Triple-synergistic 2D material-based dual-delivery antibiotic platform

Two-dimensional (2D) nanomaterials have raised significant interest in not only energy and environmental fields but also biomedical areas. Among these materials, one type that has many interesting properties and possesses numerous exciting applications is graphene oxide (GO)-based 2D materials. However, their poor stability in aqueous solutions and weak bioactivities limit their use in biomedical applications, especially antimicrobial fields. In this study, GO was functionalized with hydrophilic polymers and used as a vector for silver nanoparticles (Ag NPs) and sulfadiazine (SD). The stability of the material in aqueous solutions was greatly improved. The antibacterial activity of the novel hybrid antibacterial system (HAS) was enhanced by over 3 times compared to that of the system lacking SD. The antibacterial performance of the HAS was due to the triple synergy: bacterial capping, puncture, and inhibition. This study provides new insights into the design and fabrication of surface-modified GO and carbon materials and their 2D hybrid multifunctional materials for advanced applications including biomedical and especially antibacterial applications, broadening the design and application scope of carbon and 2D materials. An efficient method for harnessing the antibacterial properties of silver nanoparticles has been developed by researchers in China and the USA. Silver is frequently used in bandages because of its antibacterial effects. Recent studies have suggested that silver nanoparticles are even more effective; however, a way to prevent them clumping together is needed. Fengqi Han from the Henan Provincial People’s Hospital, Zhengzhou, and co-workers achieved this using two-dimensional graphene oxide combined with a hydrophilic polymer. While graphene oxide has already found numerous applications, its use in medicine has been limited because it is unstable in water and other aqueous solutions. The researchers increased this stability by adding the hydrophilic polymer. When loaded with silver sulfadiazine nanoparticles, the hybrid material exhibited antibacterial activity three times higher than silver sulfadiazine on its own. In this study, a novel hybrid antibacterial system (HAS) based on graphene oxide (GO) 2D materials for co-delivery of silver nanoparticles (Ag NPs) and sulfadiazine (SD) was fabricated. The antibacterial activity of HAS was enhanced over 3 times due to triple synergy: capping effect of GO, puncture effect of Ag NPs, and inhibitory effect of SD. This study provides new insights into the design and fabrication of antibiosis systems and multifunctional materials and broadens the biomedical applications of 2D materials.


Introduction
Graphene oxide (GO), an important two-dimensional (2D) nanomaterial, has attracted extensive attention due to its unique physical, chemical, and biological properties 1,2 . GO has a high specific surface area and has many carboxylic groups at its edges 3,4 . Since graphene was discovered in 2004 5 , its applications in various medical fields have been explored, including in photothermal therapy 6 , bioassays 7 , and drug delivery 8,9 , as well as in antibacterial agents 10 . Recent research has shown that graphene-based materials have low cytotoxicity and exhibit antibacterial activity [11][12][13] . However, the antibacterial properties and biomedical applications of these materials have been limited due to their low solubility in aqueous solution 14 .
Silver (Ag) has been an efficient antibacterial agent for centuries. With the development of nanotechnology, silver nanoparticles (Ag NPs) have shown better antibacterial performance than traditional bulk silver. Recent studies have shown that smaller Ag NPs have a better antimicrobial effect 15 . However, small Ag NPs coagulate easily, limiting their biomedical applications. GO was chosen for loading Ag NPs because it has a high specific surface area and unique physicochemical properties.
Silver sulfadiazine (SSD) and sulfadiazine (SD) are broad-spectrum antimicrobial agents with wide clinical applications 16 . They are widely used in the clinical treatment of burn diseases and show an excellent treatment effect. We introduced SD and Ag NPs onto the surface of GO to improve the antimicrobial activity of the hybrid materials, taking advantage of the high specific surface area of GO.
Recently, Ag/rGO was successfully synthesized. However, the material has poor aqueous stability, and its small number of functional groups available for conjugating efficient macromolecule agents has greatly limited its applications 17,18 .
In this study, hydrophilic macromolecules were introduced into carboxylated graphene oxide to improve the aqueous stability of the hybrid materials. To further enhance the antimicrobial effect, GO was surfacemodified as a vector to allow the loading of Ag NPs and SD. The facilely prepared 2D hybrid materials showed a good synergistic antibacterial effect.

Synthesis of 2D antibacterial nanomaterials
As shown in Scheme 1, polyethylene glycol-modified carboxylated graphene oxide was prepared as a carrier and release platform for Ag NPs and SD to build up the novel hybrid antibacterial system (HAS).

Synthesis of carboxylated graphene oxide (GO-COOH)
GO was prepared by the modified Hummers method with some modifications 19 . To synthesize carboxylated graphene oxide, GO suspension solution (1 mg/mL) was sonicated for 30 min, and then, ClCH 2 COONa (1.2 g, 40 mL) was mixed with GO solution (1 mg/mL, 20 mL) under ultrasonication for 30 min. Finally, sodium hydroxide solution (10 mol/mL, 40 mL) was added to the mixture under magnetic stirring for 3 h. The mixture solution was neutralized by a HCl solution (1 M), and the precipitate was washed with distilled, deionized water and alcohol by centrifugation to remove the unreacted ClCH 2 COONa 20,21 .

Synthesis of PEGylated graphene oxide (GO-PEG)
The GO-COOH solution (1 mg/mL, 20 mL, DMF) was mixed with 5 mg of hydrophilic PEG-OH under magnetic stirring for 10 min; then, DCC (10 mg) and DMAP (2 mg) were added to the mixture, which was stirred for 3 h at 60°C. Excess PEG in the as-synthesized GO-PEG sample and DMF were removed by dialysis against water using a dialysis bag (Mw cut-off: 3.5 kDa) for 3 days 22 .

Synthesis of Ag NPs decorated on PEGylated graphene oxide nanocomposites (GO-PEG/Ag)
The GO-PEG/Ag and HAS SD-nanocomposites were synthesized by a microwave irradiation route. In this synthesis process, GO solution (0.25 mg/mL, 28 mL) was mixed with AgNO 3 (5 mM, 5 mL) under ultrasonication for 30 min. Then, sodium hydroxide (1 M, 10 mL), as a deoxygenating and reducing agent, was added to the mixture under magnetic stirring. Finally, the mixture was transferred into a microwave oven (XO-SM 100, 2.45 GHz, Galanz, Shunde, Guangdong, China). The microwave frequency was 2.45 GHz, and the input power was 200 W at different reaction times during the microwave irradiation process. The solution was then cooled to room temperature. The products were collected by centrifugation and washed with distilled deionized water and alcohol until the pH value was 7.0.

Synthesis of GO-PEG/Ag-SD (HAS)
The SD (30 mg/mL, 100 μL, DMSO) solution was added dropwise into 10 mL of GO-PEG/Ag (0.25 mg/mL) under ultrasonication using a Type 60 Sonic Dismembrator (Thermo Fisher Scientific, Waltham, MA, USA). The DMSO was then completely removed by dialysis. Finally, the resulting dispersion was centrifuged for 10 min at 1000 rpm to remove free SD. Subsequently, the obtained sample was dried by a freeze-drying method.

Characterizations
Scanning electron microscopy (SEM) images were obtained using a JEOL field-emission scanning electron microscope (JSM-7001 F, Japan). A model JEM-2100 instrument (JEOL, Tokyo, Japan) provided highresolution transmission electron microscopy (HRTEM) images. X-ray diffraction was performed using an XD-3 system (Purkinje General Instrument Co., Ltd., Beijing) with Cu-Kα radiation (λ = 0.15406 nm). The UV-visible absorption spectra were recorded on the UV-1800 spectrophotometer in the range of 200-800 nm, and SD had an absorption peak at 310 nm. Fourier transform infrared (FT-IR) spectra were recorded on a TENSOR II FT-IR system (Germany) with a wavenumber range of 3800-600 cm −1 at a resolution of 4 cm −1 . The antibacterial property test was investigated by a 2104 Multilabel Microplate Reader (PerkinElmer, Kentucky, USA) using MTT to signal living bacteria. Fluorescence images of bacteria were obtained using a fluorescence microscope (Nikon 80i, Japan).

Antibacterial test
The antibacterial activity of HAS nanocomposites was investigated using a gram-negative bacterium, Escherichia coli (E. coli), and gram-positive bacterium, Staphylococcus aureus (S. aureus), as two model microorganisms 23 . The effects of different sizes of Ag NPs to HAS SD-, HAS, and SSD on the antibacterial activity of this hybrid material and the mechanism of antibacterial activity were systematically investigated.

Microdilution methods
The hybrid materials were accurately weighed, sterilized, and then diluted with LB broth. The LB broth was used for bacterial activation, which was placed in a shaker (170 rpm/min) and incubated at 37°C for 12 h. Then, 5 mL of the LB broth medium was mixed with 1% inoculum of activated bacteria, adding 1 mL of phosphate buffer, mixing evenly and diluting to a concentration of 10 5 CFU/mL to test the antibacterial properties of E. coli and S. aureus.
To examine the minimum inhibitory concentration (MIC) of HAS, 100 μL of 0.9% saline was first injected into the 96-well plate; then, 100 μg/mL of the materials solution was added by pipette into the first well, and a 50 μg/ mL sample was added to the second well with the double dilution method. Finally, 10 5 CFU/mL of bacteria was added by pipette. Seven wells had a certain amount of sample and bacteria, while 200 μL of LB broth and 0.9% saline with bacteria were injected into the eleventh and twelfth wells, respectively. Through mixing, the mixture was uniform and placed in the incubator with a plastic wrap at 37°C to measure the OD value. Then, 20 μL of MTT was added to every well. The OD value of different bacteria was determined after 4 h. In this process, to obtain a more accurate OD value, the absorbance of materials was subtracted, and samples were measured three times for each group. The averaged OD value was useful, and the control group only had 0.9% saline added.
The living microbial cells in the abovementioned 96well plate were analyzed using the colony-forming units (CFU) method. Each culture was diluted and plated on LB-agar plates and incubated at 37°C for 12 h.

Diffusion method
The diffusion method is another recognized technique to determine the in vitro susceptibility of bacteria to various drugs. The disk concentration (10 mg/disk) was based on preliminary experiments in which blank disks were impregnated with 10 mL of suspension containing 1 mg/mL drug. Specifically, MH agar plates were inoculated with a bacterial suspension of 10 5 CFU/mL by swabbing the agar surface. After the plates were dry, sterile paper disks containing the corresponding drug, HAS, HAS SD-and SSD were placed on the agar surface. The diameters (in mm) of the zone of complete inhibition were determined after 25 h of incubation at 37°C.
Fluorescence-based live/dead bacterial assay E. coli and S. aureus cells were inoculated in fresh LB broth at 10 5 CFU/mL with HAS (50 μg/mL). After growth for 1 h at 37°C in the shaking incubator, the bacteria were collected by centrifugation, stained with propidium iodide (PI, 1 μg/mL) for 15 min, counterstained with 4′-6-diamidino-2-phenylindole (DAPI, 5 μg/mL) for 5 min in the dark, and then imaged using a Nikon 80i fluorescence microscope.

Synthesis and characterization of HAS
The modified Hummers method was applied to prepare the graphite oxide 24 ; GO sheets with carboxylic groups, epoxide groups, and phenol hydroxyl at its edges and basal plane were produced by the oxidation process 25 . To improve the stability of the material in water, hydroxyl groups were converted to carboxylic groups via carboxylation, which could provide more reactive sites for covalent modifications such as the esterification of carboxyl groups 26 . The existence of oxygen-containing groups on the surface of GO was examined by FT-IR spectroscopy (Fig. 1). As shown in Fig. 1a, the presence of an absorption band at approximately 1625 cm −1 corresponds to the C=C bonds of the aromatic rings of the GO carbon skeleton structure. The absorption band at approximately 3454 cm −1 is from -OH groups, which may come from distilled water and hydroxyl groups. The vibrational peaks at approximately 1078, 1384, and 1720 cm −1 are assigned to the C=O, C-OH, and C=O stretching vibrations of COOH groups, respectively.
After the carboxylation reaction, the peak at 1720 cm −1 in the FT-IR spectrum was stronger than that in the spectrum of GO (Fig. 1a). This evidence distinctly demonstrated that the initial hydroxyl groups on the surface of the GO sheets were converted to carboxylic groups. After conjugation with PEG following the esterification reaction, a new signal attributed to ester bonds (Fig. 1b) appeared at 1735 cm −1 in the FT-IR spectrum. The result directly confirmed that PEG was successfully conjugated to GO.
The morphology of GO, GO-COOH, and GO-PEG was visualized by SEM, as shown in Fig. 2. The GO sheets were loosely stacked with some wrinkles, and the surface was smooth (Fig. 2a). The surface appearances of carboxyl-functionalized GO sheets (Fig. 2b) and PEGylated GO sheets (Fig. 2c) were similar to those of GO sheets but with more wrinkles.  The stability of GO, GO-COOH, and GO-PEG was tested in aqueous media for 96 days (Fig. 3). GO-PEG was found to be stable in water without precipitation; PEGylated graphene oxide exhibited significantly improved water stability. The unfunctionalized GO, which was suspended in water via a small number of hydrophilic groups, such as carboxylic groups, could not provide sufficient buoyancy force. When hydrophilic PEG-OH was introduced into the carboxylated graphene oxide (GO-COOH) as GO-PEG, the number of hydrophilic groups was significantly increased, which could provide enough buoyancy force to keep the GO sheets stable in aqueous solution.
The preparation of GO-PEG/Ag by a facile synthesis method via microwave irradiation is a rapid and green route. The size of Ag NPs can be easily controlled by adjusting the microwave time.
SEM was employed to characterize the morphology and microstructure of the graphene-based hybrid materials to verify the effective anchoring of Ag NPs onto large GO-PEG sheets. As shown in Fig. 4a, GO-PEG/Ag was synthesized at 200 W for 1 min, and Ag NPs with an average diameter of 8 nm were embedded on the GO-PEG sheets. When the time was changed to 2 min, the size of the Ag NPs was~50 nm (Fig. 4b). The Ag NPs did not agglomerate. TEM, HRTEM, and XRD images of the GO-PEG/Ag composite (200 W, 1 min) are shown in Fig. 5. Ag NPs with a size of 8 nm were highly monodispersed on the GO-PEG sheet (Fig. 5a). The TEM image shows that Ag NPs were embedded on the surface of the GO-PEG sheets. The HRTEM (Fig. 5a) lattice distance was 0.236 nm, indicating a lattice plane of Ag (111) 27 , which is in accordance with the XRD (Fig. 5b) profiles of the GO and GO-PEG/Ag samples (200 W, 1 min). The (001) diffraction peak of graphene oxide at 10.8°was retained after microwave irradiation, indicating that the hybrid materials still have a large number of functional groups on the GO surface. The face-centered cubic Ag crystal planes of (111), (200), (220), and (311) correspond to the peaks at 2θ = 38.2°, 44.2°, 64.5°, and 77.3°, respectively, proving that the metallic Ag NPs were formed under the microwave irradiation power applied for 1 min at 200 W.
As shown in Fig. 6a, the FT-IR spectrum of GO-PEG/ Ag-SD was different from that of GO-PEG. New signals appeared at 3424 cm −1 , 3356 cm −1 , 2874 cm −1 , 2136 cm −1 , 1654 cm −1 , 1156 cm −1 and 1097 cm −1 in the FT-IR spectrum of GO-PEG/Ag-SD, which were attributed to SD and indicated the successful loading of SD onto GO-PEG/Ag.
The drug loading ability of GO-PEG/Ag was verified by UV-vis spectroscopy. Figure S1 shows the standard  UV-vis curve of the SD solution, with an absorption wavelength at 310 nm. With an increase in the SD: GO-PEG ratio (0.5:1, 1:1, 2:1, and 4:1, by weight), the drug loading content increased (59.24%, 78.83%, 135.26%, and 138.24%, respectively) and the entrapment rate decreased (93.00%, 44.08%, 21.56%, and 10.88%), as shown in Fig. 6b; the drug loading rate decreased and then gradually leveled off, but the entrapment rate was opposite to the drug loading rate. The 2:1 SD:GO-PEG ratio shows the maximal drug loading and best entrapment rate, and this ratio was chosen to synthesize the GO-PEG/Ag-SD composite.

In vitro evaluation of the antibacterial activity
The effect of Ag NP size on the antibacterial activity Ag NPs have been extensively explored for their excellent antibacterial activity 28 In this study, Ag NPs of different sizes were synthesized by changing the microwave irradiation time. The smaller Ag NPs GO-Ag1 (8 nm) showed better antibacterial activity than the larger Ag NPs GO-Ag2 (50 nm) (Fig. 7a), with 6.25 μg/mL and 12.5 μg/mL MIC values, respectively (Fig. 7b). This trend is consistent with previous research 15,29 . Based on this result, GO-Ag1 was chosen as the precursor to load SD.

Antibacterial activity of HAS
The antibacterial activity of HAS against E. coli was determined by MTT assay using HAS SD-and SSD as control groups. HAS showed better antibacterial activity (Fig. 8a), with almost complete inhibition of the growth of E. coli. As shown in Fig. 8b, c, the HAS MIC was~0.78 μg/ mL for E. coli, showing over 3 times improved antibacterial activity compared to that of HAS SD-(3.12 μg/ mL). The results showed that HAS presents the best synergistic antibacterial activity towards E. coli.
The paper disk method was also studied to further compare the antibacterial activity of HAS with that of HAS SD-and SSD (Fig. 9). Compared to that of HAS SDand SSD, a larger inhibition zone diameter against E. coli was observed for the HAS, indicating that it has the best antimicrobial activity.
The antibacterial activity of HAS, HAS SD-and SSD towards the gram-positive bacterium S. aureus was also  explored. As shown in Figs. S2a and S3a, the results of the bacterial colony counts showed that there was no significant difference in antibacterial activity between HAS and HAS SD-. Figs. S2b and S3b show the same antibacterial activity results for HAS and HAS SD-, and the MICs were both 6.25 μg/mL. The paper disk method results presented the same trend (Fig. S4).

Antimicrobial mechanism of HAS
To study the antibacterial mechanism of HAS, two fluorescent nucleic acid dyes, DAPI and PI, were employed to stain the DNA of bacteria. DAPI labels both live and dead cells, while PI can only penetrate cells with compromised or damaged membranes 30 , which can be used to test whether the bacteria are live (blue) or dead  (red). As shown in Fig. 10, the E. coli and S. aureus cells were almost completely stained by PI, which shows damaged cell walls and cell membranes and/or mass cell death after treatment with HAS (50 μg/mL) for 1 h, and the efficiency of sterilization was above 98%, showing efficient and fast antibacterial activity.
Scanning electron microscopywas used to study the interactions between HAS and bacteria. As shown in Fig. 11, HAS served as a net to wrap E. coli and S. aureus in the treated samples, which limited bacteria movement. The large size of HAS ensured that it could trap or wrap many bacteria and kill or inhibit bacterial growth. The surfaces of E. coli and S. aureus were obviously wrinkled and damaged. Cells were observed to be leaking cytoplasm, and bacteria were in contact with the Ag NPs.
The above results indicated that the HAS presented excellent antibacterial activity against E. coli. The hybrid system, which consisted of GO-PEG, Ag NPs, and SD, exhibited superior synergistic antimicrobial activity via three effects (Fig. 12b): (1) The capping effect of graphene oxide. Adsorption of the antibacterial system to the bacteria is the most important step in killing or inhibiting bacterial growth. As shown in Fig. 12a, bacteria were wrapped or trapped by the thin sheets of HAS via intermolecular forces 31 . The outer membrane of bacteria contains sugars, phosphates, and lipids, which can form hydrogen bonds with HAS and subsequently form agglomerates. HAS adsorption on the bacteria served two antibacterial functions: (i) it significantly increased the concentration of Ag NPs and SD around the bacteria and (ii) blocked nutrient transport. The entry of nutrients into the cell and the delivery of excreta out of the cell are critical for cell and bacterial growth 32 . It has been demonstrated that it is hard for nutrients to pass across GO-based hybrid materials, which indicates that bacterial nutritional supplementation and metabolism will be inhibited once bacteria are trapped by GO-based hybrid materials 33,34 . (2) The puncture effect of Ag NPs. Small Ag NPs (8 nm) with positive surface potential can easily be adsorbed onto the bacterial surface, which can affect the permeability of the bacterial cell membranes and rupture the membranes 35,36 . (3) The inhibitory effect of SD. SD could directly inhibit nucleic acid synthesis in bacterial cells. SD has a similar chemical structure to that of para-aminobenzoic acid, which is a necessary substance for adsorbing folic acid and can inhibit bacterial adsorption of folic acid, thus further inhibiting the growth of bacteria. Additionally, SD can weaken the E. coli cell wall to some extent 37,38 and further enhance the cell membrane rupture ability of Ag NPs, highlighting the significant synergistic antibacterial effect of Ag NPs and SD.

Conclusion
A novel 2D hybrid-material-based antibacterial system was developed via a facile, rapid, microwave-assisted green route and efficient antibacterial drug loading. The HAS possessed superior water phase stability through surface hydrophilic modification. The high specific     12 Schematic illustration of the interaction between HAS and microorganisms a and the synergistic antibacterial mechanism of HAS b. The antibacterial mechanism could be divided into three parts (Fig. 12B): (1) The capping effect of graphene oxide via intermolecular forces has two antibacterial functions: (i) significantly enhancing the concentration of Ag NPs and SD around bacteria and (ii) blocking nutrient transport; (2) The puncture effect of Ag NPs; and (3) The inhibitory effect of SD, which could directly inhibit nucleic acid synthesis in bacterial cells and could weaken the E. coli cell wall. In this study, a novel hybrid antibacterial system (HAS) based on graphene oxide (GO) 2D materials for co-delivery of silver nanoparticles (Ag NPs) and sulfadiazine (SD) was fabricated. The antibacterial activity of HAS was enhanced over 3 times due to triple synergy: capping effect of GO, puncture effect of Ag NPs, and inhibitory effect of SD. This study provides new insights into the design and fabrication of antibiosis systems and multifunctional materials and broadens the biomedical applications of 2D materials. effect of GO, puncture effect of Ag NPs, and inhibitory effect of SD. This study provides new insights into the design and fabrication of surface-modified GO and carbon materials [39][40][41][42][43] and their 2D hybrid multifunctional materials for advanced applications including biomedical 44 especially antibacterial applications, broadening the design and application scope of carbon and 2D materials.