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Dosimetric quantification of coating-related uptake of silver nanoparticles Dajana Lichtenstein, Thomas Meyer, Linda Böhmert, Sabine Juling, Christoph Fahrenson, Soeren Selve, Andreas F. Thünemann, Jan Meijer, Irina Estrela-Lopis, Albert Braeuning, and Alfonso Lampen Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b01851 • Publication Date (Web): 18 Sep 2017 Downloaded from http://pubs.acs.org on September 18, 2017

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Dosimetric quantification of coating-related uptake of silver nanoparticles Dajana Lichtenstein1, Thomas Meyer4, Linda Böhmert1, Sabine Juling1, Christoph Fahrenson3, Sören Selve3, Andreas Thünemann2, Jan Meijer5, Irina Estrela-Lopis4, Albert Braeuning1, Alfonso Lampen1 1

German Federal Institute for Risk Assessment, Dept. Food Safety, Max-Dohrn-Str. 8-10, 10589,

Berlin, Germany 2

German Federal Institute for Materials Research and Testing, Unter den Eichen 87, 12205 Berlin,

Germany 3

ZELMI, Technical University Berlin, Straße des 17. Juni 135, 10623 Berlin, Germany

4

Institute for Medical Physics and Biophysics, Leipzig University, Härtelstraße 16-18, 04107 Leipzig,

Germany 5

Nuclear Solid State Physics, Leipzig University, Linnéstraße 5, 04103 Leipzig, Germany

Corresponding author: [email protected] Tel.: +4930 18412 3158

1 ACS Paragon Plus Environment

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Abstract The elucidation of mechanisms underlying cellular uptake of nanoparticles is an important topic in nanotoxicological research. Most studies dealing with silver nanoparticle uptake provide only qualitative data about internalization efficiency and do not consider nanoparticle-specific dosimetry. Therefore, we performed a comprehensive comparison of the cellular uptake of differently coated silver nanoparticles of comparable size in different human intestinal Caco-2 cell-derived models to cover also the influence of the intestinal mucus barrier and uptake-specialized M-cells. We used a combination of the TranswellTM system, transmission electron microscopy (TEM), atomic absorption spectroscopy (AAS) and ion beam microscopy (IBM) techniques. The computational in vitro sedimentation, diffusion and dosimetry (ISDD) model was used to determine the effective dose of the particles in vitro based on their individual physico-chemical characteristics. Data indicate that silver nanoparticles with similar size and shape show coating-dependent differences in their uptake into Caco-2 cells. The internalization of silver nanoparticles was enhanced in uptakespecialized M-cells, while the mucus did not provide a substantial barrier for nanoparticle internalization. ISDD modeling revealed a fivefold underestimation of dose-response relationships of nanoparticles in in vitro assays. In summary, the present study provides dosimetry-adjusted quantitative data about the influence of nanoparticle coating materials in cellular uptake into human intestinal cells. Underestimation of particle effects in vitro might be prevented by using dosimetry models and by considering cell models with greater proximity to the in vivo situation such as the M-cell model.

Keywords: dosimetry; ISDD-model; Ion Beam Microscopy; PIXE; RBS; Transwell-system


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Introduction Silver and silver nanoparticles are used in a wide range of food contact products and dietary supplements and are therefore increasingly investigated in nanotoxicology. Especially, the oral uptake is of major relevance due to the increasing market of food-related products and the rising environmental burden.1-2 Therefore, data concerning the mechanism and efficiency of gastrointestinal nanoparticle uptake, intracellular localization of absorbed particles and the distribution of such materials are essential for risk assessment. In vivo studies with orally administered silver nanoparticles are rare and difficult to compare due to variations in the type of particles, dosing regimen, exposure period and organism.3-6 Additionally, published literature suggests that different chemical entities of silver exist during distribution in the organism. Particles agglomerate or dissolve, release ions and form de novo particles.6-8 These complex interactions between particles and the surrounding conditions can only be investigated ex vivo. Therefore, in vitro models are appropriate to answer complex questions concerning the identification of nanoparticle characteristics which determine particle uptake efficiency in human enterocytes. A widely used and well-characterized intestinal cell model is the Caco-2 cell line. Following their differentiation into a tight monolayer of polarized enterocyte-like cells, they can be used in a TranswellTM system for particle uptake and transport studies. To expand the possibilities and significance of this in vitro model, improved variants have been developed. The most relevant models in the context of nanotoxicology are the M-cell model and the mucus-producing model. Intestine-derived mucus-producing cells like HT-29 MTX in coculture with Caco-2 cells have been investigated extensively and recently became commercially available.9-10 This model provides the additional intestinal mucus barrier present in vivo which potentially influences cellular nanoparticle adsorption and absorption.1113

A more recent model implements a special intestinal cell type called M-cells. These cells

are not an independent cell type, but differentiate from normal epithelial Caco-2 cells which are in contact with immune cells.14-16 M-cells phagocytize particles like pathogens from the intestinal lumen and transport them to the subjacent tissue, thus initiating an immune response.17-18 For both, nano- and micro-particles, M-cells are the most common uptake route into the gastrointestinal tract.19 This specialization makes M-cells attractive for cellular uptake studies. M-cells can be generated in vitro by culturing Caco-2 cells with immune cells like the B-lymphocyte cell line Raji B.20 A topic that should receive more attention in in vitro studies is the dosimetry of nanoparticles. Due to their size, nanoparticles diffuse very slowly and sediment over time, which can cause an over- or underestimation of the particle dose in vitro when the calculation of particle uptake is based on the initial concentration of particles in cell culture medium. One


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way to estimate the effective dose (the amount of administered particles that encounters the cells) is by computational calculations. The in vitro sedimentation, diffusion and dosimetry model (ISDD) considers agglomeration, diffusion and sedimentation of nanoparticles in cell culture medium during in vitro experiments, which are linked to the unique physicochemical properties of each particle, namely size, shape, stability, charge and density, as well as to culture media characteristics such as viscosity.21-24 These parameters influence the effective dose of the particles and thus lead to dosimetry-caused distortion of results. Therefore, particle dosimetry is of greatest importance to avoid under- or over-estimation of nanoparticle uptake. The majority of in vitro studies have investigated silver nanoparticle uptake via transmission electron microscopy (TEM), scanning electron microscopy (SEM) or confocal laser scanning microscopy (CLSM) and thus mostly focused on qualitative rather than quantitative determination of particle uptake. While quantitative uptake data are available for non-silver particles based on their fluorescent signals,25-27 this approach is not practicable in the case of silver due to the fluorescence-quenching properties of this material. Therefore, element analysis like ion beam microscopy, inductive coupled plasma-mass spectrometry (ICP-MS) or atomic absorption spectroscopy are the methods of choice. All in all, however, only few studies deal with silver nanoparticles and their uptake into cells in a quantitative way and included suitable cell models.28-29 However, no data are available yet for particles with comparable properties as well as reference materials and their dosimetry. Therefore, we used three differently coated silver nanoparticles to study their uptake in three different Caco-2-based models of the intestinal barrier. The particles were of comparable size and shape but differed in their coating material and included a reference material named NM300 or AgPURETM. Furthermore, powerful particle-specific dosimetry methods were applied to generate a comprehensive and quantitative comparison of silver nanoparticle particle uptake into intestinal cells.


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Materials and Methods

Chemicals All chemicals were purchased from

Merck (Darmstadt, Germany), Sigma-Aldrich

(Taufkirchen, Germany) or Carl Roth (Karlsruhe, Germany) in the highest available purity, unless mentioned otherwise.

Nanoparticles Surfactant-coated AgPURETM nanoparticles were purchased from Rent a Scientist GmbH (Regensburg, Germany) as aqueous dispersion and contain 10 % (w/w) silver stabilized with 4 % (w/w) polyoxyethylene glycerol trioleate (trade name Tagat TO) and 4 % (w/w) polyoxyethylene (20) sorbitan monolaurate (Tween 20). This material was characterized for the use as a reference material by the Federal Institute for Materials Research and Testing (BAM) in Germany, called BAM001, which is analogous to the reference material NM-300 available from the Joint Research Centre (JRC) of the European Commission.30 These particles are characterized and described in previous publications.31-32 Polyvinylpyrrolidone- (PVP, 10 kD) coated silver nanoparticles (BioPureTM) were obtained from nanoComposix (California; USA) as aqueous dispersion and was also previously described.32 Poly (acrylic acid)-coated nanoparticles were synthesized in a polyol process as described by Hu, et al.

33

using silver nitrate and poly (acrylic acid) MW =1800 g/mol.34

Detailed information of nanoparticle handling are given the Supporting Information. All concentrations are given as µg(Ag)/ml as they give the mass of silver for either nanoparticles or silver ions to enable a proper comparison by excluding the stabilizing agent and counter ion from the unit of concentration.

Nanoparticle characterization Particle characterization via small-angle X-ray scattering (SAXS) and nanoparticle tracking analysis (NTA) was performed for the stock suspension as well the diluted particles under cell culture conditions as described in the following part “cell models” (24 h in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % FCS, 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C) to simulate cell culture conditions. SAXS measurements were performed using SAXSess (Anton Paar, Graz, Austria) as previously described.31-32,

34

The measured intensity was corrected by subtracting the

intensity of a capillary filled with solvent solution. After background correction, the scattering data were deconvoluted (slit length desmearing) with SAXSquant software (Anton Paar,


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Graz, Austria). Curve fits of SAXS data were performed with SASfit software (Paul Scherrer Institute) and curve simulation by Monte Carlo methods with the McSAS software.35 NTA measurements were performed with a NanoSight LM20 (NanoSight, Amesbury, United Kingdom), equipped with a sample chamber with a 650 nm laser. All measurements were performed at room temperature. The software used for capturing and analyzing the data was the NTA 2.3. The samples were diluted with filtered water (0.02 µm syringe filters Whatman, Kent, UK) down to 107 - 109 total particles/ml and measured in minimum three times for 60 s with manual gain adjustments. Ion release was determined via ultracentrifugation and subsequent atomic absorption spectroscopy (AAS) measurements. Therefore, cell culture medium as used for all studies was incubated with silver nanoparticle concentrations as used in the cytotoxicity assay and the uptake study. The samples were incubated for 24 h at 37°C in a humidified atmosphere and afterwards centrifuged for 1 h at 100 000 x g (Ultracentrifuge Optima TLX, Beckman, Krefeld, Germany). Half of the supernatant was collected and digested via nitric acid and the amount of ionic silver in the supernatant was determined by AAS in the section “Atomic absorption spectroscopy” below. Zeta-Potential determination of particles in stock solution (pH 7) was conducted by using a Zetasizer Nano ZS (Malvern, Herrenberg, Germany). Nanoparticle stock solutions were diluted to 100 µg/mL and measured (n=6) at room temperature. Detailed information on nanoparticle characterization and device parameters are given in the Supporting Information. Cell models Caco-2 (ECACC: 86010202) and HT-29 MTX E12 (ECACC: 12040401) cells were obtained from the European collection of Cell Cultures, whereas Raji-B lymphocytes (ATCC: CCL-86) were purchased from American Type Culture Collection, USA. Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM, GE Healthcare, Freiburg, Germany) supplemented with 10 % FCS, 100 U/ml penicillin and 100 µg/ml streptomycin (PAA Laboratories GmbH, Pasching, Austria) at 37°C in a humidified atmosphere containing 5 % CO2. Additionally, 1 % non-essential amino acids were supplemented for HT 29 MTX and their coculture cultivation (NEAA, PAA Laboratories GmbH, Pasching, Austria). Caco-2 and Ht-29 MTX cells were used at passages 20–28 and 55-65, respectively. Raji cells were discarded after 10 times subcultivation. All cells were cultured in tissue culture flasks for maintaining (75 cm2) and in 96-well plates or 12-well TranswellTM-membranes for experiments.


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For uptake studies via AAS, Caco-2 cells were seeded in 12-well TranswellTM-inserts (1.12 cm2 growth area, 3.0 µm pore size, polycarbonate membranes; Corning Costar, USA) at a density of 50 000 cells/well and cultured for 20 days after seeding. Culture medium was refreshed every second day. To obtain a mucus-producing model, Caco-2 and HT-29 MTX cells were seeded at a ratio of 4:1 and cocultured. Their mucus secreting properties are show among others by Mahler, et al. 9, Walczak, et al.

36

and Walter, et al.

10

For generating

M-cells, an inverted model including Raji-B lymphocytes was used as follows: on day 7 of Caco-2 differentiation, the inserts were inverted and further cultivated as described by des Rieux, et al.

20

using silicon rubbers. On day 16, 50 000 Raji cells per well were added to the

basolateral silicon rubber. On day 21 Raji cells were removed and the inserts were placed back in their original orientation and allowed to adapt for one day. Incubation started on day 22. Functionality was shown by investigating M-cell-specific morphology and particle uptake as well as particle transport properties (Figure S4 and S5). For ion beam microscopy (IBM) experiments, Caco-2 cells (52 000 cells per cm2) were seeded on sterilized and fibronectin-coated (5 µg/ml Superfibronectin, Sigma Aldrich, Darmstadt, Germany) polypropylene foil (9.1 cm2; Goodfellow, UK) and cultured for 20 days in the same way as for AAS studies. For IBM analysis, cells were washed 4 times with PBS and fixed for 10 minutes with iced methanol at room temperature. Dried cells were stored at 4°C. Particle uptake experiments and assessment of cell barrier integrity The TranswellTM uptake experiments started by refreshing the basolateral medium and replacing the apical medium with medium containing nanoparticles. After a 24 h incubation period, samples from both chambers were collected; the TranswellTM-membranes were washed with PBS, which was also collected, followed by cutting out the membranes. All samples were immediately digested by nitric acid for AAS analysis or stored (except membranes) at -80°C. The monolayer integrity was routinely checked before and after nanoparticle exposure by measuring the transepithelial electrical resistance (TEER) and by assessing

the

paracellular

permeability

with

FITC-dextran

(10 kDa

Fluorescein

isothiocyanate dextran). Therefore, FITC-dextran was co-incubated during nanoparticle exposure (1 mg/ml). Media samples of the apical and basolateral chambers were collected and the amount of FITC-dextran was measured by fluorometry (Infinite F200 Pro, Tecan, Switzerland) at wavelengths of Ex/Em 485/535 nm. The apparent permeability coefficients (Papp, cm s-1) were calculated as follows: Papp = (∆Q/∆t) x (A x c0)-1, where ∆Q/∆t is the amount of FITC-dextran transported to the basolateral compartment over time t (s). A is the membrane area (1.12 cm2); while c0 is the initial concentration of FITC-dextran in the apical


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compartment (1 mg/ml). All experiments were performed using non-toxic concentrations of nanoparticles (cell viability > 95%). Cytotoxicity of nanoparticles has been determined in previous studies and is shown by Böhmert, et al. Lichtenstein, et al.

34

31

, Hansen and Thünemann

32

and

Detailed information on cytotoxicity testing are given in the Supporting

Information.

Atomic absorption spectroscopy An acidic microwave lysis method and atomic absorption spectroscopy (AAS; AAnalyst 800, Perkin Elmer, Massachusetts, USA) were used to determine the silver concentrations in cell culture media, stock suspensions and cells as described in Lichtenstein, et al.

34

Therefore cell culture samples diluted to 200 – 500 ng silver/ml (500 µl) as well as cells grown on TranswellTM-membranes were digested by adding 2 ml 70 % nitric acid, followed by microwave treatment for 40 min at 190°C (ETHOS, Leutkirch, Germany). For AAS measurement, all samples were diluted down to 6 % nitric acid by adding MilliQ H2O. Detailed information regarding acidic microwave lysis and atomic absorption spectroscopy as well as device parameters are given in the Supporting Information. Nanoparticle dosimetry To correlate administered silver concentration and cellular dose the estimation of the effective particle dose under cell culture conditions was needed. To calculate the effective dose of the particles we used the matlab script of the in vitro sedimentation, diffusion and dosimetry (ISDD) model provided by Justin Teeguarden and completed the script with the required data, namely; particle diameter [nm], agglomerate diameter [nm], particle density [g/cm³], effective agglomerate density [g/cm³], temperature [K], medium viscosity [Pa*s], medium height [mm], medium density [g/cm3], cell height [mm], medium volume [µl], initial particle concentration [µg/ml] and incubation time [h]. The effective agglomerate density [g/cm³] was determined as described by DeLoid, et al.

22

Therefore, cell culture-relevant

concentrations of nanoparticles were suspended and incubated in cell culture media (37°C, 24 h) and afterwards centrifuged in a PCV tube by volumetric centrifugation (1 h, 2000 x g). The silver content in the supernatant was measured by AAS and the effective density of the nanoparticle agglomerates was calculated. For further details see DeLoid, et al. 22 Transmission electron microscopy (TEM) To confirm cellular uptake of nanoparticles and to obtain additional information about nanoparticle distribution and localization within the cell TEM and energy-dispersive X-ray spectroscopy (EDX) were used. Caco-2 cells were seeded and incubated as described


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above. For sample preparation, TranswellTM-membranes were washed twice with cold PBS to remove the cell culture medium and nanoparticles after incubation and fixed with 4 % formaldehyde and 6 % glutaraldehyde. Uncut samples were postfixed with 2 % OsO4 in cacodylate buffer for 1 h at 4°C and dehydrated in a grade series of ethanol. Samples were embedded and cut into ultrathin sections (60 nm) on an Ultracut E ultramicrotome (ReichertJung, Vienna, Austria) with a diamond knife. The subsequent staining of sample sections and therefore the further use of TEM staining agents like uranyl acetate was omitted to avoid false positive results on the sections. Additionally, element analysis was performed via a conventional LaB6 200KV TECNAI G²20 (FEI, Oregon, USA), equipped with an EDAX EDX rTEM SUTW-detector. EDX measurements were applied to qualitatively verify the presence of silver, as nanoparticles from various materials might appear as dark spots in TEM bright field images. Therefore, the e-beam was focused on larger single particles or arrays of small particles in order to enhance the signal to noise ratio. Close beside the measured areas an "empty" EDX spectrum from an area without visible nanoparticles was obtained to show the absence of silver contamination inside the cell or on the specimen's surface. Diffraction patterns were also obtained in order to determine the amorphous or crystalline structure of the particles. Scanning Electron Microscopy Conversion of Caco-2 cell into M-cells was confirmed by SEM analysis of mono- and cocultures. This technique has already used by Gebert and des Rieux et al.17, 20 to verify Mcell formation. M-cells and Caco-2 cells differ in their surface properties. On Caco-2 cells, a regular brush border of similarly-formed microvilli is present, whereas M-cells show only few and sparsely rearranged microvilli. For SEM analysis mono- and cocultures were cultured as describes in the section “cell models”. 22 days after seeding the cells were washed twice with cold PBS to remove the cell culture medium and then fixed with 6 % formaldehyde and 4 % glutaraldehyde. After a fixation minimum of 1 h cells were dried via ethanol dehydration (30 % EtOH, 30 min.; 50 % EtOH, 30 min.; 70 % EtOH, overnight; 90 % EtOH, 2x 30 min.; 100 % EtOH, 2 h). Afterwards, the alcohol was replaced by carbon dioxide and the samples were dried using a Balzers Critical Point Dryer, model CPD 030 (Leica Microsystems, Wetzlar, Germany). Thus, distortions of cellular structure by surface tension forces were avoided. Pictures of cell monolayers were obtained with a DSM 982 Gemini SEM (Zeiss, Jena, Germany).

Ion Beam Microscopy


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Label-free Ion Beam Microscopy (IBM) techniques provide unique information on intracellular concentration and distribution of cellular matrix elements, trace elements and incubated nanomaterials down to the single cell level. Two different modes of IBM measurements were applied simultaneously, namely, micro-particle induced X-ray emission (µPIXE) and microRutherford backscattering spectroscopy (µRBS). A proton beam was used for scanning the sample in the xy-plane at a resolution of about 1 µm. In contrast to electron-based X-ray emission, protons are able to penetrate samples with a significantly higher depth; thus the total cellular concentration of nanoparticles in cells and tissues is quantifiable. Moreover, the proton induced X-ray emission has larger cross section of X-ray production and protons produce less bremsstrahlung than electrons, which cause a better signal-to-noise ratio, allowing for the accurate detection of cellular trace elements at very low concentrations.37-38 IBM measurements were performed at the Leipzig ion Nanoprobe LIPSION. A 2.25 MeV proton beam was applied to investigate the samples. To realize good measurement conditions, a vacuum of 5.0 x 10-5 to 10-7 Torr was used. The PIXE detector (Canberra, Connecticut, U.S.A.) consists of a High Purity Germanium crystal (95 mm2 active area). Additionally, the detector is covered with a 60 µm polyethylene layer, in order to avoid the penetration of backscattered protons. Backscattered ions were detected with a Canberra PIPS-detector. µPIXE data were used to visualize the two-dimensional distributions of phosphor, sulfur and silver. Single cell analysis was performed by determining the region of interest (ROI) by marking the edges of the cells based on the P and S signal in displayed PIXE element maps. Out of the ROI, µPIXE and µRBS spectra were extracted. Experimental RBS data were fitted to determine the local charge, element matrix composition (C, N, O, etc.) and the thickness of the sample. Based on this information, the known cross sections and the PIXE integral peak areas, the cellular concentrations of silver were calculated. The element concentration is given in ng/cm². Silver concentration can be determined from the signal from the K-lines (AgK) and the L-lines (AgL) from the X-ray spectra.


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Results

Particle characterization We characterized nanoparticles in water (Milli-Q-grade; 18.2 MΩ x cm at 25°C) as well as under cell culture conditions (24 h in cell culture medium containing 10 % FCS, 37°C). Particle features are summarized in Table 1. All investigated particles show a spherical and negatively charged surface. No alterations in core sizes were detectable during in cell culture media, whereas the hydrodynamic diameter increased under cell culture conditions. Table 1: Particle characteristics of the three investigated, differently coated silver nanoparticles in water and cell culture conditions (24 h, DMEM +10 % FCS, 37°C, 5 % CO2). Abbreviations: SAXS small-angle X-ray scattering; NTA nanoparticle tracking analysis; FCS fetal calf serum; DMEM Dulbecco’s modified Eagle’s medium; PVP polyvinylpyrrolidone; PAA poly (acrylic acid)

nanoparticle dispersion

Ag_Pure (AgPURETM)

Ag_PVP (BioPureTM)

Ag_PAA

Coating material

Tween 20, Tagat TO V

polyvinylpyrrolidone

poly (acrylic acid)

Size in H2O SAXS (r in nm) NTA (d in nm)

9.2 ± 0.1 57.0 ± 3.0

10.2 ± 0.1 68.0 ± 1.2

3.2 ± 0.1 44.0 ± 1.0

9.5 ± 0.1 110.0 ± 8.5

11.2 ± 0.2 142.0 ± 6.4

3.6 ± 0.1 199.0 ± 2.3

-19 ± 5 spherical 2.9



Size in DMEM +10 % FCS SAXS (r in nm) NTA (d in nm) Zeta potential (mV, pH 7) Shape Ion release in DMEM (%)

During uptake experiments using the Transwell system, nanoparticles were co-incubated with FITC-Dextran to check cell monolayer integrity. Therefore, alterations in size distributions of particles in cell culture medium containing FITC-Dextran were additionally investigated. No relevant alteration in agglomeration /aggregation status was observable (representative data are shown in the Supporting Information, Figure S3).

Quantification of cellular uptake of differently coated silver nanoparticles In order to detect differences in cellular silver nanoparticle uptake we used Caco-2 cells as a model for the human intestinal epithelium. Caco-2 cells differentiated spontaneously and formed a tight epithelial barrier after 21 days by using the TranswellTM-system. For all


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particles, non-toxic concentrations (cell viability > 95%) were investigated. Cytotoxic nanoparticle concentrations has been determined in previous studies and are above 20 µg/ml as shown in Böhmert, et al.

31

, Hansen and Thünemann

32

and Lichtenstein, et al.

34

Accordingly, all integrity checks of Caco-2 monolayers showed tight barrier functions during the whole experiment (see S1-S2). The calculated Papp values for FITC dextran between 10-7 and 10-8 cm/s are described for substances transported below 1 %.39 Caco-2 cells internalized particles in a concentration-dependent manner (Figure 1). The results showed a strong increase of cellular silver after incubation with surfactant-coated nanoparticles (Ag_Pure) and maximum uptake values of about 401 ng/cm2 for treatment with Ag_Pure at the highest concentration of 20 µg(Ag)/ml. Similarly, a concentration-dependent increase in silver

internalization

was

observed

for

polyvinylpyrrolidone-coated

(Ag_PVP)

and

poly (acrylic acid)-coated particles (Ag_PAA) with maximum uptake values of 282 and 213 ng/cm2, respectively. Silver ions in the form of AgNO3 were tested in concentrations comparable with the ion release of the particles under cell culture conditions (3 – 7 %). Internalized silver was detectable only for the highest tested concentration of silver ions.

600

**

500

cellular uptake Ag [ng/cm²]

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* *

400

*

* *

*

300 200 100 0

Dosimetric quantification of coating-related uptake ... - ACS Publications

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