Wolfgang G. Kreyling

Targeted (nano-)drug delivery is essential for treating respiratory diseases, which are often confined to distinct lung regions. However, spatio-temporal profiling of drugs or nanoparticles (NPs) and their interactions with lung macrophages remains unresolved. Here, we present LungVis 1.0, an AI-powered imaging ecosystem that integrates light sheet fluorescence microscopy with deep learning-based image analysis pipelines to map NP deposition and dosage holistically and quantitatively across bronchial and alveolar (acinar) regions in murine lungs for widely-used bulk-liquid and aerosol-based delivery methods. We demonstrate that bulk-liquid delivery results in patchy NP distribution with elevated bronchial doses, whereas aerosols achieve uniform deposition reaching distal alveoli. Furthermore, we reveal that lung tissue-resident macrophages (TRMs) are dynamic, actively patrolling and redistributing NPs within alveoli, contesting the conventional paradigm of TRMs as static entities. LungVis 1.0 provides an advanced framework for exploring pulmonary delivery dynamics and deepening insights into TRM-mediated lung immunity.

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.

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.

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. 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). 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.

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. 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.

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 to 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 two hours. Immediately afterwards lungs were excised, inflated and micro-wave 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 1h to 28d 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 hours 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 gastro-intestinal-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.

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. 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. 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. 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.

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. 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. A workshop of experts systematically analyzed the available information and identified 19 key lessons that can facilitate knowledge exchange between these discipline areas. 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. 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.

To study the influence of colloidal stability on protein corona formation, gold nanoparticles are synthesized with five distinct surface modifications: coating with citric acid, bis(p‐sulfonatophenyl)phenylphosphine dihydrate dipotassium salt, thiol‐terminated methoxy‐polyethylene glycol, dodecylamine‐grafted poly(isobutylene‐alt‐maleic anhydride), and dodecylamine‐grafted poly(isobutylene‐alt‐maleic anhydride) conjugated with polyethylene glycol. The nanoparticles are incubated with serum or bronchoalveolar lavage fluid from C57BL/6 mice (15 min or 24 h) to assess the effect of differential nanoparticle surface presentation on protein corona formation in the air–blood barrier exposure pathway. Proteomic quantification and nanoparticle size measurements are used to assess protein corona formation. We show that surface modification has a clear effect on the size and the composition of the protein corona that is related to the colloidal stability of the studied nanoparticles. Additionally, differences in the composition and size of the protein corona are shown between biological media and duration of exposure, indicating evolution of the corona through this exposure pathway. Consequently, a major determinant of protein corona formation is the colloidal stability of nanoparticles in biological media and chemical or environmental modification of the nanoparticles alters the surface presentation of the functional epitope in vivo. Therefore, the colloidal stability of nanoparticles has a decisive influence on nano–bio interactions.