A kind of iron phthalocyanine-based isomeric conjugated microporous polymer with three enzyme mimicking activities and its preparation method and application

Abstract

The invention discloses an iron phthalocyanine-based isomeric conjugated microporous polymer with three enzyme simulation activities and a preparation method and application thereof, and belongs to the field of biomedical technology. An s-triazine containing a thiophthalonitrile group and a metal salt are added to quinoline for a solvothermal reaction to obtain an iron phthalocyanine-based isomeric conjugated microporous polymer with three enzyme simulation activities. The s-triazine containing a thiophthalonitrile group is 2,4,6-(3-thiophthalonitrile)-triazine or 2,4,6-(4-thiophthalonitrile)-triazine. The polymer prepared by the present invention enhances the spatial distortion due to the isomerization effect, improves the antibacterial property, and is used as a biomimetic cascade intelligent phototherapy agent for a bio-responsive self-oxygen supply system for the treatment of infected wounds, realizing photothermal / photodynamic / POD / CAT / OXD cascade synergistic treatment, and significantly accelerating the healing of open bacterial infection wounds.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to an iron phthalocyanine-based isomeric conjugated microporous polymer with three enzyme mimicking activities, and a preparation method and application thereof. Background Art

[0002] Pathogenic microbial infections, especially multidrug-resistant bacterial infections, pose an unprecedented threat to the ecological balance and have brought a huge impact on the clinical and medical systems. In addition to drug resistance, the overuse and abuse of antibiotics also favor the formation of bacterial biofilms, further exacerbating the difficulty of treatment while increasing morbidity and mortality. At the same time, compared with the normal physiological environment, the infectious microenvironment (IME) shows obvious specificity, which is characterized by hypoxia, pathological acidity, and overexpression of H2O2. Therefore, it is urgent to develop antibiotic-free antibacterial comprehensive treatment strategies, which can not only solve the problem of bacterial resistance, but also effectively regulate the infection microenvironment.

[0003] At present, bacterial inactivation methods based on reactive oxygen species (ROS) have attracted extensive research attention due to their non-invasive, strong therapeutic specificity, and low induction of drug resistance. In general, peroxidase-like (POD) is the most typical enzyme therapy, which is achieved by selectively converting low-toxic hydrogen peroxide (H2O2) into highly toxic hydroxyl radicals (·OH) in pathological acidic microenvironments. Although exogenous H2O2 (0.5-3.0%) can significantly improve the bactericidal effect, it will inevitably cause damage to normal cells and tissues. Similarly, as an oxygen-dependent treatment, the practical application of PDT also faces the challenge of hypoxia caused by severe damage to blood vessels in IME. At the same time, the commonly used organic photosensitizers (PSs) also face a major challenge, which is the severe quenching of photoactivity caused by the intrinsic self-aggregation of their large conjugated structures. In addition, hypoxic IME is also conducive to bacterial growth, thereby greatly delaying the wound healing process. If a prospective therapeutic platform that can provide O2 and H2O2 on its own is designed, the limitations of IME can be overcome.

[0004] After decades of development, there are only a handful of materials that can provide both O2 and H2O2, such as metal peroxides and natural enzymes. However, metal peroxides, such as calcium peroxide (CaO2), are unstable and easily decomposed under environmental conditions. Conjugated microporous polymers (CMPs) are a booming artificial multifunctional material with a structure similar to that of biological macromolecules, inherent biocompatibility and structural toughness, and have been successfully applied in the fields of environment, energy, catalysis, and biomedicine. Fully conjugated CMPs can not only improve light absorption, but also broaden the light absorption range, enhance photothermal and photodynamic activity. Therefore, it is necessary to design a fully conjugated CMP that can not only achieve synergistic and amplified therapeutic effects, but also play a pH-sensitive antibacterial and anti-biofilm effect to accelerate the treatment of infected wounds; thus, a pH-responsive therapeutic agent that can simultaneously produce ROS, H2O2, and O2 is obtained, paving the way for rapid treatment of infected wounds. Summary of the invention

[0005] In view of the above-mentioned prior art, the purpose of the present invention is to provide an iron phthalocyanine-based isomeric conjugated microporous polymer with three-enzyme mimetic activity and its preparation method and application. The present invention uses thiophthalonitrile-isotriazine compounds to self-polymerize cyano groups and insert metal modification to obtain metal isomeric conjugated microporous polymers with good photothermal and photodynamic activity. The isomerization effect of the polymer enhances its spatial distortion and improves its antibacterial properties. It is used as a biomimetic cascade intelligent phototherapy agent for a bio-responsive self-oxygen supply system for the treatment of infected wounds, realizing photothermal / photodynamic / POD / CAT / OXD cascade synergistic treatment, and significantly accelerating the healing of open bacterial infected wounds.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] The first aspect of the present invention provides a method for preparing an iron phthalocyanine-based isomeric conjugated microporous polymer having three enzyme mimicking activities, the preparation method comprising:

[0008] The s-triazine and metal salt containing thiophthalonitrile groups were added to quinoline for solvothermal reaction to obtain iron phthalocyanine-based isomeric conjugated microporous polymers with three enzyme mimetic activities.

[0009] The chemical formula of the s-triazine containing thiophthalonitrile group is C 27 H9N9S3.

[0010] Preferably, the s-triazine containing a thiophthalonitrile group is 2,4,6-(3-thiophthalonitrile)-triazine, and its structural formula is:

[0011] .

[0012] Preferably, the 2,4,6-(3-thiophthalonitrile)-s-triazine is prepared by the following method:

[0013] 3-nitrophthalonitrile and 1,3,5-triazine-2,4,6-trithione are added into an organic solvent for solvothermal reaction to obtain 2,4,6-(3-thiophthalonitrile)-s-triazine.

[0014] More preferably, the temperature of the solvothermal reaction is 65° C., and the time of the solvothermal reaction is 48 h.

[0015] Preferably, the s-triazine containing a thiophthalonitrile group is 2,4,6-(4-thiophthalonitrile)-triazine, and its structural formula is:

[0016] .

[0017] Preferably, the 2,4,6-(4-thiophthalonitrile)-triazine is prepared by the following method:

[0018] 4-nitrophthalonitrile and 1,3,5-triazine-2,4,6-trithione are added to an organic solvent for solvothermal reaction to obtain 2,4,6-(4-thiophthalonitrile)-s-triazine.

[0019] More preferably, the temperature of the solvothermal reaction is 65° C., and the time of the solvothermal reaction is 48 h.

[0020] Preferably, the metal salt is an iron salt, a copper salt, a nickel salt, a zinc salt or a cobalt salt.

[0021] Preferably, the iron salt is ferric chloride.

[0022] Preferably, the molar ratio of the s-triazine containing thiophthalonitrile group, the metal salt and quinoline is 1:2:50.

[0023] Preferably, the solvent thermal reaction is: reacting at 100° C. for 15 min, reacting at 120° C. for 30 min, and reacting at 160° C. for 18 h.

[0024] The second aspect of the present invention provides an iron phthalocyanine-based isomeric conjugated microporous polymer having three enzyme mimicking activities obtained by the above preparation method ( iso -CMP-1 or iso -CMP-2), the structural unit of the iron phthalocyanine-based isomeric conjugated microporous polymer having tri-enzyme mimicking activity is:

[0025] or .

[0026] The third aspect of the present invention provides the use of an iron phthalocyanine-based isomeric conjugated microporous polymer having tri-enzyme mimetic activity in the preparation of an antibacterial drug.

[0027] Beneficial effects of the present invention:

[0028] (1) The present invention has developed an iron phthalocyanine-based isomeric conjugated microporous polymer with negligible biological toxicity and triple enzyme mimetic activity ( iso -CMP-1 and iso -CMP-2), used for auxiliary antibacterial and anti-biofilm treatment. Through cyano self-polymerization and metal ions inserted into the center of the structure, the synthesized photosensitive sample has enzyme-induced biological cascade enzyme catalytic activity, which can achieve efficient regulation and specific utilization of the infectious microenvironment (IME), and realize light-induced synergistic amplification precision treatment.

[0029] (2) The iron phthalocyanine-based isomeric conjugated microporous polymer of the present invention converts Fe 3+ And functional phthalocyanine units are integrated into the entire porous skeleton, which can be used as a bionic cascade intelligent phototherapy agent. It can be used in the bioresponsive self-oxygen supply system for the treatment of infectious wounds, realizing photothermal / photodynamic / POD / CAT / OXD cascade synergistic treatment, and cascade enzyme therapy of iron phthalocyanine-based heterogeneous conjugated microporous polymers. It overcomes the limitations of single-mode enzyme therapy, achieves an amplification effect, and significantly accelerates the healing of open bacterial infected wounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Picture 1 : Picture 1 :a) 2,4,6-(3-thiophthalonitrile)-s-triazine and iso - Fourier transform infrared spectrum of CMP-1; b) 2,4,6-(4-thiophthalonitrile)-triazine and iso - Fourier transform infrared spectrum of CMP-2; c) iso -CMP-1 13 C NMR; d) iso - Powder XRD of CMP-1; e) iso -XPS spectrum of CMP-1; f) XPS spectrum of C 1s; g) XPS spectrum of N 1s; h) XPS spectrum of S 2p; i) High-resolution XPS spectrum of Fe 2p;

[0031] Picture 2 : a) Scale bar: 1 μm iso -SEM of CMP-1; b) Scale bar: 500 nm iso -SEM of CMP-1; c) Scale bar 100 nm iso -SEM of CMP-1; d) Scale bar: 500 nm iso -TEM of CMP-1; e) Scale bar 200 nm iso -TEM of CMP-1; f) Scale bar: 50 nm iso -TEM of CMP-1) Scale bar: 5 nm iso -HR-TEM of CMP-1; h) iso -HADDF-STEM image of CMP-1, scale bar 1μm; i) iso -CMP-1 C element distribution map; j) iso -N element distribution map of CMP-1; k) iso -S element distribution map of CMP-1; l) iso -Fe element distribution map of CMP-1;

[0032] Picture 3 :a) At different concentrations iso - Temperature curve of CMP-1; b) at different concentrations iso - Temperature curve of CMP-2; d) under different power density iso -Photothermal curve of CMP-1; c) iso -Infrared thermal imaging of CMP-1 at different concentrations; d) at different power densities iso -Photothermal curves of CMP-1; e) at different power densities iso -Photothermal curve of CMP-2; f) iso -CMP-1 aqueous solution (200 μg / mL) was irradiated with four-shot laser (λ = 638 nm, 1.5 W cm -2 ) Temperature variation curve of irradiation on / off cycle; g) iso -CMP-2 aqueous solution (200 μg / mL) was irradiated with four-shot laser (λ = 638 nm, 1.5 W cm -2 ) Temperature variation curve of irradiation on / off cycle; h) iso -The relationship curve between the cooling time and negative natural logarithm of temperature of CMP-1 aqueous solution; i) iso -The relationship curve between the cooling time and negative natural logarithm of temperature of CMP-2 aqueous solution; j) iso -CMP-1 and iso -∆T curve of CMP-2;

[0033] Picture 4 : a) UV-visible spectrum of DPBF under laser irradiation; b) 638 nm (1.5 W cm -2 ) Under laser irradiation iso -UV-visible spectrum of CMP-1 + DPBF; c) 638 nm (1.5 W cm-2 ) Under laser irradiation iso -UV-visible spectrum of CMP-2 + DPBF; d) UV-visible spectrum of MB under laser irradiation; e) 638 nm (1.5 W cm -2 ) Under laser irradiation iso UV-visible spectrum of CMP-1 + MB; f) 638 nm (1.5 W cm -2 ) Under laser irradiation iso -UV-visible spectrum of CMP-2 + MB; g) fluorescence spectrum of DPBF under laser irradiation; h) 638 nm (1.5 W cm -2 ) Under laser irradiation iso -Fluorescence spectra of CMP-1 +DHR123; i) 638 nm (1.5 W cm -2 ) Under laser irradiation iso -Fluorescence spectrum of CMP-2 + DHR123; j) 638 nm (1.5 W cm -2 ) Under laser irradiation iso -CMP-1 and iso -Comparison of DPBF decay rates induced by CMP-2; k) 638 nm (1.5 W cm -2 ) Under laser irradiation iso -CMP-1 and iso -Comparison of MB decay rates induced by CMP-2; l) 638 nm (1.5 W cm -2 ) Under laser irradiation iso -CMP-1 and iso -Comparison of the decay rates of DHR123 induced by CMP-2; m) Electron paramagnetic resonance (EPR) spectrum of •OH; n) O2 •- Electron paramagnetic resonance (EPR) spectrum; o) 1 Electron paramagnetic resonance (EPR) spectrum of O2;

[0034] Picture 5 : a) iso a) UV absorption spectra of TMB oxidized by CMP-1 and iso-CMP-2; b) under oxygen and nitrogen saturated conditions iso -UV absorption spectra of TMB oxidation catalyzed by CMP-1; c) Effects of different scavengers on iso -CMP-1 + TMB reaction system absorbance; d) three groups (TMB, TMB + hydrogen peroxide, iso -CMP-1+hydrogen peroxide+TMB) UV-visible spectrum; e) 200 μg / mL iso-CMP-1 solution decomposes the UV absorption of hydrogen peroxide to oxidize TMB in PBS at different pH values; f) different concentrations of iso -CMP-1 solution decomposes the ultraviolet absorption of TMB by hydrogen peroxide in PBS at pH 5.5; g) iso -CMP-1 UV absorption of iso-CMP-1 in the presence or absence of light; h) different concentrations of iso -CMP-2 solution decomposes the UV absorption of hydrogen peroxide oxidizing TMB in PBS at pH 5.5; i) Effect of different scavengers on the iso -CMP-1 + hydrogen peroxide + TMB reaction system absorbance; j) UV-visible absorption spectra of CAT activity in different reaction systems; k) at different concentrations iso -UV-visible spectrum of hydrogen peroxide produced by CMP-1; l) iso -UV-visible absorption spectra of hydrogen peroxide produced by CMP-1 at different incubation times;

[0035] Picture 6 : a) iso -Photos of Staphylococcus aureus bacterial colonies formed after CMP-1 was treated with each group; b) iso - Bacterial survival rate of Staphylococcus aureus after CMP-1 treatment in each group; c) iso -Photos of Escherichia coli bacterial colonies formed after CMP-1 was treated with each group; d) iso - Bacterial survival rate of Escherichia coli after CMP-1 treatment in each group; e) iso -Photographs of Staphylococcus aureus bacterial colonies formed after CMP-2 was treated with each group; f) iso -Photos of Escherichia coli bacterial colonies formed after CMP-2 was treated in each group; g) iso - Bacterial survival rates of Staphylococcus aureus and Escherichia coli after CMP-2 treatment in each group;

[0036] Picture 7 :a) Fluorescence images of Staphylococcus aureus treated in each group after co-staining; b) Live-dead bacterial ratio of Staphylococcus aureus treated in each group c) Fluorescence images of Escherichia coli treated in each group after co-staining; d) Live-dead bacterial ratio of Escherichia coli treated in each group;

[0037] Picture 8 :a) TEM images of Staphylococcus aureus treated in various groups after co-staining; b) TEM images of Escherichia coli treated in various groups after co-staining;

[0038] Picture 9 :a) Different concentrations iso-CMP-1 hemolysis graph; b) MTT test of NIH / 3T3 after 24 h of treatment with different concentrations; c) Biofilm ratio of Staphylococcus aureus biofilm experiment; d) Biofilm ratio of Escherichia coli biofilm experiment; e) Different concentrations iso -Cell migration after 24 h of CMP-1 treatment; f) Different concentrations iso -Cell rate after 24 h of CMP-1 treatment;

[0039] Picture 10 :a) Photos of the back wounds of mice infected with Staphylococcus aureus after treatment in each group; b) Changes in the weight of mice during treatment; c) Curve of the change of wound area and time;

[0040] Picture 11 : H&E and Masson trichrome staining images of wound tissues in each group on the 11th day of wound healing;

[0041] Picture 12 :a) Hematocrit of mice on day 11; b) Hemoglobin of mice on day 11; c) Average hemoglobin content of mice on day 11; d) Average hemoglobin concentration of mice on day 11; e) Average corpuscular volume of mice on day 11; f) Platelets of mice on day 11; j) Red blood cells of mice on day 11; h) White blood cells of mice on day 11;

[0042] Picture 13 :H&E staining of the main organs of mice: heart, liver, spleen, lung and kidney;

[0043] Picture 14 : iso -CMP-1 and iso -Schematic diagram of the structure of CMP-2. DETAILED DESCRIPTION

[0044] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0045] As introduced in the background technology section, the currently commonly used organic photosensitizers (PSs) face a major challenge, namely, the severe quenching of photoactivity caused by the intrinsic self-aggregation of their large conjugated structures; and the hypoxic infectious microenvironment is conducive to bacterial growth, which greatly delays the wound healing process.

[0046] Based on this, the purpose of the present invention is to provide an iron phthalocyanine-based heterogeneous conjugated microporous polymer with three enzyme mimicking activities and its preparation method and application. The present invention designs two heterogeneous conjugated microporous polymers (CMPs) based on iron phthalocyanine through the self-polymerization of two isomeric monomers 2,4,6-(3-thiophthalonitrile)-s-triazine and 2,4,6-(4-thiophthalonitrile)-s-triazine, namely iso -CMP-1 and iso -CMP-2. Its unique structure and composition make the heterogeneous conjugated microporous polymer have photoresponsiveness and triple enzyme activity. The congestion of the active groups in the isomers has a significant impact on the structure and performance of the polymer. High congestion will produce greater steric hindrance, resulting in greater spatial deformation of the porous skeleton structure, which will effectively inhibit the aggregation of phthalocyanines, leading to photoactivity quenching, thereby further improving enzyme activity. Picture 14 It can be seen that iso -CMP-1 has a greater degree of structural distortion than iso -CMP-2, so the isomerism effect can be used to enhance the spatial distortion of the polymer, thereby improving the antibacterial performance.

[0047] Specifically, iso -CMP-1 can be used as a type I PS, synergizing with PTT to exert an O2-independent PDT effect. iso -CMP-1 also has the activity of simulating peroxidase (POD), oxidase (OXD) and catalase (CAT), which can realize cascade enzyme therapy. It can not only provide H2O2 and O2 by itself to overcome the limitations of IME, but also generate sufficient ROS at the pathological acid IME, synergistically with PTT and PDT to achieve the purpose of amplifying the therapeutic effect. iso -CMP-1 can not only effectively inhibit bacterial reproduction and biofilm formation, but also accelerate the healing of infected wounds. The iron phthalocyanine-based heterogeneous conjugated microporous polymer with triple enzyme mimetic activity of the present invention paves the way for the manufacture of pH-responsive therapeutic agents that can simultaneously generate ROS, H2O2 and O2 to quickly treat infected wounds.

[0048] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments.

[0049] Note: Unless otherwise stated, the pH of PBS used in the present invention is 7.0. The control group in the present invention refers to the group without drug addition (e.g. iso -CMP-1, iso -CMP-2 and H2O2), a group with only PBS and no laser treatment, and the rest of the treatments were the same as the other groups. The laser group refers to the group with laser treatment.

[0050] The test materials used in the examples of the present invention are all conventional test materials in the art and can be purchased through commercial channels.

[0051] Embodiment 1: iso - Preparation of CMP-1

[0052] (1) Preparation of 2,4,6-(3-thiophthalonitrile)-triazine:

[0053] 3-nitrophthalonitrile (4.40 g, 25.4 mmol) and 1,3,5-triazine-2,4,6-trithione (1.0 g 8.46 mmol) were placed in a reaction bottle, mixed and dissolved with N,N-dimethylformamide (50 mL), then charged with Ar protection, the mixture was stirred for 10 min to dissolve, and then potassium carbonate (3.50 g, 25.42 mmol) was added, and the reaction was heated at 65 ° C for 48 h. After the reaction was completed, the reactant was cooled to room temperature. Then poured into ice water, filtered, washed with water and ethanol, and dried to obtain a crude yellow solid product, namely 2,4,6-(3-thiophthalonitrile)-triazine. The synthesis route is as follows:

[0054] .

[0055] (2) iso - Preparation of CMP-1

[0056] 2,4,6-(3-thiophthalonitrile)-s-triazine (993.4 mg, 1.00 mmol), ferric chloride (344.0 mg, 2.0 mmol) and quinoline (7 mL, 50 mmol) were placed in a 25 mL reactor, and the reactor was placed in an oven. The temperature was first raised to 100 ° C for 15 min, then raised to 120 ° C for 30 min, and finally raised to 160 ° C for 18 h. After the reaction was completed, the precipitate was filtered out and the product was washed with dichloromethane, DMF, THF, DMSO, methanol, water, and ethanol in turn, and vacuum dried at 100 ° C to obtain black powder iso-CMP-2 with a yield of 61%. The synthetic route is as follows:

[0057] .

[0058] Embodiment 2: iso - Preparation of CMP-2

[0059] (1) Preparation of 2,4,6-(4-thiophthalonitrile)-triazine:

[0060] 4-nitrophthalonitrile (4.40 g, 25.4 mmol) and 1,3,5-triazine-2,4,6-trithione (1.0 g 8.46 mmol) were placed in a reaction bottle, mixed and dissolved with N,N-dimethylformamide (50 mL), then charged with Ar protection, the mixture was stirred for 10 min to dissolve, and then potassium carbonate (3.50 g, 25.42 mmol) was added, heated at 65 ° C for 48 h, and the reactants were cooled to room temperature after the reaction was completed. Then poured into ice water, filtered, washed with water and ethanol, and dried to obtain a crude yellow solid product, namely 2,4,6-(4-thiophthalonitrile)-triazine. The synthetic route is as follows:

[0061] .

[0062] (2) iso- Preparation of CMP-2

[0063] 2,4,6-(4-thiophthalonitrile)-s-triazine (993.4 mg, 1.00 mmol), ferric chloride (344.0 mg, 2.0 mmol) and quinoline (7 mL) were placed in a 25 mL reactor, and the reactor was placed in an oven. The temperature was first raised to 100°C for 15 min, then raised to 120°C for 30 min, and finally raised to 160°C for 18 h. After the reaction was completed, the reaction mixture was cooled and the precipitate was filtered out. The product was washed with dichloromethane, DMF, THF, DMSO, methanol, water, and ethanol, and vacuum dried at 100°C to obtain black powder iso-CMP-2 with a yield of 54%. The synthetic route is as follows:

[0064] .

[0065] Example 3: Characterization

[0066] (1) Using infrared spectroscopy analysis iso -CMP-1 structure, 2,4,6-(3-thiophthalonitrile)-s-triazine prepared in step (1) of Example 1 and 2,4,6-(3-thiophthalonitrile)-s-triazine prepared in step (2) of Example 1 iso -CMP-1 for infrared detection. Picture 1 a) -CN (2230 cm) in 2,4,6-(3-thiophthalonitrile)-s-triazine -1 ) peak attenuates significantly, which indicates that iso -CN disappears in -CMP-1. At the same time, -C=N- (1720cm -1 ) peaks are enhanced and shifted, which is due to the formation of -C=N- in the phthalocyanine ring and the formation of Fe-N (1010 cm -1) . The benzene ring in 2,4,6-(3-thiophthalonitrile)-s-triazine (1450 cm-1 ) and -S- (742 cm -1 ) peak still exists, which indicates that the material has been successfully polymerized and the monomer structure remains intact. At the same time, the structure of iso-CMP-2 was analyzed by infrared spectroscopy. The 2,4,6-(4-thiophthalonitrile)-s-triazine prepared in step (1) of Example 2 and the iso -CMP-2 for infrared detection. Picture 1 b) -CN (3090 cm) in 2,4,6-(3-thiophthalonitrile)-s-triazine -1 ) peak attenuates significantly, which indicates that iso -CN disappears in CMP-2. At the same time, -C=N- (1670cm -1 ) peaks are enhanced and shifted, which is due to the formation of -C=N in the phthalocyanine ring and the formation of Fe-N (1010 cm -1) . The benzene ring in 2,4,6-(4-thiophthalonitrile)-s-triazine (1450 cm -1 ) and -S- (742 cm -1 ) still exists, which indicates that the material has been successfully polymerized and the monomer structure remains intact.

[0067] (2) Solid-state 13C-NMR further confirmed iso -CMP-1 composite carbon skeleton structure. Picture 1 As shown in Figure c), characteristic strong signals of imine and aromatic carbon appeared between 145-120 ppm, and the peak at 168.03 ppm was attributed to -NC=N- in the phthalocyanine ring; these results further confirmed the successful polymerization of phthalocyanine polymers. iso -The X-ray diffraction (XRD) pattern of CMP-1 is as follows Picture 1 d) shows only a broad peak between 20 and 30, indicating iso -CMP-1 is an amorphous structure.

[0068] X-ray photoelectron spectroscopy (XPS) was used to investigate iso -Surface elemental composition of CMP-1. Picture 1 e) iso -CMP-1 measured spectrum, there are clear peaks of Fe, N, C, S elements, the atomic ratio of 2.03%, 16.2%, 76.31% and 5.46% respectively. X-ray photoelectron spectroscopy (XPS) was used to characterize iso - The composition of CMP-1 confirmed the presence of C, N, S and Fe elements. Picture 1f) The C1 s spectrum is decomposed into two peaks: CC (284.8 eV) and C=C (286.8 eV). Picture 1 g) is divided into three peaks, including pyridinic N (398.5 eV), pyrrolic N (399.8 eV) and Fe-N (404.2 eV), indicating that iso -In CMP-1, Fe atoms are successfully coordinated with N elements. Picture 1 h) S 2p spectrum can be observed 3 / 2 (163.7 eV), S 2p 1 / 2 (165.4 eV) two -SC- signal peaks. Picture 1 i) Fe 2p spectrum shows iso -The two signal peaks of CMP-1 at 710.0 and 720.8 eV belong to Fe 3+ Fe2p 3 / 2 and Fe2p 1 / 2 , the two characteristic peaks at 714.2 and 724.9 eV belong to Fe 2+ Fe2p 3 / 2 and Fe2p 1 / 2 The above characterization proves iso -Successful synthesis of CMP-1.

[0069] (3) Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) iso The morphology and internal structure of CMP-1 were characterized. Picture 2 a) ~ Picture 2 f), it can be seen that iso-CMP-1 is composed of tightly packed blocks and macropores that grow together. Meanwhile, high-resolution transmission electron microscopy (HR-TEM) shows the coexistence of micropores. Picture 2 The light and dark contrast shown in g) is also very obvious. The elemental map shows that Fe, N and S are evenly distributed in the porous matrix dominated by C, as shown in Figure 2. Picture 2 h)~ Picture 2 l). Through inductively coupled plasma emission spectroscopy (ICP) and elemental analysis, the contents of C, N, S and Fe were determined to be 61.33%, 9.43%, 18.51% and 10.73%, respectively.

[0070] (4) Photothermal conversion performance test

[0071] Configure different concentrations iso -CMP-1 and iso -CMP-2 aqueous solution (0, 50, 100, 150, 200, 250 μg / mL) was added to 1.5 mL centrifuge tubes. iso -CMP-1 and iso The total volume of the CMP-2 aqueous solution was 1 mL. Each centrifuge tube was placed in a vacuum chamber with a wavelength of 638 nm and a power density of 1.5 W cm -2 The samples were irradiated with infrared laser for 10 min. The temperature values ​​were displayed on a thermal imaging display, and the temperature rise within 10 min was taken every 30 s for plotting. iso -CMP-1 and iso -CMP-2 aqueous solution, with power densities of 0.5, 1.0, 1.5, and 2.0 W cm -2 The iso-CMP-1 and iso-CMP-2 were irradiated with 638 nm laser for 10 min, and data were taken every 30 s for plotting. iso -Thermal stability of CMP-2, the configured iso -CMP-1 and iso -CMP-2 aqueous solution (200 μg / mL) was irradiated by infrared laser (power density 1.5 W cm -2 ), after 10 min of irradiation, turn off the laser and iso -CMP-1 was cooled naturally to room temperature. Then the same method was repeated for 3 times, and the temperature changes during the entire cycle were recorded. One of the cycles was taken out separately for plotting, and the photothermal conversion efficiency was calculated based on the temperature changes during the descent process.

[0072] from Picture 3 a) and Picture 3 b) It can be seen that iso -CMP-1 and iso The temperature rise behavior of -CMP-2 is related to its concentration. -2 After 10 minutes of laser irradiation, iso -CMP-1 and iso - The concentration of CMP-2 aqueous solution increased from 50 μg / mL to 250 μg / mL, iso -CMP-1 temperature increment increased from 16.8°C to 43.9°C, while iso - The temperature of CMP-2 increased from 11.6°C to 28.7°C. However, the temperature of pure water remained almost constant regardless of whether the laser was irradiated or not. Picture 3 c) It can be seen that iso -CMP-1 showed a wide temperature change region from light blue (22.1°C) to bright red (62.4°C) within 10 min, indicating iso -CMP-1 has good photothermal conversion performance. Picture 3 d) and Picture 3 e) as shown, iso -CMP-1 and iso The temperature increase of -CMP-2 is also positively correlated with the laser density power. When the concentration is 200 μg / mL and the laser power is 0.5, 1.0, 1.5, and 2.0 W cm -2 hour, iso The temperature increments of -CMP-1 were 13.5°C, 24.6°C, 37.6°C and 41.8°C, respectively. iso The temperature increments of -CMP-2 were 9.7°C, 17.5°C, 23.4°C and 31.9°C respectively. Picture 3 f) and Picture 3 g), iso -CMP-1 and iso -CMP-2 has good durability after 4 consecutive laser irradiation cycles with no obvious attenuation. iso -CMP-2 showed slight attenuation after 4 consecutive laser irradiation cycles. These results indicate that iso -CMP-1 is a durable and efficient photothermal agent for repeated photothermal treatments. Picture 3 j) It can be seen that when other conditions are the same iso -CMP-1 temperature increment is higher than iso -CMP-2.

[0073] iso -CMP-1 photothermal conversion efficiency: η (%) = 79.96%; iso-CMP-2 photothermal conversion efficiency: η (%) = 52.64%. The photothermal conversion efficiency calculation formula is calculated with reference to the photothermal conversion efficiency formula in "A Photothermal-Fenton-like Reaction Artificial Nanozyme and Its Preparation Method and Application" disclosed in application number CN202310175497.X. Picture 3 h) and Picture 3 i) shows iso -CMP-1 and iso -τS and θ values ​​of CMP-2 in the light-to-heat conversion efficiency calculation formula.

[0074] (5) Testing of Type I and Type II photodynamic performance

[0075] The type I and type II photodynamic properties of the nanozyme were investigated. Since the DPBF probe is easily oxidized and further decomposed into 1,2-diphenylmethylbenzene when it comes into contact with reactive oxygen species (ROS), the absorbance decreases. iso -CMP-1 and iso -Type II photodynamic properties of CMP-2.

[0076] according to Picture 4a) It can be seen that pure DPBF is irradiated with laser for 10 min (638 nm, 1.5 W / cm 2 ) after addition, the absorbance decreased only slightly (17.72%). iso -CMP-1 and iso -CMP-2, the absorbance decreased evenly with the extension of laser irradiation time. Picture 4 b) and Picture 4 j), when the laser irradiation lasted for 10 min, iso -CMP-1 absorbance decreased to 96.01% of its initial value; Picture 4 c) and Picture 4 j), iso -CMP-2's absorbance dropped to 76.92% of its initial value. iso -CMP-1 and iso -CMP-2 can be induced under laser irradiation 1 The generation of O2 leads to a decrease in the absorbance of DPBF.

[0077] right iso -CMP-1 and iso -CMP-2 was tested for its type I photodynamic properties. Methylene blue (MB) was used as a probe. Under the action of •OH, methylene blue can be specifically converted into colorless methylene blue (Leuco-MB) and exhibit a characteristic 665 nm ultraviolet absorption peak under laser irradiation. As can be seen from Figure 4d), pure MB exhibits a high absorption peak under laser irradiation (638 nm, 1.5 W / cm 2 ) After 10 minutes, the absorbance only decreased slightly. Picture 4 e) and Figure 4f), after adding iso -CMP-1 and iso -CMP-2, the decrease in absorbance was greater with the extension of irradiation time. Picture 4 k) The quantitative results show that compared with pure MB (18.29%), iso -CMP-1 and iso -CMP-2 showed a significant decrease of 72.21% and 53.01% within 10 minutes. Similarly, in Figure 4g) to Figure 4i), compared with the small ΔI value change of pure DHR123, the introduction of iso -CMP-1 and iso -CMP-2, the absorbance soared rapidly. Specifically, as shown in Figure 4l), after 10 minutes of laser irradiation, iso -CMP-1 and isoThe ΔI values ​​of -CMP-2 were 4003.6 and 2651.86, which were much higher than those of pure DHR123 (394.7). Picture 4 m) and Picture 4 n) Electron paramagnetic resonance (EPR) spectroscopy directly detects the generated •OH and O2 • - ; It can be seen iso -OH and O2 of CMP-1 •- The EPR of both showed strong signals, proving that •OH and O2 •- At the same time Picture 4 o)'s electron paramagnetic resonance (EPR) spectroscopy also detected 1 O2 signal, but the intensity is lower than OH and O2 • - weak, indicating that type I photodynamics is dominant. iso -CMP-1 and iso -CMP-2 has both Type I and Type II photodynamics, and iso -The photodynamic properties of CMP-1 are stronger.

[0078] (6) Determination of oxidase (OXD) activity

[0079] To evaluate iso -CMP-1 and iso -OXD-like activity of CMP-2 nanozyme, selecting TMB as chromogenic substrate in the absence of H2O2. Picture 5 a) as shown, iso -CMP-1 and iso -CMP-2 has UV absorption at 650nm, and iso -CMP-1 has the highest OXD-like activity.

[0080] To study the effect of O2 on TMB oxidation, PBS (pH 5.5) was bubbled with O2 and N2 for 20 min. Picture 5 b) It can be seen that the oxidation of TMB is significantly improved under O2 saturation conditions, while it is significantly weakened when filled with N2, indicating that iso -CMP-1 catalyzes the oxidation of the substrate while consuming O2. In other words, iso -CMP-1 does not act as an oxidant to directly oxidize TMB, but rather acts as an OXD mimetic, utilizing O2 to generate ROS for the oxidation of TMB. To investigate the types of ROS generated by OXD-like activity, free radical scavengers (5 mM, 50 μL) were introduced: isopropanol (IPA, 5 mM), D-histidine (HD, 5 mM), p-benzoquinone (BQ, 5 mM), and ethylenediaminetetraacetic acid (EDTA, 5 mM), and the reaction mixture ( iso-CMP-1 +TMB + free radical scavenger + PBS) was incubated at room temperature for 20 min, and then the UV absorption spectrum of the reaction system was recorded. Picture 5 c) shows that the absorbance of the OXD-like active catalytic system only slightly decreased with IPA, indicating that iso -CMP-1 + TMB catalytic system produces a small amount of •OH. However, when BQ is added iso -CMP-1 +TMB system, iso -CMP-1 activity was significantly inhibited, indicating that the main ROS type that oxidizes TMB is O2 •- When HD and EDTA are added iso -CMP-1 + TMB, the absorbance also decreased, indicating that there is still a small amount of ROS type that oxidizes TMB 1 O2 and H + (hole). The above experiment shows iso -CMP-1 has OXD-like activity.

[0081] To prove the generation of H2O2, the Solarbio superoxide anion scavenging capacity test kit was used for detection. Reagent three (hydroxylamine hydrochloride, 250 μL) and reagent four (p-aminobenzenesulfonic acid, 250 μL) were mixed with different concentrations of iso -CMP-1 (0, 50, 100, 150, 200 μg / mL) was incubated at 37°C for 30 min, and then reagent five (1-naphthylamine, 250 μL) was added and incubated at 37°C for 20 min, and the UV absorption spectrum of the reaction system was recorded. Picture 5 k), in the absence of iso -CMP-1 nanozyme, the reaction system did not change color. iso -CMP-1 (50, 100, 150, 200 μg / mL), the reaction system turned light purple. iso -As the concentration of CMP-1 increases, the color gradually darkens, and the absorbance gradually increases from the UV absorption curve at 540nm. iso -CMP-1 nanozyme exerts OXD enzyme activity to generate O2 •- The effects of different incubation times (1h, 2h, 3h, 4h, 5h) were then determined. Picture 5 l) It can be seen that as the incubation time increases, the absorbance gradually decreases, indicating that iso -CMP-1 nanozyme in generating O2 •- Then it is further converted into H2O2, which is iso -CMP-1 nanozyme exerts POD and CAT activities to provide H2O2.

[0082] (7) Peroxidase (POD) oxidative activity assay

[0083] 3,3',5,5'-Tetramethylbenzidine (TMB) was used as an indicator of the production of ROS by the decomposition of H2O2 catalyzed by the material. Free radical scavengers (5mM, 50μL) were introduced: isopropyl alcohol (IPA, 5mM), D-histidine (HD, 5mM), p-benzoquinone (BQ, 5mM) and ethylenediaminetetraacetic acid (EDTA, 5mM), and then the free radical scavengers were added to iso -CMP-1 (200 μg / mL) + H2O2 (3.0 mM) + TMB (1.5 mM) reaction mixture, incubate at room temperature for 1 min, and then record the UV absorption spectrum of the reaction system. Picture 5 i) shows that the absorbance of the reaction system with the addition of isopropanol (IPA), D-histidine (HD), and ethylenediaminetetraacetic acid (EDTA) only slightly decreased, but the absorbance of the reaction system with the addition of p-benzoquinone (BQ) decreased significantly, indicating that iso -CMP-1 catalyzes the decomposition of H2O2 to produce a large amount of O2 •- , and a small amount of •OH. O2 • - It has lower toxicity than •OH and has less effect on the vitality of normal cells, thus it can play a mild bactericidal role in the wound healing process.

[0084] The color change of TMB and the peak at 650 nm confirmed the presence of O2 •- The production of. Picture 5 d) It can be observed iso -CMP-1 + H2O2 + TMB group showed an obvious absorption peak at 650nm, and the colorless TMB was oxidized to blue. In contrast, the H2O2 + TMB group and the simple TMB group did not show an absorption peak at 650nm, nor did they show color changes. The above research results show that iso -CMP-1 can catalyze the production of a large amount of O2 in the presence of H2O2 •- On this basis, the solution pH and iso -CMP-1 concentration iso -The effect of CMP-1 enzyme activity was investigated and tested iso - UV absorption of CMP-1 at different pH (1.5, 2.5, 3.5, 4.5, 5.5, 6.5), Picture 5 e) Display from pH 6.5-3.5 iso -CMP-1 enzyme activity gradually increased, and the highest enzyme activity was at pH 3.5. As the pH decreased, the enzyme activity also decreased. iso-CMP-1 enzyme activity is correlated with pH. In addition, it can be seen that pH 6.5 also has strong enzyme activity, which broadens the pH range of enzyme response. iso - UV absorption of CMP-1 at different concentrations (50 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL), Picture 5 f) Display as iso -The concentration of CMP-1 gradually increases, iso -The enzyme activity of CMP-1 also gradually increased, indicating iso -CMP-1 enzyme activity is concentration-dependent. In addition, the activity of CMP-1 with and without laser irradiation was compared. iso - Enzymatic activity of CMP-1, Picture 5 g) shows that under near-infrared illumination, iso The catalytic activity of -CMP-1 was improved, which was due to the fact that it produced more active oxygen and promoted the oxidation process of TMB. iso -CMP-2 was also tested, from Picture 5 h) shows that at pH 5.5, iso When the concentration of -CMP-2 was 200 μg / mL, the UV absorption of iso-CMP-2 at 650 nm was lower than iso -CMP-1, Description iso -CMP-1 has a iso -CMP-2 has strong POD enzyme activity.

[0085] (8) Catalase-like activity assay

[0086] For the purpose of research iso -CMP-1 simulates the catalytic activity of catalase (CAT) and measures the concentration change of H2O2 based on the decomposition of H2O2 into O2. H2O2 and titanium tetrachloride can react to produce a yellow product with characteristic absorption at 412nm. iso -CMP-1 and iso -H2O2 (30mM, 100μL) was added to CMP-2 to make a total volume of 3mL solution, and then incubated for 20min. During the reaction, a large number of bubbles were seen. After the incubation, 20μL of 5% TiCl4 hydrochloric acid solution was added for color development, and the UV absorption curve was measured. iso -CMP-1 catalytically decomposes H2O2 to generate O2. Picture 5 j), after incubation for 20 min iso -The absorbance of the CMP-1 system dropped significantly. iso -CMP-2 system also showed a downward trend, but iso -CMP-1 system absorbance decreased more significantly. The above results show iso -CMP-1 has very strong CAT mimetic activity, iso -CMP-2 has a lower CAT mimetic activity iso -CMP-1 is weak. iso -CMP-1 has excellent photodynamic properties, and iso -The CAT-mimicking activity of CMP-1 provides O2 for PDT, promoting the bactericidal effect of PDT.

[0087] Test Example 1: In vitro antibacterial performance test

[0088] Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) were used as representatives of Gram-positive and Gram-negative bacteria to detect iso -CMP-1 and iso - Antibacterial properties of CMP-2.

[0089] (1) iso -The antibacterial test of CMP-1 was divided into 8 groups: I: control (PBS); II: H2O2; III: iso -CMP-1; IV: H2O2+ iso -CMP-1; V: PBS + laser; VI: H2O2 + laser; VII: iso -CMP-1 + Laser; VIII: iso -CMP-1 +H2O2+laser. In the above grouping, the total volume of each mixed solution was 1mL, the bacterial solution concentration was 100 μL / mL (the number of colonies was 10 8 CFU mL -1 ), iso The concentration of -CMP-1 was 200 μg / mL, the concentration of H2O2 was 10 μL / mL, and the pH of PBS in each group was 5.5. The laser group was irradiated with 638 nm infrared laser with a power density of 1.5 W cm -2 , the irradiation time was 10 min. Finally, the mixed solution of each group was placed in a shaker and incubated at 37°C for 24 h. 100 μL of the mixed solution of each group was transferred to the solid culture medium, the morphology was observed, and the number of colonies was calculated. And compared with the control group (PBS group), the antibacterial effect was evaluated.

[0090] As shown in Figure 6a) Picture 6 d) As shown in the figure, with or without laser irradiation, isoThe number of colony forming units (CFU) in the H2O2 groups (II and VI) of -CMP-1 was similar to that in the control groups (I and IV), indicating that low concentrations of H2O2 (10 μL / mL) had little effect on the elimination of bacteria. iso -CMP-1 group (III), the number of CFU was only slightly lower than that of the PBS group, indicating that the use of iso -CMP-1 (200 μg / mL) had a weaker antibacterial effect. iso -The bacterial colony count in the CMP-1+laser group (VII) was significantly reduced, as evidenced by the survival rates of 1.37% for Staphylococcus aureus and 1.85% for Escherichia coli. iso The bacterial survival rates of S. aureus and E. coli in the CMP-1+H2O2 group (IV) were 73.32% and 69.97%, respectively, indicating that iso -CMP-1 can catalyze the decomposition of low concentration H2O2 into toxic •OH to kill bacteria. When H2O2 and laser irradiation are used simultaneously, iso -The survival rates of Staphylococcus aureus and Escherichia coli in the CMP-1 + H2O2 + laser group (X) dropped sharply to 0.39% and 0.68%, respectively, demonstrating the great advantage of the PTT / PDT / POD / CAT / OXD cascade synergistic treatment in effective sterilization.

[0091] (2) iso -The antibacterial test of CMP-2 was divided into 3 groups: I: control (PBS); II: iso -CMP-2 + Laser; III: iso -CMP-2 + H2O2 + laser. Other test conditions are the same as iso -CMP-1 is exactly the same.

[0092] from Picture 6 e) ~ Picture 6 g) It can be seen that iso - The bacterial survival rates of Staphylococcus aureus and Escherichia coli in the CMP-2 + laser group (II) were 13.39% and 14.64%, respectively, iso -The bacterial survival rates of Staphylococcus aureus and Escherichia coli in the CMP-2 + H2O2 + laser (III) group were 9.68% and 10.20%, respectively. iso -The antibacterial effect of CMP-2 is better iso -CMP-1 is weak.

[0093] Test Example 2: Bacterial staining test

[0094] (1) Bacterial live / dead staining

[0095] The mixture was divided into 8 groups according to the grouping of Experiment 1 (1) (the total volume of the mixed solution in each group was 1 mL). 400 μL of the solution was taken from each group and 20 μL of SYTO-9 (1.0×10 -3 M) and 20 μL PI (1.5 × 10 -3 M) and incubated at 37°C in the dark for 15 min. Afterwards, the excess SYTO-9 and PI were removed by centrifugation. The bacteria in each solution were then resuspended with 50 μL PBS and the images were observed under an inverted fluorescence microscope.

[0096] Figure 7a) and Picture 7 c) As shown in the control group, PBS+laser group, H2O2 group, H2O2+laser group and iso -CMP-1 group, iso -Staphylococcus aureus and Escherichia coli in the CMP-1 + laser group survived, and the red fluorescence disappeared. iso -CMP-1+ laser, iso -CMP-1+ H2O2 and iso -After treatment with CMP-1+ H2O2+ laser group, obvious red fluorescence can be detected. This is consistent with the results obtained by plate counting method. iso -The bacteria treated with CMP-1+H2O2+laser showed the strongest red fluorescence, indicating that the number of bacterial deaths increased significantly after multi-mode treatment. In addition, for a clearer comparison iso -The bactericidal effect of CMP-1 was quantitatively analyzed by live and dead bacterial staining. Picture 7 b) and Picture 7 d) It can be clearly seen that the bacterial survival rate of group VII of Staphylococcus aureus is 6.68%±1.40, and the bacterial survival rate of group VIII is 2.85%±0.24. The bacterial survival rate of group VII of Escherichia coli is 13.97±1.76, and the bacterial survival rate of group VIII is 5.74%±0.97.

[0097] (2) The morphology of bacteria under projection

[0098] Divide into 8 groups according to the grouping of Experiment 1 (1). Add 2.5wt% glutaraldehyde solution to each group of solutions, fix at 4℃ for 24h, then wash with PBS 3 times, embed and seal with agar. Treat the bacteria with 30 wt%, 50 wt%, 70 wt%, 90 wt%, 95 wt% and 100 wt% ethanol solutions in sequence for 10min to dehydrate them, then treat with acetone for 3h, and embed by gradient penetration. Finally, the bacteria were negatively stained, fixed on a nickel grid and observed by transmission electron microscopy.

[0099] like Picture 8As shown, regardless of the laser irradiation intensity, the bacterial surface treated with PBS and H2O2 was smooth and intact, with Staphylococcus aureus in a spherical shape and Escherichia coli in a rod shape. iso -CMP-1 group (Group III), only one week of damage on the bacterial cell surface was observed, which means that only iso The antibacterial effect of the -CMP-1 group was minimal. iso The cell walls of the -CMP-1+H2O2 group (Group IV) were also damaged, indicating that the Fenton effect of this material also has a certain bactericidal effect. iso -After CMP-1 + laser treatment (Group VII), the cell membrane was destroyed and cytoplasm outflow occurred. iso -The cell membrane of the CMP-1+laser+H2O2 group (Group VII) was almost completely destroyed, and a large amount of cytoplasm flowed out, which was consistent with the antibacterial results.

[0100] Test Example 3: In vitro biocompatibility test

[0101] (1) Cell hemolysis experiment

[0102] The blood used in the experiment was taken from 5-week-old female KM mice (purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd.). The fresh blood was centrifuged (10000rpm, 20 min) and the supernatant was removed to collect the red blood cells. PBS buffer was then added for washing. The volume ratio of the added PBS buffer to the red blood cells was about 1:1. The supernatant was removed after centrifugation. This was repeated 4 times. The red blood cells were then mixed with PBS buffer at a volume ratio of 3:11. iso -CMP-1 (concentrations of 50, 100, 150, 200, 300 μg / mL) was mixed at a ratio of 1:9 (v / v) and incubated at 37°C for 4 h. 100 μL of the supernatant from each tube was added to a 96-well plate and the absorbance was measured using an ELISA reader. Distilled water was used as a positive control and the PBS group was used as a negative control. The calculation formula is as follows:

[0103] Hemolysis volume (%) = (A-An) / (Ap-An) × 100%;

[0104] A: Add to red blood cells iso -The absorbance obtained by taking the supernatant after CMP-1;

[0105] An: absorbance obtained by taking the supernatant after adding PBS to red blood cells (negative control);

[0106] Ap: absorbance of the supernatant after adding distilled water to red blood cells (positive control).

[0107] like Picture 9 a), after co-culture with blood, iso -CMP-1 exhibited concentration-dependent hemolysis behavior, and the hemolysis rate increased with the increase of concentration; within the experimental concentration range (50, 100, 150, 200, 300 μg / mL), the hemolysis rate remained below the standard value (4.0%), indicating that iso -CMP-1 has good blood compatibility.

[0108] (2) Cytotoxicity assay

[0109] NIH / 3T3 cells (from the Cell Bank of the Chinese Academy of Sciences) were plated in a 96-well plate at 5 × 10 3 The cells were inoculated at a density of 100 μL per well, and 100 μL of PBS was used to seal the edges of the plate to prevent excessive evaporation. After incubation for 24 h, the control group was treated with PBS, and the experimental group was treated with different concentrations of iso -CMP-1 (concentrations of 50, 100, 150, 200, 300 μg / mL), discard the supernatant after 24 hours of rest, add 10 μL MTT (methyl tetrazolyl blue, 5 mg / mL) to each well, incubate in the incubator for 4 hours, aspirate the supernatant, add 100 μL dimethyl sulfoxide, and rest for about 10 minutes before measuring the absorbance at 540 nm with an enzyme marker. Each group of experiments was repeated 3 times.

[0110] like Picture 9 b) shows that the cytotoxicity evaluation results showed iso -CMP-1 has good cell compatibility, and the cell survival rate is over 80% at a given dose (50-300μg / mL).

[0111] (3) Cell scratch test

[0112] Prepare three 6-well plates, each with three wells. Divided into 0μg / mL, 50μg / mL, 100μg / mL, 150μg / mL, 200μg / mL, the 0μg / mL group was treated the same as the other groups except that no drug was added. NIH / 3T3 cells (from the Cell Bank of the Chinese Academy of Sciences) were inoculated into 6-well plates and cultured for 24h. Then, the cells were scraped with the tip of a sterile pipette and cultured for 12h, and then irradiated with laser for 10min. The cells were then rinsed with PBS, and phase contrast images were taken using a microscope at 0h, 12h, and 24h. Image J software was used to measure the distance of cell migration and calculate the percentage of wound healing.

[0113] like Picture 9 e) and Picture 9f) As shown in Figure 2, with the extension of culture time, the scratch area of ​​the three experimental groups decreased significantly. The scratch healing rates of the 50μg / mL group to the 200μg / mL group were basically the same as those of the 0μg / mL group, indicating that iso -CMP-1 had little effect on normal cell migration function. All these results indicate iso -CMP-1 has good biocompatibility and can be used as a therapeutic agent for infections in vivo.

[0114] (4) Bacterial biofilm experiment

[0115] Staphylococcus aureus and Escherichia coli were used for modeling: 150 μL of liquid culture medium and 50 μL of bacterial solution were added to each well of a 96-well plate, and the plate was incubated in an incubator for 72 hours. The experiment was divided into a control group, i.e., PBS group (I), an iso-CMP-1 group (II), iso -CMP-1+H2O2 group (III), iso -CMP-1+Laser Group (IV), iso -CMP-1+Laser+H2O2 Group (V). iso -CMP-1 group were added with 50 μL of iso -CMP-1; the group with H2O2 was treated with 10 μL of H2O2; the group with laser was treated with 638 nm, 1.5 W / cm 2 Laser irradiation for 10 minutes. The above groups were added to the incubated bacterial solution and incubated for another 5 hours. After incubation, it was stained with crystal violet (CV): washed with PBS, fixed with 100μL methanol for 30 minutes, then washed with PBS, and finally added 50μL of 0.1% crystal violet for 20 minutes. After incubation, it was washed with PBS until clear, and 100μL of glacial acetic acid was added, and then incubated for 30 minutes. The supernatant was aspirated to a blank plate to measure the OD value (490nm). Biofilm ratio = OD value of groups II~V / OD value of group I.

[0116] like Picture 9 c) and Picture 9 d), the color of the pure iso-CMP-1 group (II) did not change significantly compared with the control group (I), indicating that pure iso -CMP-1 had little effect on biofilm. iso -CMP-1+H2O2 group (III) is lighter in color than pure iso -CMP-1(II) group, description iso -The peroxidase (POD)-like activity of CMP-1 can destroy biofilms. iso -CMP-1+ Laser Group (IV) and iso-The color of the CMP-1+laser+H2O2 group (V) was significantly lighter than that of the other groups, and iso -CMP-1+laser+H2O2(V) group was the shallowest. By measuring the CV absorbance, further evaluation iso -The elimination effect of CMP-1 biofilm can be clearly seen iso -CMP-1+ Laser Group (IV) and iso In the -CMP-1+laser+H2O2 group (V), the biofilm elimination rates of Staphylococcus aureus were 80.82 ± 1.63% and 84.39 ± 1.09%, and the biofilm elimination rates of Escherichia coli were 67.57 ± 6.03% and 72.42 ± 5.97%, respectively, indicating that iso-CMP-1, as a photosensitizer, has a significant effect on destroying bacterial biofilms, and its biofilm destruction effect on Staphylococcus aureus is more significant.

[0117] Test Example 4: Wound healing experiment

[0118] The wound trauma model was established using 5-week-old female KM mice weighing about 25 g (purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd.). The mice were randomly divided into 6 groups: I: blank; II: control; III: iso-CMP-1; IV: iso-CMP-1+H2O2; V: iso -CMP-1+Laser;VI: iso -CMP-1+laser+H2O2, 6 mice per group. Normally raised mice were used as the blank group (6 mice) for comparison with the other 5 groups mentioned above. Before formal infection, the weight of each group of mice was weighed. Except for the blank group, the mice in other groups were anesthetized, the hair on the back was shaved, and the wound with a radius of about 5 mm was formed. Staphylococcus aureus (1×10 6 CFU / mL), 24 hours after infection, the mice were divided into groups and treated according to the groups. iso -CMP-1 concentration was 200 μg / mL, H2O2 concentration was 10 μL / mL; laser irradiation power was 1.5 W cm -2 The irradiation time was 10 min. The changes in the back wounds of the mice were recorded on days 1, 3, 5, 7, 9, and 11, and the weight of the mice was monitored every day. The weight changes and back wound area of ​​the mice were plotted. On the 11th day, the mice were anesthetized, the eyeballs were removed and blood was collected. After the blood was collected, the mice were killed, and the back wounds and major organ tissues (heart, liver, spleen, lung, kidney) were fixed with 10% formalin, and then H&E staining and Masson trichrome staining were performed. 2 mL of blood was taken from each group for routine blood tests.

[0119] Picture 10 The wound areas of mice under different treatments were shown on days 1, 3, 5, 7, 9, and 11. On day 1, obvious wound exudate was found in all groups, indicating that the establishment of the animal model was successful. As the treatment time prolonged, the wounds in each group gradually healed, but the healing rates of different groups varied greatly. iso - The wound recovery of the CMP-1 group was slower than that of the iso -The wound repair process in the CMP-1+H2O2 group was slightly accelerated, while iso -CMP-1+ laser and iso -CMP-1+laser+H2O2 showed the fastest wound healing. On the 11th day, all wounds in each group showed obvious healing. iso -CMP-1+ laser and iso - All the cases in the CMP-1+laser+H2O2 group were basically healed.

[0120] like Picture 10 As shown in Figure a) and Figure c), after 11 days of treatment, iso -The wound area of ​​mice in the CMP-1+laser+H2O2 group was almost completely healed, and the remaining wound area was 3.8% of the initial wound area, which was much smaller than that in the other groups. Picture 10 b) It can be seen that similar to the blank group mice, the body weight of the mice in the other groups did not change significantly during the entire treatment process, which indicates that iso -CMP-1 has excellent biosafety. In order to directly estimate the healing efficiency of infected wounds, histological analysis of the newly formed skin at the wounds of mice after different treatments was performed.

[0121] Picture 11 Histological analysis and Masson staining showed that new skin layers and capillaries were generated in all treatment groups. However, compared with groups I-IV, the epidermal layer of wounds treated with groups V and VI was clearer and the wound area was smaller. iso -The skin in the CMP-1+laser+H2O2 group was almost completely healed, with the appearance of hair follicles and connective tissue. These results indicate that iso -CMP-1+laser+H2O2 is very important for accelerating wound healing.

[0122] As can be seen from Figure 12, similar to the mice in the blank group, there were almost no significant differences in all the blood routine indicators. Picture 13 As can be seen from the results, after different treatments, there was no obvious organ damage or abnormality in the mouse tissues. All these results confirm that iso -CMP-1 has good in vivo biocompatibility and can be used as a safe and efficient material-based antimicrobial agent.

[0123] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing an iron phthalocyanine-based isomeric conjugated microporous polymer having triple enzyme mimicking activity, characterized in that: The preparation method is: The s-triazine containing thiophthalonitrile groups and metal salt are added to quinoline for solvothermal reaction to obtain an iron phthalocyanine-based isomeric conjugated microporous polymer having three enzyme mimicking activities; the metal salt is ferric chloride; The chemical formula of the s-triazine containing thiophthalonitrile group is C 27 H9N9S3.

2. The preparation method according to claim 1, characterized in that: The s-triazine containing a thiophthalonitrile group is 2,4,6-(3-thiophthalonitrile)-triazine, and its structural formula is: 。 3. The preparation method according to claim 1, characterized in that: The s-triazine containing a thiophthalonitrile group is 2,4,6-(4-thiophthalonitrile)-triazine, and its structural formula is: 。 4. The preparation method according to claim 1, characterized in that: The molar ratio of the s-triazine containing thiophthalonitrile group, the metal salt and quinoline is 1:2:

50.

5. The preparation method according to claim 1, characterized in that: The solvent thermal reaction is: reacting at 100° C. for 15 min, reacting at 120° C. for 30 min, and reacting at 160° C. for 18 h.

6. The iron phthalocyanine-based isomeric conjugated microporous polymer having tri-enzyme mimicking activity obtained by the preparation method according to any one of claims 1 to 5, characterized in that: The structural unit of the iron phthalocyanine-based isomeric conjugated microporous polymer having tri-enzyme mimicking activity is: or .

7. Use of the iron phthalocyanine-based isomeric conjugated microporous polymer having triple enzyme mimicking activity as claimed in claim 6 in the preparation of antibacterial drugs.

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