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Chapter 13 Cytotoxicity of graphene and Graphene-based Biosensors

13.1 Anti-bacterial graphene-based materials

13.2 Cytotoxicity of graphene and graphene-related materials

13.3 Graphene-based biosensors

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13.1 Anti-bacterial graphene-based materials [13-1]

GO nanosheets were prepared according to a modified Hummer method, resulting in a brown colloidal suspension. The thickness of the GO sheets was 1.1 nm as measured via atomic force microscopy (AFM), suggesting the formation of a single-layer 2-D nanomaterial (Figure 1a). Hydrazine reduction of GO led to a black rGO suspension, a more conductive version of GO nanosheets with less surface defects. AFM measurements revealed that rGO had a reduced sheet thickness of 1.0 nm (Figure 1b), which was possibly attributed to partial removal of oxygen functional groups on the surface of GO nanosheets during the reduction process.

Preparation of GO and rGO

Figure 1

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Cellular uptake and cytotoxicity of GO nanosheets (85 g/mL) with a mammalian cell line, A549. TEM studies demonstrated that GO nanosheets were inside the endosome of the cytoplasm (Figure 2a), suggesting that GO nanosheets could be internalized within A549 cells via endocytosis. GO nanosheets (20 g/mL) exhibited no cytotoxicity to A549 within 2 h incubation and a slight decrease in cell viability (20%) within 24 h. GO nanosheets of higher concentration (85 g/mL) led to an increased cytotoxicity (50%) within 24 h (Figure 2b).

Fig. 2c depicts the distribution of A549 cells without GO nanosheet treatment (left) and treated with 20 g/mL (middle) and 85 g/mL (right) GO for 24 h.

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The number of untreated cells was 3.11-fold that of seeded cells (Table S1), while the number of cells treated with GO nanosheets of 85 g/mL proliferated 2.78-fold. The results implied that the observed small decrease in cell viability might arise from GO-retarded cell cycles and thus slightly decreased proliferation rates, rather than from apoptosis or death of cells. Therefore, we concluded that GO nanosheets were relatively biocompatible nanomaterials with mild cytotoxicity.

人類周邊血中的顆粒球 (G) ，單核球 (M)

* 流式細胞技術 Flow Cytometry 謝世良

Table S1*

Flow cytometry 技術主要是為了快速偵測一顆接著一顆流動於液體水柱（ fluid stream ）中的顆粒或細胞，因此 flow cytometry 所偵測的訊號是以一個（而非一群）細胞或顆粒，被雷射光激發後產生的光學訊號，再轉換電子訊號由電腦分析細胞或顆粒的特性。

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Antibacterial Activity of GO Nanosheets

The metabolic activity of E. coli DH5 cells in the presence of GO nanosheets was measured via a luciferase-based ATP assay kit. (Luciferase is a term for the class of oxidative enzymes used in bioluminescence)

After 2 h incubation with GO nanosheets of 20 g/mL at 37 °C, the cell metabolicactivity for E. coli deceased to 70% and to 13% at a GO nanosheet concentration of 85 g/mL (Figure 3a), suggesting the strong inhibition ability of GO nanosheets to E. coli.

A classic colony counting method was to measure the microbial viability of E. coli treated with 85 g/mL GO for 2 h. Significantly, GO almost completely suppressed the growth of E. coli, leading to a viability loss up to 98.5% (Figure 3b).

TEM studies revealed that E. coli largely lost cellular integrity, with the cell membrane being severely destroyed and the cytoplasm flowing out (Figure 3c,d).

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Figure 4. shows antibacterial activity and cytotoxicity of rGO nanosheets. (a) Metabolic activity of E. coli treated with 85 g/mL GO and rGO nanosheets, respectively. (b) Antibacterial activity of 85 g/mL GO and rGO nanosheets against E. coli. (c) TEM image of E. coli exposed to 85 g/mL rGO nanosheets at 37 °C for 2 h; TEM studies revealed that rGO nanosheets destroyed the membrane of E. coli in a way similar to that of GO nanosheets (Figure 4c). (d) Viability of A549 cell incubated with 20 and 85 g/mL rGO nanosheets, respectively.

Figure 4.

rGO nanosheets possessed antibacterial properties that were only slightly lower than those of GO nanosheets while their cytotoxicity was significantly higher than GO’s. Such difference in cytotoxicity might arise from different surface charges and functional groups of GO and rGO nanosheet surfaces.

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After overnight incubation at 37 °C, we could not find any cell growth on the GO paper (Figure 5a) and only a limited number of E. coli colonies on the rGO paper (Figure 5b), implying the superior antibacterial effect of such graphene-based papers. In contrast, control studies in the absence of either GO or rGO paper led to a great number of colony-forming units (CFU). SEM studies further confirmed that E. coli cells on the paper lost the integrity of membranes (Figure 5c,d), which was responsible for the bacteria-killing effect of the graphene-based paper.

Fig. 5 Photographs of E. coli growth on GO (a) and rGO (b) paper (overnight incubation at 37 °C). SEM images of E. coli attached to GO (c) and rGO (d) paper (12 h incubation at 37 °C).

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13.2 Cytotoxicity of graphene and graphene-related materials [13-2]

Most reports show that GO materials, including GO films (paper), are superiorbiocompatible materials that allow the effective proliferation of human and mammalian cells with limited or no cytotoxicity. Such characteristics seem to indicate that GO materials may be used in tissue engineering, tissue implants, wound therapy, and drug delivery applications.

Recently, several reports have shown that GO paper promotes the adhesion and proliferation of L-929 cells, osteoblasts, kidney cells, and embryonic cells.However, additional studies have shown that cellular internalization of GOnanosheets applied to the culture media at a concentration of 20 μg/mL can causea 20% decrease in mammalian cell viability, while a concentration of 50 μg/mL can lead to a 50% loss in cell viability, indicating that some inhibitory effect can be observed if a GO suspension is applied to the growth media.

A recent study showed that graphene and graphene oxide materials are cytotoxic to human erythrocytes and skin fibroblasts. Another study showed that films developed from a suspension of reduced graphene oxide and polyoxyethylene sorbitan laurate (TWEEN) were noncytotoxic to three different types of mammalian cells. These combined results appear to support that GO materials are biocompatible with mammalian cells by promoting cell adhesion and proliferation as effectively as commercial polystyrene tissue culture materials. On the other hand, colloidalGO solutions appeared to be mildly cytotoxic at high concentrations.

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On the other hand, recent studies have also indicated that GO is not cytotoxic and also lacks any antibacterial effect. Das et al. showed that, when GO was placed in the center of a nutrient media plate previously inoculated with bacteria, a growth inhibition zone was not formed. Alternatively, when silver decorated GO was used, a clear inhibition zone was formed.

It shows that graphene oxide materials do not adversely impact microbial and mammalian cell growth. Furthermore, graphene oxide materials tend to produce a dramatic increase in microbial and mammalian cell proliferation, indicating that graphene oxide is not a bactericidal or bacteriostatic material, but instead a general growth enhancer that acts as a scaffold for cell surface attachment and proliferation.

The results showed that the GO-containing samples achieved an average absorbance of 1.7 in 16 h of incubation while the bacteria growing in LB broth only achieved an absorbance of 1.3 (Figure 1a) These results indicated that bacteria in the presence of GO grew faster than bacteria in LB media and were able to achieve cell saturation sooner.

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The culture tubes containing graphene oxide did not visually show any apparent reduction in bacterial growth (Figure 1b). Furthermore, they appeared more turbid than the control culture (Figure 1c), and a dense dark precipitate was observed at the bottom of the tube (Figure 1b). The dark precipitate was not produced in the control cultures without GO (Figure 1c).

We proceeded to determine growth level in the bacteria cultures by measuring the absorbance at 600 nm. Samples were taken from the supernatant without disturbing the dark precipitates at the bottom of the samples containing GO. It was possible that the dark precipitate observed in samples containing GO was responsible for enhancing bacterial growth in the media or harboring bacterial growth itself.

SEM analysis showed that the dark precipitate was formed by a thick bacterial biofilm(Fig.1f,g) containing a large mass of aggregated cells (Fig. 1g) and extracellular polymeric material (Fig. 1f).

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The results showed that the precipitation of GO in the culture media may be acting as a scaffold for bacterial attachment, proliferation, and biofilm formation. Studies have shown that carbon nanomaterials could act as attachment surfaces where small colonies grow around tubular carbon nanostructures. Further, it seems that precipitated GO induced massive cell growth, aggregation, and secretion of extracellular polymeric substance (EPS) (Figure 1f,g).

Quantitative real-time PCR was used to assess bacterial growth in filterswith and without GO (a). PVDF filters coated with 0 (c), 25 (d), and 75 μg (e) of GO were inoculated with E. coli and incubated at 37 C for 18 h.

The results shown in Figure 2a indicate that GO not only lacks antimicrobial properties, but that it actually enhances microbial growth when coated onto another surface.

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Small 1 cm∼ 2 pieces of PVDF filter (f), GO film (g), and Ag-GO film were inoculated with E. coli and culture for 18 h at 37 C. Bacterial growth was quantified by real-time PCR (b).

Bacteria growth was observed with the naked eye in all samples, but the filters containing GO presented large bacteria colonies around specific areas that seem to contain more GO (Figure 3a-d).

An area of dense bacterial growth in the LB media was produced around all neat filter replicates (Figure 2c). This halo of cells was not observed in any of the GO-coated filters (Figure 2d,e). This was an interesting observation that implied that there is an inherent preference by bacteria to attach and grow in areas containing GO, especially those areas containing the highest GO levels (Figure 3).

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Figure 3 Bacteria interaction with graphene oxide. Black arrows indicate some of the areas with increased bacterial growth observed on filters coated with 25 (a,b) and 75 μg (c,d) of GO. Bacterial colonies can be easily observed as elongated features in GO-coated filters but not in a neat PVDF filter.

Graphene oxide films were placed onto LB culture plates that were previously inoculated with 1 x106. Then, Then, 1 x106 E. coli cells were directly inoculated on top of the film pieces and allowed to dry. The purpose of this type of inoculation was to observe growth over the GO film and also to determine if any growth inhibition zone was formed around the GO film. Growth inhibition zones around GO film have been reported in the past. Inhibition areas would indicate that the material has some toxic effect on the bacteria.

Bacteria Interaction with GO and Ag-GO Films.

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Figure 4 shows graphene oxide and silver-coated graphene oxide characterization. (a) TEM image of neat GO, (b) TEM image of Ag decorated GO, (c) XRD spectrum of Ag-decorated GO and ICDD 00-004-0783 card data for face-centered cubic Ag, and (d) size distribution studies performed using TEM for Ag-decorated GO.

Figure 4

Results showed that growth inhibition zones were not detected in the plate containing either GO film or filter paper (Figure 2f,g). However, Ag-decorated GO showed large growth inhibition zones characterized by a clear area with no cell growth (Figure 2h). These results clearly demonstrate that GO does not have any antimicrobial effects capable of producing a toxic effect in the area surrounding the GO film.

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A study was performed to test the role of GO film on mammalian cell attachment and proliferation. Control glass slides and glass slides coated with 10 μg of GO (Figure 5a) were placed onto a culture dish to which culture media and 6 x105 mammalian colorectal adenocarcinoma HT-29 cells were added. The cells were allowed to attach and develop on the slides. At various time intervals, cell attachment was assessed by light microscopy. Shown in Figure 5b,c are representative images of cell morphology after incubation for 6 h. The results indicated that the mammalian cells attached more efficiently to the GO-coated glass slides and grew (Figure 5c).

Mammalian Cell Attachment and Proliferation onto GO Film.

The results of this study clearly demonstrate that graphene oxide does not have antibacterial properties. Furthermore, graphene oxide lacks any bacteriostatic property as shown by the prolific growth observed on all forms of GO tested. It seems that GO acts as an enhancer of life, increasing not only mammalian cell growth but also bacterial growth.

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Cytotoxicity of Graphene Oxide and Graphene in Human Erythrocytes and Skin Fibroblasts [13-4]

One method of toxicity assessment was based on measurement of the efflux of hemoglobin from suspended red blood cells. At the smallest size, graphene oxide showed the greatest hemolytic activity, whereas aggregated graphene sheets exhibited the lowest hemolytic activity. Coating graphene oxide with chitosan nearly eliminated hemolytic activity. Together, these results demonstrate that particle size, particulate state, and oxygen content/surface charge of graphene have a strong impact on biological/toxicological responses to red blood cells.

The compacted graphene sheets are more damaging to mammalian fibroblasts than the less densely packed graphene oxide. Clearly, the toxicity of graphene and graphene oxide depends on the exposure environment (i.e., whether or not aggregation occurs) and mode of interaction with cells (i.e., suspension versus adherent celltypes).

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Figure 4. (a) Percent hemolysis of RBCs incubated with different concentrations (3.125 to 200 μg mL1) of GO (red), bGO (blue), pGO-5 (green), pGO-30 (purple), and GS (black) for 3 h at 37 C with agitation. Data represent mean(SD from at least five independent experiments. Also included is the percent hemolysis of RBCs incubated with pGO-30/chitosan at 100 μg mL1 for 3 h at 37 C with agitation. (b) Photographs of RBCs after 3-h exposure to GO, bGO, pGO-5, pGO-30, and GS at different concentrations (3.125 to 200 μgmL1). The presence of red hemoglobin in the supernatant indicates RBCs with membrane damage. (+) and () symbols represent positive control and negative control, respectively.

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Figure 5. Optical microscographs of RBCs in the presence of (a) PBS (control), (b) pGO-5, (c) pGO-30, and (d) GS at 25 μgmL1 for 3 h at 37 C with agitation. Inset images are magnified RBCs. Scale bars in the inset images represent 10 μm. The arrows in b and c show lysed RBCs. The arrows in d represent the hemagglutination caused by GS aggregates.

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Figure 6. Cell viability of human skin fibroblast cells determined from MTT and WST-8 assay after 24-h exposure to different concentrations of pGO-5 and GS. Data represent mean ( SD.)

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13-1 Wenbin Hu et al. ACS nano (2010) 4 7 4317

13-2 Oscar N. Ruiz et al. ACS nano (2011) 5 10 8100

13-3 Omid A et al. Biomaterials (2012) 33 8017

13-4 Ken-Hsuan Liao et al. ACS Applied Materials& Interfaces (2011) 3 2607

13-5 Yuyan et al. Electroanalysis (2010) 22 10 1027

13-6 Tapas Kuila et al. Biosensora and Bioelectronics (2011) 26 4637

13-7 Yi Wan et al. (2011) 83 648

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