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Eingeladener Vortrag
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A systematic study of the luminescence properties of monodisperse β-NaYF4: 20% Yb3+, 2% Er3+ upconversion nanoparticles (UCNPs) with sizes ranging from 12–43 nm is presented utilizing steady-state and time-resolved fluorometry.
Special emphasis was dedicated to the absolute quantification of size- and environment-induced quenching of upconversion luminescence (UCL) by highenergy O–H and C–H vibrations from solvent and ligand molecules at different excitation power densities (P). In this context, the still-debated Population pathways of the 4F9/2 energy level of Er3+ were examined. Our results highlight the potential of particle size and P value for color tuning based on the pronounced near-infrared emission of 12 nm UCNPs, which outweighs the red Er3+ emission under “strongly quenched” conditions and accounts for over 50% of total UCL in water. Because current rate equation models do not include such emissions, the suitability of these models for accurately simulating all (de)population pathways of small UCNPs must be critically assessed. Furthermore, we postulate population pathways for the 4F9/2 energy level of Er3+, which correlate with the size-, environment-, and P-dependent quenching states of the higher Er3+ energy levels.
Nanomaterials are used in many different applications in the material and life sciences. Examples are optical reporters, barcodes, and nanosensors, magnetic and optical contrast agents, and catalysts. Due to their small size and large surface area, there are also concerns about their interaction with and uptake by biological systems. This has initiated an ever increasing number of cyctoxicity studies of nanomaterials of different chemical composition and surface chemistry, but until now, the toxicological results presented by different research groups often do not address or differ regarding a potential genotoxicity of these nanomaterials. This underlines the need for a standardized test procedure to detect genotoxicity.1,2
Aiming at the development of fast, easy to use, and automatable microscopic methods for the determination of the genotoxicity of different types of nanoparticles, we assess the potential of the fluorometric γH2AX assay for this purpose. This assay, which can be run on an automated microscopic detection system, relies on the determination of DNA double strand breaks as a sign for genotoxicity.3 Here, we present first results obtained with broadly used nanomaterials like CdSe/CdS and InP/ZnS quantum dots as well as iron oxide, gold, and polymer particles of different surface chemistry with previously tested colloidal stability. These studies will be also used to establish nanomaterials as positive and negative genotoxicity controls or standards for assay performance validation for users of this fluorometric genotoxicity assay. In the future, after proper validation, this microscopic platform technology will be expanded to other typical toxicity assays.
References. (1) Landsiedel, R.; Kapp, M. D.; Schulz, M.; Wiench, K.; Oesch, F., Reviews in Mutation Research 2009, 681, 241-258. (2) Henriksen-Lacey, M.; Carregal-Romero, S.; Liz-Marzán, L. M., Bioconjugate Chem. 2016, 28, 212-221. (3) Willitzki, A.; Lorenz, S.; Hiemann, R.; Guttek, K.; Goihl, A.; Hartig, R.; Conrad, K.; Feist, E.; Sack, U.; Schierack, P., Cytometry Part A 2013, 83, 1017-1026.
A systematic study of the luminescence properties of monodisperse β-NaYF4: 20% Yb3+, 2% Er3+ upconversion nanoparticles (UCNPs) with sizes ranging from 12–43 nm is presented utilizing steady-state and time-resolved fluorometry.
Special emphasis was dedicated to the absolute quantification of size- and environment-induced quenching of upconversion luminescence (UCL) by highenergy O–H and C–H vibrations from solvent and ligand molecules at different excitation power densities (P). In this context, the still-debated Population pathways of the 4F9/2 energy level of Er3+ were examined. Our results Highlight the potential of particle size and P value for color tuning based on the pronounced near-infrared emission of 12 nm UCNPs, which outweighs the red Er3+ emission under “strongly quenched” conditions and accounts for over 50% of total UCL in water. Because current rate equation models do not include such emissions, the suitability of these models for accurately simulating all (de)population pathways of small UCNPs must be critically assessed. Furthermore, we postulate population pathways for the 4F9/2 energy level of Er3+, which correlate with the size-, environment-, and P-dependent quenching states of the higher Er3+ energy levels.
Surface functionalization of nanomaterials is nowadays at the core of many applications of functional materials in the life and material sciences. Examples range from membranes and microarrays over bead-based assays, and next generation sequencing to nanometer-sized optical reporters, nanosensors, and magnetic and optical contrast agents. Typical function-nalization steps include silanization and grafting reactions with reactive monomers to introduce functional groups like amino or carboxylic acid groups or the attachment of ligands like polyethylene glycol (PEG) molecules and biomolecules. [1-3] This enables to tune e.g., dis-persibility, hydrophilicity and biocompatibility, minimize unspecific interactions, improve biofunctionalization efficiencies, and enhance blood circulation times and allows for the use of nanomaterials as reporters in assays or the design of targeted probes for bioimaging.
At the core of all functionalization strategies are reliable and validated methods for surface group and ligand quantification that can be preferably performed with routine laboratory instrumentation, require only small amounts of substances, and are suitable for many different types of nanomaterials. [3] There is meanwhile a considerable need to make these methods traceable. We present here versatile and simple concepts for the quantification of common functional groups, ligands, and biomolecules on different types of organic and inorganic nanomaterials, using conventional and newly developed cleavable and multimodal reporters, that can be detected with optical spectroscopy. [4-7] These reporters are chosen to enable method validation with the aid of method comparisons and mass balances. Also, strategies how to make these simple assays traceable to SI units using quantitative nuclear resonance spectroscopy (qNMR) and X-ray photoelectron spectroscopy (XPS) are derived.
Biophotonics and analytics - Photoluminescence properties of nanocrystals and surface group analysis
(2018)
Correlating the photoluminescence (PL) properties of nanomaterials like semiconductor nanocrystals (QDs) and upconversion nanocrystals (UCNPs) assessed in ensemble studies and at the single particle level and studying their surface chemistry is increasingly relevant for applications of these nanomaterials in the life and material sciences. Here we present a comparison of the spectroscopic properties of ensembles and single nanocrystalline emitters and simple methods for the quantification of functional groups and ligands on particle surfaces. The overall goal of this study was to derive particle architectures and surface chemistries well suited for spectroscopic and microscopic applications.
In this work, we describe a simple solvothermal route for the synthesis of Eu3+-doped gadolinium orthovanadate nanocrystals (Eu:GdVO4−PAA) functionalized with poly(acrylic)acid (PAA), that are applicable as cell labeling probes for multimodal cellular imaging. The Eu3+ doping of the vanadate matrix provides optical functionality, due to red photoluminescence after illumination with UV light. The Gd3+ ions of the nanocrystals reduce the T1 relaxation time of surrounding water protons, allowing these nanocrystals to act as a positive MRI contrast agent with a r1 relaxivity of 1.97 mM−1 s−1. Low background levels of Eu3+, Gd3+, and V5+ in biological systems make them an excellent label for elemental microscopy by Laser Ablation (LA)-ICP-MS. Synthesis resulted in polycrystalline nanocrystals with a hydrodynamic diameter of 55 nm and a crystal size of 36.7 nm, which were further characterized by X-ray diffraction (XRD), photoluminescence spectroscopy (PL) and transmission electron microscopy (TEM). The multifunctional nanocrystals were subsequently used for intracellular labeling of both human adipose-derived stem cells (MSCs) and A549 (adenocarcinomic human alveolar basal epithelial) cells.
Imaging of Eu doped very small iron oxide nanoparticle in atherosclerotic plaques via LA-ICP-MS
(2018)
Atherosclerotic lesions can be visualized by magnetic resonance imaging (MRI) using very small iron oxide nanoparticles (VSOP). VSOP accumulate in atherosclerotic plaques and thus serve as an atherosclerosis probe.
The aim of this project was to image the distribution of europium doped VSOP (Eu-VSOP) by laser ablation ICP-MS in histological thin cuts of the aortic root region of ApoE knockout mouse model that is rich in atherosclerotic plaques. In addition, it was investigated whether VSOP accumulation in the plaques correlates with other biomarkers of inflammation for example macrophages and altered endothelium to assess whether it correlates with instability or vulnerability of the plaque regions. For this purpose, antibodies were labeled with various lanthanide elements and correlated with the Eu-VSOP distribution using LA-ICP-MS in a multiplex measurement mode.
A possible correlation of reactive nitrogen species (RNS) with endogenous iron or Eu-VSOP can also be detected by LA-ICP-MS. For this purpose, RNS-specific antibodies were also labeled with lanthanides.
Imaging of elemental distributions in single cell assays as well as tissue sections can be performed by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). This powerful technique offers precise spatially resolved measurements at trace and ultratrace levels and has been established as an excellent tool to answer analytical, biological and biomedical questions. To date, mass cytometry is already able to simultaneously detect up to 40 cellular targets due to conjugation of isotopically pure lanthanides to affinity binders, e.g. antibodies.
To further enhance the ability of multiparametric analysis to more than 100 analytes at once, we investigated lanthanide nanocrystals as new, highly sensitive metal tags for identification of targets in clinical cell assays and tissue samples. Multiparametric analysis will be possible by encoding the lanthanide composition of nanocrystals associated to the affinity binders. Nanocrystals showed remarkable potential for sensitive detection in MS due to high stability and signal amplification compared to e.g. polymer tags, carrying fewer metal atoms. Furthermore, the nanocrystals allow multimodal imaging due fluorescence of Eu3+ as well as contrast enhancing properties of Gd3+ in magnetic resonance imaging.
Synthesis of functionalized lanthanide nanocrystals for further bioconjugation was performed with high reproducibility and monodisperse size distribution. For proof of principle, the uptake and distribution of these nanocrystals within the monolayered cell line A549 were investigated by mapping the intensities at subcellular resolution using LA-ICP-MS. It could be shown, that the cells were efficiently labeled with the nanocrystals. Additionally, the bioconjugation of the nanocrystals to antibodies and particularly the preservation of the antibody specificity was investigated using Dot Blot experiments. All in all, the results imply high sensitivity and the possibility of multiparametric analysis by doting various lanthanides into the nanocrystals.
Imaging of elemental distributions in single cell assays as well as tissue sections can be performed by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). This powerful technique offers precise spatially resolved measurements at trace and ultratrace levels and has been established as an excellent tool to answer analytical, biological and biomedical questions. To date, mass cytometry is already able to simultaneously detect up to 40 cellular targets due to conjugation of isotopically pure lanthanides to affinity binders, e.g. antibodies.
To further enhance the ability of multiparametric analysis to more than 100 analytes at once, we investigated lanthanide nanocrystals as new, highly sensitive metal tags for identification of targets in clinical cell assays and tissue samples. Multiparametric analysis will be possible by encoding the lanthanide composition of nanocrystals associated to the affinity binders. Nanocrystals showed remarkable potential for sensitive detection in MS due to high stability and signal amplification compared to e.g. polymer tags, carrying fewer metal atoms. Furthermore, the nanocrystals allow multimodal imaging due fluorescence of Eu3+ as well as contrast enhancing properties of Gd3+ in magnetic resonance imaging.
Synthesis of functionalized lanthanide nanocrystals for further bioconjugation was performed with high reproducibility and monodisperse size distribution. For proof of principle, the uptake and distribution of these nanocrystals within the monolayered cell line A549 were investigated by mapping the intensities at subcellular resolution using LA-ICP-MS. It could be shown, that the cells were efficiently labeled with the nanocrystals. Additionally, the bioconjugation of the nanocrystals to antibodies and particularly the preservation of the antibody specificity was investigated using Dot Blot experiments. All in all, the results imply high sensitivity and the possibility of multiparametric analysis by doting various lanthanides into the nanocrystals.
Nanoparticles (NPs) have found a wide range of applications in research and industry. Thereby the interaction of NPs with biological systems like cells has become a major field of interest, ranging from medical applications to nanotoxicology. Size, shape and surface modification of the nanomaterials determine the uptake rate and pathway into the cells, and therefore impact specific cell components and processes.
Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) is an established quantitative multi-elemental analysis and mapping technique. In recent years, it was shown that LA-ICP-MS can provide quantitative as well as distribution information of metal containing nanoparticles (NPs) in cell samples.
Here LA-ICP-MS was applied for the imaging of individual fibroblast cells to study the uptake and intracellular processing of NPs. Our results show that LA-ICP-MS can be used to localize nanoparticle aggregates within cellular compartments. The studied NPs accumulate in the perinuclear region in the course of intracellular processing, but do not enter the cell nucleus. The uptake efficiency depends strongly on the physico-chemical properties of the nanostructures as well as on the incubation conditions like concentration and incubation time.
ICP-MS was used to determine the composition of the nanomaterials as well as the number of NPs in cells after acid digestion of the samples.
Common approaches to improve the optical properties of semiconductor quantum dots and lanthanide doped nanophosphors present core/shell structures as radiationless deactivation at the particle surface is usually the main energy loss mechanism. This has led to increasingly sophisticated particle architectures using multishell systems with shells of different chemical composition and thickness and initiated an increasing number of quantitative spectroscopic studies focusing on the key performance parameter photoluminescence quantum yield to identify optimum particle structures. This is particularly challenging in the long wavelength region > 1000 nm and for nonlinear emitters like upconversion nanocrystals. Here, we present suitable absolute methods to quantify the photoluminescence of these different emitters in the vis/NIR/IR and as function of excitation power density and underline the impact of such measurements on a profound mechanistic understanding of the nonradiative deactivation pathways in semiconductor and upconversion nanocrystals of different size and particle architecture in different environments.
DNA origami nanostructures provide a platform where dye molecules can be arranged with nanoscale accuracy allowing to assemble multiple fluorophores without dye−dye aggregation. Aiming to develop a bright and sensitive ratiometric sensor system, we systematically studied the optical properties of nanoarrays of dyes built on DNA Origami platforms using a DNA template that provides a high versatility of label choice at minimum cost. The dyes are arranged at distances, at which they efficiently interact by Förster resonance energy transfer (FRET). To optimize array brightness, the FRET efficiencies between the donor fluorescein (FAM) and the acceptor cyanine 3 were determined for different sizes of the array and for different arrangements of the dye molecules within the array. By utilizing nanoarrays providing optimum FRET efficiency and brightness, we subsequently designed a ratiometric pH nanosensor using coumarin 343 as a pH-inert FRET donor and FAM as a pHresponsive acceptor. Our results indicate that the sensitivity of a ratiometric sensor can be improved simply by arranging the dyes into a well-defined array. The dyes used here can be easily replaced by other analyte-responsive dyes, demonstrating the huge potential of DNA nanotechnology for light harvesting, signal enhancement, and sensing schemes in life sciences.
Nanoparticles (NPs) have found a wide range of applications in research and industry. Thereby the interaction of NPs with biological systems like cells has become a major field of interest, ranging from medical applications to nanotoxicology. Size, shape and surface modification of the nanomaterials determine the uptake rate and pathway into the cells, and therefore impact specific cell components and processes.
In recent years, elemental imaging of biological samples using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) is gaining more and more importance. Improvements concerning both spatial resolution (down to 1 µm) and signal-to-background ratio due to low-dispersion LA chambers make LA-ICP-MS particularly interesting for single cell analysis.
Here LA-ICP-MS was applied for the imaging of individual cells to study the uptake and intracellular processing of metal-containing nanostructures. The cells were incubated with different NPs under varying experimental conditions and afterwards fixed with para-formaldehyde and dried for LA analysis. High-spatial resolution LA-ICP-MS was achieved by careful optimisation of the laser ablation parameters.
Our findings show, that LA-ICP-MS is applicable to localize NP aggregates within cellular compartments. The uptake efficiency depends strongly on the physicochemical properties of the nanostructures as well as on the incubation conditions like concentration and incubation time.
The results demonstrate the potential of LA-ICP-MS providing insight into nanoparticle-cell interaction dependent on experimental parameters.
Upconversion core/shell nanocrystals with different mean sizes ranging from 15 to 45 nm were prepared via a modified synthesis procedure based on anhydrous rare‐earth acetates. All particles consist of a core of NaYF4:Yb,Er, doped with 18 % Yb3+ and 2 % Er3+, and an inert shell of NaYF4, with the shell thickness being equal to the radius of the core particle. Absolute measurements of the photoluminescence quantum yield at a series of different excitation power densities show that the quantum yield of 45 nm core/shell particles is already very close to the quantum yield of microcrystalline upconversion phosphor powder. Smaller core/shell particles prepared by the same method show only a moderate decrease in quantum yield. The quantum yield of 15 nm core/shell particles, for instance, is reduced by a factor of three compared to the bulk upconversion phosphor at high power densities (100 W cm−2) and by approximately a factor of 10 at low power densities (1 W cm−2).
The analytical potential of a nanosecond laser ablation inductively coupled plasma mass spectrometer (ns-LA-ICP-SFMS) system, equipped with an ultra-fast wash-out ablation chamber, is critically investigated for fast and highly spatially resolved (∼μm) qualitative elemental distribution within single cells. Initially, a low surface roughness (< 10 nm) thin In–SnO2 layer (total coating thickness ∼200 nm) deposited on glass is employed to investigate the size, morphology and overlapping of laser-induced craters obtained at different laser repetition rates, making use of Atomic Force Microscopy (AFM). Conical craters with a surface diameter of about 2 µm and depths of about 100 nm were measured after a single laser shot. Furthermore, the influence of the sampling distance (i.e. distance between the sample surface and the inner sniffer of the ablation chamber) on the LA-ICP-MS ion signal wash-out time is evaluated. A significant decrease of the transient 120Sn+ ion signal is noticed after slight variations (±200 μm) around the optimum sampling position. Ultra-fast wash-outs (< 10 ms) are achieved reducing the aerosol mixing from consecutive laser shots even when operating the laser at high repetition rates (25 – 100 Hz). Fast and highly spatially resolved images of elemental distribution within mouse embryonic fibroblast cells (NIH/3T3 fibroblast cells) and human cervical carcinoma cells (HeLa cells), incubated with gold nanoparticles (Au NPs) and Cd-based quantum dots (QDs), respectively, are determined at the optimized operating conditions. Elemental distribution of Au and Cd in single cells is achieved using a high scanning speed (50 µm/s) and high repetition rate (100 Hz). The results obtained for the distribution of fluorescent Cd-based QDs within the HeLa cells are in good agreement with those obtained by confocal microscopy. The size, morphology and overlapping of laser-induced craters in the fixed cells are also investigated using AFM, observing conical craters with a surface diameter of about 2.5 µm and depths of about 800 nm after a single laser shot.
We synthesized and characterized a set of ultrasmall hexagonal-phase NaGdF4: 20% Yb3+, 2% Er3+ upconversion nanoparticles with core diameters of 3.7 ± 0.5 nm. In order to assess passivation effects and the influence of possible core−shell intermixing and to identify optimum particle structures for combined imaging in the visible and near-infrared (vis−NIR: 410−850 nm) and short-wave infrared (SWIR: 1520 nm), NaYF4 shells of varying thicknesses (monolayer to 10 nm) were introduced and the influence of this parameter on the upconversion and downshifting photoluminescence of these particles was studied at different excitation power densities. This included excitation power-dependent emission spectra, slope factors, quantum yields, and excited state decay kinetics. These measurements revealed enhancement factors of the upconversion quantum yield of >10 000 in the low power region and an excitation power density-independent quantum yield of the downshifted emission at 1520 nm between 0.1 and 14%. The optimized shell thickness for combined vis and SWIR imaging was identified as 5 nm. Moreover, lifetimes and quantum yields can be continuously tuned by shell thickness which can be exploited for lifetime multiplexing and encoding. The fact that we did not observe a saturation of the upconversion quantum yield or the excited state decay kinetics with increasing shell thickness is ascribed to a strong intermixing of the active core with the inert shell during the shelling procedure. This indicates the potential of spectroscopic tools to detect cation intermixing.
Imaging of Eu doped very small iron oxide nanoparticle in atherosclerotic plaques via LA ICP-MS
(2018)
Atherosclerotic lesions can be visualized by magnetic resonance imaging (MRI) using very small iron oxide nanoparticles (VSOP). VSOP accumulate in atherosclerotic plaques and thus serve as an atherosclerosis probe.
The aim of this project was to image the distribution of europium doped VSOP (Eu-VSOP) by laser ablation ICP-MS in histological thin cuts of the aortic root region of ApoE knockout mouse model that is rich in atherosclerotic plaques. In addition, it was investigated whether VSOP accumulation in the plaques correlates with other biomarkers of inflammation for example macrophages and altered endothelium to assess whether it correlates with instability or vulnerability of the plaque regions. For this purpose, antibodies were labeled with various lanthanide elements and correlated with the Eu-VSOP distribution using LA-ICP-MS in a multiplex measurement mode.
A possible correlation of reactive oxygen species (ROS) with endogenous iron or Eu-VSOP can also be detected by LA-ICP-MS. For this purpose, ROS-specific antibodies were also labeled with lanthanides.
Imaging of Eu doped very small iron oxide nanoparticle in atherosclerotic plaques via LA-ICP-MS
(2018)
Atherosclerotic lesions can be visualized by magnetic resonance imaging (MRI) using very small iron oxide nanoparticles (VSOP). VSOP accumulate in atherosclerotic plaques and thus serve as an atherosclerosis probe. The aim of this project was to image the distribution of europium doped VSOP (Eu-VSOP) by laser ablation ICP-MS in histological thin cuts of the aortic root region of ApoE knockout mouse model that is rich in atherosclerotic plaques. In addition, it was investigated whether VSOP accumulation in the plaques correlates with other biomarkers of inflammation for example macrophages and altered endothelium to assess whether it correlates with instability or vulnerability of the plaque regions. For this purpose, antibodies were labeled with various lanthanide elements and correlated with the Eu-VSOP distribution using LA-ICP-MS in a multiplex measurement mode.
The interaction of nanoparticles (NPs) with cells has become a major field of interest, ranging from medical applications to nanotoxicology. Size, shape and surface modification of the NPs determine the uptake rate and pathway into the cells, and therefore impact specific cell components and processes.
Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) is an established quantitative multi-elemental analysis and mapping technique. In recent years, it was shown that LA-ICP-MS can provide quantitative as well as distribution information of NPs in cell samples.
Here LA-ICP-MS was applied for the imaging of individual cells to study the uptake and intracellular processing of metal-containing nanostructures. Additionally, the local distribution of naturally occurring elements in cells like P was measured to indicate cell morphology. The cells were incubated with different types of NPs under varying experimental conditions. For LA analysis, the cells were fixed and dried.
Our findings show, that LA-ICP-MS is suitable for the localisation of nanoparticle aggregates within cellular compartments. The studied NPs accumulate in the perinuclear region in the course of intracellular processing, but do not enter the cell nucleus. The uptake efficiency depends strongly on the physicochemical properties of the nanostructures as well as on the incubation conditions like concentration and incubation time.
The results demonstrate the potential of LA-ICP-MS providing insight into NP uptake, intracellular distribution and cell-to-cell variation dependent on experimental parameters.
Nanoparticles (NPs) have found a wide range of applications in research and industry. Thereby the interaction of NPs with biological systems like cells has become a major field of interest, ranging from medical applications to nanotoxicology. Size, shape and surface modification of the nanomaterials determine the uptake rate and pathway into the cells, and therefore impact specific cell components and processes.
Inductively coupled plasma mass spectrometry (ICP-MS) is a well-established analytical method offering high sensitivity and multi-element capability. By coupling a laser ablation (LA) system to an ICP-MS the analysis of different kinds of solid samples is possible. In recent years, it was shown that LA-ICP-MS can provide quantitative as well as distribution information of metal containing nanoparticles (NPs) in cell samples.
Here LA-ICP-MS was applied for the imaging of individual fibroblast cells to study the uptake and intracellular processing of NPs. Our results show that LA-ICP-MS can be used to localize nanoparticle aggregates within cellular compartments. The studied NPs accumulate in the perinuclear region in the course of intracellular processing, but do not enter the cell nucleus. The uptake efficiency depends strongly on the physico-chemical properties of the nanostructures as well as on the incubation conditions like concentration and incubation time.
ICP-MS was used to determine the composition of the nanomaterials as well as the number of NPs in cells after acid digestion of the samples.