A porous organic polymer rich in heteroatoms and its preparation method and application

Abstract

The present invention discloses a porous organic polymer rich in heteroatoms, a preparation method and an application thereof. Under a protective atmosphere, 5, 10, 15, 20-tetrakis(4-(2,4-diaminotriazine)phenyl)porphyrin and a monoaldehyde monomer are added to an organic solvent, and then a solvent thermal reaction is carried out. After cooling, the obtained solid is washed and dried to obtain a porous organic polymer rich in heteroatoms. The monoaldehyde monomer is ferrocene carboxaldehyde, 3,5-dimethoxybenzaldehyde, p-hydroxybenzaldehyde, p-bromomethylbenzaldehyde or 1H-pyrazole-4-carboxaldehyde; the organic solvent is dimethyl sulfoxide; the protective atmosphere is nitrogen. The present invention uses a low-cost monomer containing a single aldehyde group and porphyrin as a reaction monomer, and synthesizes a series of heteroatom-rich porous organic polymers (POPs) through Schiff base polycondensation reaction, which can achieve three antibacterial effects of PTT, PDT and enzyme-like.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a porous organic polymer rich in heteroatoms, a preparation method and application thereof. Background Art

[0002] As the largest organ in the human body, the skin is the first line of defense to protect the body from physical, chemical, mechanical and pathogen damage. Human health and even life are closely related to the integrity of the skin. The loss of skin integrity will destroy the function of the skin and make the body more susceptible to infection by pathogens, especially bacterial infections. However, traditional antibiotic treatment faces the challenge of antimicrobial resistance due to abuse. In order to achieve the therapeutic purpose, excessive drugs and long treatment cycles are usually required, which may cause fatal harm to patients. To cope with this crisis, there is an urgent need to explore new low-cost, antibiotic-free antimicrobial agents or treatment strategies to effectively treat infected wound healing.

[0003] Among these newly developed drugs, organic antimicrobial agents with structural compositions similar to proteins are considered to be the most promising alternatives. Among them, high molecular weight organic antimicrobial agents with photothermal, photodynamic and enzymatic synergistic effects are widely used. Synergistic antimicrobial therapy can be roughly divided into three categories: photothermal therapy (PTT), photodynamic therapy (PDT) and enzyme-like effects. The first two methods require a specific light source to irradiate the photosensitizer to exert a phototherapy effect. The latter method is that metal catalyzes hydrogen peroxide to produce hydroxyl radicals to exert a bactericidal effect. Photothermal therapy refers to the photosensitizer converting light energy into heat energy by receiving light of a certain wavelength to kill bacteria. Photodynamic therapy refers to the photosensitizer transferring light energy at a specific wavelength to the surrounding oxygen in a certain way, thereby producing singlet oxygen to kill bacteria. Enzyme-like effects usually occur in materials containing metal structures. Metals can play a catalase-like role in acidic or weakly acidic environments, catalyzing hydrogen peroxide to produce hydroxyl radicals to inactivate bacteria. Patent application number CN202310601592.1 discloses a ferrocene-based metal organic framework material, its preparation method and application, wherein two aldehyde groups of ferrocene dicarboxaldehyde are respectively combined with two porphyrin units to polymerize to obtain a ferrocene-based metal organic framework material, but the material can only be antibacterial through PTT and CDT. Therefore, a new porous material based on ferrocene and porphyrin is needed to realize three synergistic antibacterial materials of PTT, PDT and enzyme-like. Summary of the invention

[0004] In view of the above-mentioned prior art, the purpose of the present invention is to provide a heteroatom-rich porous organic polymer and its preparation method and application. The present invention uses a low-cost monomer containing a single aldehyde group and porphyrin as reaction monomers, and synthesizes a series of heteroatom-rich porous organic polymers (POPs) through Schiff base polycondensation reaction, which can achieve three antibacterial effects of PTT, PDT and enzyme-like.

[0005] To achieve the above object, the present invention adopts the following technical solution:

[0006] The first aspect of the present invention provides a method for preparing a porous organic polymer rich in heteroatoms, the preparation method comprising:

[0007] Under a protective atmosphere, 5, 10, 15, 20-tetrakis(4-(2,4-diaminotriazine)phenyl)porphyrin and a monoaldehyde monomer are added to an organic solvent, and then a solvothermal reaction is carried out. After cooling, the obtained solid is washed and dried to obtain a porous organic polymer rich in heteroatoms.

[0008] Preferably, the monoaldehyde monomer is ferrocenecarboxaldehyde, 3,5-dimethoxybenzaldehyde, p-hydroxybenzaldehyde, p-bromomethylbenzaldehyde or 1H-pyrazole-4-carboxaldehyde; the organic solvent is dimethyl sulfoxide; and the protective atmosphere is nitrogen.

[0009] Preferably, the 5, 10, 15, 20-tetrakis(4-(2,4-diaminotriazine)phenyl)porphyrin is prepared by the following method:

[0010] Under nitrogen environment, 5, 10, 15 and 20-tetrachloro(4-cyanophenyl)porphyrin, dicyandiamide and potassium hydroxide are mixed, added into dry 2-methoxyethanol, heated to reflux, the product is washed and dried to obtain 5, 10, 15, 20-tetrakis(4-(2,4-diaminotriazine)phenyl)porphyrin.

[0011] Preferably, the molar ratio of 5, 10, 15 and 20-tetrachloro(4-cyanophenyl)porphyrin, dicyandiamide and potassium hydroxide is 0.82:4.1:5.3.

[0012] Preferably, the heating reflux temperature is 130° C. and the time is 48 h; the washing is washing with warm water at 50° C.; and the drying is vacuum drying in an oven at 70° C. overnight.

[0013] Preferably, the ratio of the added amount of the 5, 10, 15, 20-tetrakis(4-(2,4-diaminotriazine)phenyl)porphyrin, the monoaldehyde monomer, and dimethyl sulfoxide is 1 mmoL, 4 mmoL, and 20 ml.

[0014] Preferably, the temperature of the solvent thermal reaction is 180° C. and the time is 48 h; the washing is washing with N,N-dimethylformamide, methanol and tetrahydrofuran until the washing liquid is colorless; and the drying is vacuum drying at 120° C. overnight.

[0015] The second aspect of the present invention provides a heteroatom-rich porous organic polymer prepared by the above preparation method.

[0016] The third aspect of the present invention provides use of a porous organic polymer rich in heteroatoms in the preparation of antibacterial drugs.

[0017] Preferably, the heteroatom-rich porous organic polymer has synergistic antibacterial effects through photothermal, photodynamic and enzyme-like effects.

[0018] Beneficial effects of the present invention:

[0019] (1) The present invention uses low-cost monomers containing a single aldehyde group and porphyrin as reaction monomers to synthesize a series of heteroatom-rich porous organic polymers (POPs) through Schiff base condensation reaction; the heteroatom-rich porous organic polymers prepared by the present invention produce a good photothermal conversion effect when irradiated by a laser with a wavelength of 638nm, and can also catalyze oxygen and convert it into singlet oxygen, thereby achieving PTT, PDT and enzyme-like synergistic antibacterial effects.

[0020] (2) The heteroatom-rich porous organic polymer prepared by the present invention has high biocompatibility, has little effect on the cell viability of L929 cells, has a blood cell dissolution rate of less than 1% at the concentration used, and has almost no cytotoxicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 : Infrared spectra of TPI-POP, TPB-POP, TPH-POP and TPD-POP, wherein (a) infrared spectrum of TPI-POP; (b) infrared spectrum of TPH-POP; (c) infrared spectrum of TPD-POP; (d) infrared spectrum of TPB-POP;

[0022] Figure 2 : Infrared light characterization of TPF-POP, including (a) infrared spectrum of TPF-POP; (b) solid carbon spectrum of TPF-POP 13 C NMR; (c) low temperature N2 absorption isotherm of TPF-POP at 77K; (d) pore size distribution curve of TPF-POP;

[0023] Figure 3 : XRD and thermogravimetric curves of TPF-POP, where (a) XRD of TPF-POP; (b) thermogravimetric curve of TPF-POP;

[0024] Figure 4 : SEM and TEM images of TPF-POP, where (ac) SEM images of TPF-POP at 100nm and 1, 10μm scales, (dg) TEM images of TPF-POP at 500, 200, 50 and 10nm;

[0025] Figure 5 : Element mapping of TPF-POP, including (a) HAADF map of TPF-POP; (b) distribution map of iron in TPF-POP; (c) distribution map of carbon in TPF-POP; (d) distribution map of nitrogen in TPF-POP;

[0026] Figure 6 : EDS diagram and mapping of TPF-POP;

[0027] Figure 7 : Characterization of the stability of TPF-POP, including (a) water stability of TPF-POP; (b) light stability of TPF-POP;

[0028] Figure 8 :Photothermal performance of TPF-POP, including (a) photothermal effect of TPF-POP suspensions of different concentrations at 638nm for 10min; (b) photothermal effect of TPF-POP (200μg / mL) under 638nm laser irradiation of different powers for 10min; (c) thermal imaging of TPF-POP irradiation at a certain concentration; (d) thermal cycle diagram of TPF-POP; (e) temperature change curve of TPF-POP (200μg / mL) after laser irradiation at 638nm, 1.0W / cm2; (f) photothermal conversion efficiency of TPF-POP;

[0029] Fig. 9 : At a wavelength of 638nm and a power density of 1W / cm 2 Photodynamic diagram of TPF-POP under laser irradiation;

[0030] Fig.10 :Verification of the photodynamic, photothermal, and enzyme-like effects of TPF-POP, including (a) the ultraviolet absorbance of TMB, TMB+H2O2, and TPF-POP+TMB+H2O2 at a wavelength of 650nm; (b) the effect of 638nm laser irradiation on the catalytic ability of TPF-POP; (c) the absorbance of a certain concentration of TPF-POP (200μg / mL) in a PBS environment with different pH values; (d) the absorbance of different concentrations of TPF-POP at a wavelength of 650nm when the pH value is 5.5;

[0031] Fig.11: Biocompatibility of TPF-POP, including (a) hemolytic effect of different concentrations of TPF-POP; (b) cytotoxicity of different concentrations of TPF-POP;

[0032] Fig.12 :The synergistic antibacterial effect of TPF-POP+H2O2+laser, including (a) photos of the synergistic antibacterial treatment of each group, (b) statistics of bacterial survival rate of the synergistic antibacterial treatment of each group;

[0033] Fig.13 :The ability of each group to treat against Escherichia coli and Staphylococcus aureus, including (a) TEM images of each group of Escherichia coli and Staphylococcus aureus after treatment; (b) SYTO 9 / PI staining images of 8 groups of Escherichia coli and Staphylococcus aureus;

[0034] Fig.14 : Changes in wounds and body weight of mice after treatment, including (a) wound images of mice on days 1, 3, 6, and 9 after treatment; (b) wound healing rates of mice in each group on days 1-9; (c) daily body weight changes of mice;

[0035] Fig.15 :Skin staining of mouse wounds and organ sections after treatment, including (a) H&E staining of the skin wounds of mice in the control group and TPF-POP+H2O2+laser group; (b) schematic diagram of mouse organ sections;

[0036] Fig.16 :The results of routine blood tests in different groups on the 9th day; (a) red blood cell test results; (b) mean red blood cell volume test results; (c) hemoglobin test results; (d) mean hemoglobin concentration test results; (e) white blood cell test results; (f) mean hemoglobin content test results;

[0037] Fig.17 :NMR data of 5, 10, 15, 20-tetrakis(4-(2,4-diaminotriazine)phenyl)porphyrin, 1H-NMR: (500MHz DMSO-d6): δ=8.92 (s, 8H, H-1); 8.71 (d, 8H, H-2); 8.35 (d, 8H, H-3); 6.89 (s, 16H, H-4);

[0038] Fig.18 : Schematic diagram of reaction synthesis. DETAILED DESCRIPTION

[0039] 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.

[0040] As introduced in the background technology section, compared with traditional inorganic metal materials, organic metal photothermal agents have good biocompatibility and low toxicity, which makes them safer for use in organisms. So far, many antibacterial metal organic polymers have been successfully prepared for antibacterial applications. There are reports on the combined use of PTT and CDT for antibacterial purposes, but there are no reports on the synergistic antibacterial effects of PTT, PDT and enzyme-like agents.

[0041] Based on this, the object of the present invention is to provide a porous organic polymer rich in heteroatoms and a preparation method and application thereof. Fig.18 As shown, the present invention uses 5, 10, 15, 20-tetrakis(4-(2,4-diaminotriazine)phenyl)porphyrin as a skeleton material for polymerization, and then reacts the aldehyde group on the monoaldehyde monomer with the two amino groups on 2,4-diaminotriazine, so that the monoaldehyde monomer is connected to the skeleton material to form a polymerization network, and a porous organic polymer rich in heteroatoms is obtained. The combination of photoactive triazine porphyrin and redox active ferrocene carboxaldehyde gives the special persistent organic pollutant (TPF-POP) self-enhanced photothermal, photodynamic and peroxidase-like activities. The triazine porphyrin and the monoaldehyde monomer can play a synergistic role and can act as an intelligent synergistic antibacterial platform. It is worth noting that in vitro experiments have verified that TPF-POP can not only effectively excite oxygen and low levels of hydrogen peroxide (H2O2) in the environment to generate reactive oxygen species (ROS), but also convert light energy into local heat, exerting a photothermal effect, and has an effective bactericidal effect on Staphylococcus aureus and Escherichia coli at low concentrations (200 μg / ml). It also has excellent biocompatibility and almost no cytotoxicity at antibacterial concentrations.

[0042] 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.

[0043] 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.

[0044] Example:

[0045] (1) Under nitrogen atmosphere, a mixture of 5, 10, 15 and 20-tetrachloro(4-cyanophenyl)porphyrin (586 mg, 0.82 mmol), dicyandiamide (345 mg, 4.1 mmol) and potassium hydroxide (300 mg, 5.3 mmol) was added to dry 2-methoxyethanol (20 ml) and heated under reflux at 130°C for 48 hours. The product was then washed with 100 mL of warm water and dried in a vacuum oven at 70°C overnight to obtain 5, 10, 15, 20-tetrakis(4-(2,4-diaminotriazine)phenyl)porphyrin (H2TDPP), the NMR data of which are shown in the figure. Fig.17 : 1H-NMR: (500MHz DMSO-d6): δ = 8.92 (s, 8H, H-1); 8.71 (d, 8H, H-2); 8.35 (d, 8H, H-3); 6.89 (s, 16H, H-4).

[0046] (2) Preparation of TPF-POP: Ferrocene carboxaldehyde (817 mg, 4 mmol, denoted as F-CHO), 3,5-dimethoxybenzaldehyde (664 mg, denoted as D-CHO), p-hydroxybenzaldehyde (488 mg, denoted as H-CHO), p-bromomethylbenzaldehyde (796 mg, denoted as B-CHO) and 1H-pyrazole-4-carboxaldehyde (384 mg, denoted as I-CHO) were added to five 100 ml round-bottom flasks, respectively. H2TDPP (126 mg, 1 mmol) prepared in step (1) was added to each flask, and nitrogen was introduced three times, followed by the addition of DMSO (20 ml). The reaction system was degassed again and maintained for 10 min, and then the reaction mixture was transferred to an autoclave and purged with nitrogen. The reaction mixture was heated at 180°C for 48 h under nitrogen protection. After cooling to room temperature, the reactants were filtered to obtain a black fluffy substance, which was washed with methanol, DMF and THF in sequence. The synthesized solid was dried in a vacuum oven at 120°C overnight to obtain black powders, which were successively recorded as TPF-POP, TPD-POP, TPH-POP, TPB-POP and TPI-POP. See the specific synthesis route for details. Fig.18 .

[0047] Characterization:

[0048] (1) Determination of infrared spectrum of catalyst: The structure of the catalyst was roughly determined by infrared spectrum. About 2 mg of H2TDPP, Fc-CHO, I-CHO, Br-CHO, Hy-CHO and DMe-CHO as well as TPI-POP, TPH-POP, TPD-POP, TPB-POP and TPF-POP were respectively ground with dry potassium bromide powder in a mortar and kept dry at all times. Then, the mixture was placed in a tabletting mold and pressed into a transparent, crack-free tablet. The tablet was placed in an infrared spectrum scanner at 4000-400 cm-2 The infrared spectra of TPF-POP, TPD-POP, TPH-POP, TPB-POP and TPI-POP prepared in the embodiment are as follows: Figure 1 and Figure 2 (a) shown.

[0049] The construction of porous polyamide networks based on TP was preliminarily studied using Fourier transform infrared spectroscopy (FTIR). Figure 2 and Figure 3 As shown in (a), the Fourier transform infrared spectra of the synthesized TP-based porous polyaromatic compounds all integrate the porphyrin characteristics belonging to the TP unit (at 1590-1300, 3500-3300 and 970 cm -1 ), revealing that the TP unit was successfully introduced into the porous framework. For example, in addition to the characteristic vibration of the TP unit, the TPF-POP obtained by copolymerization of TP and Fc-CHO also showed the characteristic peaks of ferrocene (1490, 1103 and 809 cm -1 Meanwhile, distinct NH vibration bands can be clearly detected from the synthesized POPs, from primary amines (3315 and 3188 cm -1 ) to secondary amines (3401 and 1200 cm -1 ), and the disappearance of aldehydes due to the polymerization of acetal ammonia (1679 cm -1 ) and the appearance of methylene (2917cm -1 ).

[0050] (2) Figure 2 (b) shows the solid state of TPF-POP 1 H- 13 C NMR spectrum, from which strong carbon signals at 160, 130, 39 and 15 ppm can be clearly observed. The former belongs to the carbon on the triazine ring. The signal at 130 ppm belongs to the aromatic carbon in the porphyrin and ferrocene units. The remaining two peaks belong to the resonance of the saturated tertiary carbon formed by the polymerization of the primary amine group and the aldehyde group, which further provides strong evidence for the formation of the amino connection.

[0051] (3) The pore distribution of the framework is understood through the N2 adsorption-desorption curve and pore size distribution curve. Figure 2 As can be seen in (c), TPF-POP shows a typical type IV isotherm with a large hysteresis loop at the branch of the adsorption-desorption curve, indicating that its pore structure is mainly mesoporous. In addition, the rapid adsorption in the low pressure range (P / P0 < 0.05) shows the coexistence of macropores in the porous framework. Figure 2The pore size distribution based on the BJH model in (d) also verifies the hierarchical pore structure. It can be seen that the pore size distribution is mainly distributed in the mesopore range (2-50nm), with the main peak at 5m and two secondary peaks at 2.5nm and 3.0nm respectively.

[0052] (4) XRD and thermogravimetric analysis were used to further understand the structure and stability of TPF-POP. Figure 3 As shown in (a), similar to previous reports, the powder X-ray diffraction of TPF-POP only shows a large broad peak around 25°, indicating that it has an amorphous structure. Figure 3 Thermogravimetric analysis in (b) shows that TPF-POP has excellent thermal stability, with no weight loss below 100°C and only 10% weight loss at around 200°C.

[0053] (5) Scanning electron microscope (SEM) and transmission electron microscope (TEM): TPF-POP was adhered to a mica substrate with conductive glue and sprayed with gold, and then the sample morphology was observed under SEM. The TPF-POP methanol dispersion after ultrasonic dispersion was dripped onto a copper mesh, and the observation sample was obtained after air drying. The sample was loaded into TEM to observe the sample morphology and take pictures, and the sample element analysis diagram and the atomic content table of various elements were derived.

[0054] Scanning electron microscopy images ( Figure 4 ac) and transmission electron microscopy (TEM, Figure 4 dg) The particle morphology of the prepared TPF-POP was also studied. Scanning electron microscope images showed that TPF-POP was a bulk material composed of interlaced nearly spherical particles with inherent macroporous voids. At the same time, TEM results also showed that TPF-POP was spherical in shape ( Figure 4 g), has a loose and porous microstructure.

[0055] (6) Elemental spectrum ( Figure 5 ) and the corresponding energy dispersive X-ray spectroscopy (EDS) results ( Figure 6 ) showed that C (92.17%), N (3.75%) and Fe (4.06%) elements coexisted and were evenly distributed on the TPF-POP matrix.

[0056] (7) The photostability and water stability of TPF-POP were also measured to evaluate its practical applicability: Figure 7 As shown in (a), the UV-visible absorbance of TPF-POP dispersed in water (200 μg / mL) did not change much at different time periods (0 day and 15 day), indicating that it has good water stability. 2) The UV-visible absorption of TPF-POP collected after irradiation is almost overlapping. These results indicate that TPF-POP has excellent light stability and water stability.

[0057] (8) Photothermal performance test of TPF-POP: 1 mL of 0, 100, 200, 300, 400, and 500 μg / mL TPF-POP aqueous suspension was added to a 1.5 mL EP tube, and then the power density was 1 W / cm 2 The 638nm laser was irradiated for 10 minutes. The temperature change during the irradiation process was measured using a thermal imager and the temperature change at different time points was recorded. Data was recorded at different time points. In addition, 0.5, 0.8, 1, 1.2 and 1.5W / cm 2 The near-infrared laser was irradiated with a 200μg / mL TPF-POP aqueous suspension for 10 minutes, and the temperature change at a specific time at each power density was recorded with a thermal imager. The data was recorded every 50 seconds and a graph was drawn. The photothermal stability of TPF-POP was evaluated by an on / off cycle irradiation experiment. 1mL of a 200μg / mL TPF-POP aqueous suspension was irradiated with a 638nm laser for 10 minutes, and then the laser was turned off and the TPF-POP material was naturally cooled to the starting temperature.

[0058] like Figure 8 (a) shows that at 1.0W / cm 2 At a fixed laser power, the temperature of the TPF-POP water suspension increased significantly with the increase of irradiation time (0-10 minutes) or TPF-POP concentration. When the material concentration was 100 μg / mL, the temperature reached 50°C after 10 minutes of laser irradiation, and when the concentration was 500 μg / mL, the temperature quickly rose to 68°C within 10 minutes. Since pure water has no light and heat conversion ability, under the same experimental conditions, the temperature of pure water hardly changed. Figure 8 As shown in (b), at a fixed concentration (200 μg / mL), the laser power increased from 0 to 1.5 W / cm 2 The final temperature range is from 0 to 65°C. To further evaluate the photothermal performance of TPF-POP, a digital thermal imager was used to monitor the temperature curve. Figure 8 As can be seen in (c), TPF-POP exhibits a wide temperature change region from light blue (33.3°C) to bright brown (56.5°C) within 10 minutes of irradiation, indicating that TPF-POP has good photothermal conversion performance. In order to evaluate the photothermal stability of TPF-POP, a heating / cooling cycle was also performed, in which the laser was turned off after 10 minutes of irradiation to allow natural cooling. Figure 8 The corresponding results in (d) demonstrate that TPF-POP has superb stability, and its heating / cooling curves are almost overlapped after 4 consecutive switching cycles.

[0059] During the whole experiment, the temperature change was recorded at different time points, and the photothermal conversion efficiency was calculated using the following formula (S1):

[0060] η(%)=[hS(T max -T surr )-Q dis ] / I(1-10 -A 638 )(Formula 1)

[0061] Where h is the heat transfer coefficient, S is the surface area of ​​the container, Tmax is the equilibrium temperature after 10 minutes of irradiation, I represents the 638nm laser power (1W / cm 2 ); A638 is the absorbance of TPF-POP suspension aqueous solution at 638 nm. The value of hS is determined according to the following formula (S2):

[0062] hS=m H2O C H2O / τS(Formula 2)

[0063] In the formula, m H2O is the mass of water solvent (1g), C H2O is the heat capacity of water solvent (4.2 J / g); S is the negative natural logarithm of the cooling time and temperature slope, determined by the following formula (S3):

[0064] t=-τS(Inθ)(Formula 3)

[0065] "θ" is defined as the ratio of ΔT to ΔTMax, and t is the time it takes to cool from the maximum temperature to room temperature.

[0066] In summary, for further verification, a single cycle cooling process was selected for analysis, and the light-to-heat conversion efficiency (η) of TPF-POP was obtained based on the cooling data obtained from the above formula. Figure 8 As shown in (f), the η of TPF-POP is calculated to be 54.6%.

[0067] (9) Photodynamic performance of TPF-POP: TPF-POP at 638nm wavelength and power density of 1W / cm 2 The photodynamic properties under irradiation were studied.

[0068] First, 1,3-diphenylisobenzofuran (DPBF) was used as a photodynamic probe and dissolved in dimethyl sulfoxide. The singlet oxygen ( 1O2). DPBF will produce a peak at around 400nm. The more singlet oxygen is produced, the more DPBF is consumed, and the lower the peak is. On this basis, 3mL of 200μg / mL TPF-POP DMSO solution was prepared, ultrasonically dispersed, and 50μL of DPBF probe was added.

[0069] like Fig. 9 As shown, with the increase of irradiation time, the curve gradually decreases, indicating that within 5 minutes of irradiation, TPF-POP produces a large amount of singlet oxygen, further indicating that TPF-POP has good photodynamic performance.

[0070] (10) Fenton performance test of TPF-POP: Taking advantage of the fact that TMB can change color with active oxygen and has a specific absorption peak at a wavelength of 652 nm, the active oxygen released by TPF-POP polymer can be detected in vitro. First, different concentrations of TPF-POP (100, 200, 300, 400, 500 μg / mL) were mixed with TMB (75 μl), PBS (pH = 5.5) and H2O2 (75 μl), and PBS was added to 1 mL. The mixture was kept in the dark for 10 minutes, and then the filter membrane was aspirated with a 1 mL syringe and added to a cuvette. The wavelength of 500-800 nm was measured under a UV spectrophotometer. The absorbance was measured at a wavelength of 800 nm. Similarly, the concentration of TPF-POP was fixed at 200 μg / mL, the pH value of PBS was set to 1.5, 2.5, 3.5, 4.5, 5.5, 6.5, mixed with TMB and H2O2 into 1 mL static filter membrane, and then the absorbance at 500-800 nm was measured under ultraviolet light.

[0071] like Fig.10 As shown in (a), in the absence of TPF-POP, TMB and TMB+H2O2 groups have no absorption at 650nm and no color change. After adding TPF-POP, obvious absorption peaks and obvious color changes appeared, indicating that H2O2 generated hydroxyl radicals. Fig.10 As shown in (b), compared with the group without laser, the peak around 650nm showed a sharp increase in absorption under laser irradiation. This indicates that laser irradiation can significantly increase the activity of the enzyme. Fig.10 (c) It can be seen that the enzyme activity of TPF-POP is related to the pH value, and its color changes with the change of pH value. It is worth noting that TPF-POP shows stronger catalytic activity under weakly acidic conditions, which is similar to the optimal microenvironment for bacterial growth, and reaches its maximum at a pH of 4.5. At the same time, the enzyme activity of TPF-POP is also closely related to its concentration. Fig.10(d) It can be seen that as the concentration of TPF-POP increases, the absorption value also increases. The above results show that TPF-POP has a good antibacterial effect in the microenvironment where bacteria grow, providing a good foundation for the next application.

[0072] Test Example 1: In vitro biocompatibility test

[0073] (1) Hemolysis test

[0074] Place fresh blood from mice into a blood collection tube, centrifuge at 15,000 rpm for 10 min, discard the supernatant to collect red blood cells, add an equal amount of PBS and wash evenly three times until colorless. Add PBS to the centrifuged red blood cells at a volume ratio of 3:11 to obtain a mixed solution. Add the mixed solution to TPF-POP suspensions of different concentrations (prepared with PBS), the volume ratio of the mixed solution to the TPF-POP suspension is 1:9, incubate at 37°C for 3 h, centrifuge at 12,000 rpm for 15 min, and measure the absorbance at 540 nm using a UV-visible spectrometer. Using distilled water as the positive control and PBS as the negative control, use the following formula to calculate the amount of hemolysis:

[0075] Hemolysis amount (%)=(A-An) / (Ap-An)×100%.

[0076] "A" is the absorbance of the supernatant after adding TPF-POP to erythrocytes. "An" is the absorbance of the supernatant after adding PBS to erythrocytes (negative control). "Ap" is the absorbance of the supernatant after adding distilled water to erythrocytes (positive control).

[0077] from Fig.11 (a) It can be seen that the hemolysis rate of TPF-POP at different concentrations (50-400 μg / mL) is less than 1%, indicating that it has good blood biocompatibility.

[0078] (2) Cytotoxicity assay

[0079] In a 96-well plate, the seeding density per well was 8 × 10 3 L929 cells were added to each well, and 100 μL of cell suspension was added to the surrounding wells to prevent excessive evaporation of cells. After 24 hours of culture, different concentrations of TPF-POP in PBS solution (0, 50, 100, 150, 200, 300, 400 μg / μL) were added. After 24 hours, the supernatant was discarded, and then the same volume of PBS solution containing 10 μL MTT was added. After 4 hours, the supernatant was aspirated and 100 μL DMSO was added to dissolve MTT. After about 5 minutes, it was placed in an ELISA reader and measured at a wavelength of 490 nm. Each group was measured three times in parallel.

[0080] like Fig.11 As shown in (b), with the increase of TPF-POP concentration, the cell viability decreased slightly, but even at a high concentration of 400 μg / mL, the cell viability could still be maintained above 80%, indicating that the cytotoxicity of TPF-POP was negligible.

[0081] Test Example 2: In vitro antibacterial test

[0082] (1) Bacterial culture: This experiment used E. coli and S. aureus as representatives of Gram-negative and Gram-positive bacteria, and subcultured them to the second generation of bacteria to complete the following experiments. The specific culture method of the second generation of bacteria is: revive the bacteria frozen in the refrigerator, thaw the frozen bacteria at 37°C, take 100μL of the bacterial solution in a shaking tube containing 5mL LB liquid culture medium, place it on a shaking table and culture it at a constant temperature for 12h, take 100μL of the cultured bacterial solution and place it in a 2mL EP tube containing 900μL LB liquid culture medium, and then dilute it according to the gradient method of 10 -2 Dilute 5-10 tubes, take 100μL of the bacterial solution in each tube, use a coating rod to evenly spread it on a culture dish containing LB solid culture medium, incubate at 37℃ for 24h, observe the clone morphology and colony count, and take the culture dish with about 1000 colonies as the first generation of bacteria. Use a bacteria picking stick to pick out a colony from the first generation of bacteria and add it to a shaking tube containing 5mL liquid culture medium. Cultivate it according to the method of culturing first generation bacteria to obtain a culture dish with about 1000 colonies as the second generation of bacteria.

[0083] (2) The plate count method was used to evaluate the in vitro antibacterial activity of TPF-POP. Escherichia coli and Staphylococcus aureus were used as models of Gram-negative and Gram-positive bacteria. Single colonies of these two bacteria were transferred to lysogeny broth (LB) tubes containing 5 mL of liquid culture medium shake tubes and incubated overnight at 37°C. The in vitro antibacterial test was divided into 7 groups as required: PBS group (I), H2O2 group (II, 10 μL), H2O2+laser group (III, H2O2 was added with 10 μL, and the laser parameters were 638 nm, 1.0 W / cm 2, irradiation time of 10min), TPF-POP group (IV, 200μg / mL), TPF-POP+laser group (V, TPF-POP concentration of 200μg / mL, laser parameters refer to the laser group), TPF-POP+H2O2 group (VI, TPF-POP concentration of 200μg / mL, H2O2 added 10μL), and TPF-POP+H2O2+laser group (VII, refer to the above), the above groups were filled to 1mL with PBS (TPF-POP concentration of 200μg / mL is the final concentration). 1mL of each of the above groups was used to treat bacteria, and each group was placed in a constant temperature shaking window at 37℃ for incubation for 12 hours. Then after the bacterial solution was incubated, 80μL was taken from each tube and transferred to LB solid culture medium and spread evenly with a spreader, incubated in an incubator at 37℃ for 24 hours, and the morphology of the colonies was observed and the colonies were counted.

[0084] like Fig.12 As shown in (a), similar to the PBS group (I), the bacterial survival rates of H2O2 (II), H2O2 + laser (III), and TPF-POP (IV) were above 90%, and there was no obvious bactericidal effect. The bacteria in the H2O2 (II) and H2O2 + laser (III) groups decreased slightly, probably due to the inherent bactericidal effect of hydrogen peroxide itself, but there was almost no change. This is because laser irradiation alone (638nm, 1.0W / cm 2 , irradiation time of 10min, PBS made up to 1mL) sterilization, there is no sterilization effect when no drugs are added, the sterilization rate is basically 0%, so the sterilization rate of H2O2 (II) and H2O2 + laser (III) has almost no change. And it can also be explained that the effect of laser cannot catalyze hydrogen peroxide to kill more bacteria. However, in the case of TPF-POP + laser, due to the combined use of PDT and PTT, the colony forming units (cfu) of Staphylococcus aureus and Escherichia coli dropped significantly to 33% and 21%, respectively. At the same time, due to the enzymatic catalysis of TPF-POP, the number of CFU in the TPF-POP + H2O2 treatment group was also significantly reduced, and the survival rates of Staphylococcus aureus and Escherichia coli were 51% and 56%, respectively. It is worth noting that in the TPF-POP+H2O2+laser treatment group, the detected bacterial survival rate was very low, almost all bacteria died, and the bactericidal rates of Staphylococcus aureus and Escherichia coli were both greater than 99.9%, which was attributed to the powerful antibacterial effect synergistically induced by PDT, PTT, and enzyme therapy. Fig.12 (b) The histogram can more intuitively show the bactericidal effects of different groups.

[0085] Test Example 3: Bacterial Transmission Electron Microscopy

[0086] The cells were grouped according to the method in Experimental Example 2. Afterwards, the bacterial cells of Staphylococcus aureus and Escherichia coli were fixed with 2.5wt% glutaraldehyde solution at 4°C for 2 hours, rinsed three times with PBS, embedded with agar, and sealed. The bacteria were continuously dehydrated for about 10 minutes at room temperature with ethanol solutions of different concentrations (30wt%, 50wt%, 70wt%, 90wt%, 95wt% and 100wt%), and then the ethanol was removed by acetone at room temperature for about 3 hours, embedded with embedding medium gradient infiltration, negatively stained, and sliced ​​on a nickel mesh, which was placed under TEM for observation to capture the bacterial morphology.

[0087] like Fig.13 As shown in (a), the surfaces of Staphylococcus aureus and Escherichia coli treated with PBS, TPF-POP, H2O2 and H2O2+laser were similar to those of the PBS group, still smooth, without signs of rupture, and with clearly visible flagella. However, after treatment with TPF-POP+H2O2, TPF-POP+laser and TPF-POP+H2O2+laser, different degrees of obvious morphological changes occurred. Due to the synergistic bactericidal effect of local illumination and enzymes, the bacteria treated with TPF-POP+H2O2+laser were most severely damaged, with cell lysis combined with cell outflow, so the bactericidal effect of TPF-POP+H2O2+laser was the most significant.

[0088] Test Example 4: Bacterial live / dead staining test:

[0089] The groups were divided according to the method in Experimental Example 2. Then, 100 μL of bacterial suspension from each group was mixed with 20 μL of SYTO-9 (1.0×10 -3 M) and 20 μL PI (1.5×10 -3 M) and incubated in the dark at 37°C for 15 min. After staining, the samples were centrifuged in PBS to remove excess SYTO-9 and PI. The bacteria were then resuspended in 50 μL PBS and placed on the surface of the slide. Images of E. coli or S. aureus were then captured using a fluorescent inverted microscope.

[0090] from Fig.13 (b) It can be seen that, consistent with the results of imaging analysis and plate counting, the bacteria treated with (I) PBS, (II) H2O2, (III) H2O2+laser, and (IV) TPF-POP only showed strong green fluorescence. However, under the treatment of the other three groups, the red fluorescent spots increased significantly. In particular, the bacteria cultured with the TPF-POP+H2O2+laser treatment group only showed red fluorescence. This further proves the superiority of the self-enhanced PDT / PTT / enzyme activity antibacterial effect.

[0091] Test Example 5: Animal Test

[0092] (1) In vitro wound healing experiment:

[0093] 6-week-old bal b / c mice (purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd.) were randomly divided into 8 groups, 6 in each group. Before treatment, except for the blank group (no treatment mice), all the hair on the back of each group of mice was shaved, a wound with a diameter of about 6mm was punched out with a hole puncher, and 25 μL of cultured Staphylococcus aureus was dripped in. After 24 hours of infection, each group of mice was set according to different groups (divided into control group (PBS group), H2O2 group, H2O2+ laser group, TPF-POP group, TPF-POP+ laser group, TPF-POP+H2O2 group and TPF-POP+H2O2+ laser group, specifically referring to Test Example 2), and treated on the 1st, 3rd, 6th and 9th days respectively. On the second day, each group of mice was treated according to the requirements of different group settings, and the wounds of each group of mice were photographed and weight changes were monitored on the 1st, 3rd, 6th and 9th days respectively. On the 9th day, about 2mL of blood was collected from the ophthalmic artery, and each blood sample was subjected to routine blood tests. The mice were sacrificed, and the skin tissues and important organs (heart, liver, spleen, lungs, and kidneys) at the wounds were excised and immersed in 4% paraformaldehyde solution in a 50 mL centrifuge tube, followed by H&E staining and Masson staining.

[0094] like Fig.14 As shown in (a), each group had different degrees of wound healing on the 3rd and 6th days. Similar to the PBS treatment group, the TPF-POP group, hydrogen peroxide group, and H2O2+laser group also showed weak wound healing. In contrast, the wounds in the TPF-POP+laser, TPF-POP+H2O2, and TPF-POP+laser+H2O2 groups healed well, especially the TPF-POP+laser+H2O2 treatment group, which was similar to the previous antibacterial results. In addition, the wound healing rate of the TPF-POP+laser+H2O2 group reached more than 90% on the 9th day, and there was still obvious scab on the wound sites of the other groups. At the same time, Fig.14 The wound area quantification graph in (b) also shows the antibacterial effect of the TPF-POP+laser+H2O2 group, which can significantly promote wound healing. Fig.14 (c) shows the weight changes of mice during treatment. It can be seen that, except for the blank group, the weight of mice in other groups decreased slightly after the first day of the infection model establishment. After that, the weight quickly returned to normal or even increased, which shows that TPF-POP has super strong biosafety and also illustrates the accuracy of the cytotoxicity experiment.

[0095] H&E staining was used for histological evaluation. Fig.15As shown in (a), the PBS-treated group showed incomplete skin wounds with obvious scabs, unhealed subcutaneous tissue, and a large number of inflammatory cells under a high-power microscope. Compared with the PBS group, the number of inflammatory cells in the wounds of the TPF-POP+laser+H2O2 group was significantly reduced. Fig.15 (b) Under high magnification, significant regeneration of epidermal tissue and even skin structure, as well as a large number of blood vessels, blood cells, and collagen fibers were observed, indicating that the wound healing effect was good.

[0096] (2) Routine blood test

[0097] In order to study the in vivo biocompatibility of TPF-POP, blood was drawn from the eyes of the above mice for routine blood tests. Fig.16 As shown, no significant differences were found in the blood routine values ​​among the groups.

[0098] 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. Use of a porous organic polymer rich in heteroatoms in the preparation of antibacterial drugs, characterized in that: The heteroatom-rich porous organic polymer synergistically fights bacteria through photothermal, photodynamic and enzyme-like effects; The heteroatom-rich porous organic polymer is prepared by the following method: Under a protective atmosphere, 5,10,15,20-tetrakis(4-(2,4-diaminotriazine)phenyl)porphyrin and a monoaldehyde monomer are added to an organic solvent, and then a solvent thermal reaction is carried out. After cooling, the obtained solid is washed and dried to obtain a porous organic polymer rich in heteroatoms; the monoaldehyde monomer is ferrocenecarboxaldehyde, 3,5-dimethoxybenzaldehyde, p-hydroxybenzaldehyde, p-bromomethylbenzaldehyde or 1H-pyrazole-4-carboxaldehyde; the organic solvent is dimethyl sulfoxide; the ratio of the added amounts of 5,10,15,20-tetrakis(4-(2,4-diaminotriazine)phenyl)porphyrin, the monoaldehyde monomer and dimethyl sulfoxide is 1mmoL:4mmoL:20ml.

2. The use according to claim 1, characterized in that The protective atmosphere is nitrogen.

3. The use according to claim 1, characterized in that The 5,10,15,20-tetrakis(4-(2,4-diaminotriazine)phenyl)porphyrin is prepared by the following method: Under a nitrogen environment, 5,10,15,20-tetrachloro(4-cyanophenyl)porphyrin, dicyandiamide and potassium hydroxide are mixed, added into dry 2-methoxyethanol, heated to reflux, and the product is washed and dried to obtain 5,10,15,20-tetrakis(4-(2,4-diaminotriazine)phenyl)porphyrin.

4. The use according to claim 3, characterized in that The molar ratio of the 5,10,15,20-tetrachloro(4-cyanophenyl)porphyrin, dicyandiamide and potassium hydroxide is 0.82:4.1:5.

3.

5. The use according to claim 3, characterized in that: The heating reflux temperature is 130° C. and the time is 48 hours; the washing is washing with warm water at 50° C.; and the drying is vacuum drying in an oven at 70° C. overnight.

6. The use according to claim 1, characterized in that: The temperature of the solvent thermal reaction is 180° C. and the time is 48 hours; the washing is washing with N,N-dimethylformamide, methanol and tetrahydrofuran until the washing liquid is colorless; the drying is vacuum drying at 120° C. overnight.

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