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.


Background
By range of applications, silver nanoparticles (AgNP) are the most frequently used nanomaterial due to their antimicrobial, cytotoxic and electrical properties. In 2014 the Nanotechnology Consumer Products Inventory (CPI) of the Woodrow Wilson International Center for Scholars listed 435 (out of 1814) products containing nano-silver [1]. Silver nanoparticles (AgNP) have been used extensively (a) in electronic products (b) as antimicrobial and anti-bacterial agents used in wound dressings, sprays, textiles, and medical devices, (c) in food storage and food packaging, (d) as textile coatings and (e) in a number of environmental applications [1][2][3] such as water treatment [4]. Moreover, the textile industry has started to use AgNP in different textile fabrics [2,3]. Recently, inkjet technology has been used to produce flexible electronic circuits, using nano-sized metal particles such as Au or Ag of high electrical conductivity [2,3,5] uniformly dispersed in the inks. Overall 25% of the above-mentioned products apply or contain nanomaterials in a possibly inhalable way for example as sprays [1], which deserves special attention since AgNP may penetrate the air-blood barrier (ABB) and even subsequent barriers such as the placental barrier [6].
A recent review [7] summarizes the numerous ways to generate AgNP: currently, Ag-NP are being fabricated on an industrial scale utilizing physico-chemical techniques such as chemical reduction, gamma-ray radiation, microemulsion techniques, electrochemical methods, laser ablation, autoclaving, microwaving, and photochemical reduction. These methods are all effective but suffer from several limitations such as the use of toxic ingredients, high operational cost, and high energy consumption. In order to address these weaknesses, recently "green methods" to synthesize AgNP have been advanced. These methods make use of the capabilities of some microorganisms such as certain bacteria, fungi, yeasts, algae, or plant extracts to reduce and/or stabilize certain silver compounds thereby forming AgNP. Depending on the synthesis methods and the synthesis conditions, AgNP produced by green methods vary in size, shape, surface electric charge, and in other physicochemical characteristics. Like other nanomaterials, nanosized Ag-particles are several times more reactive than the corresponding bulk particles and exhibit much more pronounced catalytic effects. However, these desired properties may also increase the toxicity of the nanoforms due to their capability to generate reactive oxygen species (ROS).
In vivo and in vitro toxicity of AgNP has been reported in numerous research papers and reviews [8][9][10][11][12][13]. The present work deals with the effects of pristine AgNP, generated by spark ignition aerosol generarion between two pure silver electrodes and immediately used for inhalation experiments with rats. Therefore, we refer mainly to investigations that refrained from applying coatings or capping agents in order to prevent AgNP agglomeration in suspension. Here we investigate rather the interactions of inhaled pristine AgNP with the biological environment, and their subsequent fate in the lungs and the entire organism of healthy rats [14][15][16][17][18][19][20][21][22].
Substantial evidence exists to suggest that the adverse effects induced by AgNP are predominantly mediated via Ag + ions that are released from the particle surface [7,21,23,24]. Therefore, and in contrast to our earlier experiments with inhaled 20 nm nanoparticle-sized aerosols of gold [25], iridium [26][27][28], elemental carbon [29], or titanium dioxide [30] we expect clearly observable differences in the translocation across the air-blood-barrier (ABB), the biodistribution in the entire organism and the excretion kinetics of AgNP due to the instability of AgNP which is mediated by gradual oxidation leading to Ag + ion release and AgNP dissolution [31]. However, the thermodynamically possible full oxidation and dissolution of AgNP has never been observed in biological systems, and the fast, initial Ag + ion release gradually slows down [32]. Therefore, these authors conclude that AgNP are protected against complete dissolution by the formation of very stable Ag 6 O octahedra on their surface when oxygen radicals penetrate the nanoparticle surface.
Furthermore, Li and co-workers [33] showed that in biological fluids containing high Cl − concentrations the released Ag + ions precipitate as solid AgCl (with a very low solubility constant (Ksp = 1.77 10 − 11 mol/L)) on the AgNP. The precipitated AgCl can form both, nano-sized silver clusters in the body fluid or they can form smooth surface layers on AgNP thereby dramatically altering the AgNP morphology [31]. TEM images confirmed by Xray diffraction showed clusters formed by partially dissolved Ag which adhere to each other and form agglomerates of irregular shape and size. The smooth surface of the adhered Ag-clusters is attributed to continued AgCl precipitation on their surface [31]. Therefore, the decrease in the soluble silver species concentration in body fluids containing chloride ions is attributed to the formation of AgCl clusters and to AgCl(s) precipitation as shells on persistent cores of AgNP [34,35]. When exposed to human synthetic stomach fluid containing HCl and pepsin, a pronounced release of Ag + ions has been reported as well as a pronounced agglomeration of the AgNP with AgCl being present on the surfaces and interfaces between the AgNP [36,37]. Levard and coworkers [38] also reported sulfidation of AgNP surfaces since silver readily reacts with sulfide to form Ag(0)/Ag 2 S core−shell particles; i.e. elemental silver in the AgNP is oxidized to Ag + , which then reacts with inorganic sulfide abundantly present in biological fluids to form secondary Ag 2 S NPs or core −shell Ag:Ag 2 S particles. As the concentration ratio of sulfur to silver increases, the release of Ag + ions from the AgNP decreases [3,31].
The formation of secondary nanoparticles containing silver has been reported by Juling et al. [39] after oral delivery of 15 nm AgNP to rats as well as after intravenous injection of silver ions in the form of silver acetate. By TEM and EDX analyses of liver tissue after oral AgNP delivery, particles containing silver and sulfur in a size range of 5 to 12 nm were observed inside different types of liver cells. Small silver particles of about 6 nm were also detected at high numbers in the livers of rats even after intravenous injection of silver acetate, which shows that nano-sized Ag-clusters can be formed in vivo from Ag + ions de novo [39].
Further evidence for the formation of secondary nanoparticles comes from an oral exposure study using either 20 nm AgNP or AgNO 3 solution [40]. In this study, Agclusters were detected using single-particle ICPMS in organs of both groups of animals that were orally instilled with either 20 nm AgNP or with AgNO 3 solution. Similar results were found by [31,41] as well as in argyria patients who had ingested soluble forms of silver only [42,43] where also selenium-rich nanoparticles were detected. The concentration of Se in living organisms is much lower than that of S, but Se binds more strongly to Ag than S and may, therefore, replace S in Ag 2 S gradually over time forming more stable Ag 2 Se nanoparticles. The capability of Ag + ions to form nanoparticulate Ag 2 S and Ag 2 Se in vivo and the capability of AgNP to dissolve implies that the toxicity of Ag is subject to an interplay between various chemical processes. The formation of secondary nanoparticles and nano-sized silver salt clusters [44] from Ag + ions, consisting of precipitated, poorly soluble AgCl 3 , Ag 2 S, Ag 2 Se, Ag 2 PO 4 [44], explains well why similar effects are observed irrespective whether Ag + ions or AgNP are applied [43]. This is also in line with similar organ distribution patterns of Ag found in rats after oral exposure to AgNP and to Ag acetate [40,41].
In an early study published in 2001, Takenaka and coworkers [45] described similar clearance kinetics patterns of inhaled AgNPgenerated in a manner similar to the methodology of the current studyand an AgNO 3 -solution instilled into the lungs of rats. Since at that time the authors were not aware of the subsequent work discussed above, they were not able to fully interpret the results of their study.
In order to analyze the results and deduce the biokinetics and competitive clearance pathways of AgNP after inhalation of freshly produced, pristine, radiolabeled, 20 nm-sized [ 105 Ag]AgNP by healthy rats, three auxiliary biokinetics studies were additionally performed in the present study by applying [ 110m Ag]AgNO 3 -solutions, (i) by intravenous injection (IV) into blood, (ii) by intratracheal instillation (IT) into the lungs and (iii) by oral instillation (GAV) into the gastro-intestinal-tract (GIT).
This biokinetics study on inhaled 20 nm [ 105 Ag]AgNP is the fifth in a series that used the same inhalation apparatus and methodology, the same strain of adult healthy rats and the same biokinetics analysis methodology as for four different, 20-nm-sized, poorly soluble radio-labeled NP materials applied in exactly the same way. This allowed us the unique possibility of comparing the in vivo fate of inhaled AgNP -which are expected to dissolve partially during the experimentswith that of the poorly soluble, more stable nanoparticles.

Aims and rationale
The present study was designed to investigate the quantitative biokinetics of AgNP after a single 1½ -hour intratracheal inhalation exposure of 105 Ag radio-labeled, 20 nm [ 105 Ag]AgNP over a period of up to 28 days. Female, adult Wistar-Kyoto rats inhaled an [ 105 Ag]AgNP aerosol freshly generated by spark ignition between two pure, proton-irradiated silver electrodes. The study design is presented in Table 1. The study is part of a series of studies comparing the biokinetics and accumulation of five different inhaled 20-nm NP-materials -AgNP (this study), IrNP [26][27][28], AuNP [25], elemental carbon NP [29] and TiO 2 -NP [30] including their translocation kinetics across the air-blood-barrier (ABB) and their subsequent gradual uptake from the blood into secondary organs and tissues up to 28 days post-exposure (p.e.). In order to ensure comparability, the same strain of rats, the same inhalation technology and the same analytical methodology of the biokinetics was used for all experiments. From the previous biokinetics results, it is known that nanoparticle translocation and accumulation occurs rather rapidly during the first 24 h. Therefore, the present investigation covers early accumulation with three time-points of 0.75 h, 4 h, and 24 h, followed by two time-points after 7 days and 28 days to investigate possibly slower processes of accumulation, redistribution, and clearance of nanoparticles.
The added value of these studies is related to the small size of the agglomeration-controlled nanoparticles in the aerosols which were inhaled immediately after generation. In many other inhalation studies, the applied NP agglomerates were much larger than 20 nm when inhaled by the rats. Moreover, the use of radiolabeled NP in this study provides the required precision and aneasy-to-use analytical methodology to study translocation kinetics across the ABB and also to quantify minor NP accumulations in secondary organs and tissues and in excretions.
Since [ 105 Ag]Ag + ions in body fluids with their abundant presence of chloride, phosphate and sulfide ions, it is rather unlikely to find 105 Ag activity in purely ionic form as summarized in the Background section. It should be noted, however, that from radioactivity measurements of 105 Ag or 110m Ag in organs, tissues and excretions it is not possible to distinguish between Ag + -ions or primary or secondary nanoparticles, which is a major difference with earlier experiments using de facto unsoluble nanoparticles.
[ 105 Ag]AgNP aerosol exposure and deposition Table 2 compiles the key characterization parameters of the [ 105 Ag]AgNP aerosols used for each group of rats. These were derived from in situ measurements during inhalation exposure using a Scanning Mobility Particle Sizer (SMPS) Spectrometer and a Condensation Particle Counter (CPC) synchronized with the flight time required until the inhalation by the rats (assuring the correct measurement of the contemporary size distribution at inhalation), as well as γ-spectrometry results on a filter collecting a fraction of the aerosol in a bypass line. As described in detail in the Supplementary Information the count median diameter (CMD) and its geometric standard deviation (GSD) are obtained from the as-measured particle size spectra. In Fig. S1 of the Supplementary Information aerosol parameters during the inhalation exposures for all groups of rats are presented together with S/TEM images of the AgNP. Additionally, the experimentally determined particle size spectra were averaged and then fitted to a log-normal distribution, applying a least-squares method. The fitted log-normal distribution was extrapolated down to a particle size of 1 nm in order to overcome the operative threshold of the SMPS of 10 nm and to estimate the contributions of smaller particles. In order to examine the dissolution behavior of the spark ignition generated [ 105 Ag]AgNP, we performed a simple in vitro test in which the NP collected on a filter during the exposure of the group of rats dissected 24 h p.e. were tightly covered with a plain sandwich filter in a filter holder and submersed in distilled water; the dissolved [ 105 Ag]Ag fraction in the water was measured γspectrometrically after submersion times of 15 min, 1 h, 1 day and 3 days. More experimental details and the data are presented in Fig. S2 of the Supplementary Information. Basically, the [ 105 Ag]AgNP used in the present study showed a similar pattern of partial oxidative dissolution previously reported by Kittler and Lonza and their co-workers [46,47].
Intratracheal inhalation exposure allowed deep breath ventilation and avoided head airway deposition, thus leading to enhanced intrathoracic conducting airway  Table 3 for each group of rats. In addition, the activity fractions cleared from the lungs that can be attributed to early clearance into the gastro-intestinal-tract (GIT) and feces up to 2 days p.e. and the long-term clearance of [ 105 Ag]AgNP and their degradation products formed in the retention period from 3 days up to 28 Table 3 are consistently lower (0.3-0.4 for 24 h until 28d groups of rats) than the total deposited fraction of 0.6 as calculated by the MPPD software 3.04 (see Fig. S5 of the Supplementary Information). This may have resulted from lowpressure ventilation in the plethysmograph.

Auxiliary studies of the biokinetics after intravenous injection and intratracheal instillation of soluble [ 105 Ag]AgNO 3 salt solutions
In the auxiliary biokinetics study after IV injection of an [ 110m Ag]AgNO 3 -solution in rats, the silver was mainly cleared into the GIT and almost completely excreted in the feces with negligible urinary excretion. After IT instillation of an [ 110m Ag]AgNO 3 -solution, we observed an Ag-fraction in the GIT and feces which was much higher than expected for fast mucociliary clearance. Figure 1 shows the biodistributions of intravenously injected, (IV, panel A) and of intratracheally instilled (IT, panel B), and of intraesophageal instilled (GAV, panel C) [ 110m Ag]AgNO 3 salt solutions (fully dissociated at the time of application) in four groups of rats (n = 4) after 24 h and 7 days, respectively. Already 24 h after IV injection (Fig. 1a), a fraction of 0.96 was eliminated from the blood circulation. Small fractions (< 0.04) were retained in secondary organs and the tissues of the remaining carcass. In contrast, predominant fractions were found in GIT (0.63) and feces (0.24). When distinguishing the GIT into its compartments -stomach, small intestine, and hindgut -, 110m Ag activity fractions of 0.002, 0.017 and 0.61, respectively, were found 24 h p.e. These data confirm that after IV-injection the passage into and through the small intestine is fast and almost complete within 24 h p.e. Noteworthy is the minimal urinary excretion of 0.005 24 h p.e. (and 0.0005 after GAV). After 1 week the fractions in all secondary organs and the carcass had decreased tenfold and more, but in the GIT the decrease was even 1000-fold due to almost complete fecal excretion of [ 110m Ag]Ag.
Only an [ 110m Ag]Ag fraction of about 0.14 of the intratracheally instilled material is retained in the lungs and BAL 24 h p.e. (Fig. 1b) which continues to slightly decrease during the following week to 0.07. Thus 24 h after IT instillation, a fraction of 0.86 has been translocated across the ABB into blood and fractions of 0.48 and 0.22 were found in the GIT in feces, respectively. In contrast, retained fractions in the liver, kidneys (~0.02 each) and  105 Ag activity as a fraction of the inhaled 105 Ag activity. Additionally, the retained alveolar fraction at the time of dissection, the early clearance during the first two days p.e. and the integral long-term clearance of [ 105 Ag]AgNP including their secondary products from day 3-28 after inhalation is given; the latter is calculated based on fecal excretion and retention in the gastro-intestinaltract (GIT). All fractional data are normalized to the Initial Lung Dose (ILD), i.e. the sum of 105 Ag radioactivities of all organs and tissues including excretion carcass (0.06), and even lower fractions in other secondary organs were found, indicating rapid elimination from blood and all secondary organs and tissues into the GIT. The translocated fraction across the ABB into blood increased to 0.9 at 7 days p.e. while the fractions in all secondary organs and tissues decreased sharply. Noteworthy is the fact that the urinary excretions (< 0.01) are minimal 24 h and 7 days p.e. After gavage fractions of secondary organs and tissues fractions are even lower than after IV injection (Fig. 1c).

Retention of [ 105 Ag]AgNP and their degradation products in the lungs and BAL
Fractional lung retention and BAL data relative to ILD are shown in Fig. 2a and b and in Table 4. According to Fig. 1, the sharp drop of the retention fraction in Fig. 2a of 0.4 at 24 h p.e. comprises not only mucociliary clearance from the conducting airways towards the larynx but also [ 105 Ag]Ag translocation across the ABB into the blood. The lung retained fraction continues to decline rapidly during the first week p.e. such that already 0.9 of the deposited [ 105 Ag]Ag activity is eliminated from the lungs at 7 days. Thereafter, the lung retention decreases moderately until 28 days p.e. The rather low retentions in BAL cells and BAL fluid, both diminishing rapidly in the same fashion after 4 h p.e., indicate rapid conformational changes of the inhaled [ 105 Ag]AgNP due to dissolution. These data differ strikingly from those obtained previously from poorly soluble 20 [30] as will be considered in the Discussion section.
In the caption of Fig. 2, the matrix of significances is given. This indicates highly significant changes between the early biodistributions determined within the first 24 h p.e. and the late biodistributions obtained 7 days and 28 days p.e. It is noteworthy that the independent analyses of two sets of rats 0.75 h p.e. and 24 h p.e., respectively, show no significant differences, emphasizing the high reproducibility between the two groups of rats of each time point (see also Table 4.)

Long-term [ 105 Ag]AgNP clearance
The daily fecal excretion per ILD is shown in Fig. 3a and the cumulative fecal excretion in Fig. 3b, based on the data retrieved from the two groups of rats dissected at 7 and 28 days p.e.
The observed cumulative fecal excretion of a fraction of more than 0.8 of the [ 105 Ag]Ag activity deposited in the lungs as [ 105 Ag]AgNP after the first week p.e. indicates that fecal excretion not only comprised the clearance of [ 105 Ag]AgNP to the larynx but also the degradation products of the nanoparticles. The auxiliary biokinetics studies after intratracheal instillation of soluble Ag + solutions (see Fig. 1) suggest that the nano-sized precipitates formed in the alveolar region may translocate across the ABB into the blood followed by rapid hepato-biliary transport into the duodenum of the GIT. Therefore, the dissolution of We also cannot distinguish between particulate transport from the lungs to the larynx and the GIT versus the translocation across the ABB into blood and GIT.
In Fig. 4a and b the daily urinary excretion is plotted, derived from the data retrieved from the rats of the 7days-and the 28-day groups. These rates remain low but reach a maximum 2 days p.e of 0.006 d − 1 and drop sharply thereafter to around 0.001 d − 1 . It remains speculative whether these fractions comprise soluble [ 105 Ag]Ag + ions and/or nano-sized [ 105 Ag]AgCl 3 precipitates of a small enough size (6-8 nm) to pass kidney clearance into the urine. Interestingly, after the first week p.e. urinary rates increase again and show a second maximum of 0.002 d − 1 at 18 days p.e.
In Figs. 4c the fraction of [ 105 Ag]AgNP and their degradation products that were translocated across the ABB are presented as stacked columns with accumulation in major secondary organs, the carcass, and cumulative urine. The urinary fractions are low and become only visible on the linear scale 28 days p.e. The carcass shows the highest retained fractions at all time points followed by the liver and blood.

Biokinetics of translocated [ 105 Ag]AgNP into secondary organs and tissues
In Table 4 the retention of [ 105 Ag]AgNP and/or [ 105 Ag]Agsalt precipitates in the lungs and in all secondary organs and tissues are presented as mean ± SEM for all dissection time points (note, the analyses at 0.75 h and 24 h p.e. were repeated in a second group of rats for each time point; in order to present the results separately, the groups have been labeled as 0.75 h/1st, 0.75 h/2nd and 24 h/1st, 24 h/ 2nd). The [ 105 Ag]Ag -activity data are corrected for the activity in the residual blood volume of the organs and tissues and given as fractions and mass concentrations normalized to ILD, which is the total deposited lung dose applying the mathematical procedure described in the Supplementary Information. No number concentration data are provided due to the fact that the rapid transformation of the deposited [ 105 Ag]AgNP starts to occur immediately after   Table 3. Figure 5 displays the retained [ 105 Ag]Ag -activity fractions (per ILD) of each organ or tissue corrected for residual [ 105 Ag]Ag blood activity (according to the 1st line in Table 4 of each organ or tissue). Immediately after inhalation a rapid translocation into the blood is observed followed by fast uptake in the carcass, which declines until 28 days p.e. by two orders of magnitude. The increasing [ 105 Ag]Ag blood activity (Fig. 5a) leads to steep increases in kidneys and moderate increases in the liver and brain. In the heart and uterus the [ 105 Ag]Ag activities remain constant over the first 24 h p.e. after which they decline more than tenfold until 28 days p.e. (Fig. 5b and c). The [ 105 Ag]Ag activity percentages in liver and kidneys reach about 1% of [ 105 Ag]AgNP ILD while those in the heart and uterus are smaller than 0.1% of ILD. The [ 105 Ag]Ag activities in the carcass reach even higher values (4%) 24 h p.e. which is more than the sum of all secondary organs. The total fraction of [ 105 Ag]Ag activity translocated across the ABB reaches nearly 0.1 of ILD 28 days p.e. (Fig. 5d) which is tenfold more than the ABB-translocation of same-sized [ 195 Au]AuNP after intratracheal inhalation [25].    Table 4) indicating rapid and uniform uptake from blood and accumulation in organs and tissues. Thereafter, Fig. 6a shows a tenfold increase of the concentration in blood at 4 h p.e. followed by a continuous decline by two orders of magnitude by 28 days p.e., which results in an approximately fivefold lower concentration than initially determined 0.75 h p.e. The initially steep increase was also found in liver, kidneys, heart, and uterus indicating rapid uptake by the MPS cells of these secondary organs when the blood concentration is still high. The decrease of the concentration between 24 h p.e. and 28 days p.e. is similar in the aforementioned organs as in the blood. The spleen shows a different behavior characterized by a gradual tenfold decrease over 28 days p.e. The [ 105 Ag]Ag concentration pattern in the carcass ( Fig. 6d and Table 4) follows basically the concentration pattern of blood but with lower accumulation and clearance rates.
In Fig. 7  Ag]Ag-salt precipitates) is translocated across the ABB and remains circulating in the blood indicating that there is an ongoing interaction between the blood and the organs, mainly with liver and kidneys, but as well as with the carcass and other secondary organs. During the period from 7 days to 28 days p.e., the [ 105 Ag]Ag activity declines in these organs and the carcass and is mirrored by the parallel decline of the [ 105 Ag]Ag activity in the blood. In contrast, much less of the translocated 105 Ag activity fraction is retained in spleen, heart, brain, and uterus ranging between 0.001 and 0.01.
In Fig. 8 the ratios of the [ 105 Ag]Ag activity in the residual blood, remaining after exsanguination at the time of In most secondary organs about onetenth of the [ 105 Ag]Ag activity can be attributed to the residual blood volume. This ratio remains rather constant throughout the 28 days observation period (see Fig. 8a and  b). For the brain, the residual blood contribution is about one-tenth during the first 24 h after which it drops sharply by two orders of magnitude. In Fig. 8c the residual blood content of the tissues of the carcass is almost identical to those of all secondary organs. In contrast, residual blood contributes minimally and rather constant over time to the lung activity indicating that in the lungs [ 105 Ag]AgNP and/or particulate [ 105 Ag]Ag-salt precipitates dominate the retention throughout the 28 days observation period.

Previous scientific results
In this section, we will compare our experimental results after the inhalation of 20   The partial [ 105 Ag]AgNP dissolution that we discuss is in agreement with previous studies [47] on commercially available AgNP; it results from oxidative dissolution and depends on several parameters such as temperature, Ag-ion concentration, and oxygen availability as shown earlier [46]. The literature presented in the Background section on the release of [ 105 Ag]Ag + ions from the surfaces of [ 105 Ag]AgNP retained in the lungs [7,21,23,24], indicate a "rich set of biochemical transformations occurring with Ag-NP in biological media, including accelerated oxidative dissolution, thiol binding and exchange to secondary zero-valent Ag-NP" as emphasized by [31]. The literature summarized in the Background section provides evidence that the release of [ 105 Ag]Ag + ions from the surfaces of [ 105 Ag]AgNP immediately forms  Table 4; data are presented as mean ± SEM; n = 4 rats per time point. Data points for both groups of rats at 0.75 and 24 h are set slightly aside from each other for easier distinction of the highly reproducible data obtained. Statistical one-way ANOVA analysis with the post-hoc Bonferroni test in between all timepoints are given in the matrix below  The literature discussed in the Background section notes that the fast, initial Ag + ion release gradually slows down [32] due to poorly soluble Ag-salt layers which are formed on the surface of the remaining [ 105 Ag]AgNP [31,38,40].
The complex biokinetics of inhaled [ 105 Ag]AgNP leads to the clearance of two slowly dissolving particle species -[ 105 Ag]AgNP and clusters of [ 105 Ag]Ag-salt. This is schematically illustrated in Fig. 9 summarizing dissolution, transformation, and precipitation of [ 105 Ag]AgNP in several consecutive steps.
As indicated in the schematics (Fig. 9) the Ag-salt layers around In addition, translocation across the ABB may be mediated by naturally occurring exosomal nanovesicles (ENV) initially synthesized in the endosomal compartment of many eukaryotic cells including macrophages and cells of the lung epithelium. When ENV fuse ]Ag-salt clusters are phagocytized by lung surface macrophages (step 7) which will gradually transport them to the distal end of the ciliated airways for mucociliary transport to the larynx where they are swallowed into the GIT (step 8). Alternatively both particulate species may be endocytosed by cells of the alveolar epithelium (e.g. epithelial type 1 + 2 cells, fibroblasts et.) which may exocytose them in exosomes for translocation across the ABB (step 9). Translocation across the ABB of both particulate species may also occur directly from the ELF as indicated by the arrows of translocation. Hence this series of steps highlights the fate of [ 105 Ag]AgNP and their degradation products, which results in the clearance of two slowly dissolving particle speciespersistent cores of [ 105 Ag]AgNP and clusters of low solubility [ 105 Ag]Ag-salt. Once arrived in the blood both particulate species may accumulate in secondary organs and tissues as indicated schematically by liver, spleen, and kidneys and discussed below. Note that we focus here on the alveolar epithelium due to our interest in longterm particle clearance. We hypothesize that steps 1 to 6 are similarly occurring in the airway epithelium leading predominantly to mucociliary clearance with the inner cell surface they are released extracellularly. They are considered to serve "as a mechanism to discharge unwanted material from the cells, but they also could form the basis of an efficient cell-cell communication mechanism" [49]. For example, when 20 nm AuNP were applied to cultured primary human macrophages the AuNP were rapidly taken up intracellularly and released within ENV [50]. Moreover, very recently ENV received much attention as natural, non-cytotoxic, nanotherapeutic carriers for specific cell targeting [51].

Comparison of 24 h excretions after IV injection, IT instillation and GAVage of soluble ions of 192 Ir, 48 V, 198 Au and of [ 110m Ag]Ag ions
In Fig. 1a we showed that 24 h p.e. IV injected soluble [ 110m Ag]AgNO 3 was rapidly eliminated from the blood into the GIT and feces (fraction of 0.87) with only small fractions found in secondary organs and tissues. Even more surprising, in Fig. 1b we found that 24 h p.e. a fraction of IT instilled soluble [ 110m Ag]AgNO 3 was also rapidly eliminated (0.70) from the lungs into the blood and further into the GIT and feces with only small fractions found in secondary organs and tissues and also in urine. Therefore, the question arises, how such a rapid elimination from the blood to the GIT is possible after IV injection of soluble [ 110m Ag]AgNO 3 , and what leads to such a rapid translocation across the ABB into the blood and further into the GIT after IT instillation of soluble [ 110m Ag]AgNO 3 The literature cited in the Background section suggests rapid precipitation of low-solubility Agsalts in blood and/or ELF, respectively, [31,39,41] which is sketched in Fig. 9. It basically excludes the presence of significant amounts of [ 110m Ag]Ag-ions in solution due to the abundance of salt-ions in blood and ELF which lead to the precipitation of nano-sized clusters.
However, nano-sized clusters smaller than 6-8 nm and/or macromolecules in blood would be subject to renal glomerular filtration and urinary excretion [52], which we did not observe. Instead, the [ 110m Ag]Ag-salt precipitates were rapidly eliminated into the GITmost likely by liver uptake and the hepato-biliary clearance pathway. The size of those precipitates may play an important role. After IT instillation of monodisperse, triphenylphosphine surface-coated gold NP of 1.4 nm, 2.8 nm, 5 nm, 18 nm, 80 nm, and 200 nm diameter the translocated fraction across the ABB during the first 24 h p.e. declined rapidly with increasing NP size from almost 0.1 (of the initially delivered NP mass) for 1.4 nm NP by a hundred-fold decline for 200 nm sized particles [53]. Furthermore, after IV injection of the same set of gold NP resulting in predominant liver retention, we quantitated the hepato-biliary cleared fraction (HBC) of 1.4 nm gold NP to be 0.05 while the HBC of 2.8 nm gold NP was 0.008 and all larger-sized gold NP from 5 nm to 80 nm were only cleared by 0.005 during the first 24 h p.e [54]. This is in clear contrast to the predominant elimination of the nano-sized [ 110m Ag]Ag-salt clusters from the blood into the GIT. The results of the IT instilled and IV injected gold NP together with the absence of urinary [ 110m Ag]Ag excretion implies that the Ag-salt precipitates may (a) either have translocated across the ABB as extremely small particulates (a fewnanometers) and were scavenged rapidly in the liver or (b) the small Ag-salt precipitates increased their size during circulation to larger than about 10 nm (which seems not very plausible) or (c) that such extremely small Ag-salt precipitates are protected against renal filtration by unknown surface modifications of their biomolecular corona and/or (d) by exosomal mediation. However, the current literature does not provide any suitable candidate biomolecules blocking renal filtration and concurrently allowing hepatocytes to transcytose the [ 110m Ag]Ag-salt clusters into the Space of Dissé for further elimination through the gall-bladder into the small intestine. These remain urgent questions for future investigations.
In Table 5 the urinary and fecal excretion data after application of [ 110m Ag]Ag + ions are compared with the corresponding data after application of soluble 192 198 Au-and 48 V-ions. The comparison is done for the first 24 h after intratracheal instillation and intravenous injection. The GIT retention found within the first 24 h is added to the fecal excretion data in order to obtain the fractions that have de facto already been cleared from the lungs and are 'ready' for excretion. The data are compiled from auxiliary studies of previous inhalation investigations of poorly soluble [ 192 Ir]IrNP [28], [ 198 Au]AuNP [25] and [ 48 V]TiO 2 NP [30].
After IV injection of 192 Ir-, 198 Au-and 48 V-ions these are minimally excreted in feces but more prominantly excreted via urine as indicated by their ratio (urinary: fecal) in Table 5. In contrast, IV injected [ 110m Ag]Ag ions are almost completely (fraction of0.87) excreted in feces and only a 100-fold lower amount in urine. This implies that [ 110m Ag]Ag + ions do not remain in a form that allows renal clearance, which means that they either are no longer ions that could be excreted by renal clearance or that they have formed secondary nanoparticles which were not accessible to renal filtration. Instead, they were eliminated into the GIT for fecal excretion. This is in line with the findings in the literature as presented in the Background section and summarized above. Several reports [33,39,40,55,56] noted precipitation of Ag + -ions in biological fluids and subsequent formation of Ag-salt precipitates such as poorly soluble AgCl, Ag 2 S, Ag 2 PO 4, and Ag 2 Se. According to these observations and, since renal clearance of [ 110m Ag]Ag ions is negligible, after IV injection a large fraction of the Ag + -ions must have formed poorly soluble, nano-sized Ag-salt precipitates. The predominant fecally excreted fraction indicates that these precipitates were cleared via the hepato-biliary pathway; i.e. they were metabolized mainly by liver hepatocytes and released into the bile fluid of the Space of Dissé for further elimination via the gall bladder into the small intestine [57,58] as sketched in Fig. 9. Note, hepatocytes do not metabolize metallic cations like [ 110m Ag]Ag [57,58].
Twenty-four hours after IT instillation of 192 Ir, 198 Au and 48 V ions only small fractions of between 0.06 and 0.2 (Table 5) are fecally excreted (including GIT retention), while 24 h after IT instillation of [ 110m Ag]Ag-ions, a fraction of 0.7 of the instilled dose is excreted in feces. This is almost the same fraction as after IV injection (0.87). Urinary excretion was similarly low after IV injection.
After IT instillation, rapid mucociliary clearance of [ 110m Ag]Ag deposited on the airway epithelium is expected to contribute to the fecally excreted fraction. However, based on the 24 h data after the inhalation of [ 105 Ag]AgNP compiled in Table 3 it is not plausible to attribute a fraction of more than 0.3 to fast clearance up to this time point. According to literature presented in the Background section, it appears reasonable to attribute the additional fecal fraction of 0.3-0.4 to the rapid clearance of poorly soluble, nano-sized Ag-salt precipitates which had been formed with abundant Cl − -, S 2− -, PO 4 2− -, ions as well as less abundant but more stably binding Se 2− -ions present in ELF. For their rapid elimination via feces within 24 h, they were first translocated across the ABB into the blood and from there via the hepato-biliary pathway into the GIT and feces. Furthermore, after intratracheal instillation of [ 110m Ag]Ag -ions the differential fraction of 0.21 between the fecal excretion after 24 h (0.70, including the GIT content) and that after 7 days (0.91) is likely due to the hepato-biliary clearance pathway (HBC). Hence, there is a biphasic clearance after either IT instillation or IV injection of soluble [ 110m Ag]AgNO 3 . Additional confirmation comes from the daily fecal excretion measurements after all three instillation applications (IV, IT, GAV) which are shown in Fig. S7 of the Supplementary Information.
Since mucociliary clearance to the larynx and ABB translocation into blood lead both to fecal excretion of [ 110m Ag]Ag it is not directly possible to distinguish between both clearance pathways after IT instillation of soluble [ 110m Ag]Ag. The negligible amount of urinary excretion indicates that free 110m Ag-ions are virtually absent due to their precipitation in ELF, while a slightly higher fraction of 198 Au-ions and large fractions of 192 Irand 48 V-ions were found in urine 24 h p.e. (see Table 5). Additionally, the third auxiliary biokinetics study in Fig.  1c after oral instillation of an [ 110m Ag]Ag-solution showed almost exclusive fecal and minimal urinary excretion, which is compatible with results reported earlier [40], and suggests the dominant precipitation of Ag-salt in the GIT and negligible uptake from the GIT through the intestinal barrier into the blood.
Therefore it is plausible to conclude that the differential 0.21 fraction is the upper limit of [ 105 Ag]Ag-salt precipitates after [ 105 Ag]AgNP inhalation which may have been translocated across the ABB from day 2 to day 7 into the blood and eliminated via HBC into the GIT and feces.   Figure 1) applied in the auxiliary studies in the present work. Note, in the data of 24 h fecal excretion, the 24 h GIT fractions are added due to the delayed passage through the GIT into feces. Data are given as mean ± SD, n ≥ 4 rats per group.
Figs. 10, 11, 12, 13 and 14 since both NP show a similar NP condensation dynamics immediately after spark ignition and evaporation. Initially, in the hot zone close to the igniting spark both, vaporized Ag and Au, condense and coalesce, forming liquid droplets up to 5-8 nm. Thereafter, when the droplets have escaped towards colder zones downstream, they solidify while continuing to coagulate until agglomeration is essentially stopped by dilution with clean air adjusted to maintain NP aerosol concentrations of about 1•10 7 NP/cm 3 . The inhalation of these aerosols by the rats after sufficient cooling -occurs within 5-10 s after generation (see the experimental setup in Fig. S2 of Supplemental Information). Since we cannot distinguish later between the retention and clearance of the inhaled [ 105 Ag]AgNP and their degradation products formed after inhalation we simply write "[ 105 Ag]Ag activities".  (Fig. 10c) during the first 24 h after inhalation is more than 100 times higher than the corresponding [ 195 Au]Au activity after [ 195 Au]AuNP inhalation (Fig. 10d), we conclude that translocation of [ 105 Ag]AgNP and their degradation products across the ABB accounts for this cleared fraction. From the blood, the material is eliminated via hepato-biliary clearance into the GIT and subsequently excreted in feces similar to the fecal clearance after IT instillation of soluble [ 110m Ag]AgNO 3 .
Similarly, Fig. 10a and b show that from day 2 until day 7, long-term macrophage-mediated clearance (

Comparison of the fecal excretion after inhalation of [ 105 Ag]AgNP and [ 195 Au]AuNP
The daily fecal excretion per ILD and the cumulative fecal excretion after inhalation of [ 105 Ag]AgNP are shown in Fig. 3a and Fig. 1), we postulate that besides mucociliary clearance from the conducting airways a second clearance pathway was possible after inhalation of [ 105 Ag]AgNP via ABB translocation into the blood and subsequent clearance into the duodenum  [31,40,41] and discussed in the Background section of this report.

Comparison of the urinary excretion after inhalation of [ 105 Ag]AgNP and [ 195 Au]AuNP
As shown in Fig. 4, [ 105 Ag]Ag activity fractions in urinary excretion are very low and accumulate after 28 days to a fraction of only 0.015 of the inhaled [ 105 Ag]AgNP dose. When comparing these data with those after inhalation of poorly soluble [ 195 Au]AuNP in Fig. 12 there are clear differences in the kinetics of urinary excretion. After inhalation of [ 105 Ag]AgNP, a maximal urinary excretion rate of 0.003 d − 1 is observed 2 days p.e. followed by a decrease of a factor of 10 until the end of the observation period 28 days p.e. We hypothesize that the urinary excretion pattern during the first week p.e. relates to the urinary excretion of very small amounts of nano-sized precipitated [ 105 Ag]Ag-salt which are smaller than 6-8 nm and will pass through the kidneys into urine [52,59].
In contrast, after [ 195 Au]AuNP inhalation the urinary excretion rates remain very low at about 0.00005 d − 1 throughout the first week p.e. and start to increase thereafter with a peak fraction of 0.005 at 12 days p.e. As a result, the cumulative urinary excretion of inhaled [ 195 Au]AuNP in Fig. 12d is almost negligible during the first week p.e. but increases steadily up to 0.015. In contrast, cumulative urinary excretion of inhaled [ 105 Ag]AgNP in Fig. 12c steadily increases after inhalation up to 0.017 at 28 days p.e., which is similar to that after [ 195 Au]AuNP inhalation in Fig. 12d.    Fig. 13a Fig. 14c and d and Fig. 14e and f). This may indicate rather a rather slow clearance dynamics in the secondary organs. Along this line the similar fractional concentrations of both types of nanoparticles in the brain suggests that only a small amount of precipitated [ 105 Ag]Ag-salt accumulates in the brain during the first 4 h p.e. But 24 h and 7 days p.e. the [ 105 Ag]Ag accumulation in the brain increases rather rapidly which differs from the rather constant fractional concentrations of the other secondary organs. This may suggest that the blood-brain-barrier is tight for [ 105 Ag]Ag-salt precipitates only for the first 4 h p.e.

Conclusion
Intratracheal inhalation of freshly generated [ 105 Ag]AgNP aerosols for 1½ hours allows low dose deposition in the lungs of adult healthy rats thereby avoiding nasal and pharyngeal deposition. The sensitivity obtained with radiolabeled NP is extremely high. Highly sensitive γ-ray-spectrometry, allows a dynamic, analytical range over five orders of magnitude across the different specimens. The methodology of radio-analysis is easy to manage for the large number of samples in order to study quantitative biodistributions of [ 105 Ag]AgNP and their possible degradation products in the rat organism, as well as complete excretional clearance out of the rat organism after five different retention time intervals up to 28 days p.e. The repeatability of the initially rapidly evolving biokinetics proved to be convincingly good when comparing the two independently investigated groups of four rats studied at 0.75 h and 24 h p.e., respectively. Rapid

Study design
Twenty-eight healthy, adult, female Wistar Kyoto rats (WKY/Kyo@Rj rats, Janvier, Le Genest Saint Isle, France) were randomly assigned to seven groups of four rats and subjected to intratracheal inhalation of an [ 105 Ag]AgNP aerosol for 1½ hours via an endotracheal tube [60]. The biodistribution was analyzed 0.75 h, 4 h, 24 h, 7d and 28d after exposure. The first group of rats, which was exsanguinated and dissected immediately after the 1½-hour intratracheal inhalation exposure, was assigned to the retention time-point 0.75 h since the nanoparticles brought into the rats' lungs over 1½ hours had an estimated average time of only 45 min for deposition, uptake, and distribution. In order to test the repeatability of the rapidly changing biokinetics directly after inhalation, two additional groups of four rats were inhalation exposed and analyzed at 0.75 h and 24 h p.e., respectively. The additional two groups were exposed 1 day after the inhalation exposures of the initial 0.75 h and 24 h groups, respectively.
Furthermore, twenty-four healthy, adult, female Wistar Kyoto rats (WKY/Kyo@Rj rats, Janvier, Le Genest Saint Isle, France) were randomly assigned to six groups of four rats and subjected to intratracheal instillation, or intravenous injection, or oral instillation (gavage) of soluble [ 110m Ag]AgNO 3 solutions and biokinetically studied at either 24 h or 7 days p.e.

Animals and maintenance
All Wistar-Kyoto rats (WKY/Kyo@Rj rats, Janvier, Le Genest Saint Isle, France) were housed in relativehumidity and temperature-controlled ventilated cages (VentiRack Bioscreen TM, Biozone, Margate, UK) on a 12-h day/night cycle. Rodent diet and water were provided ad libitum. The rats were adapted for at least 2 weeks after purchase and then randomly attributed to the experimental groups. When starting the studies, the rats were 8-10 weeks old and exhibited a mean body weight of 204 ± 13 g. Some physiological parameters of the rats are given in Table 3.
All experiments were conducted under German federal guidelines for the use and care of laboratory animals in accordance with EU Directive 2010/63/EU for animal experiments. The studies were approved by the Regierung von Oberbayern (Government of District of Upper Bavaria, Approval No. 211-2531-94/04) and by the Institutional Animal Care and Use Committee of the Helmholtz Centre Munich.

Synthesis and characterization of the [ 105 Ag]AgNP aerosol
Radiolabeled AgNP aerosols were produced continuously during the experiments by spark ignition between two high-purity (99.997%), cylindrical silver electrodes (diameter 3.0 mm, length 5 mm; Cat. No. AG007912; Goodfellow GmbH, Hamburg, Germany) which had been proton irradiated in the cyclotron at JRC (Ispra, Italy). The protons impinging on one of the flat ends of each electrode had an energy of about 33.5 MeV in order to achieve the highest possible 105 Ag-activity near the surface. This radioactivity was distributed nearly homogeneously throughout a layer thickness that would theoretically be consumed by the spark ignition process during the experiments. At the time the inhalation experiments were performed, the specific 105 Ag radioactivity was 2.60 MBq/mg in a surface layer of about 250 μm thickness. The radioactive 105 Ag decays back to 105 Pd via electron capture and positron emission, with a half-life of 41.3 days, thereby emitting γ-rays with different energies; one γ-emission line of 0.28 MeV with a fractional emission probability of 0.302 was selected for the γspectrometrical analyses.
For each group of rats, the [ 105 Ag]AgNP aerosol was freshly generated in the spark ignition aerosol generator (GFG100, Palas, Karlsruhe, Germany) at 250 Hz spark frequency in an argon (Ar) gas stream of 3 L/min. During spark ignition between the two Ag electrodes, small amounts of Ag evaporate. The vaporized Ag coalesces, forming liquid droplets up to 5-8 nm. When the droplets have escaped towards colder zones downstream, they solidify to solid spheres. The electrically charged aerosol of these primary spheres is immediately quasineutralized by an inline radioactive 85 Kr source and the highly concentrated and continuously agglomerating [ 105 Ag]AgNP pass through a 30 cm long tubular furnace that is kept at a temperature of 600°C to form single, densely packed agglomerated particles. Coagulation continues until it is stopped by dilution with clean air when the [ 105 Ag]AgNP reach a size of 20 nm. Since densely packed spheres will occupy 74% and more randomly packed spheres about 64% of the agglomerate volume we suggest a density of 7.3 g/cm 3 (= 0.7 • 10.49) of the generated and heat-treated AgNP.
Downstream of the furnace the aerosol was cooled and diluted in a copper tube (inner diameter 8 mm) by mixing with humidified oxygen and nitrogen to obtain a fractional oxygen concentration of 0.2-0.25. After dilution to concentrations of about 1•10 7 NP/cm 3 further agglomeration was negligible within the few seconds prior to inhalation by the rats. The generated 20 nmsized [ 105 Ag]AgNP still have a chain agglomerated/aggregated structure even after 600°C heat-treatment albeit more compact than the non-heat-treated NP. The flow rate was typically 10 L/min and the fractional relative humidity of the aerosol was set to about 0.7 before entering the inhalation apparatus; the whole inhalation apparatus and the inhalation methodology including the pre-set fractional relative humidity were described earlier [61] and is schematically displayed in Fig. S3 of the Supplementary Information. The aerosol particle concentration and size distribution were continuously sampled and controlled by a condensation particle counter (CPC 3022A, TSI, Aachen, Germany) and a scanning mobility particle spectrometer (SMPS; consisting of a model 3071 differential mobility analyzer and a CPC model 3010, TSI, Aachen, Germany), respectively. Averages of the count median diameters (CMD), volume median diameters (VMD) and geometric standard deviations (GSD) as well as number concentrations and volume concentrations are given as mean ± SD in Table 2. Since the SMPS instrument exhibited a lower particle size detection limit of 10 nm, the averaged spectra were fitted to a lognormal size distribution using the leastsquares method and the fits were extrapolated to a size of 1 nm (for details see Supplementary Information). These corrections led to slightly lower CMDs while the GSD changed only negligibly (see Table 2). The characteristic parameters of the freshly generated [ 105 Ag]AgNP aerosol were the same as those generated without the radio-label using non-irradiated, pure silver electrodes; TEM images were analyzed from the latter. Similarly, the chemical composition was determined by Electron Energy Loss Spectrometry (EELS). The specific 105 Ag activity of the aerosol particles was determined by γspectrometric analysis of absolute filters onto which [ 105 Ag]AgNP had been collected at an aerosol flow (0.3 L/min) throughout each 1½-h exposure period. From the activity deposited on the filter, an activity concentration of the [ 105 Ag]AgNP aerosol of 2.60 kBq•μg − 1 was derived. At this activity concentration the atomic ratio of 105 Ag: Ag in the nanoparticles is about 1.3 × 10 − 6 . Hence, statistically, every second AgNP will contain a 105 Ag -radiolabel. Therefore, the 105 Ag-radiolabeling involves a minimal impurity of the stable silver matrix which would be extremely unlikely to affect the stability and the physicochemical characteristics of the [ 105 Ag]AgNP.

Intratracheal inhalation exposure
The four slightly anesthetized adult rats in each group were ventilated individually via a flexible endotracheal tube and placed on their left lateral side in an air-tight plethysmograph box of our tailor-made inhalation apparatus and connected to the aerosol system, (see Fig. S3 of Supplementary Information). They were exposed to the freshly generated aerosol for 1½ hours. In this report, this exposure method will be called "intratracheal inhalation" (see reference [60]).

Treatment of the rats after inhalation
Anesthesia of each rat was antagonized immediately post-exposure (p.e.) as described in the Supplementary Information and previously in [25,62]. Thereafter, each rat was kept individually in a metabolic cage and excreta were collected separately and quantitatively. For ethical reasons, the rats of the 28 days group were maintained individually in a normal cage on cotton cloths starting immediately after [ 105 Ag]AgNP inhalation. Each cloth was replaced with a new one every 3-4 days (2 cloths per week), and the fecal droppings were quantitatively separated from the previous one. After separation, the cloth contained only [ 105 Ag]Ag activity originating from urine which had soaked and dried. In Table S2 the list of collected organs, tissues, body fluids, and excretion is given. Since the cages of the four rats of each group were located next to each other, the rats had the continuous sensory perception of each other.

Evaluation and statistical analysis of [ 105 Ag]AgNP biokinetics
At 0.75 h, 4 h, 24 h, 7d, and 28d p.e., rats were anesthetized (by 5% isoflurane inhalation) and euthanized by exsanguination via the abdominal aorta. Blood, all organs, tissues, and excretions were collected and the 105 Ag radioactivity was determined by γ-spectrometry without any further physicochemical processing of the samples, as described in the Supplementary Information and in earlier works [25,[62][63][64]. Throughout this report nanoparticle quantities are calculated from the 105 Ag activity determined with γ-scintillation detectors, properly calibrated in γ-ray energy and detection efficiency for 105 Ag, and corrected for background and radioactive decay during the experiments (see Supplementary Information). Samples yielding background-corrected counts in the 280 keV region-of-interest of the 105 Ag γ-spectrum were defined to be below the detection limit (<DL; 0.1 Bq) when the number of counts was less than three standard deviations of the background counts collected during 200 min without any sample in the γ-scintillation detector.
BALs were performed by applying 6 × 5 ml of phosphate-buffered-saline solution (PBS without Ca 2+ or Mg 2+ ) under the gentle massage of the thorax. The recovered fractional BAL fluid (BALF) (about 0.8 of instilled PBS) was centrifuged at 500 g for 20 min at room temperature to separate the lavaged cells from the supernatant. The [ 105 Ag]AgNP content was determined by γ-ray-spectrometry.
Up to 24 h p.e. early clearance was measured in the GIT and feces comprising of (a) MCC-cleared [ 105 Ag]AgNP and of precipitated, low-solubility [ 105 Ag]Ag-salt precipitates from the conducting airways and (b) of precipitated, low-solubility [ 105 Ag]Ag-salt precipitates which had translocated across the respiratory ABB into blood and via liver and gall-bladder into the small intestine. The early clearance data of the 7 days-and 28 days-group were derived from fecal excretion measurements during the first 3 days p.e. The clearance contribution of the [ 105 Ag]Ag + -ion release -according to the literature discussed in the Background section -precipitating as low-solubility salt precipitates was calculated from the 24 h data of the auxiliary study after the intratracheal instillation of [ 110m Ag]Ag solution.
After 3 days p.e. fecal excretion was considered as long-term clearance from the peripheral lungs comprising of macrophage-mediated LT-MC of (a) persistent, low-solubility Ag-salt-layer protected cores of [ 105 Ag]AgNP in the lung periphery towards the larynx into the GIT and (b) low-solubility [ 105 Ag]Ag-salt precipitates translocated across the ABB into blood and via liver and gall-bladder into the small intestine. The clearance contribution of the long-term [ 105 Ag]Ag + -ion release precipitating to low-solubility salt precipitates was estimated from the difference of the 7 days minus 24 h data of the auxiliary study after the intratracheal instillation of [ 110m Ag]Ag solution.
About 0.7 of the fractional blood volume was recovered by exsanguination. Thus, organs and tissues contain residual blood whose 105 Ag radioactivity needs to be subtracted to obtain the true content of nanoparticles and their degradation products. For this purpose, the residual blood contents of organs and tissues after exsanguination were calculated by making use of the findings of Oeff & König [48], and the true radioactivities of the organs and tissues were obtained by subtracting the blood-related 105 Ag-radioactivity values. The procedure is outlined (see Eqns. (4)(5)(6)(7)(8)) in the Supplementary Information.
The measured [ 105 Ag]Ag-activity values were expressed as fractions of the initial lung dose (ILD) i.e. the [ 105 Ag]AgNP radioactivity deposited in the lungs. Fractions were normalized to the sum of all sampled 105 Ag-radioactivities of a given rat (see Supplementary  Information). The mathematical procedure is derived in Eqns. 11 and 14 of the Supplementary Information. The fractions for each organ or tissue were averaged over the group of rats and were presented with the standard error of the mean (SEM). All calculated significances are based on One-Way-ANOVA analyses with the post-hoc Bonferroni test. In the case of direct comparisons of two groups, the unpaired t-test was used. Significance was considered at p ≤ 0.05.
The biokinetics data of lung-applied [ 105 Ag]AgNP were further normalized to the [ 105 Ag]AgNP fraction which had translocated across the ABB (see Eqns. 23 and 24 in the Supplementary Information).

Biokinetics of soluble [ 110m Ag]Ag after intratracheal instillation, intravenous injection or oral instillation (gavage)
Since [ 105 Ag]AgNP reportedly dissolve in body fluids a certain fraction of the [ 105 Ag]Ag may be released from the [ 105 Ag]AgNP, thus affecting the AgNP biokinetics analysis. In order to estimate such a release from the [ 105 Ag]AgNP surface and to quantify its effect, auxiliary experiments with carrier-free ionic 110m Ag were carried out. Note, that we could not use 105 Ag for these studies since this radioisotope was produced during proton bombardment of the metallic matrix of the solid silver electrodes using the JRC cyclotron; instead, we used 110m Ag + in a 0.1 m HNO 3 solution (5 mg Ag + in 0.5 ml) which was neutron-activated at the nuclear research reactor of Research Centre Rez (Husinec-Rez, Czech Republic) yielding 24 MBq of 110m Ag + . The 110m Ag isotope has a half-life of 249.9 d and is a gamma emitter emitting with several gamma emission lines of which we used those in the range of 650-900 keV. The use of both 110m Ag and 105 Ag isotopes of the chemical element Ag is expected to be equivalent in these biokinetics studies since both are isotopes of the chemical element silver. As reported previously [62], auxiliary studies (AUX) were performed at 24 h and 7 days after intratracheal instillation (IT) or intravenous injection (IV) or oral instillation (gavage) with the purpose of correcting the biodistributions of inhaled [ 105 Ag]AgNP for [ 105 Ag]Ag -ion release. In order to mimic [ 105 Ag]Ag released by [ 105 Ag]AgNP in the auxiliary studies, the following [ 110m Ag]Ag + -solutions were prepared: 0.33 μg•μL − 1 ionic Ag(NO 3 ) was added to carrier-free ionic 110m Ag + . The pH value was adjusted to 5. For the experiments, 60 μL of a solution containing 100 kBq ionic 110m Ag + and 20 μg of ionic, non-radioactive Ag + were administered per rat.