Toxicity Evaluation in

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FeO@SiO Nanoparticles Concurrently Coated with Chitosan and GdOF:Ce ,Tb Luminophore for Bioimaging: Toxicity Evaluation in Zebrafish Model Latif Ullah Khan, Gabriela Helena Da Silva, Aline M. Z. de Medeiros, Zahid Ullah Khan, Magnus Gidlund, Hermi Felinto Brito, Oscar Moscoso-Londoño, Diego Muraca, Marcelo Knobel, Carlos A Pérez, and Diego Stéfani Teodoro Martinez ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.9b00339 • Publication Date (Web): 09 May 2019 Downloaded from http://pubs.acs.org on May 10, 2019

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ACS Applied Nano Materials

Fe3O4@SiO2 Nanoparticles Concurrently Coated with Chitosan

and

GdOF:Ce3+,Tb3+

Luminophore

for

Bioimaging: Toxicity Evaluation in Zebrafish Model Latif U. Khana*, Gabriela H. da Silvaa,b, Aline M. Z. de Medeirosa,b, Zahid U. Khanc, Magnus Gidlundc, Hermi F. Britod, Oscar Moscoso-Londoñoe,f, Diego Muracae, Marcelo Knobele, Carlos A. Pérezg*, and Diego Stéfani T. Martineza,b* aBrazilian

Nanotechnology National Laboratory (LNNano), Brazilian Center for Research in

Energy and Materials (CNPEM), Zip Code 13084-970, Campinas-SP, Brazil. bCentre

for Nuclear Energy in Agriculture (CENA), University of São Paulo (USP), Zip Code

13416-000, Piracicaba-SP, Brazil. cDepartment

of Immunology, Institute of Biomedical Sciences-IV, University of Sao Paulo (USP),

Zip Code 05508-000, São Paulo-SP, Brazil. dDepartment

of Fundamental Chemistry, Institute of Chemistry, University of Sao Paulo (USP),

Zip Code 05508-000, São Paulo-SP, Brazil. eInstitute

of Physics “Gleb Wataghin”, University of Campinas (UNICAMP), Zip Code 13083-

859, Campinas-SP, Brazil. fFaculty

of Engineering, Autonomous University of Manizales, Antigua Estación del Ferrocarril,

Manizales, Colombia. gBrazilian

Synchrotron Light Laboratory (LNLS), Brazilian Center for Research in Energy and

Materials (CNPEM), Zip Code 13084-970, Campinas-SP, Brazil. KEYWORDS: Multifunctional nanoparticles, magnetism, green-emission, nanotoxicity, nanosafety and -XRF imaging.

ACS Paragon Plus Environment

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ABSTRACT: In this work, design and physiochemical characterization of biocompatible nanoplatform with integrated photoluminescence and magnetic properties were reported. The proactive in vivo toxicity was assessed, exploring nanoparticles biodistribution by synchrotron Xray fluorescence (SXRF) imaging in zebrafish embryos as a biological model. Their synthesis is accessible through combining magnetic iron oxide nanoparticles with Ce3+ and Tb3+ doped GdOF luminophore,

concurrently

capping

in

situ

with

chitosan

biopolymer.

The

Fe3O4@SiO2/GdOF:xCe3+,yTb3+ nanoparticles manifested near superparamagnetic behavior at 300 K, displaying green emission lines, arising from the characteristic 5D47FJ transitions (J = 60) of Tb3+ ion. The limited permeability of chorion membrane is a critical factor in toxicity screening, a potential approach to remove the chorion and expose the chorion-off zebrafish embryos to nanoscale materials. Accordingly, multifunctional nanoparticles exhibited no acute toxicity to the zebrafish embryos with-chorions and chorion-off ones up to 100 mg L-1 exposure concentration, suggesting remarkable in vivo biocompatibility. Exploiting the nano-bio interaction via deep-tissue SXRF imaging, it was visualized the distribution of Gd and Fe elements with roughly constant relative ratio in the whole body of early stage embryos. However, the elements mapping data revealed predominantly localization of Gd and Fe in gastrointestinal tract, manifesting bioaccumulation of magneto-luminescent nanoparticles as integrated nanoplatform in the respective region. This result demonstrated that the particles’ uptake by embryos were mostly through oral exposer then the dermal pathway, offering a new route to nanoparticles administration orally for future nanomedicine and biological applications.

1. INTRODUCTION Recent advances in nanobiotechnology have attracted strong research interest in exploring the interactions of nanoengineered materials with biosystems,1 enabling a perspective to evaluate in


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vivo the toxicity of nanomaterials.2,3 There is substantial evidence that nanomaterials exhibit distinct nano-bio interactions when compared to the molecular compounds or bulk materials.1 However, few information is available in literature, exploring the bio-nanointerface,4 and there is no consensus on the toxicity5 of magneto-luminescent nanosystems. Therefore, considerable research interest must be dedicated to advance the fundamental understanding of bio-nano interactions1,6 and in turn, tailor the design, physiochemical characterizations and biosafety of nanomaterials for potential biological applications. Multifunctional nanosized materials co-assembling optical and magnetic properties7 in single entity nanostructures have been shown great promise in variety of biotechnological and environmental applications, such as multimodal imaging and cancer therapy,8,9 diagnostic and nanothermometry,10 cell tracking and labeling,11 quantitative DNA analysis,12 biochemical separation,13 pollutants removal and sensing,14 among others. As a matter of fact, to fabricate effective magneto-luminescent nanomaterials, various synthetic approaches, utilizing iron oxide as magnetic entity and RE3+ complexes/quantum dots as luminescent center have been reported.15 The magnetite imparts magnetic characteristics to bifunctional nanomaterials, but on the other hand it acts as a strong luminescence quencher. Therefore, intermediate layer or spacer, such as silica, organic macrocycles, polymers etc.,7,15 is usually added in between the Fe3O4 and luminophore, in order to overcome this drawback. The silica coating confers encapsulated iron oxide nanoparticles hydrophilicity and chemically modifiable surface, which can be further functionalized with RE3+ compounds16 to produce bifunctional nanomaterials. Trivalent lanthanide ions based nanomaterials show luminescence from UV-visible to nearinfrared regions due to the intraconfigurational transitions in their 4f energy level structures.7 These ions can act as efficient activators, if they are doped in rare earth oxyfluorides (e.g., GdOF)


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host matrices, featured with low-phonon cut-off energy.17 The existence of partial rare earth-oxides in their structure may improve the emission intensity via O2−Ln3+ charge transfer (CT) process.18 The Ce3+ and Tb3+ ions codoped GdOF nanomaterials contain paramagnetic Gd3+ (4f7), bearing large energy gap (32000 cm−1) between the fundamental 8S7/2 and first excited 6P7/2 energy level that facilitates and bridges the energy transfer cascade from Ce3+ to Tb3+ ions.19 In addition, gadolinium based compounds (T1 imaging agents) are also used as remarkable contrast agents for magnetic resonance imaging.20 The co-assembling of the Fe3O4 and GdOF in single nanostructures might be efficient candidate for T1and T2 bi-modal magnetic resonance imaging. Extensive research studies concerning the biological and technological applications of magnetic and luminescent nanomaterials are published every year. However, an insufficient amount of toxicological data2,3 on these nanoparticles makes it difficult to properly assess their impact on human and environmental health. Moreover, the emerging applications of magnetic nanoparticles with controlled morphology and tuned surface chemistry in diagnostic and therapy have made a serious concern regarding their nanosafety.21–23 In previous study,22 uncoated-iron oxide nanoparticles have been reported to present higher level of toxicity to zebrafish embryos when compared to flavin mononucleotide (FMN) and guanosine monophosphate (GMP) coated ones. However, very insufficient knowledge is available in experimental literature on internalization and distribution of modified surface chemistry Fe3O4 nanoparticles in zebrafish, 21–23 as well as on their underlying nanotoxicity effect. Therefore, systematic, relevant and efficient testing approaches must be adopted to reach at conclusions with respect to the toxicity of iron oxide-based functional nanomaterials. Zebrafish (Danio rerio) has been applied as a promising biological model for in vivo nanotoxicity evaluation,24,25 nanomedicine and nanosafety studies.26,27 The important features of


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Danio rerio is easily sustainable in laboratory, its eggs are 1.0–1.2 mm in diameter, developed rapidly2,3,28 and transparent that facilitates the observation of toxic effects of testing materials on their internal organs, using optical microscopy. Zebrafish embryo assay offers an interesting test for in vivo toxicity screening,24,25,29–32 in a cost-effective manner, bridging the recent gap between ex-vivo cells models and in vivo mice studies,26 which is less expensive than toxicity screening in mice, but more complex than cells culture.22 Danio rerio embryo is surrounded by an acellular chorion membrane that may act as a barrier for nanomaterials and chemicals.33 The surface charge of the chorion is usually negative, attracting and blocking positively charged heavy metal ions.30 The chorion contains pore channels with a diameter of ∼200 nm,30 thus, the size of nanoparticles and molecules should be adequately smaller to pass through the chorion membrane, for instance, ≤ 4000 Da has been reported for the polyethylene glycols.31 There are few evidences in experimental literature that embryonic development affects the permeability of chorion membrane, which turns less permeable after toughening,32 and works as a barrier in the zebrafish embryo, even for small molecular size compounds. The most potential approach is to remove the chorion membrane and use the dechorionated embryos34 for in vivo nanotoxicity screening of nanoscale materials. The biodistribution of metal ions and nanoparticles in zebrafish embryos are usually visualized with various bioimaging techniques, in order to probe their hazardous effects. Accordingly, X-ray fluorescence spectroscopy, optical and transmission electron microscopy and laser ablation inductively coupled plasma mass spectrometry (ICP-MS) have been considerably employed for this sort of studies in the literature.30 X-ray fluorescence (XRF) imaging is considered a remarkable technique for chemical mapping of elements and nanoparticles, qualitatively and quantitatively in biological systems.35 Synchrotron light source generates a micro-focused monochromatic X-ray


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beam36 that allows XRF micro-spectroscopic imaging with high spatial resolution and chemically map the various elements with high sensitivity at precise histological level.37 This can facilitate our current understanding on the toxicity pathway of nanoparticles in biological system, studying in vivo the bio-nano interaction. Herein, we report syntheses, a rather complete physiochemical characterization and in vivo toxicity evaluation of multifunctional Fe3O4@SiO2/GdOF:xCe3+,yTb3+ nanoplatform, using zebrafish embryos with chorion and chorion-off (dechorionated) ones as model organism for the very first time. Synchrotron X-ray Fluorescence (SXRF) mapping was acquired to follow the nanoparticles uptake and bioaccumulation in embryos, by exploring the correlation between gadolinium and iron elements present in the same nanostructure. 2. EXPERIMENTAL SECTION 2.1. Materials preparation The following reagents were purchased from commercial sources and used directly in nanoparticles syntheses. The FeCl2.4H2O, NaOH and tetraethyl orthosilicate (TEOS) were bought from Sigma-Aldrich. Hexane, octanol, NH4OH and oleic acid were supplied by Synth. Ce(NO3)3 was prepared from Ce2O3 in nitric acid (VETEC), whereas GdCl3 and TbCl3 were prepared through reported literature procedure,38 from Gd2O3 and Tb2O3 (99.99 % Rhodia) in aqueous hydrochloric acid (VETEC) solution. Fe3O4 nanoparticles: The magnetite nanoparticles were synthesized with hydrothermal method, as we reported in previous work.19 In a typical procedure, 0.4 g (10 mmol) of NaOH was welldissolved in 5.0 mL of ethanol under vigorous stirring, then added 4.0 g (12.72 mmol) of oleic acid (90 wt. %), stirred at room temperature until white viscous solution was formed. Finally, 7 mL solution of 0.2 g FeCl2⋅4H2O in water (0.143 mol L-1) was mixed with the above viscous


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suspension under continuous stirring, and a dark color suspension was appeared instantly. The resulting homogeneous mixture was transferred to a Teflon-vessel containing stainless-steel autoclave (25 mL), tightly closed and heated at 180 °C temperature for 16 h. The reaction vessel was then cooled to ambient temperature, and a black color mixture was obtained at the bottom of the vessel. The resulting magnetite nanoparticles were washed with n-butanol and water, collected with applying external magnet and dried in vacuum desiccator. Fe3O4@SiO2: These nanoparticles were prepared by microemulsion method, according to the literature reported procedure.39 Typically, 80 mg of Fe3O4 nanocrystals were well-dispersed in 120 mL of n-Hexane by sonication at room temperature, then added 30 mL Octanol as surfactant, stirring the resultant suspension for 10 min. Thereafter, 2.6 mL ammonium hydroxide (28 wt/wt%) and 1.0 mL H2O were added to form reverse microemulsion solution and allowed to stir for 30 min. Then, 0.30 mL tetraethyl orthosilicate was dropwise added to the homogenous suspension and stirred at room temperature for 24 h. The final Fe3O4@SiO2 nanoparticles were washed with ethanol and water, isolated by applying external magnet, and dried in vacuum desiccator. Fe3O4@SiO2/GdOF:xCe3+,yTb3+: These nanocomposites were prepared through chitosan assisted co-precipitation method, as we reported in previous work.19 The concentration of the Ce3+ dopant ion was maintained constant at 5 mole percent, while the Tb3+ was varied from 5 to 10 mole percent of the total Gd3+ amount. Typically, for the synthesis of Fe3O4@SiO2/GdOF:xCe3+,yTb3+ (x and y = 5 mol%) nanomaterial, 0.462 g (1.24 mmol) GdCl3∙6H2O, 0.030 g (0.069 mmol) Ce(NO3)3∙6H2O and 0.026 g (0.069 mmol) TbCl3∙6H2O were mixed in 50 mL solution of 0.25 g chitosan in Acetic acid/Milli-Q water (5 v/v %) and stirred at room temperature to give homogenous solution. Thereafter, pH was neutralized to ~7 by dropwise addition of NH3∙H2O (28 wt/wt %) and freshly prepared solution of Fe3O4@SiO2 (0.1 g) nanoparticles in 20 mL Milli-Q water was injected under


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continuous N2 flow. After a while when the two solutions were completely mixed, an aqueous solution of 0.153 g (4.13 mmol) NH4F in 10 mL Milli-Q water was injected slowly to the reaction mixture and heated at reflux under continuous N2 flow for 3 hours. The resulting brown suspension was cooled to ambient temperature and the synthesized magnetic and optical nanomaterials were isolated by centrifugation, washed with Milli-Q water and ethanol, and dried to obtain a brown colored

Fe3O4@SiO2/GdOF:xCe3+:yTb3+

(x

and

y

=

5

mol%)

nanomaterial.

The

Fe3O4@SiO2/GdOF:xCe3+,yTb3+ (x = 5; y = 10 mol%) nanomaterial was also prepared by same procedure, using the corresponding stoichiometric concentrations of the Ce3+ and Tb3+ dopant ions. 2.2. Materials Characterization The magnetic and luminescent nanomaterials were structurally analyzed by measuring XRD patterns with a Rigaku Miniflex II diffractometer in the 2θ range of 10 to 90 degrees, using CuKα1 radiation (: 1.5406 Å). FTIR absorption spectra were measured with a Bomem MB100 FTIR spectrometer in the spectral range of 400-4000 cm−1, using KBr pellet-based sample preparation technique. The surface chemical compositions of the bifunctional nanomaterials were characterized using K-AlphaTM X-ray Photoelectron Spectrometer system, Thermo Fisher Scientific Inc., equipped with a monochromatic Al Kα X-rays (: 1486.6 eV) of small spot size (300 µm). The survey spectra were measured by scanning three selected regions of the sample in the energy range of 0-1350 eV at 200 eV pass energy and spatial resolution of 400 μm. Highresolution spectra of Gd 4d, F 1s, C 1s, N 1s and O 1s were measured in their respective energy ranges, scanning the sample at 50 eV pass energy, with an incremental step size of 0.1 eV. The deconvolution of peaks and subtraction of background were performed using Avantage 5.89 software (Thermos scientific).


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TEM images of the magnetic and luminescent nanomaterials were measured using transmission electron microscope TEM-FEG, Jeol (JEM 2100F), equipped with field-emission electron gun (acceleration voltage 200 kV), using Spot Size 1 and Alpha Selector 3. The samples were prepared by gently placing a small drop of aquous dispersion of nanoparticles on a carbon-coated copper grid (ultrathin carbon/holey carbon, 400 mesh, Ted Pella, Inc.), using a single-tilt sample holder. TEM images were captured with Gatan 831.J45M0 camera, using Gatan Digital Micrograph and EMMENU programs. M-H hysteresis and Zero Field Cooling/Field Cooling (ZFC/FC) magnetization curves were measured at 300 and 5 K temperatures with a Quantum Design, MPMS XL, SQUID magnetometer. Magnetization-magnetic field (M-H) curves were measured at 5 and 300 K under a maximum applied magnetic field of H = 20 kOe. Temperature dependent magnetization measurements (ZFC/FC curves) were recorded by cooling down the samples from 300 to 5 K, initially in the absence of applied magnetic field. Then 50 Oe static magnetic field was applied and measured magnetization by increasing the temperature till 300 K. Thereafter, the samples were cooled down to 5 K under the same 50 Oe applied magnetic field and the magnetization was measured by heating up the samples from 5 to 300 K temperature. The excitation and emission spectra and luminescence decay curves of the magnetoluminescent Fe3O4@SiO2/GdOF:xCe3+,yTb3+ (x and y = 5 and 10 mol%) nanomaterials were measured in solid state at room temperature, using HORIBA Jobin Yvon Fluorolog-3 spectrofluorometer. This instrument is equipped with 450 W Xenon lamp and pulsed Xenon flash lamp as excitation sources, double-grating monochromators, CCD detector and phosphorimeter accessory. The luminescence data is processed in FluorEssence™ software that merges the acquisition of data from spectrofluorometer directly to the OriginTM software.


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2.3. Toxicity Assays with Zebrafish Embryos Toxicity of magneto-luminescent nanoparticles were assessed in zebrafish embryos, applying similar protocol, as reported in the literature.24,25 Danio rerio embryos were provided by the Brazilian Biosciences National Laboratory (LNBio), CNPEM. In a typical toxicity screening protocol, adult zebrafishes (male and female: 2 and 1 ratio) were placed and allowed to stay overnight separately in the same breeding tank a day before performing experiment. Then, they were brought together and mat at early morning of the assay in the dark to produce eggs. Subsequently, embryos were collected after 1 h post-fertilization (hpf), then they were washed with reconstituted water, examined under optical microscope and viable eggs were chosen for nanomaterials toxicity experiment. A standardized OECD fish embryo toxicity (FET) assay was performed. Briefly, 24 organisms per group (3 replicates) were exposed to 100, 10 and 1 mg L-1 of Fe3O4@SiO2/GdOF:xCe3+,yTb3+ (x = 5; y = 10 mol%) nanoparticles for 96 hours, examining the embryos and larvae under an optical microscope (Stereo Discovery V20, Zeiss) on daily basis. The parameters assessed were mortality, malformation, edema, hatching rate, total length and yolk sac size. After the exposure period, total length and yolk sac size were measured with Axion Vision software. Additionally, this result was further well interpreted via performing the modified version of standardized FET assay using chorion-off (dechorionated) and with-chorion embryos.40 For this purpose, 24 hpf embryos were collected and dechorionated mechanically according to the reported procedure,40 using Dumont™ no. 5 forceps. The egg chorion is usually act as a barrier that avoids the nanoparticles direct contact with the undeveloped embryo. Therefore, it is important to remove this barrier to increase the contact, which can only be achieved without injure the embryo at 24 hours post fertilization.40 In this assay, the chorion-off and with-chorion embryos at 24 hpf were exposed to 100, 10, 1 mg L-1 of Fe3O4@SiO2/GdOF:xCe3+,yTb3+ nanoparticles, assessing the same


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parameters of the traditional FET, except the hatching rate. Zebrafish experiments were permitted by the Institutional Committee for Ethics in the Uses of Animals (Protocol number 40/2017) at LNBio/CNPEM, according to the ethical principles for animal research recognized by the National Council for Control of Animal Experimentation (CONCEA). 2.4. Synchrotron X-ray Fluorescence (SXRF) Mapping and Imaging The SXRF elemental mapping and images of the zebrafish embryos exposed to nanoparticles at 0, 24 and 72 hpf were recorded at the D09-XRF beamline of the Brazilian synchrotron light source, using a set-up that has been described previously.36 The embryos exposed to nanoparticles at 0, 24 and 72 hpf were selected for SXRF imaging, because these life stages are crucial in the toxicity assay of zebrafish development. Accordingly, at 0 hpf exposure, one can observe completely if the fish development is affected by hazardous effect of nanoparticles. The chorion barrier can be mechanically removed without injury at 24 hpf,40 to increase the contact of nanoparticles with embryo. After, 72 hpf, the larval mouth is fully developed and ready to start ingesting food.41 Therefore, we exposed the zebrafish embryos to nanoparticles dispersion at 72 hpf, to assure their uptake via oral exposer and subsequent information on the fate of magnetoluminescent nanocomposites, whether they are disintegrated in the gastrointestinal tract or remain intact. For, SXRF analyses, the sample was prepared by placing the zebrafish embryo in between two Ultralene® thin films and mounted in an acrylic holder at 45o from the incoming beam direction for x-ray fluorescence detection. XRF elemental mapping and images were measured by raster scanning the zebrafish embryos with the white X-ray beam. The step size was 0.03 mm and the dwell time per pixel was 0.6 sec. All XRF spectra were then processed using the PyMca42 software. 3. RESULTS AND DISCUSSION


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The multifunctional nanomaterials were synthesized through a multi-step synthetic protocol, using magnetic Fe3O4 core nanoparticles, which were functionalized with thin layer of silica via reverse microemulsion to produce Fe3O4@SiO2. These nanoparticles were further coated with GdOF:RE3+ phosphor through new facile chitosan assisted high temperature co-precipitation method. The overall stepwise preparation procedure of the magneto-luminescent nanomaterials is shown in the Scheme 1. Scheme 1. Schematic illustration of the synthetic procedure of magneto-luminescent Fe3O4@SiO2/GdOF:xCe3+,yTb3+ (x = 5; y = 5 and 10 mol%) nanoparticles. OH O

O

O

HO HO

NH3

O

HO

OH

NH3

Fe3O4

TEOS/NH3·H2O

RE3+/Chitosan

Hexane/Octanol

NH4F/H2O

O

O H 3N

HO

O OH

NH3 O HO

OH

H 3N

Fe3O4@SiO2

O

OH NH3

OH

O

O

H 3N O HO

OH O

O

HO

Fe3O4@SiO2/GdOF:xCe3+,yTb3+

FTIR absorption spectroscopy (Supporting Information Figure S1) was employed to characterize the successful coating of Fe3O4 core nanoparticles with silica and further functionalization with chitosan capped GdOF:xCe3+,yTb3+ luminophore. TG/DTG curves suggested the presence of about 6% organic content in multifunctional nanoparticles, manifesting from the larger exothermic event occurred in the temperature interval of 390-720 K (Supporting Information Figure S2), attributing to the liberation of CO2 due to the thermal decomposition of chitosan biopolymer. The surface elements compositions of these nanoparticles were studied by X-ray photoelectron spectrometry (XPS) technique (Supporting Information Figure S3). The survey spectrum shows the presence of most abundant F and Gd elements as well as trace of C,


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Fe, N, O and rare earth dopants. It is worth highlighting the detection of negligibly small signal in the energy range of Fe 2p (Figure S3a), when compared to Gd and F peaks suggest the presence of abundant surface GdOF on Fe3O4 nanoparticles. The peaks in the binding energies ranges of C 1s and N 1s signals are aroused from the surface hydrocarbons. The deconvoluted C 1s core spectrum presents peaks at binding energies: 285, 286.4, 287.9 and 289.3 eV, attributing to the CC, C-O, C=O and O-C=O bonds, respectively (Figure S3b), indicating the presence of surface chitosan and adsorbed acetic acid. The appearance of peak in N 1s spectrum of Fe3O4@SiO2/GdOF:xCe3+,yTb3+ at relatively higher binding energy with broad feature centered at 402.58 eV (Figure S3c) is attributed to the protonated amine (N+-H),43 corroborated through zeta potential (see supporting information Table S1), which shows positive surface charge for these nanomaterials. The protonation of amine (NH2) might be caused by acetic acid during synthesis in preparation of chitosan solution. The Gd 4d spectrum (Figure S3d) exhibits main peaks in the characteristic Gd3+ binding energies that can be assigned as: Gd3+ 4d5/2 (BE = 143.9 eV) and Gd3+ 4d3/2 (BE = 150.0 eV)44 for the Fe3O4@SiO2/GdOF:xCe3+,yTb3+ nanoparticles. The peaks from Tb 3d5/2, and 3d3/2 at binding energies:1243.8 and 1278.6 eV, respectively suggest the presence of Terbium ion (Figure S3e). Ce 3d XPS spectrum exhibits peaks from 3d5/2 (BE = 885.1 eV) and 3d3/2 (BE = 903.5 eV), attributing to the +3 oxidation state of cerium ion (Figure S3f). In addition, the high intensity peak in energy region of F 1s centered at 685.8 eV (Figure S3g) is attributed to the Gd-F bond. The deconvoluted O 1s spectrum shows a peak at ~529.8 eV, assigned to the Gd3+ (Gd-O), and overlapped dominant broad peak located at higher binding energy (BE ≥532 eV) corresponding to the C-O bond (Figure S3h). However, the relative intensity of O 1s peak is quite lower when compared to the F 1s one,


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as shown in survey spectrum, suggesting the formation of Gadolinium oxyfluoride, as verified by the XRD pattern (Gd4O3F6, P12/c1 monoclinic).45 X-ray diffraction pattern (Supporting Information Figure S4) of magnetite nanocrystals shows diffraction peaks, which are indexed as a face-centered cubic (fcc) lattice structure of Fe3O4 phase (ICDD/PDF 19-629). Accordingly, the Fe3O4@SiO2 nanoparticles retain the main diffraction peaks of the Fe3O4 spinel structure, as exhibited by the XRD pattern. However, additional coating of these magnetic nanoparticles with the Ce3+ and Tb3+ doped GdOF matrix shows the XRD patterns of the similar diffraction peaks, attributing to the monoclinic phase of Gd4O3F6 (space group: P2/c),45 as reported in the literature. Transmission electron microscopy (TEM) images of Fe3O4 show that most of the synthesized nanoparticles are quasi-spherical shape with dominant mean diameter of ~ 8 nm, as suggested by the particles size distribution histogram (Figure 1 and Supporting Information Figure S5). The average diameter was obtained by counting more than one and a half hundreds of Fe3O4 nanoparticles, using imageJ free software and nonlinearly Lognormal fit of the size distribution curve in OriginTM 8.5. However, a small fraction (< 10 %) of bigger nanoparticles between 17 and 39 nm, with mean size of ~ 22 nm were also observed. The high resolution TEM images suggest good crystallinity for these nanoparticles, corroborated by means of XRD patterns. Moreover, functionalization of Fe3O4 with silica gave Fe3O4@SiO2 nanoparticles of >1.0 nm SiO2 shell thickness (Figure 1 and Supporting Information Figure S5). Then concurrent coating of these nanoparticles with chitosan and GdOF:xCe3+,yTb3+ luminophore resulted in irregular shape and morphology aggregated nanoplatform of size between 30 and 100 nm (Figure 1 and Supporting Information Figure S5).


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Figure 1. Transmission electron microscopy images of Fe3O4 (a), Fe3O4@SiO2 (b) and Fe3O4@SiO2/GdOF:xCe3+,yTb3+ nanomaterials (c); HAADF, STEM image and EDS elemental mappings of the Fe3O4@SiO2/GdOF:xCe3+,yTb3+ (x and y = 5 mol%), showing the distribution of Fe, O, Si, Gd, O, F, Ce and Tb elements.


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High-angle annular dark-field (HAADF), STEM image and elemental analysis of EDS mapping (Figure 1) illustrate the uniform and homogenous distributions of Gd, O and F along with Ce and Tb dopants, validating the formation of GdOF:Ce3+,Tb3+ nanostructure. The Fe element was also distributed in the whole magneto-luminescent nanostructure; however, it was found highly concentrated near middle region, a similar result was also manifested from the EDS mapping of O, confirming the presence of Fe3O4 nanoparticles. The EDS mapping also shows the presence of Si element concentrated very close to the Fe3O4, suggesting the presence of Fe3O4@SiO2 nanoparticles. Additionally, dynamic light scattering data display nanoparticles mean size (hydrodynamic diameter) of 166.8 (±7) nm with good colloidal stability in deionized water suspensions (100 mg L-1) for the Fe3O4@SiO2/GdOF:xCe3+,yTb3+ nanomaterials (see supporting information Figure S6 and Table S1). The surface charge was found positive (+24.0 mV ζpotential), probably due to the presence of protonated amino (NH3+) group of the chitosan functionalizing the RE3+ doped GdOF, corroborated through XPS analysis.

3.1. Magnetic and Photoluminescence Properties The multifunctional nanomaterials exhibit near superparamagnetic behavior at room temperature (Figure 2), owing to the existence of well crystalline/stoichiometric magnetite nanoparticles. The magnetic moment of RE3+ ions are also shown considerable contribution to the magnetic properties of these nanoparticles.38 The detail magnetic properties of the magnetoluminescent nanomaterials are described in the supporting information (Figure S7, Table S2 and Figure 2). These hybrid nanoparticles can be rapidly magnetically confined, allowing simultaneously

efficient

visible

emission.

Accordingly,

the

aqueous

dispersion

of

Fe3O4@SiO2/GdOF:xCe3+,yTb3+ nanomaterials in cuvette shows significant green emission by


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irradiating with UV lamp (254 nm). After placing the external magnet bar close to the cuvette, the nanoparticles are rapidly magnetically confined and stacked at a single place along one side of the cuvette (Figure 3a), indicating that they possess an integration of magnetic and luminescent properties in a nanoplatform. This characteristic makes magneto-luminescent nanoparticles, remarkable candidates for multimodal imaging, for example. en emu por gramo de Fe3O4

-1 0,5

-1

M / emu g

1,0

0.3 0.0

-0.3

0,0

-0.6

-0,5

b)

-1,0

-0.9 -6 -4 -2 0 2 4 6 8 10 -500 -1,5 -20

-10

c)

H / kOe 0 10 20

-250

250

500

0.9

5K 300K Langevin Fit

0.5

-1

1.0

M / emu g

-1

0.6

M / emu g

-1

3+

0

H / Oe

H / kOe

10 Fe3O4@SiO2/GdOF:xCe ,yTb 8 ((x = 5; y = 10 mol%) FC 6 0.08 4 2 0.06 0 -2 0.04 -4 -6 0.02 -8 ZFC d) -10 0 50 100 150 200 250 300 -10-8 3+

M / emu g

-1

0.6

1,5

T/K 0.10

0.9

5K 300K Langevin Fit

M / emu g

M / emu g

-1

10 3+ 3+ Fe3O4@SiO2/GdOF:xCe ,yTb 8 (x = 5; y = 5 mol%) 6 0.08 FC 4 2 0.06 0 -2 0.04 -4 ZFC -6 TV 0.02 -8 a) -10 0 50 100 150 200 250 300 -10-8

M / emu g

-1

0.10

M / emu g

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.3 0.0

-0.3

0.0

-0.6

-0.5

e)

-1.0

H / kOe 10 20

-0.9 -6 -4 -2 0 2 4 6 8 10 -500

T/K

-20

-10

0

H / kOe

f) -250

0

H / Oe

250

500

Figure 2. ZFC/FC and M-H curves of Fe3O4@SiO2/GdOF:xCe3+,yTb3+ (x and y = 5 mol%) (a-c) and x = 5; y = 10 mol% (d-f) nanomaterials. The excitation spectra of the magnetic Fe3O4@SiO2/GdOF:xCe3+,yTb3+ (x = 5; y = 5 and 10 mol%) nanophosphors were recorded in the spectral range of 200-500 nm at 300 K temperature (Figure 3b), under emission from the higher intensity 5D47F5 transition (545 nm) of the Tb3+ ion. Excitation spectra of the magneto-luminescent nanomaterials present high intensity broad band at


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280 nm, corresponding to the 4f(2F5/2)5d transition of the Ce3+ ion. Besides, all the nanocomposites show narrow absorption lines, which are attributed to the 4f8‒4f8 intraconfigurational transitions of the Tb3+ ion such as: 7F65L8,7,6 and 5G2 (339 nm), 7F65L9 and 5G4(351nm), 7F65L10 (369 nm) and 7F65G6 (377 nm) as well as 4f7‒4f7 transitions of Gd3+ ion: 8S7/26I15/2,13/2 (272 nm). However, the relative intensities of these narrow absorption bands are very low when compared to the strong broad band of the Ce3+ ion. This result indicates that the sensitization of Tb3+ ion by energy transfer from the 4f(2F5/2)5d interconfigurational transition of Ce3+ ion, is more operative than under direct 4f8-4f8 transitions of the terbium ion in the nanophosphors. The emission spectra of the Fe3O4@SiO2/GdOF:xCe3+,yTb3+ (x = 5; y = 5 and 10 mol%) nanoparticles were measured in the spectral range from 300 to 740 nm at 300 K temperature (Figure 3c), monitoring excitation at 280 nm assigned to the 4f(2F5/2)5d transition of Ce3+ ion. The emission spectra in the range of 475-700 nm present distinct narrow emission lines, attributed to the 5D47FJ transitions (J = 6-0) of the Tb3+ ion. The high intensity 5D47F5 transition at 545 nm was found dominant one for both the Fe3O4@SiO2/GdOF:xCe3+,yTb3+ nanophosphors. In addition, the weak broad emission band observed in spectral range from 300 to 400 nm (inset, Figure 3c) is assigned to the 4f(2F5/2)5d transition (Ce3+ ion). The very weak emission line at 312 nm was attributed to the 6P7/28S7/2 transition of the Gd3+ ion. This result indicates that the Ce3+ ion is an efficient luminescence sensitizer for the Tb3+ ion (luminescence activator) through nonradiative energy transfer process for these nanophosphors. The nonradiative energy transfer (ET) pathways among the Ce3+, Gd3+ and Tb3+ ions can be described

from

the

excitation/emission

data

of

the


nanophosphors in an efficient and simplified way, as shown in schematic energy level diagram


18

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(Figure 3d). Ce3+ ion is excited from the 2F5/2 ground state to the 5d excited state, under 280 nm excitation and gives very weak radiative emission (5d4f(2F5/2) transition). It transfers the excited energy through nonradiative pathway to the 6I7/2 excited state of Gd3+ ion that undergoes nonradiative decay to 6P7/2 energy level and results in a very weak radiative emission (6P7/28S7/2 transition). Moreover, 5d and 6P7/2 excited states of Ce3+ and Gd3+ ions, respectively transfer their exited energies directly to the 4f8 intraconfigurational excited energy levels of the Tb3+ ion, which results in dominant green emission (5D47FJ transitions, J = 6-0). In this mechanism, the Gd3+ ion would provide an alternate energy transfer pathway, bridging the sensitizer (Ce3+) and activator (Tb3+) ions.

Figure 3. Digital photographs of nanoparticles aqueous dispersion under UV irradiation lamp and external magnet (a), excitation spectra (b), emission spectra (c), partial energy level diagrams, blue color dashed arrows denote nonradiative decays, and the gray, bluish and green color downward wide arrows represent the radiative decays from the Ce3+, Gd3+and Tb3+ ions, respectively (d), luminescence decay curves (e) and CIE chromaticity diagram, displaying chromatic coordinates (f) for the magneto-luminescent Fe3O4@SiO2/GdOF:xCe3+,yTb3+ nanomaterials.


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The luminescence decay curves (Figure 3e) of the magneto-luminescent nanomaterials were recorded at 300 K temperature under emission at 545 nm from the 5D47F5 transition (Tb3+) and excitation at 280 nm, assigned to the 4f(2F5/2)5d transition (Ce3+ ion). The lifetime () of the Tb3+ emitting level (5D4) was derived by fitting the luminescence decay curves with monoexponential function: 𝐼(𝑡) = 𝐼0exp (

―𝑡

𝜏), where I(t) and I0 are the emission intensities at time

t and 0, respectively, and  is the radiative decay time. The effective lifetime () values of the bifunctional Fe3O4@SiO2/GdOF:xCe3+,yTb3+ nanomaterials were obtained from the fit and a regular increase in  was observed from 6.30 to 7.56 ms by increasing the concentration of the activator dopant (Tb3+) from 5 to 10 mol%. The color coordinates of the green emitting magnetic nanocomposites were calculated, using Commission Internationale de l’Eclairage (CIE) chromatic diagram. These nanomaterials display chromatic coordinates in the well-defined green visible region, owning to the contribution of the green emission from the high emission intensity 5D47FJ transitions (J = 6-3) of the Tb3+ ion (Figure 3f). As a result, similar (x,y) chromatic coordinates, (x = 0.3805; y = 0.5195) and (x = 0.3911; y = 0.5225) were observed for the Fe3O4@SiO2/GdOF:xCe3+,yTb3+ (x = 5; y = 5 mol%) and (x = 5; y = 10 mol%) nanophosphors, respectively. 3.2. In Vivo Toxicity Assessment in Zebrafish Embryos The in vivo toxicity evaluation of multifunctional nanoparticles that can be rapidly magnetically confined, concurrently allow visible emission, is an important step towards their safe and potential biomedical applications. Notably, no previous toxicological data on the magneto-luminescent nanomaterials have been reported in the experimental literature. Zebrafish has been used as a biological model for several decades to assess the biosafety of environmental agents and


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nanoparticles, indeed certain pathways leading to drug toxicity in mammals are also conserved in D. rerio.26,27 The advantages of using this model are to assess simultaneously several parameters, such as embryonic development, genotoxicity, oxidative stress, etc., which lead us to evaluate not only the potential environmental risks in aquatic systems, but also to human health, because it has a genetic similarity24–27 to the human being.

Figure 4. Toxicity evaluation of magneto-luminescent Fe3O4@SiO2/GdOF:xCe3+,yTb3+ nanomaterial in Danio rerio embryos, employing standard FET, with-chorion and without-chorion assays: percentage of edema (a) malformation (b) and optical microscopy images (c) of embryos captured after 96 hours exposition to 100 mg L-1 of nanoparticles. Capital letters (A, B and C) indicate variances among groups (two – way ANOVA and Bonferroni post-test, p < 0.05). Accordingly, zebrafish embryos were exposed to 0, 1, 10 and 100 mg L-1 of Fe3O4@SiO2/GdOF:xCe3+,yTb3+ nanoparticles for 96 hours, employing OECD standardized fish


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embryo toxicity (FET) and its modified version assays, based on embryos with and without chorion (24 hpf). In this study, no mortality was detected in the control groups, and no significant difference was found regarding the total length and yolk sac size among any treatment groups (Figure 4). Though, hatching rate was varied among all the groups at 48 hours post fertilization (hpf) that was higher for treatment with 50 and 100 mg L-1 of nanoparticles (Figure 4), nevertheless, at 72 hpf all embryos were hatched, suggesting no significant hazardous effect. In addition, the exposure to 100 mg L-1 incubation concentration results in malformation (5.53%) and edema (19.44%) in zebrafish larvae, as shown in Figure 4. The significant data observed for hatching rate can be associated with adsorption of nanoparticles to the surface of the chorion. It has been reported that nanomaterials have tendency to aggregate in the chorion, blocking O2-exchange and decreasing the oxygen levels that can affect the hatching rate.46 Consequently, the chorion is unable to sustain the mechanical barrier and compromise the osmotic balance, which results in appearance of edema and malformations,47 as was observed for nanoparticles at 100 mg L-1 concentration. In order to test this hypothesis, we performed an embryo toxicity test with and without chorion. In these studies, no significant results for mortality, total length, yolk sac size and edema were observed. However, we observed 2.7% of malformation and 5.5% of edema with chorion and 7.0% of malformation and 8.4% of edema without chorion for the 100 mg L-1 of Fe3O4@SiO2/GdOF:xCe3+,yTb3+ nanoplatform. The edema formation with and without chorion and the malformation of embryos with chorion was significantly reduced when compared with the traditional FET (Figure 4). This result might occur because the embryos were exposed to nanoparticles at 24 hpf, which were less sensitive compared with traditional FET. However, malformation in dechorionated embryos was 2% higher than the traditional FET.


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Figure 5. Scanning electron microscopy images of zebrafish embryos exposed to 1 (a), 10 (b) and 100 (c) mg L-1 concentrations of magneto-luminescent Fe3O4@SiO2/GdOF:xCe3+,yTb3+ nanoplatform, exhibiting aggregation of the particles on their chorions membranes. The chorion is an acellular barrier, composed of mainly proteins and glycoproteins and traversed by pore channels with a diameter of ∼200 nm30 that protect the embryo from external hazards. Our results also demonstrate the significance of chorion membrane regarding protection of the larval development. The magneto-luminescent Fe3O4@SiO2/GdOF:xCe3+,yTb3+ nanoparticles have mean size of 166.8 (±7) nm (see supporting information Table S1), smaller than the chorion pores, thus the chorion shouldn’t prevent the internalization of nanoparticles. Though, the mechanism of blocking nanoparticles internalization through the chorion pores is still unknown, we hypothesize that it may be due the aggregation of particles in zebrafish medium, leading to increase the overall size of particles aggregates, as manifested from the DLS measurements (see supporting information Table S1). In addition, bifunctional nanomaterials are also functionalized with chitosan biopolymer, which contains hydroxyl (-OH) and primary amine (-NH2) groups, allowing the attraction of nanocomposites to the proteins of chorion membrane via van der Waals interactions and hydrogen bonding that caused the aggregation of nanoparticle on the chorion membrane,30 as suggested by the SEM images (Figure 5). It is noteworthy, that none of the parameters assessed for the high dose of Fe3O4@SiO2/GdOF:xCe3+,yTb3+ nanomaterials (100 mg L-1) exceed 20%, in comparison to the control. As a result, this study indicates that the


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magneto-luminescent nanoparticles show no acute toxicity and minimal chronic effects to zebrafish embryos. In order, to validate further this result, synchrotron X-ray fluorescence (SXRF) elemental mapping was employed to localize and follow the nanoparticles uptake and distribution in zebrafish embryos. 3.3. Synchrotron X-Ray Fluorescence (SXRF) Imaging of Zebrafish Embryos The micro-focused X-ray beam produced by a pair of curved mirrors allows to perform spatially-resolved XRF measurements for elemental mapping of various elements and nanoparticles in biological specimen with very efficient sensitivity.37 The deep penetration capability and high spatial resolution make the SXRF one of the few techniques to localize the distribution of nanoparticles by mapping the chemical elements in thin histological sections of biological tissues, enabling a perspective to study in-situ toxicity of nanoparticles. The elemental distributions were obtained by raster scanning the zebrafish embryos through a micro-focused x-ray beam of 25 µm in diameter while collecting the K-K, Fe K and Gd L fluorescence intensities coming from the samples. An optical microscope placed inside the experimental hutch allows the location of specific areas on the samples before measurements start. The -XRF images (Figure 6) of the zebrafish embryos exposed to 100 mg L-1 concentration of multifunctional Fe3O4@SiO2/GdOF:xCe3+,yTb3+ (x = 5; y = 10 mol%) nanoparticles at life stages of 0, 24 and 72 hpf were acquired in order to investigate the toxicity pathway of particle uptake and distribution, whether if they occur through oral exposition or dermal route (adsorption through epithelial layer). The top panel of the figure exhibits the optical micrograph (Figure 6a) and XRF image of the control embryo at 24 hours post-fertilization, showing no gadolinium signal but displaying the distribution of potassium and iron in the whole body (Figure 6b). A scatterplot for K-Fe elemental pair was established (Figure 6c), showing a clear co-localization (high correlation)


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of these elements in the corresponding region with a constant K/Fe ratio. This result was further validated when plotting the K K and Fe K fluorescence intensities across the vertical line scan, passing through the trunk of embryo (Figure 6d), where, it is possible to observe a similar behavior for both the K and Fe elements.

Figure 6. -XRF images of K, Fe and Gd in zebrafish embryos exposed to 100 mg L-1 of Fe3O4@SiO2/GdOF:xCe3+,yTb3+ nanoparticles. Optical micrograph of control sample (a); Bicolor (RB) image of K and Fe for the control (b); Correlation plot (log scale) for counts in K K and Fe K channels of the bicolor image (c); Line scans for K K and Fe K fluorescence intensities


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along a vertical line at the trunk of the control after 24 hours post-fertilization (d); Optical micrographs of zebrafishes exposed to nanoparticles at 24 and 72 hpf (e,i); Tricolor (RGB) images of K, Gd and Fe of Danio rerio exposed to nanoparticles at 24 and 72 hpf (f,j); Scatterplots (log scale) for Gd and Fe showing the trend in their distributions between the whole body and gastrointestinal tracts (g,k) and line scans for Gd-L and Fe-K fluorescence intensities along the vertical line passing through gastrointestinal tracts (h,l) for zebrafish embryos exposed to 100 mg L-1 nanoparticles at 24 and 72 hpf. On the other hand, the middle and bottom panels (Figure 6e-l) display the optical micrographs (Figure 6e,i) and -XRF images of zebrafish embryos (side view), exposed to a solution containing 100 mg L-1 of Fe3O4@SiO2/GdOF:xCe3+,yTb3+ nanoparticles at 24 and 72 hours post fertilization (hpf). The XRF maps show clearly the accumulation of highest levels of gadolinium and iron in the gastrointestinal tract (Figure 6f,j). Interestingly after analyzing the correlation between these two elements in the region of interest, the scatterplots of Gd L vs. Fe K counts (Figure 6g,k), show two different kind of populations in the dataset. The two regions show a roughly constant Gd/Fe ratio in each population. As can be seen in the Figure 6g and k, the high Gd/Fe points (purple lines) come from the region where the gastrointestinal tract of the zebrafish is located. The Gd/Fe ratios found in these populations are roughly in good agreement with the chemical composition expected from the synthesis of this material, thus suggesting a co-localization of these elements as a

nanoplatform

of

magnetic

and

luminescent


intact

nanoparticles. It is noteworthy that the levels of Gd and Fe are increased in the gastrointestinal tract as the life stage of the exposed embryos was changed from 24 to 72 hpf due to ingestion of more particles through oral exposer by the grown embryo. Because, at 72 hpf, the larvae mouth is fully developed and ready to intake nanoparticles in medium.41 This result is also quite visible when compared the Gd L and Fe K fluorescence intensities of the zebrafish embryo exposed to nanoparticles at 72


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hpf with the one exposed at 24 hpf (Figure 6h,l). However, the relative emission intensity of the Gd was found to be higher than Fe, suggesting the presence of more Gd for this case. This result can be confirmed by microstructural and magnetic characterizations, which support the presence of larger quantity of GdOF deposited on Fe3O4 nanoparticles in the multifunctional nanocomposite. In addition, the SXRF mapping was also measured for the zebrafish embryos exposed to nanoparticles at zero hour post fertilization, however, no difference was observed in comparison with the control. Therefore, these data have not shown here. Nevertheless, the obtained XRF results indicate that mostly nanoparticles were uptake orally and bioaccumulated in gastrointestinal tract as an intact magneto-luminescent Fe3O4@SiO2/GdOF:xCe3+,yTb3+ nanoplatform. This manifests that SXRF can be an efficient noninvasive technique that can map the chemical distributions and precise co-localizations of the specific elements to assess the fate and toxicity of nanoparticles inside biological specimen. 4. CONCLUSION In summary, the magneto-luminescent nanomaterials were prepared by multistep synthesis approach, using SiO2 as a spacer between the magnetic Fe3O4 nanoparticles and GdOF:Ce3+,Tb3+ luminophore, functionalized with chitosan biopolymer. The multifunctional nanoparticles are well dispersible in water, displaying efficient green emission of the Tb3+ ion when excited at 280 nm assigned to the 4f-5d interconfigurational transition of Ce3+, simultaneously manifesting near superparamagnetic behavior. They are biocompatible, presenting no acute toxicity to the zebrafish embryos in both states, with chorion and dechorionated embryo at higher concentration exposure (100 mg L-1). The synchrotron X-ray fluorescence (SXRF) mapping visualized the distribution of Gd and Fe in embryos and predominantly localized in the gastrointestinal tract as intact Fe3O4@SiO2/GdOF:xCe3+,yTb3+ nanoplatforms. This result supported the nanoparticles uptake


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mostly via oral exposition, which was confirmed by observing considerable increase in the level of multifunctional nanoparticles in gastrointestinal tract as the life stage of the exposed embryos was increased from 24 to 72 hpf. Additionally, it is a new contribution towards the toxicity assessment of complex nanohybrid materials. In future, the ideal GdOF host matrix can be doped efficiently with Nd3+ ion, tailoring the emission and excitation in near infrared (NIR) region, lying in the biological window and allow deep-tissue optical imaging. This will impart an additional feature to the biocompatible magneto-luminescent nanoparticles, to be remarkable candidates for multimodal deep tissue imaging. ASSOCIATED CONTENT The following supporting information files are available free of charge. FTIR absorption spectra, thermogravimetric analysis (TG/DTG curves), XPS spectra and XRD patterns of Fe3O4@SiO2/GdOF:xCe3+,yTb3+ nanomaterials. Histogram of the Fe3O4 nanoparticles size distribution, hydrodynamic size and zeta potential determination study and detailed magnetic properties of the magneto-luminescent nanoparticles. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]; [email protected]; [email protected]. Phone: +55 19 35183140 Notes The authors declare no competing financial interest. ACKNOWLEDGMENT


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The authors are acknowledged the financial support by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) (No. 88882.143477/2017-01), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (No. 150104/2017-0), Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Instituto Nacional de Ciência, Tecnologia e Inovação em Materiais Complexos Funcionais (INCT-Inomat), and Sistema Nacional de Laboratórios em Nanotecnologias (SisNANO-MCTIC). The World Academy of Sciences for the advancement of science in developing countries (TWAS) (No. 190932/2015-5). We also thank the CNPEM open facilities for users (LMN, LME, LAM, LMG, -XRF beamline and NBT). REFERENCES (1)

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GRAPHICAL ABSTRACT

SYNOPSIS: The multifunctional magneto-luminescent nanoparticles are biocompatible, showing no acute toxicity to the zebrafish embryos. Following the nanoparticles uptake and biodistribution via synchrotron X-ray fluorescence imaging, which manifests their uptake predominantly through oral exposition and localizes the bioaccumulation in embryo’s gastrointestinal tract as an integrated nanoplatform, suggesting a new route to nanoparticles administration orally for future nanomedicine applications. These nanocomposites can be magnetically confined, concurrently allow visible green emission that might be tunable in NIR region, suggesting good candidatures for multimodal deep tissue imaging.


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Toxicity Evaluation in

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