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Harnessing the Cancer Radiation Therapy by LanthanideDoped Zinc Oxide-Based Theranostic Nanoparticles Behnaz Ghaemi, Omid Mashinchian, Tayebeh Mousavi, Roya Karimi, Sharmin Kharrazi, and Amir Amani ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b10056 • Publication Date (Web): 15 Jan 2016 Downloaded from http://pubs.acs.org on January 16, 2016

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ACS Applied Materials & Interfaces

Harnessing the Cancer Radiation Therapy by Lanthanide-Doped Zinc Oxide-Based Theranostic Nanoparticles Behnaz Ghaemia, Omid Mashinchiana,b, Tayebeh Mousavic, Roya Karimid, Sharmin Kharrazia*and Amir Amania,e* a

Department of Medical Nanotechnology, School of Advanced Technologies in Medicine (SATiM),

Tehran University of Medical Sciences, Tehran, Iran b

Institute of Bioengineering, School of Life Sciences, École polytechnique fédérale de Lausanne

(EPFL), Lausanne, Switzerland c

Department of Materials, University of Oxford, Oxford, OX1 3PH, UK

d

Department of Tissue Engineering, School of Advanced Technologies in Medicine (SATiM), Tehran

University of Medical Sciences, Tehran, Iran e

Medical Biomaterials Research Center (MBRC), Tehran University of Medical Sciences, Tehran,

Iran *Corresponding authors: [email protected] (S.K.) and [email protected] (A.A.)

ACS Paragon Plus Environment


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Abstract: In this paper, doping of Europium (Eu) and Gadolinium (Gd) as high-Z elements into the ZnO NPs was designed to optimize restricted energy absorption from a conventional radiation therapy by XRay. Gd/Eu-doped ZnO NPs with a size of 9 nm were synthesized by chemical precipitation method. Cytotoxic effects of Eu/Gd-doped ZnO NPs were determined using MTT assay in L929, HeLa and PC3 cell lines under dark conditions as well as exposure to UV, X-ray and Gamma radiations. Doped NPs at 20 µg/ml concentration under X-ray dose of 2 Gy, were as efficient as 6 Gy X-radiation on untreated cells. It is thus suggested that the doped NPs may be used as a photo inducer to increase efficacy of X-rays within the cells, consequently, cancer cell death. The doped NPs also could reduce the received dose by normal cells around the tumor. Additionally, we evaluated the diagnostic efficacy of doped nanoparticles as CT/MRI nanoprobes. Results showed an efficient theranostic nanoparticulate system for simultaneous CT/MR imaging and cancer treatment.

Keywords: Doped ZnO, Photocatalytic activity, ROS generation, Radiation therapy, Dose enhancement, dual-mode imaging


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1. Introduction Cancer is the second cause of death in the world. Among different cancers, Prostate cancer is the most common cancer in men which includes 27% of incident cases. Cervical cancer has been introduced as a leading cause of cancer death in women in the USA and the most common solid cancer diagnosed in pregnancy1. Accordingly, there is a desperate need for a reliable approach to provide image-guided therapy. In this context, radiation therapy is one of the most widespread approaches in cancer treatments2. It is worth noting that an adjuvant radiotherapy needs to be applied following the surgery for most of the patients with metastatic cancer, depending on the malignancy3. Recent advances in radiation therapy have led to the ability of delivering specified radiation doses to precisely control/destroy malignant cancer cells within the body. However, this method is associated with drawbacks due to damage of intracellular compounds (e.g., DNA) within the normal cells around the tumor area4. Consequently, clinical management of individual patients as well as the need for assessment of therapies during different clinical trials highlight the importance of targeted radiation therapy in order to reduce damages to the normal cells. Nanoscale structures and materials as novel potential platforms have shown capable of drastically changing the cancer therapeutic systems. Increasing localized effects of ionizing radiation, increasing oxidation rate through production of reactive oxygen species (ROS) and providing a noninvasive access to interior parts of the cells are some of the advantages of using nanoparticles (NPs)5. In this regards, semiconductor NPs and quantum dots (QDs) look promising in cancer tracing/treatment. QDs can be activated by light to induce damage to targeted cells by employing free radicals6. However, production of free radicals from QDs (e.g., ZnO nanoparticles) is performed by excitation using low-energy photons (from visible to ultraviolet (UV) regions) 7. As UV radiation cannot penetrate into the deeper parts of the



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human body, these NPs cannot be utilized to eradicate the deep tumors8. An alternative for UV radiation is X-ray which is able to penetrate into the human body. The radiation efficiency of X-ray may also be increased by interaction of photoactive NPs with X-ray through adding lanthanides as dopants. When the lanthanide as a high Z element (i.e., high atomic number element) is exposed to photons with energy levels similar or above that of binding energy in innermost orbital layer (K edge), a selective photon absorption may occur9 to emit Auger electrons and photoelectrons10. It is worth mentioning that the choice of dopants is crucial for a successful X-ray interaction with nanoparticles11. Based on these assumptions, the Lanthanides-doped ZnO-NPs can be introduced as an efficient agent for the treatment of deep tumors. They have the potential of being accumulated in different tumors for specific therapies and controlled activation which may preferentially discriminate cancerous cells from healthy ones12. A previous report demonstrated that the required time for the blood clearance from ZnO-NPs is 6 h after administration13. Moreover, it can provide a potential of greater NPs accumulation in the tumor than the normal cells by the enhanced permeability and retention (EPR) effect14. Additionally, nano-based platforms could provide a valuable opportunity in improving the dynamic contrast-enhanced imaging techniques for diagnosis of cancers by multimodal nanoparticles (i.e., multimodal Theranostic agents)15. Therefore, lanthanides-doped ZnO nanoparticles (e.g., Europium/Gadolinium-doped ZnO-NPs) could be introduced as tumorspecific contrast agents for MR (Magnetic Resonance)/CT (Computed Tomography) -dualmode imaging16. In our study, Eu/Gd-doped ZnO-NPs were synthesized to significantly enhance efficiency of X-radiation therapy on prostate and cervix cancers. The particles were characterized by X-ray diffraction (XRD), UV-Vis absorption spectroscopy and high resolution transmission electron microscopy (HRTEM). The targeted cancer cells were exposed to different radiation


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conditions (i.e., dark, UV, gamma-ray and X-ray) to optimize and efficiently evaluate the radiation effects on activation of the trapped nanoparticles within the cells. Moreover, to study the potential of Eu/Gd-doped ZnO-NPs on enhancement of the radiation effects, X-ray was applied at different radiation doses. The cytotoxic effects of Eu/Gd-doped ZnO NPs were determined using MTT assay in both cancerous as well as normal cells. Additionally, we investigate efficiency of the Eu/Gd-doped ZnO NPs as a simple and versatile contrast agent for CT/MRI.

2. Experimental Details 2.1. Synthesis of ZnO Nanoparticles ZnO nanoparticles were synthesized through a chemical precipitation methodology using NaOH (NaOH, 99%, Merck Chemicals, Germany) as a precipitation precursor in methanol solvent (Dr. Mojallali Chemical Complex Co, Iran). Then, Zinc acetate (Merck Chemicals, Germany) was dissolved in methanol and stirred for 1 h at a concentration of 0.2 M. Subsequently, NaOH pellets were dissolved in methanol at 1.2 M and stirred for 1 h at a constant rotation speed of 800 rpm. Then, Zinc acetate solution dropped was added to the NaOH solution at the rate of 5 mL per minute. The final mixture was vigorously stirred (1500-rpm) for 3 h at ambient temperature. The obtained ZnO nanoparticles were centrifuged and washed to remove unreacted precursors and dried at room temperature for 48 h (washing process was repeated five times). 2.2. Preparation of Doped ZnO Nanoparticles Optimized Zinc oxide NP with a good stability was selected for doping by Europium and Gadolinium elements with doping rate of 5% mol. In this context, rare earth salts (REEs, e.g., Gadolinium (III) nitrate hexahydrate; Europium (III) nitrate pentahydrate, Sigma Aldrich,



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USA) were suspended in Zinc acetate solution and added to the NaOH solution. The final mixture was washed, dried and ground to a fine powder after 3 h stirring. 2.3. Material Characterization 2.3.1. UV-Vis absorption Spectroscopy UV-Vis absorption spectra of samples were recorded by using a Labomed UVD 2950 (Culver City, USA) Ultraviolet-visible spectrophotometer with an optical resolution of 0.01nm full width at half maximum (FWHM). The spectrum response was taken from 200 to 800 nm with 1 nm steps in a 4×1×1 cm path quartz cuvette. 2.3.2. X-ray Diffraction (XRD) XRD analysis was carried out using a fully automated EQ uniox 3000 (INEL, France) powder diffractometer employing Cu-Kα radiation (ƛ= 1.541874 Ȧ) at 40 mA and 40 kV and a secondary monochromator. Phase analysis was carried out by comparing diffraction patterns acquired from the sample with a standard database of International Centre for Diffraction Data (ICDD). The average crystallite size was calculated from the XRD data using Scherrer formula; d=0.89λ/βcosƟ, where d is the average crystallite size and λ is the Xray wavelength (CuKα, 0.154 nm). β and Ɵ are FWHM and Bragg diffraction angle of the strongest diffraction peak, respectively. 2.3.3. High-resolution Transmission Electron Microscopy (HRTEM) Morphology (i.e., particle size, crystallinity, lattice parameters and particle size distribution) of the ZnO nanoparticles was characterized by imaging and SAED (Selected Area Electron Diffraction) analysis using high resolution transmission electron microscopy (HRTEM, JEOL 2100, Japan) at an operating voltage of 200 kV. TEM samples were prepared by homogenising a suspension of nanoparticle/ethanol with 1 mM concentration in an ultrasonic vibrator and dropping a few droplets of the mixed suspension onto the lacy carbon film of a 200-mesh Cu-grid followed by the solvent evaporation at room temperature.


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2.3.4. Energy-Dispersive Analysis of X-rays (EDAX) The EDAX data was obtained using a Bruker XFlash-5030, liquid nitrogen-free silicon drift detector with an optimized collection solid angle of ∼0.13 sr. Cathodo-luminescence spectra have been measured with JEOL JSM-6500F scanning electron microscope (SEM) equipped with Gatan MonoCL3 worked at 15 kV. 2.3.5. X-ray Photoelectron Spectroscopy (XPS) XPS measurements were performed at a vacuum of 2 × 10-9 Torr in an Escalab5 (U.K.) spectrometer, employing Al-Kα radiation (1486.6 eV) as the X-ray excitation source. The XPS data were analyzed utilizing the XPSPEAK software (Version 4.1) to deconvolute the detailed spectra of Zn 2p, Eu 3d and Gd 3d. The binding energy (BE) of the samples was corrected by setting the measured BE of C 1s to 284.6 eV. 2.4. Determination of ROS Generation 2.4.1. DPPH Assay In order to measure the amount of reactive oxygen species (ROS) and other free radicals, which were produced by doped and dopant-free ZnO NPs, a stable free radical scavenger [DPPH (1,1-Diphenyl-2-picryl-hydrazyl) (sigma-Aldrich, USA)], was used at different conditions (i.e., darkness, ultraviolet, x-ray and gamma radiations). 1 ml DPPH was dissolved in 2.5 ml methanol in 1 h, then, added to each sample immediately after radiation exposure. Trapping the generated radicals by DPPH changes the color of the solution from purple to yellow17. Color intensity may then be measured by ELISA reader at 490 (purple) and 630 (yellow) nm wavelengths. In this experiment the purple color has been assessed (i.e., absorption increments at 490 nm indicating ROS generation enhancement). DPPH radical scavenging activity was calculated from the following equation in which H and Ho indicate optical density of solvent with and without sample, respectively18. Radical scavenging activity (%) = [(H - Ho)/Ho] × 100



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2.4.2. Flow-cytometry Oxidative stress was induced by exposing Gd-doped ZnO (20 µgr/ml, 6 hours) treated PC3 cells, to UV, X and gamma radiations and in dark condition. The intracellular ROS were evaluated using DCFH-DA and DHE, specific probes for H2O2 and O2- respectively, which are cell-permeable stains. DCFH is oxidized selectively by the free intracellular H2O2 into DCF that binds to DNA and emits green fluorescence. DHE is oxidized by the free intracellular O2- into ethidium bromide which binds to the DNA and emits red fluorescence19. DCFH-DA (25 mM) and DHE (1.25 mM; Sigma) were added to the NP-treated PC3 cells after exposure of each radiations and incubated at 25 ̊ C for 45 minutes with DCFH-DA and 25 minutes with DHE separately. We used hydrogen peroxide (H2O2) and O2 radical as a positive controls in these experiments. Aliquots were subsequently analyzed using a flow cytometer. Green fluorescence (DCF) was assessed in 500-530 nm, while red fluorescence (HE) was evaluated in 590-700 nm (excitation, 488 nm; emission, 525–625 nm in the FL-2 channel). Data were presented as the percentage of fluorescent PC3 cells. Apoptotic PC3 cells were excluded by using counter nucleic acid stains. Propidium iodide (PI) and YOPRO-1 were used as a counter-stain dye for DCFH-DA and HE respectively. The population of PC3 cells were gated using 90 ̊ and forward angle light scatter to discount debris and aggregates. The excitation wavelength (λ=488 nm) was supplied by an argon laser at 15mW. DCF/ YO-PRO-1 emitting green fluorescence and PI/ HE emitting red fluorescence (580–630 nm) were recorded in the FL-1 and FL-2 channels, respectively. The percentage of HE-/PI positive cells and the mean fluorescence were calculated on a 1023-channel scale and analyzed using the flow cytometer software FlowJo version 7.2.2 (FlowJo, Ashland, OR). 2.5. Confocal Microscopy Study 2.5.1. Fluorescent Labelling of Nanoparticles


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Rhodamine B fluorescent dye (Merck, Germany) was used in order to assess NPs uptake. At the first step, 3 mg rhodamine B was diluted in 10 ml methanol for 10 m at 50 ºC. Then, 50 mg NP powder was dissolved in 5 ml methanol, and the solution was dropped to the dye solution. The final mixture was stirred for 5 m at 50 ºC at dark conditions. As a final step, the dye-labelled NPs were centrifuged, washed, and dried at ambient temperature. Rhodaminelabelled-NPs were dissolved in phosphate-buffered saline (PBS) at 50 µg/ml concentrations and added to targeted cell lines (as discussed below). The targeted cells were incubated for 34 h with the labelled NPs. 2.5.2. Cell Staining Analysis The stained cells were assessed by inverted confocal microscopy (Leica, TCS SP5, and Germany). Prior to imaging, cells were fixed in 4% formaldehyde for 15 min and permeabilized in a solution containing Triton X-100 in PBS for 5−10 min at room temperature.

Cell

nuclei

were

stained

by

DAPI

(4",6-diamidino-2"-phenylindole

dihydrochloride; Sigma-Aldrich, USA). Control samples were cultured on 12 mm diameter circular glass coverslips. Afterwards, Leica Application Suite Advanced Fluorescence (LAS AF) software (Leica Microsystems, Germany) was used to analyze the confocal microscope pictures. 2.6. Cell Culture Three human cell lines were assessed in vitro: L929 (fibroblast cell) as a normal cell model and HeLa (cervix cancer cell) and PC3 (prostate cancer cell). All cells were purchased from the National Cell Bank of Pasteur Institute (Iran) and cultured at a density of 1×104 cells per well (96-well plate) and cultivated in growth medium of Dulbecco’s Modified Eagle Medium (DMEM F12, Gibco Invitrogen, UK) and RPMI 1640 (Merck, Germany), which were supplemented with 10% fetal bovine serum (Sigma, USA) and 1% penicillin-streptomycin-



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glutamine (Gibco Invitrogen, UK). All cells were stored in a humidified incubator at 37°C and 5% CO2 atmosphere. 2.7. In vitro Radiation Exposures Prior to irradiate of the NPs-treated cells, they were exposed to UV, X-ray and gamma ray to analyse the radiation effects on the cells as control samples. NP solutions were then added to the cells media after 24 h of culture and incubated overnight with 5, 10, 15 and 20 µg/ml of ZnO NPs, Eu-doped ZnO NPs and Gd-doped ZnO NPs. The NPs-treated cells were subsequently assessed to analyse the toxicity effects of the NPs at dark condition (second control samples). NPs-treated cells were subsequently exposed to different radiation sources (i.e., UV, x-ray and gamma ray) in order to evaluate the toxicity effects (i.e., each variation was performed in triplicate). In this context, UV test was carried out under UV-C (254 nm) radiation for 300s, which was provided by a germicidal lamp (average intensity 0.2 mW/cm2, BMG Labtech, Korea) with 15 cm SSD (source to surface distance). In another experiment, NPs-treated cells were exposed to a broad spectrum of X-ray energies up to 200 kVp (1mmCu) from orthovoltage therapy machine (Stubilipan, Siemens, Germany) with an average energy of approximately 60–70 keV (i.e. Near K-edge of dopant elements). The monitor units (Doses) of NP-treated cells were 1.5 and 2 Gy which were acquired from a single fraction radiation, with 15 cm diameter collimator and SSD distance of 30 cm. Subsequently, untreated cells were exposed to dose of 4 Gy (tow fraction of 2 Gy in 48 hours, Ctrl3) and 6 Gy (three fraction of 2 Gy in 72 hours, Ctrl4) as control groups. The last group contained treated cells with NPs which were exposed to gamma rays by a medical linear accelerator (simulator Co60 Simax, SHINVA, Japan). The samples were irradiated in single fractions gamma radiation with monitor unit (dose) of 2 Gy and set up at SSD of 80 cm using a field size of 15 × 15 cm2. The samples were located at the centre of the gamma radiation beam to ensure that all of the targeted cells received a constant and uniform radiation dose.


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2.8. Cell Viability Assessment Apoptosis induction of the NPs was determined by MTT assay where 20 µl of MTT (5 mg/ml, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Sigma, Germany) was added to each well and incubated for 4 h at 37°C, according to the previous reports. The colour absorbance at 590 nm was then quantified using an ELISA Reader (Biotek, ELX808) and presented as percentile of controls. 2.9. CT/MR Images contrast enhancement ability of Nanoparticles T1-weighted MR images of the Eu/Gd-doped ZnO Nanoparticles were obtained by 3.0 T MRI (General Electric Medical Systems, USA). The nanoparticles (Aqueous dispersions) were prepared at 2 different concentrations (0.05mM and 0.1 mM) for imaging procedure which were prepared in 2.0 mL micro tubes and located in a self-designed water phantom. In this experiment, OMNISCAN™ (Gadodiamide, 287mg/ml, GE Healthcare Inc., USA) and deionized water were employed as positive control and negative control, respectively. The acquired parameters were as follows: human head coil, TR/TE= 400ms/7ms, T=25C, number of acquisitions (NEX) = 1, field of view (FOV) = 225×225 mm2, matrix size = 512×512 mm2, slice thickness = 5 mm, pixel spacing = 0.625 mm. CT scans were performed using a 16 Slice Toshiba Imaging System (TOSHIBA, Medical Systems, Japan) operated at 120 kV and 80 mA, with a slice thickness of 0.5 mm. The samples were prepared as described in previous section for MRI studies. Contrast enhancement was determined in Hounsfield units for each sample. Deionized water and VISIPAQUE™ (Iodixanol, 270mgI/ml, GE Healthcare Inc., USA) were utilized as negative and positive controls, respectively. 2.10. Statistical Analysis All experiments were performed at least in triplicate. The cell viability assessment was analysed by SPSS 19.0 statistical package (SPSS Inc., USA) and in case of normally



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distributed data, a tow-tailed Student's t-test was performed. P values of less than 0.001 (***), 0.001 to 0.01 (**) and 0.01 to 0.05 (*) were considered as significant. 3. Results & Discussion Our work considered cell viability when two cancer and one normal cell lines were treated with Eu/Gd-doped ZnO NPs and exposed to different radiations. Motivated by previous reports20-21, we hypothesized that absorption of X-radiations with the lanthanides may induce energy transfer to Zinc oxide crystals (ZnO NPs), leading to generation of ROS. As illustrated in Figure 1, Eu/Gd elements could absorb the X- radiations, and an efficient relaxation

of

Eu/Gd

electronic

excitation

was

occurred

through

transfer

of

the excess energy to neighboring atoms or molecules of ZnO NPs. When ZnO NPs were excited by radiations with energy levels higher than that of ZnO bad gap, an electron–hole pair (exciton) is formed. ROS are then expected to be generated from ZnO NPs by either reductive pathways (involving the transferring of electron to an acceptor, O2) or oxidative pathways (involving hole transferring to a donor, H2O) (Figure. 1). The generated ROS may cause DNA breakage or provoke genome instability in targeted tumor cells. Moreover, this energy transfer may improve the efficiency of cancer treatments22. 3.1. Size, Composition, Structure and Analytical Characterization of Fabricated NPs In order to fabricate an effective “theranostic nanoparticle” as a novel platform with therapeutic and imaging properties, spherical ZnO nanoparticles with narrow size distributions were synthesized by chemical precipitation methodology

23

. Subsequently, an

average size of 4 nm (Figure 2A) was chosen as an optimum sample for doping procedure with rare earth elements (REEs). The stability of Gd-doped sample was evaluated during 9 months. Results demonstrated that the nanoparticles have an appropriate stability with no size change after this period (Figure S1). It is worth noting that the appreciable stability in nanostructures is an important factor for practical applications of theranostic agents. Eu/Gd-


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doped ZnO-NPs were fabricated by replacing 5% Zinc acetate with lanthanide salts, with size of about 8-9 nm (Figures 2B and C). During the fabrication procedure, doping ratio was fixed at 5%, as an optimum rate for higher photocatalytic activity without disruption of crystalline structure of ZnO NPs

21

. It has been

previously demonstrated that higher amounts of Gd ions in ZnO NPs, decrease the photocatalytic activity and quantum yield

15

. The nanoparticles’ size, shape and structure

were also determined by UV-Vis absorption spectroscopy, XRD and HRTEM. HRTEM images demonstrated an average size of 4.0 and 8.5 nm for ZnO NPs and Eu/Gd-doped ZnO NPs, respectively, with spherical morphologies and mono-crystal structures (Figures 2 A1, B1 and C1). Several well-defined diffraction rings may be distinguished in the typical selected area electron diffraction (SAED) patterns of the Gd-doped ZnO NPs and ZnO NPs (Figure 2 A2, B2 and C2). This reveals high crystallinity of the fabricated NPs. SAED patterns and obtained FFT showed lattice spacing of ~0.26 nm for ZnO NPs and Eu/Gd doped ZnO NPs, which were well consistent with the lattice spacing in the ZnO wurtzite phase24. The results suggest that Eu/Gd doping procedure has not induced any lattice distortions in the fabricated ZnO NPs. A previously published study has demonstrated that the crystallinity of ZnO NPs became so weak that no clear lattice fringe could be distinguished when doping concentration increased to 0.3%15. The XRD patterns (Figure 2D) show seven distinct peaks and represent crystal structures of NPs consistent with SAED pattern. XRD patterns of purely synthesized NPs exhibit all the reflection peaks which can be indexed to ZnO wurtzite structure25. ZnO NPs have a noticeable diffraction angle peak at 2θ = 36.8˚ attributed to (101) plane (Figure 2D). These results are consistent with previously published report26 . Additionally, there is no significant reflection peak for Gd in the XRD patterns due to formation of amorphous species. Average crystallite size which is estimated on the basis of Scherer formula (i.e. 8.5 nm for Eu-doped



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ZnO and 9 nm for Gd-doped ZnO NPs) is in agreement with TEM results and size estimated based on UV-Vis absorption spectra. UV-Vis absorption spectra of ZnO (dopant-free NP) and Eu/Gd-doped ZnO NPs in the region of 200-800 nm exhibited an absorption peak at ~320 nm27 , which is attributed to the excitonic absorption peak of ZnO NPs (Figure 2E). Furthermore, energy-dispersive analysis of X-rays (EDAX) and X-ray photoelectron spectroscopy (XPS) were employed to reveal the Eu/Gd doping and its oxidation state. For ZnO NPs, FESEM and the EDAX pattern demonstrated a strong correlation of Zn element with the ZnO NPs. No Gd or other impurity elements were detected (Figure 3 A). Whereas, significant peaks of Gd element were observed in the EDAX patterns of Gd-doped ZnO NPs, as an important approval for Gd doping (Figure 3 B). Figure 3 C shows the XPS spectrum of Eu 3d region for Eu-doped ZnO NPs. Two intense peaks at 1164.5 and 1134.7 eV point to Eu 3d1/2 and Eu 3d3/2 core levels, respectively. Furthermore, XPS spectrum of Gd-doped ZnO NPs showed two obvious peaks positioned at 1192 eV and 1225 eV, corresponding to Gd 3d5/2 and Gd 3d3/2, respectively (Figure 3 D). Obtained results indicate that the oxidation states of Eu/Gd ions are mainly trivalent for the Gd/Eu-doped ZnO NPs. The atomic ratio of Eu to Zn and Gd to Zn were quantitatively evaluated by XPS analysis and the results demonstrated a ratio of 0.09 for Eu-doped ZnO NPs and 0.10 for Gd-doped ZnO NPs. 3.2. Determination of Reactive-Oxygen Species (ROS) Production Subsequent to oxidative stress, defined as a state of redox disequilibrium, generated ROS may overwhelm the antioxidant defense capacity of the cells28. In our study, synthesized nanoparticles were found to be capable of generating ROS when assessed by DPPH. NPs may generate ROS at their surface due to surface chemistry and/or characteristics. They are able to generate free radicals when interacting with cellular components (e.g., mitochondrial damage, Bax/Bcl2 modulation, caspases activation and apoptosis29). In this context, the electrons and holes could react with the oxygen and hydroxyl ions, respectively, which are


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present in the aqueous environment of the ZnO NPs. So, this procedure could produce highly reactive free radicals (i.e. ROS) including superoxide anion radicals (from electrons) and hydroxyl radicals (from holes)30. Subsequently, the generated radicals can oxidize and reduce the biological macromolecules (i.e., DNA, lipids and proteins), resulting in significant oxidative damage to the cells. For instance, increment in ROS may result in a redox adaptation and upregulate the antioxidant capacity in cancer cells31. In order to characterize different levels of ROS formation for employed NPs (e.g., ZnO and Eu/Gd -doped ZnO NPs) under UV, X-Ray and gamma radiations were employed. The exposed samples were evaluated using DPPH assay (Figure 4). ROS generation was quantified by neutralization of DPPH in methanol solutions. In our study, results of ZnO and Eu/Gd -doped ZnO NPs showed no considerable change in levels of produced ROS in comparison with control sample in the absence of the different radiation sources (Figure 4 A). Obtained results under UV radiation illustrated a concentration-dependent increase in the radical production procedure for ZnO NPs as well as Eu/Gd -doped ZnO NPs (Figure 4 B). This consequence was particularly more pronounced for ZnO NPs due to a high photocatalytic activity. Previous studies have revealed that ZnO NPs can be stimulated by UV light to produce ROS for photodynamic therapy32. The synthesized NPs were subsequently examined for X-ray-activated ROS generation. A concentration-dependent increase in ROS generation after X-radiation (2 Gy) may also be observed for Eu/Gd-doped ZnO NPs (Figure 4 D). Mechanism of action for the X-ray-excited fabricated ZnO NPs would be expected to be similar to that found for photo-excited ZnO NPs33. Moreover, the dopant elements in the structure of particles could successfully convert the X-ray energy into ROS, which may destroy the cancerous cells/tissues. Previous studies showed that ROS production increases under X-radiation in lanthanides-doped titania (TiO2@RE@Si)21 and gold nanoparticles34 . On the other hand, a small increase in free-



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radical generation after X-radiation for ZnO NPs may also be observed. However, in comparison with dark conditions, it may be concluded that the increase of ROS generation under X-ray radiation is negligible. This confirms lack of ability of ZnO NPs to produce ROS under X-ray radiation (Figure 4 D). Studying DPPH absorbance under gamma radiation indicates an enhancement in ROS generation but with no concentration dependence pattern (Figure 4 C). Reports show that ROS generation due to gamma radiation is mediated through interaction of ionizing radiation and environmental solutions, proteins, lipids and nucleic acids. Also, these radicals may be generated from primary ionization or through secondary amplification systems35. Therefore, in this study, it appears that the ROS generation has occurred accidentally due to high ionization potential of gamma radiation, which is almost independent of the NPs activation. To define the mechanisms involved in cytotoxic effects of ZnO NPs, we studied the effect of Gd-doped ZnO NPs on generation of H2O2 and O2 radicals as important intracellular ROS in the PC3 cells at dark condition and under UV, X and gamma radiations. The results of all NPs and radiations-treated cells show an increase in the fluorescence intensity of DCF and HE as scavenger materials for H2O2 and O2 radicals in comparison with dark conditions. Figure 5 illustrates the flow cytometry marker histogram in Gd-doped ZnO NPs treated PC3 cells at different exposure conditions. Obtained results of ROS generation at dark condition, show no significant radical production in Gd-doped ZnO NPs treated cells in comparison with untreated cells. Also, the results of ROS generation under UV radiation (Figure 5 B1, B2) show that the percentage of apoptotic cells was positively associated to the intracellular levels of H2O2 and O2 radicals. In this regards, Gd-doped ZnO NPs generated radicals under UV radiation in comparison with control cells. Subsequently, we found that the Gd-doped ZnO NPs induced generation of H2O2 and O2 under X-radiation (Figure 5 C1, C2), whereas Flow cytometry in dark condition exhibited lower levels of ROS is in the highest level close


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to the positive controls. In this context, percentage of intracellular ROS is close to positive controls and is in the highest level. We have noted that intracellular H2O2 levels are associated to the intracellular O2 radical levels as both are final products of photocatalytic activity of ZnO NPs under UV and X-radiations36. In addition, ROS generation under gamma radiation (Figure 5 D1, D2) show overall effects in control and NP-treated cells which is comparable with positive controls. Gamma radiation as ionizing radiation triggers ROS generation in cells which is independent to treatment with Gd-doped ZnO NPs. Furthermore, In order to evaluate the role of ZnO as host matrix in enhancement of radiations and ROS generation, ZnO NPs, Gd ions (0.5, 10 and 20 µgr/ml) and Gd-doped ZnO NPs were exposed to different radiation before MTT (on PC3 cells) and DPPH assays. The results demonstrated that Gd ions are potentially toxic for cells even in low concentrations whereas they cannot generate significant ROS under radiations (Supporting Information, Section 2 and Figure S2). 3.3. Uptake of Gd-Doped ZnO NPs by cells The inverted confocal microscopy was applied to assess cellular uptake of fabricated nanoparticles in L929 and HeLa cells. In this test, we analysed only one type of nanoparticles (Gd-doped ZnO) as both Eu/Gd-doped and ZnO NPs were expected to show similar physiochemical characteristics (Figure 6). Prepared rhodamine B labeled Gd-doped ZnO NPs were characterized by UV-Vis spectroscopy to confirm the presence of Rhodamin B conjugation on Gd-doped ZnO NPs. The results of normalized UV-Vis absorbance spectrum indicates that non-covalently bonded Rhodamine B is formed on the surface of NPs 37. The spectrum exhibits a strong excitonic absorption peak at about 320 nm for free Gd-doped ZnO and a single broad peak for rhodamine B at 520 nm. In case of Rhodamin B conjugated Gd-doped ZnO NP, both peaks of NP and dye are noticeable which suggests a successful bonding of dye to NPs (Figures 6 A and B)38. The red fluorescence of internalized rhodamine



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B in the treated cells, clearly illustrates that the Gd-doped ZnO NPs could be efficiently internalized into the cancer cells (Figures 6 D and C). Recent studies have revealed that ZnO NPs indicate a high degree of cancer cell selectivity39. The NPs have different effects on mammalian cell viability due to different cell uptake and cytotoxicity mechanisms (i.e., killing cancer cells with minimum effect on the normal cells40). Based on previous experimental reports, there is a noticeable difference in the uptake between cancer cells and normal cells which suggests a potential for ZnO NPs as a novel alternative for cancer therapy41. In addition, studies have shown that intracellular pH increases by cell cycle progression and proliferation42 which could affect electrostaticallydriven interactions with charged particles at the cell membrane. Thus, interactions with positively charged ZnO nanoparticles are expected to be driven by electrostatic interactions, thereby, promoting cellular uptake, phagocytosis and ultimate cytotoxicity30. 3.4. Induced cell death and Cytotoxicity effects of different radiations To evaluate the damage of radiations itself on cells, viability of untreated cells were assessed under different radiation conditions (Dark, UV, X and Gamma). Figure 7 shows cell viability of each radiation, without adding NPs. For this evaluation, viability of PC3 and L929 cells under darkness was considered as controls. The energy-dependence of radiation interaction with the cells shows that cell death under X and gamma radiations is extremely higher (i.e. ~60% to ~90%) than non-ionizing UV radiation which is ~24%. Moreover, viability of the cells slightly decreased with increasing X-radiation doses. Literature implies that UV photons have enough energy to destroy chemical bounds causing photochemical effect43. However, UV light can ionize only certain types of molecules under specific conditions and is generally not considered as ionizing radiation. Thus, usually cells are able to tolerate the UV induced DNA lesions44.


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Gamma-rays and X-rays as ionizing radiations can generate energetically charged particles such as electrons in the biological systems. Primary effects of such radiations are ionization, dissociation and excitation45. Excitation may cause a weak interaction, while ionization and dissociation result in stronger interactions. This ionizing radiation could be absorbed by critical targets in the cells (e.g. DNA). Furthermore, they may interact with other biological molecules, particularly water molecules, to produce free radicals which can diffuse far enough to reach and damage different important compounds of cells. They could damage cell membrane and cause apoptosis and autophagy46, metabolism/functional disruption, mitochondrial dysfunction and ROS generation35. Overall, our findings showed no significant different cell death between healthy and cancer cells under exposure of various radiations. Lack of radiation specificity between cancerous and healthy cells is the main problem in the conventional radiation therapies. 3.5. In-Vitro Cytotoxicity Assay and photocatalytic Properties of ZnO and Eu/Gddoped ZnO NPs under Dark, UV and Gamma Radiations Low toxicity of each designed nanomaterial is a key criterion for their biomedical applications. Cytotoxicity effects of ZnO and Eu/Gd-doped ZnO NPs were assessed under darkness, UV and gamma radiations. MTT assays were performed to investigate the cytotoxicity of designed NPs on PC3 prostate cancer cells, HeLa cervix cancer cells and L929 normal fibroblast cells. As shown in Figure 8A, cytotoxicity Results under dark conditions demonstrate a dose-dependent manner for all NPs. However, the decrease in viability is small and no significant differences at 5, 10 and 15 µgr/ml concentrations of samples may be obtained. NPs-treated cell lines remained more than 85% viable at concentrations up to 20 µgr/ml under dark condition (Figure 8 A). The findings demonstrate that ZnO and Eu/Gd-doped ZnO NPs do not cause considerable cell death in cancerous and healthy cell lines without excitation radiations, suggesting a low cytotoxic effect from NPs.



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Also, in the absence of radiations and after treatment with NPs, there was a slight decrease in proliferation of cells without any significant reduction, indicating that the particles are nontoxic in dark condition. A previous research illustrated low toxicity of ZnO NPs due to high natural abundance of Zn+ ions in the body which make it a suitable nanomaterial in biomedical applications47. Cytotoxicity results under UV radiation demonstrated a concentration-dependence cell viability when cells were exposed to ZnO and Eu/Gd-doped ZnO NPs (Figure 8 B). At a NPs concentration of 20 µg/ml, cell viability reduced down to ~20%. This demonstrates the excellent photocatalytic performance of the synthesized NPs which is consistent with previous observations20, 48. Also, the details show that at 5 and 10 µg/ml concentrations of NPs, viability % of L929 healthy cells is more than the cancerous cells. Other studies demonstrate that the healthy cells could tolerate mild oxidative stress by upregulating synthesis of antioxidant defense agents49. At higher concentrations of NPs, both healthy and cancer cells appear not to be able to tolerate the oxidative stress due to extensive free radicals reacting directly with DNA or indirectly through oxidative modification of biomolecules29. However, UV-sensitive nanomaterials may not be utilized for treatment of deep tumors due to inability of UV radiations to penetrate deep into the body29. The results of treating cells with different NPs and applying gamma radiation have been given in Figure 8 C. A slight concentration-dependence cell death is observed which is comparable with the dark condition that is not significant for concentrations up to 20 µg/ml. The results demonstrate that ZnO and Eu/Gd-doped ZnO NPs could not be activated under gamma radiation. Having mentioned that other works show the ability of high ionizing gamma radiation to destroy the proliferative capacity of the cells50 and induce high level of DNA damages51 which causes cell death.


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3.6. Evaluation of Eu/Gd-doped ZnO NPs-mediated cell death under X-Radiation To determine the capacity of Eu/Gd-doped ZnO NPs for amplification of X-radiation effects, viability of the NP-treated cells was assessed under different doses of X-ray. Cell lines (i.e. PC3, HeLa and L929) were incubated overnight in presence of ZnO and Eu/Gd-doped ZnO NPs and subsequently exposed to X-ray. Three different cell lines were used due to the fact that differential susceptibility among different types of cells (healthy, cancerous) could introduce variability in the results. These properties could be resulted from differences in metabolic rate/capacity, antioxidant enzymes, and DNA repair competences52. In a clinical setting, radiotherapy doses are typically administered fractionally over several weeks. This allows healthy cells to recover radiation-induced damages before the next fraction. Employed doses in this experiment are a conservative demonstration of a curative treatment. Samples were irradiated at 0.58 Gy min-1 to give exposures up to 2 Gy, and MTT cell viability assay was performed immediately. Results demonstrated that cell viability decreases in a dose dependent way down to ~25% due to response of the Eu/Gd-doped ZnO NPs to Xray radiation (Figure 9 A). Results of ZnO NPs-treated cells under X-ray showed the inability of ZnO NPs to be activated under X-radiation. Reduction in cell viability for these samples is not different from that of ZnO-NPs treated samples under dark condition. There is only a small difference between the two groups which is probably from weakly activation of ZnO NPs by low energy X-ray scattered radiations53. When introducing Eu/Gd-doped ZnO NPs to the systems, cell proliferation is reduced strongly in Figure 9 A. All cell lines showed significant reductions in cell proliferation depending on the concentration, whereas the effect of X-radiation itself on the cell death has been excluded based on the results from Figure 7. These results show that the particular combination of Eu/Gd-doped ZnO NPs in cells enhances the radiation effects to produce a biological destructive process and organize consecutive apoptotic cascades. Previous study



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showed that using surface-modified gold particles resulted in 46% inhibition of cell growth in DU-145 cells54. Towenly et al. demonstrated 75% decrease in cell proliferation after treatment of cells by Lanthanides-doped TiO2 NPs under 2 fraction of 3 Gy X-rays in 48h 21. Studies by Liu et al. examined the use of PEGylated gold NPs for improved radiotherapy in CT26 cells and confirmed an increase in cell death of about 15% at 3 Gy, relative to control, after 14 days incubation55. A recent study shows 80% cell death in-vitro and in-vivo for transferrin-coated TiO2 nanoparticles treated cells under 633 nm laser light to break the depth dependency of cancer treatment

56

. The utilized cell lines in these experiments arise from

different types of cancer and healthy cells and could therefore be expected to respond differently to the treatment. However, all studied cells could not tolerate the high amounts of ROS generation at this stage. It is worth noting that this method may be applied for the treatment of all types of cancer. In clinical settings, total dose for radiation therapy is fractionated to give sufficient time to the healthy cells to recover. The main purposes of this experiment are evaluating the enhancement of radiation effects on cancer cells by adding NPs, together with elimination of the excess exposed radiations to normal cells around the tumor. In order to illustrate this ability of designed NPs, un-treated PC3 cells were exposed to fractionized radiation doses of 4Gy (2 fractions of 2 Gy in 24h: cells were exposed to 2 Gy X-radiation, incubated for 24 hours, then, the radiation was repeated once again) and 6 Gy (3 fractions of 2 Gy in 48h) as controls. Obtained results were compared with cytotoxicity of Eu/Gd-doped ZnO NPs under 2 Gy X-radiation. Figure 9 B illustrates the efficiency of Eu/Gd-doped ZnO NPs-treated cells to absorb and enhance the X-radiation effects for a consequent cell death. It is apparent that cell death at Eu/Gd -doped ZnO NPs concentration of 20 µg/ml under 2 Gy X-ray is similar to 6 Gy exposure to untreated samples (i.e. samples without NPs). Furthermore, the Xradiation induced-cell death under 4 Gy is similar to 5 µg/ml Eu/Gd-doped ZnO NPs-treated


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cells under 2 Gy X-radiation. It is noteworthy that accumulating Eu/Gd-doped ZnO NPs to cancer cells with enhanced permeation and retention effect (EPR) could also augment the radiation effects on cancer cells along with decrease of received dose to the normal cells around the tumor. Figure 10 shows the relationship between cell death and x-radiation doses in ZnO and Eu/Gddoped ZnO NPs-treated PC3 cells in comparison with untreated control samples. As shown in Figure 10 A, for ZnO-treated cells, maximum cell death was found to be 40% for 20 µg/ml ZnO NPs under 2 Gy X-radiation. Figures 10 B and C demonstrate that Eu/Gd-doped ZnO NPs-mediated cell death under 2 Gy x-radiation is about 85% at 20 µg/ml concentration which is equal to the cell death under 6 Gy x-radiation on un-treated cells. Results illustrated that addition of designed NPs to the cancer cells leads to around 3 times enhancement in the radiation effects. Consequently, untreated healthy cells around the tumors could survive from an excess exposure of 4 Gy. This represents higher efficiency due to the addition of NPs in comparison with the current treatment with only radiation. 3.7. CT / MRI contrast Properties of Eu/Gd-doped ZnO NPs To examine diagnostic efficacy and function of Eu/Gd-doped ZnO NPs, the synthesized NPs were evaluated as in-solution MR/CT imaging contrast agents. To estimate the capability of Eu/Gd-doped ZnO NPs probe for MRI application, longitudinal proton relaxation times (T1) were determined as a function of NPs concentration (0.05 and 1.0 mM). T1-weighted MR image was evaluated at a 3T human clinical scanner. The images (Figure 11) demonstrate a positive enhancement of the MRI signal for Gd-doped ZnO colloid compared to water and OMNISCAN®. The T1-weighted MR images became brighter, corresponding to the increase in concentrations. Gd ions in Gd-doped ZnO QDs can accelerate longitudinal (T1) relaxation of water protons and exert bright contrast in regions where the nanoprobes localize15. These results show that the Gd-doped ZnO QDs could serve as effective T1-MRI contrast agents57.



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Studies demonstrated that conjugation58, doping15, composition59 of NPs with Gd ions as well as encapsulation60 and coating61 of Gd oxide NPs, make them capable to enhance T1-waited MRI signals at low concentrations in comparison with commercial and molecular Gd-based contrast agents62. Whereas, ZnO and Eu-doped ZnO NPs did not show any important enhancement in MRI signals at the concentrations studied (0.05 and 0.1 mM). Subsequently, we proceeded to examine the capability of synthesized NPs as CT contrast agents. Among the other elements, lanthanide (i.e. Gd and Eu) ions have high atomic number, high X-ray absorption coefficient, and large K-edge energy. Therefore, lanthanide-based NPs have been introduced as potential candidates for X-ray CT contrast agents. Since Eu and Gd exhibit high X-ray attenuation, CT contrast increment ability of Eu/Gd-doped ZnO NPs were examined by applying a clinical 16 slice CT imaging system. To assess the CT contrast efficiency, X-ray attenuation ability of synthesized NPs was compared to Visipaque®, the most widely used CT contrast agent in clinic. The results in Figure 11 demonstrate a concentration-dependent enhancement in brightness and contrast of CT images for Eu/Gddoped ZnO NPs in comparison with water. Also, synthesized NPs showed an extensive improvement in contrast enhancement at 0.1 mM concentrations compared with traditionally used contrast agent (i.e. Visipaque at 225 mM concentration). The findings recommend that Eu/Gd-doped ZnO NPs have great potential for X-ray attenuation compared with conventional iodine-based molecular CT contrast agents under very low concentrations. This will decrease the conventional side effects of contrast agents63. Previous studies have illustrated that high atomic number elements, for example Au NPs64, bismuth sulphide NPs65, Tungsten oxide nanorods66, Gd-coated/conjugated Au NPs67, Au‐Fe alloy nanoparticles68, Fe3O4/Au Nanocomposite69, hybrid BaYbF5 nanoparticles70 and Yb3+ and Gd-doped ZnO NPs16 could enhance contrast of CT images . Overall, doping of ZnO NPs with Eu/Gd, high


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atomic number elements, make them capable of being traced under MRI as well as CT imaging at a potentially safe concentrations. 4. Conclusions: Development of multifunctional platforms for simultaneous diagnostic and therapeutic applications has recently attracted a great deal of interest. This study demonstrates the advantages of using lanthanide-doped Zinc Oxide-based theranostic nanoparticles in treatment and diagnosis of cervix and prostate cancers. Synthesized Eu/Gd-doped ZnO NPs showed high radio-sensitizing properties and efficiently decreased cell viability of cancer cells under X-radiation in-vitro. This study provides a new therapeutic option as radiation enhancer in treatment of deep tumors under clinical X-radiation therapy. NPs could extremely decrease the required treatment doses due to an increase in radiation effects and ROS generation and, subsequently, decrease total received dose of the normal cells around the tumor. Furthermore Eu/Gd-doped ZnO NPs enabled multimodal detection by MR/CT imaging. Based on these preclinical results, Eu/Gd-doped ZnO NPs are anticipated to have potential applications as a simultaneous imaging and therapy modality for deep tumors as image-guided therapy.

Supporting Information: UV-Vis absorption spectra of Gd-doped ZnO NPs at 1st and 9th month of synthesis, MTT and DPPH assays of Gd ion, ZnO NPs and Gd-doped ZnO NPs under dark condition, UV and Xradiation, SEM and related EDAX spectrum of ZnO and Gd-doped ZnO NPs.

Acknowledgement: This research has been supported by Tehran University of Medical Sciences and health Services under grant No: 92-03-87-23370. The authors wish to thank Narges Pashmforoosh



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and Seyed Mohammad Amini for their assistance with the CT/MR imaging and Akram Vatannejad for her assistance with the Flow cytometry experiments.


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Figures:

Figure 1. Schematic illustration of proposed “activation mechanism” for Eu/Gd-doped ZnO nanoparticles under X-ray radiation. Eu/Gd elements could generate "ejected K-shell electrons" and "photoelectron wave packets" due to the activation by X-ray. Subsequently, this phenomenon could activate ZnO nanoparticles to generate ROS from environmental biological molecules within the cells.



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Figure 2. Size distribution, HRTEM images and selected area electron diffraction (SAED) patterns of ZnO NPs (A, A1 and A2), Gd-doped ZnO NPs (B, B1 and B2) and Eu-doped ZnO NPs (C, C1 and C2) respectively. XRD patterns of Eu/Gd-doped ZnO and ZnO NPs (D). UVVis absorbance of ZnO and Eu/Gd-doped ZnO NPs (E).


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Figure 3. EDX spectrum taken from the marked area in SEM images of ZnO (A) and Gddoped ZnO NPs (B). XPS spectra Eu 3d core-level of Eu-doped ZnO NPs (C) and Gd 3d core-level of Gd-doped ZnO NPs (D).



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Figure 4. DPPH radical scavenging activity and ROS generation of the ZnO and Eu/Gddoped ZnO NPs in methanol at different radiation conditions: dark (A), UV (B), Gamma (C) and X (D) radiations.


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Figure 5. Representative flow cytometry marker histogram for Gd-doped ZnO NPs treated PC3 cells, positive DCF fluorescence (represents intracellular H2O2) and positive ·

fluorescence for HE fluorescence (represents intracellular O2 ) at dark condition (A1 and A2), under 5 minute UV radiation (B1 and B2), 2Gy X-radiation (C1 and C2) and 2Gy gamma radiation (D1 and D2) respectively.



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Figure 6. Schematic illustration of Rhodamin B conjugated Gd-doped ZnO NPs (A), UV-Vis spectra of Free Gd-doped ZnO, Rhodamine and Rhodamine-conjugated Gd-doped ZnO NPs with photograph insect of the NPs suspension (B), Representative confocal microscopy images of (C) HeLa and (D) PC3 cells incubated with Rhodamin-labelled Gd-doped ZnO NPs for 2 hour. Scale bar: 50 µm.


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Figure 7. HeLa and PC3 cell viabilities after exposure to different radiations (i.e. UV, X and Gamma radiations)



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Figure 8. Concentration-dependent cell viability of PC3, HeLa and L929 cells after incubation with ZnO, Eu/Gd-doped ZnO NPs: Dark condition (A), under UV (B) and gamma (C) radiations.


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Figure 9. Survival of PC3, HeLa and L929 cell at different concentrations under 2Gy Xradiation (A) and comparison of the viability of the Eu/Gd-doped ZnO NPs exposed to 2 Gy x-radiations with un-treated Control cells under 4 (Control3) and 6 (Control4) Gy Xradiations (B).



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Figure 10. Effect of different doses of x-radiation on NPs-treated and un-treated cells and total cell death (%). (A) ZnO-treated PC3 cells under 1.5 and 2 Gy X-radiations, (B,C) Eu/Gd- doped ZnO NP- treated PC3 cells under 1.5 and 2 Gy X-radiation, in comparison with cell death of control cells under 1.5, 2, 4 and 6 Gy X-radiations.


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Figure 11. (Left) T1 weighted MR images of ZnO and Eu/Gd-doped ZnO NPs at tow concentrations in comparison with water as negative control and OMNISCAN as positive control. Signal strength is indicated by brightness of the images. (Right) 16 slice clinical-CT images of ZnO and Eu/Gd-doped ZnO NPs at different concentrations in comparison with water and Visipaque as negative and positive controls. Signal strength is indicated by the brightness of the images.



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