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Eingeladener Vortrag
- nein (22)
Objectives: Using a murine model of multiple sclerosis, we previously showed that repeated administration of gadopentetate dimeglumine led to retention of gadolinium (Gd) within cerebellar structures and that this process was enhanced with inflammation. This study aimed to compare the kinetics and retention profiles of Gd in inflamed and healthy brains after application of the macrocyclic Gd-based contrast agent (GBCA) gadobutrol or the linear GBCA gadopentetate. Moreover, potential Gd-induced neurotoxicity was investigated in living hippocampal slices ex vivo.
Materials and Methods: Mice at peak of experimental autoimmune encephalomyelitis (EAE; n = 29) and healthy control mice (HC; n = 24) were exposed to a cumulative dose of 20 mmol/kg bodyweight of either gadopentetate dimeglumine or gadobutrol (8 injections of 2.5 mmol/kg over 10 days). Magnetic resonance imaging (7 T) was performed at baseline as well as at day 1, 10, and 40 post final injection (pfi) of GBCAs. Mice were sacrificed after magnetic resonance imaging and brain and blood Gd content was assessed by laser ablation-inductively coupled plasma (ICP)-mass spectrometry (MS) and ICP-MS, respectively. In addition, using chronic organotypic hippocampal slice cultures, Gd-induced neurotoxicity was addressed in living brain tissue ex vivo, both under control or inflammatory (tumor necrosis factor α [TNF-α] at 50 ng/μL) conditions.
Results: Neuroinflammation promoted a significant decrease in T1 relaxation times after multiple injections of both GBCAs as shown by quantitative T1 mapping of EAE brains compared with HC. This corresponded to higher Gd retention within the EAE brains at 1, 10, and 40 days pfi as determined by laser ablation-ICP-MS. In inflamed cerebellum, in particular in the deep cerebellar nuclei (CN), elevated Gd retention was observed until day 40 after last gadopentetate application (CN: EAE vs HC, 55.06 ± 0.16 μM vs 30.44 ± 4.43 μM). In contrast, gadobutrol application led to a rather diffuse Gd content in the inflamed brains, which strongly diminished until day 40 (CN: EAE vs HC, 0.38 ± 0.08 μM vs 0.17 ± 0.03 μM). The analysis of cytotoxic effects of both GBCAs using living brain tissue revealed an elevated cell death rate after incubation with gadopentetate but not gadobutrol at 50 mM. The cytotoxic effect due to gadopentetate increased in the presence of the inflammatory mediator TNF-α (with vs without TNF-α, 3.15% ± 1.18% vs 2.17% ± 1.14%; P = 0.0345).
Conclusions: In the EAE model, neuroinflammation promoted increased Gd retention in the brain for both GBCAs. Whereas in the inflamed brains, efficient clearance of macrocyclic gadobutrol during the investigated time period was observed, the Gd retention after application of linear gadopentetate persisted over the entire observational period. Gadopentetate but not gadubutrol appeared to be neurotoxic in an ex vivo paradigm of neuronal inflammation.
We have efficiently produced collagen-rich microstructures in fibroblast multicellular spheroids (MCSs) as a three-dimensional in vitro tissue analog to investigate silver (Ag) nanoparticle (NP) penetration. The MCS production was examined by changing the seeding cell number (500 to 40,000 cells) and the growth period (1 to 10 days). MCSs were incubated with Ag NP suspensions with a concentration of 5 μg/mL for 24 h. For this study, laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) was used to visualize Ag NP localization quantitatively. Thin sections of MCSs were analyzed by LA-ICP-MS with a laser spot size of 8 μm to image distributions of 109Ag, 31P, 63Cu, 66Zn, and 79Br. A calibration using a NP suspension was applied to convert the measured Ag intensity into the number of NPs present. The determined numbers of NPs ranged from 30 to 7200 particles in an outer rim of MCS. The particle distribution was clearly correlated with the presence of 31P and 66Zn and was localized in the outer rim of proliferating cells with a width that was equal to about twice the diameter of single cells. Moreover, abundant collagens were found in the outer rim of MCSs. For only the highest seeding cell number, NPs were completely captured at the outer rim, in a natural barrier reducing particle transport, whereas Eosin (79Br) used as a probe of small molecules penetrated into the core of MCSs already after 1 min of exposure.
We applied high resolution laser ablation inductively coupled plasma time-of-flight mass spectrometry (LA-ICP-TOF-MS) with cellular spatial resolution for bioimaging of nanoparticles uptaken by fibroblast multicellular spheroids (MCS). This was used to quantitatively investigate interactions of silver nanoparticles (Ag NPs) and the distributions of intrinsic minerals and biologically relevant elements within thin sections of a fibroblast MCS as a three-dimensional in vitro tissue model. We designed matrix-matched calibration standards for this purpose and printed them using a noncontact piezo-driven array spotter with a Ag NP suspension and multielement standards. The limits of detection for Ag, Mg, P, K, Mn, Fe, Co, Cu, and Zn were at the femtogram (fg) level, which is sufficient to investigate intrinsic minerals in thin MCS sections (20 μm thick). After incubation for 48 h, Ag NPs were enriched in the outer rim of the MCS but not detected in the core. The localization of Ag NPs was inhomogeneous in the outer rim, and they were colocalized with a single-cell-like structure visualized by Fe distribution (pixel size of elemental images: 5 × 0.5 μm). The quantitative value for the total mass of Ag NPs in a thin section by the present method agreed with that obtained by ICP-sector field (SF)-MS with a liquid mode after acid digestion.
We investigated the penetration of silver nanoparticles (Ag NPs) into a three-dimensional in vitro tissue analog using NPs with various sizes and surface coatings, and with different incubation times. A high-Resolution laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) time-of-flight (TOF) instrument was applied for imaging the distributions of elements in thin sample sections (20 μm thick). A fibroblast multicellular spheroid (MCS) was selected as the model system and cultured for more than 8 days to produce a natural barrier formed by the extracellular matrix containing collagen. The MCS was then exposed for up to 48 h to one of four types of Ag NPs (∅ 5 nm citrate coated, ∅ 20 nm citrate coated, ∅ 20 nm polyvinylpyrrolidone coated, and ∅ 50 nm citrate coated). Imaging showed that the penetration pathway was strongly related to steric networks formed by collagen fibrils, and Ag NPs with a hydrodynamic diameter of more than 41 nm were completely trapped in an outer rim of the MCSs even after incubation for 48 h. In addition, we examined the impact of these NPs on essential elements (P, Fe, Cu, and Zn) in
areas of Ag NP accumulation. We observed a linear increase at the sub-femtogram level in the total concentration of Cu (fg per pixel) in samples treated with small or large Ag NPs (∅ 5 nm or ∅ 50 nm) for 48 h.
Nearly all disease processes are associated with variations of components of the extracellular matrix (ECM) that are typically observed during the development of inflammation. This concerns for example proteoglycans and their associated glycosaminoglycans (GAG), which have been shown to bind to cationic metal imaging probes due to their strong complexing activity. The complexing activity largely depends on the degree of GAG sulfation and/or carboxylation as well as on the GAG isomericity. In this central project, we investigate GAG structures from inflammatory disorders (namely cardiovascular diseases, inflammatory intestinal diseases and neuroinflammation) provided by researchers of the Collaborative Research Center at the molecular disaccharidic level using chromatographic and mass spectrometric methods. In parallel, the spatial localization and quantification of metal-based imaging probes are evaluated by LA-ICP-MS imaging.
Imaging Keratan Sulfate in Ocular Tissue Sections by Immunofluorescence Microscopy and LA-ICP-MS
(2022)
Carbohydrate-specific antibodies can serve as valuable tools to monitor alterations in the extracellular matrix resulting from pathologies. Here, the keratan sulfate-specific monoclonal antibody MZ15 was characterized in more detail by immunofluorescence microscopy as well as laser ablation ICP-MS using tissue cryosections and paraffin-embedded samples. Pretreatment with keratanase II prevented staining of samples and therefore demonstrated efficient enzymatic keratan sulfate degradation. Random fluorescent labeling and site-directed introduction of a metal cage into MZ15 were successful and allowed for a highly sensitive detection of the keratan sulfate landscape in the corneal stroma from rats and human tissue.
Multifunctional composite nanoprobes consisting of iron oxide nanoparticles linked to silver and gold nanoparticles, Ag–Magnetite and Au–Magnetite, respectively, were introduced by endocytic uptake into cultured fibroblast cells. The cells containing the non-toxic nanoprobes were shown to be displaceable in an external magnetic field and can be manipulated in microfluidic channels. The distribution of the composite nanostructures that are contained in the endosomal system is discussed on the basis of surfaceenhanced Raman scattering (SERS) mapping, quantitative laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) micromapping, and cryo soft X-ray tomography (cryo soft-XRT). Cryo soft-XRT of intact, vitrified cells reveals that the composite nanoprobes form intra-endosomal aggregates. The nanoprobes provide SERS signals from the biomolecular composition of their surface in the endosomal environment. The SERS data indicate the high stability of the nanoprobes and of their plasmonic properties in the harsh environment of endosomes and lysosomes. The spectra point at the molecular composition at the surface of the Ag–Magnetite and Au–Magnetite nanostructures that is very similar to that of other Composite structures, but different from the composition of pure silver and gold SERS nanoprobes used for intracellular investigations. As shown by the LA-ICP-MS data, the uptake efficiency of the magnetite composites is approximately two to three times higher than that of the pure gold and silver nanoparticles.
The cellular response to nanoparticle exposure is essential in various contexts, especially in nanotoxicity and nanomedicine. Here, 14-nm gold nanoparticles in 3T3 fibroblast cells are investigated in a series of pulse-chase experiments with a 30-min incubation pulse and chase times ranging from 15 min to 48 h. The gold nanoparticles and their aggregates are quantified inside the cellular ultrastructure by laser ablation inductively coupled plasma mass spectrometry micromapping and evaluated regarding the surface-enhanced Raman scattering (SERS) signals. In this way, both information about their localization at the micrometre scale and their molecular nanoenvironment, respectively, is obtained and can be related. Thus, the nanoparticle pathway from endocytotic uptake, intracellular processing, to cell division can be followed. It is shown that the ability of the intracellular nanoparticles and their accumulations and aggregates to support high SERS signals is neither directly related to nanoparticle amount nor to high local nanoparticle densities. The SERS data indicate that aggregate geometry and interparticle distances in the cell must change in the course of endosomal maturation and play a critical role for a specific gold nanoparticle type in order to act as efficient SERS nanoprobe. This finding is supported by TEM images, showing only a minor portion of aggregates that present small interparticle spacing. The SERS spectra obtained after different chase times show a changing composition and/or structure of the biomolecule corona of the gold nanoparticles as a consequence of endosomal processing.
An immunohistochemical method is described to visualize the distribution of metallothioneins 1/2 (MT 1/2) and metallothionein 3 (MT 3) in human ocular tissue. It is making use of (a) antibodies conjugated to gold nanoclusters (AuNCs) acting as labels, and (b) laser ablation (LA) coupled to inductively coupled plasma – mass spectrometry (ICP-MS). Water-soluble fluorescent AuNCs (with an average size of 2.7 nm) were synthesized and then conjugated to antibody by carbodiimide coupling. The surface of the modified AuNCs was then blocked with hydroxylamine to avoid nonspecific interactions with biological tissue. Immunoassays for MT 1/2 and MT 3 in ocular tissue sections (5 μm thick) from two post mortem human donors were performed. Imaging studies were then performed by fluorescence using confocal microscopy, and LA-ICP-MS was performed in the retina to measure the signal for gold. Signal amplification by the >500 gold atoms in each nanocluster allowed the antigens (MT 1/2 and MT 3) to be imaged by LA-ICP-MS using a laser spot size as small as 4 μm. The image patterns found in retina are in good agreement with those obtained by conventional fluorescence immunohistochemistry which was used as an established reference method.
An immunohistochemical method is described to visualize the distribution of metallothioneins 1/2 (MT 1/2) and metallothionein 3 (MT 3) in human ocular tissue. It is making use of (a) antibodies conjugated to gold nanoclusters (AuNCs) acting as labels, and (b) laser ablation (LA) coupled to inductively coupled plasma – mass spectrometry (ICP-MS).Water-soluble fluorescent AuNCs (with an average size of 2.7 nm) were synthesized and then conjugated to antibody by carbodiimide coupling. The surface of the modified AuNCs was then blocked with hydroxylamine to avoid nonspecific interactions with biological tissue. Immunoassays for MT 1/2 and MT 3 in ocular tissue sections (5 μm thick) from two post mortem human donors were performed. Imaging studies were then performed by fluorescence using confocal microscopy, and LA-ICP-MS was performed in the retina to measure the signal for gold. Signal amplification by the >500 gold atoms in each nanocluster allowed the antigens (MT 1/2 and MT 3) to be imaged by LA-ICP-MS using a laser spot size as small as 4 μm. The image patterns found in retina are in good agreement with those obtained by conventional fluorescence immunohistochemistry which was used as an established reference method.
Directing nanoparticles to the nucleus by attachment of nuclear localization sequences (NLS) is an aim in many applications. Gold nanoparticles modified with two different NLS were studied while crossing barriers of intact cells, including uptake, endosomal escape, and nuclear translocation. By imaging of the nanoparticles and by characterization of their molecular interactions with surface-enhanced Raman scattering (SERS), it is shown that nuclear translocation strongly depends on the particular incubation conditions. After an 1 h of incubation followed by a 24 h chase time, 14 nm gold particles carrying an adenoviral NLS are localized in endosomes, in the cytoplasm, and in the nucleus of fibroblast cells. In contrast, the cells display no nanoparticles in the cytoplasm or nucleus when continuously incubated with the nanoparticles for 24 h. The ultrastructural and spectroscopic data indicate different processing of NLS-functionalized particles in endosomes compared to unmodified particles. NLS functionalized nanoparticles form larger intraendosomal aggregates than unmodified gold nanoparticles. SERS spectra of cells with NLS-functionalized gold nanoparticles contain bands assigned to DNA and were clearly different from those with unmodified gold nanoparticles. The different processing in the presence of an NLS is influenced by a continuous exposure of the cells to nanoparticles and an ongoing nanoparticle uptake. This is supported by mass-spectrometry-based quantification that indicates enhanced uptake of NLS-functionalized nanoparticles compared to unmodified particles under the same conditions. The results contribute to the optimization of nanoparticle analysis in cells in a variety of applications, e.g., in theranostics, biotechnology, and bioanalytics.
Gold nanostructures that serve as probes for nanospectroscopic analysis of eukaryotic cell cultures can be obtained by the in situ reduction of tetrachloroauric acid (HAuCl4). To understand the formation process of such intracellularly grown particles depending on the incubation medium, the reaction was carried out with 3T3 fibroblast cells in three different incubation media, phosphate buffer, Dulbecco's Modified Eagle Medium (DMEM), and standard cell culture medium (DMEM with fetal calf serum). The size, the optical properties, the biomolecular corona, and the localization of the gold nanoparticles formed in situ vary for the different conditions. The combination of surface-enhanced Raman scattering (SERS) and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) microscopic mapping and transmission electron microscopy (TEM) provides complementary perspectives on plasmonic nanoparticles and non-plasmonic gold compounds inside the cells. While for the incubation with HAuCl4 in PBS, gold particles provide optical signals from the nucleus, the incubation in standard cell culture medium leads to scavenging of the toxic molecules and the formation of spots of high gold concentration in the cytoplasm without formation of SERS-active particles inside the cells. The biomolecular corona of nanoparticles formed in situ after incubation in buffer and DMEM differs, suggesting that different intracellular molecular species serve for reduction and stabilization. Comparison with data obtained from ready-made gold nanoparticles suggests complementary application of in situ and ex situ generated nanostructures for optical probing.
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.
By adding a gold core to silica nanoparticles (BrightSilica), silica-like nanoparticles are generated that, unlike unmodified silica nanoparticles, provide three types of complementary information to investigate the silica nano-biointeraction inside eukaryotic cells in situ. Firstly, organic molecules in proximity of and penetrating into the silica shell in live cells are monitored by surface-enhanced Raman scattering (SERS). The SERS data show interaction of the hybrid silica particles with tyrosine, cysteine and phenylalanine side chains of adsorbed proteins. Composition of the biomolecular corona of BrightSilica nanoparticles differs in fibroblast and macrophage cells. Secondly, quantification of the BrightSilica nanoparticles using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) micromapping indicates a different interaction of silica nanoparticles compared to gold nanoparticles under the same experimental conditions. Thirdly, the metal cores allow the investigation of particle distribution and interaction in the cellular ultrastructure by cryo nanoscale X-ray tomography (cryo-XT). In 3D reconstructions the assumption is confirmed that BrightSilica nanoparticles enter cells by an endocytotic mechanism. The high SERS intensities are explained by the beneficial plasmonic properties due to agglomeration of BrightSilica. The results have implications for the development of multi-modal qualitative and quantitative characterization in comparative nanotoxicology and bionanotechnology.
This work demonstrates the sub-cellular resolution bio-imaging capabilities of the NWRimage system with Dual Concentric Injector (DCI) technology in a practical application. 3T3 fibroblast cells incubated with gold nanoparticles were imaged, demonstrating the sub-cellular imaging capabilities. A laser ablation system specifically designed for rapid, high resolution imaging was employed. The system´s optical layout is optimized to minimize crater diameters. Sensitivity and speed were achieved using the DCI technology for ultra-fast washout times.
The traceability and availability of nanoparticles enables their use to enhance a variety of nano-biological and nano-medicinal applications. The particular size and shape of nanoparticles determine the uptake rate and pathway into the cell, and therefore impact specific cell components and processes. Selecting specific particle types allows researchers to target the process or structure of interest, with minimal additional impact. This can be used for drug or DNA delivery, and is being explored for use in oncology. Understanding the different uptake mechanisms and impacted processes requires sub-cellular Imaging resolution to determine, for example, whether or not the nanoparticles are reaching the nucleus.
Sub-cellular imaging has traditionally been challenging to achieve with laser ablation ICP-MS due to a lack of sensitivity at small spots. Bioimaging using LA-ICP-MS is a well-established technique, but usually applied on the tissue scale, which depends on larger spot areas where sensitivity is less problematic. The improved sensitivity and washout from the NWRimage has allowed faster imaging of smaller spots. The NWRimage also provides the possibility of true sub-micron spot sizes. This work compares the capabilities of standard laser Ablation (NWR213 system) with results from the NWRimage platform, which has been optimized for imaging applications.
Inflammatory bowel diseases (IBD) comprise mainly ulcerative colitis (UC) and Crohn´s disease (CD). Both forms present with a chronic inflammation of the (gastro) intestinal tract, which induces excessive changes in the composition of the associated extracellular matrix (ECM). In UC, the inflammation is limited to the colon, whereas it can occur throughout the entire gastrointestinal tract in CD. Tools for early diagnosis of IBD are still very limited and highly invasive and measures for standardized evaluation of structural changes are scarce. To investigate an efficient non-invasive way of diagnosing intestinal inflammation and early changes of the ECM, very small superparamagnetic iron oxide nanoparticles (VSOPs) in magnetic resonance imaging (MRI) were applied in two mouse models of experimental colitis: the dextran sulfate sodium (DSS)-induced colitis and the transfer model of colitis. For further validation of ECM changes and inflammation, tissue sections were analyzed by immunohistochemistry. For in depth ex-vivo investigation of VSOPs localization within the tissue, Europium-doped VSOPs served to visualize the contrast agent by imaging mass cytometry (IMC). VSOPs accumulation in the inflamed colon wall of DSS-induced colitis mice was visualized in T2* weighted MRI scans. Components of the ECM, especially the hyaluronic acid content, were found to influence VSOPs binding. Using IMC, colocalization of VSOPs with macrophages and endothelial cells in colon tissue was shown. In contrast to the DSS model, colonic inflammation could not be visualized with VSOP-enhanced MRI in transfer colitis. VSOPs present a potential contrast agent for contrast-enhanced MRI to detect intestinal inflammation in mice at an early stage and in a less invasive manner depending on hyaluronic acid content.
ICP-MS is a well-established analytical method which excels by high accuracy, high dynamic range and extremely low limits of detection for most metals. Furthermore ICP-MS offers a very high multi-element coverage so that many elements of the periodic table can be detected simultaneously. In this series of lectures, we want to focus on the historical developments, fundamentals, instrumentation and novel applications of ICP-MS in the life and material sciences.
ICP-MS is a well-established analytical method which excels by high accuracy, high dynamic range and extremely low limits of detection for most metals. Furthermore ICP-MS offers a very high multi-element coverage so that many elements of the periodic table can be detected simultaneously. In this series of lectures, we want to focus on the historical developments, fundamentals, instrumentation and novel applications of ICP-MS in the life and material sciences.