Development of a multi-route physiologically based pharmacokinetic (PBPK) model for nanomaterials: a comparison between a traditional versus a new route-specific approach using gold nanoparticles in rats.

BACKGROUND: Physiologically based pharmacokinetic (PBPK) modeling is an important tool in predicting target organ dosimetry and risk assessment of nanoparticles (NPs). The methodology of building a multi-route PBPK model for NPs has not been established, nor systematically evaluated. In this study, we hypothesized that the traditional route-to-route extrapolation approach of PBPK modeling that is typically used for small molecules may not be appropriate for NPs. To test this hypothesis, the objective of this study was to develop a multi-route PBPK model for different sizes (1.4-200 nm) of gold nanoparticles (AuNPs) in adult rats following different routes of administration (i.e., intravenous (IV), oral gavage, intratracheal instillation, and endotracheal inhalation) using two approaches: a traditional route-to-route extrapolation approach for small molecules and a new approach that is based on route-specific data that we propose to be applied generally to NPs. RESULTS: We found that the PBPK model using this new approach had superior performance than the traditional approach. The final PBPK model was optimized rigorously using a Bayesian hierarchical approach with Markov chain Monte Carlo simulations, and then converted to a web-based interface using R Shiny. In addition, quantitative structure-activity relationships (QSAR) based multivariate linear regressions were established to predict the route-specific key biodistribution parameters (e.g., maximum uptake rate) based on the physicochemical properties of AuNPs (e.g., size, surface area, dose, Zeta potential, and NP numbers). These results showed the size and surface area of AuNPs were the main determinants for endocytic/phagocytic uptake rates regardless of the route of administration, while Zeta potential was an important parameter for the estimation of the exocytic release rates following IV administration. CONCLUSIONS: This study suggests that traditional route-to-route extrapolation approaches for PBPK modeling of small molecules are not applicable to NPs. Therefore, multi-route PBPK models for NPs should be developed using route-specific data. This novel PBPK-based web interface serves as a foundation for extrapolating to other NPs and to humans to facilitate biodistribution estimation, safety, and risk assessment of NPs.

Quantitative biokinetics over a 28 day period of freshly generated, pristine, 20 nm silver nanoparticle aerosols in healthy adult rats after a single 1½-hour inhalation exposure.

BACKGROUND: There is a steadily increasing quantity of silver nanoparticles (AgNP) produced for numerous industrial, medicinal and private purposes, leading to an increased risk of inhalation exposure for both professionals and consumers. Particle inhalation can result in inflammatory and allergic responses, and there are concerns about other negative health effects from either acute or chronic low-dose exposure. RESULTS: To study the fate of inhaled AgNP, healthy adult rats were exposed to 1½-hour intra-tracheal inhalations of pristine 105Ag-radiolabeled, 20 nm AgNP aerosols (with mean doses across all rats of each exposure group of deposited NP-mass and NP-number being 13.5 ± 3.6 µg, 7.9 ± 3.2•1011, respectively). At five time-points (0.75 h, 4 h, 24 h, 7d, 28d) post-exposure (p.e.), a complete balance of the [105Ag]AgNP fate and its degradation products were quantified in organs, tissues, carcass, lavage and body fluids, including excretions. Rapid dissolution of [105Ag]Ag-ions from the [105Ag]AgNP surface was apparent together with both fast particulate airway clearance and long-term particulate clearance from the alveolar region to the larynx. The results are compatible with evidence from the literature that the released [105Ag]Ag-ions precipitate rapidly to low-solubility [105Ag]Ag-salts in the ion-rich epithelial lining lung fluid (ELF) and blood. Based on the existing literature, the degradation products rapidly translocate across the air-blood-barrier (ABB) into the blood and are eliminated via the liver and gall-bladder into the small intestine for fecal excretion. The pathway of [105Ag]Ag-salt precipitates was compatible with auxiliary biokinetics studies at 24 h and 7 days after either intravenous injection or intratracheal or oral instillation of [110mAg]AgNO3 solutions in sentinel groups of rats. However, dissolution of [105Ag]Ag-ions appeared not to be complete after a few hours or days but continued over two weeks p.e. This was due to the additional formation of salt layers on the [105Ag]AgNP surface that mediate and prolonge the dissolution process. The concurrent clearance of persistent cores of [105Ag]AgNP and [105Ag]Ag-salt precipitates results in the elimination of a fraction > 0.8 (per ILD) after one week, each particulate Ag-species accounting for about half of this. After 28 days p.e. the cleared fraction rises marginally to 0.94 while 2/3 of the remaining [105Ag]AgNP are retained in the lungs and 1/3 in secondary organs and tissues with an unknown partition of the Ag species involved. However, making use of our previous biokinetics studies of poorly soluble [195Au]AuNP of the same size and under identical experimental and exposure conditions (Kreyling et al., ACS Nano 2018), the kinetics of the ABB-translocation of [105Ag]Ag-salt precipitates was estimated to reach a fractional maximum of 0.12 at day 3 p.e. and became undetectable 16 days p.e. Hence, persistent cores of [105Ag]AgNP were cleared throughout the study period. Urinary [105Ag]Ag excretion is minimal, finally accumulating to 0.016. CONCLUSION: The biokinetics of inhaled [105Ag]AgNP is relatively complex since the dissolving [105Ag]Ag-ions (a) form salt layers on the [105Ag]AgNP surface which retard dissolution and (b) the [105Ag]Ag-ions released from the [105Ag]AgNP surface form poorly-soluble precipitates of [105Ag]Ag-salts in ELF. Therefore, hardly any [105Ag]Ag-ion clearance occurs from the lungs but instead [105Ag]AgNP and nano-sized precipitated [105Ag]Ag-salt are cleared via the larynx into GIT and, in addition, via blood, liver, gall bladder into GIT with one common excretional pathway via feces out of the body.

Quantitative biokinetics over a 28 day period of freshly generated, pristine, 20 nm titanium dioxide nanoparticle aerosols in healthy adult rats after a single two-hour inhalation exposure.

BACKGROUND: Industrially produced quantities of TiO2 nanoparticles are steadily rising, leading to an increasing risk of inhalation exposure for both professionals and consumers. Particle inhalation can result in inflammatory and allergic responses, and there are concerns about other negative health effects from either acute or chronic low-dose exposure. RESULTS: To study the fate of inhaled TiO2-NP, adult rats were exposed to 2-h intra-tracheal inhalations of 48V-radiolabeled, 20 nm TiO2-NP aerosols (deposited NP-mass 1.4 ± 0.5 µg). At five time points (1 h, 4 h, 24 h, 7d, 28d) post-exposure, a complete balance of the [48V]TiO2-NP fate was quantified in organs, tissues, carcass, lavage and body fluids, including excretions. After fast mucociliary airway clearance (fractional range 0.16-0.31), long-term macrophage-mediated clearance (LT-MC) from the alveolar region is 2.6-fold higher after 28d (integral fraction 0.40 ± 0.04) than translocation across the air-blood-barrier (integral fraction 0.15 ± 0.01). A high NP fraction remains in the alveoli (0.44 ± 0.05 after 28d), half of these on the alveolar epithelium and half in interstitial spaces. There is clearance from both retention sites at fractional rates (0.02-0.03 d- 1) by LT-MC. Prior to LT-MC, [48V]TiO2-NP are re-entrained to the epithelium as reported earlier for 20 nm inhaled gold-NP (AuNP) and iridium-NP (IrNP). CONCLUSION: Comparing the 28-day biokinetics patterns of three different inhaled NP materials TiO2-NP, AuNP and IrNP, the long-term kinetics of interstitial relocation and subsequent re-entrainment onto the lung-epithelium is similar for AuNP and Ir-NP but slower than for TiO2-NP. We discuss mechanisms and pathways of NP relocation and re-entrainment versus translocation. Additionally, after 28 days the integral translocated fractions of TiO2-NP and IrNP across the air-blood-barrier (ABB) are similar and become 0.15 while the translocated AuNP fraction is only 0.04. While NP dissolution proved negligible, translocated TiO2-NP and IrNP are predominantly excreted in urine (~ 0.1) while the urinary AuNP excretion amounts to a fraction of only 0.01. Urinary AuNP excretion is below 0.0001 during the first week but rises tenfold thereafter suggesting delayed disagglomeration. Of note, all three NP dissolve minimally, since no ionic radio-label release was detectable. These biokinetics data of inhaled, same-sized NP suggest significant time-dependent differences of the ABB translocation and subsequent fate in the organism.

Early pulmonary response is critical for extra-pulmonary carbon nanoparticle mediated effects: comparison of inhalation versus intra-arterial infusion exposures in mice.

BACKGROUND: The death toll associated with inhaled ambient particulate matter (PM) is attributed mainly to cardio-vascular rather than pulmonary effects. However, it is unclear whether the key event for cardiovascular impairment is particle translocation from lung to circulation (direct effect) or indirect effects due to pulmonary particle-cell interactions. In this work, we addressed this issue by exposing healthy mice via inhalation and intra-arterial infusion (IAI) to carbon nanoparticles (CNP) as surrogate for soot, a major constituent of (ultrafine) urban PM. METHODS: Equivalent surface area CNP doses in the blood (30mm2 per animal) were applied by IAI or inhalation (lung-deposited dose 10,000mm2; accounting for 0.3% of lung-to-blood CNP translocation). Mice were analyzed for changes in hematology and molecular markers of endothelial/epithelial dysfunction, pro-inflammatory reactions, oxidative stress, and coagulation in lungs and extra-pulmonary organs after CNP inhalation (4 h and 24 h) and CNP infusion (4 h). For methodological reasons, we used two different CNP types (spark-discharge and Printex90), with very similar physicochemical properties [≥98 and ≥95% elemental carbon; 10 and 14 nm primary particle diameter; and 800 and 300 m2/g specific surface area] for inhalation and IAI respectively. RESULTS: Mild pulmonary inflammatory responses and significant systemic effects were observed following 4 h and 24 h CNP inhalation. Increased retention of activated leukocytes, secondary thrombocytosis, and pro-inflammatory responses in secondary organs were detected following 4 h and 24 h of CNP inhalation only. Interestingly, among the investigated extra-pulmonary tissues (i.e. aorta, heart, and liver); aorta revealed as the most susceptible extra-pulmonary target following inhalation exposure. Bypassing the lungs by IAI however did not induce any extra-pulmonary effects at 4 h as compared to inhalation. CONCLUSIONS: Our findings indicate that extra-pulmonary effects due to CNP inhalation are dominated by indirect effects (particle-cell interactions in the lung) rather than direct effects (translocated CNPs) within the first hours after exposure. Hence, CNP translocation may not be the key event inducing early cardiovascular impairment following air pollution episodes. The considerable response detected in the aorta after CNP inhalation warrants more emphasis on this tissue in future studies.

Toxic effects and biodistribution of ultrasmall gold nanoparticles.

Gold nanoparticles (AuNPs) have been extensively explored in biomedical applications, for example as drug carriers, contrast agents, or therapeutics. However, AuNP can exhibit cytotoxic profile, when the size is below 2 nm (ultrasmall AuNP; usAuNP) and when the stabilizing ligands allow for access to the gold surface either for the direct interaction with biomolecules or for catalytic activity of the unshielded gold surface. Furthermore, usAuNP exhibits significantly different biodistribution and enhanced circulation times compared to larger AuNP. This review gives an overview about the synthesis and the physico-chemical properties of usAuNP and, thereby, focusses on 1.4 nm sized AuNP, which are derived from the compound Au55(PPh3)12Cl6 and which are the most intensively studied usAuNP in the field. This part is followed by a summary of the toxic properties of usAuNP, which include in vitro cytotoxicity tests on different cell lines, electrophysiological tests following FDA guidelines as well as studies on antibacterial effects. Finally, the biodistribution and pharmacokinetics of ultrasmall AuNP are discussed and compared to the properties of more biocompatible, larger AuNP.

Discovery of unique and ENM- specific pathophysiologic pathways: Comparison of the translocation of inhaled iridium nanoparticles from nasal epithelium versus alveolar epithelium towards the brain of rats.

The biokinetics of inhaled nanoparticles (NP) is more complex than that of larger particles since NP may NP deposited on the nasal mucosa of the upper respiratory tract (URT) may translocate to the olfactory bulb of the brain and also via the trigeminus (URT neuronal route); and (b) NP deposited in the lower respiratory tract (LRT) may cross the ABB into blood and enter the brain across the blood-brain-barrier (BBB) or take a neuronal route from enervated tracheo-bronchial epithelia via the vagus nerve. Translocation from both - the URT and the LRT - are quantified during the first 24h after a 1-hour aerosol inhalation of 20nm-sized, (192)Ir radiolabeled iridium NP by healthy adult rats using differential exposures: (I) nose-only exposure of the entire respiratory tract or (II) intratracheal (IT) inhalation of intubated and ventilated rats, thereby bypassing the URT and extrathoracic nasal passages. After nose-only exposure brain accumulation (BrAcc) is significantly nine-fold higher than after IT inhalation since the former results from both pathways (a+b) while the latter exposure comes only from pathway (b). Interestingly, there are significantly more circulating NP in blood 24h after nose-only inhalation than after IT inhalation. Distinguishing translocation from URT versus LRT estimated from the differential inhalation exposures, the former is significantly higher (8-fold) than from the LRT. Although the BrAcc fraction is rather low compared to total NP deposition after this short-term exposure, this study proofs that inhaled insoluble NP can accumulate in the brain from both - URT and LRT which may trigger and/or modulate adverse health effects in the central nervous system (CNS) during chronic exposure.

Nanoparticle delivery in infant lungs.

The lung surface is an ideal pathway to the bloodstream for nanoparticle-based drug delivery. Thus far, research has focused on the lungs of adults, and little is known about nanoparticle behavior in the immature lungs of infants. Here, using nonlinear dynamical systems analysis and in vivo experimentation in developing animals, we show that nanoparticle deposition in postnatally developing lungs peaks at the end of bulk alveolation. This finding suggests a unique paradigm, consistent with the emerging theory that as alveoli form through secondary septation, alveolar flow becomes chaotic and chaotic mixing kicks in, significantly enhancing particle deposition. This finding has significant implications for the application of nanoparticle-based inhalation therapeutics in young children with immature lungs from birth to 2 y of age.

Differences in the biokinetics of inhaled nano- versus micrometer-sized particles.

Researchers need to study the biokinetics of inhaled biopersistent nano- and micrometer-sized particles (NPs and µPs) to assess their toxicity and to develop an understanding of their potential risks. When particles are inhaled, they do not necessarily remain at their sites of deposition in the respiratory tract. Instead they can undergo numerous transport processes within the various tissues of the lungs, including clearance from the lungs. In this context, we would like to understand how the biokinetic studies performed in animals can be extrapolated to humans. Interestingly, the particle retention is much shorter in rodent lungs and declines much faster than it does in human, simian, and canine lungs. The predominant long-term clearance pathway for both NPs and µPs in humans and other animal species is macrophage-mediated particle transport from the peripheral lungs toward ciliated airways and the larynx. However, the transport rate is 10 times higher in rodents than in other species. In addition to particle clearance out of the lung, we also observe particle redistribution from the epithelium toward and within the interstitium and lymph nodes of the lung and particle translocation to blood circulation leading to subsequent accumulation in secondary organs. While µPs have limited access to interstitial spaces in the rodent lungs, NPs rapidly relocate in the epithelium and the underlying interstitium. By contrast, indirect evidence shows that both NPs and µPs are relocated into the epithelium and interstitial spaces of the human, simian, and canine lungs. Only NPs translocate into the circulatory system and subsequently accumulate in the secondary organs and tissues of the body. Translocated NP fractions are rather low, but they depend strongly on the physicochemical properties of the NP and their surface properties. Growing evidence indicates that the binding and conjugation of proteins to NPs play an essential role in translocation across cellular membranes and organ barriers. In summary, particle biokinetics result from a multitude of highly dynamic processes, which depend not only on physicochemical properties of the particles but also on a multitude of cellular and molecular responses and interactions. Given the rather small accumulation in secondary organs after acute inhalation exposures, it appears likely that adverse effects caused by NPs accumulated in secondary organs may only occur after chronic exposure over extended time periods. Therefore adverse health effects in secondary organs such as the cardiovascular system that are associated with chronic exposure of ambient urban air pollution are less likely to result from particle translocation. Instead, chronic particle inhalation could trigger or modulate the autonomous nervous system or the release of soluble mediators into circulation leading to adverse health effects.

Size dependent translocation and fetal accumulation of gold nanoparticles from maternal blood in the rat.

BACKGROUND: There is evidence that nanoparticles (NP) cross epithelial and endothelial body barriers. We hypothesized that gold (Au) NP, once in the blood circulation of pregnant rats, will cross the placental barrier during pregnancy size-dependently and accumulate in the fetal organism by 1. transcellular transport across the hemochorial placenta, 2. transcellular transport across amniotic membranes 3. transport through ~20 nm wide transtrophoblastic channels in a size dependent manner. The three AuNP sizes used to test this hypothesis are either well below, or of similar size or well above the diameters of the transtrophoblastic channels. METHODS: We intravenously injected monodisperse, negatively charged, radio-labelled 1.4 nm, 18 nm and 80 nm ¹98AuNP at a mass dose of 5, 3 and 27 µg/rat, respectively, into pregnant rats on day 18 of gestation and in non-pregnant control rats and studied the biodistribution in a quantitative manner based on the radio-analysis of the stably labelled ¹98AuNP after 24 hours. RESULTS: We observed significant biokinetic differences between pregnant and non-pregnant rats. AuNP fractions in the uterus of pregnant rats were at least one order of magnitude higher for each particle size roughly proportional to the enlarged size and weight of the pregnant uterus. All three sizes of ¹98AuNP were found in the placentas and amniotic fluids with 1.4 nm AuNP fractions being two orders of magnitude higher than those of the larger AuNP on a mass base. In the fetuses, only fractions of 0.0006 (30 ng) and 0.00004 (0.1 ng) of 1.4 nm and 18 nm AuNP, respectively, were detected, but no 80 nm AuNP (<0.000004 (<0.1 ng)). These data show that no AuNP entered the fetuses from amniotic fluids within 24 hours but indicate that AuNP translocation occurs across the placental tissues either through transtrophoblastic channels and/or via transcellular processes. CONCLUSION: Our data suggest that the translocation of AuNP from maternal blood into the fetus is NP-size dependent which is due to mechanisms involving (1) transport through transtrophoblastic channels - also present in the human placenta - and/or (2) endocytotic and diffusive processes across the placental barrier.

Biokinetics of nanoparticles and susceptibility to particulate exposure in a murine model of cystic fibrosis.

BACKGROUND: Persons with cystic fibrosis (CF) are at-risk for health effects from ambient air pollution but little is known about the interaction of nanoparticles (NP) with CF lungs. Here we study the distribution of inhaled NP in a murine CF model and aim to reveal mechanisms contributing to adverse effects of inhaled particles in susceptible populations. METHODS: Chloride channel defective CftrTgH (neoim) Hgu mice were used to analyze lung function, lung distribution and whole body biokinetics of inhaled NP, and inflammatory responses after intratracheal administration of NP. Distribution of 20-nm titanium dioxide NP in lungs was assessed on ultrathin sections immediately and 24 h after a one-hour NP inhalation. NP biokinetics was deduced from total and regional lung deposition and from whole body translocation of inhaled 30-nm iridium NP within 24 h after aerosol inhalation. Inflammatory responses were assessed within 7 days after carbon NP instillation. RESULTS: Cftr mutant females had moderately reduced lung compliance and slightly increased airway resistance compared to wild type mice. We found no genotype dependent differences in total, regional and head deposition or in secondary-organ translocation of inhaled iridium NP. Titanium dioxide inhalation resulted in higher NP uptake by alveolar epithelial cells in Cftr mutants. Instillation of carbon NP induced a comparable acute and transient inflammatory response in both genotypes. The twofold increase of bronchoalveolar lavage (BAL) neutrophils in Cftr mutant compared to wild type mice at day 3 but not at days 1 and 7, indicated an impaired capacity in inflammation resolution in Cftr mutants. Concomitant to the delayed decline of neutrophils, BAL granulocyte-colony stimulating factor was augmented in Cftr mutant mice. Anti-inflammatory 15-hydroxyeicosatetraenoic acid was generally significantly lower in BAL of Cftr mutant than in wild type mice. CONCLUSIONS: Despite lacking alterations in lung deposition and biokinetics of inhaled NP, and absence of significant differences in lung function, higher uptake of NP by alveolar epithelial cells and prolonged, acute inflammatory responses to NP exposure indicate a moderately increased susceptibility of lungs to adverse effects of inhaled NP in Cftr mutant mice and provides potential mechanisms for the increased susceptibility of CF patients to air pollution.

Serum protein identification and quantification of the corona of 5, 15 and 80 nm gold nanoparticles.

When nanoparticles (NP) enter the body they come into contact with body fluids containing proteins which can adsorb to their surface. These proteins may influence the NP interactions with the biological vicinity, eventually determining their biological fate inside the body. Adsorption of the most abundantly binding proteins was studied after an in vitro 24 hr incubation of monodisperse, negatively charged 5, 15 and 80 nm gold spheres (AuNP) in mouse serum by a two-step analysis: proteomic protein identification and quantitative protein biochemistry. The adsorbed proteins were separated from non-adsorbed proteins by centrifugation and gel electrophoresis and identified using a MALDI-TOF-MS-Proteomics-Analyzer. Quantitative analysis of proteins in gel bands by protein densitometry, required the focus on predominantly binding serum proteins. Numerous proteins adsorbed to the AuNP depending on their size, e.g., apolipoproteins or complement C3. The qualitative and quantitative amount of adsorbed proteins differed between 5, 15 and 80 nm AuNP. Band intensities of adsorbed proteins decreased with increasing AuNP sizes based not only on their mass but also on their surface area. Summarizing, the AuNP surface is covered with serum proteins containing transport and immune related proteins among others. Hence, protein binding depends on the size, surface area and curvature of the AuNP.

Pulmonary surfactant is indispensable in order to simulate the in vivo situation.

The article of Gasser et al. [Part Fibre Toxicol. 24; 9:17, 2012] describes the interaction of carbon nanotubes with cells within a complex cell culture model. Besides various toxicity parameters, the influence of coating with pulmonary surfactant was investigated. Pulmonary surfactant covers the entire alveolar region with the main function of decreasing the surface tension in the alveoli to prevent alveolar collapse. Although each inhaled nanoparticle, reaching the alveoli, will come into contact with pulmonary surfactant which will probably lead to a surfactant coating, pulmonary surfactant components are not commonly integrated in in vitro systems. Gasser and co-workers have shown that this surfactant coating is able to influence the further interaction with cellular systems. Hence, each scientist, working with in vitro systems and nanoparticles, should think of integrating pulmonary surfactant structures in order to harmonize the in vitro systems with the in vivo situation. In the present commentary we discuss the most important points of the manuscript of Gasser et al. and discuss where the usage of pulmonary surfactant can be further optimized.

Cellular uptake and localization of inhaled gold nanoparticles in lungs of mice with chronic obstructive pulmonary disease.

BACKGROUND: Inhalative nanocarriers for local or systemic therapy are promising. Gold nanoparticles (AuNP) have been widely considered as candidate material. Knowledge about their interaction with the lungs is required, foremost their uptake by surface macrophages and epithelial cells. METHODS: Scnn1b-Tg and Wt mice inhaled a 21-nm AuNP aerosol for 2 h. Immediately (0 h) or 24 h thereafter, bronchoalveolar lavage (BAL) macrophages and whole lungs were prepared for stereological analysis of AuNP by electron microscopy. RESULTS: AuNP were mainly found as singlets or small agglomerates of ≤ 100 nm diameter, at the epithelial surface and within lung-surface structures. Macrophages contained also large AuNP agglomerates (> 100 nm). At 0 h after aerosol inhalation, 69.2±4.9% AuNP were luminal, i.e. attached to the epithelial surface and 24.0±5.9% in macrophages in Scnn1b-Tg mice. In Wt mice, 35.3±32.2% AuNP were on the epithelium and 58.3±41.4% in macrophages. The percentage of luminal AuNP decreased from 0 h to 24 h in both groups. At 24 h, 15.5±4.8% AuNP were luminal, 21.4±14.2% within epithelial cells and 63.0±18.9% in macrophages in Scnn1b-Tg mice. In Wt mice, 9.5±5.0% AuNP were luminal, 2.2±1.6% within epithelial cells and 82.8±0.2% in macrophages. BAL-macrophage analysis revealed enhanced AuNP uptake in Wt animals at 0 h and in Scnn1b-Tg mice at 24 h, confirming less efficient macrophage uptake and delayed clearance of AuNP in Scnn1b-Tg mice. CONCLUSIONS: Inhaled AuNP rapidly bound to the alveolar epithelium in both Wt and Scnn1b-Tg mice. Scnn1b-Tg mice showed less efficient AuNP uptake by surface macrophages and concomitant higher particle internalization by alveolar type I epithelial cells compared to Wt mice. This likely promotes AuNP depth translocation in Scnn1b-Tg mice, including enhanced epithelial targeting. These results suggest AuNP nanocarrier delivery as successful strategy for therapeutic targeting of alveolar epithelial cells and macrophages in COPD.

The procoagulant effects of fine particulate matter in vivo.

Inhalation of fine particulate matter (<2.5 µm; fine PM) has been shown to increase the risk for cardiovascular events. In this letter, we reappraise the role of tissue factor (TF) antigen and we also summarize changes in measured coagulation proteins in humans and rodents by other studies with fine PM. By considering all studies including ours, we conclude that monitoring the overall coagulation state by measuring capacity assays such as thrombin generation, and quantification of TF activity would be more suitable than determining single coagulation proteins (such as TF antigen) in order to better assess the systemic prothrombotic effects of fine PM.

Binding of polystyrene and carbon black nanoparticles to blood serum proteins.

CONTEXT: Once inhaled, nanoparticles (NP) deposit on the lung surface and have first contact with the epithelial lung lining fluid (ELF) rich in proteins, which may bind to NP. OBJECTIVE: In this study, we investigate the parameters that influence the binding between NP and proteins. MATERIALS AND METHODS: We used the proteins albumin, transferrin (TF), and apolipoprotein A-1 (all known as proteins from ELF) and different NP (polystyrene NP with negative, positive, and neutral surface coatings, Printex G and Printex 90) as models. RESULTS: In all cases, a linear correlation of the added NP amount and the amount of bound proteins was found and was described quantitatively by binding indices. Bovine serum albumin (BSA), TF, and apo A-1 were bound to the largest extent to hydrophobic NP, which shows the extraordinary importance of the NP's surface properties. DISCUSSION: The binding index indicates the relevance of primary particle size and surface properties, including hydrophobicity. CONCLUSION: Size and surface modifications of NP determine their protein binding. Our results suggest that the formation of conjugates of BSA, TF, and Apo A-1 with NP may play an important role in their translocation across the air-blood-barrier and subsequent biokinetics.

Deposition and biokinetics of inhaled nanoparticles.

Particle biokinetics is important in hazard identification and characterization of inhaled particles. Such studies intend to convert external to internal exposure or biologically effective dose, and may help to set limits in that way. Here we focus on the biokinetics of inhaled nanometer sized particles in comparison to micrometer sized ones.The presented approach ranges from inhaled particle deposition probability and retention in the respiratory tract to biokinetics and clearance of particles out of the respiratory tract. Particle transport into the blood circulation (translocation), towards secondary target organs and tissues (accumulation), and out of the body (clearance) is considered. The macroscopically assessed amount of particles in the respiratory tract and secondary target organs provides dose estimates for toxicological studies on the level of the whole organism. Complementary, microscopic analyses at the individual particle level provide detailed information about which cells and subcellular components are the target of inhaled particles. These studies contribute to shed light on mechanisms and modes of action eventually leading to adverse health effects by inhaled nanoparticles.We review current methods for macroscopic and microscopic analyses of particle deposition, retention and clearance. Existing macroscopic knowledge on particle biokinetics and microscopic views on particle organ interactions are discussed comparing nanometer and micrometer sized particles. We emphasize the importance for quantitative analyses and the use of particle doses derived from real world exposures.

Cardiovascular and inflammatory effects of intratracheally instilled ambient dust from Augsburg, Germany, in spontaneously hypertensive rats (SHRs).

RATIONALE: Several epidemiological studies associated exposure to increased levels of particulate matter in Augsburg, Germany with cardiovascular mortality and morbidity. To elucidate the mechanisms of cardiovascular impairments we investigated the cardiopulmonary responses in spontaneously hypertensive rats (SHR), a model for human cardiovascular diseases, following intratracheal instillation of dust samples from Augsburg. METHODS: 250 µg, 500 µg and 1000 µg of fine ambient particles (aerodynamic diameter <2.5 µm, PM2(.)5-AB) collected from an urban background site in Augsburg during September and October 2006 (PM2(.)5 18.2 µg/m³, 10,802 particles/cm3) were instilled in 12 months old SHRs to assess the inflammatory response in bronchoalveolar lavage fluid (BALF), blood, lung and heart tissues 1 and 3 days post instillation. Radio-telemetric analysis was performed to investigate the cardiovascular responses following instillation of particles at the highest dosage based on the inflammatory response observed. RESULTS: Exposure to 1000 µg of PM2(.)5-AB was associated with a delayed increase in delta mean blood pressure (ΔmBP) during 2(nd)-4(th) day after instillation (10.0 ± 4.0 vs. -3.9 ± 2.6 mmHg) and reduced heart rate (HR) on the 3rd day post instillation (325.1 ± 8.8 vs. 348.9 ± 12.5 bpm). BALF cell differential and inflammatory markers (osteopontin, interleukin-6, C-reactive protein, and macrophage inflammatory protein-2) from pulmonary and systemic level were significantly induced, mostly in a dose-dependent way. Protein analysis of various markers indicate that PM2(.)5-AB instillation results in an activation of endothelin system (endothelin1), renin-angiotensin system (angiotensin converting enzyme) and also coagulation system (tissue factor, plasminogen activator inhibitor-1) in pulmonary and cardiac tissues during the same time period when alternation in ΔmBP and HR have been detected. CONCLUSIONS: Our data suggests that high concentrations of PM2(.)5-AB exposure triggers low grade PM mediated inflammatory effects in the lungs but disturbs vascular homeostasis in pulmonary tissues and on a systemic level by affecting the renin angiotensin system, the endothelin system and the coagulation cascade. These findings are indicative for promotion of endothelial dysfunction, atherosclerotic lesions, and thrombogeneis and, thus, provide plausible evidence that susceptible-predisposed individuals may develop acute cardiac events like myocardial infarction when repeatedly exposed to high pollution episodes as observed in epidemiological studies in Augsburg, Germany.

Deep pulmonary lymphatics in immature lungs.

Recently, we found that the translocation of inhaled nanoparticles from the air space to secondary organs is age dependent and substantially greater in neonates than in adults (J Respir Crit Care Med 177: A48, 2008). One reason for this difference might be age-dependent differences in alveolar barrier integrity. Because the neonate lung is undergoing morphogenetic and fluid balance changes, we hypothesize that the alveolar barrier of developing lungs is more easily compromised and susceptible to foreign material influx than that of adult lungs. On the basis of these hypotheses, we predict that the postnatally developing lung is also more likely to allow the translocation of some materials from the air space to the lymphatic lumens. To test this idea, we intratracheally instilled methyl methacrylate into immature and adult lungs and compared lymphatic filling between these two age groups. Scanning electron microscopy of the resultant corrosion casts revealed peribronchial saccular and conduit lymphatic architecture. Deep pulmonary lymphatic casts were present on the majority (58.5%) of airways in immature lungs, but lymphatic casting in adult lungs, as anticipated, was much more infrequent (21.6%). Thus the neonate lung appears to be more susceptible than the adult lung to the passage of instilled methyl methacrylate from the air space into the lymphatics. We speculate that this could imply greater probability of translocation of other materials, such as nanoparticles, from the immature lung as well.

Soluble iron modulates iron oxide particle-induced inflammatory responses via prostaglandin E(2 )synthesis: In vitro and in vivo studies.

BACKGROUND: Ambient particulate matter (PM)-associated metals have been shown to play an important role in cardiopulmonary health outcomes. To study the modulation of PM-induced inflammation by leached off metals, we investigated intracellular solubility of radio-labeled iron oxide ((59)Fe(2)O(3)) particles of 0.5 and 1.5 mum geometric mean diameter. Fe(2)O(3 )particles were examined for the induction of the release of interleukin 6 (IL-6) as pro-inflammatory and prostaglandin E(2 )(PGE(2)) as anti-inflammatory markers in cultured alveolar macrophages (AM) from Wistar Kyoto (WKY) rats. In addition, we exposed male WKY rats to monodispersed Fe(2)O(3 )particles by intratracheal instillation (1.3 or 4.0 mg/kg body weight) to examine in vivo inflammation. RESULTS: Particles of both sizes are insoluble extracellularly in the media but moderately soluble in AM with an intracellular dissolution rate of 0.0037 +/- 0.0014 d(-1 )for 0.5 mum and 0.0016 +/- 0.0012 d(-1 )for 1.5 mum (59)Fe(2)O(3 )particles. AM exposed in vitro to 1.5 mum particles (10 mug/mL) for 24 h increased IL-6 release (1.8-fold; p < 0.05) and also PGE(2 )synthesis (1.9-fold; p < 0.01). By contrast, 0.5 mum particles did not enhance IL-6 release but strongly increased PGE(2 )synthesis (2.5-fold, p < 0.005). Inhibition of PGE(2 )synthesis by indomethacin caused a pro-inflammatory phenotype as noted by increased IL-6 release from AM exposed to 0.5 mum particles (up to 3-fold; p < 0.005). In the rat lungs, 1.5 but not 0.5 mum particles (4.0 mg/kg) induced neutrophil influx and increased vascular permeability. CONCLUSIONS: Fe(2)O(3 )particle-induced neutrophilic inflammatory response in vivo and pro-inflammatory cytokine release in vitro might be modulated by intracellular soluble iron via PGE(2 )synthesis. The suppressive effect of intracellular released soluble iron on particle-induced inflammation has implications on how ambient PM-associated but soluble metals influence pulmonary toxicity of ambient PM.

Expert elicitation on ultrafine particles: likelihood of health effects and causal pathways.

BACKGROUND: Exposure to fine ambient particulate matter (PM) has consistently been associated with increased morbidity and mortality. The relationship between exposure to ultrafine particles (UFP) and health effects is less firmly established. If UFP cause health effects independently from coarser fractions, this could affect health impact assessment of air pollution, which would possibly lead to alternative policy options to be considered to reduce the disease burden of PM. Therefore, we organized an expert elicitation workshop to assess the evidence for a causal relationship between exposure to UFP and health endpoints. METHODS: An expert elicitation on the health effects of ambient ultrafine particle exposure was carried out, focusing on: 1) the likelihood of causal relationships with key health endpoints, and 2) the likelihood of potential causal pathways for cardiac events. Based on a systematic peer-nomination procedure, fourteen European experts (epidemiologists, toxicologists and clinicians) were selected, of whom twelve attended. They were provided with a briefing book containing key literature. After a group discussion, individual expert judgments in the form of ratings of the likelihood of causal relationships and pathways were obtained using a confidence scheme adapted from the one used by the Intergovernmental Panel on Climate Change. RESULTS: The likelihood of an independent causal relationship between increased short-term UFP exposure and increased all-cause mortality, hospital admissions for cardiovascular and respiratory diseases, aggravation of asthma symptoms and lung function decrements was rated medium to high by most experts. The likelihood for long-term UFP exposure to be causally related to all cause mortality, cardiovascular and respiratory morbidity and lung cancer was rated slightly lower, mostly medium. The experts rated the likelihood of each of the six identified possible causal pathways separately. Out of these six, the highest likelihood was rated for the pathway involving respiratory inflammation and subsequent thrombotic effects. CONCLUSION: The overall medium to high likelihood rating of causality of health effects of UFP exposure and the high likelihood rating of at least one of the proposed causal mechanisms explaining associations between UFP and cardiac events, stresses the importance of considering UFP in future health impact assessments of (transport-related) air pollution, and the need for further research on UFP exposure and health effects.