationic iron porphyrin porous organic polymer according to claim 1, characterized in that, The bispirocyclic cationic diamine is a bispirocyclic dicationic diamine dibromide; the structural formula of the octaaldehyde porphyrin is: The acetal-amine bond is an NCN acetal-amine bond.

3. The spirocyclic engineered cationic iron porphyrin porous organic polymer according to claim 2, characterized in that, The bispirocyclic dicationic diamine dibromide is 3,12-diaza-6,9-diaza-onium bispiro[5.2.5]. 9 .2 6 Hexadecane dibromide, its structural formula is: 。 4. The spirocyclic engineered cationic iron porphyrin porous organic polymer according to claim 1, characterized in that, Its structural formula is: 。 5. The method for preparing the spirocyclic engineered cationic iron porphyrin porous organic polymer according to any one of claims 1 to 4, characterized in that, Includes the following steps: (1) Combine octaaldehyde porphyrin and 3,12-diaza-6,9-diaza-onium dispirol [5.2.5] 9 .2 6 Hexadecane dibromide was added to o-xylene, mixed evenly, and then subjected to a solvothermal reaction in a vacuum-sealed environment. After cooling, centrifugation, washing, and drying were performed to obtain a spirocyclic engineered cationic porphyrin porous organic polymer. (2) The spirocyclic engineered cationic porphyrin porous organic polymer and iron salt were added to a mixed solvent and heated under vacuum. The resulting mixture was added to water to form a brown precipitate. The precipitate was filtered, washed and dried to obtain the spirocyclic engineered cationic iron porphyrin porous organic polymer.

6. The preparation method according to claim 5, characterized in that, In step (1), the octaaldehyde porphyrin and 3,12-diaza-6,9-diaza-onium dispiro[5.2.5] 9 .2 6 The molar ratio of hexadecane dibromide is 1:(4-7); the mixing is homogenized by ultrasonic treatment for 5 min; the temperature of the solvothermal reaction is 110~130℃ and the time is 3 days.

7. The preparation method according to claim 5, characterized in that, In step (1), the 3,12-diaza-6,9-diaza-onium dispirol [5.2.5] 9 .2 6 Hexadecane dibromide is prepared by the following method: S1. Dissolve triethylenediamine in tetrahydrofuran in an ice bath, add cyanogen bromide, collect the precipitated white solid, wash, and recrystallize in an alcohol-water mixture to obtain white crystals, namely 3,12-dicyano-3,6λ. 5 ,9λ 5 ,12-Tetraazabicyclo[5.2.5] 9 .2 6 Hexadecane-6,9-bis(methylene)dibromide; S2. 3,12-dicyano-3,6λ 5 ,9λ 5 ,12-Tetraazabicyclo[5.2.5] 9 .2 6 Hexadecane-6,9-bis(methylene) dibromide was mixed with concentrated hydrochloric acid and water, and the mixture was refluxed. After cooling, it was neutralized with saturated sodium bicarbonate solution, and the solvent was removed under reduced pressure to obtain a white crystalline solid, which is 3,12-diaza-6,9-diaza-onium dispirol [5.2.5]. 9 .2 6 Hexadecane dibromide.

8. The preparation method according to claim 5, characterized in that, In step (2), the iron salt is FeCl3·6H2O; the mass ratio of the spirocyclic engineered cationic porphyrin porous organic polymer to the iron salt is 75:19; the heating treatment temperature is 80℃ and the time is 24h.

9. The use of the spirocyclic engineered cationic iron porphyrin porous organic polymer according to any one of claims 1 to 4 in the preparation of antibacterial drugs.

10. The application according to claim 9, characterized in that, The spirocyclic engineered cationic iron porphyrin porous organic polymer exhibits photothermal activity, photodynamic activity, and enzyme-like activity; the enzyme-like activity includes POD-like activity and CAT-like activity.

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