Targeting Nanodiamonds to the Nucleus in Yeast Cells

Abstract

Nanodiamonds are widely used for drug delivery, labelling or nanoscale sensing. For all these applications it is highly beneficial to have control over the intracellular location of the particles. For the first time, we have achieved targeting the nucleus of yeast cells. In terms of particle uptake, these cells are challenging due to their rigid cell wall. Thus, we used a spheroplasting protocol to remove the cell wall prior to uptake. To achieve nuclear targeting we used nanodiamonds, which were attached to antibodies. When using non-targeted particles, only 20% end up at the nucleus. In comparison, by using diamonds linked to antibodies, 70% of the diamond particles reach the nucleus.

Keywords: fluorescence nanodiamonds; nucleus targeting; yeast cells.

Figure 1

Schematic representation of the targeting strategy in a yeast cell. An antibody with a binding domain for the nuclear pore complex (NPC) was linked to fluorescent nanodiamonds (FNDs). After using a spheroplasting protocol, FNDs–antibodies (FND–AB) are ingested and will accumulate on the nucleus.

Figure 2

Confocal microscope images of FND–AB. Fluorescein (FITC) labelled antibodies (A), FNDs (B), and merged images (C). (D–F) show FITC, FND and merged images in magnification.

Figure 3

3-(4,5-dymethylthiazol-2-yl)-2,5-dyphenyltetrazolium bromide (MTT) assay for analyzing metabolic activity of yeast cells. Yeasts without any treatment were used as a comparison (grey dots) and 1% of H₂O₂ was used as positive control. Compared to yeasts without any treatment, both FND and FND–AB group have no significant effect on metabolic activity in yeast cells. Both groups only show significant difference with the positive control. Data were analyzed by using one-way ANOVA and followed by Tukey post hoc test. The significance level was set at 0.05 and **** indicated p < 0.0001.

Figure 4

Position of particle (red) inside the cell at different time points. FND (a) and FND–AB (b) groups were followed between 0 and 24 h after the particles were taken up by the cells. Scale bars in all images are 0.75 µm.

Figure 5

Distance measurement from particles to the border of the nucleus. Experiments were performed in 10 cells. Significance level was set at 0.05. **** indicates p < 0.0001. Error bars represent standard errors.

Figure 6

Proportion of particles targeted to the nucleus at the 24 h time point. Fifty particles inside cells were used from both groups.