Determination of selenium-containing species, including nanoparticles, in selenium-enriched Lingzhi mushrooms.

Mushrooms are considered a valuable food source due to their high protein and fibre and low fat content, among the other health benefits of their consumption. Selenium is an essential nutrient and is renowned for its chemo-preventative properties. In this study, batches of selenium-enriched Lingzhi mushrooms were prepared by growing mycelium and fruit in substrates containing various concentrations of sodium selenite. The mushroom fruit accumulated low levels of selenium with selenomethionine being the most abundant form in all enriched samples. Conversely, the mycelium showed significant selenium accumulation but relatively low proportions of selenomethionine. The red colour of the selenium-enriched mycelia indicated the probable presence of selenium nanoparticles, which was confirmed by single-particle inductively coupled plasma-mass spectrometry. Mean particle diameters of 90-120 nm were observed, with size distributions of 60-250 nm. Additional analysis with transmission electron microscopy confirmed this size distribution and showed that the biogenic selenium nanoparticles were roughly spherical in shape and contained elemental selenium.

Investigating the behavior of ultratrace levels of nanoparticulate and ionic silver in a seawater mesocosm using single particle inductively coupled plasma - mass spectrometry.

Silver nanoparticles (AgNPs) nowadays appear in close to 24% of consumer products that contain engineered nanomaterials. Thus, they are expected to be released into the environment, where their fate and effect are still undetermined. Considering the evidenced efficacy of the single particle Inductively Coupled Plasma - Mass Spectrometry (sp ICP-MS) technique in the study of nanomaterials, this work reports on the use of sp ICP-MS along with an online dilution sample introduction system for the direct analysis of untreated and spiked seawater samples, as part of a larger scale experiment studying the fate of Ag (ionic and nanoparticles) in seawater mesocosm systems. Silver nanoparticles coated with branched polyethyleneimine (BPEI@AgNPs) or ionic silver (Ag+) were introduced gradually into the seawater mesocosm tanks at very low, environmentally relevant concentrations (50 ng Ag L-1 per day, for 10 consecutive days, up to a total of 500 ng Ag L-1), and samples were collected and analyzed daily, within a consistent time window. Using very low detector dwell time (75 µs) and specialized data treatment, information was obtained on the nanoparticles' size distribution and particle number concentration, as well as the ionic silver content, of both the AgNPs and the Ag+ treated seawater mesocosm tanks. The results for the AgNP treated samples indicated the rapid degradation of the added silver particles, and the subsequent increase of ionic silver, with recoveries close to 100% for the first days of the experiment. On the other hand, particle formation was observed in the Ag+ treated seawater tanks, and even though the number concentration of silver-containing nanoparticles increased throughout the experiment, the amount of silver per particle remained relatively constant from the early days of the experiment. In addition, the online dilution sample introduction system for the ICP-MS proved capable of handling the untreated seawater matrix without significant contamination issues and downtime, while the low dwell time and data treatment procedure developed were shown to be suitable for the analysis of nanomaterials at the low nm-scale, despite the complex and heavy matrix introduced into the ICP-MS.

Tracking soluble and nanoparticulated titanium released in vivo from metal dental implant debris using (single-particle)-ICP-MS.

BACKGROUND: This work studies the presence of the Ti, Al and V metal ions and Ti nanoparticles released from the debris produced by the implantoplasty, a surgical procedure used in the clinic, in rat organs. METHODS: The sample preparation for total Ti determination was carefully optimized using microsampling inserts to minimize the dilution during the acid attack of the lyophilized tissues by a microwave-assisted acid digestion method. An enzymatic digestion method was optimized and applied to the different tissue samples in order to extract the titanium nanoparticles for the single-particle ICP-MS analysis. RESULTS: A statistically significant increase was found for Ti concentrations from control to experimental groups for several of the studied tissues, being and particularly significant in the case of brain and spleen. Al and V concentrations were detected in all tissues but they were not different when comparing control and experimental animals, except for V in brain. The possible presence of Ti-containing nanoparticles mobilized from the implantoplasty debris was tested using enzymatic digestions and SP-ICP-MS. The presence of Ti-containing nanoparticles was observed in all the analyzed tissues, however, differences on the Ti mass per particle were found between the blanks and the digested tissue and between control and experimental animals in some organs. CONCLUSION: The developed methodologies, both for ionic and nanoparticulated metal contents in rat organs, have shown the possible increase in the levels of Ti both as ions and nanoparticles in rats subjected to implantoplasty.

Capabilities of Single Cell ICP-MS for the Analysis of Cell Suspensions from Solid Tissues.

Single cell elemental (SC) analysis of isogenic cell cultures can be done using inductively coupled plasma (ICP-MS) detection. However, 2D cell cultures are just models to simplify the complexity of real tissue samples. Here, we show for the first time the capabilities of the technique (SC-ICP-MS) to analyze single cell suspensions of isolated cells from tissues. An optimized cocktail of proteolytic and collagenolytic enzymes was applied in a single preparation step with cellular yields up to 28% using 0.5 g of fresh rat spleen and liver, respectively. The retrieved cells revealed adequate morphology and stability to be examined by SC-ICP-MS. Quantitative elemental analysis of P, S, Cu, and Fe from disaggregated cells from rat spleen and liver tissues revealed levels of Fe of 7-16 fg/cell in the spleen and 8-12 fg/cell in the liver, while Cu was about 3-5 fg/cell in the spleen and 1.5-2.5 fg/cell in the liver. Evaluation of the transmembrane protein transferrin receptor 1 (TfR1) expression levels in disaggregated cells was also conducted by using a Nd-labelled antibody against this cell surface biomarker. Quantitative results showed significantly lower expression in the disaggregated cells than in the cell model HepG2, in agreement with the overexpression of this biomarker in tumor cells. In this proof of concept study, the tissue disaggregation protocol has shown to maintain the elemental intracellular content of cells as well as the presence of relevant antigens. This opens a completely new area of research for SC-ICP-MS in tissue samples as a complementary strategy with validation capabilities.

Ultra-Small Iron Nanoparticles Target Mitochondria Inducing Autophagy, Acting on Mitochondrial DNA and Reducing Respiration.

The application of metallic nanoparticles (materials with size at least in one dimension ranging from 1 to 100 nm) as a new therapeutic tool will improve the diagnosis and treatment of diseases. The mitochondria could be a therapeutic target to treat pathologies whose origin lies in mitochondrial dysfunctions or whose progression is dependent on mitochondrial function. We aimed to study the subcellular distribution of 2-4 nm iron nanoparticles and its effect on mitochondrial DNA (mtDNA), mitochondrial function, and autophagy in colorectal cell lines (HT-29). Results showed that when cells were exposed to ultra-small iron nanoparticles, their subcellular fate was mainly mitochondria, affecting its respiratory and glycolytic parameters, inducing the migration of the cellular state towards quiescence, and promoting and triggering the autophagic process. These effects support the potential use of nanoparticles as therapeutic agents using mitochondria as a target for cancer and other treatments for mitochondria-dependent pathologies.

Relating the composition and interface interactions in the hard corona of gold nanoparticles to the induced response mechanisms in living cells.

Understanding the formation of the intracellular protein corona of nanoparticles is essential for a wide range of bio- and nanomedical applications. The innermost layer of the protein corona, the hard corona, directly interacts with the nanoparticle surface, and by shielding the surface, it has a deterministic effect on the intracellular processing of the nanoparticle. Here, we combine a direct qualitative analysis of the hard corona composition of gold nanoparticles with a detailed structural characterization of the molecules in their interaction with the nanoparticle surface and relate both to the effects they have on the ultrastructure of living cells and the processing of the gold nanoparticles. Cells from the cell lines HCT-116 and A549 were incubated with 30 nm citrate-stabilized gold nanoparticles and with their aggregates in different culture media. The combined results of mass spectrometry based proteomics, cryo soft X-ray nanotomography and surface-enhanced Raman scattering experiments together revealed different uptake mechanisms in the two cell lines and distinct levels of induced cellular stress when incubation conditions were varied. The data indicate that the different incubation conditions lead to changes in the nanoparticle processing via different protein-nanoparticle interfacial interactions. Specifically, they suggest that the protein-nanoparticle surface interactions depend mainly on the surface properties of the gold nanoparticles, that is, the ζ-potential and the resulting changes in the hydrophilicity of the nanoparticle surface, and are largely independent of the cell line, the uptake mechanism and intracellular processing, or the extent of the induced cellular stress.

Combined single cell and single particle ICP-TQ-MS analysis to quantitatively evaluate the uptake and biotransformation of tellurium nanoparticles in bacteria.

Assessing the impact of nanoparticles in living systems implies a proper evaluation of their behaviour at single-cell level. Due to the small size of nanoparticles, their accumulation, transformation and location within single cells is challenging. In this work, the combination of single cell/single particle triple quadrupole inductively coupled plasma mass spectrometry (SC/SP-ICP-TQ-MS) analysis along with X-ray diffraction (XRD) and transmission electron microscopy (TEM) measurements has been applied to go deeper into the uptake and biotransformation of tellurium nanoparticles (TeNPs) in two bacterial model organisms, S. aureus and E. coli. The use of SC-ICP-TQ-MS enabled the individual introduction of bacterial cells where tellurium and phosphorous (as constitutive element) were monitored and detected at concentration levels down to femtogram (fg) per cell. S.aureus uptake of TeNPs was 0.5-1.9 fg Te cell-1 and 7-30 fg Te cell-1 in presence of 0.5 and 15 mg Te L-1 of TeNPs, respectively, whereas for E. coli, the amount of Te ranged from 0.08 to 0.88 fg Te cell-1 and from 2 to 36 fg Te cell-1 in presence of 0.5 and 15 mg Te L-1 of TeNPs, respectively. TEM and XRD analysis confirmed the occurrence of TeNPs biotransformation (from nanospheres to nanorods) as the nanoparticles were incorporated into both bacterial strains. Finally, SP-ICP-MS analysis after cell lysis was applied to determine the number of particles/rods per bacteria cell and to perform the dimensional characterization of the rod-shaped TeNPs. The results obtained clearly confirmed high cell-to-cell variability in terms of Te nanorods dimensions and TeNPs uptake. To the best of our knowledge, this is the first time that SC/SP-ICP-TQ-MS along with TEM and XRD analysis have been applied to investigate, quantitatively, nanoparticle uptake in bacterial cells and to estimate the dimensions of biogenic Te nanorods.

Fragmentation of Proteins in the Corona of Gold Nanoparticles As Observed in Live Cell Surface-Enhanced Raman Scattering.

Surface-enhanced Raman scattering (SERS) can provide information on the structure, composition, and interaction of molecules in the proximity of gold nanoparticles, thereby enabling studies of adsorbed biomolecules in vivo. Here, the processing of the protein corona and the corresponding protein-nanoparticle interactions in live J774 cells incubated with gold nanoparticles was characterized by SERS. Samples of isolated cytoplasm, devoid of active processing, of the same cell line were used as references. The occurrence of the most important SERS signals was compared in both types of samples. The comparison of signal abundances, supported by multivariate assessment, suggests a decreased nanoparticle-peptide backbone interaction and an increased contribution of denatured proteins in endolysosomal compartments, indicating an interaction of protein fragments with the gold nanoparticles in the endolysosome of the living cells. To study the protein fragmentation in a model and to confirm the assignment of specific spectral signatures in the live cell spectra, SERS data were collected from a solution of bovine serum albumin (BSA) digested by trypsin as an enzymatic model and from solutions of intact BSA and trypsin. The spectra from the enzymatic model confirm the strong interaction of protein fragments with the gold nanoparticles in the endolysosomal compartments. By proteomic analysis, using combined sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry of the extracted hard corona, we directly identified protein fragments, some originating from the culture medium. The results illustrate the use of appropriate models for the validation of SERS spectra and have potential implications for further developments of SERS as an in vivo analytical and biomedical tool.

In vitro and in situ experiments to evaluate the biodistribution and cellular toxicity of ultrasmall iron oxide nanoparticles potentially used as oral iron supplements.

Well-absorbed iron-based nanoparticulated materials are a promise for the oral management of iron deficient anemia. In this work, a battery of in vitro and in situ experiments are combined for the evaluation of the uptake, distribution and toxicity of new synthesized ultrasmall (4 nm core) Fe2O3 nanoparticles coated with tartaric/adipic acid with potential to be used as oral Fe supplements. First, the in vitro simulated gastric acid solubility studies by TEM and HPLC-ICP-MS reveal a partial reduction of the core size of about 40% after 90 min at pH 3. Such scenario confirms the arrival of the nanoparticulate material in the small intestine. In the next step, the in vivo absorption through the small intestine by intestinal perfusion experiments is conducted using the sought nanoparticles in Wistar rats. The quantification of Fe in the NPs suspension before and after perfusion shows Fe absorption levels above 79%, never reported for other Fe treatments. Such high absorption levels do not seem to compromise cell viability, evaluated in enterocytes-like models (Caco-2 and HT-29) using cytotoxicity, ROS production, genotoxicity and lipid peroxidation tests. Moreover, regional differences in terms of Fe concentration are obtained among different parts of the small intestine as duodenum > jejunum > ileum. Complementary transmission electron microscopy (TEM) images show the presence of the intact particles around the intestinal microvilli without significant tissue damage. These studies show the high potential of these NP preparations for their use as oral management of anemia.

Mass spectrometric approach for the analysis of the hard protein corona of nanoparticles in living cells.

The diagnostic and therapeutic application of nanoparticles requires comprehensive knowledge of their interaction with the biomolecular surroundings. The formation of the protein corona on nanoparticles that were internalized by living cells is yet to be understood. In this study, we present a robust approach for the electrophoretic and mass spectrometric analysis of the hard protein corona composition formed in living cells on ~30 nm citrate-stabilized gold nanoparticles, i.e., the proteins with the highest affinity towards the gold nanoparticle surface. The gold nanoparticles were internalized by MCF-7 cells for 24 h followed by the extraction of the hard protein corona­gold nanoparticle bioconjugates from living cell cultures. The extracted proteins were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by ESI-Q-TOF-MS, which allowed to identify 108 hard corona proteins. The experiments were repeated with J774 macrophage cells with incubation times of 1.5 h, 3 h, 6 h, and 24 h, and the results showed that the hard protein corona remained unchanged over time. Therefore, the proposed experimental approach proved to be a valuable tool for identifying hard corona proteins of nanoparticles internalized by living cells.

Quantitative Analysis of Transferrin Receptor 1 (TfR1) in Individual Breast Cancer Cells by Means of Labeled Antibodies and Elemental (ICP-MS) Detection.

Cells are able to precisely control the amount of iron they acquire in the form of transferrin (TF)-bound iron by modulating the synthesis of transferrin receptor 1 (TfR1). In tumor cells, elevated TfR1 seems to be related to poorer outcome for patients. Thus, the direct measurement of this biomarker in breast cancer tissues and cells might serve as a prognosis biomarker. In this work, we have used Nd-labeled antibodies to tag the TfR1 present on the cell surface of two cell models of breast cancer with different malignancy (MCF7 and MDA-MB 231). For this aim, monoclonal antibody anti-TfR1 is first labeled with a polymeric chelator (MAXPAR) with the subsequent incorporation of several isotopic 143Nd atoms. The characterization of the labeled antibody revealed a stoichiometry of 21 Nd atoms per antibody molecule that can be used for further quantification experiments. This antibody is used for cell tagging followed by single-cell analysis using inductively coupled plasma mass spectrometry (ICP-MS) detection. In this case, cell introduction is conducted using a high-efficiency nebulizer and spray chamber to achieve transport efficiencies of up to 55% for cells. Quantitative results revealed a number of receptors per cell significantly higher in the case of the most malignant phenotype (MDA-MB-231). Absolute and relative TfR1 concentration values are obtained in individual cells for the first time using the proposed system.

Complementary techniques (spICP-MS, TEM, and HPLC-ICP-MS) reveal the degradation of 40 nm citrate-stabilized Au nanoparticles in rat liver after intraperitoneal injection.

BACKGROUND: Due to the increased use of engineered nanoparticles (NPs), their tracing in environmental and biological systems is of utmost importance. Besides their accumulation within a biological specimen, little is known about their degradation and transformation into corresponding low-molecular species that might influence any toxicological impact. ANALYTICAL METHODS: Wistar rats underwent intraperitoneal injections of 40 nm citrate-stabilized gold nanoparticles. Different liver samples were analysed for the occurrence of nanoparticles and potential degradation products by means of spICP-MS, TEM and HPLC-ICP-MS. MAIN FINDINGS: Studies using spICP-MS revealed the presence of the originally administrated Au NPs (40 nm diameter) and some evidences of other Au-containing species due to the increased background signal. Images obtained by transmission electron microscopy (TEM) showed the predominant presence of particles of significantly smaller diameter (6 ± 2 nm). As complementary method, HPLC-ICP-MS confirmed the presence of both particle types indicating a degradation of the Au NPs accompanied by detection of low-molecular Au species. CONCLUSIONS: This study underlines that degradation of gold nanoparticles to low-molecular gold species might have to be taken into account in future for studies on their toxicological behaviour and their potential use in clinical applications.

Evaluation of the uptake, storage and cell effects of nano-iron in enterocyte-like cell models.

The therapy with nanocompounds is widely used to treat Fe deficiency and an emerging trend to inhibit tumor growth. The present work aims to address the management of different FeONP, comparing sucrose covered FeONP and Fe nanoparticles in the form of the ferritin with non-particulated inorganic Fe (II) by enterocytes-like colon cancer cell lines (Caco-2 and HT-29). Iron uptake results revealed significantly higher Fe incorporation in the case of nanoparticulated Fe, first in the form of FeONP and second in the form of ferritin with respect to inorganic Fe (II). Furthermore, the intracellular Fe fractionation, conducted by size exclusion chromatography coupled on line to inductively coupled plasma mass spectrometry (SEC-ICP-MS) showed a significant increase of the Fe-ferritin peak upon exposure of cells to the following compounds ferritin > FeONP > FeSO4. Such results point out that the sucrose coated FeONP released Fe into the cell cytosol that was used to replenish the existing cytosolic ferritin without inducing changes in the protein concentration. On the other hand, the increase of the Fe-ferritin peak in cells exposed to ferritin as iron source is due to a significant increase on the intracellular protein concentration, as proved by using an ICP-MS linked ferritin sandwich immune assay. Cell viability experiments conducted with concentrations up to 1000 µmol L-1 (as Fe) of each compound under scrutiny did not reveal significant differences among Fe species regarding global cellular toxicity. However, significant cell DNA damage was detected when treating the cells with FeONP (500 µmol L-1).

The Use of Stable Isotopic Tracers in Metallomics Studies.

Mass spectrometry represents an essential technique in Metallomics studies. It permits the identification of metal-containing molecules as part of the metallome as well as their quantification at low concentration levels. The technique becomes even more powerful in combination with the use of isotopically enriched species. Provided that they are stable, these isotopically labelled species can be easily distinguished from their natural counterparts by mass spectrometric techniques. This capability permits that these species are used for accurate and precise quantitative experiments and/or metabolic studies applying inductively coupled plasma as ionization source. In this chapter, we present the different concepts of using stable isotope tracers and isotope dilution analysis as quantification strategy. Besides some fundamental aspects, various examples from Metallomics studies, for instance, on the preparation of isotopically enriched metalloproteins and determination by isotope dilution analysis or the exploration of the biological pathways of Se species, are shown in order to demonstrate the usefulness of isotopes.

Gold nanoparticles: Distribution, bioaccumulation and toxicity. In vitro and in vivo studies.

Concerns about the bioaccumulation and toxicity of gold nanoparticles inside humans have recently risen. HT-29 and HepG2 cell lines and Wistar rats were exposed to 10, 30 or 60 nm gold nanoparticles to determine their tissue distribution, subcellular location and deleterious effects. Cell viability, ROS production and DNA damage were evaluated in vitro. Lipid peroxidation and protein carbonylation were determined in liver. ICP-MS measurements showed the presence of gold in intestine, kidney, liver, spleen, feces and urine. Subcellular locations of gold nanoparticles were observed in colon cells and liver samples by transmission electron microscopy. Inflammatory markers in liver and biochemical parameters in plasma were measured to assess the inflammatory status and presence of tissue damage. The size of the nanoparticles determined differences in the biodistribution and the excretion route. The smallest nanoparticles showed more deleterious effects, confirmed by their location inside the cell nucleus and the higher DNA damage.

Determination and speciation of cadmium in microcosms with Bunodosoma caissarum and Perna perna using isotopically enriched 116Cd.

The study of the uptake and distribution of elements in marine environments is of great interest for understanding their pathways and accumulation. Here, we investigated in laboratory experiments the accumulation behavior of Cd in the sea anemone Bunodosoma caissarum and the mussel Perna perna. Specimens were incubated with isotopically enriched 116Cd in aquariums. Cd concentrations in the seawater and in the tissues of B. caissarum and P. perna were followed by inductively coupled plasma-mass spectrometry (ICP-MS) by means of isotope dilution analysis. Bioconcentration factors for B. caissarum and P. perna exposed to 0.9µg·L-1 of 116Cd were determined to be 80.5 and 850, respectively. P. perna specimens exposed to 4.5µg·L-1 of 116Cd reached 530. Cytosolic proteins associated with Cd from the tissues were extracted and further analyzed by size-exclusion chromatography coupled to ICP-MS. Cd accumulation could be detected in both organisms ranging from high-molecular to low-molecular species.

Separation and quantification of silver nanoparticles and silver ions using reversed phase high performance liquid chromatography coupled to inductively coupled plasma mass spectrometry in combination with isotope dilution analysis.

A reversed phase high performance liquid chromatography coupled to an inductively coupled plasma mass spectrometer (HPLC-ICP-MS) approach in combination with isotope dilution analysis (IDA) for the separation and parallel quantification of nanostructured and ionic silver (Ag) is presented. The main focus of this work was the determination of the ionic Ag concentration. For a sufficient stabilization of the ions without dissolving the nanoparticles (NPs), the eluent had to be initially optimized. The determined Ag ion concentration was in a good agreement with results obtained using ultrafiltration. Further, the mechanism of the NP separation in the HPLC column was investigated. Typical size exclusion effects were found by comparing results from columns with different pore sizes. Since the recovery rates decreased with increasing Ag NP size and large Ag NPs did not elute from the column, additional interactions of the particles with the stationary phase were assumed. Our results reveal that the presented method is not only applicable to Ag NPs, but also to gold and polystyrene NPs. Finally, IDA-HPLC-ICP-MS experiments in single particle mode were performed to determine the particle cut-off size. The comparison with conventional spICP-MS experiments resulted in a similar diameter and particle size distribution.

Complementary mass spectrometric techniques for the quantification of the protein corona: a case study on gold nanoparticles and human serum proteins.

Once nanoparticles enter a biological system, it is known that their surface is instantly covered by the biomolecules present with preference to proteins. This protein corona has been a subject of numerous studies in order to reveal its composition. Besides that, growing interest exists in its quantitative determination in order to gain a deeper insight into the nature of these nanoparticle-protein bioconjugates. Only a few analytical methods are available nowadays, so the aim of this study is to provide a reliable and alternative methodology for the quantification of the protein corona. The suggested approach is based on the assumption that the total protein content within the corona can be correlated to its sulfur concentration due to the presence of cysteine and methionine as sulfur-containing amino acids. Once the most abundant proteins had been identified with the use of gel electrophoresis with subsequent peptide analysis by electrospray ionization-tandem mass spectrometry (ESI-MS/MS), the isolated nanoparticle-protein conjugates were subjected to total analysis of sulfur and the corresponding metal being present in the nanoparticles by inductively coupled plasma-mass spectrometry (ICP-MS). The concept is exemplarily demonstrated on citrate-stabilized gold nanoparticles (GNPs) incubated with human serum. Two different purification procedures were tested in order to isolate the sought bioconjugates. 26 most abundant proteins could be identified and an average of approximately 40 S atoms per protein was calculated and used for further studies. ICP-MS analyses of S/Au ratios served for the quantification of the protein corona revealing an absolute number of proteins bound to the incubated GNPs. Two main results could be obtained for this specific system under the chosen experimental conditions: the number of proteins per GNP decreased with their size from 10 nm to 60 nm and the obtained values suggested that the protein corona in this specific case was theoretically formed either as a monolayer (60 nm GNPs) or as a multilayer (5-7 protein layers per 10 nm GNP). Studies with bovine serum albumin (BSA) as the model protein showed similar results.