Engineered nanoparticles are becoming increasingly ubiquitous and their toxicological effects on human health, as well as on the ecosystem, have become a concern. Since initial contact with nanoparticles occurs at the epithelium in the lungs (or skin, or eyes),
A novel air-liquid interface cell exposure system (ALICE) for nanoparticles in liquids is presented and validated. The ALICE generates a dense cloud of droplets with a vibrating membrane nebulizer and utilizes combined cloud settling and single particle sedimentation for fast (~10 min; entire exposure), repeatable (<12%), low-stress and efficient delivery of nanoparticles, or dissolved substances, to cells cultured at the air-liquid interface. Validation with various types of nanoparticles (Au, ZnO and carbon black nanoparticles) and solutes (such as NaCl) showed that the ALICE provided spatially uniform deposition (<1.6% variability) and had no adverse effect on the viability of a widely used alveolar human epithelial-like cell line (A549). The cell deposited dose can be controlled with a quartz crystal microbalance (QCM) over a dynamic range of at least 0.02-200 μg/cm2. The cell-specific deposition efficiency is currently limited to 0.072 (7.2% for two commercially available 6-er transwell plates), but a deposition efficiency of up to 0.57 (57%) is possible for better cell coverage of the exposure chamber.
Dose-response measurements with ZnO nanoparticles (0.3-8.5 μg/cm2) showed significant differences in mRNA expression of pro-inflammatory (IL-8) and oxidative stress (HO-1) markers when comparing submerged and air-liquid interface exposures. Both exposure methods showed no cellular response below 1 μg/cm2 ZnO, which indicates that ZnO nanoparticles are not toxic at occupationally allowed exposure levels.
The ALICE is a useful tool for dose-controlled nanoparticle (or solute) exposure of cells at the air-liquid interface. Significant differences between cellular response after ZnO nanoparticle exposure under submerged and air-liquid interface conditions suggest that pharmaceutical and toxicological studies with inhaled (nano-)particles should be performed under the more realistic air-liquid interface, rather than submerged cell conditions.
Humans and other organisms are constantly exposed to a diverse set of exogenous substances. Ambient and occupational exposure to gases and particles are recognized as severe health risks, mainly via the lungs (inhalation), but also potentially via the skin or even the eyes [
Several
In this study, a new exposure system (ALICE) is presented and validated, for dose-controlled delivery of nanoparticles in liquids or solutions to cell systems cultured at the air-liquid interface. The uniformity, efficiency, repeatability and accuracy of the exposure method is determined with various solutions and nanoparticle suspensions and its applicability to toxicological and pharmacological studies is verified by examining the response of a widely used human epithelial-like cell line (A549) after exposure to dilute salt solutions and zinc oxide nanoparticles.
The ALICE utilizes cloud settling, in combination with single particle sedimentation, as the droplet deposition mechanism.
The principle of the operation of the ALICE is schematically depicted in Figure
The critical design aspects are: i) The cloud has to be dense enough for rapid "fall-out" so that most of the cloud remains in the chamber, ii) the air flow introducing the cloud into the chamber has to be chosen such that the falling cloud encounters the bottom of the chamber near its center (uniform distribution of the cloud) and the droplets have to be large enough for rapid single particle sedimentation to the ground. Since vibrating membrane nebulizers are characterized by high mass output and large particle diameter, this type of nebulizer is ideal for the ALICE.
The
where
where
The ALICE consists of four main components: 1) a
As seen in Figure
The liquid substance to be investigated is nebulized by a vibrating membrane generator (investigational eFlow, Pari Pharma GmbH, Germany), which was customized for the ALICE as described below [
In contrast to the commercially available eFlow rapid, the applied investigational eFlow used i) a membrane with larger holes resulting in larger mass output and larger particles (here: mass median diameter MMD = 4.4-5.4 μm), which is important for rapid cloud settling and short droplet sedimentation time and ii) an aerosol chamber with an air inlet at the side and an aerosol outlet through the front (directly opposite of the membrane), which can be directly connected to the exposure chamber. With this setup, the distance between the vibrating membrane and the exposure chamber was 5 cm, the inner diameter of the connector to the chamber was 1.9 cm and the flow rate was 5 liter/min.
Exposure of the cells with the substance under investigation occurs in the exposure chamber, designed to hold up to two standard transwell plates (1 cm away from the walls of the chamber) containing cells cultured at the air-liquid interface. Immediately after exposure to the nebulized substance, the transwell plates can be easily removed (within a few seconds) from the chamber via a drawer for further analytical processing or post-incubation of the cells.
The exposure chamber is a 12 liter box (bottom plate: 20 × 20 cm2; height: 30 cm) made of polycarbonate (Makrolon™) with plates held in place by an aluminum frame. Makrolon is durable enough for repeated sterilization with alcohol and its transparent nature allows visual inspection of the motion of the cloud and the extent to which the chamber is filled with droplets (denser clouds appear more opaque). The highly concentrated cloud enters through the left side wall (in the center, 20 cm above the bottom) and gravitates swiftly to the ground (within ~1 s) due to
Mass deposition onto the cells was measured with a quartz crystal microbalance (QCM 200/25, Stanford Research Systems, Sunnyvale, CA, USA) placed on the ground plate of the exposure chamber. The QCM is typically placed in one of the corners of the chamber, but the exact location is irrelevant, since the droplet deposition is spatially uniform, as is shown below. Mounting the QCM on a movable sledge allows for fast (within one second) and easy removal and insertion of the QCM, with minimum perturbation inside the exposure chamber.
The QCM determines the particle mass deposited onto a vibrating piezoelectric quartz crystal from the linear decrease in the resonance frequency of the crystal with increasing deposited mass. The QCM 200/25 uses a circular, AT-cut, α-quartz crystal with a resonance frequency of 5 MHz and operates at a sampling rate of 1 Hz. From the exposed crystal surface area, of 1.37 cm2, only the inner 0.4 cm2 is active (experiences a displacement within the plane of the crystal). The change in mass per unit area (Δ
where
For liquid films the Sauerbrey equation is not valid, since in this case the observed frequency change not only depends on the deposited mass of the film, but also on the viscosity and density of the liquid, as well as other factors such as the layer thickness, adsorption of material to the crystal and formation of sublayers within the film [
According to the manufacturer, surface-specific masses below 1 ng/cm2 (Δf < 0.05 Hz) can be detected. Although this is close to the observed zero point stability of <0.1 Hz, a more conservative lower detection limit of 18 ng/cm2 was adopted (Δ
Solutions and nanoparticle suspensions can be used in the ALICE. The characterization of the ALICE was performed with aqueous sodium chloride (NaCl), ammonium sulfate ((NH4)2SO4) and citrate solutions and with aqueous suspensions of gold (Au), ZnO and carbon black nanoparticles. The aqueous suspension of 15 nm Au nanoparticles, which was stabilized by 10 mM citrate (500 μg/mL), was purchased from British Biocell (EM.GC15, Batch 7894, British Biocell International, Plano GmbH, Wetzlar, Germany). The nominal particle concentration was 1.4 × 1012 particles/mL (mass concentration: 40 μg/mL = 40 ppm with a gold density of 19 g/cm3). Additionally, a 10 fold enriched Au suspension was prepared by centrifugation of the suspension at 18,626 RCF (relative centrifugal force) for 20 min and by removing 90% of the (particle-free) supernatant. The ZnO and carbon black nanoparticle suspensions were prepared in our lab from commercially available powders (ZnO: AlfaAesar, Ward Hill, MA, USA Id# 43141; primary diameter: 24-71 nm (manufacturer information); BET surface area: 13 m2/g, agglomerated. Carbon Black: Printex 90, Degussa (now Evonik), Germany; primary diameter: 14 nm; agglomerated). The ZnO and carbon black suspensions were prepared, as well as vortexed and sonicated twice for 1 min intermittently immediately prior to spraying the suspension with the nebulizer.
The uniformity of the droplet deposition in the exposure chamber was determined by placing 12 pieces of aluminum foil (3 × 3 cm) on the ground plate of the exposure chamber. Before and after exposure of the foils to the nebulized substances in the ALICE, their dry weight was determined by a gravimetric microbalance (Model r160p, Sartorius, Germany, accuracy ≤± 0.02 mg) and the deposited (dry) mass was determined from the change in foil mass. Adding all foil deposited salt masses, and scaling to the total area of the exposure chamber, yielded the total deposited salt (or nanoparticle) mass. The deposition efficiency was determined from the ratio of the total deposited (dry) mass and the mass filled into the nebulizer reservoir. The spatial uniformity of the deposition was determined from masses deposited onto the 12 foils. A qualitative representation of the spatial uniformity was obtained by transmission electron microscopy (TEM, CM12, FEI Co. Philips Electron Optics, Zürich, Switzerland) using a primary magnification of 3400× and 25,000× for Au and ZnO nanoparticles collected on TEM grids in the ALICE.
Time-resolved mass deposition was obtained for NaCl and (NH4)2SO4 solutions as well as for Au and ZnO suspensions by placing the QCM on the ground plate of the exposure chamber during an ALICE run.
For Au nanoparticles, the deposited mass was not only determined indirectly with the QCM (using the mass ratio of citrate and Au), but directly with gamma spectroscopy performed by the Helmholtz Zentrum Berlin in Berlin, Germany. The latter involved neutron activation of Au-197 into Au-198 (neutron flux was about 6 × 1012 cm-2s-1 for 1 hour) and subsequent determination of the Au mass on the aluminum foils from the intensity of the 412 keV gamma line of Au-198, relative to a known standard. As the obtained Au mass from QCM and gamma spectroscopy agreed within experimental uncertainties, both methods are considered equivalent.
For the ALICE experiments, ZnO suspension of 0.3, 1.5 and 7.5 mg ZnO/mL sterile H2O (Braun, Melsungen) were prepared immediately prior to use from three stock suspensions of 1 mg/ml, 2 mg/ml and 10 mg/ml, respectively. The stock suspensions were produced, vortexed and sonicated twice for 1 min intermittently and then diluted with water to obtain 1 ml of ZnO suspension for nebulization. For control purposes, cells were also exposed to dilute (10 mM) aqueous citrate (stabilization agent in Au suspension) or NaCl solutions, which were also prepared immediately prior to nebulization.
For ZnO exposure under submerged conditions, the desired amount of ZnO was incorporated directly into the cell culture medium by adding the appropriate volume of a 1 mg ZnO/mL H2O stock suspension. Within 30 min ZnO agglomerates of about 900 nm (mobility diameter) had formed in the cell culture medium as determined by dynamic light scattering measurements (HPPS 5001, Malvern Instruments Ltd, Worcestershire, UK). As agglomerates of this size are known to efficiently deposit (near 100%) due to sedimentation [
All exposure experiments were performed with a human epithelial-like cell line (A549) from a lung adenocarcinoma (obtained from ATTC, Manassas, VA, USA) representing the alveolar type II phenotype [
Then the medium in the lower chamber was replaced with 3 mL serum-free culture medium and the cells were placed in the exposure chamber of the ALICE system for exposure to ZnO nanoparticles (or salt solutions) as described above. After 10 min in the ALICE, the cells were removed and incubated for 3 h in the cell incubator. Immediately after the post-incubation period, the cells were washed with PBS and directly lysed on the insert membrane by adding 350 μl of a cell lysis buffer, suitable for isolation of total RNA (Qiagen) (further details see RT-PCR section), or 2 mL WST-1 containing medium was added to the upper compartment (insert) to measure cell viability (further details are given below).
Adopting one of the most frequently used cell handling procedures for toxicological experiments [
Gene expression at the mRNA level of interleukin-8 (IL-8) and hemeoxygenase-1 (HO-1) was measured, 3 h after exposure, by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The exposed cells were lysed and total RNA was purified using the Qiagen RNeasy Mini Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's instructions. First-strand cDNAs were synthesized by reverse transcription from 0.5 μg total DNase I-treated RNA with a random nonamer primer (Metabion, Martinsried, Germany) and Superscript II reverse transcriptase (Invitrogen, Karlsruhe, Germany). For PCR amplification, the cDNA was mixed with the specific 5' and 3'primers and transcript levels were quantified using Absolute QPCR SYBR Green Mix plus ROX kit (ABgene, Hamburg, Germany) with the ABI Prism 7000 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). The housekeeping gene GAPDH was used as internal reference to normalize the mRNA levels of the genes being studied, and IL-8 and HO-1 induction is reported after normalization to control conditions. The following primers were used: IL-8 5'primer: IL-8 5'primer: 5'-ATG ACT TCC AAG CTG GCC GTG GCT-3'; IL-8 3'primer: 5'-TCT CAG CCC TCT TCA AAA ACT TCT C-3'; HO-1 5'primer: 5'-AAG ATT GCC CAG AAA GCC CTG GAC-3'; HO-1 3'primer: 5'-AAC TGT CGC CAC CAG AAA GCT GAG-3'; GAPDH 5'primer: 5'-CCA TGA GAA GTA TGA CAA CAG CC-3'; GAPDH 3'primer: 5'-TGG CAG GTT TTT CTA GAC GG- 3'.
Cell viability was measured with the cell proliferation reagent WST-1 (Roche Applied Sciences, Germany). The ready-to-use WST-1 reagent was mixed with cell culture medium (100 μl/mL) and was added to the apical side of the cells for both air-liquid interface and submerged culture conditions. After 30 min incubation at 37°C, the light absorbance at 450 nm was measured.
The mean volume (or mass) output of the nebulizer was determined by measuring the nebulization time for a known amount of liquid, which was accomplished by observing the clearly visible dense cloud of droplets generated by the nebulizer. For nebulization of 1 mL of salt solution or nanoparticle suspension, the nebulizer needed between 90 and 150 s and the corresponding volume (mass) production rates were between 0.40 and 0.67 mL/min (or 0.40 and 0.67 g/min). The small amount of residual liquid in the nebulizer was disregarded (5-10% for 1 ml of liquid filled into the reservoir volume; measured by gravimetric analysis of the nebulizer before and after discharge). Consequently, the aerosol (droplet) mass concentration was approximately 80-130 g/m3 for a sample flow rate of 5 liter/min.
For consecutive nebulizer runs with 1 mL of 1% NaCl solution and various nanoparticle suspensions, the nebulization times were constant within ± 10 s, which means that the short term repeatability was better than 7%. However, a gradual increase in nebulization time was observed with increasing use of the membrane. For further details on the characteristics of the eFlow nebulizer, please refer to [
The droplet deposition on the cells during ALICE exposure was monitored by placing the QCM in the exposure chamber next to the transwell plates containing the cells. As mentioned previously, for liquid films the quantitative interpretation of the change in resonance frequency (
The response of the QCM during a typical ALICE exposure is depicted in Figure
The data in Figure
Hence, the timing of the ALICE experiments can be optimized as follows: The cells and the QCM can be removed from the ALICE after 300 s (5 min) and drying of the QCM deposit outside of the exposure chamber can be accomplished with dry air within a few minutes. Therefore, if the QCM does not need to be cleaned (or if a second clean quartz crystal is available) an entire ALICE run can be performed within ~10 min.
The relevance of cloud settling for the ALICE becomes evident, if the cloud settling and the single particle settling speed are compared. According to Figure
As mentioned, the dry nanoparticle/solute mass deposited on the cells can be determined from the dried deposit on the QCM. This was verified by comparing the QCM with gravimetric data for dry (NH4)2SO4 and NaCl, as well as carbon black and ZnO nanoparticles, sprayed in the ALICE, as described above. As seen in Figure
This confirms the validity of the Sauerbrey equation (equation 3) and shows that the QCM can be used for accurate mass measurements in the ALICE. The validity of the Sauerbrey equation also implies that the prerequisite of the Sauerbrey are met, namely the formation of a uniform, rigid and thin layer on the quartz crystal after exposure in the ALICE.
The good agreement between the QCM and the gravimetric mass already suggests that the droplet deposition is spatially homogeneous on the sensitive part of the quartz crystal (0.4 cm2), since this is a pre-requisite for the validity of the Sauerbrey equation. This important issue was investigated more rigorously by distributing 12 rectangular pieces of aluminum foils (3 × 3 cm2) over the exposure chamber (see insert in Figure
The high degree of homogeneity was also confirmed for Au and ZnO nanoparticles by placing a TEM grid in the ALICE. Since similar results were obtained for both nanoparticle types, only the ZnO data are shown here. As seen from Figure
It is also evident from Figure
Another important characteristic of the ALICE is the deposition efficiency of the nebulized material on the bottom of the exposure chamber or even more importantly on the cells. Since the deposition efficiency on the cells depends on cell coverage and hence on the type of transwell plates used, the deposition efficiency was initially investigated in the exposure chamber, which is defined as the ratio of solute (or nanoparticles) mass deposited on the bottom plate and solute/nanoparticle mass filled into the nebulizer. As seen from Figure
The standard deviation (0.07) of the deposition efficiencies (n = 30) represents the repeatability of substance delivery to the cells in the ALICE (Figure
It is noteworthy that the deposition efficiency is independent of the amount of sprayed material as was confirmed for 1 mL to 5 mL salt solutions and nanoparticle suspensions. This indicates that all "loss mechanisms" are independent of time including the depletion of the sample flow due to cloud settling.
Potential adverse effects of cell handling on cell viability (WST-1 test) during ALICE experiments were investigated with A549 lung epithelial cells. Although the viability of the cells was slightly impaired (87 ± 2% relative to submerged cells; Figure
As an application of the ALICE system, A549 cells were exposed to various doses of ZnO nanoparticles and the mRNA expression of the pro-inflammatory cytokine IL-8 and the oxidative stress marker HO-1 were investigated. Cell viability (WST-1) was not impaired for any of the particle concentrations used here (data not shown). The ratio of mRNA levels from the air-liquid interface and submerged conditions in the absence of ZnO was 1.9 and 1.4 for IL-8 and HO-1, respectively. This reflects the effect of transferring the cells to the air-liquid interface conditions. Furthermore, since there was no significant difference between non-exposed and 10 mM citrate-exposed cells at the air-liquid interface (data not shown), both non-exposed and 10 mM citrate-exposed cells can be used as negative control for ALICE experiments.
ALICE experiments were performed with ZnO nanoparticle doses of 0.3, 1.9 and 8.5 ZnO μg/cm2 (n ≥ 3). For reference, ZnO exposures under submerged conditions were also conducted at 0.7, 2.5 and 5.0 ZnO μg/cm2 (n ≥ 4). As discussed above, the ZnO dose was determined from the amount of ZnO nanoparticles added to the culture medium. As seen in Figure
Due to the well-known shortcomings of submerged cell exposure systems for pharmaco-toxicological studies on pulmonary epithelial cells (such as uncertain effective biological dose for nanoparticles and unrealistic exposure scenario due to missing air-liquid interface and absence of mucus or lining fluid on respiratory epithelial cells), there have been several approaches to develop exposure systems for cells at the air-liquid interface. In the CULTEX system, particles are deposited from a continuous aerosol flow by diffusional and gravitational deposition onto cells [
Previously described methods for deposition of micron-sized particles or droplets onto cells have relied on inertial impaction as the deposition mechanism. The use of impactors or impingers [
The ALICE utilizes a completely different, more "gentle" technique of particle generation and deposition and it provides direct dose measurements. As discussed above, micron-sized droplets (MMD = 4.5-5.4 μm) are generated at a very high concentration (80-130 g/m3) by a vibrating membrane nebulizer and are uniformly deposited via cloud and single particle settling onto cells at the air-liquid interface. It is noteworthy that the cloud settling speed of 13-17 cm/s is too small for impaction to occur, as evidenced by the absence of enhanced droplet deposition near the center of the chamber, where the cloud speed is largest (see foils 5 and 8 in Figure
In contrast to jet nebulizers or ultrasonic generators, vibrating membrane nebulizers do not exert high shear forces and do not heat the nebulized liquid during the generation process. Hence, it does not jeopardize the biological activity of potentially delicate therapeutic agents, such as biopharmaceuticals [
The QCM is a highly sensitive, fast-response instrument for real-time determination of the deposited substance mass on the cells. The QCM has a response time of ~1 s, a large dynamic range (0.018-1800 μ
The repeatability of 12% in mass dose delivery is similar to the value of about 10% reported for the spray exposure unit RHINOCON ([
Although the deposition efficiency in the exposure chamber of the ALICE is relatively large (0.57 ± 0.07), the cell-specific deposition efficiency is currently limited to 0.072 (for two 6-well plates) due to the poor fractional cell coverage of standard transwell plates. For comparison with previously described cell exposure systems, it is important to note that the deposition efficiency of 0.072 represents the
Unfortunately, previous exposure systems have either not been characterized in terms of deposition efficiency (no deposition efficiencies are given for any of the cell exposure systems for liquid substances described in the literature [
After nebulization of 1 mL of liquid, a 14 μm liquid layer is formed on the cells in the ALICE. Since the layer thickness is small compared to the diameter of the transwell inserts for the cells (>6.4 mm for standard 6-, 12-, and 24-well plates), nearly 100% of the nanoparticles (or solute molecules) interact with the cells due to diffusional motion and diffusional losses to lateral walls are small. Furthermore,
Although the nebulizer used in the current study can spray up to 5 mL of liquid per filling, the authors recommend using 1 mL for the following reasons: The QCM can be used as real-time indicator for 1 mL, or less, of sprayed liquid (frequency shift <715 Hz), the deposited liquid layer is sufficiently thin (14 μm) for efficient nanoparticle-cell interaction and the corresponding exposure time is short (5 min, which increases by about 2 min per additionally sprayed mL of liquid). Using less liquid reduces the liquid layer thickness and exposure time, but it also enhances the residual liquid fraction remaining in the nebulizer (0.05-0.1 mL; 10-20% for 0.5 mL) and hence the cell deposition efficiency.
The suitable concentration range of the nanoparticle suspension used in the ALICE is determined by the detection limit of the QCM. For nebulization of 1 mL of liquid, the lower detection limit of the QCM (0.018 μ
Although air-liquid interface exposures have become more widely used in recent years, there are very few quantitative comparisons between air-liquid interface and submerged (conventional) dose-response curves after nanoparticle exposure. Since the ALICE provides direct accurate dose measurements, the data set provided here can be used for such a comparison. For both exposure conditions, A549 cells showed no significant response in IL-8 and HO-1 mRNA expression after exposure to less than 1.0 μg/cm2 ZnO nanoparticles (Figure
To put the dose levels obtained during
The air-liquid interface cell exposure system (ALICE) allows uniform, efficient and dose-controlled deposition of solutions and nanoparticles in liquids on cell systems. The ALICE is a closed system suitable for up to two commercially available cell culture plates, which can be operated by a technical assistant without expert knowledge in aerosol technology. The deposition mechanism of the ALICE is based on gravimetric sedimentation, which simulates the
Cell exposure in the ALICE had no adverse effect on the viability of a widely used alveolar epithelial cell line (A549) for the recommended amount of liquid to be sprayed (1 mL). Dose delivery was repeatable (12%), uniform (<1.6% spatial variability) and occurred at a total (net) cell-specific deposition efficiency of up to 0.072 (for two 6-well plates). While this may seem small, it exceeds the total deposition efficiency obtained with most other exposure systems, if one includes all possible losses in the balance including residual liquid in the nebulizer, transport losses and losses during the turn-on/turn-off phase. Currently, the deposition efficiency of the ALICE is mainly limited by the low fractional cell coverage of standard transwell plates. For ideal cell coverage (100%) in the exposure chamber, the deposition efficiency can be improved to 0.57.
Dose-measurements with a quartz crystal microbalance (QCM) allow fast (~10 min for exposure and mass determination) and accurate (<7.3%) determination of the cell deposited dose. For the recommended 1 mL of sprayed liquid, a single ALICE exposure can provide 0.02-200 μg/cm2 of substance to the cells depending on the mass concentration of the nanoparticles/solute (0.0012-12%).
The applicability of the ALICE to obtain dose-response curves was demonstrated by exposing A549 cells to various doses of ZnO nanoparticles. The mRNA expression of the pro-inflammatory cytokine IL-8 and the oxidative stress marker HO-1 indicated substantial differences depending on the exposure method (submerged versus air-liquid interface) for doses larger than 1 μg/cm2. However, both methods suggest that ZnO nanoparticles are not toxic, if occupationally allowed exposure levels are obeyed.
The small amount of liquid to be used (1 mL), the gentle method of droplet generation, the realistic deposition process (for the alveolar region) as well as the competitive deposition efficiency (0.072) make the ALICE a useful
The authors declare that they have no competing interests.
GAF, BL, HS, EK and OS designed the exposure chamber, AGL, GAF, OS, EK conceived and designed the experiments; AGL, GAF, BL, VD, and CB performed experiments; AGL, EK and OS performed data analysis; AGL, CB, BRR, and OS made substantial contributions to writing the manuscript. All authors read and approved the final manuscript.
We would like to thank Mr. Bukalis and Mrs. Alber from the Helmholtz-Centre Berlin for Materials and Energy for determining the mass of the gold nanoparticles deposited in the ALICE using neutron activation analysis. We are grateful to Helga Hinze-Heyn and Gabriele Schumann for their excellent technical assistance and Kirsten Dobson for proof reading the English.
This study was supported by grants from the Animalfree Research Foundation, the Doerenkamp-Zbinden Foundation and the Helmholtz Association.