Preparation of GO dispersion
The GO dispersion was synthesized as follows. Graphite (15 g, FUJIFILM Wako Pure Chemicals Co., Ltd., Osaka, Japan) and 640 g of sulfuric acid (FUJIFILM Wako Pure Chemicals Co., Ltd.) were mixed, and then 45 g of potassium permanganate (FUJIFILM Wako Pure Chemicals Co., Ltd.) was added at room temperature. The mixture was allowed to react for 2 h, and then 1070 mL of water and 42 mL of 30% aqueous hydrogen peroxide (FUJIFILM Wako Pure Chemicals Co., Ltd.) were added to stop the reaction. The product was purified by static sedimentation to remove the supernatant and repeated redispersion in ion-exchanged water. After purification, the reaction solution was homogenized using a homogenizer (10,000 rpm, HG-200, AS ONE Corporation, Osaka, Japan) to obtain the GO dispersion.
Preparation of GO ultrathin film coating combined with CSAA
A fused quartz polishing plate (20 mm ´ 20 mm ´ 1 mm, AS ONE Corporation) was heated at 700°C for 2 h under an air atmosphere to burn off surface organic matter. The concentration of the GO dispersion was adjusted to 0.1%, and 0.4 mL of the GO dispersion was cast on the heat-treated quartz substrate to completely cover the quartz substrate. After drying under vacuum at room temperature, the substrate was vigorously sprayed with water to remove excess GO, and then vacuum dried again.
Benzalkonium chloride (BAC, FUJIFILM Wako Pure Chemicals Co., Ltd.), cetylpyridinium chloride (CPC, Tokyo Chemical Industry Co., Ltd., Tokyo, Japan), and benzethonium chloride (BZC, Tokyo Chemical Industry Co., Ltd.) were used as CSAAs. Each CSAA was prepared as a 0.1% aqueous solution, and the GO-coated quartz substrate was immersed in each solution for 10 s. The substrate was then immediately rinsed thoroughly with water and vacuum dried to obtain a GO ultrathin film coating combined with CSAA (denoted as GO/CSAA, e.g., GO/BAC, GO/CPC and GO/BZC). For comparison purposes, we prepared an untreated quartz substrate, a substrate with a GO coating only, and substrates immersed only in each CSAA solution.
Characterization of GO/CSAA
Each quartz substrate was investigated by Raman spectroscopy (excitation wavelength 532 nm, NSR-3100, JASCO Corporation, Hachioji, Japan) to detect the GO film. In addition, the morphology of the GO film with/without CSAA, coated on quartz substrates, was observed using field emission scanning electron microscopy (SEM, JSM-7600F, JEOL Ltd., Akishima, Japan). The ratio of the GO-coated area to the total substrate area was calculated using image analysis software (ImageJ, ver. 1.41, National Institutes of Health, Bethesda, MD, USA).
To measure the transmittance of the GO film, the absorbance at 660 nm for untreated, GO alone, BAC alone, and GO/BAC treated substrates was measured using a UV-visible spectrophotometer (UV-3100, Shimadzu Corporation, Kyoto, Japan). The transmittance for each substrate was calculated by setting the transmittance of the untreated quartz substrate as 100%.
The elemental composition and chemical states of the materials on each quartz substrate were investigated by X-ray photoelectron spectroscopy (XPS, Al-Kα, AXIS-NOVA, Shimadzu Corporation). The C1s, N1s, O1s, and Cl2p XPS spectra were analyzed to detect the presence of GO and CSAA. C1s spectra show the bonding states of GO, such as C-C, C-O, and CO-O- bonds, and N1s spectra show the quaternary amine structure of the CSAA.
The hydrophilicity of each quartz substrate was investigated by the sessile drop method using a contact angle meter (DMs-200; Kyowa Electronic Instruments Co., Ltd., Tokyo, Japan). To confirm the formation of ultrathin films on different substrate surfaces, we used a polyethylene terephthalate (PET) film (T-60, 50 μm, Toray Industries, Inc., Tokyo, Japan) as an experimental model and measured the contact angle in the same way.
ζ-Potential measurements of GO solutions with different benzyldodecyldimethylammonium chloride (BAC12) concentrations were performed at 25°C using a Zetasizer Nano ZSP (Malvern Panalytical, Ltd., Malvern, UK). The average values of six measurements were estimated in the ζ-potential measurements.
Antibacterial effect of GO/CSAA
The same procedure as used for the quartz substrate was employed to coat the bottom of a 48-well culture plate with GO/CSAA. A GO dispersion (100 µL, 0.1%) was added to each of the 48 wells to completely cover the bottom of the well. After drying at room temperature, the bottom of each well was vigorously sprayed with water to remove excess GO, and then dried. Next, 100 µL of 0.1% CSAA (BAC, CPC or BZC) was dropped into the GO-coated wells. After rinsing with water three times and drying, we obtained GO/CSAA on the bottoms of the 48 wells and examined their antibacterial properties in comparison to untreated, only GO-coated, and only CSAA-treated wells.
We prepared bacterial suspensions of two oral pathogens. Suspension (1) comprised Streptococcus mutans (ATCC 35668) (1.0×107 CFU/mL) in culture medium (brain heart infusion medium, Eiken Chemical Co., Ltd., Tokyo, Japan) with antibiotics (0.1% gramicidin (FUJIFILM Wako Pure Chemicals Co., Ltd.), 0.1% bacitracin (FUJIFILM Wako Pure Chemicals Co., Ltd.), and 1% sucrose (FUJIFILM Wako Pure Chemicals Co., Ltd.). Suspension (2) comprised Actinomyces naeslundii (ATCC 27039) (1.0×107 CFU/mL) in culture medium (Actinomyces broth, Becton Dickinson and Company, Franklin Lakes, NJ, USA). Each suspension was added to individual wells. After anaerobic incubation at 37°C for 24 h, the turbidity of each culture medium was measured at 590 nm using a colorimeter (CO7500 Colourwave, Funakoshi Co., Ltd., Tokyo, Japan). Relative turbidity was calculated based on the turbidity of an untreated well to detect bacterial growth.
We also conducted comparable testing of GO ultrathin and thick films. A GO dispersion (100 µL of 0.1%) was dried in a well and not treated with water spray to prepare a thick GO film. Turbidity tests were conducted for untreated, GO ultrathin film-treated and GO thick film-treated wells to compare their antibacterial activities in the same manner as described above.
Durability of GO/BAC during water storage
To evaluate the sustained antibacterial effect of each preparation in water, GO/BAC-treated 48-well plates were stored in 5 L of water for 1, 3 and 7 days before antibacterial evaluation. The plates were removed from the water and dried, then a bacterial suspension (as described above) was added to each well. After 24 h of anaerobic incubation at 37°C, the relative turbidity of each culture medium was determined based on the turbidity of an untreated well (not stored in water).
GO/BAC-treated 48-well plates were also kept in 5 L of water for 28 days, then incubated with the bacteria as described above, and the turbidity was then measured after 24 h of incubation and compared with untreated and GO/BAC-treated wells (both not stored in water). In addition, to investigate the CSAA re-binding activity of GO ultrathin films after long-term storage in water, 0.1% BAC was dropped again (re-processing) on the GO/BAC-treated wells already stored in water for 28 days. After rinsing with water three times and drying, the wells were inoculated with S. mutans or A. naeslundii in culture medium and the relative turbidity after 24 h of incubation was calculated.
Antibacterial effect of GO/CSAA coating on human teeth
Human tooth substrates were prepared from teeth provided by patients attending Hokkaido University Hospital who had provided informed consent. The teeth were extracted as part of routine treatment. The protocol for the clinical study was reviewed and approved by the Hokkaido University Hospital Institutional Review Board for Clinical Research (Approval No. 17-222). Experiments on human teeth were carried out in accordance with relevant guidelines and regulations.
To investigate the clinical effectiveness of GO/CSAA, the GO ultrathin film coating of tooth surfaces (by modifying the method for quartz substrate) was combined with CPC, which is clinically used as a mouthrinse agent . This allowed CPC to be retained on the tooth surfaces and act as an antibacterial agent. After trimming and ultrasonic cleaning, a human tooth (dentin) block substrate was immersed in 0.01% GO dispersion for 10 s for surface coating, and immediately dried with high-pressure air. The tooth substrate was then immersed in 0.1% CPC solution and immediately rinsed thoroughly with water. Untreated, GO-treated only, and CPC-treated only tooth substrates were prepared for comparison.
The surface of each tooth substrate was evaluated by XPS elemental analysis (for C1s, Ca2p, N1s, Na1s, O1s, and P2p) and Raman spectroscopy, and then the antibacterial activity of each tooth substrate was evaluated by seeding S. mutans (using the same culture conditions as described above). Viable bacteria adhering and proliferating on each dentin substrate were fluorescently stained using a LIVE/DEAD BacLight Bacterial Viability kit (Thermo Fisher Scientific, Waltham, MA, USA), and the region stained for live cells was quantified using a fluorescence microscope (BZ-9000 BioRevo, Keyence Corporation, Osaka, Japan) and ImageJ analysis software.
Cytocompatibility of GO/BAC
GO/BAC was prepared on glass-based dishes (AGC Techno Glass Co. Ltd., Haibara, Japan) for fluorescence staining, and the bottom of a 96-well culture plate (for cell viability and cytotoxicity assays) by the same method as for turbidity assessments (with slight modification of the GO and BAC liquid volume). Fibroblastic NIH3T3 cells (1×104, RIKEN BioResource Center, Tsukuba, Japan) and culture medium (MEM alpha, GlutaMAX-I; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (Qualified FBS; Thermo Fisher Scientific) and 1% antibiotics (penicillin-streptomycin; Thermo Fisher Scientific) were inoculated on each substrate to examine its biocompatibility properties. After 24 h of incubation at 37°C in a 5% CO2 environment, the cultured cells were fluorescently stained using a LIVE/DEAD Viability/Cytotoxicity kit (for mammalian cells, Thermo Fisher Scientific) and observed using fluorescence microscopy. Furthermore, cell viability and cytotoxicity were examined using a water-soluble tetrazolium salt (WST)-8 assay kit (Cell Counting Kit-8, Dojindo Laboratories, Mashiki, Japan) and a lactate dehydrogenase (LDH) assay kit (Cytotoxicity LDH Assay Kit-WST, Dojindo Laboratories), respectively. The absorbance at 450 nm (WST-8 activity) or 490 nm (LDH activity) was measured using a microplate reader (Multiskan FC, Thermo Fisher Scientific).
Quantitative parameters were calculated as mean + standard deviation. A two-tailed one-way analysis of variance with Tukey’s HSD post hoc test was used to determine statistical significance, with P<0.05 being considered significantly different. The analysis was performed using the SPSS software package (version 11.0; IBM Corporation, Armonk, NY, USA).