Hydrothermally synthesized lanthanide-incorporated multifunctional zirconia nanoparticles: Potential candidate for multimodal imaging

2.1 Chemicals and reagents

Research grade Zirconium chloride, Dysprosium oxide, Holmium oxide, Nitric acid, and Urea were purchased from Sigma Aldrich. Nitrates of Dy and Ho were obtained upon reacting their oxides with nitric acid. Other reagents used for dark cytotoxicity and phototoxicity include Minimum Essential Medium (MEM), Fetal Bovine Serum (FBS), Dimethyl sulfoxide (DMSO), Phosphate Buffered Saline (PBS), and Rd cell lines from the National Institute of Health (NIH).

2.2 Synthesis of Dy/Ho-ZrO₂ nanoparticles

Schematics of the synthesis procedure are shown in Fig. 1. Dy/Ho-ZrO₂ NPs were prepared by a hydrothermal method as reported previously for Eu doped ZrO₂ NPs (Atabaev, 2018). ZrCl₄ and Dy(NO₃)₃·6H₂O were taken in mole ratios 0.89:0.11 and dissolved in 70 ml deionized water in a beaker with vigorous magnetic stirring. 1.75 g Urea was then added to the solution. After 20mins of stirring, the solution was transferred into a 100 ml Teflon-lined stainless autoclave reactor which was then placed into a wind drying oven at 140 °C for 2 hrs. Pressurized heating facilitated the production of fine Zirconia nanoparticles. The autoclave was allowed to cool down to room temperature. The white crystalline product was obtained upon opening the Teflon flask which was washed three times with deionized water by centrifuging at 6000 rpm. Obtained 11% Dy-ZrO₂ nanoparticles were dried overnight at 65 °C and were further calcinated for 1 h and at 700 °C. The same procedure was followed using Ho(NO₃)₃·5H₂O in place of Dy(NO₃)₃ to obtain 11% Ho-ZrO₂ NPs. The resulting nanoparticles when ultrasonicated for 10 min lead to homogenous suspension of nanoparticles in deionized water.

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Fig. 1

Schematics of Synthesis for Dy/Ho-Zirconia NPs.

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2.3 Characterization of Dy/Ho-ZrO₂

Physical characterizations for the prepared nanoparticles were performed using powder X-Ray diffraction (Cu-Kα radiations, 0.154 nm), Scanning electron microscopy, and Energy Dispersive X-ray spectroscopy. Tyndall test was performed to examine the colloidal stability of NPs (Stetefeld et al., 2016). The optical properties of the samples were studied using an F-98 fluorescence spectrometer. CT scans of synthesized samples were performed using General Electric’s Discovery SPECT/CT at NORI hospital Islamabad. MRI contrast properties were studied using 1.5 T GE MRI scanner. Cell viability and phototoxicity test were conducted on Rd cancer cell lines. Absorbance data was recorded using a microplate reader. All bio characterizations were performed in a sterile environment.

2.4 Cell culture and cell viability test

Human Rhabdomyosarcoma (Rd) cell lines representing the muscle cancer cell (Stevens et al., 2008) were received from the National Institute of Heal (NIH). Cells were sub-cultured in minimum essential medium (MEM) non-essential amino acids solution containing 10% Fetal Bovine Serum (FBS). Moist environment and 37 °C temperature were provided for facilitating the growth and to ensure adequate adhesion to the substrate. Upon achieving the confluence state, the cells were seeded in a 96 well plate and were placed in an incubator at 37 °C for further growth and adhesion. After 24 h of subculturing, cells were treated with various concentrations (60–240 μg/ml in FBS) of prepared nanoparticles and were further incubated for 24 h. Cell viability was determined by performing an MTT assay in a 96 well plate by a method as described in previous studies (AlSalhi et al., 2020; Atif et al., 2019, 2016, 2012, 2011b,a,c, 2010, 2009; Fakhar-E-Alam et al., 2012; Fakhar-e-Alam et al., 2020; Iqbal et al., 2020; Hayat et al., 2013). All cell culture experiments were performed in a sterile environment inside a laminar flow hood.

3.1 Photoluminescence, CT contrast and MRI contrast study

Optical characteristics were evaluated using an F-98 fluorescence spectrophotometer. Emission wavelength scan shows the photoluminescence spectra of Dy and Ho enabled zirconia nanoparticles recorded in the range of 400–560 nm is shown in Fig. 4(a). Both samples show a prominent and intense emissions spectrum near 419 nm when excited at the wavelength of 310 nm.

CT scans of the suspensions of Dy/Ho-ZrO₂ NPs in a water phantom were performed at three different energies 80 KV, 100 KV, and 120 KV as shown in Fig. 5. These energies are normally used for patient scans in clinical settings. Contrast materials present in glass vials were placed in a water-filled phantom. Different concentrations of contrast material (0, 2.1, 2.8, 4.2, 6.3 and 7 mg/ml) were scanned at three energies while the rest of the parameters were similar. Contrast (Hounsfield units) is decreasing with increasing energy as shown in Fig. 5.

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Fig. 5

The plot of HU vs Different concentrations of prepared samples at three different energies. (a) for Ho-ZrO_2; (b) for Dy-ZrO_2.

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As energy is increased, more and more photons are passing through the material without making any interaction as a cross-section for photoelectric, and Compton's effect is low at high energies. Also, increasing behavior of the X-ray attenuation coefficient can be observed with the concentration of the NPs. The highest X-ray attenuation i.e. high contrast was observed at 80 KV while the lowest contrast was at 120 KV. These optical and X-ray attenuation results indicate the superior potential of Dy/Ho = ZrO₂ NPs to be used as CT contrast agents.

For the study of MRI contrast properties, a 1.5 T GE MRI scanner was used. Inside a cylindrical water-filled phantom, a series of Dy-ZrO₂ samples with varying Dysprosium ion concentrations were placed. The water phantom was placed inside the scanner using the knee flex coil. Scans were taken using the Fast Spin Echo (FSE) sequence. T₂ weighted scans were conducted since Dysprosium increase the T₂ relaxation rate. The recovery time TR was set to 3000 ms, and the echo times TE were 50 ms, 90 ms, 130 ms, 170 ms, 190 ms, 500 ms, and 900 ms, respectively. The slice thickness was 3.5 mm, and the length of the eco train was 35. The results were presented as a series of DICOM images. IMAGEJ software was used to open DICOM images. The appropriate Region of Interest (ROI) was chosen, and the intensity variation with echo time was recorded. T₂ time for a specific ROI or vial was calculated using a plot of intensity versus echo time and an exponential curve fit. T₂ time for the remaining vials was computed in the same way. Finally, T₂ durations were plotted against the concentration of paramagnetic ions, yielding a relaxation rate in mM⁻¹s⁻¹.

The T₂ weighted images become darker due to a high concentration of paramagnetic ions. In order to depict clinical conditions, contrast vials were placed in a deionized water medium. Relaxivity rate R₂ in units of mM^-1S^-1 is obtained by plotting 1/T₂ times against Dy concentration. The R₂ value obtained after the linear fit is 64.4 mM⁻¹s⁻¹ as shown in Fig. 6, which is close to values obtained by Tegafaw et al. (2015).

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Fig. 6

(a) Plot of 1/T₂ time vs Dy³⁺ ion concentration; (b) Contrast vials placed in water medium having concentration 0 to 1 mM Dy concentration.

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3.2 Dark cytotoxicity and photo-cytotoxicity studies

MTT Assay and phototoxicity studies were performed after treating the cells with Dy/Ho-ZrO2 NPs. The viability of RD cells, incubated with increasing concentrations of the NPs (60–240 µg/ml) was evaluated using the MTT colorimetric assay. Culture media was replaced with 100 μL PBS solution containing 20 μL MTT stock solution (5.0 mg/mL) and cells were further incubated for 4 h. The MTT working solution was then aspirated and 100 µL of DMSO was added per well to solubilize the formazan crystals formed within the viable cells. The absorbance data was recorded using a microplate reader. The percentage of cell viability was determined from the absorbance data using the following relation. $$\mathrm{C}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{v}\mathrm{i}\mathrm{a}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}\mathrm{p}\mathrm{e}\mathrm{r}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{g}\mathrm{e}=\frac{\mathrm{T}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{d}\mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{s}\mathrm{a}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}}{\mathit{Untreated}cellsabsorbance}\times 100$$

Cell viability was found to be 88% or more even at higher concentrations up to 240 µg/ml while much better cell viability was observed at low concentrations which is indicative of very less dark cytotoxicity of these NPs as shown in Fig. 7.

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Fig. 7

MTT Assay cytotoxicity Results.

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For phototoxicity cells were exposed to different doses of red laser after 24 h of treatment with NPs. Cells were further incubated for 24 h before performing MTT assay. The data of phototoxicity at a fixed dose of 60 μg/ml is plotted against varying doses of light as shown in Fig. 8. Cell viability for both samples was found to be more than 80% even at higher light doses which is indicates the low phototoxicity of these samples.

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Fig. 8

Phototoxicity results, both samples have a concentration of 60 μg/ml.

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