Nanoparticles and their physicochemical properties
For the purpose of the study, TiO2 NPs (AEROXIDE® P25; Sigma Aldrich, Saint Louis, MO) were utilized with a primary particle size of 21 nm. The measurement of the particles was accomplished by dynamic light scattering (DLS) on a Wyatt DynaPro Plate-Reader II (Wyatt Technology Europe GmbH, Dernbach, Germany) and a Malvern Zetasizer Nano-ZSP (Malvern Instruments GmbH, Herrenberg, Germany) in 96-well plates at room temperature. In this regard, the samples were irradiated with a laser (semiconductor laser with a λ of 830 nm [Wyatt] or a HeNe laser with a λ of 632.8 nm [Malvern]). Subsequently, the intensity fluctuations of the scattered light (detected at a backscattering angle of 156° [Wyatt] or 173° [Malvern]) were analyzed to obtain the autocorrelation function. The device software (Wyatt: DYNAMICS 7.1.9 or Malvern: Zetasizer Software 7.11) outputted the mean particle size using cumulant analysis and a size distribution using a regularization scheme by intensity or number. The mean hydrodynamic diameter was expressed as the log-normal distribution for the intensity or number density, and the dispersity, p, was calculated using the following formula:
$$ (p)=\frac{\sigma }{\mu } $$
where μ signifies the mean, and σ represents standard deviation.
It was assumed that the suspension viscosity was similar to that of water, corrected for temperature. In addition, the suspension refractive index was considered to be equal to that of water (n = 1.33). The refractive index of the NPs calculated as 2.4900 with the absorption of 0.01 was applied in the study. The characterization of the NPs was performed using the transmission electron microscopy (TEM). To this end, the NP suspensions were dried at room temperature on pioloform TEM grids and then analyzed with a Tecnai 12 FEI Biotwin TEM setup (Fig. S1).
Animals
In line with the study objectives, nine-week-old female, wild-type, BALB/c-mice were obtained from the Janvier Labs (Le Genest-Saint-Isle, France) and kept in a 12-h dark/light cycle at 22 °C with laboratory food and tap water ad libitum. The mice were acclimatized for 2 weeks prior to initiating the study. All animal experiments were performed in strict accordance with the German animal protection laws under the approval of the appropriate governmental authority. In addition, every experimental procedure was carried out following the ethical regulations and the animal welfare protocols of the state of Saarland. In order to generate an ovalbumin (OVA) mouse model, the BALB/c mice were intraperitoneally sensitized to OVA (i.e., an allergen), along with aluminum hydroxide-adsorbed OVA (2 mg AlOH3 with 20 μg OVA). On the other hand, the control animals received phosphate-buffered saline (PBS) on days 0 and 7. Afterward, the mice were subjected to OVA challenges on days 17, 18, 19, and 20 via the intranasal route (Fig. S2).
In order to prepare the NPs, the treated TiO2 NPs were dispersed in double distilled water (Milli-Q®), and the suspensions were ultrasonicated for 15 min to keep the maximum dispersed state. In the NPs groups, each of the BALB/c mice was intranasally treated with 25 μl TiO2 NPs suspension (50 mg/mL) 1 h after OVA exposure on days 17 and 20 (Fig. S2). To ensure the homogeneity of the suspension, the stock solutions were vortexed shortly before nasal installation for each mouse. Day 21 was considered the study endpoint. Prior to sacrificing the mice, they were weighed and prepared for pulmonary function testing. Subsequently, the bronchoalveolar lavage fluid (BALF) and some organs were isolated for the implementation of different experiments. Each of the three untreated (i.e., PBS/PBS, OVA/PBS, and OVA/OVA) and three treated groups (i.e., PBS/TiO2/PBS, OVA/TiO2/PBS, and OVA/TiO2/OVA) consisted of 5 mice and 10 mice, respectively.
Lung function testing
The lung function analysis was performed in our lab and included a non-invasive measurement with conscious animals. Specific airway resistance (sRaw) was performed using a double-chamber head-out plethysmograph (DSI Buxco FinePointe NAM, MN, USA). In addition, the enhancement of doses (0, 12.5, 25, and 50 mg/mL) was accomplished using methacholine (MCh) via an aerosol nebulizer. In this regard, after inserting the mice in the device, they were granted an acclimation period of 5 min to calm down. The aerosol volume was amounted to 0.02 ml and delivered within 1 min. Different MCh concentrations were applied within an interval of 6 min (i.e., 3 min for response time and 3 min for recovery period).
Tissue sampling and inductively coupled plasma mass spectrometry measurements
After the implementation of airway resistance measurements, the BALB/c-mice were sacrificed by bleeding, and their organs were removed. The lungs were subjected to histological analysis, and BALF analysis was conducted for cell counts. In order to perform ICP-MS screening for titanium (47Ti), some portions of the main organs were cut, weighed, and dissolved in 5 mL concentrated ultrapure HNO3. Subsequently, a 4% (v/v) solution of ultrapure HCl was added to a final volume of 10 ml. After a few days, the samples were dissolved and analyzed at room temperature using the Agilent 7500cx (Agilent Technologies, Santa Clara, CA). In addition, scandium (45Sc) was used as an internal standard.
Staining and histological analysis
To assess the lung histopathology and airway inflammation, lung cryosections (10 μm) were prepared by means of a cryostat (CM1950, Leica, Germany). Lung tissue cryosections were stained with hematoxylin and eosin (H&E) and periodic acid Schiff (PAS) as previously described [19, 20]. In the next stage, the sections were examined using the Zeiss Axio Imager M2 microscope (Carl Zeiss AG, Oberkochen, Germany). The number of goblet cells in the airways was counted manually after PAS staining under the same light microscope. Furthermore, immunofluorescence (IF) staining was performed with the Shandon Sequenza system (Thermo Scientific, MA, USA). The lung sections of every mouse were dried at room temperature for 15 min. To reduce the nonspecific cross-reactions, the sections were blocked with 5% donkey serum diluted in PBS. Afterward, they were incubated with primary antibodies (i.e., antimouse F4/80 [eBioscience, San Diego, CA], antimouse Ly6G [Abcam, Cambridge, UK], antimouse Siglec-F [eBioscience, San Diego, CA], and antimouse CD3ε [Biolegend, San Diego, CA]) for 1 h at 20 °C and then incubated overnight at 4 °C.
On the second day, the sections were rinsed twice with PBS and then incubated with secondary fluorescein-conjugated antibodies (donkey antirabbit IgG cyanine Cy3, donkey antirat IgG Cy5, and goat anti-Armenian hamster IgG Cy3) for 2 h at room temperature (all secondary antibodies were obtained from Jackson Immunoresearch, West Grove, PA). The cryosections were counterstained with 80 μL 4, 6-diamidino-2-phenylindole (DAPI; 0.5 μg mL− 1, Carl Roth, Karlsruhe, Germany) for 15 min, washed several times with PBS and once with double-distilled water, and mounted with Fluoroshield™ fluorescence mounting medium (Sigma-Aldrich, St Louis, MI). Additionally, fluorescence microscopy was performed by means of the Zeiss Axio Imager M2 microscope (Carl Zeiss AG).
Bronchoalveolar lavage fluid collection
For the collection of BALF, the trachea was exposed by a midline incision in the neck. Subsequently, 1 ml of ice-cold PBS (pH = 7.4) containing protease inhibitors was injected into the lungs through the trachea and withdrawn after 10 s as described previously [21, 22]. In the following stages, the recovered fluid was centrifuged at 1200 rpm for 10 min at 4 °C, the supernatants were removed, and the pellets were resuspended in 0.5 mL PBS. To determine the total cell number, the cells were enumerated by means of a Neubauer cell counting chamber. Afterward, the cytospots were prepared and stained using the Diff-Quick (Medion Diagnostics AG) staining solution in order to discriminate and count the immune cells, including macrophages, neutrophils, eosinophils, and lymphocytes.
Enzyme-linked immunosorbent assay
After collecting blood samples from the sacrificed animals, they were centrifuged, and the obtained sera were stored at − 80 °C until analysis. Serum concentrations of total immunoglobulin (Ig) E were measured using the commercially available enzyme-linked immunosorbent assay (ELISA) kits (885,046,022, Invitrogen, Vienna, Austria). To determine the protein level in the homogenates, a Pierce BCA protein assay (23,227, ThermoFisher Science, Germany) was performed on the homogenized snap-frozen lungs. After adjusting the protein level in each sample, the ELISA was conducted to analyze the cytokine levels of IL-4 (DY404–05, R&D Systems Inc., USA), IL-5 (DY405–05, R&D Systems Inc., USA), IL-13 (DY413–05, R&D Systems Inc., USA), and interferon-gamma (IFN-γ) (DY485–05, R&D Systems Inc., USA) according to the manufacturer’s protocol.
Investigation of nanoparticle phagocytosis
The phagocytic ability of primary murine macrophages was analyzed in vitro. To this end, alveolar macrophages were isolated from the BALF of control BALB/c mice (without OVA neither TiO2 NPs). The samples were then grown as adherent cultures in RPMI 1640 medium containing 10% FBS and 1% penicillin/streptomycin in eight-chamber culture dishes at 37 °C. The macrophages were treated with 0.0125, 0.025, and 1 mg/mL freshly prepared TiO2 NP dispersions for 1, 2, 4, 8, and 24 h. Subsequently, they were fixed with ice-cold acetone and stained with DAPI. Several images were randomly generated using an epifluorescence microscope (Zeiss Axio Imager M2).
Scanning electron microscopy and energy-dispersive X-ray spectroscopy
The sections obtained from the heart, lung, brain, stomach, kidney, spleen, and liver were scanned for TiO2 NPs using an SEM-EDX electron microscope (FEI/Philips XL 30 FEG ESEM; Eindhoven, NL). Moreover, the macrophages in the BALF samples were first stained with Diff-Quick and then scanned for NPs with the same microscope. The tissues, fixed in 2.5% glutardialdehyde and dehydrated in an ascending series of ethanol, dried in 1,1,1,3,3,3-hexamethyldisilazane (Sigma-Aldrich; Taufkirchen, Germany), and coated with carbon, were also evaluated with this setup.
Statistical analysis
The data were presented as mean ± SEM. Statistical analyses were carried out in GraphPad Prism 5.02 (GraphPad Software, Inc., La Jolla, CA) using one-way ANOVA, followed by Tukey’s test (comparing all pairs of columns). A p-value less than 0.05 was considered statistically significant.