Daniela Drescher

Quantitative Imaging of Gold and Silver Nanoparticles in Single Eukaryotic Cells by Laser Ablation ICP-MS

Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) was utilized for spatially resolved bioimaging of the distribution of silver and gold nanoparticles in individual fibroblast cells upon different incubation experiments. High spatial resolution was achieved by optimization of scan speed, ablation frequency, and laser energy. Nanoparticles are visualized with respect to cellular substructures and are found to accumulate in the perinuclear region with increasing incubation time. On the basis of matrix-matched calibration, we developed a method for quantification of the number of metal nanoparticles at the single-cell level. The results provide insight into nanoparticle/cell interactions and have implications for the development of analytical methods in tissue diagnostics and therapeutics.

Surface-Enhanced Raman Scattering Hybrid Nanoprobe Multiplexing and Imaging in Biological Systems

Surface-enhanced Raman scattering (SERS) labels and probes consisting of gold and silver nanoaggregates and attached reporter molecules can be identified by the Raman signature of the reporter molecule. At the same time, SERS hybrid probes deliver sensitive molecular structural information on their nanoenvironment. Here we demonstrate full exploitation of the multifunctional and multiplexing capabilities inherent to such nanoprobes by applying cluster methods and principal components approaches for discrimination beyond the visual inspection of individual spectra that has been practiced so far. The reported results indicate that fast, multivariate evaluation of whole sets of multiple probes is feasible. Spectra of five different reporters were shown to be separable by hierarchical clustering and by principal components analysis (PCA). In a duplex imaging approach in live cells, hierarchical cluster analysis, K-means clustering, and PCA were used for imaging the positions of different types of SERS probes along with the spectral information from cellular constituents. Parallel to cellular imaging experiments, cytotoxicity of the SERS hybrid probes containing aromatic thiols as reporters is assessed. The reported results suggest multiplexing applications of the nontoxic SERS nanoprobes in high density sensing and imaging in complex biological structures.

Toxicity of amorphous silica nanoparticles on eukaryotic cell model is determined by particle agglomeration and serum protein adsorption effects

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.

Trends in single-cell analysis by use of ICP-MS

The analysis of single cells is a growing research field in many disciplines such as toxicology, medical diagnosis, drug and cancer research or metallomics, and different methods based on microscopic, mass spectrometric, and spectroscopic techniques are under investigation. This review focuses on the most recent trends in which inductively coupled plasma mass spectrometry (ICP-MS) and ICP optical emission spectrometry (ICP-OES) are applied for single-cell analysis using metal atoms being intrinsically present in cells, taken up by cells (e.g., nanoparticles), or which are artificially bound to a cell. For the latter, especially element tagged antibodies are of high interest and are discussed in the review. The application of different sample introduction systems for liquid analysis (pneumatic nebulization, droplet generation) and elemental imaging by laser ablation ICP-MS (LA-ICP-MS) of single cells are highlighted. Because of the high complexity of biological systems and for a better understanding of processes and dynamics of biologically or medically relevant cells, the authors discuss the idea of “multimodal spectroscopies.”

Potent and Selective Inhibition of Acid Sphingomyelinase by Bisphosphonates

The acid sphingomyelinase (aSMase) is emerging as an important drug target for a variety of diseases. Inhibition of aSMase prevents bacterial infections in a rat model of cystic fibrosis and formation of acute lung injury (ALI) elicited by endotoxin, acid instillation, or platelet-activating factor (PAF). Moreover, aSMase is essential for infection of non-phagocytotic cells with Neisseria gonorrhoeae and formation of pulmonary emphysema. Pharmacological or genetic inhibition of aSMase prevents apoptosis and degeneration of liver cells in a mouse model for Wilson s disease. In addition, there are several reports that aSMase significantly contributes to the formation of atherosclerotic plugs. This promising progress in aSMase research, based on sophisticated animal models and cultured cells from patients, is thwarted, however, by the lack of potent and selective inhibitors of this enzyme. Phosphatidylinositol-3,5-bisphosphate (PtdIns3,5P2), to date the most potent inhibitor (KM = 0.53 mm), is not suitable for cell culture studies, because of its fivefold negative charge and its two long fatty acid chains which cause it to stack in cellular membranes. Last but not least, this inhibitor is labile towards phospholipases A1, A2, C, and D and phosphoinositide phosphatases. The aSMase is a soluble lysosomal sphingolipid hydrolase, which constitutively degrades sphingomyelin from internalized membrane fragments. Upon stimulation, however, a portion of this enzyme can be found on the outer side of the plasma membrane. This membrane-associated enzyme shows biochemical activity in serum and urine and has been termed secretory sphingomyelinase (sSMase), although it is virtually identical to the lysosomal variant. Its activity is elevated in several diseases. The secretory form of aSMase is believed to play an important role in signal transduction, since it alters the composition of the plasma membrane within putative sphingolipidand cholesterol-rich membrane microdomains. These so-called “lipid rafts” have been suggested to act as “signaling platforms”, and there is significant evidence that the cleavage of sphingomyelin to ceramide can dramatically alter the biophysical properties of the putative rafts. In addition, it is well established that ceramide is a potent inductor of apoptosis, which is the main reason for cell degeneration in many of the diseases mentioned above. However, it is unknown whether ceramide acts by remodeling the plasma membrane or by interacting with proteins like cathepsin B, which is involved in cellular signaling. Beside aSMase, two cytosolic, magnesium-dependent and membrane-bound neutral sphingomyelinases (nSMase1 and nSMase2) and an alkaline sphingomyelinase are known, whose cellular function is rather unclear. Recently nSMase has been shown to be essential for the formation of exosomes, lipid vesicles that play a key role in the infection by retroviruses. In contrast to aSMase, there are some potent small-molecule inhibitors for nSMase. Our attempts at synthesizing phosphonate analogues of PtdIns3,5P2 as potential inhibitors of aSMase yielded only moderately active substances. However, we also gained access to a collection of (bis)phosphonates that had been synthesized in the GDR Academy of Sciences and that contained some compounds that are structurally related to our phosphoinositide analogues. When we initially tested these substances at a concentration of 20 mm, we were surprised that some of them were potent inhibitors of aSMase (Tables 1 and 2). Among these substances, a-amino-

Iodine as an elemental marker for imaging of single cells and tissue sections by laser ablation inductively coupled plasma mass spectrometry

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.

Near-infrared-emitting nanoparticles for lifetime-based multiplexed analysis and imaging of living cells.

The increase in information content from bioassays and bioimaging requires robust and efficient strategies for the detection of multiple analytes or targets in a single measurement, thereby addressing current health and security concerns. For fluorescence techniques, an attractive alternative to commonly performed spectral or color multiplexing presents lifetime multiplexing and the discrimination between different fluorophores based on their fluorescence decay kinetics. This strategy relies on fluorescent labels with sufficiently different lifetimes that are excitable at the same wavelength and detectable within the same spectral window. Here, we report on lifetime multiplexing and discrimination with a set of nanometer-sized particles loaded with near-infrared emissive organic fluorophores chosen to display very similar absorption and emission spectra, yet different fluorescence decay kinetics in suspension. Furthermore, as a first proof-of-concept, we describe bioimaging studies with 3T3 fibroblasts and J774 macrophages, incubated with mixtures of these reporters employing fluorescence lifetime imaging microscopy. These proof-of-concept measurements underline the potential of fluorescent nanoparticle reporters in fluorescence lifetime multiplexing, barcoding, and imaging for cellular studies, cell-based assays, and molecular imaging.

Intracellular SERS hybrid probes using BSA-reporter conjugates.

AbstractSurface-enhanced Raman scattering (SERS) hybrid probes are characterized by the typical spectrum of a reporter molecule. In addition, they deliver information from their biological environment. Here, we report SERS hybrid probes generated by conjugating different reporter molecules to bovine serum albumin (BSA) and using gold nanoparticles as plasmonic core. Advantages of the BSA-conjugate hybrid nanoprobes over other SERS nanoprobes are a high biocompatibility, stabilization of the gold nanoparticles in the biological environment, stable reporter signals, and easy preparation. The coupling efficiencies of the BSA–reporter conjugates were determined by MALDI-TOF-MS. The conjugates’ characteristic SERS spectra differ from the spectra of unbound reporter molecules. This is a consequence of the covalent coupling, which leads to altered SERS enhancement and changes in the chemical structures of the reporter and of BSA. The application of the BSA–reporter conjugate hybrid probes in 3T3 cells, including duplex imaging, is demonstrated. Hierarchical cluster analysis and principal components analysis were applied for multivariate imaging using the SERS signatures of the incorporated SERS hybrid nanoprobes along with the spectral information from biomolecules in endosomal structures of cells. The results suggest more successful applications of the SERS hybrid probes in cellular imaging and other unordered high-density bioanalytical sensing. FigureSingle pixel spectrum obtained with SERS hybrid nanoprobes (here: BSA-AO conjugate on gold nanoparticles) inside living 3T3 cells. The distribution of SERS hybrid nanoprobes in 3T3 fibroblast cells can be obtained from chemical mapping, and from hierarchical cluster analysis (HCA) mapping employing the full spectral range from 300–1700 cm-1

Chemical Mapping of Leishmania Infection in Live Cells by SERS Microscopy

We report the direct probing of the molecular composition of Leishmania-infected macrophage cells in vitro by surface-enhanced Raman scattering (SERS). The microscopic mapping data indicate local abundance and distribution of molecular species that are very characteristic of the infection and that are observed here simultaneously. As revealed by electron microscopy, the gold nanoprobes used for SERS microspectrosopy have access to the parasitophorous vacuoles (PV) through the endosomal system. SERS nanoprobes located in the direct proximity to the parasite, in the greater volume of the PV, and in endolysosomal compartments in other cellular regions, respectively, report a characteristic chemical composition for each respective location. The data enable assessment of the distribution of ergosterol and cholesterol in the amastigote stage of the parasite and its immediate surroundings in the vacuole. Proteophosphoglycans of parasite origin, an important hallmark of the infection, are identified throughout the PV.