We have used zinc nitrate hexahydrate (Zn(NO3)2·6 H2O, Alfa Aesar, 98%), zinc acetate dihydrate (Zn(CH3COO)2·2 H2O, Alfa Aesar, > 98%), poly(N-vinylpyrrolidone) (PVP K 55; Sigma-Aldrich, p.a., M = 55,000 g mol−1), diethylene glycol (DEG, Sigma-Aldrich, 99%), ethylene glycol (EG; Sigma-Aldrich, 99.8%), dimethylformamide (DMF; Fischer Chemicals, > 99.5%), and ultrapure water (Purelab ultra instrument from ELGA). All chemicals were used as obtained without further purification. Before the experiments, all glassware was cleaned with boiling aqua regia and twice with boiling ultrapure water. Finally, all glassware was sterilized at 200 °C for 3 h. All synthesized particles were purified by centrifugation with a Heraeus Fresco 21 centrifuge (Thermo Scientific).
PVP-coated ZnO nanorods were prepared by a polyol method according to Lee et al. (2008) with slight modifications. Zinc acetate dihydrate (8.78 g), PVP (0.233 g), and 4.32 mL water were added to 96 mL of diethylene glycol (DEG) and stirred for 10 min at room temperature. Then, the reaction mixture was heated under vigorous stirring to 180 °C. The solid zinc acetate had completely dissolved at 120 °C. The reaction mixture was stirred for 30 min at 180 °C and then quenched to room temperature in an ice bath. The nanoparticles were purified by triple centrifugation (3500 rpm, 60 min) and redispersion in ethanol and then dried at 80 °C for 4 h. For the synthesis of PVP-coated ZnO nanospheres, the solvent was changed to ethylene glycol (EG). All other parameters were the same as with the nanorods.
A one-pot synthesis of PVP-coated ZnO microspheres in DMF was developed based on the method reported by Yao et al. (Yao and Zeng 2007). 1.485 g of zinc nitrate hexahydrate and 3 g of PVP were completely dissolved in 200 mL of DMF under vigorous stirring at room temperature. After stirring for 10 min at room temperature, the solution was rapidly heated to 100 °C. After 20 min, the reaction mixture had assumed a turbid color, indicating a nucleation of ZnO particles. After another 20 min, the reaction mixture was heated to 120 °C and stirred for 2 h. Finally, the mixture was quenched to room temperature with an ice bath. The particles were collected by centrifugation (3500 rpm, 30 min), washed with ethanol several times, and dried at 80 °C for 4 h. A one-pot synthesis of PVP-coated ZnO microrods was performed by adding water to DMF in a volume ratio of 5 mL:95 mL. All other parameters remained the same.
The synthesis of PVP-coated ZnO submicroparticles was performed in the same way as the synthesis of PVP-coated ZnO microspheres, but in this case, the reaction time was shortened. Without changing the concentration of reactants, the reaction mixture was heated to 120 °C and the reaction time was reduced from 120 to 20 min. After synthesis, the oil bath was removed and quickly replaced by an ice bath. Purification of the particles was performed by triple centrifugation (3500 rpm, 30 min) and redispersion in ethanol, followed by drying of the particles at 80 °C for 4 h.
Ten milligrams of PVP-coated ZnO particles were redispersed in 50 mL of four different media: ultrapure water, RPMI medium (Gibco, supplemented with 10% fetal bovine serum, FBS), simulated lysosomal medium, and citrate-free acetate buffer.
The simulated lysosomal medium (pH = 4.5) was prepared according to (Henderson et al. 2014). We used sodium chloride (NaCl, 3.21 g L−1, Bernd Kraft, > 99.5%) sodium hydroxide (NaOH, 6.0 g L−1, Baker, 99%), citric acid (20.08 g L−1, Fluka, > 99.5%), calcium chloride dihydrate (0.097 g L−1, GrisChem, > 99%), sodium phosphate dibasic heptahydrate (Na2HPO4·7 H2O, 0.179 g L−1, Riedel-de Haën, 99%), sodium sulfate heptahydrate (Na2SO4·10 H2O, 0.039 g L−1, Fluka, 99%), magnesium chloride hexahydrate (MgCl2·6 H2O, 0.106 g L−1, GrisChem, 99%), glycine (0.059 g L−1, Biomol, > 99%), sodium citrate dihydrate (0.077 g L−1, Sigma-Aldrich, 99%), sodium hydrogen L-tartrate (0.090 g L−1, Alfa Aesar, 98%), sodium L-lactate (0.085 g L−1, Sigma-Aldrich, >99%), sodium pyruvate (0.85 g L−1, Sigma-Aldrich, >99%), and formaldehyde (0.3 mL L−1, Fluka, p.a.). The solution was filled with water to 300 mL. The citrate-free acetate buffer (pH = 4.8) was prepared with an aqueous solution of acetic acid (300 mL, 1 mol L−1, Carl Roth) and sodium acetate trihydrate (1.22 mol L −1, Sigma-Aldrich) instead of citric acid/sodium citrate. All other compounds were the same as above.
The particle dispersion (10 mg in 50 mL medium) was placed into a closed round bottom flask (200 mL) and stirred at 25 °C (water) or at 37 °C (RPMI/FCS) for 5 days under sterile conditions. After 30 min and then after each day, 1 mL of the particle dispersion was taken and filtered (nanoparticles: inorganic membrane filter, Whatman Anotrop 10 Plus; 0.02 μm; submicro- and microparticles: inorganic membrane filter, Whatman Anotrop 25; 0.2 μm) to separate zinc ions from ZnO particles. When the particle dispersion was taken, its pH was measured (pH meter HANNA HI 991001). The pH increased slightly with time due to the dissolution of zinc oxide (in water: 6.9 to 7.9, RPMI/FCS: 6.9 to 7.6). For the dissolution tests of ZnO particles in simulated lysomal media, the particle solution was stirred only for 1 h due to the rapid dissolution at pH = 4.5 (citrate-buffered) and pH = 4.8 (acetate-buffered). Finally, the zinc content in the isolated particles and the zinc ion concentration in the filtrates were determined by AAS.
The biological effect of the particles was studied with the cell line NR8383 (rat alveolar macrophages, LGC Standards GmbH, Wesel, Germany). The cells were cultivated in Ham’s F12 medium containing 15% fetal calf serum (FCS, GIBCO, Invitrogen, Karlsruhe, Germany) in 175 cm2 cell culture flasks (BD Falcon, Becton Dickinson GmbH, Heidelberg, Germany) at standard cell culture conditions (humidified atmosphere, 37 °C, 5% CO2). The NR8383 cells were only partly adherent. The ratio between adherent and non-adherent cells was about 1:1. For cell experiments, adherent cells were detached from the cell culture flasks with a TPP cell scraper (TPP Techno Plastic Products AG, Trasadingen, Switzerland), subsequently combined with non-adherent cells, and seeded into 24-well cell culture plates (BD Falcon) at a concentration of 2.4 × 105 cells cm−2.
The intracellular concentration of zinc ions after 2 h of exposure of NR8383 cells to different ZnO particles at various concentrations (80, 40, 20 μg ZnO mL−1) was measured with the Zn2+-selective indicator FluoZin-3 (Invitrogen) and flow cytometry. After incubation with the ZnO particles, the cells were collected in 5-mL tubes (BD Biosciences) as described above and stained with 100 μM of FluoZin-3 for 30 min at room temperature. For the discrimination of non-viable cells, staining with 50 μg mL−1 propidium iodide (PI, Sigma-Aldrich, Taufkirchen, Germany) was also performed (10 min, room temperature).
The cytotoxicity of different ZnO particles at various concentrations (80, 40, 20, 10, 5 μg ZnO mL−1) for NR8383 cells was analyzed by flow cytometry. A solution of zinc acetate (Alfa Aesar, Karlsruhe, Germany, 98%) was used as control (40, 20, 15, 5 μg Zn2+ mL−1). After 16 h of particle or zinc acetate exposure, adherent and non-adherent cells were combined in 5 mL as described above. Non-viable cells were labeled with 50 μg mL−1 PI for 10 min at room temperature. The number of non-viable cells (PI positive) was determined by flow cytometry.
The induction of apoptosis in NR8383 cells by ZnO particles was investigated with the Annexin V apoptosis assay and flow cytometry. The cells were incubated for 16 h with ZnO particles (80, 40, 20, 10, 5 μg ZnO mL−1) as well as with zinc acetate solution (40, 20, 10, 5 μg Zn2+ mL−1). After incubation, adherent and non-adherent cells were combined in 5 mL tubes as described above. Staining of early apoptotic cells was performed with FITC-conjugated Annexin V (BioLegend GmbH, Koblenz, Germany) according to the manufacturer’s protocol in Annexin V Binding Buffer containing CaCl2 and MgCl2 (BioLegend GmbH), while necrotic and late apoptotic cells with damaged membranes were excluded by counterstaining with 50 μg mL−1 of PI (15 min, room temperature).
The formation of ROS in NR8383 cells after 2 h of incubation with 80 μg ZnO mL−1 of different ZnO particles was investigated qualitatively with the cell-permeant ROS indicator CellROX Green (Thermo Fisher Scientific, Waltham, USA) which gives a strong fluorescence after oxidation. After particle exposure, the cells were stained with 5 μM CellROX Green for 30 min under cell culture conditions and analyzed by confocal laser scanning microscopy. Cells exposed to 100 μM H2O2 (Sigma-Aldrich) for 30 min under cell culture conditions served as a positive control for elevated ROS levels.
The quantitative analysis of ROS formation was performed by the DCFDA assay with flow cytometry. Cells were incubated with different concentrations of ZnO particles (80, 40, 20 μg ZnO mL−1) as well as a zinc acetate solution (60, 40, 20 μg Zn2+ mL−1) for 2 h under cell culture conditions. Next, 20 μM of the cell-permeant ROS indicator 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA, Thermo Fisher Scientific) was added, and the cells were incubated for 30 min at 37 °C. The non-fluorescent H2DCFDA diffuses into cells, where it is deacetylated by intracellular esterases and converted to highly fluorescent 2′,7′-dichlorofluorescein (DCF) by oxidation. For discrimination between viable and non-viable cells, an additional PI staining was performed (50 μg mL−1, 10 min, room temperature).
After incubation of NR8383 cells with different ZnO particles (10 μg ZnO mL−1) for 16 h, the supernatants were collected and centrifuged at 300g for 10 min and stored at − 20 °C until microarray analysis (Profiler Array Rat XL Cytokine Array Kit, Bio-Techne GmbH, Wiesbaden-Nordenstadt, Germany). The assay detected 79 different cytokines, growth factors, and other mediators and permitted a semi-quantitative analysis. The membrane-based sandwich immunoarray consisted of a nitrocellulose membrane on which the capture antibodies were spotted as duplicated dots. The target proteins in the sample were bound to the capture antibodies and detected with biotinylated detection antibodies, followed by visualization with chemiluminescent detection reagents. For analysis, the manufacturer’s instructions were observed and the chemiluminescence signals were detected and quantified by a microarray imager and the ImageQuantTL software (Amersham Imager 600 RGB, GE Healthcare Bio-Science, Uppsala, Schweden). For the subsequent detailed analysis, 27 factors were selected based on the proteomic repertoire of NR8383 cells (Duhamel et al. 2015).
Quantitative analyses were performed with cell culture supernatants after 16 h of exposure to ZnO particles (5 to 10 μg ZnO mL−1) with Sandwich-ELISA Kits (R&D Systems Quantikine, Bio-Techne GmbH, Wiesbaden, Germany).
NR8383 cells were cultivated at 37 °C, 100% humidity, and 5% CO2 in Ham’s F-12 + 15% FCS medium (Biochrom KG, Berlin, Germany), 2 mM l-glutamine, 100 g L−1 penicillin, and 100 U mL−1 streptomycin. In 25 mL (175 cm2) medium, approximately 3 × 106 cells were seeded.
HL-60 cells were obtained from DSMZ (Braunschweig, Germany). Trans-retinal differentiated HL-60 cells (dHL-60) were used to induce the chemotaxis. For this, the HL-60 cells were cultivated for 3 days in RPMI 1640 medium (Biochrom), 10% FSC, 2 mM l-glutamine, 100 g L−1 penicillin, 100 U mL−1 streptomycin, and 1 μM trans-retinoic acid at 37 °C, 100% humidity and 5% CO2 (Breitman et al. 1980). In conventional culture dishes, the dHL-60 cells grow adherent.
For the particle-induced cell migration assay, we suspended NR8383 rat macrophages (3∙106 cells mL−1) with a vortex in 1 mL Ham’s F-12 medium containing 15% FCS, 2 mM l-glutamine, 100 mg L−1 penicillin, and 100 U mL−1 streptomycin. Then, the cells were seeded in 12.5-cm2 cell culture flasks to a final volume of 3 mL (2.4 × 105 cells cm−2). Note that it is also possible to perform the assay in a smaller volume at constant surface to volume ratio.
As negative control, we used a sample of cells without particles. We repeated the subsequent experiments up to the concentrations which gave the maximum induction of chemotaxis. The cells were incubated with the particles for 16 h at 37 °C, 100% humidity, and 5% CO2. Afterwards, we removed the cells by centrifugation at 300g for 5 min. The particles were removed by centrifugation at 15,000g for 10 min at room temperature. We used the supernatants immediately thereafter for the cell migration tests.
We investigated the cell migration according to Boyden (1962), but with the modifications described earlier by Westphal et al. (2015) and Schremmer et al. (2016). For this, we exclusively applied permanent cell lines in the following way. We added 2 × 105 unchallenged dHL-60 cells to 200 μL RPMI 1640 medium without FCS and seeded the cells in each plate well insert (THINCERT, 3-μm pore size, Greiner bio-one, Frickenhausen, Germany) and placed the insert into the cavities of 24 black well plates (Krystal, Dunn Labortechnik, Asbach, Germany). A total of 500 μL of the supernatants of the particle-incubated NR8383 cells was added to the lower chamber. The migration of dHL-60 cells across the membrane was observed for 24 h at 37 °C, 100% humidity, and 5% CO2. 105 HL-60 cells were seeded directly into four-plate wells that were left without inserts for calibration.
Calcein-AM was used to stain migrated cells. Cell calibration was performed for 60 min at 37 °C, 5% CO2 and 100% humidity by adding 500 μL calcein-AM to the plate wells (> 90% HPLC, Sigma-Aldrich, Steinheim, Germany). Calcein-AM was used as 4 mM solution in DMSO, stored in aliquots at − 18 °C, and diluted to the final concentration of 4 μM in PBS.
After that, the cell suspensions were removed from the plate wells and collected by centrifugation at 300g for 5 min at room temperature. The cells were re-suspended in 150 μL while 850 μL of the supernatant was discarded. Furthermore, the adherent cells at the outside of the inserts were detached with 500 μL trypsin/EDTA (0.05%/0.02%, Biochrom) for 10 min at 37 °C, 5% CO2, and 100% humidity. Then, the inserts were removed from the plate wells. The 150 μL containing the collected cells were added to the plate wells that contained 500 μL of trypsin/EDTA-detached cells. Cell counting was done by fluorescence spectroscopy at 490/520 nm and related to the cell calibration (SpectraMax M3, Molecular Devices, Sunnyvale, USA).
In terms of statistical significance, acceptance criteria for a valid test were positive control (nanosized silica) and negative control within the range of the established controls as established in our laboratory. In this context, we defined a positive response as a dose-dependent increase of cell migration across at least two consecutive concentrations, with a maximum that exceeded the base rate by at least twice the highest concentration (Westphal et al. 2015).
As reference compound, we used a silica reference sample (CAS No. 7631-86-9, Lot MKBF2889V, 99.5%, 10–20 nm; Sigma-Aldrich, Steinheim, Germany). These particles were previously characterized in detail and consisted of agglomerated X-ray amorphous silica particles with a primary particle size of about 50 nm and an agglomerate size of about 2 μm (Westphal et al. 2015).
Dynamic light scattering for particle size analysis and zeta-potential determination were carried out with a Malvern Zetasizer Nano ZS ZEN 3600 instrument (Malvern Panalytical Ltd.; 25 °C, laser wavelength 633 nm). The light scattering was monitored at a fixed angle of 173° in backward scattering mode. The peak profile of the size distribution was analyzed by a log-normal distribution fit. The average diameter was taken as the mean value of the maximum of the size distribution xc from the log-normal distribution fit analysis and the empirical standard deviation.
Ultraviolet-visible spectroscopy (UV/vis) was performed with a Varian Cary 300 instrument (Agilent Technologies, Inc.). Suprasil® quartz cuvettes with a sample volume of 3 mL were used after dilution and background correction.
The ZnO particles were dissolved in concentrated nitric acid before the atomic absorption spectroscopy (AAS). AAS was carried out with a Thermo Electron M-Series instrument (Thermo Fisher Scientific) according to DIN EN lSO/lEC 17025:2005.
Thermogravimetric analysis (TGA) was performed with a Netzsch STA 449 F3 Jupiter instrument to determine the content of coating polymer in the samples. The purified and dried particles were heated in an open alumina crucible with 5 K min−1 from 20 to 1000 °C under dynamic oxygen atmosphere.
Scanning electron microscopy of the particles was performed with a FEI ESEM Quanta 400 FEG microscope. Prior to the SEM investigation, the samples were sputter-coated with a thin conductive AuPd 80:20 layer.
For X-ray powder diffraction (XRD), the particle dispersion was shock-frozen with liquid nitrogen and lyophilized at 0.31 mbar and − 10 °C in a Christ Alpha 2-4 LSC instrument. XRD measurements were performed with a Bruker D8 Advance instrument in Bragg-Brentano geometry with Cu Kα radiation (λ = 1.54 Å, 40 kV and 40 mA) with a single-crystalline silicon sample holder in the crystallographic (911) plane to minimize scattering. The powder samples were investigated from 5 to 90° 2Θ with a step size of 0.01° and a counting time of 0.6 s at each step. The instrumental peak broadening was determined with lanthanum hexaboride (LaB6) from NIST (National Institute of Standards and Technology; reference compound) as internal standard. Rietveld refinement was performed with the program package TOPAS 5.0 from Bruker to determine the lattice parameters (a and c), the isotropic and anisotropic crystallite size (D and DA), and the microstrain (ε). For the calculation of D, the Scherrer and Stokes-Wilson equations were used (Klug and Alexander 1974). The diffraction pattern of hexagonal zinc oxide was taken from the ICDD database (International Centre of Diffraction Data) as reference (#36-1451) and used for the qualitative phase analysis with Diffrac.Suite EVA V1.2 (Bruker).
Flow cytometric analyses were carried out with an FACSCalibur flow cytometer (BD Bioscience, Heidelberg, Germany). For each measurement, 10,000 cells were analyzed, and the data were quantified with the CELLQuest 1.2.2 software (BD Biosciences).
Confocal laser scanning microscopy was performed with a Zeiss LSM 700 instrument (Carl Zeiss Microscopy GmbH, Jena, Germany). Fluorescence images were taken (Zeiss LSM 700 microscope and Zen 2010 software) and digitally processed using Adobe Photoshop 7 (Adobe Systems GmbH, CA, USA).
The number of particles in 1 g of solid material was computed from the average particle mass of one sphere and one rod, respectively:
$$ {\displaystyle \begin{array}{c}m\mathrm{spheres}=\frac{4}{3}\ \uppi\ r3\ \uprho \\ {}m\mathrm{rods}=\pi\ {r}^2L\ \rho \end{array}} $$
with r the particle radius and L the particle length, both obtained from SEM (Table 1), and ρ the density of ZnO (4030 kg m3). The specific surface areas of the particles (m2 g−1) were computed as follows:
$$ {\displaystyle \begin{array}{c}S\mathrm{spheres}=4\uppi\ {r}^2 Nparticles\ per\ 1g\\ {} Srods=\left(2\pi\ {r}^2+2\pi rL\right)\ Nparticles\ per\ 1g\end{array}} $$
Data are expressed as the mean ± SD (n = 3) and given as the percentage of the control (cells not exposed to particles). For statistical evaluation, one-way analysis of variance (ANOVA) with Bonferroni’s multiple comparison test was applied using the GraphPad Prism software (GraphPad Software, Inc., CA, USA), while p values ≤ 0.05 were considered statistically significant.
EC50 values (PICMA): To illustrate the dose-response relation more precisely, four-parameter log-logistic models were used. According to Van der Vliet and Ritz (Van der Vliet and Ritz 2013), the four-parameter log-logistic model is defined as:
$$ f\left(x,\left(b,c,d,e\right)\right)=c+\frac{d-c}{1+\exp \left(b\left(\log (x)-\log (e)\right)\right)} $$
with b, c, d, and e used as corresponding parameters. b represents the slope of the curve, c indents the lower and d the upper asymptote, and e is the effective concentration EC50.
The R-package drc developed by Ritz et al. (2016) provides specialized analyses for such dose-response relations. Especially, the function drm is used for fitting dose-response models.
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