GO-TAPP microporous composite material as well as preparation method and application thereof

Abstract

The invention discloses a GO-TAPP microporous composite material as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The preparation method comprises the following steps: adding 5, 10, 15, 20-tetra (4-ethynylphenyl)-porphyrin, bis (triphenylphosphine) palladium chloride and cuprous iodide into tetrahydrofuran, uniformly mixing in a dark place, adding graphene oxide and triethylamine, mixing in a dark place, carrying out a reaction to obtain a black solid after the reaction is finished, and washing and drying the black solid to obtain the GO-TAPP microporous composite material. The GO-TAPP microporous composite material taking the graphene oxide and the porphyrin as structural units can resist bacteria synergistically through PTT and PDT, and is good in biocompatibility and free of toxic and side effects, and the hemolysis rate on cells is lower than 2%. The GO-TAPP is in a sheet shape and can be directly cut, pasted and directly used, post-processing is not needed, and the application prospect of the GO-TAPP is improved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a GO-TAPP microporous composite material and a preparation method and application thereof. Background Art

[0002] Bacteria are ubiquitous in nature and in all living things, including humans, and we are exposed to different types of bacteria all the time. To date, bacterial infections account for 25% of all deaths each year, and in some countries, especially developing countries in the third world, the proportion is even higher. The most important of these is the emergence of drug-resistant bacteria caused by the misuse of antibiotics, which causes 700,000 deaths each year and brings great difficulties and challenges to public health systems around the world. Therefore, people are very eager to have some alternative antimicrobial strategies that can reduce costs, improve efficacy, and reduce drug resistance.

[0003] So far, various new materials that can be used as antimicrobial agents alone or in combination with other drugs have been explored as alternatives to traditional antibiotics. Among them, phototherapy antibacterial is a low-risk, non-invasive alternative treatment strategy for the treatment of microbial infections. Phototherapy antimicrobial therapy can be roughly divided into photothermal therapy (PTT) and photodynamic therapy (PDT), both of which require a specific light source to irradiate the photosensitizer to exert a phototherapy effect. Different photosensitizers exert different antimicrobial effects under different light source conditions. Photothermal therapy can use photosensitizers with high photothermal conversion efficiency to kill bacteria by converting light energy into heat energy by exposure to a suitable external light source. Photodynamic therapy can use photosensitizers with high light reactions, which are irradiated by a suitable external light source to transfer light energy to the surrounding oxygen, produce highly active singlet oxygen, and can undergo strong oxidation reactions, thereby killing bacteria. At present, 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 and its preparation method and application, in which the organic metal framework material ZIF-8 is embedded into the porphyrin structure to prepare a nanoagent with a bacterial-like structure, and the flagella of bacteria are imitated by the nanotubes grown on the surface of the carrier to simulate bacterial therapy, thereby achieving efficient photothermal-Fenton combined therapy. However, these photosensitizers have some disadvantages, such as the excitation wavelength is not in the optimal light transmittance area, and there is a large amount of phototoxicity and other problems. Therefore, how to prepare a phototherapeutic antibacterial agent with high antibacterial activity and high biocompatibility is an urgent problem to be solved. Summary of the invention

[0004] In view of the above-mentioned prior art, the purpose of the present invention is to provide a GO-TAPP microporous composite material and its preparation method and application. The present invention uses graphene oxide and porphyrin as structural units to provide a graphene oxide porphyrin microporous composite material (GO-TAPP), which can synergistically resist bacteria through PTT and PDT, and has good biocompatibility and no toxic side effects.

[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 GO-TAPP microporous composite material, the preparation method comprising:

[0007] 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 and 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.

[0008] Preferably, 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.

[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 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.

[0011] Preferably, the tetrahydrofuran is redistilled anhydrous tetrahydrofuran.

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

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

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

[0015] The second aspect of the present invention provides a GO-TAPP microporous composite material obtained by the above preparation method.

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

[0017] The third aspect of the present invention provides use of the GO-TAPP microporous composite material in the preparation of antibacterial drugs.

[0018] Beneficial effects of the present invention:

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

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

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

[0022] Figure 1 :Monomer NMR data: 5,10,15,20-Tetrakis(4-ethynylphenyl)-porphyrin, 1 H-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(bH,s,8H);

[0023] Figure 2 :(a) FTIR spectra of GO, TAPP and GO-TAPP; (b) FTIR spectra of GO-TAPP 13 CNMR; (c) low temperature N2 absorption isotherm of GO-TAPP at 77K; (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 5μm scale; (b) SEM of GO-TAPP at 3μm scale; (c) SEM of GO-TAPP at 1μm scale; (d) TEM of GO-TAPP at 500nm scale; (e) TEM of GO-TAPP at 200nm scale; (f) HRTEM of GO-TAPP at 20nm scale;

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

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

[0027] Figure 6 :(a) at 1.0W / cm 2 (a) The concentration-dependent photothermal effect of GO-TAPP under 638 nm laser irradiation; (b) Thermal imaging of the heating process of GO-TAPP (50-800 μg / mL) within 10 min; (c) The photothermal effect of GO-TAPP under 638 nm laser irradiation; (d) The photothermal effect of GO-TAPP under 638 nm laser irradiation; (e) The photothermal effect of GO-TAPP under 638 nm laser irradiation; (f) The photothermal effect of GO-TAPP under 638 nm laser irradiation; (g) The photothermal effect of GO-TAPP under 638 nm laser irradiation; (g) The photothermal effect of GO-TAPP under 638 nm laser irradiation; (h ... 2 (d) The relationship between the concentration of GO-TAPP and the temperature rise.

[0028] Figure 7 :(a) 638nm laser at 1.0W / cm 2 Temperature variation curve of GO-TAPP (600 μg / mL) after five times of photo-cooling under 638 nm laser irradiation (1.0 W / cm 2 ), where irradiation is continued to reach an equilibrium temperature and then the laser is turned off; (c) The negative natural logarithm of the cooling cycle and temperature; (d) GO-TAPP, GO, and TAPP at 1.0 W / cm 2 Comparison chart under 638nm laser irradiation;

[0029] Figure 8:(a) UV-visible spectra of GO-TAPP, GO and TAPP (solvent is DMSO), (b) DPBF, GO+DPBF, TAPP+DPBF and GO-TAPP+DPBF under red light (λ=638nm, 1W / cm 2 ) UV-visible spectra of GO, TAPP and GO-TAPP under red light (λ=638nm, 1W / cm 2 )Comparison of the decay rate of induced DPBF under irradiation (%);

[0030] Fig. 9 : GO-TAPP of different concentrations was exposed to red light (λ=638nm,1W / cm 2 ) Antibacterial ability after 10 minutes of irradiation: Staphylococcus aureus (a) and Escherichia coli (b) culture images of GO-TAPP with different concentrations treated with red light;

[0031] Fig.10 : The plate count method was used to measure the viability (%) of Staphylococcus aureus (a) and Escherichia coli (b) treated with red light at different concentrations of GO-TAPP (n=3, error bars represent standard deviation);

[0032] Fig.11 :TAPP, GO and GO-TAPP in the presence or absence of red light (λ=638nm,1W / cm 2 ) Antibacterial ability under irradiation conditions: 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, and (VIII) GO-TAPP + laser (concentrations: GO-TAPP = 600 μg / mL, GO = 300 μg / mL, TAPP = 300 μg / mL, λ = 1 W / cm 2 , 10 minutes);

[0033] Fig.12 : Fluorescence images of Staphylococcus aureus (a) and Escherichia coli (b) incubated with live / dead stains by PBS, TAPP, GO, and GO-TAPP with and without laser treatment, respectively. Bacteria were co-stained with SYTO-9 and PI (concentrations: 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] Fig.13TEM images of Staphylococcus aureus (a) and Escherichia coli (b) incubated 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 tip represents the damage position (concentrations: 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] Fig.14 : Hemolysis experiment of GO-TAPP: Hemolysis rate of GO-TAPP at different concentrations (n=3, error bars indicate standard deviation);

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

[0037] Fig.16 :Preparation route of graphene oxide porphyrin microporous composite materials;

[0038] Fig.17 : Representative photos of dorsal wounds of mice infected with Staphylococcus aureus at each time point treated with 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] Fig.18 :(a) Corresponding wound contraction versus time curve (%); (b) Changes in mouse body weight during treatment (n=5, error bars indicate standard deviation, *p<0.05, **p<0.01, ***p<0.001);

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

[0041] Fig. 20 : Organ staining results;

[0042] Fig.21:The results of routine blood tests (WBC, RBC, MCV, PLT, MCHC, MCH) in different groups (on the 8th day); the data are expressed as mean ± standard error (n = 3); (a) the results of white blood cell (WBC) in different groups (on the 8th day); (b) the results of red blood cell (RBC) in different groups (on the 8th day); (c) the results of mean corpuscular volume (MCV) in different groups (on the 8th day); (d) the results of platelet (PLT) in different groups (on the 8th day); (e) the results of mean hemoglobin concentration (MCHC) in different groups (on the 8th day); (f) the results of mean hemoglobin content (MCH) in different groups (on the 8th day). DETAILED DESCRIPTION

[0043] 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.

[0044] As introduced in the background technology 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 is adsorbed onto the surface of the template ZIF-8, and in the presence of a catalyst, covalent bond oxidative coupling is performed to obtain a ZIF-8 complex (PZCo-CMP) covered with a cobalt-porphyrin conjugated porous polymer (CMP). However, this artificial nanozyme is antibacterial through photothermal-Fenton combined antibacterial, 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 by the present invention is a graphene oxide and porphyrin microstructure unit, and GO-TAPP is prepared by an oxidative coupling method. Fig.16 As shown, the present invention uses graphene oxide as a template to coat several porphyrin structures with graphene oxide, forming a porphyrin-coupled polymerization-coated graphene oxide structure, which has good biocompatibility and phototherapy properties that traditional photosensitizers do not have. GO-TAPP can achieve local heating through photothermal conversion to achieve the effect of rupturing the bacterial membrane. In addition, after laser irradiation, GO-TAPP will convert oxygen elements in the surrounding environment into singlet oxygen that is harmful to bacteria, thereby causing bacterial lysis. 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 in conjunction with specific embodiments.

[0047] 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.

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

[0049] Prepare a 500mL round-bottom flask with a magnetic stirrer, add 200mL of tetrahydrofuran to it, then add 2-3g of calcium hydride to the tetrahydrofuran, then connect a 250mL constant pressure separatory funnel to the mouth of the round-bottom flask, and turn off the knob of the constant pressure separatory funnel so that it is not in circulation with the round-bottom flask below. Install a spherical condenser at the upper end of the constant pressure separatory funnel. Place the above device in a water bath at 80°C for redistillation. When there is no significant liquid residue in the round-bottom flask below the device, the redistillation process of tetrahydrofuran is completed, and the tetrahydrofuran obtained by redistillation is condensed and stored in a constant pressure separatory funnel. In addition, prepare about 100 molecular sieves 4A (sodium-A molecular sieve, spherical 3mm-5mm, Sinopharm Chemical Reagent Co., Ltd.), use a microwave oven to heat for 3 minutes on medium heat to remove water, use a vacuum pump to evacuate the molecular sieve after 3 minutes until it cools, repeat three times to ensure that there is no water in the molecular sieve, add it to a 500ml brown reagent bottle, add the above-mentioned redistilled tetrahydrofuran, and seal it for storage to obtain redistilled anhydrous tetrahydrofuran.

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

[0051] 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.

[0052] Example

[0053] (1) Synthesis of 5,10,15,20-tetrakis(4-ethynylphenyl)porphyrin (TAPP): 5,10,15,20-tetrakis(4-[(trimethylsilyl)ethynyl]-phenyl)porphyrin (1.5 g, 1.5 mM) was weighed and placed in a 250 mL three-necked flask with rubber stoppers on both sides and a magnet, and then redistilled anhydrous tetrahydrofuran (100 mL) was added. Then, a 25 mL constant pressure separatory funnel was equipped in the middle hole, and the knob of the constant pressure separatory funnel was closed so that it was not connected to the three-necked flask below. The reaction system was placed at a temperature of -78°C and adapted for 5 min, and then tetrabutylammonium fluoride (7.5 mL) was added to the constant pressure separatory funnel. Subsequently, the knob of the constant pressure separatory funnel was opened, and the liquid flow rate was adjusted to add tetrabutylammonium fluoride to the three-necked flask at a rate of 4 drops / s. Afterwards, the reaction system was slowly heated from -78°C (heating rate 0.43°C / min) to room temperature (25°C) for 10 hours. After 10 hours, the reaction solution was mixed with distilled water (100 mL), and the aqueous phase was washed three times with dichloromethane (200 mL). The organic phase was collected and filtered after dehydration with anhydrous magnesium sulfate, and then vacuum dried at 60°C for 10 hours. After drying, a dark purple solid (0.75 g, yield 70.3%) was obtained. 1 H-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(bH,s,8H).

[0054] (2) Preparation of GO-TAPP composite material: Weighed 5,10,15,20-tetrakis(4-(ethynylphenyl)-porphyrin (31 mg, 0.04 mM), bis(triphenylphosphine)palladium chloride (2.8 mg, 0.004 mM) and cuprous iodide (0.8 mg, 0.004 mM) were added to a 5 mL brown vial, and then dry tetrahydrofuran (2 mL) was added. The reaction system was ultrasonically treated for 5 min, and then graphene oxide (31 mg) and triethylamine (34 μL, 0.25 mM) were added. The reaction system was ultrasonically treated for 5 min again, and then the reaction was started at room temperature (25°C) for 48 h. A black composite was obtained after the reaction was completed, and then the composite was immersed in redistilled anhydrous tetrahydrofuran (2 mL) for 24 h. After 24 h, the solvent was removed, and the composite was washed with acetone until the eluate was colorless, and then vacuum dried at 60°C for 6 h to obtain the target composite.

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

[0056] Characterization:

[0057] (1) Determination of infrared spectrum of catalyst: The structure of the catalyst was determined by infrared spectrum. 3 mg of GO-TAPP, graphene oxide (GO) and 5,10,15,20-tetrakis(4-(ethynylphenyl)-porphyrin (TAPP) were taken and ground with dry potassium bromide powder in a mortar and kept dry at all times. Then, the mixture was placed in a tablet pressing mold to form a transparent, crack-free tablet. The tablet was placed in an infrared spectrum scanner at 500-400 cm -2 Scan 36 times within the range.

[0058] Fourier transform infrared spectroscopy (FT-IR) was used to verify the construction of the GO-TAPP complex. Figure 2 As shown in (a), GO-TAPP integrates the characteristics of GO and TAPP. The characteristic absorption bands of GO can be clearly observed, namely at 3340-3350, 3230, 1725 and 1620 cm -1 The observed wavelengths at 3270 and 1100 cm-1 belong to -OH, -C=O and -C=C-, as well as porphyrin. -1 The stretching vibration at 3270cm -1 The stretching vibration band of -C≡CH at 2105cm -1 The presence of the stretching vibration band of -C≡C- at the site indicates that -C≡CC≡C- is formed by the oxidation of -C≡CH. This proves that GO is successfully coated by TAPP coupling polymerization to form a stable GO-TAPP complex.

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

[0060] (3) The pore distribution of GO-TAPP composite materials was understood through N2 adsorption-desorption curve and pore size distribution curve. Figure 2 (cd) The low-temperature N2 adsorption / desorption isotherms of the GO-TAPP composite show a typical mesopore-dominated pore structure. The calculated surface area of ​​GO-TAPP was determined to be 8.14 m 2 g -1 , the total pore volume is 0.057 cm 3 g -1The corresponding pore size distribution (PSD) of GO-TAPP realized by nonlocal density functional theory (NLDFT) further revealed that it was a typical type II isotherm with broad mesopores distributed between 2.31–19.05 nm.

[0061] (4) The thermal stability of GO-TAPP composites was investigated by thermogravimetric analysis. Figure 2 As shown in (e), GO-TAPP exhibits high thermal stability and has almost no weight loss when heated below 121°C. GO-TAPP degrades rapidly at 121-271°C, which may be caused by the continuous decomposition of the oxygen-containing groups of GO in GO-TAPP. GO-TAPP degrades slowly at 271-800°C and retains 49% of its initial weight at temperatures up to 800°C.

[0062] (5) The crystal morphology of GO-TAPP composites was understood through X-ray diffraction patterns, such as Figure 2 (f) as shown.

[0063] (6) Scanning electron microscopy (SEM) and transmission electron microscopy (TEM): GO-TAPP and graphene oxide were adhered to a mica substrate with conductive glue and sprayed with gold, and then the sample morphology was observed under SEM. The GO-TAPP methanol dispersion after ultrasonic dispersion was dripped onto a copper mesh, and the observation sample was obtained after air drying. The sample was placed in a 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.

[0064] The morphology of GO-TAPP composites was observed by SEM. Figure 3 As shown, Figure 3 (ac) shows the morphology of GO-TAPP composite materials at 5μm, 3μm and 1μm, showing large interconnected flakes, covered with a rough mesh structure, and a large number of mesopores and micropores. Observe the TEM morphology of GO-TAPP, Figure 3 (de) shows the morphology of the GO-TAPP composite material at 500nm and 200nm, in which the transparent paper-like structure is a graphene oxide sheet, indicating that TAPP successfully coated GO after coupling. After high-resolution TEM observation (f), there is no obvious lattice fringes, and no metallic crystal form was found by bright field and dark field contrast. In addition, the morphology of graphene oxide was observed using SEM, such as Figure 4 As shown, Figure 4 (ab) show the morphology of graphene oxide at 5 μm and 1 μm, showing large flakes. Figure 5 The element distribution of GO-TAPP and its EDX are shown, showing that its various elements are evenly distributed.

[0065] (7) Photothermal performance of GO-TAPP composites: The photothermal performance of GO-TAPP composites was investigated by changing the concentration of GO-TAPP composites (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 composite materials with different concentrations is as follows: first, 10 mg of GO-TAPP is weighed and fully dispersed in 1 mL of distilled water using an ultrasonic instrument to prepare a 10 mg / mL mother solution, and then 5, 10, 20, 40, 60 and 80 μL of the mother solution are respectively drawn into 995, 990, 980, 960, 940 and 920 μL of distilled water, and finally 50, 100, 200, 400, 600 and 800 μg / mL of GO-TAPP aqueous dispersion are prepared.

[0066] First, under laser irradiation (λ = 638nm, 1W / cm 2 , 10min), the temperature rise behavior of GO-TAPP with different concentrations was studied. Figure 6 (a)- Figure 6 As shown in (b), GO-TAPP exhibits concentration-dependent photothermal conversion ability, and its temperature increases significantly with the increase of GO-TAPP concentration. For example, the minimum temperature rise (ΔT) of the GO-TAPP composite material at a concentration of 50 μg / mL is 3.2°C. 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 image obtained from the thermal imager can also intuitively reflect the concentration-dependent temperature rise behavior, see Figure 6 (c). Figure 6 (d) shows the temperature rise of GO-TAPP (600 μg / mL) under 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 rises rapidly from 25.4 ° C to 37.7 ° C, 52.2 ° C and 61.4 ° C, and the power change range is 0.5 W / cm 2 , 1W / cm 2 and 1.5W / cm 2 .at last, Figure 7 (a) shows the estimated photothermal stability of GO-TAPP through five consecutive laser on / off cycles. It can be clearly seen that GO-TAPP exhibits an efficient photothermal response 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 is calculated according to the photothermal conversion efficiency formula 1:

[0068] η(%)=[hS(T max -T surr )-Q dis ] / I(1-10 -A638 ) (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 after 10 minutes of irradiation (51.6°C); "T surr " is the ambient temperature during the experiment (24.3℃);" dis " is the heat dissipation of the test unit (25.03mW); "I" represents the 638nm laser power (1W / cm 2 ). "A638" is the absorbance of GO-TAPP aqueous solution at 638 nm (0.512).

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

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

[0072] The meaning of each element in the formula is: H2O " is the mass of the aqueous 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 formula 3:

[0074] t=-τS(Inθ) (Formula 3)

[0075] τS is the GO-TAPP time constant (274); "θ" is the time constant between ΔT and T Max ratio. Figure 7 (bc) shows the τS and θ values ​​in the formula.

[0076] In summary, the photothermal conversion efficiency (η) of GO-TAPP was determined to be 57.5%. Figure 7 (d) shows the 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, which indicates that the photothermal effect of the GO-TAPP composite mainly comes from the GO component.

[0077] (8) Photodynamic performance of GO-TAPP: GO-TAPP was subjected to 638nm laser (1W / cm 2 ) were studied.

[0078] First, 1,3-diphenylisobenzofuran (DPBF) was used as a photodynamic probe. It can be mixed with a photosensitizer (GO-TAPP group) and dissolved in dimethyl sulfoxide. By changing the laser irradiation time of the photosensitizer, the singlet oxygen ( 1 O2). 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 600μg / mL, and the concentration of GO or TAPP in the GO group and TAPP group was 300μg / mL. The preparation method is as follows: weigh 1 mg of GO-TAPP, and use an ultrasonic instrument to fully disperse GO or TAPP in 1 mL of dimethyl sulfoxide to prepare a 1 mg / mL mother solution. 600 μL of the GO-TAPP mother solution is added to 400 μL of DMSO to prepare a 600 μg / mL GO-TAPP dimethyl sulfoxide dispersion. 300 μL of the GO mother solution is added to 700 μL of DMSO to prepare a 300 μg / mL GO dimethyl sulfoxide dispersion. 300 μL of the TAPP mother solution is added to 700 μL of DMSO to prepare a 300 μg / mL TAPP dimethyl sulfoxide dispersion.

[0079] In order to study the photodynamic properties of GO-TAPP, Figure 8 As shown in (a), the absorbance of GO-TAPP, GO and TAPP in dimethyl sulfoxide was measured using a UV spectrophotometer, and the photodynamic properties of the materials were explored on this basis. Figure 8 (b)- Figure 8 As shown in (e), the pure DPBF group is the control group, that is, no photosensitizer is added, and only the DPBF probe is present. Except that the absorbance of the pure DPBF solution has almost no change, the UV-visible absorption light intensity at ~412nm of the other three groups, GO group, TAPP group and GO-TAPP group, shows a significant downward trend with the increase of laser irradiation time, indicating that DPBF is continuously consumed under light. For example, after 10 minutes of 638nm laser irradiation, the absorbance decrease rate of the GO-TAPP group reached 69.49±0.29% (relative to 9.56±0.68% of the pure DPBF group), which is 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. From the curve of absorption intensity changing with time Figure 8(f) can more intuitively reflect the performance difference. Such results verify that GO-TAPP can produce toxic substances that can be used for photodynamics under laser excitation. 1 O2, through the combination of GO and TAPP, its production amount and rate are significantly improved.

[0080] Test Example 1: In vitro antibacterial test

[0081] (1) Bacterial culture: This experiment used two bacteria, E. coli and S. aureus, and used second-generation bacteria to complete the following experiments. The specific culture method of the second-generation bacteria is as follows: first, thaw the frozen bacteria at 37°C, take 100 μL of the bacterial solution in a shaking tube containing 5 mL of liquid culture medium, place it in a constant temperature shaker (110 rpm, 37°C) and culture it for 12 hours, take 100 μL of the cultured bacterial solution and place it in a 2 mL EP tube containing 900 μL of 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 apply it to a culture dish containing solid culture medium, incubate at 37℃ for 24h, observe the clone morphology and colony count, and take a 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. According to the method of culturing first generation bacteria, a culture dish with about 1000 colonies is cultured as the second generation of bacteria. The culture medium in the bacterial solution (E.coli or S.aureus) is a liquid culture medium. The specific configuration method is to take 5g of LB broth, disperse it in 200mL of distilled water, and then sterilize it by high pressure sterilization to obtain a bacterial liquid culture medium. The specific configuration method of solid culture medium is to take 5g of LB broth and 3g of agar, disperse them in 200mL of distilled water, and then sterilize them by high pressure sterilization to obtain a solid culture medium.

[0082] (2) Determination of the antibacterial activity of GO-TAPP by plate counting method: After testing the performance of GO-TAPP on PTT and PDT, it can be concluded that GO-TAPP has certain antibacterial potential. The plate counting method was used to study the antibacterial ability of GO-TAPP induced by laser irradiation. The specific preparation method of bacterial dispersions of different concentrations of GO-TAPP groups is as follows: 10 mg of GO-TAPP powder was fully dispersed in 1 mL PBS to prepare a 10 mg / mL GO-TAPP stock solution, and 100 μL of 10 was added to 6 2 mL EP tubes respectively. 8 CFU mL -1Then 820, 840, 860, 880, 890 and 900 μL of PBS were added, and then 80, 60, 40, 20, 10 and 0 μL of 10 mg / mL GO-TAPP stock solution were added, respectively, to prepare 800, 600, 400, 200, 100 and 0 μg / mL GO-TAPP and 10 8 CFU mL -1 The PBS dispersion is the GO-TAPP solution. Then the GO-TAPP solution of different concentrations is irradiated with laser (laser parameters: λ = 638nm, 1W / cm 2 , 10min), GO-TAPP solutions of different concentrations were placed in a constant temperature shaker (110rpm, 37℃) for 12h, and then the cultured bacterial solution was gradiently diluted 10 5 Transfer 100 μL of the blown bacterial solution to the solid culture medium and spread it evenly. Incubate at 37°C for 24 hours to observe the morphology of the clones. Count the colonies and compare the bacterial activity of each group. Figure 9-10 As shown in the figure, with the increase of the concentration of GO-TAPP solution, the bactericidal performance against Staphylococcus aureus (S.aureus) and Escherichia coli (E.coli) is greatly enhanced. At a concentration of 100μg / mL, there are only slight bactericidal effects of 51.63±3.45% and 66.89±2.48% against Staphylococcus aureus and E. coli. However, at a concentration of 600μg / mL, the antibacterial rate of GO-TAPP solution against these two bacteria reaches more than 96.5% (96.88±0.33% for E. coli and 98.46±0.11% for Staphylococcus aureus). At a concentration of 800μg / mL, the antibacterial rate of GO-TAPP solution against these two bacteria is close to 100%, with the antibacterial rate of E. coli being 99.42±0.06% and the antibacterial rate of Staphylococcus aureus being 99.06±0.10%.

[0083] For comparison, the in vitro antibacterial ability of GO-TAPP, GO and TAPP was also evaluated. The plate count method was still used to observe the antibacterial effects of GO-TAPP (600μg / mL), GO (300μg / mL) and TAPP (300μg / mL) under the condition of light or not. The in vitro bacterial experiment was divided into eight groups, namely PBS group, TAPP group, GO group, GO-TAPP group, PBS+laser group, TAPP+laser group, GO+laser group and GO-TAPP+laser group. The preparation method of co-culture solution in different groups is as follows: first prepare GO-TAPP, GO or TAPP mother solution, that is, take 10 mg of GO-TAPP, GO or TAPP respectively, fully disperse it in 1 mL PBS, and prepare it into 10 mg / mL PBS dispersion 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, then add 840 μL of PBS, and then add 60 μL of 10 mg / mL GO-TAPP stock solution to make 600 μg / mL GO-TAPP and 10 8 CFU mL -1 The PBS dispersion was obtained to obtain the treated GO-TAPP group.

[0085] The treatment method of GO-TAPP+laser group was as follows: 600 μg / mL GO-TAPP and 10 8 CFU mL -1 The PBS dispersion was then irradiated with laser to obtain the treated GO-TAPP+laser group. The treatment method for the GO group was to add 100 μL 10 8 CFU mL -1 Bacterial solution, then add 870 μL of PBS, and then add 30 μL of 10 mg / mL GO stock solution to make 300 μg / mL GO and 10 8 CFU mL -1 PBS dispersion was used to obtain the treated GO group.

[0086] The treatment method of GO+laser group was to prepare 300 μg / mL GO and 10 8 CFU mL -1 The PBS dispersion was then irradiated with a laser to obtain the treated GO+laser group. The TAPP group was treated by adding 100 μL of 10 8 CFU mL -1 Bacterial solution, add 870 μL of PBS, and then add 30 μL of 10 mg / mL TAPP stock solution to make 300 μg / mL TAPP and 10 8 CFU mL -1 PBS dispersion was added to obtain the treated TAPP group.

[0087] The treatment method of TAPP+laser group was as follows: 300 μg / mL TAPP and 10 8 CFU mL -1 The PBS dispersion was then irradiated with a laser to obtain a treated TAPP+laser group.

[0088] Laser parameters: λ = 638nm, 1W / cm2 , 10min; then placed in a constant temperature shaker (110rpm, 37℃) for 12h according to the group, and then the cultured bacterial solution was gradient diluted 10 5 Transfer 100 μL of the blown bacterial solution to the solid culture medium and spread it evenly. Incubate at 37°C for 24 hours to observe the morphology of the clones. Count the colonies and compare the bacterial activity of each group. Fig.11 It can be seen that the bacteria treated with (V) PBS, (VI) TAPP, (VII) GO, (VIII) GO-TAPP and (I) PBS+laser groups 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 efficiency of the (II) TAPP+laser group reached 36.73±6.61% and 57.8±4.29% for the survival rates of Staphylococcus aureus and Escherichia coli, respectively. However, 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 capabilities of GO and TAPP to achieve a synergistic photothermal (PTT) and photodynamic (PDT) sterilization effect. In conclusion, GO-TAPP has good synergistic PTT and PDT antibacterial capabilities and can be used as a spectrum antibacterial agent with potential antibacterial capabilities to replace antibiotics.

[0089] Test Example 2: Bacterial live / dead staining test:

[0090] SYTO-9 and PI are used to distinguish between live and dead microbial cells. SYTO-9 penetrates all bacterial membranes (intact and damaged), thus marking the bacteria green. On the other hand, PI only penetrates the injured bacterial membrane, marking the bacteria red while reducing the green color of SYTO-9.

[0091] The 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 Experimental Example 1. Then, 100 μL of the bacterial suspension of each group was mixed with 20 μL of SYTO-9 (1.0×10 -3 M) and 20 μL PI (1.5×10 -3M) and co-incubated for 15 min in the dark at 37°C. 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.

[0092] from Fig.12 It can be seen that the results of live / dead staining are consistent with the results of the aforementioned co-culture experiment, and the two bacteria under different treatments show different fluorescence signals. Fig.13 Group I and Group II in (a), Fig.13 As shown in Group I and Group II in (b), the bacteria treated with PBS and PBS+laser only showed strong green fluorescence. However, for the bacteria treated with other groups, such as Fig.13 Group III and Group VIII in (a), Fig.13 As shown in Group III and Group VIII in (b), the group irradiated with laser exhibits much stronger red fluorescence than the single material. Fig.13 Group VIII in (a) and Fig.13 As shown in group VIII in (b), the GO-TAPP+laser group showed the most prominent sterilization effect, in which almost all Staphylococcus aureus and Escherichia coli were marked in red. Fig.13 (a) Group III, Group V and Fig.13 As shown in Group III and Group V in (b), only a portion of Staphylococcus aureus and Escherichia coli were stained red in the TAPP+laser and GO+laser groups, further demonstrating the superiority of GO-TAPP in synergizing PTT and PDT in antibacterial treatment.

[0093] Test Example 3: Bacterial transmission electron microscopy:

[0094] The bacterial liquid of PBS group, TAPP group, GO group, GO-TAPP group, PBS+laser group, TAPP+laser group, GO+laser group and GO-TAPP+laser group was prepared according to the method in Experimental Example 1. Afterwards, 100 μL of bacterial liquid was fixed in 2.5wt% glutaraldehyde solution (4°C, 2h), washed three times with PBS, embedded in agar and blocked. Then, ethanol solution (30wt%, 50wt%, 70wt%, 90wt%, 95wt% and 100wt%) was continuously treated at room temperature for 10min to dehydrate the bacteria, followed by acetone treatment at room temperature for 3h, embedded with embedding medium (epoxy resin) gradient infiltration (immersed for 1 hour with acetone and epoxy resin mass ratio of 3:1, 1:1, 1:3, and finally immersed in pure epoxy resin overnight) negative staining, and sectioned on nickel mesh. The nickel mesh was placed under TEM for observation to capture the bacterial morphology.

[0095] TEM was used to observe the integrity of bacterial membranes in different groups. Fig.13 (a) and Fig.13 As shown in (b), in the PBS and PBS+laser groups, the bacterial membranes of Staphylococcus aureus and Escherichia coli were intact in the presence or absence of light, indicating that the viability of the bacteria was not affected by light. The bacterial membranes under the TAPP treatment group (IV) were slightly damaged, as shown in Fig.13 Groups III and IV in (a) Fig.13 As shown in Groups III and IV in (b), the damage caused was minimal, probably because TAPP scratched the bacterial membrane. Similar to the live / dead staining results, the bacterial membranes of the other groups were also damaged to varying degrees after treatment. Fig.13 (a) Groups VII and VIII and Fig.13 As shown in groups VII and VIII in (b), GO-TAPP+laser caused the most serious damage to its bacterial membrane, and a large amount of bacterial contents flowed out. Therefore, the GO-TAPP composite material with synergistic PDT and PTT antibacterial ability has great application potential as a broad-spectrum antibacterial agent without antibiotics, which can effectively kill bacteria.

[0096] Test Example 4: In vitro biocompatibility test:

[0097] (1) Hemolysis test

[0098] The experiment was conducted under the guidance of the Ethics Committee and strictly in accordance with the protocol of Weifang Medical University. Fresh blood was obtained from KM female mice (Beijing Weitonglihua Experimental Animal Technology Co., Ltd.).

[0099] Red blood cells were collected by centrifugation at 1500rpm for 20min and then washed three times with PBS. Red blood cells (4% w / w) were then incubated with GO-TAPP (100-800μg / mL) at a ratio of 1:9 (v / v) at 37°C for 3h and then centrifuged at 12000rpm for 20min. Then, 100μL of supernatant from each group was placed in a 96-well plate, and the absorbance of each group was measured at 570nm using a microplate reader. Distilled water was used as a positive control and PBS was used as a negative control. The amount of hemolysis was calculated using the following formula 4:

[0100] Hemolysis amount (%) = (A-An) / (Ap-An) × 100% (Formula 4).

[0101] "A" is the absorbance of the supernatant after adding GO-TAPP 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).

[0102] like Fig.14 As shown, GO-TAPP showed only a small amount (less than 2%) or no hemolytic activity within the concentration range showing antibacterial activity. The hemolysis rate of GO-TAPP varied with the concentration of GO-TAPP, and increased from 0.02±0.01% to 1.29±0.01% as the concentration increased from 100 to 800 μg / mL. This indicates that GO-TAPP has good blood compatibility and has no or negligible damage to the red blood cell membrane.

[0103] (2) Cytotoxicity assay

[0104] HEK-293 cells (established from primary human embryonic kidney transformed with adenovirus type 5 (Ad 5), German Cell Bank (DSMZ): ACC 305) were cultured at 5 × 10 per well in a 96-well plate. 3 The cells were seeded at a density of 100 cells per well, 180 μL of cells per well, and 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 incubated 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 blue tetrazolyl). After dissolving on a shaker for 10 min, the absorbance of the 96-well plate was measured at 570 nm using a microplate reader. Each group of experiments was repeated three times.

[0105] At the same time, 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. Fig.15 The cell viability of HEK-293 cells cultured for 3 days with different concentrations of GO-TAPP (100-800 μg / mL) is shown. It can be clearly seen that as the concentration of the material decreases, the relative cell viability gradually increases, and its value can be maintained at more than 85% at all experimental concentrations, close to or above 100%, indicating no toxicity to HEK-293 cells. All these results confirm that GO-TAPP with good biocompatibility is a selective agent for bacteria.

[0106] Test Example 5:

[0107] (1) In vivo wound healing experiment:

[0108] Five-week-old female KM mice (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) (n = 5 per group, 20-25 g) were used to establish the wound healing model and divided into 7 groups. The specific modeling method was as follows: after disinfection with ethanol solution (75%), the back hair of each mouse was shaved before surgery to form a wound of d = 5 mm, and then Staphylococcus aureus (1×106 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 incubate the cells with or without light (λ = 638 nm, 1 W / cm 2 The infected wounds were treated at 4 °C for 10 min and 10 min, and the wound surface was photographed on days 0, 2, 4, 6, and 8, while the weight of the mice was monitored. The changes in wound size were measured using an image analysis program (Image.J, National Institutes of Health).

[0109] To evaluate the role of GO-TAPP in wound healing, a dorsal cortical injury wound model of KM rats infected with Staphylococcus aureus was established. For comparison, the effects of PBS (100 μL, control group), TAPP (100 μL, 600 μg / mL) + laser (λ = 638 nm, 1 W / cm 2 , 10min), TAPP (100μL, 600μg / mL), GO (100μL, 600μg / mL) + laser (λ = 638nm, 1W / cm 2 , 10min), GO (100μL, 600μg / mL), GO-TAPP (100μL, 600μg / mL) + laser (λ = 638nm, 1W / cm 2 The in vivo antibacterial capacity of the GO-TAPP (100 μL, 600 μg / mL) group was evaluated. The specific method is as follows: One day after the establishment of the damaged wound model of the back cortex of KM rats infected with Staphylococcus aureus, the wounds of each group were treated with the above dosage, which is the wound treatment. Day 0 to day 1 is the modeling time, and the damaged wounds are infected with Staphylococcus aureus. In addition, the wounds of the mice were photographed and recorded on days 0, 2, 4, 6 and 8. Fig.17 As shown, the photo of the back wound of the mouse just after perforation is shown on day 0. The photo of the back wound after one day of treatment is provided on day 2. It can be seen that all wounds showed the characteristics of bacterial infection, and the wound contraction rate of all groups was almost the same (~30%). Compared with the swelling of the non-laser group (PBS, TAPP, GO and GO-TAPP), the wounds of the laser treatment groups (TAPP+laser, GO+laser and GO-TAPP+laser) had begun to scab on the 4th day. As the treatment time was extended, the wounds under different treatment methods further shrank. Fig.18(a) shows the wound contraction rates of the wounds in 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). However, even after 7 days of treatment, the wound contraction rates of the non-laser group were only 65.35±4.12% (PBS), 76.85±0.42% (TAPP), 82.75±1.45% (GO), and 84.55±1.69% (GO-TAPP). In contrast, the wounds treated in the GO-TAPP+laser group were almost completely healed on day 8, which was significantly higher than that in the other two laser irradiation groups (88.86±1.1% for TAPP+laser and 92.76±0.86% for GO+laser). Fig.17 The superimposed wound images in the last row show more clearly that the wounds in the laser-irradiated group healed much better than those in the non-laser group. All these results indicate that laser-irradiated GO-TAPP not only significantly reduced bacterial invasion but also accelerated wound contraction compared with the other groups. At the same time, the body weight of KM rats was recorded daily for further comparison. Fig.18 As shown in (b), during the 8-day treatment, the mice in each group had no obvious behavioral abnormalities and no significant changes in body weight.

[0110] (2) In vivo biocompatibility study: tissue staining experiment (HE staining and Masson's III staining)

[0111] To further investigate the wound healing status of mice in each group, hematoxylin and eosin (H&E) staining and Masson's trichrome staining were used to evaluate the wound healing status of mice in (1) in vivo wound healing assay. Fig.19 As shown in Figure 2, histological analysis of wounds infected with Staphylococcus aureus showed that all groups had new capillary and skin growth to varying degrees. The most intuitive thing is that the scab area of ​​the wounds in the light-exposed group was smaller than that in the non-light-exposed group, indicating that the healing speed of the GO-TAPP, GO, and TAPP groups after laser irradiation was significantly accelerated. In addition, the scab area of ​​the GO-TAPP+laser group was the smallest, with 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 more quickly after laser irradiation.

[0112] To investigate the in vivo biocompatibility of GO-TAPP, the heart, liver, spleen, lung, and kidney of KM mice were subjected to tissue sectioning and H&E staining. Fig. 20 As shown, the results showed that there was no obvious inflammation and morphological damage in different organs of each group.

[0113] (2) Routine blood test

[0114] On day 8, 1 to 2 ml of blood samples were collected from the retinal arteries of 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 hemoglobin concentration (MCHC), and mean hemoglobin content (MCH).

[0115] In order to study the in vivo biocompatibility of GO-TAPP, blood was drawn from the eyes of the above KM mice for routine blood tests. Fig.21 As shown, no significant differences were found in the blood routine values ​​among the groups.

[0116] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing a GO-TAPP microporous composite material, characterized in that: The preparation method is: 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 and 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.

2. The preparation method 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 preparation method according to claim 1, characterized in that: 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.

4. The preparation method according to claim 1 or 3, characterized in that: The tetrahydrofurans are all redistilled anhydrous tetrahydrofurans.

5. The preparation method according to claim 1, characterized in that: The mixing was all carried out by ultrasonic mixing, and the mixing time was all 5 minutes.

6. The preparation method 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.

7. The preparation method according to claim 1, characterized in that: The washing is performed by washing with acetone until the washing liquid is colorless; and the drying is performed by vacuum drying at 60° C. for 6 hours.

8. The GO-TAPP microporous composite material obtained by the preparation method according to claims 1 to 7.

9. The GO-TAPP microporous composite material according to claim 8, characterized in that: The hemolysis rate of the GO-TAPP microporous composite material on cells is less than 2%.

10. Use of the GO-TAPP microporous composite material according to claim 8 or 9 in the preparation of antibacterial drugs.

Images