Radiolabelling of engineered nanoparticles for in vitro and in vivo tracing applications using cyclotron accelerators.

We present in this article an outline of some cyclotron-based irradiation techniques that can be used to directly radiolabel industrially manufactured nanoparticles, as well as two techniques for synthesis of labelled nanoparticles using cyclotron-generated radioactive precursor materials. These radiolabelled nanoparticles are suitable for a range of different in vitro and in vivo tracing studies of relevance to the field of nanotoxicology. A basic overview is given of the relevant physics of nuclear reactions regarding both ion-beam and neutron production of radioisotopes. The various issues that determine the practicality and usefulness of the different methods are discussed, including radioisotope yield, nuclear reaction kinetics, radiation and thermal damage, and radiolabel stability. Experimental details are presented regarding several techniques applied in our laboratories, including direct light-ion activation of dry nanoparticle samples, neutron activation of nanoparticles and suspensions using an ion-beam driven activator, spark-ignition generation of nanoparticle aerosols using activated electrode materials, and radiochemical synthesis of nanoparticles using cyclotron-produced isotopes. The application of these techniques is illustrated through short descriptions of some selected results thus far achieved. It is shown that these cyclotron-based methods offer a very useful range of options for nanoparticle radiolabelling despite some experimental difficulties associated with their application. For direct nanoparticle radiolabelling, if care is taken in choosing the experimental conditions applied, useful activity levels can be achieved in a wide range of nanoparticle types, without causing substantial thermal or radiation damage to the nanoparticle structure. Nanoparticle synthesis using radioactive precursors presents a different set of issues and offers a complementary and equally valid approach when laboratory generation of the nanoparticles is acceptable for the proposed studies, and where an appropriate radiolabel can be incorporated into the nanoparticles during synthesis.

Multifunctional nanoparticles for dual imaging.

For imaging with different modalities, labels, which provide contrast for all modalities, are required. Colloidal nanoparticles composed out of an inorganic core and a polymer shell offer progress in this direction. Both, the core and the polymer shell, can be synthesized to be fluorescent, magnetic, or radioactive. When different cores are combined with different polymer shells, different types of particles for dual imaging can be obtained, as for example, fluorescent cores with radioactive polymer shells. Properties and perspectives of such nanoparticles for multimodal imaging are discussed.

The allergen Bet v 1 in fractions of ambient air deviates from birch pollen counts.

BACKGROUND: Proof is lacking that pollen count is representative for allergen exposure, also because allergens were found in nonpollen-bearing fractions of ambient air. OBJECTIVE: We monitored simultaneously birch pollen and the major birch pollen allergen Bet v 1 in different size fractions of ambient air from 2004 till 2007 in Munich, Germany. METHODS: Air was sampled with a ChemVol high-volume cascade impactor equipped with stages for particulate matter (PM)>10 microm, 10 microm>PM>2.5 microm, and 2.5 microm>PM>0.12 microm. Allergen was determined with a Bet v 1-specific ELISA. Pollen count was assessed with a Burkard pollen trap. We also measured the development of allergen in pollen during ripening. RESULTS: About 93 +/- 3% of Bet v 1 was found in the PM > 10 microm fraction, the fraction containing birch pollen. We did not measure any Bet v 1 in 2.5 microm > PM > 0.12 microm. Either in Munich no allergen was in this fraction or the allergen was absorbed to diesel soot particles that also deposit in this fraction. Pollen released 115% more Bet v 1 in 2007 than in 2004. Also within 1 year, the release of allergen from the same amount of pollen varied more than 10-fold between different days. This difference was explained by a rapidly increasing expression of Bet v 1 in pollen in the week just before pollination. Depending on the day the pollen is released during ripening, its potency varies. CONCLUSION: In general, pollen count and allergen in ambient air follow the same temporal trends. However, because a 10-fold difference can exist in allergen potency of birch pollen, symptoms might be difficult to correlate with pollen counts, but perhaps better with allergen exposure.

Long-term responses of canine lungs to acidic particles.

Sixteen beagle dogs were housed in four large chambers under minimum restraint. They were exposed for 16 months to clean air and individual baseline data of markers were obtained. For 13 months, eight dogs were further exposed to clean air and eight dogs for 6 h/d to 1-microm MMAD (mass median aerodynamic diameter) acidic sulfate particles carrying 25 micromol H(+) m(-3) into their lungs. To establish functional responses (lung function, cell and tissue integrity, redox balance, and non-specific respiratory defense capacity), each exposed animal served as its own control. To establish structural responses, the eight non-exposed animals served as controls. Acidic particles were produced by nebulization of aqueous sodium hydrogen sulfate at pH 1.5. Only subtle exposure-related changes of lung function and structure were detected. A significant increase in respiratory burst function of alveolar macrophages points to a marginal inflammatory response. This can be explained by the significant production of prostaglandin E(2), activating cyclooxygenase-dependent mechanisms in epithelia and thus inhibiting lung inflammation. The non-specific defense capacity was slightly affected, giving increased tracheal mucus velocity and reduced in vivo dissolution of moderately soluble test particles. Hypertrophy and hyperplasia of bronchial epithelia were not observed, but there was an increase in volume density of bronchial glands and a shift from neutral to acidic staining of epithelial secretory cells in distal airways. The acidic exposure had thus no pathophysiological consequences. It is therefore unlikely that long-term inhalation of acidic particles is associated with a health risk.

Dosimetry and toxicology of inhaled ultrafine particles.

Both epidemiological and toxicological studies indicate that inhalation and subsequent deposition of airborne particles into the lungs have adverse health effects. Recently, the ultrafine particle (UfP) fraction (diameter < 100 nm) has received particular attention, as their small size may lead to more toxic properties. In this study we summarize the current knowledge on the dosimetry of inhaled particles (including UfPs) with a focus on recent data on translocation of UfPs into secondary target organs (such as brain and heart) suggesting that the lifetime dose of ambient UfPs in secondary target organs is about 10(11) particles. Furthermore, we highlight the main pathways of particle induced toxicity and the reasons for the potentially higher toxicity of UfPs. Finally, we discuss recent evidence indicating that (BET) surface area is the single most relevant dose metric for the toxicity of UfPs, which has important implications for regulatory measures on the toxicity of ambient and engineered particles.

Dose-controlled exposure of A549 epithelial cells at the air-liquid interface to airborne ultrafine carbonaceous particles.

The geometry of commercially available perfusion chambers designed for harbouring three membrane-based cell cultures was modified for reliable and dose-controlled air-liquid interface (ALI) exposures. Confluent A549 epithelial cells grown on membranes were integrated in the chamber system and supplied with medium from the chamber bottom. Cell viability was not impaired by the conditions of ALI exposure without particles. Expression of the inflammatory cytokines interleukin 6 and interleukin 8 by A549 cells during ALI exposure to filtered air for 6h and subsequent stimulation with tumor necrosis factor was not altered compared to submersed controls, indicating that the cells maintained their functional integrity. Ultrafine carbonaceous model particles with a count median mobility diameter of about 95+/-5 nm were produced by spark discharge at a stable concentration of about 2 x 10(6) cm(-3) and continuously monitored for accurate determination of the exposure dose. Delivery to the ALI exposure system yielded a homogeneous particle deposition over the membranes with a deposition efficiency of 2%. Mid dose exposure of A549 cells to this aerosol for 6h yielded a total particle deposition of (2.6+/-0.4) x 10(8) cm(-2) corresponding to (87+/-23) ng cm(-2). The 2.7-fold (p < or = 0.05) increased transcription of heme oxygenase-1 indicated a sensitive antioxidant and stress response, while cell viability did not reveal a toxic mechanism.

Distribution pattern of inhaled ultrafine gold particles in the rat lung.

The role of alveolar macrophages in the fate of ultrafine particles in the lung was investigated. Male Wistar-Kyoto rats were exposed to ultrafine gold particles, generated by a spark generator, for 6 h at a concentration of 88 microg/m3 (4 x 10(6)/cm3, 16 nm modal mobility diameter). Up to 7 days, the animals were serially sacrificed, and lavaged cells and lung tissues were examined by transmission electron microscopy. The gold concentration/content in the lung, lavage fluid, and blood was estimated by inductively coupled plasma-mass spectrometry. Gold particles used were spherical and electron dense with diameters of 5-8 nm. The particles were individual or slightly agglomerated. By inductively coupled plasma-mass spectrometry analysis of the lung, 1945 +/- 57 ng (mean +/- SD) and 1512 +/- 184 ng of gold were detected on day 0 and on day 7, respectively, indicating that a large portion of the deposited gold particles was retained in the lung tissue. In the lavage fluid, 573 +/- 67 ng and 96 +/- 29 ng were found on day 0 and day 7, respectively, which means that 29% and 6% of the retained gold particles were lavageable on these days. A low but significant increase of gold (0.03 to 0.06% of lung concentration) was found in the blood. Small vesicles containing gold particles were found in the cytoplasm of alveolar macrophages. In the alveolar septum, the gold particles were enclosed in vesicles observed in the cytoplasm of alveolar type I epithelial cells. These results indicate that inhaled ultrafine gold particles in alveolar macrophages and type I epithelial cells are processed by endocytotic pathways, though the uptake of the gold particles by alveolar macrophages is limited. To a low degree, systemic particle translocation took place.

Negligible clearance of ultrafine particles retained in healthy and affected human lungs.

Ambient particles are believed to be a specific health hazard, although the underlying mechanisms are not fully understood. There are data in the literature indicating fast and substantial systemic uptake of particles from the lung. The present authors have developed an improved method to produce ultrafine particles with more stable radiolabelling and defined particle size range. Fifteen subjects inhaled technetium 99m (99mTc)-labelled carbonaceous particles of 100 nm in size. Radioactivity over the lung was followed for 70 h. The clearance of these ultrafine particles from the lungs and specifically translocation to the circulation was tested. Lung retention for all subjects at 46 h was mean+/-sd 99+/-4.6%. Cumulative leaching of 99mTc activity from the particles was 2.6+/-0.96% at 70 h. The 24-h activity leaching in urine was 1.0+/-0.55%. No evidence of a quantitatively important translocation of 100-nm particles to the systemic circulation from the lungs was found. More research is needed to establish if the approximately 1% cleared activity originates from leached activity or insoluble translocated particles, and whether a few per cent of translocated particles is sufficient to cause harmful effects.

Improved ventricular function during inhalation of PGI(2) aerosol partly relies on enhanced myocardial contractility.

Inhaled prostacyclin (PGI(2)) aerosol induces selective pulmonary vasodilation. Further, it improves right ventricular (RV) function, which may largely rely on pulmonary vasodilation, but also on enhanced myocardial contractility. We investigated the effects of the inhaled PGI(2) analogs epoprostenol (EPO) and iloprost (ILO) on RV function and myocardial contractility in 9 anesthetized pigs receiving aerosolized EPO (25 and 50 ng.kg(-1).min(-1)) and, consecutively, ILO (60 ng.kg(-1).min(-1)) for 20 min each. We measured pulmonary artery pressure (PAP), RV ejection fraction (RVEF) and RV end-diastolic-volume (RV-EDV), and left ventricular end-systolic pressure-volume-relation (end-systolic elastance, E(es)). EPO and ILO reduced PAP, increased RVEF and reduced RVEDV. E(es) was enhanced during all doses tested, which reached statistical significance during EPO(25 ng) and ILO, but not during EPO(50 ng). PGI(2) aerosol enhances myocardial contractility in healthy pigs, contributing to improve RV function.

Sources and elemental composition of ambient PM(2.5) in three European cities.

Source apportionment of urban fine particle mass (PM(2.5)) was performed from data collected during 1998-1999 in Amsterdam (The Netherlands), Erfurt (Germany) and Helsinki (Finland), using principal component analysis (PCA) and multiple linear regression. Six source categories of PM(2.5) were identified in Amsterdam. They were traffic-related particles (30% of the average PM(2.5)), secondary particles (34%), crustal material (7%), oil combustion (11%), industrial and incineration processes (9%), and sea salt (2%). The unidentified PM(2.5) fraction was 7% on the average. In Erfurt, four source categories were extracted with some difficulties in interpretation of source profiles. They were combustion emissions related to traffic (32%), secondary PM (32%), crustal material (21%) and industrial processes (8%). In Erfurt, 3% of PM(2.5) remained unidentified. Air pollution data and source apportionment results from the two Central European cities were compared to previously published results from Helsinki, where about 80% of average PM(2.5) was attributed to transboundary air pollution and particles from traffic and other regional combustion sources. Our results indicate that secondary particles and local combustion processes (mainly traffic) were the most important source categories in all cities; their impact on the average PM(2.5) was almost equal in Amsterdam and Erfurt whereas, in Helsinki, secondary particles made up for as much as half of the total average PM(2.5).

Radionuclide biokinetics database (RBDATA-EULEP): an update.

The main activity of the RBDATA-EULEP project is the development of an electronic database of information on the biokinetics of radionuclides after intake by inhalation, ingestion or injection. It consists of linked tables of publications and experiments, with details and comments on the materials, procedures and results. By March 2004 it contained information on more than 1600 experiments from 600 publications. It will be extended and Internet access will also be provided.

Daily variation in fine and ultrafine particulate air pollution and urinary concentrations of lung Clara cell protein CC16.

BACKGROUND: Daily variations in ambient particulate air pollution have been associated with respiratory mortality and morbidity. AIMS: To assess the associations between urinary concentration of lung Clara cell protein CC16, a marker for lung damage, and daily variation in fine and ultrafine particulate air pollution. METHODS: Spot urinary samples (n = 1249) were collected biweekly for six months in subjects with coronary heart disease in Amsterdam, Netherlands (n = 37), Erfurt, Germany (n = 47), and Helsinki, Finland (n = 47). Ambient particulate air pollution was monitored at a central site in each city. RESULTS: The mean 24 hour number concentration of ultrafine particles was 17.3x10(3) cm(-3) in Amsterdam, 21.1x10(3) cm(-3) in Erfurt, and 17.0x10(3) cm(-3) in Helsinki. The mean 24 hour PM2.5 concentrations were 20, 23, and 13 microg/m3, respectively. Daily variation in ultrafine particle levels was not associated with CC16. In contrast, CC16 concentration seemed to increase with increasing levels of PM2.5 in Helsinki, especially among subjects with lung disorders. No clear associations were observed in Amsterdam and Erfurt. In Helsinki, the CC16 concentration increased by 20.2% (95% CI 6.9 to 33.5) per 10 microg/m3 increase in PM2.5 concentration (lag 2). The respective pooled effect estimate was 2.1% (95% CI -1.3 to 5.6). CONCLUSION: The results suggest that exposure to particulate air pollution may lead to increased epithelial barrier permeability in lungs.

Translocation of inhaled ultrafine particles to the brain.

Ultrafine particles (UFP, particles <100 nm) are ubiquitous in ambient urban and indoor air from multiple sources and may contribute to adverse respiratory and cardiovascular effects of particulate matter (PM). Depending on their particle size, inhaled UFP are efficiently deposited in nasal, tracheobronchial, and alveolar regions due to diffusion. Our previous rat studies have shown that UFP can translocate to interstitial sites in the respiratory tract as well as to extrapulmonary organs such as liver within 4 to 24 h postexposure. There were also indications that the olfactory bulb of the brain was targeted. Our objective in this follow-up study, therefore, was to determine whether translocation of inhaled ultrafine solid particles to regions of the brain takes place, hypothesizing that UFP depositing on the olfactory mucosa of the nasal region will translocate along the olfactory nerve into the olfactory bulb. This should result in significant increases in that region on the days following the exposure as opposed to other areas of the central nervous system (CNS). We generated ultrafine elemental (13)C particles (CMD = 36 nm; GSD = 1.66) from [(13)C] graphite rods by electric spark discharge in an argon atmosphere at a concentration of 160 microg/m(3). Rats were exposed for 6 h, and lungs, cerebrum, cerebellum and olfactory bulbs were removed 1, 3, 5, and 7 days after exposure. (13)C concentrations were determined by isotope ratio mass spectroscopy and compared to background (13)C levels of sham-exposed controls (day 0). The background corrected pulmonary (13)C added as ultrafine (13)C particles on day 1 postexposure was 1.34 microg/lung. Lung (13)C concentration decreased from 1.39 microg/g (day 1) to 0.59 microg/g by 7 days postexposure. There was a significant and persistent increase in added (13)C in the olfactory bulb of 0.35 microg/g on day 1, which increased to 0.43 microg/g by day 7. Day 1 (13)C concentrations of cerebrum and cerebellum were also significantly increased but the increase was inconsistent, significant only on one additional day of the postexposure period, possibly reflecting translocation across the blood-brain barrier in certain brain regions. The increases in olfactory bulbs are consistent with earlier studies in nonhuman primates and rodents that demonstrated that intranasally instilled solid UFP translocate along axons of the olfactory nerve into the CNS. We conclude from our study that the CNS can be targeted by airborne solid ultrafine particles and that the most likely mechanism is from deposits on the olfactory mucosa of the nasopharyngeal region of the respiratory tract and subsequent translocation via the olfactory nerve. Depending on particle size, >50% of inhaled UFP can be depositing in the nasopharyngeal region during nasal breathing. Preliminary estimates from the present results show that approximately 20% of the UFP deposited on the olfactory mucosa of the rat can be translocated to the olfactory bulb. Such neuronal translocation constitutes an additional not generally recognized clearance pathway for inhaled solid UFP, whose significance for humans, however, still needs to be established. It could provide a portal of entry into the CNS for solid UFP, circumventing the tight blood-brain barrier. Whether this translocation of inhaled UFP can cause CNS effects needs to be determined in future studies.

Long-term clearance kinetics of inhaled ultrafine insoluble iridium particles from the rat lung, including transient translocation into secondary organs.

Recently it was speculated that ultrafine particles (UFP) may translocate from deposition sites in the lungs to systemic circulation and whether long-term clearance differs between ultrafine and micrometer-sized particles. We have studied lung retention and clearance kinetics in 12 healthy male adult WKY rats up to 6 mo after an inhalation of (192)Ir-radiolabeled, insoluble, ultrafine 15- to 20-nm iridium particles. Whole-body retention was followed by external gamma counting, and particle clearance kinetics were determined by excretion radioanalysis. Four rats each were sacrificed after 3 wk and 2 and 6 mo; all organs as well as tissues and the carcass were radioanalyzed to balance the entire deposited radioactivity of the particles. The most prominent fraction was retained in the lungs at each time point of sacrifice (26%, 15%, 6%, respectively), and clearance out of the body was solely via excretion. Extrapulmonary particle uptake did not continue to increase but decreased with time in liver, spleen, heart, and brain when compared to previous data obtained during the first 7 days after inhalation (Kreyling et al., 2002). UFP long-term lung retention derived from whole-body measurements was comparable to previously reported data using insoluble micrometer-sized particles (Bellmann et al., 1994; Lehnert et al., 1989). In addition, differential analysis including daily excretion data revealed a pattern of fractional particle clearance rate of the ultrafine iridium particles similar to that of micrometer-sized particles reported by Snipes et al. (1983) and Bailey et al. (1985).

Fate and toxic effects of inhaled ultrafine cadmium oxide particles in the rat lung.

Female Fischer 344 rats were exposed to ultrafine cadmium oxide particles, generated by spark discharging, for 6 h at a concentration of 70 microg Cd/m(3) (1 x 10(6)/cm(3)) (40 nm modal diameter). Lung morphology and quantification of Cd content/concentration by inductively coupled plasma (ICP)-mass spectrometry were performed on days 0, 1, 4, and 7 after exposure. Cd content in the lung on day 0 was 0.53 +/- 0.12 microg/lung, corresponding to 19% of the estimated total inhaled cumulative dose, and the amount remained constant throughout the study. In the liver no significant increase of Cd content was found up to 4 days. A slight but statistically significant increase was observed in the liver on day 7. We found neither exposure-related morphological changes of lungs nor inflammatory responses in lavaged cells. Another group of rats were exposed to a higher concentration of ultrafine CdO particles (550 microg Cd/m(3) for 6 h, 51 nm modal diameter). The rats were sacrificed immediately and 1 day after exposure. The lavage study performed on day 0 showed an increase in the percentage of neutrophils. Multifocal alveolar inflammation was seen histologically on day 0 and day 1. Although the Cd content in the lung was comparable between day 0 and day 1 (3.9 microg/lung), significant elevation of Cd levels in the liver and kidneys was observed on both days. Two of 4 rats examined on day 0 showed elevation of blood cadmium, indicating systemic translocation of a fraction of deposited Cd from the lung in this group. These results and comparison with reported data using fine CdO particles indicate that inhalation of ultrafine CdO particles results in efficient deposition in the rat lung. With regard to the deposition dose, adverse health effects of ultrafine CdO and fine CdO appear to be comparable. Apparent systemic translocation of Cd took place only in animals exposed to a high concentration that induced lung injury.

Comparison of predicted with observed biokinetics of inhaled plutonium nitrate and gadolinium oxide in humans.

The absorption kinetics to blood of plutonium and gadolinium after inhalation as nitrate and oxide in humans and animals has been studied. For each material, values describing the time dependence of absorption were derived from the studies in animals and used with the ICRP human respiratory tract model to predict lung retention and cumulative amounts to blood for the volunteers inhaling the same materials. Comparison with the observed behaviour in the volunteers suggests that absorption of plutonium and gadolinium is reasonably species independent, and that data obtained from animal studies can be used to assess their biokinetic behaviour in humans.

RBDATA-EULEP: providing information to improve internal dosimetry.

The overall aim of the concerted action RBDATA-EULEP is to provide information to improve the assessments of intakes of radionuclides and of the resulting doses. This involves a review of the behaviour of radionuclides following intake, and the transfer of expertise on methodology by organising small training workshops. The main activity is the development of an electronic database, effectively an annotated bibliography, but the electronic format used facilitates extension, updating and information retrieval. It consists of linked tables of references and experiments, with details and comments on the materials, procedures and results. By June 2002 it contained information on 524 inhalation, 282 ingestion and 164 injection experiments from 391 references. It will be extended, and Internet access provided. Prospective users include groups developing standards for internal dosimetry, scientists conducting research on radionuclide biokinetics and health physicists assessing the consequences of accidental intakes.

Elemental composition and sources of fine and ultrafine ambient particles in Erfurt, Germany.

We present the first results of a source apportionment for the urban aerosol in Erfurt, Germany, for the period 1995-1998. The analysis is based on data of particle number concentrations (0.01-2.5 microm; mean 1.8 x 10(4) cm(-3), continuous), the concentration of the ambient gases SO(2), NO, NO(2) and CO (continuous), particle mass less than 2.5 microm (PM(2.5)) and less than 10 microm (PM(10)) (Harvard Impactor sampling, mean PM(2.5) 26.3 micro/m(3), mean PM(10) 38.2 microg/m(3)) and the size fractionated concentrations of 19 elements (impactor sampling 0.05-1.62 microm, PIXE analysis). We determined: (a) the correlations between (i) the 1- and 24-h average concentrations of the gaseous pollutants and the particle number as well as the particle mass concentration and (ii) between the 24-h elemental concentrations; (b) Crustal Enrichment Factors for the PIXE elements using Si as reference element; and (c) the diurnal pattern of the measured pollutants on weekdays and on weekends. The highly correlated PIXE elements Si, Al, Ti and Ca having low enrichment factors were identified as soil elements. The strong correlation of particle number concentrations with NO, which is considered to be typically emitted by traffic, and the striking similarity of their diurnal variation suggest that a sizable fraction of the particle number concentration is associated with emission from vehicles. Besides NO and particle number concentrations other pollutants such as NO(2), CO as well as the elements Zn and Cu were strongly correlated and appear to reflect motor vehicle traffic. Sulfur could be a tracer for coal combustion, however, it was not correlated with any of the quoted elements. Highly correlated elements V and Ni have similar enrichment factors and are considered as tracers for oil combustion.