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- 2011 (4) (entfernen)
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- Agglomeration (2)
- Nanoparticles (2)
- BSA (1)
- Cell imaging (1)
- Characterisation (1)
- Cytotoxicity (1)
- FLIM (1)
- Fibroblast cells (1)
- Fluorescence (1)
- Fluorescence lifetime imaging microscopy (1)
Cell cultures form the basis of most biological assays conducted to assess the cytotoxicity of nanomaterials.
Since the molecular environment of nanoparticles exerts influence on their physicochemical properties, it can have an impact on nanotoxicity. Here, toxicity of silica nanoparticles upon delivery by fluid-phase uptake is studied in a 3T3 fibroblast cell line. Based on XTT viability assay, cytotoxicity is shown to be a function of (1) particle concentration and (2) of fetal calf serum (FCS) content in the cell culture medium.
Application of dynamic light scattering shows that both parameters affect particle agglomeration. The DLS Experiments verify the stability of the nanoparticles in culture medium without FCS over a wide range of particle concentrations.
The related toxicity can be mainly accounted for by single silica nanoparticles and small agglomerates. In contrast, agglomeration of silica nanoparticles in all FCS-containing media is observed, resulting in a decrease of the associated toxicity. This result has implications for the evaluation of the cytotoxic potential of silica nanoparticles and possibly also other nanomaterials in standard cell culture.
Increasing the information content from bioassays which requires robust and efficient strategies for the detection of multiple analytes or targets in a single measurement is an important field of research, especially in the context of meeting current security and health concerns. An attractive alternative to spectral multiplexing, which relies on fluorescent labels excitable at the same wavelength, yet sufficiently differing in their emission spectra or color presents lifetime multiplexing. For this purpose, we recently introduced a new strategy based on 'pattern-matching' in the lifetime domain, which was exemplary exploited for the discrimination between organic dyes and quantum dot labels revealing multi-exponential decay kinetics and allowed quantification of these labels. Meanwhile, we have succeeded in extending this lifetime multiplexing approach to nanometer-sized particle labels and probes absorbing and emitting in the visible (vis) and near-infrared (NIR) spectral region. Here, we present a first proof-of-principle of this approach for a pair of NIR-fluorescent particles. Each particle is loaded with a single organic dye chosen to display very similar absorption and emission spectra, yet different fluorescence decay kinetics. Examples for the lifetime-based distinction between pairs of these fluorescent nanoparticles in solution and in cells are presented. The results underline the potential of fluorescenc lifetime multiplexing in life science and bioanalysis.
A new laser ablation (LA)-ICP-MS method for single cell and cell nucleus imaging was developed. Therein, iodine was employed as an elemental dye for fibroblast cells and for thin tissue sections. At an incubation time of 60 s, iodine is located mainly within the cell nuclei. This effect was illustrated in fibroblast cells, and iodine signal within the cell nucleus was as high as 5 × 104 cps at 4 µm laser spot size. The surrounding cytoplasm was iodinated as well, but to a lesser extent. The spatial resolution attained was sufficient to detect even smaller cell nuclei within a liver biopsy tissue. Furthermore, iodine was successfully employed for biomolecule labeling and we demonstrated that iodine signal increased with increasing thickness of a palatine tonsil tissue. Thus, the use of iodine as an internal standard to correct for tissue inhomogeneities in LA-ICP-MS was investigated for the simultaneous detection of two tumor markers (Her 2 and CK 7) in breast cancer tissue. Additionally, lanthanide background resulting from glass ablation can be corrected for by Eu standardization.
The size, surface charge and agglomeration state of nanoparticles under physiological conditions are fundamental parameters to be determined prior to their application in toxicological studies. Although silica-based materials are among the most promising candidates for biomedical applications, more systematic studies concerning the characterisation before performing toxicological studies are necessary. This interest is based on the necessity to elucidate the mechanisms affecting its toxicity. We present here TEM, SAXS and SMPS as a combination of methods allowing an accurate determination of single nanoparticle sizes. For the commercial material, Ludox TM50 single particle sizes around 30 nm were found in solution. DLS measurements of single particles are rather affected by polydispersity and particles concentration but this technique is useful to monitor their agglomeration state. Here, the influence of nanoparticle concentration, ionic strength (IS), pH and bath sonication on the agglomeration behaviour of silica particles in solution has been systematically investigated. Moreover, the colloidal stability of silica particles in the presence of BSA has been investigated showing a correlation between silica and protein concentrations and the formation of agglomerates. Finally, the colloidal stability of silica particles in standard cell culture medium has been tested, concluding the necessity of surface modification in order to preserve silica as primary particles in the presence of serum. The results presented here have major implications on toxicity investigations because silica agglomeration will change the probability and uptake mechanisms and thereby may affect toxicity.