Ensuring the safety and sustainability of advanced materials (AdMas) is critical for fostering innovation while protecting human health and the environment. As industries integrate AdMas into commercial products to innovate in the next stage of the value chains, there is an urgent need for robust methodologies to detect, characterize, and assess their potential risks throughout their life cycle. The MACRAMÉ Project addresses this challenge by advancing standardized testing and regulatory frameworks, supporting the EU's vision for a toxic-free environment. Through cutting-edge research and international collaboration, MACRAMÉ lays the groundwork for reliable hazard assessment, regulatory compliance, and the responsible development of next-generation materials. The MACRAMÉ Project aims to enhance the detection, characterization, and quantification of Advanced Materials (AdMas) throughout their life cycle, assessing potential human and environmental health impacts during exposure. By developing, demonstrating, and standardizing advanced methodologies, MACRAMÉ ensures their broad applicability across market-relevant AdMas-containing products. Fully aligned with EU strategies such as the Chemical Strategy for Sustainability and the European Green Deal, the project extends nanosafety approaches to the broader AdMas category, focusing on inhalable carbon-based materials - graphene-related materials, carbon nanofibers, and poly lactic-co-glycolic acid nanoparticles. Building on over 15 years of research, MACRAMÉ integrates knowledge from major European and international initiatives to establish harmonized test guidelines, guidance documents, and standards. Through five industrial Use-Cases, the project applies innovative sample preparation, detection, and toxicity assessment techniques to develop a tiered approach for AdMa safety testing. Centralized in the MACRAMÉ Information Hub, all data will support regulatory frameworks and future research. The project's outcomes - harmonization and pre-standardization proposals - will contribute to a unified European assessment framework, reinforcing the continent's leadership in safe and sustainable materials innovation.

The detection of biomedical organic nanocarriers in cells and tissues is still an experimental challenge. Here we developed an imaging strategy for the label-free detection of poly (ethylbutyl cyanoacrylate) (PEBCA) particles. Experiments were carried out with phagocytic NR8383 macrophages exposed to non-toxic and non-activating concentrations of fluorescent (PEBCA NR668 and PEBCA NR668/IR), non-fluorescent (PEBCA), and cabazitaxel-loaded PEBCA particles (PEBCA CBZ). Exposure to PEBCA NR668 revealed an inhomogeneous particle uptake similar to what was obtained with the free modified Nile Red dye (NR668). In order to successfully identify the PEBCA-loaded cells under label-free conditions, we developed an imaging strategy based on enhanced darkfield microscopy (DFM), followed by confocal Raman microscopy (CRM) and time-of-flight secondary ion mass spectrometry (ToF–SIMS). Nitrile groups of the PEBCA matrix and PEBCA ions were used as suitable analytes for CRM and ToF–SIMS, respectively. Masses found with ToF–SIMS were further confirmed by Orbitrap-SIMS. The combined approach allowed to image small (< 1 µm) PEBCA-containing phagolysosomes, which were identified as PEBCA-containing compartments in NR8383 cells by electron microscopy. The combination of DFM, CRM, and ToF–SIMS is a promising strategy for the label-free detection of PEBCA particles. Graphical Abstract

Time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS) is a powerful tool for surface analysis, but fragmentation of molecular species during the SIMS process may lead to complex mass spectra. While the fragmentation pattern is typically characteristic for each compound, industrial samples are engineered materials, and, thus, may contain a mixture of many compounds, which may result in a variety of overlapping peak patterns in ToF‐SIMS spectra. Consequently, the process of data evaluation is challenging and time‐consuming. Principal component analysis (PCA) can be used to simplify data analysis for complex sample systems. Especially, correlation loadings were observed as an ideal tool to identify relevant signals in PCA results, which induce the separation of different sample groups. This is because correlation loadings show the relevance of signals independent from their intensity in the raw data. In correlation loadings, however, fragmentation patterns are no longer observed and the identification of peaks' sum formulas is challenging. In this study, a new approach is presented, which simplifies peak identification and assignment in ToF‐SIMS spectra after PCA is performed. The approach uses a mathematical transformation that projects PCA results, in particular loadings and correlation loadings, in the direction of specific sample groups. The approach does not change PCA results but rather presents them in a new way. This method allows to visualize characteristic spectra for specific sample groups that contain only relevant signals and, additionally, visualize fragmentation patterns. Data analysis is simplified and helps the user to focus on data interpretation rather than processing.

Amorphous silica nanoparticles comprise a class of widely used industrial nanomaterials, which may elicit acute inflammation in the lung. These materials have a large specific surface to which components of the pulmonary micro-milieu can bind. To conduct appropriate binding studies, paramagnetic Fe2O3/SiO2 core/shell nanoparticles (Fe-Si-NP) may be used as an easy-to-isolate silica surrogate, if several prerequisites are fulfilled. To this end, we investigated the distribution of Fe, Si, protein and phosphatidylcholine (PC) by Time-of-Flight secondary ion mass spectrometry (ToF-SIMS) in cryo-sections from the rat lungs to which Fe-Si-NP had been administered for 30 min. Regions-of-interest were identified and analyzed with incident light and enhanced dark-field microscopy (DFM). Fe-Si-NP particles (primary particle size by electron microscopy: 10–20 nm; aggregate size by tracking analysis: 190 ± 20 nm) and agglomerates thereof were mainly attached to alveolar walls and only marginally internalized by cells such as alveolar macrophages. The localization of Fe-Si-NP by DFM was confirmed by ToF-SIMS signals from both, Fe and Si ions. With respect to an optimized signal-to-noise ratio, Fe+, Si+, CH4N+ and the PC head group (C5H15NO4P+) were the most versatile ions to detect iron, silica, protein, and PC, respectively. Largely congruent Fe+ and Si+ signals demonstrated that the silica coating of Fe-Si-NP remained stable under the conditions of the lung. PC, as a major lipid of the pulmonary surfactant, was colocalized with the protein signal alongside alveolar septa, but was not detected on Fe-Si-NP, suggesting that silica nanoparticles do not adsorb lipids of the lung surfactant under native conditions. The study shows that ToF-SIMS is a valuable technique with adequate spatial resolution to analyze nanoparticles together with organic molecules in the lung. The paramagnetic Fe-Si-NP appear well suited to study the binding of proteins to silica nanomaterials in the lung.

The increasing use of nanoparticles (NP) in commercial products requires elaborated techniques to detect NP in the tissue of exposed organisms. However, due to the low amount of material, the detection and exact localization of NP within tissue sections is demanding. In this respect, Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) and Ion Beam Microscopy (IBM) are promising techniques, because they both offer sub-micron lateral resolutions along with high sensitivities. Here, we compare the performance of the non-material-consumptive IBM and material-consumptive ToF-SIMS for the detection of ZrO2 NP (primary size 9–10 nm) in rat lung tissue. Unfixed or methanol-fixed air-dried cryo-sections were subjected to IBM using proton beam scanning or to three-dimensional ToF-SIMS (3D ToF-SIMS) using either oxygen or argon gas cluster ion beams for complete sample sputtering. Some sample sites were analyzed first by IBM and subsequently by 3D ToF-SIMS, to compare results from exactly the same site. Both techniques revealed that ZrO2 NP particles occurred mostly agglomerated in phagocytic cells with only small quantities being associated to the lung epithelium, with Zr, S, and P colocalized within the same biological structures. However, while IBM provided quantitative information on element distribution, 3D ToF-SIMS delivered a higher lateral resolution and a lower limit of detection under these conditions. We, therefore, conclude that 3D ToF-SIMS, although not yet a quantitative technique, is a highly valuable tool for the detection of NP in biological tissue.

The description of nanoparticle distributions in tissue and associated effects is an important goal of nanotoxicology.

The direct detection of nanoparticles in tissues at high spatial resolution is a current goal in nanotoxicology. Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) is widely used for the direct detection of inorganic and organic substances with high spatial resolution but its capability to detect nanoparticles in tissue sections is still insufficiently explored. To estimate the applicability of this technique for nanotoxicological questions, comparative studies with established techniques on the detection of nanoparticles can offer additional insights. Here, we compare ToF-SIMS imaging data with sub-micrometer spatial resolution to fluorescence microscopy imaging data to explore the usefulness of ToF-SIMS for the detection of nanoparticles in tissues. SiO2 nanoparticles with a mean diameter of 25 nm, core-labelled with fluorescein isothiocyanate, were intratracheally instilled into rat lungs. Subsequently, imaging of lung cryosections was performed with ToF-SIMS and fluorescence microscopy. Nanoparticles were successfully detected with ToF-SIMS in 3D microanalysis mode based on the lateral distribution of SiO3- (m/z 75.96), which was co-localized with the distribution pattern that was obtained from nanoparticle fluorescence. In addition, the lateral distribution of protein (CN-, m/z 26.00) and phosphate based signals (PO3-, m/z 78.96) originating from the tissue material could be related to the SiO3- lateral distribution. In conclusion, ToF-SIMS is suitable to directly detect and laterally resolve SiO2 nanomaterials in biological tissue at sufficient intensity levels. At the same time, information about the chemical environment of the nanoparticles in the lung tissue sections is obtained.

A powerful tool for high resolution surface, interface, and thin-film analysis is the time-of-flight secondary ion mass spectrometry (ToF-SIMS). In principle, SIMS is a destructive technique since bombarding a biomaterial probe with high energetic primary cations leads to the desorption of secondary ions from the surface. This chapter outlines ToF-SIMS fundamentals and instrumentation, as well its manifold applications and perspectives, especially in the field of lung surfactant and biomembrane research. One of the grand advantages of the ToF-SIMS technique is the consolidation of chemical characterization and lateral imaging of heterogeneous sample surfaces. Lipids and also proteins can easily be analyzed in the bulk state or as dried films on a solid support to obtain ToF-SIMS mass spectra. ToF-SIMS therefore presents a key for understanding how biomolecules assemble and which parameters influence their localization in biological assemblies.

The present study describes the detection and localization of silica particles with diameters between 2 µm and 150 nm within the cytoplasm of mammalian cells by means of ToF‐SIMS 3D analysis. The particles were selected as model objects for non‐luminescent, unlabeled particles that are hard to localize by other experimental approaches. ToF‐SIMS analysis proved the uptake of the particles into the cell body, provided images of their distribution around the cell nucleus and indications that the cell membranes are undulated by the µm‐sized particles beneath the membrane. Two ion sources (Cs+ and O2+) were applied for sputtering the organic material and expose deeper sections of the cells. The resulting images are presented and compared. Copyright © 2012 John Wiley & Sons, Ltd.

In this work, we have quantified for the first time the fluorescence and singlet oxygen quantum yields of a silicon(IV) phthalocyanine bound to the surface of zeolite L nanocrystals. The photophysical properties were correlated with the absorption spectra and the morphology of the nanoparticles, and most importantly, with the fraction of photoactive chromophores. By comparison with the fluorescence and singlet oxygen quantum yields of the free phthalocyaninate in dilute solution (ΦF = 0.50 and Φ∆ = 0.50, respectively), we conclude that for the most efficient nanoparticles nearly 80% of chromophores are active as monomeric units on the surface, as indicated by the corresponding quantum yields (ΦF = 0.40 and Φ∆ = 0.40). We further functionalized and raised the ζ‐potential of the best performing nanomaterial to improve its water dispersibility. The functionalization was monitored by thermogravimetric analysis and time‐of‐flight secondary‐ion mass spectrometry, and its influence on the photophysical properties was assessed. The resulting nanomaterials are capable of establishing stable suspensions in water while retaining the ability to form reactive oxygen species upon irradiation with red light. This provides a basis for the rational design of photoactive nanomaterials for photodynamic therapy or water decontamination.

One of the main determinants of lung surfactant function is the complex interplay between its protein and lipid components. The lipid specificity of surfactant protein B (SP-B), however, and the protein's ability to selectively squeeze out lipids, has remained contradictory. In this work we present, for the first time to our knowledge, by means of time-of-flight secondary ion mass spectrometry chemical imaging, a direct evidence for colocalization of SP-B as well as its model peptide KL(4) with negatively charged dipalmitoylphosphatidylglycerol under absolute calcium free conditions. Our results prove that protein/lipid localization depends on the miscibility of all surfactant components, which itself is influenced by subphase ionic conditions. In contrast to our earlier studies reporting SP-B/KL(4) colocalization with zwitterionic dipalmitoylphosphatidylcholine, in the presence of even the smallest traces of calcium, we finally evidence an apparent reversal of protein/lipid mixing behavior upon calcium removal with ethylene diamine tetraacetic acid. In addition, scanning force microscopy measurements reveal that by depleting the subphase from calcium ions the protrusion formation ability of SP-B or KL(4) is not hampered. However, in the case of KL(4), distinct differences in protrusion morphology and height are visible. Our results support the idea that calcium ions act as a "miscibility switch" in surfactant model systems and probably are one of the major factors steering lipid/protein mixing behavior as well as influencing the protein's protrusion formation ability.

Studies of different fragments and mutants of SP-B suggest that the function related structural and compositional characteristics in SP-B are its positive charges with intermittent hydrophobic domains. KL4 ([lysine-(leucine)4]4-lysine) is a synthetic peptide based on SP-B structure and is the major constituent of Surfaxin®, a potential therapeutic agent for respiratory distress syndrome in premature infants. There is, however, no clear understanding about the possible lipid-KL4 interactions behind its function, which is an inevitable knowledge to design improved therapeutic agents. To examine the phase behavior, topography, and lipid specificity of KL4/lipid systems, we aimed to study different surfactant model systems containing KL4, neutral dipalmitoylphosphatidylcholine (DPPC) and/or negatively charged dipalmitoylphosphatidylglycerol (DPPG) in the presence of Ca2+ ions. Surface pressure-area isotherms, fluorescence microscopic images, scanning force microscopy as well as time-of-flight secondary ion mass spectrometry suggest (i) that KL4 is not miscible with DPPC and therefore forms peptide aggregates in DPPC/KL4 mixtures; (ii) that KL4 specifically interacts with DPPG via electrostatic interactions and induces percolation of DPPG-rich phases; (iii) that existing DPPG-Ca2+ interactions are too strong to be overcome by KL4, the reason why the peptide remains excluded from condensed DPPG domains and passively colocalizes with DPPC in a demixed fluid phase; and (iv) that the presence of negatively charged lipid is necessary for the formation of bilayer protrusions. These results indicate that the capability of the peptide to induce the formation of a defined surface-confined reservoir depends on the lipid environment, especially on the presence of anionic lipids.

Studies of different fragments and mutants of SP-B suggest that the function related structural and compositional characteristics in SP-B are its positive charges with intermittent hydrophobic domains. KL4 ([lysine-(leucine)4]4-lysine) is a synthetic peptide based on SP-B structure and is the major constituent of Surfaxin, a potential therapeutic agent for respiratory distress syndrome in premature infants. There is, however, no clear understanding about the possible lipid-KL4 interactions behind its function, which is an inevitable knowledge to design improved therapeutic agents. To examine the phase behavior, topography, and lipid specificity of KL4/lipid systems, we aimed to study different surfactant model systems containing KL4, neutral dipalmitoylphosphatidylcholine (DPPC) and/or negatively charged dipalmitoylphosphatidylglycerol (DPPG) in the presence of Ca2+ ions. Surface pressure-area isotherms, fluorescence microscopic images, scanning force microscopy as well as time-of-flight secondary ion mass spectrometry suggest (i) that KL4 is not miscible with DPPC and therefore forms peptide aggregates in DPPC/KL4 mixtures; (ii) that KL4 specifically interacts with DPPG via electrostatic interactions and induces percolation of DPPG-rich phases; (iii) that existing DPPG-Ca2+ interactions are too strong to be overcome by KL4, the reason why the peptide remains excluded from condensed DPPG domains and passively colocalizes with DPPC in a demixed fluid phase; and (iv) that the presence of negatively charged lipid is necessary for the formation of bilayer protrusions. These results indicate that the capability of the peptide to induce the formation of a defined surface-confined reservoir depends on the lipid environment, especially on the presence of anionic lipids.

Angewandte ChemieVolume 119, Issue 28 p. 5427-5431 Zuschrift Chemische Analyse tierischer Zellen und ihrer intrazellulären Kompartimente durch 3D-Massenspektrometrie† Daniel Breitenstein Dr., Daniel Breitenstein Dr. Tascon GmbH, Heisenbergstraße 15, 48149 Münster, DeutschlandSearch for more papers by this authorChristina E. Rommel Dr., Christina E. Rommel Dr. Institut für Biochemie, Westfälische Wilhelms-Universität Münster, Wilhelm-Klemm Straße 2, 48149 Münster, Deutschland, Fax: (+49) 251-833-3206Search for more papers by this authorRudolf Möllers Dr., Rudolf Möllers Dr. IonTof GmbH, Heisenbergstraße 15, 48149 Münster, DeutschlandSearch for more papers by this authorJoachim Wegener PD Dr., Joachim Wegener PD Dr. [email protected] Institut für Biochemie, Westfälische Wilhelms-Universität Münster, Wilhelm-Klemm Straße 2, 48149 Münster, Deutschland, Fax: (+49) 251-833-3206Search for more papers by this authorBirgit Hagenhoff Dr., Birgit Hagenhoff Dr. Tascon GmbH, Heisenbergstraße 15, 48149 Münster, DeutschlandSearch for more papers by this author Daniel Breitenstein Dr., Daniel Breitenstein Dr. Tascon GmbH, Heisenbergstraße 15, 48149 Münster, DeutschlandSearch for more papers by this authorChristina E. Rommel Dr., Christina E. Rommel Dr. Institut für Biochemie, Westfälische Wilhelms-Universität Münster, Wilhelm-Klemm Straße 2, 48149 Münster, Deutschland, Fax: (+49) 251-833-3206Search for more papers by this authorRudolf Möllers Dr., Rudolf Möllers Dr. IonTof GmbH, Heisenbergstraße 15, 48149 Münster, DeutschlandSearch for more papers by this authorJoachim Wegener PD Dr., Joachim Wegener PD Dr. [email protected] Institut für Biochemie, Westfälische Wilhelms-Universität Münster, Wilhelm-Klemm Straße 2, 48149 Münster, Deutschland, Fax: (+49) 251-833-3206Search for more papers by this authorBirgit Hagenhoff Dr., Birgit Hagenhoff Dr. Tascon GmbH, Heisenbergstraße 15, 48149 Münster, DeutschlandSearch for more papers by this author First published: 02 July 2007 https://doi.org/10.1002/ange.200604468Citations: 6 † Diese Arbeit wurde finanziell im Rahmen der Förderrichtlinie Nanobiotechnologie des BMBF (Nr. 0312002A) sowie durch das sechste Rahmenprogramm der EU (FP6-513698/Toxdrop & FP6-005045/Nanobiomaps) unterstützt. Wir danken M. Fartmann für die Durchsicht des Manuskriptes, S. Grunewald für ihre Unterstützung bei der Zellpräparation, R. Kersting und E. Tallarek für technische Ratschläge, E. Niehuis für hilfreiche Diskussionen und J. Zehnpfenning für die Software-Unterstützung. Read the full textAboutPDF ToolsRequest permissionAdd to favorites ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat Graphical Abstract Massenweise Informationen: Eine in ein ToF-Sekundärionen-Massenspektrometer integrierte C60+-Sputter-Quelle ermöglicht die schichtweise Abtragung tierischer Zellen und die ortsaufgelöste Analyse der chemischen Zusammensetzung der nach jedem Sputter-Zyklus freigelegten Probenoberfläche. Wiederholung der Sputter- und Analyse-Schritte liefert molekulare, markerfreie Informationen aus dem Zellinneren (siehe Bild; rot: Aminosäuren, grün: Phospholipide, blau: Substrat). References 1A. Benninghoven, F. G. Rüdenauer, H. W. Werner, Wiley, New York, 1987, S. 761. Google Scholar 2J. C. Vickerman in ToF-SIMS—Surface analysis by mass spectrometry (Hrsg.: ), IMPublications, Manchester/Chichester, 2001. Google Scholar 3F. Kollmer, Appl. Surf. Sci. 2004, 231–232, 153. 10.1016/j.apsusc.2004.03.101 CASWeb of Science®Google Scholar 4A. M. Belu, D. J. Graham, D. G. 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A peek inside: A C60+ sputter ion source integrated into a time-of-flight secondary-ion mass spectrometer was used to dissect animal cells in a layer-by-layer fashion and analyze the spatial distribution of endogenous molecules. Repeated cycles of sputter erosion and SIMS analysis provided molecular information from the inside of the cells (see picture; red: amino acids, green: phospholipids, blue: glass substrate).