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.

Unpredictable Nanoparticle Retention in Commonly Used Plastic Syringes Introduces Dosage Uncertainties That May Compromise the Accuracy of Nanomedicine and Nanotoxicology Studies.

In recent animal experiments with suspensions of radiolabeled TiO2 nanoparticles large and highly variable radioactivity fractions were retained in disposable plastic syringes. After unloading between 10% and up to 70% of the loaded dose were still present in the syringes. As a consequence the effectively delivered nanoparticle dose to the animals was frequently much smaller than the nominal dose of the nanoparticles loaded into the syringe. The high variability of this nanoparticle retention challenges the application of a precise, predefined dose and creates a major error source when normalizing organ and tissue contents to the dose loaded into the syringe, which is usually set as the applied dose. A control study was performed employing six commonly used syringe types with seven types of radiolabeled oxide and metallic nanoparticles. For this purpose the syringes were loaded with a given volume of nanoparticle suspension, the radioactivity was measured, the syringe was unloaded and the activity measurement was repeated with the empty syringe. The highest retention values were found when using TiO2 nanoparticle suspensions with Tuberkulin type syringes. In the worst case between 6.6% and 79.1% of the nanoparticles were retained in the syringe. When using the same nanoparticle suspension with an insulin-type syringe the retention was reduced to 1.4% to 20.6%. For amorphous silica nanoparticles the maximum observed retention was 8% and for Au nanoparticles it was 5.1%. Further data gathered from in vivo animal imaging studies show that nanoparticle retention in syringes also affects experiments with nanoparticles such as exosomes, polymersomes, and protein-based nanoparticles investigated for possible applications in nanomedicine. Since the retention is highly variable the effectively applied dose cannot be determined by applying a simple syringe retention factor. The present work shall alert to the problem and illustrate its possible magnitude and unpredictable variability. As mitigation strategy adequate checks with different syringe types are proposed in order to find out whether a given combination of syringe type and nanoparticle suspension is affected by nanoparticle retention and, if necessary, to select a different syringe type that minimizes retention.

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.

Correction to: Particle toxicology and health - where are we?

After the publication of this article [1] it was hihglighted that the number of deaths related to natural disasters was incorrectly reported in the second paragraph of the Hazards from Natural particulates and the evolution of the biosphere section. This correction article shows the correct and incorrect statement. This correction does not change the idea presented in the article that from an evolutionary view point, natural disasters account only for a small fraction of the people on the planet. The original article has been updated.

Particle toxicology and health - where are we?

BACKGROUND: Particles and fibres affect human health as a function of their properties such as chemical composition, size and shape but also depending on complex interactions in an organism that occur at various levels between particle uptake and target organ responses. While particulate pollution is one of the leading contributors to the global burden of disease, particles are also increasingly used for medical purposes. Over the past decades we have gained considerable experience in how particle properties and particle-bio interactions are linked to human health. This insight is useful for improved risk management in the case of unwanted health effects but also for developing novel medical therapies. The concepts that help us better understand particles' and fibres' risks include the fate of particles in the body; exposure, dosimetry and dose-metrics and the 5 Bs: bioavailability, biopersistence, bioprocessing, biomodification and bioclearance of (nano)particles. This includes the role of the biomolecule corona, immunity and systemic responses, non-specific effects in the lungs and other body parts, particle effects and the developing body, and the link from the natural environment to human health. The importance of these different concepts for the human health risk depends not only on the properties of the particles and fibres, but is also strongly influenced by production, use and disposal scenarios. CONCLUSIONS: Lessons learned from the past can prove helpful for the future of the field, notably for understanding novel particles and fibres and for defining appropriate risk management and governance approaches.

Age-Dependent Rat Lung Deposition Patterns of Inhaled 20 Nanometer Gold Nanoparticles and their Quantitative Biokinetics in Adult Rats.

The increasing use of gold nanoparticles leads to a possible increase of exposure by inhalation. Therefore, we have studied the deposition patterns of inhaled 20 nm gold nanoparticles (AuNP) in 7-90 day old rats and their biokinetics in 60 day old ones. Wistar-Kyoto rats inhaled intratracheally 20 nm 195Au-radiolabeled AuNP by negative pressure ventilation over 2 h. Immediately afterward lungs were excised, inflated and microwave dried. AuNP deposition was analyzed by single-photon emission computed tomography, computed-tomography and autoradiography. Completely balanced, quantitative biodistributions in major organs and all body tissues and total excretion were analyzed from 1 h to 28 d after inhalation. Intratracheal inhalation caused AuNP deposition predominately in the caudal lungs, independent of age. About 30% AuNP were deposited on airway epithelia and rapidly cleared by mucociliary clearance. About 80% of AuNP deposited in alveoli was relocated from the epithelium into the interstitium within 24 h and was inaccessible to broncho-alveolar lavage. During interstitial long-term retention, re-entrainment within macrophages back onto the lung epithelium and to the larynx and gastrointestinal tract (GIT) dominated AuNP clearance (rate 0.03 d-1) In contrast, AuNP-translocation across the air-blood barrier was much smaller leading to persistent retention in secondary organs and tissues in the ranking order liver > soft issue > spleen > kidneys > skeleton > blood > uterus > heart > brain. The age-independent, inhomogeneous AuNP deposition was probably caused by the negative pressure ventilation. Long-term AuNP clearance was dominated by macrophage-mediated transport from the interstitium to the larynx and GIT. Translocation across the rat air-blood barrier appeared to be similar to that of humans for similar sized AuNP.

Protein corona: implications for nanoparticle interactions with pulmonary cells.

BACKGROUND: We previously showed that cerium oxide (CeO2), barium sulfate (BaSO4) and zinc oxide (ZnO) nanoparticles (NPs) exhibited different lung toxicity and pulmonary clearance in rats. We hypothesize that these NPs acquire coronas with different protein compositions that may influence their clearance from the lungs. METHODS: CeO2, silica-coated CeO2, BaSO4, and ZnO NPs were incubated in rat lung lining fluid in vitro. Then, gel electrophoresis followed by quantitative mass spectrometry was used to characterize the adsorbed proteins stripped from these NPs. We also measured uptake of instilled NPs by alveolar macrophages (AMs) in rat lungs using electron microscopy. Finally, we tested whether coating of gold NPs with albumin would alter their lung clearance in rats. RESULTS: We found that the amounts of nine proteins in the coronas formed on the four NPs varied significantly. The amounts of albumin, transferrin and α-1 antitrypsin were greater in the coronas of BaSO4 and ZnO than that of the two CeO2 NPs. The uptake of BaSO4 in AMs was less than CeO2 and silica-coated CeO2 NPs. No identifiable ZnO NPs were observed in AMs. Gold NPs coated with albumin or citrate instilled into the lungs of rats acquired the similar protein coronas and were cleared from the lungs to the same extent. CONCLUSIONS: We show that different NPs variably adsorb proteins from the lung lining fluid. The amount of albumin in the NP corona varies as does NP uptake by AMs. However, albumin coating does not affect the translocation of gold NPs across the air-blood barrier. A more extensive database of corona composition of a diverse NP library will develop a platform to help predict the effects and biokinetics of inhaled NPs.

Nanomaterials Versus Ambient Ultrafine Particles: An Opportunity to Exchange Toxicology Knowledge.

BACKGROUND: A rich body of literature exists that has demonstrated adverse human health effects following exposure to ambient air particulate matter (PM), and there is strong support for an important role of ultrafine (nanosized) particles. At present, relatively few human health or epidemiology data exist for engineered nanomaterials (NMs) despite clear parallels in their physicochemical properties and biological actions in in vitro models. OBJECTIVES: NMs are available with a range of physicochemical characteristics, which allows a more systematic toxicological analysis. Therefore, the study of ultrafine particles (UFP, <100 nm in diameter) provides an opportunity to identify plausible health effects for NMs, and the study of NMs provides an opportunity to facilitate the understanding of the mechanism of toxicity of UFP. METHODS: A workshop of experts systematically analyzed the available information and identified 19 key lessons that can facilitate knowledge exchange between these discipline areas. DISCUSSION: Key lessons range from the availability of specific techniques and standard protocols for physicochemical characterization and toxicology assessment to understanding and defining dose and the molecular mechanisms of toxicity. This review identifies a number of key areas in which additional research prioritization would facilitate both research fields simultaneously. CONCLUSION: There is now an opportunity to apply knowledge from NM toxicology and use it to better inform PM health risk research and vice versa. https://doi.org/10.1289/EHP424.

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.

Silver nanoparticles inhaled during pregnancy reach and affect the placenta and the foetus.

Recently, interest for the potential impact of consumer-relevant engineered nanoparticles on pregnancy has dramatically increased. This study investigates whether inhaled silver nanoparticles (AgNPs) reach and cross mouse placental barrier and induce adverse effects. Apart from their relevance for the growing use in consumer products and biomedical applications, AgNPs are selected since they can be unequivocally identified in tissues. Pregnant mouse females are exposed during the first 15 days of gestation by nose-only inhalation to a freshly produced aerosol of 18-20 nm AgNPs for either 1 or 4 h, at a particle number concentration of 3.80 × 107 part./cm-3 and at a mass concentration of 640 µg/m³. AgNPs are identified and quantitated in maternal tissues, placentas and foetuses by transmission electron microscopy coupled with energy-dispersive X-ray spectroscopy and single-particle inductively coupled plasma mass spectrometry. Inhalation of AgNPs results in increased number of resorbed foetuses associated with reduced oestrogen plasma levels, in the 4 h/day exposed mothers. Increased expression of pregnancy-relevant inflammatory cytokines is also detected in the placentas of both groups. These results prove that NPs are able to reach and cross the mouse placenta and suggest that precaution should be taken with respect to acute exposure to nanoparticles during pregnancy.

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.

Quantitative biokinetics of titanium dioxide nanoparticles after intravenous injection in rats: Part 1.

Submicrometer TiO2 particles, including nanoparticulate fractions, are used in an increasing variety of consumer products, as food additives and also drug delivery applications are envisaged. Beyond exposure of occupational groups, this entails an exposure risk to the public. However, nanoparticle translocation from the organ of intake and potential accumulation in secondary organs are poorly understood and in many investigations excessive doses are applied. The present study investigates the biokinetics and clearance of a low single dose (typically 40-400 µg/kg BW) of 48V-radiolabeled, pure TiO2 anatase nanoparticles ([48V]TiO2NP) with a median aggregate/agglomerate size of 70 nm in aqueous suspension after intravenous (IV) injection into female Wistar rats. Biokinetics and clearance were followed from one-hour to 4-weeks. The use of radiolabeled nanoparticles allowed a quantitative [48V]TiO2NP balancing of all organs, tissues, carcass and excretions of each rat without having to account for chemical background levels possibly caused by dietary or environmental titanium exposure. Highest [48V]TiO2NP accumulations were found in liver (95.5%ID after one day), followed by spleen (2.5%), carcass (1%), skeleton (0.7%) and blood (0.4%). Detectable nanoparticle levels were found in all other organs. The [48V]TiO2NP content in blood decreased rapidly after 24 h while the distribution in other organs and tissues remained rather constant until day-28. The present biokinetics study is part 1 of a series of studies comparing biokinetics after three classical routes of intake (IV injection (part 1), ingestion (part 2), intratracheal instillation (part 3)) under identical laboratory conditions, in order to test the common hypothesis that IV-injection is a suitable predictor for the biokinetics fate of nanoparticles administered by different routes. This hypothesis is disproved by this series of studies.

Quantitative biokinetics of titanium dioxide nanoparticles after oral application in rats: Part 2.

The biokinetics of a size-selected fraction (70 nm median size) of commercially available and 48V-radiolabeled [48V]TiO2 nanoparticles has been investigated in female Wistar-Kyoto rats at retention timepoints 1 h, 4 h, 24 h and 7 days after oral application of a single dose of an aqueous [48V]TiO2-nanoparticle suspension by intra-esophageal instillation. A completely balanced quantitative body clearance and biokinetics in all organs and tissues was obtained by applying typical [48V]TiO2-nanoparticle doses in the range of 30-80 µg•kg-1 bodyweight, making use of the high sensitivity of the radiotracer technique. The [48V]TiO2-nanoparticle content was corrected for nanoparticles in the residual blood retained in organs and tissue after exsanguination and for 48V-ions not bound to TiO2-nanoparticles. Beyond predominant fecal excretion about 0.6% of the administered dose passed the gastro-intestinal-barrier after one hour and about 0.05% were still distributed in the body after 7 days, with quantifiable [48V]TiO2-nanoparticle organ concentrations present in liver (0.09 ng•g-1), lungs (0.10 ng•g-1), kidneys (0.29 ng•g-1), brain (0.36 ng•g-1), spleen (0.45 ng•g-1), uterus (0.55 ng•g-1) and skeleton (0.98 ng•g-1). Since chronic, oral uptake of TiO2 particles (including a nano-fraction) by consumers has continuously increased in the past decades, the possibility of chronic accumulation of such biopersistent nanoparticles in secondary organs and the skeleton raises questions about the responsiveness of their defense capacities, and whether these could be leading to adverse health effects in the population at large. After normalizing the fractions of retained [48V]TiO2-nanoparticles to the fraction that passed the gastro-intestinal-barrier and reached systemic circulation, the biokinetics was compared to the biokinetics determined after IV-injection (Part 1). Since the biokinetics patterns differ largely, IV-injection is not an adequate surrogate for assessing the biokinetics after oral exposure to TiO2 nanoparticles.

Quantitative biokinetics of titanium dioxide nanoparticles after intratracheal instillation in rats: Part 3.

The biokinetics of a size-selected fraction (70 nm median size) of commercially available and 48V-radiolabeled [48V]TiO2 nanoparticles has been investigated in healthy adult female Wistar-Kyoto rats at retention time-points of 1 h, 4 h, 24 h, 7 d and 28 d after intratracheal instillation of a single dose of an aqueous [48V]TiO2-nanoparticle suspension. A completely balanced quantitative biodistribution in all organs and tissues was obtained by applying typical [48V]TiO2-nanoparticle doses in the range of 40-240 µg·kg-1 bodyweight and making use of the high sensitivity of the radiotracer technique. The [48V]TiO2-nanoparticle content was corrected for residual blood retained in organs and tissues after exsanguination and for 48V-ions not bound to TiO2-nanoparticles. About 4% of the initial peripheral lung dose passed through the air-blood-barrier after 1 h and were retained mainly in the carcass (4%); 0.3% after 28 d. Highest organ fractions of [48V]TiO2-nanoparticles present in liver and kidneys remained constant (0.03%). [48V]TiO2-nanoparticles which entered across the gut epithelium following fast and long-term clearance from the lungs via larynx increased from 5 to 20% of all translocated/absorbed [48V]TiO2-nanoparticles. This contribution may account for 1/5 of the nanoparticle retention in some organs. After normalizing the fractions of retained [48V]TiO2-nanoparticles to the fraction that reached systemic circulation, the biodistribution was compared with the biodistributions determined after IV-injection (Part 1) and gavage (GAV) (Part 2). The biokinetics patterns after IT-instillation and GAV were similar but both were distinctly different from the pattern after intravenous injection disproving the latter to be a suitable surrogate of the former applications. Considering that chronic occupational inhalation of relatively biopersistent TiO2-particles (including nanoparticles) and accumulation in secondary organs may pose long-term health risks, this issue should be scrutinized more comprehensively.

Biokinetic datasets of PEI F25-LMW complexed and non-complexed 32P-siRNA within different lung compartments.

Biokinetics data of lung-administered PEI F25-LMW/siRNA polyplexes within different lung compartments are presented. Thereby, at three different timepoints (1 h, 3 h, 8 h), the data was determined by calculations to the 32P-radioactivity in the whole mouse body. Additionally, data was optimized to the available PEI F25-LMW/siRNA polyplexes in the target organ and therefore normalized to the sum of all lung compartments. Methods, other biokinetics data and the discussion of the results are published in "Biokinetic studies of non-complexed siRNA versus nano-sized PEI F25-LMW/siRNA polyplexes following intratracheal instillation into mice" (Lipka et al., 2016 [1]).

A Multilaboratory Toxicological Assessment of a Panel of 10 Engineered Nanomaterials to Human Health--ENPRA Project--The Highlights, Limitations, and Current and Future Challenges.

ENPRA was one of the earlier multidisciplinary European Commission FP7-funded projects aiming to evaluate the risks associated with nanomaterial (NM) exposure on human health across pulmonary, cardiovascular, hepatic, renal, and developmental systems. The outputs from this project have formed the basis of this review. A retrospective interpretation of the findings across a wide range of in vitro and in vivo studies was performed to identify the main highlights from the project. In particular, focus was placed on informing what advances were made in the hazard assessment of NM, as well as offering some suggestions on the future of "nanotoxicology research" based on these observations, shortcomings, and lessons learned from the project. A number of issues related to the hazard assessment of NM are discussed in detail and include use of appropriate NM for nanotoxicology investigations; characterization and dispersion of NM; use of appropriate doses for all related investigations; need for the correct choice of experimental models for risk assessment purposes; and full understanding of the test systems and correct interpretation of data generated from in vitro and in vivo systems. It is hoped that this review may assist in providing information in the implementation of guidelines, model systems, validation of assessment methodology, and integrated testing approaches for risk assessment of NM. It is vital to learn from ongoing and/or completed studies to avoid unnecessary duplication and offer suggestions that might improve different aspects of experimental design.

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.

Biokinetic studies of non-complexed siRNA versus nano-sized PEI F25-LMW/siRNA polyplexes following intratracheal instillation into mice.

Successful gene therapy requires stability and sufficient bioavailability of the applied drug at the site of action. In the case of RNA interference (RNAi), non-viral vectors play a promising role for delivering intact siRNA molecules. We selected a low molecular weight polyethyleneimine (PEI F25-LMW) and investigated the biokinetics of PEI F25-LMW/siRNA polyplexes in comparison to non-complexed siRNA molecules upon intratracheal application into mice. Additionally, a bronchoalveolar lavage was performed to locate the siRNA within the different lung compartments and to analyse possible inflammatory reactions. Liquid scintillation counting of a 32P-label was used to follow the siRNA within the whole body. During the complete observation time more than 75% of the applied dose was found at the target site. The complexation with PEI F25- LMW prevented the siRNA from being degraded and cleared and prolonged its retention time. A low inflammatory reaction was observed on the basis of cell differentiation. Taken together, PEI F25-LMW meets fundamental requirements on non-viral vectors for local pulmonary siRNA delivery.

In vivo integrity of polymer-coated gold nanoparticles.

Inorganic nanoparticles are frequently engineered with an organic surface coating to improve their physicochemical properties, and it is well known that their colloidal properties may change upon internalization by cells. While the stability of such nanoparticles is typically assayed in simple in vitro tests, their stability in a mammalian organism remains unknown. Here, we show that firmly grafted polymer shells around gold nanoparticles may degrade when injected into rats. We synthesized monodisperse radioactively labelled gold nanoparticles ((198)Au) and engineered an (111)In-labelled polymer shell around them. Upon intravenous injection into rats, quantitative biodistribution analyses performed independently for (198)Au and (111)In showed partial removal of the polymer shell in vivo. While (198)Au accumulates mostly in the liver, part of the (111)In shows a non-particulate biodistribution similar to intravenous injection of chelated (111)In. Further in vitro studies suggest that degradation of the polymer shell is caused by proteolytic enzymes in the liver. Our results show that even nanoparticles with high colloidal stability can change their physicochemical properties in vivo.