Effect of Graphene Oxide on Bacteria and Peripheral Blood Mononuclear Cells

GO preparation

We obtained graphite oxide from expanded graphite (from Nacional de Grafite, Brasil) using the modified Hummers’ method, followed by a purification process. We dispersed graphite oxide in H₂O or PBS by sonication (ultrasonic bath) for 6 hours, thus obtaining GO-H₂O and GO-PBS, respectively. The sample GO-MG was obtained by manually grinding graphite oxide using a mortar (for further details, see “Supplementary material”).

Raman spectroscopy

Raman spectroscopy analyses were carried out on a Witec Alfa 300 spectrometer, with a 532 - nm laser line in backscattering configuration using a microscope with a ×50 objective. The laser power was kept under 0.1 mW to avoid local heat damage to samples. All spectra were acquired with 10 accumulations of 10 seconds of integration time in the region between 100 cm⁻¹ and 3,600 cm⁻¹.

Atomic force microscopy

To carry out AFM imaging of the different GO samples, we deposited a diluted solution of each GO on a Si/SiO₂ wafer by drop casting. The drop was carefully rolled over the wafer to uniformly disperse single layers and a few layers of GO. Finally, the excess of solution was blown off with nitrogen (N₂). The samples were dried in ambient conditions for 24 hours. The AFM images were obtained in intermittent contact mode with a JPK Nanowizard 3 using a silicon nitride tip (spring constant of 40 N/m).

Transmission electron microscopy

Dispersed solutions of the different GO materials were used to deposit them on Cu⁻ holey carbon grids (300 mesh). A probe-corrected FEI Titan 80-300 microscope was used at 80 kV to minimize beam damage effects. Conventional bright field images and high resolution transmission electron microscopy (HRTEM) images were taken. Electron energy loss spectroscopy (EELS) measurements were done in spectroscopy mode (STEM-EELS) using a GIF 866 camera under the following experimental conditions: α = 58.5 mrad, GIF aperture = 2.5 mrad, dispersion = 0.02 eV/ch with ZLP_FWHM resolution of 0.8 eV.

Scanning electron microscopy

The GO solutions were diluted to a concentration of 5 µg/mL using deionized water. One drop of each solution was deposited on a 300-nm SiO₂/Si substrate kept at 50°C on a hot plate and allowed to dry. SEM images were obtained at 15 kV and 50 pA, with secondary electron detection using the Through Lens Detector (TLD-detector) in FEI Helios Nanolab 650.

X-ray photoelectron spectroscopy

XPS analyses were performed in an ultrahigh vacuum medium (pressure of 10⁻⁹ mbar) using an Mg, Kα (hν = 1,253.6 eV) X-ray source, with power given by emission of 20 mA, at a voltage of 15 kV. For the carbon element, the high-resolution spectra were obtained with analyzer pass energy of 20 eV in steps of 0.05 eV. The binding energies were referred to the carbon 1s level of a neat graphite sample, set as 284.8 eV.

Cell contact to GO

In the literature, many groups have tested the antibacterial activity of GO. Although the early reports were contradictory, a recent study attempted to elucidate the nature of these controversial effects (17). The authors state that the observed antibacterial effect is directly related to the availability of graphene's basal planes, and they further affirm that when GO is exposed to bacteria, its effect is determined by the media in which the 3-hour exposure takes place. If the exposure is carried out in a saline solution, then GO will have an antibacterial effect; when this exposure is carried out in LB broth growth medium, no antibacterial effect was evidenced.

Having these results in mind and to avoid masking antibacterial effects by the medium components, we decided to test the effect our GO-H₂O material for the growth of the gram-negative bacteria E. coli (DH5 α), probing 3 different exposure times: 0, 1 and 3 hours in saline solution. Briefly, the experiment consisted of growing E. coli at 37°C overnight. Then the culture was changed with fresh medium and allowed to grow again to the exponential phase. E. coli at 10⁷ colony forming units/mL was resuspended in saline solution (0.9% NaCl) and incubated to a final concentration of 200 µg/mL of GO-H₂O for 1 and 3 hours at 37°C and under agitation. As control, we used samples exposed to Milli-Q grade water. After this step (referred to as exposure), cells along with the materials were harvested by centrifugation, resuspended in 60 mL of fresh medium and incubated at 37°C under agitation (the recovery step) for 500 minutes. For the 0-hour-exposure experiments, the cells were put in contact with the material and immediately allowed to undergo the recovery step (for more details, see “Supplementary material”). After 500 minutes, the optical density at 600 nm (OD₆₀₀) of the cell cultures was measured, readings were recorded 3 times and all experiments were performed in triplicate.

We tested our materials on mammalian cells by exposing PBMCs to different concentrations of GO-H₂O, GO-PBS and GO-MG, and performed cell viability and apoptosis tests after 24 hours. PBMCs were isolated from the blood of healthy individuals, and a protocol was followed for the separation of PBMCs based on density gradient centrifugation (for further details, see “Supplementary material”). Then the cells were exposed to our materials at concentrations of 2, 20 and 200 µg/mL on multiwell culture plates for 24 hours, including a positive and a negative control. The negative control consisted of only medium and cells, and positive control was 0.3% H₂O₂. We performed trypan blue cell viability tests counting live/dead cells using a hemocytometer.

Sample characterization

We thoroughly studied the physical and chemical properties of our GO materials. AFM, SEM and TEM were used to figure out the morphological characteristics of the different samples, and EELS and XPS to understand their chemistry. Our studies revealed that the GO-H₂O sample contained small fragments of GO with average 2D sizes of 100 nm and thicknesses of 1 or 2 layers (Fig. 1A, D and G; see also “Supplementary material” for more details). The sample GO-PBS was very similar in morphology to GO-H₂O, as can be confirmed in Figure 1B and E. This was not a surprise since both samples were subjected to the same preparation treatment (6 hours in ultrasonic bath); however, the difference between the samples lay in the medium of dispersion (i.e., for GO-PBS, PBS was used). This difference resulted in many PBS residues that accompanied the GO sheets. Figure 1H shows a TEM image of GO-PBS where an impurity can be seen. Furthermore, Figure S2 (available online as supplementary material at www.jab-fm.com) shows an SEM image where the impurities are clearly localized on top of the GO, and our XPS analysis revealed signals from Na, Cl, K and P that evidently came from PBS residues.

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Fig. 1

Representative atomic force microscopy (AFM) (A-C), scanning electron microscopy (SEM) (D-F) and transmission electron microscopy (TEM) (G-I) micrographs of graphene oxide (GO) samples: (A, D, G) GO-H₂O; (B, E, H) GO–phosphate-buffered saline (GO-PBS); (C, F, I) GO manually ground and dispersed in water (GO-MG). Scale bars represent 500 nm in (A-C); 1 µm in (D-F); and 2 nm, 5 nm and 200 nm in (G), (H) and (I), respectively.

Sample GO-MG showed very different characteristics. Figure 1C, f and i reveal that GO-MG contained larger-area GO sheets, averaging several microns, showing similar morphological characteristics to those of exfoliated graphene. Nevertheless, structural damage in the honeycomb structure was detected.

Our XPS analysis of the C binding energies confirmed the presence of sp² (C = C) at 283.9 eV and carbon atoms out of regular sp² configurations (C-C/C-H) at 284.8 eV as expected for GO (31). Beyond that, other oxygenated carbon functional groups were observed, such as phenol or epoxide (C-OH/C-O-C) between 285.8-286.3 eV, carbonyl groups (C = O) at 287.1 eV, carboxyl groups (COOH) at 288.7 eV and the π-π* shake-up satellite at approximately 291.0 eV typical of aromatic delocalized electrons (32). Figure 2A, B shows XPS spectra of GO-H₂O and GO-MG, respectively. It is clear that although the nature of the bonds present in both samples is the same, their distribution is different. This was expected, as both samples were derived from GtO and dispersed in water; however, the preparation method led to more sp² hybridization in the GO-H₂O sample (18% against 9% in GO-MG; as confirmed by EELS results in Fig. S3, available online as supplementary material at www.jab-fm.com). On the other hand, the oxidized functional groups also showed differences in composition: the most intense component for GO-H₂O were the carbonyl groups (C = O) (25%) (see Suppl Tab. I for information on the EELS analysis, available online as supplementary material at www.jab-fm.com), which were preferentially located on the edges of graphene sheets, while for GO-MG, the most intense component was the third component, C-OH/C-O-C (25%), as shown in Supplemental Table I, typically located on the basal plane of graphene (33).

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Fig. 2

(A, B) X-ray photoelectron spectroscopy (XPS) spectra of graphene oxide (GO)-H₂O and GO manually ground and dispersed in water (GO-MG), respectively; (C) Raman spectra of the GO materials.

As mentioned above, the XPS analysis of the GO-PBS sample revealed the presence of elements other than C and O, which were due to the diluent.

Representative Raman spectroscopy results are included in Figure 2C. Bulk measurements of the GO materials revealed the strong presence of the D and G bands, located at ∼1,350 cm⁻¹ and ∼1,590 cm⁻¹, respectively. All spectra shared intense D bands and sharp G bands. We attribute such an intense defect-related band (D band) to the presence of sp³ hybridization and to the high amount of oxygen bonds which were perforce present on GO. However, the I_D/I_G ratio of the GO-PBS sample was the highest (1.1 compared with 0.97 for both GO-H₂O and GO-MG) probably due to the presence PBS residues, as confirmed by TEM and SEM. From a simple inspection, it is quite obvious that the spectra of the 3 different GO samples are rather similar. For samples GO-H₂O and GO-PBS, this is expected, since the morphology of the samples is comparable (see Fig. 1); however, although sample GO-MG possesses very different characteristics, its Raman spectrum also looks similar. This suggests a high level of defects due to the less regular sp² carbon structure, as detected by XPS and EELS. It is possible that the high D band is due to the symmetry breakpoints that arise from in-plane defects (see Fig. 1C), high proportion of sp³ hybridization, OH-terminated edges and C-O bonds present in the sample.

From our characterization, we can conclude that GO-H₂O and GO-PBS share morphology features (∼100 nm, 1-2 layers, amount of defects), while GO-MG presents larger areas but the same average amount of defects. Regarding surface chemistry, it can be concluded that all samples present a significant amount of sp³ hybridization and oxygen species.

Biocompatibility tests

Figure 3 shows the growth of E. coli in the presence of GO. Our results showed that the presence of GO-H₂O at such a high concentration did not have a negative impact on bacterial cell growth (Fig. 3A). For the 3 exposure times, the OD values of the cultures with GO-H₂O were quite similar to those of controls, suggesting that the amount of living cells did not decrease in contact with GO-H₂O. It is worth mentioning the slight decrease in OD₆₀₀ values for the 3-hour exposure set; both samples (control and GO-H₂O) showed lower values compared with those of the 0-hour and 1-hour exposure sets. We attribute this to a uniform decrease in the amount of living cells after 3 hours of agitation and the decrease of specific growth rate (µ) due to the lack of nutrients in the saline media. To avoid this effect, we chose to perform exposures of 1 hour for our growth kinetic experiments.

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Fig. 3

(A) E. coli exposed to graphene oxide (GO)-H₂O at different times (0, 1 and 3 hours) and incubated for 500 minutes in fresh Luria-Bertani medium for a recovery process; (B-D) growth kinetics of E. coli exposed for 1 hour to different concentrations of GO-H₂O, GO–phosphate-buffered saline (GO-PBS), GO manually ground and dispersed in water (GO-MG), respectively. Inset in (D) contains the color codes assigned to the different concentrations used in the study, which apply to graphs (B-D). Experiments were performed in triplicate, error bars stand for the standard deviation of the recorded values. OD = optical density; *p<0.05, n = 3.

To assess the effect of our different GO materials (GO-H₂O, GO-PBS and GO-MG), we performed a 7-hour kinetic study of the growth of E. coli, with an exposure to the materials of 1 hour. We tested 3 different concentrations: 2, 20 and 200 µg/mL for each material. Conditions were similar to those in the above-described experiment (see the section “Cell contact to GO”) except that during the recovery step, systematic OD₆₀₀ readings were recorded from the cultures (see more details in “Supplementary material”).

Figure 3B, C and D shows the results of our kinetic study of E. coli exposed to GO-H₂O, GO-PBS and GO-MG, respectively. From our above-described results. We did not expect to find an antibacterial effect of the GO-H₂O material, and such a conclusion was confirmed by Figure 3B which shows that the 3 concentrations of GO-H₂O tested had no adverse effect on the growth of E. coli. Our characterization showed that the morphology of the GO-PBS sample was quite similar to that of GO-H₂O. Thus no antibacterial effect was expected from this sample either. Figure 3C confirms that the presence of GO-PBS at concentrations of 2, 20 and 200 µg/mL did not interfere with the growth of E. coli.

Our characterization evidenced the marked differences in morphology that the GO-MG sample presented when compared with GO-H₂O (see Fig. 1). Such differences proved to have a strong impact on the growth of E. coli at the highest tested concentration (200 µg/mL). The results of our kinetic study (Fig. 3D) revealed that low doses of GO-MG (2 and 20 µg/mL) do not show any effect on the growth of E. coli; however, a higher concentration (200 µg/mL) proved to have an adverse effect on the growth, where µ decreased to a value of 0.021 h⁻¹ (±0.002) compared with 0.0265 h⁻¹ (±0.0009) for the control (Fig. 4A).

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Fig. 4

(A) Specific growth rate of E. coli in contact with graphene oxide (GO) materials, calculated from the 7-hour growth kinetic study. *p<0.05, n = 3; (B) viability of peripheral blood mononuclear cells exposed to GO materials after 24 hours.

We performed 1- and 2-way ANOVA with Dunnett's post test to compare the different concentrations used against the control. Values of p <0.05 were considered as significant.

We conclude that the sample GO-MG at high concentration shows an antibacterial effect which is the product of the high amount of large-area sheets that wrap around the bacteria, isolating them and disabling their proliferation, in good agreement with the reports of Liu et al (14) and Hui et al (17).

Our study revealed as well that the GO-MG antibacterial effect evidenced here was the product of the interactions with GO during the exposure step, and such an effect was not masked by the presence of LB growth medium during the recovery phase. Furthermore, the absence of this exposure step in saline solution (0-hour exposure) led to a complete suppression of the antibacterial effect of GO-MG (see Suppl Fig. 5, available online as supplementary material at www.jab-fm.com, for results for GO-MG exposed to bacteria), suggesting an adsorption of LB on the surface of the graphene and an inability of the sheets to wrap and isolate E. coli – in good agreement with the conclusions drawn by the group of Hui et al (17).

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Fig. 5

Results of the annexin V-FITC and propidium iodide (PI) assay. Representative scatter diagrams of peripheral blood mononuclear cells (PBMCs): (A) negative control, (B) positive control, (C-E) exposed to 20 µg/mL of graphene oxide (GO)-H₂O, GO–phosphate-buffered saline (GO-PBS) and GO manually ground and dispersed in water (GO-MG), respectively; (F) summary of the percentage of positive cells for annexin, PI and annexin/PI.

Cytotoxicity to PBMCs

Results for the test of cell viability are shown on Figure 4B, indicating that the presence of GO materials did not have a negative effect on the viability of cells at 24 hours of exposure. The lowest viability identified was for the GO-H₂O sample at the highest concentration tested (200 µg/mL) with a value of 89.95% ± 10.55%. For the rest of the samples, viability was above 90%, proving good hemocompatibility, which is in agreement with previous reports on the subject (22, 27).

Apoptosis tests were conducted on PBMCs exposed to our materials for 24 hours at concentrations of 2 and 20 µg/mL (see details on “Supplementary material”).

We evaluated whether our materials induced apoptosis of PBMCs, by flow cytometry using annexin V/propidium iodide markers. Representative dot plots of the apoptosis assay are shown in Figure 5A–E. Interestingly, for all of the materials for both concentrations tested, the percentage of apoptotic cells was very close to that of the negative control (Fig. 5F). In addition, we did not find any increase in necrotic cells when our materials were present, as revealed by the propidium iodide marker, and no early apoptosis was detected, which was revealed when only the annexin marker was used.

It is important to point out that the 24-hour exposure of GO samples to PBMCs described above was done in the presence of RPMI-1640 medium with L-glutamine, 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (see “Supplementary material”). The availability of nutrient molecules from the growth medium opens the possibility of absorption of proteins into the GO basal plane masking thus its toxic effect. To investigate this possibility, new sets of experiments are being designed and performed, in which exposure of GO samples to PBMCs is carried out in the absence of nutrient molecules, as in the case of bacteria.