Preparation method and application of quaternary ammonium salt and gold rod modified two-dimensional graphene oxide

Abstract

The present invention provides a preparation method and application of a quaternary ammonium salt and a gold rod modified two-dimensional oxidized graphene, the preparation method of the quaternary ammonium salt and the gold rod modified two-dimensional oxidized graphene comprising the following steps: preparation of a quaternary ammonium salt monomer VPIM-Br, preparation of a gold seed Au solution, preparation of a gold rod AuNR solution, preparation of oxidized graphene GDYO, preparation of a quaternary ammonium salt and a gold rod modified two-dimensional oxidized graphene GDYO-VPIM-AuNR. The present invention solves the problem that existing antibacterial materials cannot effectively kill drug-resistant bacteria for a long time, promote cell proliferation, and accelerate the healing of diabetic wounds. It has excellent antibacterial ability and good biosafety. It can accelerate cell proliferation, enhance the production of cell growth factors, and accelerate wound healing under the synergistic effect of photodynamic and photothermal effects.

Description

Technical Field

[0001] The invention relates to the technical field of antibacterial materials, and in particular to a preparation method and application of quaternary ammonium salt and gold rod modified two-dimensional oxidized graphene. Background Art

[0002] Diabetes is a metabolic disease characterized by high blood sugar levels and is ranked as the third largest disease in the world. Diabetic infected wounds are one of the most serious complications of diabetes and may lead to amputation in severe cases. Compared with ordinary wounds, diabetic infected wounds show excessive and persistent inflammatory responses due to increased blood sugar levels, leading to wound deterioration and delayed healing. The main obstacle to healing is chronic inflammation, which is caused by impaired cell proliferation and persistent infection with resistant bacteria, which exacerbates the inflammatory response. Apoptosis further hinders angiogenesis and tissue occurrence, posing a major challenge to diabetic wound healing. Therefore, improving cell proliferation and preventing bacterial infection during the healing process are crucial to preventing diabetic ulcer formation and wound expansion.

[0003] At present, the treatment of diabetic wound infection mainly includes taking hypoglycemic drugs, injecting insulin, and using topical antibacterial materials to promote wound healing. However, current antibacterial materials cannot effectively kill drug-resistant bacteria, promote cell proliferation, and accelerate the healing of diabetic wounds in the long term. Wounds that do not heal for a long time are prone to infection and other diseases, which can bring serious consequences to patients. Therefore, it is urgent to develop an topical antibacterial material that can effectively promote the healing of diabetic wounds. Summary of the invention

[0004] Based on this, the present invention provides a preparation method and application of quaternary ammonium salt and gold rod modified two-dimensional oxidized graphene, which solves the problem that existing antibacterial materials cannot effectively kill drug-resistant bacteria for a long time, promote cell proliferation, and accelerate the healing of diabetic wounds.

[0005] In order to solve the above technical problems, one of the objects of the present invention is to provide a method for preparing two-dimensional graphene oxide modified with quaternary ammonium salt and gold rod, comprising the following steps:

[0006] S1. Preparation of quaternary ammonium salt monomer VPIM-Br: 1-vinylimidazole and 1-bromopentane were added to cyclohexane, reacted at 50°C for 24 hours, cooled to room temperature, stored in a -20°C refrigerator for 12-24 hours, tetrahydrofuran was added, magnetic stirring was performed until a white solid precipitated, the supernatant was discarded, and vacuum dried at 30-40°C for 12-24 hours to obtain the quaternary ammonium salt monomer VPIM-Br;

[0007] S2: Preparation of gold seed Au solution: add hexadecyltrimethylammonium bromide into deionized water and fully dissolve it. Under magnetic stirring and at a temperature of 25°C, quickly add chloroauric acid solution into the hexadecyltrimethylammonium bromide solution. After mixing evenly, add sodium borohydride solution into the mixed solution, stir for 2 minutes, and then incubate in a constant temperature water bath at 25.5°C for 0.5-2 hours to obtain a gold seed Au solution.

[0008] S3: Preparation of gold rod AuNR solution: add hexadecyltrimethylammonium bromide to deionized water and fully dissolve it, add silver nitrate solution to hexadecyltrimethylammonium bromide solution, add chloroauric acid solution and shake quickly, add L-ascorbic acid solution, add the gold seed Au solution prepared in S2 at a solution temperature of 24-26°C, incubate in a constant temperature water bath at 27.5°C for 12 h, and obtain a gold rod AuNR solution;

[0009] S4: Preparation of oxidized graphene GDYO: Grind graphene GDY into powder, disperse in ultrapure water, ultrasonically treat in an ice-water bath for 10 h, centrifuge and freeze-dry for 24 h to obtain a monolayer flaky graphene GDY powder, add hydrogen peroxide and concentrated sulfuric acid to the monolayer flaky graphene GDY powder, incubate in an ice-water bath for 5 h, dialyze and freeze-dry for 24 h to obtain oxidized graphene GDYO;

[0010] S5: Preparation of quaternary ammonium salt and gold rod modified two-dimensional graphene oxide GDYO-VPIM-AuNR: Graphene oxide GDYO was ultrasonically dispersed in deionized water, and quaternary ammonium salt monomer VPIM-Br and gold rod AuNR solution were added. The mixture was ultrasonically treated for 2 h and magnetically stirred for 3 h. The quaternary ammonium salt and gold rod modified two-dimensional graphene oxide GDYO-VPIM-AuNR was obtained by centrifugal washing and vacuum freeze drying.

[0011] Preferably, the molar ratio of 1-vinylimidazole to 1-bromopentane in S1 is 1:1; the solid-liquid ratio of 1-vinylimidazole to cyclohexane is 47:500.

[0012] Preferably, the volume ratio of hexadecyltrimethylammonium bromide, chloroauric acid and sodium borohydride solution in S2 is 35:1.25:1, wherein the concentration of hexadecyltrimethylammonium bromide solution is 0.2 mol / L, the concentration of chloroauric acid solution is 15 mmol / L, and the concentration of sodium borohydride solution is 0.01 mol / L.

[0013] Preferably, the volume ratio of hexadecyltrimethylammonium bromide, silver nitrate, chloroauric acid, L-ascorbic acid, and gold seed Au solution in S3 is 88.4:4:5.2:1.24:1, wherein the concentration of hexadecyltrimethylammonium bromide solution is 0.16 mol / L, the concentration of silver nitrate solution is 4 mmol / L, the concentration of chloroauric acid solution is 15 mmol / L, the concentration of L-ascorbic acid solution is 0.08 mol / L, and the concentration of gold rod AuNR solution is 1.0 nmol / L.

[0014] Preferably, the solid-to-liquid ratio of the single-layer flaky graphyne GDY powder and the mixed solution of hydrogen peroxide and concentrated sulfuric acid in S4 is 5:4, and the volume ratio of hydrogen peroxide to concentrated sulfuric acid is 1:3.

[0015] Preferably, the particle size of the graphyne GDY powder in S4 is ≤1 μm.

[0016] Preferably, the molecular weight cutoff of the dialysis bag used for dialysis in S4 is ≤200 Da.

[0017] Preferably, the mass ratio of the graphene oxide GDYO to the quaternary ammonium salt monomer VPIM-Br is 1:10, the solid-liquid ratio of the graphene oxide GDYO to the gold rod AuNR solution is 1:0.7-1, and the solid-liquid ratio of the graphene oxide GDYO to deionized water is 1:1000.

[0018] Based on the same inventive concept, the present invention also provides an application of quaternary ammonium salt and gold rod modified two-dimensional oxidized graphene, and the quaternary ammonium salt and gold rod modified two-dimensional oxidized graphene is used as a wound healing material for diabetic patients.

[0019] The above one or more technical solutions of the present invention have the following technical effects:

[0020] The quaternary ammonium salt and gold rod modified two-dimensional oxidized Graphene prepared by the present invention has excellent antibacterial ability and good biological safety. Under the synergistic effect of photodynamic and photothermal effects, it can accelerate cell proliferation, enhance the production of cell growth factors, and accelerate wound healing. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for describing the embodiments are briefly introduced below.

[0022] Figure 1 :Preparation process of two-dimensional graphene oxide modified by quaternary ammonium salt and gold rod;

[0023] Figure 2 : Transmission electron microscopy (TEM) images of two-dimensional GO modified by quaternary ammonium salt and gold rod;

[0024] Figure 3: X-ray photoelectron spectroscopy XPS spectra of two-dimensional GO modified by quaternary ammonium salt and gold rod;

[0025] Figure 4 :Active oxygen fluorescence spectra of two-dimensional GO modified by quaternary ammonium salt and gold rod;

[0026] Figure 5 :Photothermal temperature change diagram of two-dimensional GO modified by quaternary ammonium salt and gold rod;

[0027] Figure 6 :Bacterial killing rate of quaternary ammonium salt and gold rod modified two-dimensional graphene oxide against drug-resistant Escherichia coli;

[0028] Figure 7 :Agar plate image of two-dimensional graphene oxide modified with quaternary ammonium salt and gold rod against drug-resistant Escherichia coli;

[0029] Figure 8 :Bactericidal rate of quaternary ammonium salt and gold rod modified two-dimensional graphene oxide against methicillin-resistant Staphylococcus aureus;

[0030] Fig. 9 :Agar plate image of quaternary ammonium salt and gold rod modified two-dimensional graphene oxide against methicillin-resistant Staphylococcus aureus;

[0031] Fig.10 :Biocompatibility and cell viability of two-dimensional graphene oxide modified with quaternary ammonium salt and gold rod;

[0032] Fig.11 :Quaternary ammonium salt and gold rod modified two-dimensional graphene oxide mild photothermal therapy promotes cell growth;

[0033] Fig.12 :Digital image of two-dimensional oxidized graphene modified with quaternary ammonium salt and gold rod for the treatment of diabetic wounds;

[0034] Fig.13 :Wound recovery rate diagram of diabetic wounds treated with two-dimensional oxidized graphene modified with quaternary ammonium salt and gold rod;

[0035] Fig.14 :Agar plate photos of the total number of colonies in the treatment of diabetic wounds by two-dimensional oxidized Graphene modified with quaternary ammonium salt and gold rod;

[0036] Fig.15 : The number of surviving colonies in the treatment of diabetic wounds using two-dimensional oxidized graphene modified with quaternary ammonium salt and gold rod. DETAILED DESCRIPTION

[0037] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention.

[0038] Throughout the specification, unless otherwise specified, the terms used herein should be understood as commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. If there is a conflict, the present specification takes precedence. The preparation process of quaternary ammonium salt and gold rod modified two-dimensional graphene oxide is as follows Figure 1 shown.

[0039] Example 1

[0040] A method for preparing two-dimensional graphene oxide modified by quaternary ammonium salt and gold rod comprises the following steps:

[0041] S1. Preparation of quaternary ammonium salt monomer VPIM-Br: Add 2.82 g 1-vinyl imidazole and 4.53 g 1-bromopentane to 30 ml cyclohexane, react at 50°C for 24 h, cool to room temperature, store in a -20°C refrigerator for 12-24 h, add 10 ml tetrahydrofuran, stir magnetically until white solid precipitates, discard the supernatant, and dry under vacuum at 30-40°C for 12-24 h to obtain quaternary ammonium salt monomer VPIM-Br;

[0042] S2: Preparation of gold seed Au solution: add 0.1823 g hexadecyltrimethylammonium bromide to 3.5 g deionized water and fully dissolve it. Under magnetic stirring and a temperature of 25°C, quickly add 125 μL of 15 mmol / L chloroauric acid solution to the hexadecyltrimethylammonium bromide solution. After mixing evenly, add 500 ml of 0.01 mol / L sodium borohydride solution to the mixed solution, stir for 2 min, and then incubate in a constant temperature water bath at 25.5°C for 0.5-2 h to obtain a gold seed Au solution.

[0043] S3: Preparation of gold rod AuNR solution: add 0.3645 g hexadecyltrimethylammonium bromide to 8.84 g deionized water and fully dissolve it, add 400 μL of 4 mmol / L silver nitrate solution to the hexadecyltrimethylammonium bromide solution, add 520 μL of 15 mmol / L chloroauric acid solution and shake quickly, add 124 μL of 0.08 mol / L L-ascorbic acid solution, add 100 μL of gold seed Au solution prepared in S2 at a solution temperature of 24-26°C, incubate in a constant temperature water bath at 27.5°C for 12 hours, and obtain a gold rod AuNR solution;

[0044] S4: Preparation of oxidized graphene GDYO: Grind graphene GDY into powder, disperse in ultrapure water, ultrasonically treat in an ice-water bath for 10 h, centrifuge and freeze-dry for 24 h to obtain a single-layer flaky graphene GDY powder, add 2.0 ml hydrogen peroxide and 6.0 ml concentrated sulfuric acid to 10 mg of the single-layer flaky graphene GDY powder, incubate in an ice-water bath for 5 h, dialyze and freeze-dry for 24 h to obtain oxidized graphene GDYO;

[0045] S5: Preparation of quaternary ammonium salt and gold rod modified two-dimensional graphene oxide GDYO-VPIM-AuNR: Ultrasonic disperse 10 mg of graphene oxide GDYO in 10 ml of deionized water, add 100 mg of quaternary ammonium salt monomer VPIM-Br and 7.0 ml of gold rod AuNR solution prepared in S3, ultrasonicate for 2 h, magnetically stir for 3 h, and obtain quaternary ammonium salt and gold rod modified two-dimensional graphene oxide GDYO-VPIM-AuNR by centrifugal washing and vacuum freeze drying.

[0046] Example 2

[0047] Application of quaternary ammonium salt and gold rod modified two-dimensional GO for healing materials of infected wounds in patients with diabetes: quaternary ammonium salt and gold rod modified two-dimensional GO were dissolved in deionized water and sterilized to form a solution with a concentration of 1.0 mg / ml. The solution was added dropwise to the infected wound of the diabetic patient and irradiated with laser for 5 minutes (wavelength 808 nm, power 1 W / cm 2 ), and then irradiated with LED for 30 minutes (power 15mW / cm 2 ).

[0048] Test Example 1

[0049] Morphological characterization of two-dimensional graphene oxide GDYO-VPIM-AuNR modified by quaternary ammonium salt and gold rod: The two-dimensional graphene oxide GDYO-VPIM-AuNR modified by quaternary ammonium salt and gold rod prepared in Example 1 was selected for testing. Figure 2 As shown, the quaternary ammonium salt and gold rod modified two-dimensional graphene oxide GDYO-VPIM-AuNR presents a lamellar structure, and the gold rod AuNR is uniformly loaded on GDYO.

[0050] The quaternary ammonium salt and gold rod modified two-dimensional graphene oxide GDYO-VPIM-AuNR prepared in Example 1 were selected for testing. Figure 3 As shown, the X-ray photoelectron spectrum has carbon, nitrogen, bromine and gold element signals at 286.7 eV, 404.0 eV, 67.0 eV and 87.5 eV, respectively, indicating that the quaternary ammonium salt and gold rods are both loaded on oxidized graphyne GDYO.

[0051] Test Example 2

[0052] Characterization of the photodynamic effect of quaternary ammonium salt and gold rod modified two-dimensional graphene oxide GDYO-VPIM-AuNR to produce reactive oxygen and photothermal performance: The quaternary ammonium salt and gold rod modified two-dimensional graphene oxide GDYO-VPIM-AuNR prepared in Example 1 were selected for testing. The photodynamic performance of quaternary ammonium salt and gold rod modified two-dimensional graphene oxide GDYO-VPIM-AuNR in releasing reactive oxygen under LED light was evaluated using a typical reactive oxygen species (ROS) probe 2',7'-dichlorodihydrofluorescein diacetate (2,7-DCFH-DA). The probe was converted into 2-(3,6-diacetoxy-2,7-dichloro-9-xanthen-9-yl) benzoic acid (DCF) with fluorescent properties in the presence of ROS. Figure 4 As shown, the fluorescence intensity increases with the increase of illumination time, indicating that the quaternary ammonium salt and gold rod modified two-dimensional oxidized graphyne GDYO-VPIM-AuNR can produce active oxygen under the irradiation of LED light, and has good photodynamic properties to achieve a bactericidal effect.

[0053] like Figure 5 As shown, at a power of 1.0 W / cm 2 , under laser irradiation with a laser wavelength of 808nm, the photothermal effect of quaternary ammonium salt and gold rod modified two-dimensional oxidized graphene GDYO-VPIM-AuNR was evaluated as a function of temperature over time. After 5 minutes of laser irradiation, the temperature of quaternary ammonium salt and gold rod modified two-dimensional oxidized graphene GDYO-VPIM-AuNR reached 48°C, which is a mild photothermal effect, promoting cell proliferation, sterilizing, and accelerating wound healing.

[0054] Test Example 3

[0055] Antibacterial performance test: the order of magnitude is 1.0×10 7 CFU / ml of methicillin-resistant Staphylococcus aureus (MRSA) and ampicillin-resistant Escherichia coli ( E. coli pUC 19) were contacted with the quaternary ammonium salt and gold rod modified two-dimensional graphene oxide GDYO-VPIM-AuNR prepared in Example 1, and the antibacterial properties were verified by measuring the percentage of bacterial survival after contact with different conditions. The action conditions were: (1) darkness, (2) photodynamic effect PDT (LED light for 30 minutes), (3) photothermal effect PTT (808 nm, 1.0 W / cm 2 Laser irradiation for 5 minutes, (4) Photodynamic and photothermal synergistic effect PDT & PTT (LED light irradiation for 30 minutes and 808 nm, 1.0 W / cm 2 Laser irradiation for 5 minutes).

[0056] like Figure 6 and 7As shown in the figure, the two-dimensional graphene oxide GDYO-VPIM-AuNR modified with quaternary ammonium salt and gold rods showed limited antibacterial activity in the dark. After the photodynamic effect PDT, photothermal effect PTT, and photodynamic and photodynamic synergistic effects, the antibacterial ability of the two-dimensional graphene oxide GDYO-VPIM-AuNR modified with quaternary ammonium salt and gold rods was improved. For ampicillin-resistant Escherichia coli ( E. coli pUC 19), after photodynamic therapy (PDT), the quaternary ammonium salt and gold rod modified two-dimensional graphene oxide GDYO-VPIM-AuNR was effective against ampicillin-resistant Escherichia coli ( E. coli The antibacterial rate of pUC 19 was 75%. After the photothermal effect PTT, the quaternary ammonium salt and gold rod modified two-dimensional graphene oxide GDYO-VPIM-AuNR was effective against ampicillin sodium-resistant Escherichia coli ( E. coli pUC 19) had an antibacterial rate of 86%. After the synergistic effects of photodynamic and photothermal effects, the quaternary ammonium salt and gold rod modified two-dimensional graphene oxide GDYO-VPIM-AuNR was effective against ampicillin-resistant Escherichia coli ( E. coli The antibacterial rate of pUC 19 is significantly improved, and the antibacterial rate can reach 99.99%.

[0057] like Figure 8 and 9 As shown in the figure, for methicillin-resistant Staphylococcus aureus (MRSA), after the photodynamic effect PDT, the antibacterial rate of quaternary ammonium salt and gold rod modified two-dimensional graphene oxide GDYO-VPIM-AuNR against methicillin-resistant Staphylococcus aureus (MRSA) was 64%. After the photothermal effect PTT, the antibacterial rate of quaternary ammonium salt and gold rod modified two-dimensional graphene oxide GDYO-VPIM-AuNR against methicillin-resistant Staphylococcus aureus (MRSA) was 76%. After the synergistic effect of photodynamic and photothermal, the antibacterial rate of quaternary ammonium salt and gold rod modified two-dimensional graphene oxide GDYO-VPIM-AuNR against methicillin-resistant Staphylococcus aureus (MRSA) was significantly improved, and the antibacterial rate could reach 99.99%.

[0058] In summary, the photodynamic and photothermal synergistic effects of PDT and PTT (LED light for 30 minutes and 808 nm, 1.0 W / cm 2 After laser irradiation for 5 minutes, both methicillin-resistant Staphylococcus aureus (MRSA) and ampicillin-resistant Escherichia coli ( E. coli pUC 19), quaternary ammonium salt and gold rod modified Graphene oxide GDYO-VPIM-AuNR have an antibacterial rate of 99.99%, which can be applied to diabetic bacterial infection wounds.

[0059] Test Example 4

[0060] Biocompatibility of quaternary ammonium salt and gold rod modified two-dimensional oxidized graphene GDYO-VPIM-AuNR: Mouse embryonic fibroblasts (NIH-3T3 cells) were used as model cells, and their biocompatibility was evaluated by measuring their toxicity to mammalian cells. The quaternary ammonium salt and gold rod modified two-dimensional oxidized graphene GDYO-VPIM-AuNR prepared in Example 1 were selected, and prepared into 4.0 mg / ml solutions, and diluted into 2.0 mg / ml, 1.5 mg / ml, 1.0 mg / ml, 0.75 mg / ml, and 0.5 mg / ml solutions, respectively. After incubating with NIH-3T3 cells for 24 h, the cell activity of the cells after the action of quaternary ammonium salt and gold rod modified two-dimensional oxidized graphene GDYO-VPIM-AuNR was tested using the CCK-8 kit. Fig.10 As shown, even when the concentration of quaternary ammonium salt and gold rod modified oxidized graphyne GDYO-VPIM-AuNR reached 2.0 mg / ml, the cell survival rate remained above 80%, indicating that it has good biosafety.

[0061] Test Example 5

[0062] Characterization of mild photothermal cell growth promotion by quaternary ammonium salt and gold rod modified two-dimensional graphene oxide GDYO-VPIM-AuNR: Fig.11 The ability of quaternary ammonium salts and gold rods modified two-dimensional oxidized graphene GDYO-VPIM-AuNR to promote cell proliferation was evaluated by measuring the concentration of basic fibroblast growth factor (bFGF) in NIH-3T3 cells using an enzyme-linked immunosorbent assay (ELISA). Comparing the cell growth factor (bFGF) concentration of quaternary ammonium salts and gold rods modified two-dimensional oxidized graphene GDYO-VPIM-AuNR+PTT group with mild photothermal effect PTT was higher than that of quaternary ammonium salts and gold rods modified two-dimensional oxidized graphene GDYO-VPIM-AuNR, reaching 30 pg / ml in 24 hours and 40 pg / ml in 48 hours. The experimental results show that under the photothermal effect PTT, quaternary ammonium salts and gold rods modified two-dimensional oxidized graphene GDYO-VPIM-AuNR enhanced the production of cell proliferation stimulating cell growth factor (bFGF). The photothermal effect of quaternary ammonium salt and gold rod modified two-dimensional graphene oxide GDYO-VPIM-AuNR can accelerate cell proliferation, enhance the production of cell growth factors, and accelerate wound healing.

[0063] Test Example 6

[0064] Treatment of diabetic wounds: Diabetes was established by administering streptozotocin (STZ) to C57BL / 6 mice for 7 consecutive days. The efficiency of quaternary ammonium salt and gold rod modified two-dimensional graphene oxide GDYO-VPIM-AuNR in treating diabetic skin wounds infected with methicillin-resistant Staphylococcus aureus (MRSA) was evaluated in the mouse model. The diabetic mouse wound model was established when the average blood glucose concentration of the mice reached above 16.7 mmol / L. The diabetic mice were anesthetized and shaved, and a circular full-thickness skin wound area (10 mm×10 mm) was formed on the back, and then 10 μL of methicillin-resistant Staphylococcus aureus (10 μL) solution was added. 7 CFU / ml) were dripped into the wound. The mice were randomly divided into five groups: (1) control group (methicillin-resistant Staphylococcus aureus MRSA was first dripped and then phosphate buffer solution PBS solution was dripped, which served as a blank control group without quaternary ammonium salt and gold rod modified two-dimensional graphene oxide GDYO-VPIM-AuNR treatment), (2) dark treatment group, (3) photodynamic therapy group PDT (LED light for 30 minutes), (4) photothermal therapy group PTT (808 nm, 1.0 W / cm 2 Laser irradiation for 5 minutes, (5) Photodynamic and photothermal synergistic effect treatment group PDT & PTT (LED light irradiation for 30 minutes and 808 nm, 1.0 W / cm 2 The solution was added to the infected wound and then laser irradiated for 5 minutes at 15 mW / cm 2 LED irradiation for 30 minutes, 1.0 W / cm 2 The wounds were irradiated with 808 nm laser for 5 min. The wound size was measured using a vernier digital caliper and photographs were taken at 0, 3, 7, 9 and 13 days.

[0065] Fig.12 Wound healing was monitored by photographing the wounds on days 1, 3, 7, 9, and 13 to assess the progress of healing. Fig.12 The wound photos at different time points are shown, showing the repair process and healing effect of quaternary ammonium salt and gold rod modified two-dimensional Graphene oxide GDYO-VPIM-AuNR under different treatment conditions. By the 13th day, the wound of the PDT&PTT group of quaternary ammonium salt and gold rod modified two-dimensional Graphene oxide GDYO-VPIM-AuNR photodynamic and photothermal effect synergistic treatment was almost healed, leaving only a faint white scar. Compared with other groups, the PDT&PTT group of quaternary ammonium salt and gold rod modified two-dimensional Graphene oxide GDYO-VPIM-AuNR photodynamic and photothermal effect synergistic treatment was better. This shows that the photodynamic and photothermal synergistic effect treatment has the best effect.

[0066] Fig.13Wound recovery rate and quantitative analysis of diabetic wound area. Compared with the control group (82%), dark group (85%), photodynamic effect PDT group (90%) and photothermal PTT group (95%), the wounds treated with PDT&PTT using quaternary ammonium salt and gold rod modified two-dimensional graphene oxide GDYO-VPIM-AuNR showed significant healing on the 13th day, with the highest wound recovery rate (100%). This further proves that photothermal and photodynamic synergistic therapy has the best effect.

[0067] Test Example 7

[0068] To evaluate the in vivo antibacterial effect of quaternary ammonium salt and gold rod modified 2D Graphene Oxide GDYO-VPIM-AuNR, methicillin-resistant Staphylococcus aureus (MRSA) from the wounds were collected using sterile swabs on day 13, and the number of bacteria was determined by counting the bacteria on agar plates. Fig.14 As shown in the figure, the bacterial growth on the Luria-Bertani (LB) agar plate with photothermal and photodynamic effects synergistically acting on PDT & PTT was significantly reduced, while a large number of bacterial colonies existed in other groups. Fig.15 As shown, the photodynamic and photothermal effects of quaternary ammonium salt and gold rod modified two-dimensional graphene oxide GDYO-VPIM-AuNR synergistically with PDT&PTT can effectively kill bacteria with the lowest number of methicillin-resistant Staphylococcus aureus (MRSA) colonies. This indicates that the photodynamic and photothermal effects of quaternary ammonium salt and gold rod modified two-dimensional graphene oxide GDYO-VPIM-AuNR synergistically with PDT&PTT can effectively kill bacteria.

[0069] In summary, the present invention solves the problem that existing antibacterial materials cannot effectively kill drug-resistant bacteria for a long time, promote cell proliferation, and accelerate the healing of diabetic wounds. It has excellent antibacterial ability and good biological safety. Under the synergistic effect of photodynamic and photothermal effects, it can accelerate cell proliferation, enhance the production of cell growth factors, and accelerate wound healing.

Claims

1. A method for preparing two-dimensional graphene oxide modified by quaternary ammonium salt and gold rod, characterized in that: The steps include: S1. Preparation of quaternary ammonium salt monomer VPIM-Br: 1-vinylimidazole and 1-bromopentane were added to cyclohexane, reacted at 50°C for 24 hours, cooled to room temperature, stored in a -20°C refrigerator for 12-24 hours, tetrahydrofuran was added, magnetic stirring was performed until a white solid precipitated, the supernatant was discarded, and vacuum dried at 30-40°C for 12-24 hours to obtain the quaternary ammonium salt monomer VPIM-Br; S2: Preparation of gold seed Au solution: add hexadecyltrimethylammonium bromide into deionized water and fully dissolve it. Under magnetic stirring and at a temperature of 25°C, quickly add chloroauric acid solution into the hexadecyltrimethylammonium bromide solution. After mixing evenly, add sodium borohydride solution into the mixed solution, stir for 2 minutes, and then incubate in a constant temperature water bath at 25.5°C for 0.5-2 hours to obtain a gold seed Au solution. S3: Preparation of gold rod AuNR solution: add hexadecyltrimethylammonium bromide to deionized water and fully dissolve it, add silver nitrate solution to hexadecyltrimethylammonium bromide solution, add chloroauric acid solution and shake quickly, add L-ascorbic acid solution, add the gold seed Au solution prepared in S2 at a solution temperature of 24-26°C, incubate in a constant temperature water bath at 27.5°C for 12 h, and obtain a gold rod AuNR solution; S4: Preparation of oxidized graphene GDYO: Grind graphene GDY into powder, disperse in ultrapure water, ultrasonically treat in an ice-water bath for 10 h, centrifuge and freeze-dry for 24 h to obtain a monolayer flaky graphene GDY powder, add hydrogen peroxide and concentrated sulfuric acid to the monolayer flaky graphene GDY powder, incubate in an ice-water bath for 5 h, dialyze and freeze-dry for 24 h to obtain oxidized graphene GDYO; S5: Preparation of quaternary ammonium salt and gold rod modified two-dimensional graphene oxide GDYO-VPIM-AuNR: Graphene oxide GDYO was ultrasonically dispersed in deionized water, and quaternary ammonium salt monomer VPIM-Br and gold rod AuNR solution were added. The mixture was ultrasonically treated for 2 h and magnetically stirred for 3 h. The quaternary ammonium salt and gold rod modified two-dimensional graphene oxide GDYO-VPIM-AuNR was obtained by centrifugal washing and vacuum freeze drying.

2. The method for preparing a two-dimensional graphene oxide modified by a quaternary ammonium salt and a gold rod according to claim 1, characterized in that: The molar ratio of 1-vinylimidazole to 1-bromopentane in S1 is 1:1; the solid-liquid ratio of 1-vinylimidazole to cyclohexane is 47:

500.

3. The method for preparing a two-dimensional graphene oxide modified by a quaternary ammonium salt and a gold rod according to claim 1, characterized in that: The volume ratio of hexadecyltrimethylammonium bromide, chloroauric acid and sodium borohydride solution in S2 is 35:1.25:1, wherein the concentration of hexadecyltrimethylammonium bromide solution is 0.2 mol / L, the concentration of chloroauric acid solution is 15 mmol / L, and the concentration of sodium borohydride solution is 0.01 mol / L.

4. The method for preparing a two-dimensional graphene oxide modified by a quaternary ammonium salt and a gold rod according to claim 1, characterized in that: The volume ratio of hexadecyltrimethylammonium bromide, silver nitrate, chloroauric acid, L-ascorbic acid, and gold seed Au solution in S3 is 88.4:4:5.2:1.24:1, wherein the concentration of hexadecyltrimethylammonium bromide solution is 0.16 mol / L, the concentration of silver nitrate solution is 4 mmol / L, the concentration of chloroauric acid solution is 15 mmol / L, the concentration of L-ascorbic acid solution is 0.08 mol / L, and the concentration of gold rod AuNR solution is 1.0 nmol / L.

5. The method for preparing a two-dimensional graphene oxide modified by a quaternary ammonium salt and a gold rod according to claim 1, characterized in that: The solid-to-liquid ratio of the monolayer flaky graphyne GDY powder in S4 to the mixed solution of hydrogen peroxide and concentrated sulfuric acid is 5:4, and the volume ratio of hydrogen peroxide to concentrated sulfuric acid is 1:

3.

6. The method for preparing a two-dimensional graphene oxide modified by a quaternary ammonium salt and a gold rod according to claim 1, characterized in that: The particle size of the graphyne GDY powder in S4 is ≤1 μm.

7. The method for preparing a two-dimensional graphene oxide modified by a quaternary ammonium salt and a gold rod according to claim 1, characterized in that: The molecular weight cut-off of the dialysis bag used for dialysis in S4 is ≤200 Da.

8. The method for preparing a two-dimensional graphene oxide modified by a quaternary ammonium salt and a gold rod according to claim 1, characterized in that: The mass ratio of the graphene oxide GDYO to the quaternary ammonium salt monomer VPIM-Br is 1:10, the solid-liquid ratio of the graphene oxide GDYO to the gold rod AuNR solution is 1:0.7-1, and the solid-liquid ratio of the graphene oxide GDYO to deionized water is 1:1000.

9. An application of a quaternary ammonium salt and a gold rod modified two-dimensional graphene oxide prepared by the method according to any one of claims 1 to 8, characterized in that: The quaternary ammonium salt and gold rod modified two-dimensional graphene oxide is used as a healing material for infectious wounds of diabetic patients.

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