Winfried Möller

Particle size-dependent and surface charge-dependent biodistribution of gold nanoparticles after intravenous administration

Gold nanoparticles (GNP) provide many opportunities in imaging, diagnostics, and therapies of nanomedicine. Hence, their biokinetics in the body are prerequisites for specific tailoring of nanomedicinal applications and for a comprehensive risk assessment. We administered (198)Au-radio-labelled monodisperse, negatively charged GNP of five different sizes (1.4, 5, 18, 80, and 200 nm) and 2.8 nm GNP with opposite surface charges by intravenous injection into rats. After 24h, the biodistribution of the GNP was quantitatively measured by gamma-spectrometry. The size and surface charge of GNP strongly determine the biodistribution. Most GNP accumulated in the liver increased from 50% of 1.4 nm GNP to >99% of 200 nm GNP. In contrast, there was little size-dependent accumulation of 18-200 nm GNP in most other organs. However, for GNP between 1.4 nm and 5 nm, the accumulation increased sharply with decreasing size; i.e. a linear increase with the volumetric specific surface area. The differently charged 2.8 nm GNP led to significantly different accumulations in several organs. We conclude that the alterations of accumulation in the various organs and tissues, depending on GNP size and surface charge, are mediated by dynamic protein binding and exchange. A better understanding of these mechanisms will improve drug delivery and dose estimates used in risk assessment.

Air-blood barrier translocation of tracheally instilled gold nanoparticles inversely depends on particle size.

Gold nanoparticles (AuNP) provide many opportunities in imaging, diagnostics, and therapy in nanomedicine. For the assessment of AuNP biokinetics, we intratracheally instilled into rats a suite of (198)Au-radio-labeled monodisperse, well-characterized, negatively charged AuNP of five different sizes (1.4, 2.8, 5, 18, 80, 200 nm) and 2.8 nm AuNP with positive surface charges. At 1, 3, and 24 h, the biodistribution of the AuNP was quantitatively measured by gamma-spectrometry to be used for comprehensive risk assessment. Our study shows that as AuNP get smaller, they are more likely to cross the air-blood barrier (ABB) depending strongly on the inverse diameter d(-1) of their gold core, i.e., their specific surface area (SSA). So, 1.4 nm AuNP (highest SSA) translocated most, while 80 nm AuNP (lowest SSA) translocated least, but 200 nm particles did not follow the d(-1) relation translocating significantly higher than 80 nm AuNP. However, relative to the AuNP that had crossed the ABB, their retention in most of the secondary organs and tissues was SSA-independent. Only renal filtration, retention in blood, and excretion via urine further declined with d(-1) of AuNP core. Translocation of 5, 18, and 80 nm AuNP is virtually complete after 1 h, while 1.4 nm AuNP continue to translocate until 3 h. Translocation of negatively charged 2.8 nm AuNP was significantly higher than for positively charged 2.8 nm AuNP. Our study shows that translocation across the ABB and accumulation and retention in secondary organs and tissues are two distinct processes, both depending specifically on particle characteristics such as SSA and surface charge.

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

Ambient particles are believed to be a specific health hazard, although the underlying mechanisms are not fully understood. There are data in the literature indicating fast and substantial systemic uptake of particles from the lung. The present authors have developed an improved method to produce ultrafine particles with more stable radiolabelling and defined particle size range. Fifteen subjects inhaled technetium 99m (99mTc)-labelled carbonaceous particles of 100u2005nm in size. Radioactivity over the lung was followed for 70u2005h. The clearance of these ultrafine particles from the lungs and specifically translocation to the circulation was tested. Lung retention for all subjects at 46u2005h was mean±sd 99±4.6%. Cumulative leaching of 99mTc activity from the particles was 2.6±0.96% at 70u2005h. The 24-h activity leaching in urine was 1.0±0.55%. No evidence of a quantitatively important translocation of 100-nm particles to the systemic circulation from the lungs was found. More research is needed to establish if the ∼1% cleared activity originates from leached activity or insoluble translocated particles, and whether a few per cent of translocated particles is sufficient to cause harmful effects.

No Significant Translocation of Inhaled 35-nm Carbon Particles to the Circulation in Humans

Human pulmonary retention of 35 nm 99mTc-labeled carbonaceous particles, produced with a modified Technegas generator, was followed for 24 h using a gamma camera imaging technique. Nine healthy subjects and four asthmatics inhaled the test particles. Particle labeling stability was tested in vitro during 48 h. We also measured in vivo leaching in blood and in urine for 24 h. One additional subject was exposed to particles with unstable labeling. There were no significant differences between healthy and asthmatic subjects in any of the parameters studied. Particle retention after 24 h was 102% (SD ± 4.7). Cumulative in vitro leaching of 99mTc activity from the particles was 1.7% (±1.1) after 24 h. In blood samples, 80 min after exposure, 1.1% (± 0.4) of initially deposited activity was detected and 91% of the activity was not bound to particles. In urine sampled during the first 24 h after exposure, 3.6% (± 0.9) of lung deposited activity was detected. Lung retention was 30% after 1 h in the subject exposed to the leaching aerosol (n = 1). Thirty-one percent of the deposited activity was detected in the blood after 80 min and 80% was not bound to particles. Fifty percent of the activity was excreted with urine within 24 h. On gamma camera images the activity visibly translocated from lungs to thyroid and gastrointestinal tract. In conclusion, over a 24-h period there was no significant translocation of inhaled 35-nm particles to the systemic circulation.

Measurement Techniques for Respiratory Tract Deposition of Airborne Nanoparticles: A Critical Review

Determination of the respiratory tract deposition of airborne particles is critical for risk assessment of air pollution, inhaled drug delivery, and understanding of respiratory disease. With the advent of nanotechnology, there has been an increasing interest in the measurement of pulmonary deposition of nanoparticles because of their unique properties in inhalation toxicology and medicine. Over the last century, around 50 studies have presented experimental data on lung deposition of nanoparticles (typical diameter≤100u2009nm, but here≤300u2009nm). These data show a considerable variability, partly due to differences in the applied methodologies. In this study, we review the experimental techniques for measuring respiratory tract deposition of nano-sized particles, analyze critical experimental design aspects causing measurement uncertainties, and suggest methodologies for future studies. It is shown that, although particle detection techniques have developed with time, the overall methodology in respiratory tract deposition experiments has not seen similar progress. Available experience from previous research has often not been incorporated, and some methodological design aspects that were overlooked in 30-70% of all studies may have biased the experimental data. This has contributed to a significant uncertainty on the absolute value of the lung deposition fraction of nanoparticles. We estimate the impact of the design aspects on obtained data, discuss solutions to minimize errors, and highlight gaps in the available experimental set of data.

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

BackgroundInhalative 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.Diseased lungs are of specific interest, since these are the main recipients of inhalation therapy. We, therefore, used Scnn1b-transgenic (Tg) mice as a model of chronic obstructive pulmonary disease (COPD) and compared uptake and localization of inhaled AuNP in surface macrophages and lung tissue to wild-type (Wt) mice.MethodsScnn1b-Tg and Wt mice inhaled a 21-nm AuNP aerosol for 2xa0h. Immediately (0xa0h) or 24xa0h thereafter, bronchoalveolar lavage (BAL) macrophages and whole lungs were prepared for stereological analysis of AuNP by electron microscopy.ResultsAuNP were mainly found as singlets or small agglomerates of ≤ 100xa0nm diameter, at the epithelial surface and within lung-surface structures. Macrophages contained also large AuNP agglomerates (> 100xa0nm). At 0xa0h 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 0xa0h to 24xa0h in both groups. At 24xa0h, 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 0xa0h and in Scnn1b-Tg mice at 24xa0h, confirming less efficient macrophage uptake and delayed clearance of AuNP in Scnn1b-Tg mice.ConclusionsInhaled 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.

Gold nanoparticle aerosols for rodent inhalation and translocation studies

The intensive use of nano-sized particles in many different applications necessitates studies on their risk assessment as there are still open questions on their safe handling and utilization. For reliable risk assessment, the interaction of nanoparticles (NP) with biological systems after various routes of exposure needs to be investigated using well-characterized NP. We report here on the generation of gold-NP (Au-NP) aerosols for inhalation studies with the spark ignition technique, and their characterization in terms of chemical composition, physical structure, morphology, and specific surface area, and on interaction with lung tissues and lung cells after 1xa0h inhalation by mice. The originally generated agglomerated Au-NP were converted into compact spherical Au-NP by thermal annealing at 600xa0°C, providing particles of similar mass, but different size and specific surface area. Since there are currently no translocation data available on inhaled Au-NP in the 10–50xa0nm diameter range, the emphasis was to generate NP as small as 20xa0nm for inhalation in rodents. For anticipated in vivo systemic translocation and dosimetry analyses, radiolabeled Au-NP were created by proton irradiating the gold electrodes of the spark generator, thus forming gamma ray emitting 195Au with 186xa0days half-life, allowing long-term biokinetic studies. The dissolution rate of 195Au from the NP was below detection limits. The highly concentrated, polydisperse Au-NP aerosol (1–2xa0×xa0107xa0NP/cm3) proved to be constant over several hours in terms of its count median mobility diameter, its geometric standard deviation and number concentration. After collection on filters particles can be re-suspended and used for instillation or ingestion studies.

Quantitative detection of drug dose and spatial distribution in the lung revealed by Cryoslicing Imaging

Administration of drugs via inhalation is an attractive route for pulmonary and systemic drug delivery. The therapeutic outcome of inhalation therapy depends not only on the dose of the lung-delivered drug, but also on its bioactivity and regional distribution. Fluorescence imaging has the potential to monitor these aspects already during preclinical development of inhaled drugs, but quantitative methods of analysis are lacking. In this proof-of-concept study, we demonstrate that Cryoslicing Imaging allows for 3D quantitative fluorescence imaging on ex vivo murine lungs. Known amounts of fluorescent substance (nanoparticles or fluorophore-drug conjugate) were instilled in the lungs of mice. The excised lungs were measured by Cryoslicing Imaging. Herein, white light and fluorescence images are obtained from the face of a gradually sliced frozen organ block. A quantitative representation of the fluorescence intensity throughout the lung was inferred from the images by accounting for instrument noise, tissue autofluorescence and out-of-plane fluorescence. Importantly, the out-of-plane fluorescence correction is based on the experimentally determined effective light attenuation coefficient of frozen murine lung tissue (10.0 ± 0.6 cm(-1) at 716 nm). The linear correlation between pulmonary total fluorescence intensity and pulmonary fluorophore dose indicates the validity of this method and allows direct fluorophore dose assessment. The pulmonary dose of a fluorescence-labeled drug (FcγR-Alexa750) could be assessed with an estimated accuracy of 9% and the limit of detection in ng regime. Hence, Cryoslicing Imaging can be used for quantitative assessment of dose and 3D distribution of fluorescence-labeled drugs or drug carriers in the lungs of mice.

In vivo Dynamic Phase-Contrast X-ray Imaging using a Compact Light Source

We describe the first dynamic and the first in vivo X-ray imaging studies successfully performed at a laser-undulator-based compact synchrotron light source. The X-ray properties of this source enable time-sequence propagation-based X-ray phase-contrast imaging. We focus here on non-invasive imaging for respiratory treatment development and physiological understanding. In small animals, we capture the regional delivery of respiratory treatment, and two measures of respiratory health that can reveal the effectiveness of a treatment; lung motion and mucociliary clearance. The results demonstrate the ability of this set-up to perform laboratory-based dynamic imaging, specifically in small animal models, and with the possibility of longitudinal studies.