The Response of HeLa Cells to Fluorescent NanoDiamond Uptake

Abstract

Fluorescent nanodiamonds are promising probes for nanoscale magnetic resonance measurements. Their physical properties predict them to have particularly useful applications in intracellular analysis. Before using them in intracellular experiments however, it should be clear whether diamond particles influence cell biology. While cytotoxicity has already been ruled out in previous studies, we consider the non-fatal influence of fluorescent nanodiamonds on the formation of reactive oxygen species (an important stress indicator and potential target for intracellular sensing) for the first time. We investigated the influence of different sizes, shapes and concentrations of nanodiamonds on the genetic and protein level involved in oxidative stress-related pathways of the HeLa cell, an important model cell line in research. The changes in viability of the cells and the difference in intracellular levels of free radicals, after diamond uptake, are surprisingly small. At lower diamond concentrations, the cellular metabolism cannot be distinguished from that of untreated cells. This research supports the claims of non-toxicity and includes less obvious non-fatal responses. Finally, we give a handhold concerning the diamond concentration and size to use for non-toxic, intracellular measurements in favour of (cancer) research in HeLa cells.

Keywords: biocompatibility; cellular uptake; fluorescent nanodiamonds; reactive oxygen species.

Figure 1

Confocal images of HeLa cells incubated with FNDs. To visualize internalized nanodiamond particles (in red), HeLa cells were fixed with 3.7% PFA and subsequently stained with Phalloidin-FITC and imaged using a LSM780 microscope. Phalloidin-FITC stains the actin cytoskeleton of the cells (in green). For visualization purposes, both signals are shown in white and merged. As examples, cellular uptake of the standard situation (1 µg FND₇₀), the uptake after 24 h, cells with 120 nm FNDs (1 µg FND₁₂₀) and cells with rounded FNDs are shown. The morphological differences between the cells in the images are a natural variation. As each cluster grows out of one mother cell, they resemble the closest sister cells but not by definition the cells of other clusters. The images were recorded in z-stacks and a focal plane was chosen to display here, approximately 2 μm above the cover glass, as these show the largest volume of the cells. The scale bars in these single optical section images represent 50 μm.

Figure 2

FND uptake in HeLa cells. After confocal imaging, cells are analysed using FIJI software. Our analysis counts the objects and particles inside cells, giving an arbitrary measure for the amount of diamonds taken up. Objects are adjacent FND positive pixels in cells, incorporating both single particles and aggregates or adjacent particles. Particles represent an estimation of the amount of FNDs, calculated by the intensity and size of the objects. The sample incubated with more nanodiamonds, 10 µg of 70 nm FNDs T = 24, resulted in significantly more nanodiamonds per cell (p < 0.001) in comparison to most other samples with the exception of 10 µg of 70 nm FNDs T = 0.

Figure 3

Viability of cells after FND uptake. If the viability compared to the control is between 0.8 and 1.2 it is considered to be unaffected. The samples in which 1 mM H₂O₂ was used differed significantly from the control (* p < 0.05, *** p < 0.001). Error bars show the standard deviation.

Figure 4

Mean free radical production. ROS production can be measured by the conversion of DCFDA to DCF. The more DCF there is, the higher the fluorescent signal a sample emits. As expected, adding a high concentration of hydrogen peroxide increases the signal drastically (up to 100-fold). All diamond samples do not alter the total free radical production inside cells. HeLa cells without a stimulant were used as a negative control to relate all values to. ** p < 0.01, **** p< 0.0001. Error bars show the standard deviation. The inset in this figure shows a cell in greyscale with the metabolized DCF in green.

Figure 5

Relative expression of oxidative stress-related genes. The relative expression of four different genes as a response to the uptake of diamonds or the presence of H₂O₂ has been analysed using quantitative PCR. The control is set as zero, the increase or decrease of the genetic expression is showed for all samples. Glutathion reductase and superoxide dismutase differed most often significantly from the control. * p < 0.05, ** p < 0.01. Error bars show standard deviation. Values are averages out of three independent qPCR runs that were performed in triplicate.

Figure 6

Levels of oxidative stress-related proteins. By quantitating Western Blots, protein levels as a response to the uptake of diamonds or the presence of H₂O₂ have been analysed. The control is set as 1, the protein levels are shown as fold increase or decrease for all samples. The protein levels after uptake of 40, 70 and 120 nanometre FNDs did not differ significantly from the control situation. Note that although in cells with rounded FNDs the caspase-3 levels are increased, this is not significant. **** p < 0.0001. Error bars show the standard deviations. The values are averages out of 3 independent Western Blots of samples in triplicate.