Revealing Silver Nanoparticle Uptake by Macrophages Using SR-µXRF and LA-ICP-MS.

To better study the impact of nanoparticles on both in vitro and in vivo models, tissue distribution and cellular doses need to be described more closely. Here silver nanoparticles were visualized in alveolar macrophages by means of synchrotron radiation micro X-ray fluorescence spectroscopy (SR-µXRF) with high spatial resolution of 3 × 3 µm2. For the spatial allocation of silver signals to cells and tissue structures, additional elemental labeling was carried out by staining with eosin, which binds to protein and can be detected as bromine signal with SR-µXRF. The method was compatible with immunostaining of macrophage antigens. We found that the silver distribution obtained with SR-µXRF was largely congruent with distribution maps from a subsequent laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) of the same tissue sites. The study shows a predominant, though not exclusive uptake of silver into alveolar macrophages in the rat lung, which can be modeled by a similar uptake in cultured alveolar macrophages. Advantages and limitations of the different strategies for measuring nanoparticle uptake at the single cell level are discussed.

A Ribonucleoprotein Supercomplex Involved in trans-Splicing of Organelle Group II Introns.

In the chloroplast of the green alga Chlamydomonas reinhardtii, two discontinuous group II introns, psaA-i1 and psaA-i2, splice in trans, and thus their excision process resembles the nuclear spliceosomal splicing pathway. Here, we address the question whether fragmentation of trans-acting RNAs is accompanied by the formation of a chloroplast spliceosome-like machinery. Using a combination of liquid chromatography-mass spectrometry (LC-MS), size exclusion chromatography, and quantitative RT-PCR, we provide the first characterization of a high molecular weight ribonucleoprotein apparatus participating in psaA mRNA splicing. This supercomplex contains two subcomplexes (I and II) that are responsible for trans-splicing of either psaA-i1 or psaA-i2. We further demonstrate that both subcomplexes are associated with intron RNA, which is a prerequisite for the correct assembly of subcomplex I. This study contributes further to our view of how the eukaryotic nuclear spliceosome evolved after bacterial endosymbiosis through fragmentation of self-splicing group II introns into a dynamic, protein-rich RNP machinery.

A pioneer protein is part of a large complex involved in trans-splicing of a group II intron in the chloroplast of Chlamydomonas reinhardtii.

Splicing of organellar introns requires the activity of numerous nucleus-encoded factors. In the chloroplast of Chlamydomonas reinhardtii, maturation of psaA mRNA encoding photosystem I subunit A involves two steps of trans-splicing. The exons, located on three separate transcripts, are flanked by sequences that fold to form the conserved structures of two group II introns. A fourth transcript contributes to assembly of the first intron, which is thus tripartite. The raa7 mutant (RNA maturation of psaA 7) is deficient in trans-splicing of the second intron of psaA, and may be rescued by transforming the chloroplast genome with an intron-less version of psaA. Using mapped-based cloning, we identify the RAA7 locus, which encodes a pioneer protein with no previously known protein domain or motif. The Raa7 protein, which is not associated with membranes, localizes to the chloroplast. Raa7 is a component of a large complex and co-sediments in sucrose gradients with the previously described splicing factors Raa1 and Raa2. Based on tandem affinity purification of Raa7 and mass spectrometry, Raa1 and Raa2 were identified as interacting partners of Raa7. Yeast two-hybrid experiments indicate that the interaction of Raa7 with Raa1 and Raa2 may be direct. We conclude that Raa7 is a component of a multimeric complex that is required for trans-splicing of the second intron of psaA. The characterization of this psaA trans-splicing complex is also of interest from an evolutionary perspective because the nuclear spliceosomal introns are thought to derive from group II introns, with which they show mechanistic and structural similarity.

Elemental bioimaging of thulium in mouse tissues by laser ablation-ICPMS as a complementary method to heteronuclear proton magnetic resonance imaging for cell tracking experiments.

Due to the fact that cellular therapies are increasingly finding application in clinical trials and promise success by treatment of fatal diseases, monitoring strategies to investigate the delivery of the therapeutic cells to the target organs are getting more and more into the focus of modern in vivo imaging methods. In order to monitor the distribution of the respective cells, they can be labeled with lanthanide complexes such as thulium-1,4,7,10-tetraazacyclodoecane-α,α,α,α-tetramethyl-1,4,7,10-tetraacetic acid (Tm(DOTMA)). In this study, experiments on a mouse model with two different cell types, namely, tumor cells and macrophages labeled with Tm(DOTMA), were performed. The systemic distribution of Tm(DOTMA) of both cell types was investigated by means of laser ablation-inductively coupled plasma-mass spectrometry (LA-ICPMS). Using the high resolution of 25 µm, distribution maps of Tm in different tissues such as tumor, liver, lung, and spleen as well as in explanted gel pellets were generated and the behavior of the labeled cells inside the tissue was investigated. Additionally, quantitative data were obtained using homemade matrix-matched standards based on egg yolk. Using this approach, limits of detection and quantification of 2.2 and 7.4 ng·g(-1), respectively, and an excellent linearity over the concentration range from 0.01 to 46 µg·g(-1) was achieved. The highest concentration of the label agent, 32.4 µg·g(-1), in tumor tissue was observed in the area of the injection of the labeled tumor cells. Regarding the second experiment with macrophages for cell tracking, Tm was detected in the explanted biogell pellet with relatively low concentrations below 60 ng·g(-1) and in the liver with a relatively high concentration of 10 µg·g(-1). Besides thulium, aluminum was detected with equal distribution behavior in the tumor section due to a contamination resulting from the labeling procedure, which includes the usage of an Al electrode.

Quantitative bioimaging of p-boronophenylalanine in thin liver tissue sections as a tool for treatment planning in boron neutron capture therapy.

An analytical method using laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) was developed and applied to assess enrichment of 10B-containing p-boronophenylalanine-fructose (BPA-f) and its pharmacokinetic distribution in human tissues after application for boron neutron capture therapy (BNCT). High spatial resolution (50 µm) and limits of detection in the low parts-per-billion range were achieved using a Nd:YAG laser of 213 nm wavelength. External calibration by means of 10B-enriched standards based on whole blood proved to yield precise quantification results. Using this calibration method, quantification of 10B in cancerous and healthy tissue was carried out. Additionally, the distribution of 11B was investigated, providing 10B enrichment in the investigated tissues. Quantitative imaging of 10B by means of LA-ICP-MS was demonstrated as a new option to characterise the efficacy of boron compounds for BNCT.

Highly shifted proton MR imaging: cell tracking by using direct detection of paramagnetic compounds.

PURPOSE: To explore the feasibility of tracking thulium (Tm)-1,4,7,10-tetraazacyclododecane-α,α',α'',α'''-tetramethyl-1,4,7,10-tetraacetic acid (DOTMA)-labeled cells in vivo by means of highly shifted proton magnetic resonance (MR) imaging as a potential alternative to established cell-tracking methods. MATERIALS AND METHODS: All animal experiments were approved by the local ethics committee for animal experiments. Highly shifted proton MR imaging is based on the principle that the shifted resonances on Tm and dysprosium (Dy)-DOTMA can be detected separately from the tissue water signal at MR imaging with very short echo time and radial center-out readout (UTE, or "ultrashort echo time"). MR imaging of aqueous solutions and in mice in vivo was performed at 9.4 T. Human fibrosarcoma cells (HT-1080) and murine macrophages were labeled with different amounts of Tm-DOTMA. Labeled fibrosarcoma cells were injected subcutaneously into three mice. For cell tracking, labeled macrophages were administered intravenously into eight mice bearing local granulomatous inflammation. Three-dimensional UTE MR imaging was performed during 1 week. Macrophage viability and activity and fibrosarcoma cell viability were statistically analyzed by performing an unpaired two-tailed t test for labeled versus unlabeled cells by using data of at least six independent experiments. RESULTS: The strongly shifted MR lines of Tm- and Dy-DOTMA can be separated from the tissue water signal and from each other. A detection limit of about 25 µmol/L of Tm-DOTMA was calculated from in vitro MR measurements. A mean ± standard error of the mean intracellular uptake of (4.19 ± 0.88) × 10(9) (HT-1080) and (10.1 ± 3.0) × 10(10) (macrophages) of Tm-DOTMA molecules per cell was achieved. In vivo, Tm-DOTMA signal was detectable for 1 week in both tumors and macrophages, with a detection limit of approximately 10(4) HT-1080 and 600 macrophages. Histologic examination results and elemental bioimaging confirmed labeled cells as source of MR signal. CONCLUSION: Strongly shifted proton three-dimensional UTE MR imaging of Tm-DOTMA-labeled cells is a highly specific and sensitive tool for in vivo cell tracking.

Elemental bioimaging of manganese uptake in C. elegans.

A new method for elemental bioimaging with laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) was developed and applied to study the uptake of manganese (Mn) in Caenorhabditis elegans (C. elegans). C. elegans is a well-established model organism in neuroscience, genetics and genomics, which has been extensively studied to decipher mechanisms of heavy metal induced toxicity. Knowledge about the distribution of manganese (Mn) and other metals in this organism will be helpful in elucidating pathways and mechanisms of transport, distribution and excretion. The LA-ICP-MS method requires limited sample preparation and can be used rapidly and easily to visualize the Mn distribution in C. elegans. Due to thorough optimization of the analytical parameters, intense Mn signals in C. elegans wild-type (WT) and mutants were obtained at a spatial resolution as small as 4 µm, thus proving the suitability of LA-ICP-MS to study the uptake of metals in C. elegans.

A palladium label to monitor nanoparticle-assisted drug delivery of a photosensitizer into tumor spheroids by elemental bioimaging.

In this study, the cellular uptake of the second generation photosensitizer 5,10,15,20-tetrakis(3-hydroxyphenyl)porphyrin (mTHPP) was investigated using laser ablation coupled to inductively coupled plasma mass spectrometry (LA-ICP-MS) at a spatial resolution of 10 µm. To achieve high sensitivity, the photosensitizer was tagged with palladium. As a tumor model system, a 3D cell culture of the TKF-1 cell line was used. These tumor spheroids were incubated with the Pd-tagged photosensitizer embedded in poly(lactic-co-glycolic acid) (PLGA) nanoparticles to investigate the efficiency of nanoparticle based drug delivery. An accumulation of the drug in the first cell layers of the tumor spheroid was observed. In the case of nanoparticle based drug delivery, a significantly more homogeneous distribution of the photosensitizer was achieved, compared to tumor spheroids incubated with the dissolved photosensitizer without the nanoparticular drug delivery system. The infiltration depth of the Pd-tagged photosensitizer could not be increased with rising incubation time, which can be attributed to the adsorption of the photosensitizer onto cellular components.

Elemental bioimaging of nanosilver-coated prostheses using X-ray fluorescence spectroscopy and laser ablation-inductively coupled plasma-mass spectrometry.

The distribution of different chemical elements from a nanosilver-coated bone implant was visualized, combining the benefits of two complementary methods for elemental bioimaging, the nondestructive micro X-ray fluorescence (µ-XRF), and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). Challenges caused by the physically inhomogeneous materials including bone and soft tissues were addressed by polymer embedding. With the use of µ-XRF, fast sample mapping was achieved obtaining titanium and vanadium signals from the metal implant as well as phosphorus and calcium signals representing hard bone tissue and sulfur distribution representing soft tissues. Only by the use of LA-ICP-MS, the required high sensitivity and low detection limits for the determination of silver were obtained. Metal distribution within the part of cancellous bone was revealed for silver as well as for the implant constituents titanium, vanadium, and aluminum. Furthermore, the detection of coinciding high local zirconium and aluminum signals at the implant surface indicates remaining blasting abrasive from preoperative surface treatment of the nanosilver-coated device.

Quantitative bioimaging of platinum in polymer embedded mouse organs using laser ablation ICP-MS.

A novel quantification approach for tissue imaging using laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) based on tissue embedding in cold-curing resins (Technovit 7100) is presented. With respect to massive side effects on cisplatin, the platinum distribution at different time intervals after cisplatin treatment of mice was determined quantitatively in different tissues including cochlea, testis and kidney. For this purpose, cold-curing resin blocks spiked with different amounts of platinum acetyl acetonate prior to curing were ablated after sectioning at 5 µm thickness and were analysed using ICP-MS after microwave digestion. High spatial resolution and limits of detection in the low ppb range (8 µg kg(-1)) were achieved using a simple and efficient sample preparation. External calibration using the Technovit 7100 standards proved to yield precise and reproducible quantification results. The distribution and retention behaviour of cisplatin in the organs was investigated using the new calibration method.

Identification of a chloroplast ribonucleoprotein complex containing trans-splicing factors, intron RNA, and novel components.

Maturation of chloroplast psaA pre-mRNA from the green alga Chlamydomonas reinhardtii requires the trans-splicing of two split group II introns. Several nuclear-encoded trans-splicing factors are required for the correct processing of psaA mRNA. Among these is the recently identified Raa4 protein, which is involved in splicing of the tripartite intron 1 of the psaA precursor mRNA. Part of this tripartite group II intron is the chloroplast encoded tscA RNA, which is specifically bound by Raa4. Using Raa4 as bait in a combined tandem affinity purification and mass spectrometry approach, we identified core components of a multisubunit ribonucleoprotein complex, including three previously identified trans-splicing factors (Raa1, Raa3, and Rat2). We further detected tscA RNA in the purified protein complex, which seems to be specific for splicing of the tripartite group II intron. A yeast-two hybrid screen and co-immunoprecipitation identified chloroplast-localized Raa4-binding protein 1 (Rab1), which specifically binds tscA RNA from the tripartite psaA group II intron. The yeast-two hybrid system provides evidence in support of direct interactions between Rab1 and four trans-splicing factors. Our findings contribute to our knowledge of chloroplast multisubunit ribonucleoprotein complexes and are discussed in support of the generally accepted view that group II introns are the ancestors of the eukaryotic spliceosomal introns.

Laser ablation based bioimaging with simultaneous elemental and molecular mass spectrometry: towards spatially resolved speciation analysis.

RATIONALE: Biological functions of metals are not only specified by the element itself, but also by its chemical form and by its organ, cell and subcellular location. The developed laser ablation based setup enables spatially resolved analysis with simultaneous elemental and molecular mass spectrometry (MS) and promises therefore localization, identification and quantification of metal or heteroelement-containing species in biological samples such as tissue sections. METHODS: A UV laser ablation (LA) system is hyphenated in parallel both with an elemental and a molecular mass spectrometer via flow splitted transfer lines to simultaneously obtain data from both of the mass spectrometers. Elemental MS was performed using inductively coupled plasma (ICP)-MS, whereas atmospheric pressure chemical ionization (APCI)-MS with an orbitrap mass analyzer was utilized for molecular MS. RESULTS: Simultaneous elemental and molecular MS imaging with high lateral resolution down to 25 µm was presented for the staining agents eosin Y and haematoxylin as well as for the chemotherapy drug cisplatin in thin tissue sections. For molecular MS, target compounds were identified by their exact masses and by characteristic fragment ions. CONCLUSIONS: The first simultaneous elemental and molecular MS imaging approach based on laser ablation sampling was introduced for spatially resolved speciation analysis. The combination of the advantages of LA-ICP-MS such as low detection limits and high spatial resolution with information on the chemical identity promises not only localization of metals, but also identification of metal species in biological samples. Therefore, this novel technique opens up new possibilities to address complex challenges in life science research.

Ambient molecular imaging by laser ablation atmospheric pressure chemical ionization mass spectrometry.

RATIONALE: The functions and properties of compounds are not only specified by their chemical structures, but also by their location inside a sample. Mass spectrometry is a powerful tool for imaging, whereby the kind of sample and compound depend on the used sampling and ionization methods. The developed laser ablation mass spectrometry method delivers high resolution images of small molecules in native samples. METHODS: A UV laser ablation (LA) system was combined with an atmospheric pressure chemical ionization (APCI) mass spectrometer. The spatially resolved sampling was performed by focusing the 213 nm laser beam onto a sample. The fine aerosol generated by the ns pulsed laser irradiation was then transported to the APCI mass spectrometer by a nitrogen stream. In the APCI source, post-ionization was accomplished by a corona discharge. The resulting ions were detected with an orbitrap mass spectrometer. RESULTS: The properties of the novel LA-APCI-MS setup are demonstrated by spatially resolved analysis of several samples including tablets, TLC plates and dried droplets. The target compounds are detected with high spatial and mass resolution. For higher molecular weight compounds like thyroxine, fragmentation was observed, whereas small molecules like caffeine stayed intact. CONCLUSIONS: LA-APCI-MS is introduced as an ambient molecular mass spectral imaging method for molecules with high resolution in space and mass. The combination of two independent instruments offers flexible ion source and mass analyzer exchange and therefore LA-APCI-MS opens up new possibilities for molecular imaging under ambient conditions.