Intracellular ROS Levels Investigation

Intracellular ROS levels were investigated using flow cytometry analysis after exposure of HCT116 to either RGO, CeO₂ or nano-CeO₂/RGO nanohybrid over a period of 24 h and illustrated in Figure 5.11a. Data is computed as mean fold change in DCF signal compared to cell exposed to medium only in Figure 5.11b.

Figure 5.11 Intracellular ROS levels in HCT116 cells as measured by flow cytometry after (a) 24 h of exposure to 100µg/ml Control (curve a), nano-CeO₂/RGO (curve b), CeO₂(curve c) and RGO (curve d) (b) Data are demonstrated as mean fold change in DCF signal compared to as cells exposed to medium only ±SD (n=3), ** denoted significant difference (p<0.05) compared with graphene treated cells and cells exposed to medium only ; *** denoted significant difference (p<0.05) compared with nanohybrid treated cells and cells exposed to medium only.# indicated significant difference (p<0.05) compared with cells exposed to nanohybrid and CeO₂ as determined by two tailed nonparametric t tests.

The nano-CeO₂/RGO nanohybrid and CeO₂ were noticed to significantly decrease (p < 0.05) intracellular ROS content as compared with cells exposed to medium only at 24h. It was evident from the decrease in mean fluorescence intensity profiles (Figure 5.11b). In contrast, RGO did not significantly (p < 0.05) alter the level of intracellular ROS compared to cells exposed to medium only.

This phenomenon can be justified and explained by the level of ROS production by the epithelial cells. The ROS level stimulated the signaling of growth factors, mainly vascular endothelial growth factors (VEGF) and fibroblast growth factors (FGF) which in turn induce the cell proliferation and cell survival signaling (Lord et al 2013). Besides that, ROS leads to the accumulation of oxidants either by generating free radicals, reactive anions containing oxygen atoms or molecules containing oxygen atoms. This results the production of various free radicals e.g hydroxyl radicals (•OH), superoxide (•O2^–) and peroxynitrite (ONOO⁻) (Cadenas & Davies 2000). Hence, by utilizing these free radicals ROS promotes oxidative stress within the cell and even causes apoptosis or cell death. Similar findings have been reported by Fiers et al (2000) and Nicholls & Budd (2000).

In the present study the decrease in the intracellular ROS level was noticed in nano-CeO₂/RGO nanohybrid and CeO₂ nanomaterials. Therefore, it can be stated that the cells are in low oxidative stress and effective in reducing the intracellular ROS level compared to cell exposed to medium only. Moreover the low stress on cells signifies that the nanohybrid develops an innate defence within the cell that can interplay with the stress and resulting in the contraction of stress stimuli. Thus, these cells do not undergo any further degradation mechanism which can cause the morphological alteration of the cell structure.

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Conversely, CeO₂ treated cell exhibits relatively more intracellular ROS level compared to nanohybrid (P< 0.05). This can be attributed to the fact that CeO₂ treated cells were supposed to be in medium oxidative stress where the cell adapts by remodeling the production of its stress proteins particularly heat shock proteins (HSPs). Ritossa (1962) first performed a heat shock on Drosophilia melanogaster larvae, and noted that it stimulated with unusual gene expression. The resulting products of these genes were termed as HSPs and isolated in 1974 (Tissiéres et al 1974). Further, HSPs can control the normal cellular functionality until it has acquainted its defence to the stress controlled point and thereby the appearance of irregular disrupted cellular morphology along with confluent cytoplasmic cell aggregates.

Conversely, cells exposed to RGO nanomaterials were found to be in high oxidative stress, which is evident from the increase in mean fluorescence intensity profile (Figure 5.11b). Here the cell has utilized all of its resources to survive against the stress. The cell cycle has been arrested by restricting normal protein production. Thus the cell growth was inhibited and majority of the cells has appeared as characteristics spherical shapes of apoptotic bodies. This fact can also be justified from the above mentioned morphological images of HCT 116 cells in MTT assay (Figure 5.7b).

Cellular uptake of nanomaterials using side scatter analysis

The uptake of nanomaterials into HCT116 cells was analyzed by flow cytometry employing side scatter measurements, a crucial parameter to assess cellular granularity and depicted in Figure 5.12.

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Figure 5.12 (a) Flow cytometry analysis of Control (curve a), RGO (curve b), CeO₂ (curve c) and nano-CeO₂/RGO nanohybrid (curve d) uptake into HCT 116 cells analyzed by side scatter measurement after 24 h of exposure to nanomaterials concentration of 100µg/ml. (b) Data showed statistically significant differences from the control group by the two tailed nonparametric t tests (*p<0.05).

As shown in Figure 5.12, nano-CeO₂/RGO nanohybrid and CeO₂ exhibited significantly increased side scatter profile as compared to cells exposed to medium only indicating that nanohybrid and CeO₂ has internalized by HCT 116 cells and retained inside the cell. This is attributed to the reversibility of the oxidation states of CeO₂ implies a regenerative capacity for ROS scavenging. Similar concept has also been reported by Lorda et al (2012), where the authors have studied the intracellular scavenging properties of the size-dependent CeO₂ nanoparticles to monocyte cell line U937 and showed nanoceria in the range of 7-94 nm are not toxic and potentially active to scavenge intracellular ROS. Further, Asati et al (2010) has reported the ROS internalization and localized in the cytoplasm. In contrast, the side scatter measurements in cells exposed to graphene were not significantly different to cells exposed to medium only which suggests that graphene molecule was not efficient to scavenge intracellular ROS and thereby a decrease in side scatter measurement was measurement. This result is possibly due to the flat shaped structure of RGO molecules (Zhang et al 2010).

Conversely, based on the aforementioned phenomenon, it is noted that after internalization within the cell, nanohybrid adapts a mechanism of cellular uptake where it utilizes the scavenging property of CeO₂. Subsequently, the nanohybrid can easily interact with the stress regulating proteins that can neutralize the oxidative stress thereby inducing the cell proliferation.

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Figure 5.13 Scheme of the proposed mechanism of action of nanomaterials on cell

Cytotoxicity involved in our studies was governed by oxidative stress mechanism and was also induced through metal toxicity and physical piercing causing ruptured cellular morphology (Figure 5.13). In the current study, we have proposed the cytotoxicity of the nanomaterials occurred mostly due to the oxidative stress through the generation of reactive oxygen species (ROS) (Manna et al 2005 and Pulskamp et al 2007). Additionally, the nanohybrid composed of CeO₂ and RGO. CeO₂ is well recognized for its facile cyclic oxidation states (Ce(III) as Ce₂O₃ and Ce(IV) as CeO₂) and electrocatalytic activity wherein RGO possesses flat shaped structural morphology Hence, metal toxicity can also be influence the toxic response mechanism. Thus, these two mechanisms could interplay for the cytotoxic mechanism of graphene based nanomaterials on human cells. Similar concepts have also been reported by Shvedova et al (2007) and Hull et al (2009).

Besides that, the cellular uptake studies and optical images showed that after internalization within the cell nanohybrid adapts a mechanism where it utilizes the scavenging property of CeO₂. Subsequently, it can easily interact with the stress regulating proteins that can neutralize the oxidative stress and induces cell proliferation. Present studies have shown that the nanomaterials such as RGO and CeO₂ can alter the cell membrane integrity. This can be assigned due to a stronger interaction between the more sharpened edges of RGO nanosheet with the cell membrane or a charge transfer between the membrane protein and the spherical aggregates of CeO₂ nanoparticles resulting in distinct alteration of cellular morphology (Ting et al 2013).

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Analysis of cell granularity

Flow cytometry analysis has been performed to measure the ROS scavenging and cellular uptake of the RGO, CeO₂ and nano-CeO₂/RGO nanohybrid to analyze the cell granularity and shown in Figure 5.14.

Figure 5.14 The cell granularity measurements of (a) control (b) nano-CeO₂/RGO nanohybrid (c) CeO₂ and (d) RGO nanomaterials using flow cytometry analysis

To find the change in cellular granularity the HCT 116 cells were exposed to three different nanomaterials RGO, CeO₂ and nano-CeO₂/RGO nanohybrid for about 24h. Generally, if the nanomaterials are engulfed by the cells, the granularity inside the cell increases due to the interaction of nanomaterials with the cell organelle. Here it was observed that the granularity of the cells decreases in case of nano-CeO₂/RGO nanohybrid as compared to nanomaterials graphene and CeO₂. It can be expected due to the more nanohybrid internalization as compared with other two, providing more ROS scavenging capacity.

FLUORESCENCE MICROSCOPY ANALYSIS OF NANOFILLERS EXPOSED TO BACTERIAL CELL LINE

The damage to bacterial cell membrane was also monitored with the Live/Dead Backlight kit by discriminating the live and dead bacteria under a fluorescence microscope and illustrated in Figure 5.15.

Figure 5.15 Fluorescence micrographs of Salmonella typhimurium cells treated with (a) Control (b) CeO₂ (c) RGO (d) nano-CeO₂/RGO nanohybrid

As shown in Figure 5.15, the viable cell with the unaltered cell membrane was envisioned by their green fluorescence under blue light and the dead cells with ruptured and damaged membranes by red fluorescence under green light. The fluorescence micrographs of Salmonella typhimurium control cells (Figure 5.15a) exhibited various intact green colored prominent viable cells, while the cells treated with CeO₂ (Figure 5.15b) showed the appearance of the intense red colored cell along with few healthy green colored cells. This suggests that CeO₂ can cause damage of the cell membrane resulting in the red colored dead cells. Further the cells treated with graphene displayed mixed population of live and dead cells where RGO (Figure 5.15c) can induce the cell death through cell rupturing. However, a higher population of red cells was noted in nano-CeO₂/RGO nanohybrid treated culture media (Figure 5.14d) implying the higher cell and membrane damage, leading to increase in cell death which appears as red color. Hence, these above explanations state that as compared with CeO₂ and RGO, nanohybrid reveals significant levels of antibacterial properties.

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