A GO-TAPP microporous composite material and its preparation method and application

Abstract

The present invention discloses a GO-TAPP microporous composite material and a preparation method and application thereof, belonging to the field of biomedical technology. 5,10,15,20-tetrakis(4-ethynylphenyl)-porphyrin, bis(triphenylphosphine)palladium chloride and cuprous iodide are added to tetrahydrofuran and mixed evenly in the dark, graphene oxide and triethylamine are added, mixed in the dark, and then reacted, and a black solid is obtained after the reaction is completed, and the GO-TAPP microporous composite material is obtained after washing and drying. The GO-TAPP microporous composite material with graphene oxide and porphyrin as structural units of the present invention can be synergistically antibacterial through PTT and PDT, and has good biocompatibility, no toxic side effects, and a hemolysis rate of less than 2% for cells. GO-TAPP is in sheet form and can be directly cut and applied for direct use without post-processing, thereby improving its application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a GO-TAPP microporous composite material, a preparation method thereof, and an application thereof. Background Art

[0002] Bacteria are ubiquitous in nature and all living organisms, including humans. We are constantly exposed to different types of bacteria. Among them, the most prominent is the emergence of drug-resistant bacteria caused by the abuse of antibiotics, which brings great difficulties and challenges to public health systems around the world. Therefore, there is a great hope for some alternative antibacterial strategies that can reduce costs, improve efficacy, and reduce drug resistance.

[0003] So far, various materials that can be used alone or in combination with other drugs as antibacterial agents have been explored as alternatives to traditional antibiotics. Among them, phototherapy antibacterial is a non-invasive alternative treatment strategy with relatively low risk for treating microbial infections. Phototherapy antibacterial therapy can be roughly divided into photothermal therapy (PTT) and photodynamic therapy (PDT). Both of these therapies require specific light sources to irradiate photosensitizers to exert the phototherapy effect. Different photosensitizers play different antimicrobial roles under different light source conditions. Photothermal therapy can use photosensitizers with high photothermal conversion efficiency to convert light energy into heat energy to kill bacteria by exposure to a suitable external light source. Photodynamic therapy can utilize photosensitizers with high photoreactivity to transfer light energy to surrounding oxygen through irradiation with a suitable external light source, generating highly reactive singlet oxygen, which can undergo strong oxidation reactions to kill bacteria. Currently, researchers have discovered a variety of photosensitizers with phototherapy effects. For example, the patent with application number 202310175497.X discloses a photothermal-Fenton-like reaction artificial nanozyme, a preparation method thereof, and an application thereof. An organic metal framework material ZIF-8 is embedded into a porphyrin structure to prepare a nanoagent with a bacterial-like structure. The nanotubes grown on the surface of the carrier are used to mimic the flagella of bacteria to simulate bacterial therapy; thus, efficient photothermal-Fenton combined therapy is achieved. However, these photosensitizers have some disadvantages, such as the excitation wavelength not being in the optimal light transmission region and having relatively large phototoxicity and other problems. Therefore, how to prepare a phototherapy antibacterial agent with high antibacterial activity and high biocompatibility is an urgent problem to be solved. Summary of the Invention

[0004] Aiming at the above-mentioned existing technologies, the purpose of the present invention is to provide a GO-TAPP microporous composite material, a preparation method thereof, and an application thereof. The present invention provides a graphene oxide porphyrin microporous composite material (GO-TAPP) based on graphene oxide and porphyrin as structural units. This material can synergistically antibacterial through PTT and PDT, and has good biocompatibility and no toxic side effects.

[0005] To achieve the above purpose, the present invention adopts the following technical solutions:

[0006] In the first aspect of the present invention, a method for preparing a GO-TAPP microporous composite material is provided, and the preparation method is as follows:

[0007] 5,10,15,20-Tetrakis(4-ethynylphenyl)porphyrin, bis(triphenylphosphine)palladium chloride and copper iodide are added to tetrahydrofuran and mixed evenly in the dark, then graphene oxide and triethylamine are added and mixed in the dark, and then allowed to stand and react to obtain a black solid, which is washed and dried to obtain the GO-TAPP microporous composite material.

[0008] Preferably, the addition ratio of 5,10,15,20-tetrakis(4-ethynylphenyl)porphyrin, graphene oxide, bis(triphenylphosphine)palladium chloride, copper iodide, triethylamine and tetrahydrofuran is 62 mg: 62 mg: 5.6 mg: 1.6 mg: 68 μL: 4 mL.

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

[0010] 5,10,15,20-Tetrakis(4-[(trimethylsilyl)ethynyl]-phenyl)porphyrin is added to tetrahydrofuran, tetrabutylammonium fluoride is added dropwise at -78 °C, the temperature is raised to room temperature for reaction, the reaction system is extracted with distilled water, the aqueous phase is washed and the organic phase is separated, and after removing water, it is dried to obtain a black solid, which is 5,10,15,20-tetrakis(4-ethynylphenyl)porphyrin.

[0011] Preferably, the tetrahydrofuran is anhydrous tetrahydrofuran that has been redistilled.

[0012] Preferably, the mixing is ultrasonic mixing, and the mixing time is 5 min.

[0013] Preferably, the temperature of the standing reaction is 25 °C, and the time of the standing reaction is 48 h.

[0014] Preferably, the washing is washing with acetone until the washing liquid is colorless; the drying is vacuum drying at 60 °C for 6 h.

[0015] In the second aspect of the present invention, a GO-TAPP microporous composite material obtained by the above preparation method is provided.

[0016] Preferably, the hemolysis rate of the GO-TAPP microporous composite material to cells is less than 2%.

[0017] In the third aspect of the present invention, the use of the GO-TAPP microporous composite material in the preparation of antibacterial drugs is provided.

[0018] Advantages of the present invention:

[0019] (1) The method for preparing the graphene oxide porphyrin microporous composite material GO-TAPP of the present invention is simple and can be obtained by a one-step reaction at room temperature, reducing the preparation cost.

[0020] (2) The GO-TAPP prepared by the present invention has a good photothermal conversion effect under laser irradiation at a wavelength of 638 nm and can also catalyze oxygen to convert into singlet carbon monoxide, thereby achieving a synergistic antibacterial effect.

[0021] (3) The GO-TAPP prepared by the present invention has high biocompatibility, has little effect on the cell viability of HEK-293 cells, and the hemolysis rate on blood cells is less than 2%. It can promote wound healing, which is beneficial to its application in the biological field. The GO-TAPP of the present invention is in the form of flakes and can be directly cut and applied without post-processing, improving its application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 : Monomer NMR data: 5,10,15,20-tetrakis(4-ethynylphenyl)porphyrin, 1 1H-NMR (CDCl3, ppm): -2.84 (NH, s, 2H), 3.33 (C≡CH, s, 4H), 7.89 (m-ArH, d, 8H); 8.16 (o-ArH, 8H); 8.84 (b-H, s, 8H);

[0023] Figure 2 : (a) IR spectra of GO, TAPP and GO-TAPP; (b) 13C NMR of solid-state carbon spectrum of GO-TAPP; (c) Low-temperature N2 adsorption isotherm of GO-TAPP at 77 K; (d) Pore size distribution curve of GO-TAPP; (e) Thermogravimetric curve of GO-TAPP; (f) X-ray diffraction pattern of GO-TAPP; 13 13C NMR; (c) Low-temperature N2 adsorption isotherm of GO-TAPP at 77 K; (d) Pore size distribution curve of GO-TAPP; (e) Thermogravimetric curve of GO-TAPP; (f) X-ray diffraction pattern of GO-TAPP; 2 Absorption isotherm; (d) Pore size distribution curve of GO-TAPP; (e) Thermogravimetric curve of GO-TAPP; (f) X-ray diffraction pattern of GO-TAPP;

[0024] Figure 3 : SEM and TEM of GO-TAPP: (a) SEM of GO-TAPP at a scale of 5 µm; (b) SEM of GO-TAPP at a scale of 3 µm; (c) SEM of GO-TAPP at a scale of 1 µm; (d) TEM of GO-TAPP at a scale of 500 nm; (e) TEM of GO-TAPP at a scale of 200 nm; (f) HRTEM of GO-TAPP at a scale of 20 nm;

[0025] Figure 4: SEM images of graphene oxide: (a) SEM image of graphene oxide at a scale of 10 µm; (b) SEM image of graphene oxide at a scale of 5 µm; (c) SEM image of graphene oxide at a scale of 1 µm;

[0026] Figure 5 : Element distribution maps and EDX spectra of GO-TAPP: (a) Element distribution map of GO-TAPP; (b - d) Distribution of C, N, and O elements in GO-TAPP; (e) EDX spectrum of GO-TAPP;

[0027] Figure 6 : (a) Concentration-dependent photothermal effect of GO-TAPP under 638 nm laser irradiation at 1.0 W / cm 2 ; (b) Thermal imaging pictures of the heating process of GO-TAPP (50 - 800 μg / mL) within 10 min; (c) Laser power-dependent photothermal effect of GO-TAPP under 638 nm laser irradiation at 0.5, 1.0, and 1.5 W / cm 2 respectively; (d) Relationship between the concentration of GO-TAPP and the temperature change;

[0028] Figure 7 : (a) Temperature change curve of 5 cycles of light irradiation and cooling of GO-TAPP (600 μg / mL) under 638 nm laser at 1.0 W / cm 2 ; (b) Photothermal effect of the aqueous dispersion of GO-TAPP (600 μg / mL) under 638 nm laser irradiation (1.0 W / cm 2 ), where the irradiation continues until the temperature reaches equilibrium and then the laser is turned off; (c) Cooling cycle vs. negative natural logarithm of temperature; (d) Comparison of GO-TAPP, GO, and TAPP under 638 nm laser irradiation at 1.0 W / cm 2 ;

[0029] Figure 8 : (a) UV - Vis spectra of GO-TAPP, GO, and TAPP (solvent: DMSO), (b - e) UV - Vis spectra of DPBF, GO + DPBF, TAPP + DPBF, and GO-TAPP + DPBF at different times under red light irradiation (λ = 638 nm, 1 W / cm 2 ); (f) Comparison of the decay rates (%) of DPBF induced by GO, TAPP, and GO-TAPP under red light irradiation (λ = 638 nm, 1 W / cm 2 );

[0030] Figure 9:Antibacterial ability of GO-TAPP at different concentrations after being irradiated with red light (λ = 638 nm, 1 W / cm 2 ): Bacterial culture diagrams of Staphylococcus aureus (a) and Escherichia coli (b) treated with different concentrations of GO-TAPP by red light;

[0031] Figure 10 : Viability (%) of Staphylococcus aureus (a) and Escherichia coli (b) treated with different concentrations of GO-TAPP by red light measured using the plate counting method (n = 3, error bars represent standard deviation);

[0032] Figure 11 :Antibacterial ability of TAPP, GO, and GO-TAPP under the conditions of with or without red light (λ = 638 nm, 1 W / cm 2 ): Colony photos of Staphylococcus aureus (a) and Escherichia coli (b) treated with (I) PBS, (II) TAPP, (III) GO, (IV) GO-TAPP, (V) PBS + laser, (VI) TAPP + laser, (VII) GO + laser, (VIII) GO-TAPP + laser (concentration: GO-TAPP = 600 μg / mL, GO = 300 μg / mL, TAPP = 300 μg / mL, λ = 1 W / cm 2 , 10 minutes);

[0033] Figure 12 : Fluorescence images of Staphylococcus aureus (a) and Escherichia coli (b) after incubation with live / dead stain respectively after being treated with PBS, TAPP, GO, and GO-TAPP with or without laser, bacteria co-stained with SYTO-9 and PI (concentration: GO-TAPP = 600 μg / mL, GO = 300 μg / mL, TAPP = 300 μg / mL; scale bar = 200 μm, λ = 1 W / cm 2 , 10 minutes);

[0034] Figure 13 : TEM images of Staphylococcus aureus (a) and Escherichia coli (b), cultured with (I) PBS + laser, (II) PBS, (III) TAPP + laser, (IV) TAPP, (V) GO + laser, (VI) GO, (VII) GO-TAPP + laser, and (VIII) GO-TAPP, the red tips in the figure represent the damage positions (concentration: GO-TAPP = 600 μg / mL, GO = 300 μg / mL, TAPP = 300 μg / mL; scale bar = 2 μm, λ = 1 W / cm 2 , 10 minutes);

[0035] Figure 14 Hemolysis experiment of GO-TAPP: Hemolysis rates of GO-TAPP at different concentrations (n = 3, error bars represent standard deviation);

[0036] Figure 15 Cytotoxicity experiment of GO-TAPP: Bacterial viability (%) after co-culturing GO-TAPP at different concentrations with HEK-293 cells (n = 3, error bars represent standard deviation);

[0037] Figure 16 Preparation route of graphene oxide porphyrin microporous composite material;

[0038] Figure 17 Representative photos of the back wounds of mice infected with Staphylococcus aureus at each time point by TAPP (300 μg / ml), GO (300 μg / ml), and GO-TAPP (600 μg / ml) with or without laser irradiation and the corresponding PBS control group;

[0039] Figure 18 : (a) Corresponding curve of wound contraction vs. time (%); (b) Changes in body weight of mice during treatment (n = 5, error bars represent standard deviation, *p < 0.05, **p < 0.01, ***p < 0.001);

[0040] Figure 19 Skin staining results: H&E and Masson's trichrome staining images of wound tissues in each group on the 8th day of the wound healing process (yellow circles indicate inflammatory cells, red circles indicate blood vessels, scale bar is 200μm);

[0041] Figure 20 Organ staining results;

[0042] Figure 21 Blood routine examination results of different groups (on the 8th day) (WBC, RBC, MCV, PLT, MCHC, MCH); Data are presented as mean ± standard error (n = 3); (a) White blood cell (WBC) examination results of different groups (on the 8th day); (b) Red blood cell (RBC) examination results of different groups (on the 8th day); (c) Mean corpuscular volume (MCV) examination results of different groups (on the 8th day); (d) Platelet (PLT) examination results of different groups (on the 8th day); (e) Mean corpuscular hemoglobin concentration (MCHC) examination results of different groups (on the 8th day); (f) Mean corpuscular hemoglobin (MCH) examination results of different groups (on the 8th day). Detailed implementation manners

[0043] It should be noted that the following detailed description is illustrative and aims to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present application pertains.

[0044] As introduced in the background art section, the patent with application number 202310175497.X discloses a photothermal-Fenton-like reaction artificial nanozyme and its preparation method and application. The reaction monomer cobalt-porphyrin group is adsorbed on the surface of the template ZIF-8, and in the presence of a catalyst, through covalent bond oxidative coupling, a ZIF-8 composite (PZCo-CMP) covered with cobalt-porphyrin-based conjugated porous polymer (CMP) is obtained. However, this artificial nanozyme has antibacterial activity through the combination of photothermal and Fenton reactions, and its biocompatibility needs to be improved.

[0045] Based on this, the purpose of the present invention is to provide a GO-TAPP microporous composite material and its preparation method and application. The microporous composite material GO-TAPP prepared in the present invention is prepared by an oxidative coupling method using graphene oxide and porphyrin microstructure units. As Figure 16 shown, the present invention uses graphene oxide as a template to coat several porphyrin structures on graphene oxide, forming a structure in which porphyrin is coupled and polymerized to coat graphene oxide, which has good biocompatibility and phototherapy performance that traditional photosensitizers do not have. GO-TAPP can achieve local temperature rise through photothermal conversion to achieve the effect of rupturing the bacterial cell membrane. In addition, after laser irradiation, GO-TAPP will convert the oxygen element in the surrounding environment into singlet oxygen that is harmful to bacteria, thereby lysing the bacteria. At the optimal antibacterial concentration, GO-TAPP has almost no hemolytic effect and hardly affects the growth of normal cells, making it have good biological application prospects.

[0046] 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 with specific embodiments.

[0047] The test materials used in the embodiments of the present invention are all conventional test materials in the art and can be obtained through commercial channels.

[0048] Note: The tetrahydrofuran used in the present invention is anhydrous tetrahydrofuran that has been redistilled, and its preparation method is:

[0049] Prepare a 500 mL round-bottom flask with a magnetic stir bar, add 200 mL of tetrahydrofuran to it, then add 2 - 3 g of calcium hydride to the tetrahydrofuran. Subsequently, connect a 250 mL constant-pressure dropping funnel to the mouth of the round-bottom flask, and close the knob of the constant-pressure dropping funnel to make it non-communicating with the round-bottom flask below. Install a spherical condenser at the upper end of the constant-pressure dropping funnel. Place the above device in a water bath at 80 °C for re-distillation. When there is no significant liquid residue in the round-bottom flask below the device, the re-distillation process of tetrahydrofuran ends, and the re-distilled tetrahydrofuran condenses and is stored in the constant-pressure dropping funnel. In addition, prepare about 100 molecular sieves 4A (sodium-A type molecular sieves, spherical, 3 mm - 5 mm, Sinopharm Chemical Reagent Co., Ltd.), heat them in a microwave oven at medium power for 3 min to remove water, after 3 min, use a vacuum pump to evacuate to cool the molecular sieves, repeat three times to ensure that there is no water in the molecular sieves, add them to a 500 ml brown reagent bottle, and then add the above re-distilled anhydrous tetrahydrofuran, seal and store to obtain re-distilled anhydrous tetrahydrofuran.

[0050] Graphene oxide (GO) was purchased from Shanghai Dingfen Chemical Technology Co., Ltd.

[0051] The test materials used in the embodiments of the present invention are all conventional test materials in the art and can be obtained through commercial channels.

[0052] Example

[0053] (1) Synthesis of 5,10,15,20-tetra(4-ethynylphenyl)-porphyrin (TAPP): Weigh 5,10,15,20-tetra(4-[(trimethylsilyl)ethynyl]-phenyl)-porphyrin (1.5 g, 1.5 mM) and put it into a 250 mL three-necked flask with rubber stoppers at both sides and a magnetic stir bar, and then add re-distilled anhydrous tetrahydrofuran (100 mL). Then, equip a 25 mL constant-pressure dropping funnel in the middle hole, and close the knob of the constant-pressure dropping funnel to make it non-communicating with the three-necked flask below. Place the reaction system at -78 °C for 5 min to adapt, and then add tetrabutylammonium fluoride (7.5 mL) to the constant-pressure dropping funnel. Subsequently, open the knob of the constant-pressure dropping funnel and adjust the liquid flow rate to add tetrabutylammonium fluoride to the three-necked flask at a speed of 4 drops / s. Then, slowly raise the reaction system from -78 °C (heating rate 0.43 °C / min) to room temperature (25 °C) and react for 10 h. After 10 h, mix the reaction solution with distilled water (100 mL), and wash the aqueous phase three times with dichloromethane (200 mL). Collect the organic phase, remove water with anhydrous magnesium sulfate, filter by suction, and then dry in vacuo at 60 °C for 10 h. After drying, a black-purple solid (0.75 g, yield 70.3%) is obtained. 1 H-NMR (CDCl 3, ppm): -2.84 (NH, s, 2H), 3.33 (C≡CH, s, 4H), 7.89 (m-ArH, d, 8H), 8.16 (o-ArH, 8H), 8.84 (b-H, s, 8H).

[0054] (2) Preparation of GO-TAPP composite: Weigh 5,10,15,20-tetrakis(4-(ethynylphenyl)-porphyrin (31 mg, 0.04 mM), bis(triphenylphosphine)palladium(II) chloride (2.8 mg, 0.004 mM) and copper(I) iodide (0.8 mg, 0.004 mM) and add them into a 5 mL brown vial. Then add dry tetrahydrofuran (2 mL). Ultrasonicate the reaction system for 5 min, then add graphene oxide (31 mg) and triethylamine (34 μL, 0.25 mM). Ultrasonicate the reaction system again for 5 min, and then start the reaction at room temperature (25 °C) for 48 h. After the reaction, a black composite is obtained. Then soak the composite in redistilled anhydrous tetrahydrofuran (2 mL) for 24 h. After 24 h, remove the solvent, and then wash the composite with acetone until the washing liquid is colorless. Dry the composite in vacuum at 60 °C for 6 h to obtain the target composite.

[0055] The synthetic route of this example is shown in Figure 16 .

[0056] Characterization:

[0057] (1) Determination of the infrared spectrum of the catalyst: Use infrared spectroscopy to determine the structure of the catalyst. Weigh 3 mg of GO-TAPP, graphene oxide (GO) and 5,10,15,20-tetrakis(4-(ethynylphenyl)-porphyrin (TAPP) respectively, and grind them thoroughly with dry potassium bromide powder in a mortar while keeping it dry all the time. Then place them in a tablet press mold to press into a transparent and crack-free tablet. Place the tablet in an infrared spectrum scanner and scan it 36 circles in the range of 500 - 400 cm -2 range.

[0058] Fourier transform infrared spectroscopy (FT-IR) is used to verify the construction of the GO-TAPP composite. As shown in Figure 2 (a), as can be seen, GO-TAPP integrates the characteristics of GO and TAPP simultaneously. The characteristic absorption bands of GO can be clearly observed, that is, those observed at 3340 - 3350, 3230, 1725 and 1620 cm -1 which belong to -OH, -C=O and -C=C-, and the stretching vibrations of porphyrin at 3270 and 1100 cm -1 which belong to -C≡C-H and C-N bonds. It should be noted that the 3270 cm -1The stretching vibration band of -C≡C-H at [specific position] disappeared, while the stretching vibration band of -C≡C- at 2105 cm -1 existed, indicating that -C≡C-C≡C- was formed by the oxidation of -C≡C-H. It was proved that GO was successfully coated by TAPP coupling polymerization to form a stable GO-TAPP composite.

[0059] (2)The carbon skeleton structure of the GO-TAPP composite was further confirmed by solid-state 13 C-NMR. As shown in Figure 2 (b), in addition to the characteristic peaks of the porphyrin macrocycle (121.3, 130.1, 140, and 142.7 ppm), a dense peak signal of the chemical shift of the carbon atoms of -C≡C-C≡C- at 100 - 110 ppm could also be detected, further indicating the formation of the composite network.

[0060] (3)The pore distribution of the GO-TAPP composite was understood through N 2 adsorption - desorption curves and pore size distribution curves. As shown in Figure 2 (c - d), the low-temperature N 2 adsorption / desorption isotherm of the GO-TAPP composite showed a typical mesoporous-dominated pore structure. The calculated surface area of GO-TAPP was determined to be 8.14 m 2 g -1 , and the total pore volume was 0.057 cm 3 g -1 . The corresponding pore size distribution (PSD) of GO-TAPP achieved by non-local density functional theory (NLDFT) further revealed that it was a typical type II isotherm, with a wide mesopore distribution between 2.31 - 19.05 nm.

[0061] (4)The thermal stability of the GO-TAPP composite was understood through thermogravimetric analysis. As shown in Figure 2 (e), as can be seen, GO-TAPP showed high thermal stability, with almost no weight loss when heated at temperatures below 121℃. GO-TAPP degraded rapidly at 121 - 271℃, which might be due to the continuous decomposition of the oxygen-containing groups of GO in GO-TAPP. GO-TAPP degraded slowly at 271 - 800℃, and at a temperature as high as 800℃, it could still retain 49% of the initial.

[0062] (5)The crystalline morphology of the GO-TAPP composite was understood through X-ray diffraction patterns, as shown in Figure 2 (f).

[0063] (6) Scanning electron microscopy (SEM) and transmission electron microscopy (TEM): GO-TAPP and graphene oxide were adhered to a mica substrate with conductive adhesive and sputter-coated with gold, and then the morphology of the samples was observed under SEM. The ultrasonic-dispersed methanol dispersion of GO-TAPP was dropped onto a copper grid, air-dried to obtain the observation sample, and the sample was placed in a TEM to observe the morphology of the sample and take pictures, and the elemental analysis map of the sample and the atomic content table of various elements were exported.

[0064] The morphology of the GO-TAPP composite was observed by SEM, as Figure 3 shown, Figure 3 (a-c) show the morphology of the GO-TAPP composite at 5 μm, 3 μm, and 1 μm, presenting large interconnected sheets, coated with a reticulated rough structure, and having a large number of mesopores and micropores. Observing the TEM morphology map of GO-TAPP, Figure 3 (d-e) show the morphology of the GO-TAPP composite at 500 nm and 200 nm, where the transparent paper-like structure is graphene oxide sheets, indicating that TAPP was successfully coated on GO after coupling. After observation by high-resolution TEM (f), there are no obvious lattice fringes, and no metal crystal forms were found by bright-field and dark-field contrast. In addition, the morphology of graphene oxide was observed by SEM, as Figure 4 shown, Figure 4 (a-b) show the morphology of graphene oxide at 5 μm and 1 μm, presenting large thin sheets. Figure 5 shows the elemental distribution of GO-TAPP and its EDX, showing that the distribution of various elements is uniform.

[0065] (7) Photothermal properties of the GO-TAPP composite: By changing the concentration of the GO-TAPP composite (50, 100, 200, 400, 600, and 800 μg / mL) or the power density of the laser (0.5, 1.0, and 1.5 W / cm 2 ), the photothermal conversion performance of GO-TAPP was studied in detail. Among them, the preparation method of GO-TAPP composites with different concentrations is as follows: First, 10 mg of GO-TAPP was weighed and fully dispersed in 1 mL of distilled water by an ultrasonic instrument to prepare a mother liquor of 10 mg / mL. Then, 5, 10, 20, 40, 60, and 80 μL were respectively taken from the mother liquor and added to 995, 990, 980, 960, 940, and 920 μL of distilled water, and finally, GO-TAPP aqueous dispersions of 50, 100, 200, 400, 600, and 800 μg / mL were prepared.

[0066] First, under laser irradiation (λ = 638 nm, 1 W / cm 2, (10 min), the heating behavior of GO-TAPP at different concentrations was studied. As Figure 6 (a)- Figure 6 (b) shows, GO-TAPP exhibits concentration-dependent photothermal conversion ability, and its temperature increases significantly with the increase of GO-TAPP concentration. For example, the GO-TAPP composite material has the lowest temperature rise (ΔT) of 3.2 °C at a concentration of 50 μg / mL. As the concentration increases, the ΔT of the solution increases significantly, reaching a maximum value of 30.6 °C at 800 μg / mL. In addition, the temperature pictures obtained from the thermal imager can also intuitively reflect the concentration-dependent heating behavior, as shown in Figure 6 (c). Figure 6 (d) shows the temperature rise of GO-TAPP (600 μg / mL) at different laser powers (λ = 638 nm, 10 min). It can be seen that with the increase of laser power density, the photothermal performance of GO-TAPP is significantly enhanced, and its temperature rapidly rises from 25.4 °C to 37.7 °C, 52.2 °C and 61.4 °C, and the power ranges are 0.5 W / cm 2 , 1 W / cm 2 and 1.5 W / cm 2 . Finally, Figure 7 (a) shows the estimation of the photothermal stability of GO-TAPP through five consecutive laser on / off cycles. It can be clearly seen that GO-TAPP exhibits an efficient photothermal reaction with almost no temperature fluctuation after five repeated on / off cycles, which is quite important for practical applications.

[0067] In addition, the photothermal conversion efficiency of GO-TAPP was calculated according to the photothermal conversion efficiency formula 1:

[0068] η (%) = [hS (T max – T surr ) – Q dis / I (1 – 10 – A 638 ) (Formula 1)

[0069] The meaning of each element in the formula is: "h" is the heat transfer coefficient; S is the surface area of the container; "T max " is the equilibrium temperature (51.6 °C) after 10 minutes of irradiation; "T surr " is the ambient temperature (24.3 °C) during the experiment; "Q dis " is the heat dissipation of the test unit (25.03 mW); "I" represents the laser power at 638 nm (1 W / cm 2 ). "A638" is the absorbance of the GO-TAPP aqueous solution at 638 nm (0.512).

[0070] Calculate the hS value according to Equation 2:

[0071] hS = m H2O C H2O / τS (Equation 2)

[0072] The meaning of each element in the equation is: "m H2O " is the mass of the water solvent during the experiment (1×10 -3 kg); "C H2O " is the specific heat capacity of water (4.2×10 3 J / kg℃).

[0073] Calculate the τS value according to Equation 3:

[0074] t = -τS (Inθ) (Equation 3)

[0075] τS is the GO-TAPP time constant (274); "θ" is the ratio of ΔT to T Max . Figure 7 The values of τS and θ in the equation are shown in (b - c).

[0076] In summary, the photothermal conversion efficiency (η) of GO-TAPP is determined to be 57.5%. Figure 7 (d) shows a comparison of the temperature rise behaviors of GO-TAPP, GO, and TAPP under red light (λ = 638 nm, 1 W / cm 2 ). It can be clearly seen that GO-TAPP exhibits a photothermal effect similar to that of GO but much higher than that of pure TAPP, indicating that the photothermal effect of the GO-TAPP complex mainly comes from the GO component.

[0077] (8) Photodynamic performance of GO-TAPP: The photodynamic performance of GO-TAPP under irradiation with a 638 nm laser (1 W / cm 2 ) was investigated.

[0078] First, 1,3-diphenylisobenzofuran (DPBF) was used as a photodynamic probe. It can be mixed and dissolved in dimethyl sulfoxide with a photosensitizer (GO-TAPP group). By changing the laser irradiation time of the photosensitizer, the singlet oxygen generated by the photosensitizer ( 1 O 2). The more singlet oxygen is produced, the more DPBF is consumed. On this basis, the singlet oxygen produced by the GO-TAPP group and other groups (pure DPBF group, GO group, and TAPP group) was determined. Among them, the concentration of GO-TAPP in the GO-TAPP group was 600 μg / mL, and the concentration of GO or TAPP in the GO group and TAPP group was 300 μg / mL. The preparation method was as follows: Weigh 1 mg of GO-TAPP, GO, or TAPP, and use an ultrasonic instrument to fully disperse it in 1 mL of dimethyl sulfoxide to prepare a stock solution of 1 mg / mL. Then, take 600 μL from the stock solution of GO-TAPP and add it to 400 μL of DMSO to prepare a 600 μg / mL GO-TAPP dimethyl sulfoxide dispersion; take 300 μL from the stock solution of GO and add it to 700 μL of DMSO to prepare a 300 μg / mL GO dimethyl sulfoxide dispersion; take 300 μL from the stock solution of TAPP and add it to 700 μL of DMSO to prepare a 300 μg / mL TAPP dimethyl sulfoxide dispersion.

[0079] To study the photodynamic properties of GO-TAPP, as Figure 8 shown in (a), the absorbance of GO-TAPP, GO, and TAPP in dimethyl sulfoxide was measured using a UV-visible spectrophotometer, and based on this, the photodynamic performance of the materials was explored. As Figure 8 shown in (b)-(e), where the pure DPBF group was used as the control group, that is, without adding any photosensitizer, only the DPBF probe was present. Except that the absorbance of the pure DPBF solution hardly changed, in the other three groups, namely the GO group, the TAPP group, and the GO-TAPP group, the ultraviolet-visible absorption light intensity at ~412 nm showed an obvious downward trend with the increase of laser irradiation time, indicating that DPBF was continuously consumed under light irradiation. For example, after 10 min of laser irradiation at 638 nm, the absorbance decrease rate of the GO-TAPP group reached 69.49 ± 0.29% (compared with 9.56 ± 0.68% of the pure DPBF group), which was much higher than that of the pure DPBF group. The absorbances of the GO group and the TAPP group were only 17.03 ± 0.43% and 64.45 ± 0.57% respectively. The performance differences can be more intuitively reflected from the curve Figure 8 of the absorption intensity changing with time Figure 8 shown in (f). Such results verified that GO-TAPP could produce toxic 1 O 2 for photodynamic use under laser excitation. Through the combination of GO and TAPP, both the production amount and rate were significantly improved.

[0080] Test Example 1: In vitro antibacterial test

[0081] (1) Bacterial culture: In this experiment, two types of bacteria, E. coli and S. aureus, were used, and the following experiments were completed using the second-generation bacteria. The specific method for culturing the second-generation bacteria is as follows: First, resuscitate the cryopreserved bacteria, melt the frozen bacteria at 37 °C, take 100 µL of the bacterial solution and place it in a shaking tube containing 5 mL of liquid medium, and place it in a constant-temperature shaker (110 rpm, 37 °C) for 12 h. Take 100 µL of the cultured bacterial solution and place it in a 2 mL EP tube containing 900 µL, and then dilute it stepwise according to the gradient of 10 -2 dilute 5 - 10 tubes, take 100 µL of the bacterial solution from each tube, and evenly spread it on a petri dish containing solid medium using a spreading rod. Incubate it at 37 °C for 24 h, observe the colony morphology and the number of colonies, and select the petri dish with approximately 1000 colonies as the first-generation bacteria. Use a bacterium-picking rod to pick out one colony from the first-generation bacteria and add it to a shaking tube containing 5 mL of liquid medium, and culture it according to the method of culturing the first-generation bacteria to obtain a petri dish with approximately 1000 colonies as the second-generation bacteria. The medium in the bacterial solution (E. coli or S. aureus) is a liquid medium. The specific preparation method is to take 5 g of LB broth, disperse it in 200 mL of distilled water, and then sterilize it by autoclaving to obtain the bacterial liquid medium. The specific preparation method of the solid medium is to take 5 g of LB broth, 3 g of agar, disperse it in 200 mL of distilled water, and then sterilize it by autoclaving to obtain the solid medium.

[0082] (2) Determination of the antibacterial activity of GO-TAPP by the plate counting method: After testing the performance of GO-TAPP regarding PTT and PDT, it can be concluded that GO-TAPP has certain antibacterial potential. The laser irradiation-induced antibacterial ability of GO-TAPP was studied by the plate counting method. Among them, the specific preparation method of the bacterial dispersion of different concentrations of GO-TAPP groups is as follows: Take 10 mg of GO-TAPP powder, fully disperse it in 1 mL of PBS to prepare a 10 mg / mL GO-TAPP mother solution. Add 100 µL of 10 8 CFU mL -1 bacterial solution to 6 2 mL EP tubes, and then add 820, 840, 860, 880, 890, and 900 μL of PBS respectively, and then add 80, 60, 40, 20, 10, and 0 μL of the 10 mg / mL GO-TAPP mother solution respectively to prepare GO-TAPP solutions of 800, 600, 400, 200, 100, and 0 μg / mL and 10 8 CFU mL -1 PBS dispersion is the GO-TAPP solution. Then, irradiate different concentrations of GO-TAPP solutions with laser (laser parameters: λ = 638 nm, 1 W / cm2 , 10 min), GO-TAPP solutions with different concentrations were placed in a constant temperature shaker (110 rpm, 37 °C) for 12 h. Then, the cultured bacterial solution was serially diluted 10 5 times in the way of bacterial culture. 100 μL of the well-mixed bacterial solution was transferred to a solid medium and spread evenly, and then incubated at 37 °C for 24 h to observe the colony morphology. The colonies were counted and the bacterial activities were compared with those of each group. As Figures 9 - 10 shown, with the increase in the concentration of the GO-TAPP solution, the bactericidal performance against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) was greatly enhanced. At a concentration of 100 μg / mL, there were only slight bactericidal effects of 51.63 ± 3.45% and 66.89 ± 2.48% against S. aureus and E. coli, respectively. While at a concentration of 600 μg / mL of the GO-TAPP solution, the antibacterial rates against these two bacteria reached over 96.5% (E. coli: 96.88 ± 0.33%, S. aureus: 98.46 ± 0.11%). At a concentration of 800 μg / mL of the GO-TAPP solution, the antibacterial rates against these two bacteria were close to 100%, with the antibacterial rate of E. coli being 99.42 ± 0.06% and that of S. aureus being 99.06 ± 0.10%.

[0083] For comparison, the in vitro antibacterial abilities of GO-TAPP, GO, and TAPP were also evaluated. The plate counting method was still used to study the antibacterial effects of GO-TAPP (600 μg / mL), GO (300 μg / mL), and TAPP (300 μg / mL) under light and dark conditions. The in vitro bacterial experiments were divided into eight groups, namely the PBS group, the TAPP group, the GO group, the GO-TAPP group, the PBS + laser group, the TAPP + laser group, the GO + laser group, and the GO-TAPP + laser group. The preparation methods of the co-culture solutions for different groups were as follows: First, prepare the stock solutions of GO-TAPP, GO, or TAPP, that is, take 10 mg of GO-TAPP, GO, or TAPP respectively, and disperse them well in 1 mL of PBS to prepare 10 mg / mL PBS dispersions of GO-TAPP, GO, or TAPP.

[0084] The treatment method for the GO-TAPP group was to add 100 µL of 10 8 CFU mL -1 bacterial solution into a 2 mL EP tube, then add 840 μL of PBS, and then add 60 μL of the 10 mg / mL GO-TAPP stock solution to prepare a 600 μg / mL GO-TAPP and 10 8 CFU mL -1The PBS dispersion was obtained to get the treated GO-TAPP group.

[0085] For the treatment method of the GO-TAPP + laser group, 600 μg / mL of GO-TAPP and 10 8 CFU / mL -1 The PBS dispersion was prepared, and then the dispersion was irradiated with laser to obtain the treated GO-TAPP + laser group.

[0086] For the treatment method of the GO group, 100 μL of 10 8 CFU / mL -1 bacterial solution was added to a 2 mL EP tube, then 870 μL of PBS was added, and then 30 μL of 10 mg / mL GO stock solution was added to prepare a PBS dispersion of 300 μg / mL of GO and 10 8 CFU / mL -1 to obtain the treated GO group.

[0087] For the treatment method of the GO + laser group, 300 μg / mL of GO and 10 8 CFU / mL -1 The PBS dispersion was prepared, and then the dispersion was irradiated with a laser to obtain the treated GO + laser group. The treatment method of the TAPP group was as follows: 100 μL of 10 8 CFU / mL -1 bacterial solution was added to a 2 mL EP tube, then 870 μL of PBS was added, and then 30 μL of 10 mg / mL TAPP stock solution was added to prepare a PBS dispersion of 300 μg / mL of TAPP and 10 8 CFU / mL -1 to obtain the treated TAPP group.

[0088] For the treatment method of the TAPP + laser group, 300 μg / mL of TAPP and 10 8 CFU / mL -1 The PBS dispersion was prepared, and then the dispersion was irradiated with a laser to obtain the treated TAPP + laser group.

[0089] Laser parameters: λ = 638 nm, 1 W / cm 2 , 10 min; then they were placed in a constant temperature shaker (110 rpm, 37 °C) for 12 h according to the groups, and then the cultured bacterial solution was serially diluted 10 5times, transfer 100 μL of the evenly blown bacterial solution to the solid medium, smear it evenly, and incubate at 37 °C for 24 h to observe the morphology of the clones. Calculate the colonies and compare the bacterial activities with each group. From Figure 11 It can be seen that the bacteria treated with (V) PBS, (VI) TAPP, (VII) GO, (VIII) GO-TAPP, and (I) PBS + laser all showed roughly the same number of colonies. However, the antibacterial effects of TAPP, GO, and GO-TAPP were significantly improved after laser irradiation. For example, the antibacterial efficiencies of the (II) TAPP + laser group against Staphylococcus aureus and Escherichia coli survival rates reached 36.73 ± 6.61% and 57.8 ± 4.29%, respectively. While the survival rates of Staphylococcus aureus and Escherichia coli treated with the (III) GO + laser group decreased to 14.84 ± 2.04% and 25.22 ± 5.78%, respectively. The GO-TAPP + laser group showed the most prominent antibacterial effect, with the survival rates of Staphylococcus aureus and Escherichia coli being only 3.69 ± 0.39% and 4.13 ± 0.42%, respectively. This is because GO-TAPP combines the antibacterial abilities of GO and TAPP, achieving a synergistic photothermal (PTT) and photodynamic (PDT) bactericidal effect. In summary, GO-TAPP has good synergistic PTT and PDT antibacterial abilities and can be used as a spectral antibacterial agent with potential antibacterial ability to replace antibiotics.

[0090] Experimental Example 2: Bacterial live / dead staining test:

[0091] SYTO-9 and PI were used to distinguish between live and dead microbial cells. SYTO-9 can penetrate all bacterial membranes (intact and damaged), thus labeling the bacteria green. On the other hand, PI only penetrates damaged bacterial membranes, labeling the bacteria red while reducing the green color of SYTO-9.

[0092] Prepare the bacterial solutions of the PBS group, TAPP group, GO group, GO-TAPP group, PBS + laser group, TAPP + laser group, GO + laser group, and GO-TAPP + laser group according to the method in Experimental Example 1. Then take 100 µL of the bacterial suspension from each group and incubate it with 20 µL of SYTO-9 (1.0×10 -3 M) and 20 µL of PI (1.5×10 -3 M) in the dark at 37 °C for 15 min. After staining, centrifuge the samples in PBS to remove the excess SYTO-9 and PI. Then resuspend the bacteria in 50 µL of PBS and place them on the surface of the glass slide. Then capture images of Escherichia coli or Staphylococcus aureus with a fluorescence inverted microscope.

[0093] From Figure 12It can be seen that the results of the live / dead staining are consistent with those of the aforementioned co-culture experiment, and the two bacteria under different treatments show different fluorescence signals. For example Figure 13 Groups I and II in (a), Figure 13 as shown by Groups I and II in (b), the bacteria treated with the PBS and PBS + laser groups only showed strong green fluorescence. However, for the bacteria under the treatments of other groups, such as Figure 13 Groups III and VIII in (a), Figure 13 as shown by Groups III and VIII in (b), the groups irradiated with laser showed much stronger red fluorescence than the individual materials. As Figure 13 Group VIII in (a) and Figure 13 Group VIII in (b) shown, the GO-TAPP + laser group showed the most prominent sterilization effect, in which almost all Staphylococcus aureus and Escherichia coli were labeled red. But as Figure 13 Groups III and V in (a) and Figure 13 Groups III and V in (b) shown, for the TAPP + laser and GO + laser groups, only a part of Staphylococcus aureus and Escherichia coli were stained red. This further demonstrated the superiority of GO-TAPP in synergistic PTT and PDT antibacterial aspects.

[0094] Test Example 3: Transmission electron microscopy of bacteria:

[0095] Bacterial suspensions of the PBS group, TAPP group, GO group, GO-TAPP group, PBS + laser group, TAPP + laser group, GO + laser group and GO-TAPP + laser group were prepared according to the method in Test Example 1. Then, 100 μL of the bacterial suspension was fixed in 2.5 wt% glutaraldehyde solution (4 °C, 2 h), washed three times with PBS, embedded in agar and blocked. Then, the bacteria were dehydrated by continuous treatment with ethanol solutions (30 wt%, 50 wt%, 70 wt%, 90 wt%, 95 wt% and 100 wt%) at room temperature for 10 min, followed by treatment with acetone at room temperature for 3 h, and gradient infiltration embedding with an embedding medium (epoxy resin) (soaked with acetone and epoxy resin in a mass ratio of 3:1, 1:1, 3:1 for 1 hour respectively, and finally pure epoxy resin soaked overnight), negative staining, and sectioning on a nickel mesh. The nickel mesh was placed under TEM for observation to capture the bacterial morphology.

[0096] TEM was used to observe the integrity of the bacterial membranes treated with different groups. As Figure 13 in (a) and Figure 13As shown in (b), in the PBS and PBS+laser groups, the bacterial membranes of Staphylococcus aureus and Escherichia coli were intact with or without light, indicating that the viability of the bacteria was not affected by light. The bacterial membranes in the treated TAPP group (IV) were slightly damaged, as shown in Figure 13 groups III and IV in (a) and Figure 13 groups III and IV in (b), probably because TAPP scratched the bacterial membranes, resulting in very little damage. Similar to the live / dead staining results, the bacterial membranes in other treated groups also had varying degrees of damage. Figure 13 groups VII and VIII in (a) and Figure 13 groups VII and VIII in (b), GO-TAPP+laser caused the most severe damage to their bacterial membranes, with a large amount of bacterial content flowing out. Therefore, the GO-TAPP composite material with synergistic antibacterial abilities of PDT and PTT has great application potential as a broad-spectrum antibacterial agent without antibiotics and can effectively kill bacteria.

[0097] Experimental Example 4: In vitro biocompatibility experiment:

[0098] (1) Hemolysis experiment

[0099] This experiment was conducted under the guidance of the ethics committee and strictly in accordance with the protocol of Weifang Medical University. Fresh blood was taken from KM female mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.).

[0100] Red blood cells were collected by centrifugation at 1500 rpm for 20 min and then washed three times with PBS. Then, the red blood cells (4% w / w) were incubated with GO-TAPP (100 - 800 μg / mL) at a ratio of 1:9 (v / v) at 37 °C for 3 h, and then centrifuged at 12000 rpm for 20 min. Then, 100 μL of the supernatant from each group was placed in a 96-well plate, and the absorbance of each group was measured at 570 nm using a microplate reader. Distilled water was used as the positive control, and PBS was used as the negative control. The hemolysis volume was calculated using the following formula 4:

[0101] Hemolysis volume (%) = (A - An) / (Ap - An) × 100% (Formula 4).

[0102] Where "A" is the absorbance obtained from the supernatant after adding GO-TAPP to the red blood cells. "An" is the absorbance obtained from the supernatant after adding PBS to the red blood cells (negative control). "Ap" is the absorbance obtained from the supernatant after adding distilled water to the red blood cells (positive control).

[0103] As shown in Figure 14As shown, within the concentration range where GO-TAPP exhibits antibacterial activity, it shows only a small amount (less than 2%) or no hemolytic activity. The hemolysis rate of GO-TAPP varies with the concentration of GO-TAPP. As the concentration increases from 100 to 800 μg / mL, the hemolysis rate rises from 0.02±0.01% to 1.29±0.01%. This indicates that GO-TAPP has good blood compatibility and causes no or negligible damage to the erythrocyte membrane.

[0104] (2)Cytotoxicity experiment

[0105] In a 96-well plate, HEK-293 cells (established from human primary embryonic kidney transformed by adenovirus type 5 (Ad 5), German Collection of Microorganisms and Cell Cultures (DSMZ): ACC 305) were seeded at a density of 5×10 3 cells per well, with 180 µL of cells per well. 200 µL of PBS was added to the surrounding duplicate wells for liquid sealing to prevent excessive evaporation. After incubation for 24 h, 20 µL of GO-TAPP at different concentrations (100 - 800 μg / mL) was added and incubated for 72 h. Then, 20 µL of MTT (4 mg / mL) solution was added to each well and cultured in an incubator for 4 h. After 4 h, the supernatant was aspirated and 150 µL of dimethyl sulfoxide was added to dissolve MTT (tetramethyl azo blue). After dissolving on a shaker for 10 min, the absorbance of the 96-well plate was measured at 570 nm using an enzyme-linked immunosorbent assay reader. Each experiment was repeated three times.

[0106] Meanwhile, in order to further study the adverse damage of the material itself to normal cells, the cytotoxicity test of GO-TAPP on HEK-293 was carried out. As Figure 15 shown, the cell viability of HEK-293 cells cultured with different concentrations of GO-TAPP (100 - 800 μg / mL) for 3 days is presented. It can be clearly seen that as the material concentration decreases, the relative cell viability gradually increases, and its value can remain greater than 85% at all experimental concentrations, approaching or higher than 100%, indicating no toxicity to HEK-293 cells. All these results confirm that GO-TAPP with good biocompatibility is a selective agent against bacteria.

[0107] Test Example 5:

[0108] (1)In vivo wound healing experiment:

[0109] A wound healing model was established using 5-week-old female KM mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) (n = 5 per group, 20 - 25 g) and divided into 7 groups. The specific modeling method was as follows: After disinfection with ethanol solution (75%), the dorsal hair of each mouse was shaved off before surgery to form a wound with d = 5 mm, and then infected with Staphylococcus aureus (1×10 6 CFU / mL) for 24 h. Then, GO-TAPP (100 µL, 600 µg / mL), GO (100 µL, 600 µg / mL), TAPP (100 µL, 600 µg / mL), and PBS (100 µL, control group) were used to treat the infected wounds with or without light irradiation (λ = 638 nm, 1 W / cm 2 , 10 min), respectively. The wound surface was photographed on days 0, 2, 4, 6, and 8, and the body weight of the mice was monitored simultaneously. An image analysis program (Image.J, National Institutes of Health) was used to measure the change in wound size.

[0110] To evaluate the role of GO-TAPP in wound healing, a model of Staphylococcus aureus-infected dorsal cortex injury in KM rats was established. For comparison, the in vivo antibacterial abilities of the PBS (100 µL, control group), TAPP (100 µL, 600 µg / mL) + laser (λ = 638 nm, 1 W / cm 2 , 10 min), TAPP (100 µL, 600 µg / mL), GO (100 µL, 600 µg / mL) + laser (λ = 638 nm, 1 W / cm 2 , 10 min), GO (100 µL, 600 µg / mL), GO-TAPP (100 µL, 600 µg / mL) + laser (λ = 638 nm, 1 W / cm 2 , 10 min), and GO-TAPP (100 µL, 600 µg / mL) groups were also estimated. The specific method was as follows: One day after the establishment of the Staphylococcus aureus-infected dorsal cortex injury model in KM rats, the wounds of each group were treated with the above dosages at the wound site, which was the wound treatment. Days 0 to 1 were the modeling time, and the damaged wounds were infected with Staphylococcus aureus. In addition, the wounds of the mice were photographed and recorded on days 0, 2, 4, 6, and 8. As Figure 17As shown, the photo taken on the 0th day shows the back wound of the mouse just after perforation. The photo on the 2nd day shows the back wound of the mouse one day after treatment. It can be seen that all the wounds show the characteristics of bacterial infection, and the wound contraction rates of all groups are almost the same (~30%). Compared with the swelling of the non-laser groups (PBS, TAPP, GO, and GO-TAPP), the wounds in the laser-treated groups (TAPP+laser, GO+laser, and GO-TAPP+laser) had started to scab on the 4th day. As the treatment time extended, the wounds under different treatment methods further contracted. Figure 18 (a) shows the wound contraction rates of different groups. On the 6th day, only the PBS group showed swelling. The wound contraction rates reached 50.4±3.26% (PBS), 71.24±1.7% (TAPP+laser), 66.78±0.15% (TAPP), 79.29±1.54% (GO+laser), 70.38±1.36% (GO), 92.64±0.85% (GO-TAPP+laser), and 77.99±1.53% (GO-TAPP), respectively. However, even after 7 days of treatment, the wound contraction rates of the non-laser groups were only 65.35±4.12% (PBS), 76.85±0.42% (TAPP), 82.75±1.45% (GO), and 84.55±1.69% (GO-TAPP), respectively. In contrast, the wounds treated with the GO-TAPP+laser group were almost completely healed on the 8th day, significantly higher than the other two laser-irradiated groups (88.86±1.1% for TAPP+laser and 92.76±0.86% for GO+laser). It can be seen more clearly from the Figure 17 overlay wound diagram in the last row that the wound healing of the laser-irradiated groups was much better than that of the non-laser groups. All these results indicate that compared with other groups, laser irradiation of GO-TAPP can not only significantly reduce bacterial invasion but also accelerate wound contraction. At the same time, the body weights of KM rats were recorded daily for further comparison. As Figure 18 shown in (b), during the 8-day treatment, there were no obvious behavioral abnormalities in the mice of each group, and the body weights did not change significantly.

[0111] (2) In vivo biocompatibility study: Tissue staining experiments (HE staining and Masson trichrome staining)

[0112] To further study the wound healing of the mice in each group, hematoxylin and eosin (H&E) staining and Masson trichrome staining were used to evaluate the wound healing of the mice in the (1) in vivo wound healing test. As Figure 19As shown, histological analysis of wounds infected with Staphylococcus aureus showed that new capillaries and skin growth occurred to varying degrees in all groups. Most intuitively, it can be seen that the scab area of the light-irradiated group was smaller than that of the non-light-irradiated group, indicating that the healing rate of the GO-TAPP, GO, and TAPP groups was significantly accelerated after laser irradiation. In addition, the GO-TAPP + laser group had the smallest scab area and the most newly generated capillaries and skin, indicating the fastest healing. The control group had the largest scab area, few new capillaries and skin, and a large number of inflammatory cells. Therefore, GO-TAPP can accelerate wound reconstruction faster after laser irradiation.

[0113] To study the in vivo biocompatibility of GO-TAPP, histological sections and H&E staining were performed on the heart, liver, spleen, lungs, and kidneys of KM mice. As Figure 20 shown, it was found that there were no obvious inflammations and morphological damages in different organs of each group.

[0114] (2) Blood routine experiment

[0115] On the 8th day, 1 to 2 mL of blood samples were collected from the fundus arteries of the mice. 200 μL of each blood sample was taken for routine blood analysis, including white blood cells (WBC), red blood cells (RBC), mean corpuscular volume (MCV), platelets (PLT), mean corpuscular hemoglobin concentration (MCHC), and mean corpuscular hemoglobin content (MCH).

[0116] To study the in vivo biocompatibility of GO-TAPP, blood was drawn from the eyes of the above KM mice for blood routine detection. As Figure 21 shown, no obvious differences were found in the blood routine values of each group.

[0117] The above are only the preferred embodiments of the present application and are not used to limit the present application. For those skilled in the art, various changes and modifications can be made to the present application. 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 GO-TAPP microporous composite material, characterized in that: The GO-TAPP microporous composite material uses graphene oxide as a template, so that a plurality of porphyrin structures are coated with graphene oxide to form a structure of porphyrin coupled polymerization and coated graphene oxide; the hemolysis rate of the GO-TAPP microporous composite material on cells is less than 2%; The GO-TAPP microporous composite material is prepared by the following method: 5,10,15,20-tetrakis(4-ethynylphenyl)-porphyrin, bis(triphenylphosphine)palladium chloride and cuprous iodide were added to tetrahydrofuran and mixed evenly in the dark, and then graphene oxide and triethylamine were added, mixed in the dark, and then allowed to react to obtain a black solid, which was washed and dried to obtain a GO-TAPP microporous composite material; The 5,10,15,20-tetrakis(4-ethynylphenyl)-porphyrin is prepared by the following method: 5,10,15,20-tetrakis(4-[(trimethylsilyl)ethynyl]-phenyl)-porphyrin is added to tetrahydrofuran, tetrabutylammonium fluoride is added dropwise at -78°C, the temperature is raised to room temperature for reaction, the reaction system is extracted with distilled water, the aqueous phase is washed to separate the organic phase, the organic phase is dried after dehydration, and a black solid is obtained, namely 5,10,15,20-tetrakis(4-ethynylphenyl)-porphyrin.

2. The GO-TAPP microporous composite material according to claim 1, characterized in that: The ratio of the added amounts of 5,10,15,20-tetrakis(4-ethynylphenyl)-porphyrin, graphene oxide, bis(triphenylphosphine)palladium chloride, cuprous iodide, triethylamine and tetrahydrofuran is 62 mg: 62 mg: 5.6 mg: 1.6 mg: 68 μL: 4 mL.

3. The GO-TAPP microporous composite material according to claim 2, characterized in that: The tetrahydrofurans are all redistilled anhydrous tetrahydrofurans.

4. The GO-TAPP microporous composite material according to claim 1, characterized in that: The mixing was all carried out by ultrasonic mixing, and the mixing time was all 5 minutes.

5. The GO-TAPP microporous composite material according to claim 1, characterized in that: The temperature of the static reaction is 25° C., and the time of the static reaction is 48 h.

6. The GO-TAPP microporous composite material according to claim 1, characterized in that: The washing in the washing and drying process is washing with acetone until the washing solution is colorless; and the drying in the washing and drying process is vacuum drying at 60° C. for 6 hours.

7. Use of the GO-TAPP microporous composite material according to any one of claims 1 to 6 in the preparation of antibacterial drugs.

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