Vapor phase mediated cellular uptake of sub 5 nm nanoparticles
© Serdiuk et al; licensee Springer. 2012
Received: 19 October 2011
Accepted: 11 April 2012
Published: 11 April 2012
Nanoparticles became an important and wide-used tool for cell imaging because of their unique optical properties. Although the potential of nanoparticles (NPs) in biology is promising, a number of questions concerning the safety of nanomaterials and the risk/benefit ratio of their usage are open. Here, we have shown that nanoparticles produced from silicon carbide (NPs) dispersed in colloidal suspensions are able to penetrate into surrounding air environment during the natural evaporation of the colloids and label biological cells via vapor phase. Natural gradual size-tuning of NPs in dependence to the distance from the NP liquid source allows progressive shift of the fluorescence color of labeled cells in the blue region according to the increase of the distance from the NP suspension. This effect may be used for the soft vapor labeling of biological cells with the possibility of controlling the color of fluorescence. However, scientists dealing with the colloidal NPs have to seriously consider such a NP's natural transfer in order to protect their own health as well as to avoid any contamination of the control samples.
Keywordsnanoparticles cell labeling fluorescence contamination
During the last decade, the biomedical research field has been identified as one that can greatly benefit from the advancement in nanotechnologies. In particular, the use of various kinds of nanoparticles (example: metallic, metal oxides, semiconductors, silica, etc.) in biotechnology merges successfully the fields of materials science and biology . It has been found that different nanoparticles (NPs) can enter living cells, providing specific tagging of cell components. Nowadays, NPs are preferred to be used for multicolor cell imaging due to their unique properties, such as size and composition-induced tunable spectral characteristics, narrow emission spectra, high quantum yield, and low photobleaching . In particular, II-VI NPs [3, 4] and silicon NPs  are among the most extensively studied. We have already presented studies which implemented NPs produced from SiC as fluorescent agents for living cell imaging, similar to what our colleagues did [6, 7]. We have also found preferential toxic effect of these NPs on cancer cells, which was described in detail in a previous work .
Although the potential of nanoscale objects in biology is tremendous, a number of questions concerning the safety of nanomaterials and the risk/benefit ratio of their usage remain open. Thus, nanotoxicology refers to the study of potential negative effects coming from the interaction between nanomaterials and biological objects [9, 10]. Toxicity of NPs is studied from NPs in suspensions  except for the studies of lung toxicity where NPs are studied as aerosols [10, 12].
In the following work, we show that even when NPs have been initially dispersed in liquids, they may be easily transported into the surrounding air environment at relatively long distances due to the natural evaporation of the liquids at room temperature. Such an innate transfer of NPs from the originally colloidal suspension into an aerosol state may easily lead to a significant labeling of any biological system, although it is separated from the colloidal source of NPs, by a gas phase. These results should be taken into account in order to (a) use them for soft cell labeling through the vapor phase, (b) avoid any contamination of control samples and (c) improve health protection of users.
Fluorescent NPs were formed by means of electrochemical anodization of a low-resistivity grade (< 1 Ω) bulk SiC polycrystalline wafer (NPs) . The etching process took place for 2.5 h at a current density of 25 mA/cm2 using a 1:1 HF (50%)/ethanol electrolyte. After the etching, a highly porous network constituted by interconnected SiC nanocrystals was formed. The ultraporous layer was washed several times with deionized water then naturally dried in ambient air, removed from the wafer and ground to powder. To obtain colloidal suspensions of NPs, the powder was dispersed in 1:1 deionized water/ethanol mixture or Dulbecco's Modified Eagle's Medium (DMEM) buffer for mammalian cell investigations, and then large NP crystallites were removed by centrifugation at 10,000 g for 5 min. These suspensions contained only small-sized (< 3 nm) and uniformly dispersed NPs. Typical TEM images, dynamic light scattering size distributions and zeta-potential measurement of which had already been shown in the study by Botsoa et al. .
The inner layer of a fresh thin onion epidermis was peeled off. The onion peels were neatly spread in the center of a glass slide in a drop of water. The glass slide was then mounted on a mobile holder situated above a colloidal suspension (2 mL, surface area 4 cm2) containing the NP suspension (1 g/L). Height origin was fixed at the suspension surface. At a fixed height value, the onion epidermis samples were exposed to the suspension vapors for different periods of time varying from 3 to 17 h. Depending on exposure time, evaporated volume was changing from 0.5 to 1.5 mL. All experiments were carried out at a temperature of 21°C, humidity of 60% and vapor pressure of 1,018 hPa, without air mixing. Speed of convection flow was equal to 0 m/s. The manipulations were performed under two different conditions: (a) in open air and (b) in confined air with the whole system being covered by a cap. After a certain exposure time, the onion peels were intensively rinsed in deionized water to remove the NPs deposited onto the epidermis surface and unincorporated inside the epidermal cells. Control manipulations were carried out with the use of a pure suspension without the NPs.
Suspension containing NPs was also added to the 3 T3-L1 fibroblasts (American Type Culture Collection, Manassas, VA, USA). The cells were grown on glass substrates in DMEM supplemented with 10% newborn calf serum, 4 mM glutamine, 4 nM insulin (Actrapid Human Torrent Pharmaceuticals Ltd., Ahmedabad, India; Novo Nordisk A/S, Bagsvaerd, Denmark), 10 mM HEPES, 25 μg sodium ascorbate, 100 IU penicillin, 100 μg streptomycin and 0.25 mg/L amphotericin B at 37°C in a water-saturated atmosphere with 5% CO2 in a Heraeus incubator (BB16; Thermo Scientific, Brebières, France). The cells have been incubated for 15 h in the presence of NPs (with concentration of 0.8 g/L in suspension in DMEM). Some 3 T3-L1 samples were cultured in DMEM without NPs but, in air contact, were allowed with wells containing NP suspensions. After a certain incubation time, cells were rinsed twice in pure ethanol to eliminate NPs from the environment and to fix cells.
Finally, the samples were observed under a fluorescence microscope (Leica DMI 4000B, Leica Microsystèmes SAS, NANTERRE, France) using the following filter combination: UV/violet excitation band at 2.92 to 3.5 eV with an observation spectral range of < 2.64 eV. Leica EC 3 camera (Leica Camera Sarl, Saint Jorioz, France) was used.
For each image, luminosity per cell was calculated by summing intensities (in a range from 0 to 255) of each pixel in the picture and dividing this result by the number of fluorescent cells in the picture. The MathWorks Matlab R2009b and Simulink software (Torrance, CA, USA) were used for image analyses in order to quantify luminosity per one cell.
Results and discussion
Typical fluorescence images of the onion epidermal cells exposed to the evaporating suspensions without and with NPs are shown in Figure 1c, d, respectively. As one can see, no fluorescence is visible in the absence of NPs (Figure 1c), while the cell structure of the fluorescent onion epidermis consisting of large, rectangular cells may be easily recognized in the presence of NPs (Figure 1d). The latter is due to the fluorescent labeling of the onion cells by the NPs originating from the colloidal suspension vapors. Indeed, water molecules leaving the suspension during its natural evaporation carry away the very light NPs into the surrounding air. Thus, initially colloidal NPs are transferred from liquid to vapor environment, reaching the cells, entering inside and tagging them. Similar to the fluorescent labeling of the 3 T3-L1 fibroblast cell culture reported earlier , a strong heterogeneous distribution of the fluorescent intensity through the onion cells may be observed. Indeed, one can notice that the external cell membranes and nuclei are preferentially tagged by the fluorescing NPs. The cytoplasm of the onion cells contains some small fluorescent spots as well, but their number is low, and the corresponding fluorescence intensity is weak in comparison to the fluorescence level of the cell membranes and nuclei.
In summary, the present study demonstrates that NPs in colloidal suspensions can be transported in the environment through evaporation to rather long distances and can reach biological targets. In particular, observed results need future development of deep theoretical understanding. The main conclusion is to warn scientists dealing with the colloidal NPs to seriously consider air contamination in order to take care of their own health protection as well as to avoid any contamination of their control samples sharing air environment with the colloidal suspensions containing dispersed NPs.
We would like to acknowledge BioMed Central for the waived article processing charge.
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