Single-cell ICP-MS for studying the association of inorganic nanoparticles with cell lines derived from aquaculture species

Instrumentation

Analyses have been performed using a NexION 2000 quadrupole-based inductively coupled plasma mass spectrometer (PerkinElmer, Waltham, MA, USA), equipped with a quartz torch with a quartz injector (2.5 mm i.d.), triple cone equipment, and collision/reaction cell. The instrument is also equipped with the specialized Single Cell Micro DX autosampler and with high-efficiency introduction system consisting of a CytoNeb with PFA gas line nebulizer (PerkinElmer), fitted onto the Asperon spray chamber (PerkinElmer). Microjet adapter is positioned to the Asperon spray chamber for dual make-up gas inlet to create a tangential flow. All analyses were carried out using the Syngistix™ Single Cell Application Software Module for data collection and processing. Microwave-assisted acid digestions were performed using Ethos Easy Advanced Microwave Digestion System (Milestone, Sorisole, Italy). A Laborcentrifugen 2K15 (Sigma, Osterode, Germany) was used for centrifugation (washing of cell suspension) and a USC-TH ultrasound water bath (45 Hz, 80 W) from VWR International Eurolab S.L. (Barcelona, Spain) was used for dispersing NP standards before calibrations. Cell counting was performed in a counting Neubauer chamber–improved bright-line hemocytometer (Brand, Wertheim, Germany).

Electron microscopy (EM) characterization of exposed cells was performed using a JEOL JEM 1010 transmission electron microscope (TEM) operating at voltage of 100 kV for cells from clams and a scanning electron microscope (SEM) FEI Quanta 650 FEG, operating at high vacuum, an acceleration voltage of 5 kV, and spot size set at position 3 for kidney cells from sea bream. The cell samples for SEM analysis were coated with conductive carbon using an EM ACE600 coating system (Leica microsystems). The cell samples for TEM analysis were treated using a Leica EM TP Tissue processor and the ultrathin sections with a thickness of ~70 nm were prepared using a RMC Boecketer PowerTome PC ultramicrotome system.

Reagents and standards

Ultrapure water (18.2 MΩ cm of resistivity) was obtained from a Milli-Q® IQ 7003 purification device system from Millipore (Bedford, MA, USA). Mono-elemental standards of ionic titanium [(NH4)2TiF6] and silver (AgNO3) were purchased from PerkinElmer. Hyperpure nitric acid 69% (w/v) and 33% (w/v) hydrogen peroxide were from Panreac (Barcelona, Spain). Phosphate-buffered saline (PBS) was from Thermo Fisher (Dublin, Ireland). NexION Setup Solution (Be, Ce, Fe, In, Li, Mg, Pb, U), 10 μg L−1, was from PerkinElmer. Glassware and plastic ware were decontaminated by soaking in 10% (v/v) nitric acid for at least 48 h. Material was then rinsed with ultra-pure water several times. Gold nanosphere dispersions were prepared from a N8151035 standard (nanoComposix, San Diego, CA, USA). The material consists of 49.6 ± 2.1 nm nanospheres (TEM diameter) and particle concentration of 9.89 × 106 NPs mL−1 (2% RSD) obtained by spICP-MS. The nanospheres are covered by PEG carboxyl and they are suspended in aqueous 1 mM citrate. Silver NP dispersions used for calibration were prepared from bare (citrate) Ag NP standards (aqueous 2 mM sodium citrate) from nanoComposix. The standards were 60-nm Ag NPs (nominal diameter of 59 ± 6 nm obtained by TEM, mass concentration of 0.020 mg mL−1 obtained by ICP-MS, particle concentration of 1.8 × 1010 particles mL−1, and a hydrodynamic diameter of 64 nm); 40-nm Ag NPs (nominal diameter of 41 ± 5 nm obtained by TEM, mass concentration of 0.021 mg mL−1 obtained by ICP-MS, particle concentration of 5.4 × 1010 particles mL−1, and a hydrodynamic diameter of 44 nm); and 20-nm Ag NPs (nominal diameter of 20.8 ± 3.0 nm obtained by TEM, mass concentration of 0.021 mg mL−1 obtained by ICP-MS, particle concentration of 4.2 × 1011 particles mL−1, and a hydrodynamic diameter of 27 nm). Titanium dioxide NP stock dispersions (also used for calibration) were prepared from TiO2 suspensions (mixture of rutile and anatase, 99.5%) with particle size < 150 nm (volume distribution by dynamic light scattering) at 40 wt% in water, purchased from Sigma-Aldrich (Osterode, Germany). Regarding cell exposure trials, polyvinylpyrrolidone (PVP)-coated Ag NPs with primary nominal diameter of 15 nm and 100 nm and citrate-coated TiO2 NPs with a primary nominal diameter of 5 nm, 25 nm, and 45 nm were prepared from Ag NPs and TiO2 NPs from several suppliers. A complete description of the preparation of PVP-Ag NPs and citrate-TiO2 NP dispersions can be found in the “Preparation of PVP-Ag NPs and citrate-TiO2 NP dispersions used for cell exposure trials” section.

Ammonia (99.999%) reaction gas used in the reaction cell and 99.998% argon used for plasma generation, nebulization, and as auxiliary gas were supplied by Nippon Gases (Madrid, Spain).

Leibovitz medium (L15) and fetal bovine serum (FBS) suitable for cell growth, and ACS grade dimethyl sulfoxide (DMSO) were purchased from Thermo Fisher. Sodium cacodylate buffer was prepared by dissolving 98% sodium cacodylate trihydrate (Thermo Fisher) in distilled water and adjusting the pH to 7.2 with diluted hydrochloric acid prepared from technical grade 37% hydrochloric acid (Panreac). Karnovsky fixative was prepared at 2.0% (v/v) paraformaldehyde (96% extra pure) and 2.5% (v/v) glutaraldehyde (50% solution, ≥ 48.0 to ≤ 52.0%) from Thermo in 0.1 M sodium cacodylate buffer. Propylene oxide (ReagentPlus® ≥ 99%; Sigma-Aldrich, Merck Life Science, Algés, PT), osmium tetroxide solution (2% and 4% aqueous solution; Science Services, Munich, Germany), ethanol, and EMBed-812 epoxy resin kit (Science Services, Munich, Germany) were used for the fixation, staining, dehydration, and resin embedding of the cells for TEM analysis.

Preparation of PVP-Ag NPs and citrate-TiO2 NP dispersions used for cell exposure trialsPolyvinylpyrrolidone (PVP)-coated Ag NPs with a diameter of 15 nm (15-nm Ag NPs)

Commercial PVP-coated Ag NP powder was purchased from SSNano (Houston, TX, USA; product code: 0127SH). The powder composition was 25% wt silver and 75% wt PVP. Ag NP stock dispersion at 6.2 g L−1 was prepared in ultrapure water by dispersing the powder for 15 min using a bath sonicator (37 kHz, 100%).

PVP-coated Ag NPs with a diameter of 100 nm (100-nm Ag NPs)

The 100-nm Ag NP stock dispersion with a concentration of 2.9 g L−1 was prepared from Ag ink containing 30% AgNPs with a diameter of 100 nm dispersed in ethylene glycol (Sigma-Aldrich). The ethylene glycol was removed by dialysis using a 12-kDa cellulose membrane against water. The purified Ag NPs was mixed with PVP (Mw = 40 kDa) solution to reach a Ag to PVP ratio of 1:3 wt:wt.

Citrate-coated TiO2 NPs with a primary size of 5 nm (5.0-nm TiO2 NPs)

Pristine 5-nm TiO2 NPs were purchased from Nanostructured & Amorphous Materials, Inc. (Katy, TX, USA; anatase, 5 nm size, 99%). The 5-nm TiO2 NP stock dispersion was prepared in ultrapure water by dispersing a mixture of trisodium citrate dehydrate and titanium dioxide powder at weight ratio of 1.5:1 wt:wt for 30 min using an ultrasonic probe (Branson Disintegrator Ultrasonic Model 450; 30-s pulse on/5-s pulse off, and 50% amplitude). The final concentration of citrate-coated TiO2 NPs was 13.3–15.5 g L−1 depending on the batch.

Citrate-coated TiO2 NPs with a primary size of 25 nm (25-nm TiO2 NPs)

Pristine 25-nm TiO2 NPs were supplied by Sigma-Aldrich (99.5% purity, mixture of rutile and anatase). The 25-nm TiO2 NP stock dispersion was prepared in ultrapure water by dispersing a mixture of trisodium citrate dehydrate and titanium dioxide powder at weight ratio of 0.8:1 wt:wt for 30 min using an ultrasonic probe (Branson Disintegrator Ultrasonic Model 450, 30-s pulse on/5-s pulse off, and 50% amplitude). The final concentration of citrate-coated TiO2 NPs was 13.3–15.5 g L−1 depending on the batch.

Citrate-coated TiO2 NPs with a primary size of 45 nm (45-nm TiO2 NPs)

Pristine 45-nm TiO2 NPs were purchased from Sigma-Aldrich (99.5% purity, mixture of rutile and anatase; nanoparticle size of < 100 nm (BET) and < 50 nm (XRD)) and were used without any further purification. The 45-nm TiO2 NPs were stabilized with trisodium citrate dehydrate aqueous solution reaching a weight ratio of 1:1.5 TiO2 to citrate. The mixture was dispersed for 30 min using an ultrasonic probe (Branson Disintegrator Ultrasonic Model 450; with 30-s pulse on/5-s pulse off, and 50% amplitude). The final concentration of citrate-coated TiO2 NPs was 13.3–15.5 g L−1 depending on the batch.

TEM analyses from several prepared Ag NPs and TiO2 NPs are shown in Figure S2 (electronic supplementary information, ESI).

Cytometry measurements

After appropriate dilution, 10 μL of cell suspensions was introduced in the chamber following manufacturer’s instructions and cells included in the 4 ruled areas of the corners were counted and the average number was accounted. Cells were stained with Trypan Blue classical method, to differentiate dead and alive cells. The final number of cells was calculated by multiplying average number by a factor of 104 (manufacturer’s instructions) as well as by the dilution factor.

Cell culture conditions and pre-treatments for scICP-MS and EM analysis

Assays regarding exposure tests and cell toxicity to PVP-Ag NPs and citrate-TiO2 NPs for clam gills, and sea bass and sea bream kidney cells were carried out at the facilities of the Aquaculture Cluster Technology Centre (CETGA). The species (clam (Ruditapes philippinarum), sea bass (Dicentrarchus labrax), and sea bream (Sparus aurata)) followed the standard procedure for growth under controlled conditions until they were harvested. On the one hand, sea bass and sea bream specimens were fasted for 1 day before sampling, whereas the fasting period for clams was 3 days. The specimens were sacrificed after anaesthesia overdose, and the surface of the fish and the clam shells were disinfected with 70% ethanol. In the case of sea bass and sea bream, the kidney was removed under sterile conditions. The clams were opened by sectioning the anterior and posterior adductor muscles with a scalpel, and the entire contents of the clam’s belly were then removed under sterile conditions.

Both the kidney cells (sea bass and sea bream) and the entire contents of the soft tissues of clams were disaggregated, and the cells of interest were collected using a Percoll gradient, discarding impurities and non-viable cells. A Neubauer chamber was used to count total/viable cells. After counting, the cells were adjusted to the desired concentration, distributed in the wells, and loaded in multi-well plates for different exposures with NPs (size and concentrations). The cell culture was adjusted to a concentration of 4.0 × 106 cell mL−1. The culture medium used was L-15 without phenol red supplemented with 5–10% FBS (v/v) and 1% penicillin/streptomycin (Thermo Fisher, 10,000 IU/mL−1 penicillin, 10,000 μg/mL−1 streptomycin) at 19 °C. Cell viability was tested by the MTT (tetrazolium salt (2-(4,5-dimethyl-2-thiazolyl)-3,5-diphe-nyl-2H-tetrazolium bromide) method to ensure that the cell cultures are in good condition for carrying out bioaccumulation assays. Toxicity tests were performed with an initial cell amount of 9.0 × 105 cells (volume of 3.0 mL and 3.0 × 105 cell mL−1).

Cell toxicity assays (24-h exposure) were performed under sterile conditions in a type II laminar flow cabinet by adding to each well 1.0 mL of cell culture and 3.0 mL of NP dispersions (experiment in duplicate) at several concentrations (dilutions with the cell culture medium). First NP dispersions were redispersed by sonication (10 min, 50% amplitude, pulse 15-s on/10-s off) for citrate-TiO2 NPs, and by vortexing for PVP-Ag NPs. After exposure, the cells were transferred to 15-mL tubes for further centrifugation (1500 g, 10 min), and the supernatant was removed. The pellet was mixed with 1.0 mL of freezing medium (Leibovitz medium (L15) with 20% fetal bovine serum (FBS) and 10% DMSO) to preserve the integrity of the cells during freezing and was frozen at −20 °C until analysis.

The obtained cells were further subjected to two different pre-treatments for leading scICP-MS and TEM analysis. Cells for scICP-MS analysis were suspended with 4.0 mL of L15/20% FBS/10% DMSO freezing mixture and were then frozen and kept at −20 °C. Pre-treatment for TEM analysis consisted of a centrifugation stage of the cell suspension at 320 g, 4 °C for 5.0 min and careful supernatant removal, followed by dropwise addition of 500 μL of Karnovsky fixative and keeping the mixture at 4 °C 24 h under gentle shaking. After Karnovsky fixative removal, the pellet was washed with sodium cacodylate buffer (0.1 M) for 20 min (two washing steps) and was kept at 4 °C.

Cell suspension preparation for scICP-MS

Cell suspensions were thawed and homogenised through pipette mixing, and then an aliquot was sampled and re-suspended in 1.0% (wt/v) PBS in 1.0-mL Eppendorf tubes. The diluted cell suspensions were subjected to a short and gentle centrifugation washing step (300 g, 4.0°C, 5.0 min). The supernatant was removed, and the pellet was again re-suspended in 1.0% (wt/v) PBS for scICP-MS analysis after appropriate dilution.

Microwave-assisted acid digestion

For comparison purposes, 1.0 mL of cell suspensions in 1.0% (wt/v) PBS (one replicate for each case) was subjected to microwave-assisted acid digestion by using 2.0 mL of ultrapure water, 3.0 mL of 69% nitric acid, and 1.0 mL of 33% hydrogen peroxide. The mixtures were exposed to microwave under a controlled temperature program that consisted of a ramp from room temperature to 130 °C in 20 min, and a hold stage at 130 °C for 15 min. After cool down, the acid digests were diluted to 25 mL with ultrapure water and kept at room temperature until ICP-MS analysis (operating conditions given in Table S1, electronic supplementary information, ESI).

TEM and SEM analyses

The fixated pellets of clam cells were processed following a routine methodology for TEM. Briefly, the cell pellets were post-fixated in a 1% osmium tetroxide solution. Then, they were dehydrated with increasing ethanol from 50 to 100% and with a final emersion step in propylene oxide. The infiltration was performed using mixtures of propylene oxide: epoxy resin (EMBed-812 kit) at different proportion increasing the amount of resin until having finally pure epoxy resin. Fragments of each pellet embedded were placed in the edge of a silicon template forming blocks. The blocks were cured at 60 °C for 3 days. Ultrathin sections (≈70 nm thick) were prepared using an ultramicrotome with a diamond knife (Diatome) and placed on formvar/carbon-coated 200-mesh copper grids for cells exposed to citrate-TiO2 NPs and carbon-coated 400-mesh titanium grids for cells exposed to PVP-Ag NPs to be analysed by TEM.

The fixated pellets of sea bream cells were washed in cacodylate buffer and subsequently filtered through 2-μm polycarbonate membrane. Cell-supported polycarbonate filters were placed on pin stubs (standard 12.7 mm, 8-mm pin length, Ted Pella) and subsequently, they were coated with conductive carbon for SEM analysis.

Single-cell ICP-MS measurements

Daily performance for ICP-MS was assessed (Be intensity > 2500 counts s−1, In intensity > 40,000 counts s−1, U intensity > 30,000 counts s−1, and Bkgd ≤ 3 for standards at 1.0 μg L−1, and Ce++/Ce ratio ≤ 0.05 and the CeO/Ce ratio ≤ 0.025). Daily performance (torch alignment and voltages) was performed with Ti (10 μg L−1) and Ag (3.0 μg L−1) prior to analysis. Analytical results were calculated using Syngistix™ ICP-MS 2.5 version software. Operating conditions for scICP-MS are listed in Table 1. The scICP-MS analytical data was processed using an iterative approach previously described [21, 35] aiming the cell event threshold. The transport efficiency (TE%) was automatically calculated after measuring the 49.6-nm Au NPs certified reference material at 1.00 × 105 NPs mL−1 by the particle frequency method. The TE% value obtained was from 45 to 55%.

Table 1 Operating conditions and data acquisition parameters for scICP-MS*

Determinations implied external calibrations at five level concentrations for ionic titanium (0.5–10 μg L−1) for titanium assessment, whereas calibrations with Ag NPs (20, 40, and 60 nm, 1.0 μg L−1, each one) were used for silver determinations (Ag NP dispersions were manually shaken just before measurements). Cell suspensions (after dilution in PBS) were placed in 1.0-mL cuvettes of the autosampler. The autosampler automatically mixes the cell suspensions to favour the representativeness of the sample taken and aspirates 150 μL for filling a 100-μL loop. The loaded sample is then pumped at 10 μL min−1 and nebulized with a CytoNeb nebulizer coupled to an Asperon chamber which allows a tangential flow that prevents cell damage, collisions, or cell deposition onto the chamber walls. After each injection, the sample loop is rinsed with a wash solution (1% nitric acid and 2% hydrogen peroxide).

Standard mode and mass-shift approach were used for the measurement of silver and titanium, respectively. Ammonia clusters of mass-charge ratio 131 (1.0 mL min−1 of ammonia and RPq 0.2 value [33, 34]) were used for titanium determinations. The scICP-MS analytical data were processed using an iterative approach aiming the cell event threshold. Data, graphs, and spectra analysis were performed after data being exported from 2.5 Syngistix ICP-MS software (Single Cell Application). The amount of titanium and silver in cells was determined by applying Eq. (1).

$$_c=\frac_}\cdot _}\cdot \left(_c-_\right)}$$

(1)

where mc is the mass of element of interest in single cell, ε is the transport efficiency, Qsam is the sample uptake rate, tdwell is the dwell time, m is the calibration slope, and Ic and Ibgd are the analyte and background intensity, respectively.

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