Preparation and characterisation of TiO2NPs
Three different batches of TiO2NPs were prepared in Milli-Q water: bare titania NPs (TiO2NPs), anionic NPs prepared with poly (sodium 4-styrene sulfonate) sodium salt (PSS) as TiO2NPs/PSS, and the cationic NPs prepared with poly (allylamine hydrochloride) (PAH) as TiO2NPs/PSS/PAH. The synthesis of TiO2NPs was conducted using the procedure described by Al-Awady et al. [14]—see Fig. 1a. Briefly, 1 M HNO3 was added drop-wise to 250 mL of Milli-Q water to adjust the pH to 2 followed by dropwise addition of a mixture consisting of 15 mL aliquot of isopropanol and 5.0 mL of titanium isopropoxide (TTIP) to the former solution with vigorous stirring, leading to the formation of a white turbid suspension as a result of the hydrolysis of TTIP. The suspension of Ti (OH)4 was heated to 70 °C for 20 h to form a yellow-white precipitate of titania that was filtered, washed with ethanol and further dried under vacuum (Gallenkamp vacuum oven) at 100 °C for 2 h. Aqueous dispersions of TiO2NPs was prepared by dispersing 4 mg of the titania sample in 10 mL aliquots of 20 mM aqueous solution of NaCl at pH 4 using a digital sonicator (Branson 450, 5 mm tip, 400 W maximal power) at 40% of the maximum power for 10 min at 1 s ON/1 s OFF pulse time and followed by filtration through a syringe filter of pore size 0.22 μm. The TiO2NPs were characterised in terms of size distribution and zeta potential in aqueous solutions using a Zetasizer Nano ZL (Malvern, U.K.). Transmission electron microscopy (TEM) images of the particle samples were obtained using JEM 2011 (JEOL, Japan) running at 200 kV.
Layer-by-layer polyelectrolyte-coated TiO2NPs
Polyelectrolyte-coated TiO2NPs were prepared using titania synthesised and annealed at 100 °C (anatase). 10 mL of 1500 μg mL−1 TiO2NPs dispersion in Milli-Q water was added drop-wise to an equal amount of 10 mg mL−1 of solution of PSS (M.W. ~ 70 kDa) dissolved in 1 mM NaCl solution. After shaking for 20 min, the particles were washed three times by centrifugation for 1 h at 8000 rpm to remove the excess of PSS and were finally re-dispersed in 10 mL of Milli-Q water. The PSS-coated TiO2NPs were then mixed drop-wise with 10 mL of 10 mg mL−1 PAH (M.W. 15 kDa) dissolved in 1 mM NaCl solution, shaken for 20 min and centrifuged again three times at 8000 rpm for 1 h to yield TiO2NPs/PSS/PAH. For further coating with PSS, the latter was mixed drop-wise with 10 mL of 10 mg mL−1 PSS whilst being sonicated. The mixture was shaken for 20 min, centrifuged and dispersed in Milli-Q water to produce TiO2NP/PSS/PAH/PSS. Furthermore, PSS and PAH of various molar masses (10 kDa and 70 kDa for PSS and 15 kDa and 56 kDa for PAH) were used to examine their effect on the size of the coated TiO2NPs. After each polyelectrolyte coating, the TiO2NPs were characterized by the Zetasizer Nano ZL to check their zeta potential and the particle aggregation.
Embryo exposure to TiO2NPs
Healthy D. rerio embryos (n = 10) at 0–72 hpf, with the chorion intact, were selected and exposed to a treatment dose of test media (bare TiO2NPs, TiO2NPs/PSS, or TiO2NPs/PSS/PAH) and incubated for 3 h in either dark conditions or illuminated with visible light, at particle concentrations of 0, 500 or 1000 mg/L based on published LC50 values to increase the likelihood of observable effects [25, 26] rather than environmentally-relevant levels. Healthy D. rerio embryos (n = 10) were used as a control group in parallel.
Embryo viability following exposure to TiO2NPs
Embryos (at 48–72 hpf, n = 5) from each exposure regime (control, bare TiO2NPs, TiO2NPs/PSS, and TiO2NPs/PSS/PAH) were isolated after the exposure and washed with commercially supplied (nuclease free) molecular-biology grade water (Fisher Scientific, U.K.) three times, and re-dispersed with 1 mL molecular grade water and incubated with a drop of 98% fluorescein diacetate (FDA) (Honeywell Fluka, U.K.) in acetone (0.5 mg/L) for 15 min. This assay is based on accumulation of the fluorescent by-product (fluorescein) inside the viable embryos as a result of the hydrolysis of the diffused FDA by intracellular enzymes (esterases). The embryos were then washed again with deionised water and the cell viability examined using an Olympus BX51 fluorescence microscope attached to a DP70 digital camera and FITC fluorescence filter set. Living cells were identified as having taken up FDA and fluorescent green [27].
TEM imaging of embryos after exposure to TiO2NPs
The morphology of D. rerio embryos (n = 5) after a 3 h incubation with 0, 500 or 1000 mg/L of bare TiO2NPs TiO2NPs/PSS, or TiO2NPs/PSS/PAH was examined with TEM using the following protocol. The embryos were washed with deionised water and fixed in 2.5% glutaraldehyde (0.5 ml 25% glutaraldehyde stock solution, 4.5 ml 0.1 M cacodylate buffer and glucose (20 mL 0.2 M cacodylate stock, 10 mL Milli-Q water, 0.216 g glucose, pH 7.3, and final volume made up to 40 mL) for 1 h at room temperature. Next, cacodylate buffer was removed and embryos were fixed by 1% osmium tetra-oxide in cacodylate buffer (2.5 mL 2% Osmium tetroxide, 2.5 mL 0.1 M cacodylate buffer and glucose 0.03 M) at 4 °C overnight. After the cacodylate buffer was removed, embryos were stained for 30 min with 1% uranyl acetate (2 ml 2.5% uranyl acetate stock, final volume 3 mL) and washed with solutions of ethanol of increasing concentration (30%, 50%, and 70% overnight). The embryos were washed again the next day with ethanol solutions of 90% and 100%. After standard dehydration, the embryos were embedded in fresh epoxy/araldite at 60 °C for 48 h. The embedded embryo samples were removed from the oven and allowed to stand at room temperature for 48 h, then sectioned using an ultramicrotome. The Oxford Instruments INCA Energy Dispersive Spectroscopy (EDS) was attached to the TEM and run at 120 kV to identify and semi-quantitatively characterize the TiO2NPs on the surface, or within, of the D. rerio embryo samples. The sectioned samples were imaged using a JEOL 2010 TEM (Japan) operating at 80 kV and images were captured (from one randomly selected embryo per treatment group) with a Gatan Ultrascan 4000 digital camera (Gatan, Pleasanton, U.S.A.) and the corresponding software for imaging was the Digital Micrograph.
Target gene isolation and characterization
Total RNA was extracted from pooled samples of embryos (0–72 hpf with chorion intact, n = 10) from each treatment group, using the manufacturer’s protocol (Roche Diagnostics Ltd., Burgess Hill, U.K.). The embryo pooled sample exposures consisted of bare TiO2NPs, TiO2NPs/PSS, or TiO2NPs/PSS/PAH at 500 or 1000 mg/L particle concentration for 3 h exposure duration, in dark or illuminated with visible light as well as the corresponding control treatment group (n = 10). To assess the integrity of total RNA, samples were analysed on a denaturing agarose gel stained with ethidium bromide (Life Technologies, Paisley, U.K.). 100 ng of pooled RNA was used to generate cDNA using SuperScript VILO cDNA Synthesis reagents and protocol (Life Technologies, Paisley, U.K.) with 14 μL (~ 100 ng) of total RNA. In a 0.2 mL tube, the following reagents were added: 4 μL of 5x VILO Reaction Mix (includes random primers, MgCl2, and dNTPs in a buffer formulation) 2 μL of 10x Superscript enzyme mix. Each reaction was incubated at 25 °C for 10 min, and then 60 min at 42 °C followed by 5 min at 85 °C and a holding step at 4 °C. To degrade any remaining RNA, the following reagents were added: 0.5 μL (5 units) of RNase H (supplied in 100 mM KCl, 20 mM Tris–HCl (pH 7.5), 10 mM MgCl2, 0.1 mM EDTA, 0.1 mM dithiothreitol and 50% glycerol) and 2 μL of 10x RNase H Reaction Buffer (includes 75 mM KCl, 50 mM Tris–HCl, 3 mM MgCl2, 10 mM MgCl2 in pH 8.3 at 25 °C). All reagents were mixed, incubated at 37 °C for 45 min and then stored at − 20 °C.
For the generation of SOD2, HIF1, and Pxmp2 PCR products, 1 μL of cDNA was combined with 0.5 μl of 10 mM dNTPs, 5 μL amplification buffer, 0.5 μL of 0.5–4.5 mM MgCl2, 0.5 μL of 1.5 μM for each sense and antisense primers (Table S-1) and 0.25 μL (1.25 units) of Herculase II fusion DNA polymerase (Agilent Technologies, Wokingham, U.K.) for a total reaction volume of 25 μL. Elongation factor 1 (EF), 18S rRNA (18S) and β tubulin were evaluated as potential reference genes. Amplifications were carried out using the TC-4000 Thermal Cycler (Techne, Staffordshire, U.K.) equipped with a heated lid. All reactions were initially denatured at 94 °C for 30 s then cycled 35 times with 30 s at 94 °C denaturation, 30 s at 50/55/60 °C annealing and 30 s at 72 °C for the elongation step. A final extension step of 2 min at 72 °C was conducted. The PCR fragments were sequenced commercially by Macrogen (Amsterdam, Netherlands). Identities of PCR fragments were verified using a blastn search on the NCBI database (http://blast.ncbi.nlm.nih.gov/Blast.cgi), and aligned using a multiple sequence alignment program, Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/) to determine the correct isoform.
Quantitative qPCR analysis of mRNA expression
The qPCRs analyses for each pool of embryo cDNAs from each treatment group (n = 10) were carried out using 20 μL reaction volumes consisting of 10 μL of SYBR Green Master Mix (Roche, U.K.), 7 µL of sterilised water, 1 µL of the cDNA template, and 2 µL of optimised primer concentration (EF, HIF1: 200 nM; 18S, Pxmp2: 300 nM, SOD2: 400 nM). Two reference genes (EF and 18S) were determined as the most stable across treatment groups using geNorm software. Amplifications were carried out using a CFX96 Real-time PCR system, C1000 Thermal Cycler (Bio-Rad, Hemel Hempstead, U.K.), in triplicate and with negative controls. Reactions were started with denaturation at 50 °C for 2 min, 95 °C for 10 min, followed by a three-step protocol of 40 cycles of denaturation at 95 °C for 10 s, annealing at 60 °C for 1 min, then 72 °C for 1 min. At the end, a melting/dissociation curve was conducted. A relative quantification method was used to determine changes in mRNA transcript levels of the targeted genes in the treatment group compared to untreated control samples using the geometric mean of the reference genes for normalization and the ΔΔCt method [28].
Statistical analysis
Each target gene was tested individually for significant differences among the controls and each treatment group. All data were tested for homogeneity of variances using Levene’s test in SPSS. A non-parametric test (Scheirer-Ray-Hare) was used to assess the effect of anatase TiO2NPs coating type (factor 1), TiO2NP concentration (factor 2) and the exposure condition (factor 3) and to determine the interactions among them. Significance for relative gene expression, between TiO2NP of different coatings, concentrations, or conditions was also tested individually using the Kruskal–Wallis non-parametric test. Differences were considered significant at P < 0.05.