Materials Science and Engineering C 80 (2017) 603–615

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Materials Science and Engineering C

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ZnO@Gd2O3 core/shell nanoparticles for biomedical applications:Physicochemical, in vitro and in vivo characterization

Anna Woźniak a,⁎, Bartosz F. Grześkowiak a, Nataliya Babayevska a, Tomasz Zalewski a, Monika Drobna a,b,Marta Woźniak-Budych a, Małgorzata Wiweger c, Ryszard Słomski b,d, Stefan Jurga aa NanoBioMedical Centre, Adam Mickiewicz University, Umultowska 85, 61-614 Poznan, Poland,b Institute of Human Genetics, Polish Academy of Science, Strzeszyńska 32, 60-101 Poznań, Polandc International Institute of Molecular and Cell Biology, Ks. Trojdena 4, 02-109 Warsaw, Polandd Department of Biochemistry and Biotechnology, Biocentre, University of Life Sciences, Dojazd11, 60-632 Poznan, Poland

⁎ Corresponding author.E-mail addresses: wozniaka@amu.edu.pl (A. Woźniak)

(N. Babayevska), tomekz@amu.edu.pl (T. Zalewski), Mart(M. Woźniak-Budych), mwiweger@iimcb.gov.pl (M. Wiw(R. Słomski), stjurga@amu.edu.pl (S. Jurga).

http://dx.doi.org/10.1016/j.msec.2017.07.0090928-4931/© 2017 Published by Elsevier B.V.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 27 November 2016Received in revised form 31 May 2017Accepted 8 July 2017Available online 11 July 2017

The chemical composition of nanoparticles (NPs) may be so designed as to provide measurability for numerousimaging techniques in order to achieve synergistic advantages. Innovative and unique structure of the core/shellZnO@Gd2O3 NPs possesses luminescent and magnetic properties, and is expected that they will become a newgeneration of contrast agents for Magnetic Resonance Imaging (MRI) and nanocarriers for theranostics. Thus,by surface biofunctionalization, it is possible to indicate particular nanoparticle compositions which provide ef-ficient imaging, targeted drug delivery, and biocompatibility.Novel ZnO@Gd2O3 NPswere synthesized and biofunctionalized by folic acid (FA) and doxorubicin (Doxo) to pro-vide target and anticancer functions. Physicochemical analyses of the nanoparticles were performed. The biolog-ical study included a cytotoxicity in vitro, cellular distribution evaluation, as well as toxicity analyses, performedfor the first time, on the in vivo zebrafish (Danio rerio) model.Nanoparticles were found to be effective double-function biomarkers (MRI T2 contrast agents, fluorescent imag-ing). The biological study showed that ZnO@Gd2O3 and ZnO@Gd2O3@OA-polySi@FA NPs are biocompatible in aparticular concentration ranges. Conjugationwith folic acid and/or doxorubicin resulted in effective drug deliverytargeting. The in vivo results described the toxicology profile toward the zebrafish embryo/larvae, including newdata concerning the survival, hatching ratio, and developmental malformations.

© 2017 Published by Elsevier B.V.

Keywords:ZnO@Gd2O3Core/shell nanoparticlesFolic acidDoxorubicinCytotoxicityZebrafish

1. Introduction

Recently, the design, synthesis and characterization of new multi-functional inorganic nanosystems aroused an interest of scientists inthe field of biomedicine [1–5]. One of the main directions is to developmaterial, which combines detection and therapy in a single nanocarrier.The significant advantage of this theranostic approach includes the po-tential for target drug delivery, the reduction of side effects, possibilityto use sparingly soluble and rapidly degradable drugs, thebiodistribution and drug efficacy control, as well as early detection ofcancer and monitoring the progress of the therapy.

The nanocarriers used in theranostics must meet some special re-quirements. First of all, such carrier should have optical and/ormagneticproperties and also be biocompatible and non-toxic. Furthermore, the

, natbab@amu.edu.pla.Budych@amu.edu.pleger), slomski@up.poznan.pl

drugs should be effectively delivered ensured bymeans of target specif-ic ligand attachment, and released into the cells. Therefore, finding anovel nanocarrier complying all these requirements became a challeng-ing topic of the last decade.

Inorganic and organic materials (hybrid materials - materials withcore-shell structures, inorganic-organic or inorganic-organic compos-ites, materials with high specific surface area - porous materials,nanomaterials) due to their unique properties are upcoming materialsfor potential application in drug and gene delivery/release, photody-namic therapy, bioimaging, sensors, etc. [6–9]. Almost all well-knownnanomaterials –metallic (Au), carbon-basedmaterials, semiconductorsnanoparticles (quantum dots), up-conversional NPs, polymeric andothers materials already used for biomedical purposes [9,10–12]. Thefuture lays on synthesis modification strategy (bioinspired, bioassisted)[8,13,14] as well as developing multi-functional, real-time controlledand an externally triggered delivery systems [15,16,17].

Although theranostic delivery nanosystems have been proposed be-fore, in present work we decided to develop new nanocarriers based onthe core/shell nanoparticles, due to the dual or multifaceted nature oftheir components. These structures represent a group of nanomaterials

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with a significant number of potential applications in biomedicine [18,19]. Obtaining nanomaterials which combine two or more functions(e.g. multimodal imaging, targeted drug delivery) into one system isstill a complex and difficult task. Therefore, there are some main ap-proaches to solving this issue. Firstly, the search for single-phase multi-functional nanomaterials which already exhibit desirable properties;the second approach focuses on the formation of nanomaterials,dopedwith impurities, where impurities equip thematerial with specif-ic properties; and the third approach aims at designing some materialswith core/shell structures [20,21]. Varying the core and shell parame-ters (core shape and size, and shell thickness), aswell as their properties(e.g. magnetic, optical), allow to develop of a new multimodal imagingnanosystem for an extensive, both scientific and biomedical, application[22,23].

Consequently, for the purpose of this study, a luminescent-mag-netic nanocomposite was synthetized, based on two independentoxide phases – biocompatible, resistant to photo-bleaching andnon-toxic ZnO matrix with green emissions and Gd2O3, which pos-sesses magnetic properties. This approach generates significant ad-vantages. The emission of ZnO nanocrystals at two differentwavelengths is very important for integrated biological fluorescencelabelling. Gadolinium properties resulting in an efficient shorteningof the longitudinal relaxation time and in the increasing magneticresonance signal intensity [24]. To reduce observed toxicity of thegadolinium ions in chelates [25], Gd ions were designed in the formof a shell, which is enough to obtain a better contrast in the MRIexperiment.

Neither ZnO nor Gd2O3 are stable in water. At the same time, theirsurface can be easily modified by several functional groups, like COO,NH2 [26]. Surface functionalization may provide new chemical andphysical properties, or change those already known; it also reducesthe nanoparticles agglomeration, increases their stability in aqueous, sa-line or medium solutions, and most importantly for the biomedicinepurposes, allows targeting agents to be attached. Various strategies,using organic ligands, an inorganic shell or polymer coupling agents,have been developed to stabilize and disperse nanoparticles in an or-ganic or aqueous media. Oleic acid is a well-known coupling agent,which prevents the nanoparticle agglomeration in organic media [27].One of the popular methods of stabilizing nanoparticles in water is themethod based on SiO2 compounds [28]. We proposed functionalizationby organosiloxanes as a good alternative to well-known SiO2-basedmethods for nanoparticle stabilization. The stepwise functionalizationof the nanoparticles' surface improves the compatibility between inor-ganic nanoparticles and the organic matrix. Functionalized oxide nano-particles (including ZnO and Gd2O3), stable in aqueous media wereobtained by the other groups [26,29,30].

To target nanoparticles toward cancer cells, many ligands wereinvestigated and of these, folic acid is themost common one. FA is es-sential in living organisms during the processes of cell division andgrowth, as well as in DNA repair processes. Many cancer cellsoverexpressed the folic acid receptor on the cell membrane surfaceto ensure intensified growth. This phenomenon may be used as akey factor for targeting nanoparticles. In present article, we proposedbiofunctionalization of the newly designed ZnO@Gd2O3 core/shellnanostructures by the targeting (folic acid) and chemotherapeuticagents (model anticancer drug: doxorubicin), which is achievedthrough amino groups (basically from aminosiloxanes). Thus, attrac-tive physical interactions allow to obtain a new generation ofnanocarriers for multimodal imaging and for targeted drug delivery.

The goal of the study was to characterize the physicochemicalproperties of the new core/shell ZnO@Gd2O3, ZnO@Gd2O3@OA-polySi@FA and ZnO@Gd2O3@OA-polySi@Doxo, ZnO@Gd2O3@OA-polySi@FA@Doxo nanoparticles, cytotoxicity studies at the in vitro(HeLa, KB, MSU-1.1 cell lines) and in vivo levels (zebrafish, Daniorerio). The ZnO@Gd2O3 and doped ZnO:Er3+-Yb3+@Gd2O3 nano-structures have been obtained in our group before [31,32]. The

present study was assumed to be the first which describes the bio-medical applications, as well as in vitro and in vivo studies, of thefunctionalized core/shell ZnO@Gd2O3 nanostructures.

2. Materials and methods

2.1. Materials

To synthesize ZnO@Gd2O3 core/shell nanoparticles, zinc acetatedihydrate (Zn(CH3COO)2 × 2H2O, Sigma-Aldrich), sodium hydroxide(NaOH, Stanlab), gadolinium(III) nitrate hexahydrate (Gd(NO3)3× 6H2O, 99.9%, Sigma-Aldrich) and methanol were used as startingmaterials. For ZnO@Gd2O3 NPs surface modification, oleic acid (OA,Sigma-Aldrich), tetramethylammonium hydroxide (TMAH, Sigma-Aldrich), N-(2-aminoethyl)aminopropyltrimethoxysilane (AEAPS,Sigma-Aldrich), toluene, ethanol, methanol, N-hydroxysuccinimide(NHS, 98%), N-(3-dimethylaminopropyl)-N-ethylcarbodimide hy-drochloride (EDC, 98%), folic acid (≥97%) and dimethylsulfoxide(DMSO, 99.2%) were used. All chemicals were of analytical gradeand were used without any further purification.

2.2. Synthesis of core/shell ZnO@Gd2O3 core/shell nanoparticles

The synthesis of ZnO@Gd2O3 nanoparticles consists of two stages.The core preparation involves adapted synthesis of ZnO NPs via thesimple low-temperature sol-gel route based on the study of W.Beek et al. [33]. Zn(CH3COO)2 × 2H2O) (13.4 mmol) was dissolvedin 125 ml methanol at a constant temperature of 60 °C. Next, a solu-tion of NaOH (23mmol) in 65ml of methanol was added dropwise tozinc acetate dihydrate in methanol under vigorous stirring. Afterapprox. 3 h, a white precipitate of ZnO nanoparticles was separatedfrom the mother liquor, washed with methanol twice and dried atroom temperature (RT).

The Gd2O3 shell was prepared by the “seed deposition method” de-scribed in Y. D. Han et al. [34]. A 300 mg of the ZnO@Gd2O3 nanoparti-cles were immersed in a 0.1 mol/l Gd(NO3)3 water solution for 96 h.The core-shell nanostructures obtained were separated, washed withdeionized water (18 MΩ·cm) and dried in an excicator. To reach gado-linium oxide phase, (Gd2O3), nanoparticles were annealed at 900 °C inair for 1 h.

2.3. Functionalization of ZnO@Gd2O3 core/shell nanoparticles by oleic acidand aminosiloxanes

The ZnO@Gd2O3 core/shell nanoparticles were fractionated bycentrifugation to obtain particles with a size up to 50 nm. Forfunctionalization of ZnO@Gd2O3 NPs by OA, OA (0.22 mmol) wasadded to ZnO@Gd2O3 NPs in ethanol (20 ml) under vigorous stirring.The mixture was heated to reflux, then TMAH was dissolved inrefluxing ethanol (5 ml) and rapidly injected into a flask containingZnO@Gd2O3 core/shell nanoparticles and OA. After several minutes,the mixture was diluted with ethanol (50 ml) and cooled down to0 °C in an ice bath. The resulting solution was centrifuged, andwashed several times with ethanol. Since the oleate-capped ZnO@Gd2O3 NPs obtained are not dissolvable in ethanol, the precipitatewas finally dispersed in 10 ml of toluene, leading to a clear oleate-capped ZnO@Gd2O3 core/shell NPs dispersion. Then 1 ml of AEAPSin toluene (0.1 M) was mixed with ZnO@Gd2O3 core/shell NPs in tol-uene (10 ml) under stirring at RT. After 5 min, 1 ml of TMAH in eth-anol (0.2 M) was added and the temperature was set at 85 °C for45 min. After removal of the supernatant, the precipitate waswashed with toluene, acetone and distilled water, and subsequentlydried at 60 °C.

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2.4. Biofunctionalization of ZnO@Gd2O3@OA-polySi core/shell nanoparti-cles by folic acid and doxorubicin

For biofunctionalization of ZnO@Gd2O3@OA-polySi core/shell nano-particles by folic acid, 10 mg of FA were dissolved in 2 ml DMSO undervigorous stirring at RT. After full dissolution, 10mg of NHS and 10mg ofEDS were added to the FA solution. After stirring for 15 min, ZnO@Gd2O3@OA-polySi NPs (10 mg) were added to the mixture and main-tained overnight. After that, the precipitate was washed with DMSOand distilled water, and then dried at 60 °C.

For biofunctionalization of ZnO@Gd2O3@OA-polySi nanoparticles bydoxorubicin, 5 ml of doxorubicin in water (0.7 mg/ml) were added to10mg ZnO@Gd2O3 core/shell NPs on a magnetic stirrer andmaintainedat RT overnight. For double biofunctionalization by both FA and doxoru-bicin, NPs with FA were immersed in the solution with doxorubicin andwere prepared as described above.

The detailed schematic diagram of functionalized andbiofunctionalized (example - FA-capped NPs) ZnO@Gd2O3 NPs is pre-sented in Fig. 1.

Due to the silanization process, Zn2+ added to the trimethoxysilanegroup of AEAPS, where the Zn–O–Si groups are covalent bonded withZnO surfaces. During the silanization process, TMAHwas added to initi-ate nanoparticle surface functionalization. Then the γ-carboxyl group ofFA was activated by EDC and conjugated onto a ZnO surface through

Fig. 1. Schematic diagram of the obtaini

reactions between FA and amine groups (from AEAPS) to produce thefinal stable ZnO@Gd2O3@OA-polySi@FA nanostructures.

For the sake of convenience, we refer throughout this article to thenanoparticles studied as: sample A (as-obtained ZnO NPs, synthesis at60 °C), sample B (ZnO NPs, modified by Gd2O3 for 96 h and annealedat 900 °C for 1 h), sample C (ZnO NPs, modified by Gd2O3 for 96 h andannealed at 900 °C for 1 h and capped by OA and polysiloxanes), sampleD (ZnO NPs, modified by Gd2O3 for 96 h and annealed at 900 °C for 1 hand capped by OA and polysiloxanes and FA), sample E (ZnO NPs, mod-ified by Gd2O3 for 96 h and annealed at 900 °C for 1 h and capped by OAand polysiloxanes and Doxo), and sample F (ZnO NPs, modified byGd2O3 for 96 h and annealed at 900 °C for 1 h and capped by OA andpolysiloxanes, FA and Doxo).

2.5. Physicochemical characterization

Various instruments were employed to characterize the functional-ized ZnO@Gd2O3 NPs. The size of the nanoparticles obtained was stud-ied with the method of Small Angle X-ray Scattering (SAXS) using anEmpyrean PANalytical diffractometer. Powder X-ray diffraction (XRD)studies of the powder samples were carried out on an Empyrean(PANalytical) diffractometer using Cu Kα radiation (λ = 1.54 Å), a re-flection-transmission spinner (the sample stage) and a PIXcel 3D detec-tor operating in Bragg–Brentano geometry. Scans were recorded at RT

ng functionalized ZnO@Gd2O3 NPs.

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(300 K) at angles ranging from 20 to 80 (°2Theta) with a step size of0.006, and in the continuous-scan mode. In order to examine the stabil-ity of nanoparticles, the relationship of the hydrodynamic size of nano-particles to their electrokinetic potential was measured by DynamicLight Scattering (DLS). The molecular size and zeta (ζ) potential of thenanoparticles were determined using a Zetasizer Nano (Malvern). Allmeasurements were performed at 25 °C and were done in triplicate.The Gd-layer formation, and FA and Doxo bonding were recognized bymeans of the Fourier transform infrared (FT-IR) spectra on a Tensor 27spectrometer (Bruker Optics, Ettlingen, Germany) equipped with aglobal source and an MCT detector in the range of 4500 ∼ 400 cm−1,using the KBr pelletmethod. TheUV–Vis absorption datawere recordedusing a UV–Vis spectrometer and by dispersing the nanoparticles inwater.

2.6. Magnetic Resonance Imaging

T2-weighted magnetic resonance images (MRI) were generatedusing a 9.4 Tesla MRI horizontal scanner (Agilent) with the volume RFMilipede coil (40 mm). The resolution was 0.112 × 0.112 × 2 mm3.Prior to imaging, the experiment samples with different concentrationsof nanoparticles were placed in a specially designed holder.Moreover, areference specimen with solvent (water) was placed in the central partof holder. The measurements were performed at 16 °C. In the experi-ment, a spin echo CPMG sequence was used with TE = 10–320 msand TR = 15,000 ms and with the number of echoes set to 32.

2.7. Cell culture

The examinations were conducted on HeLa (cervical cancer, ATCCCCL-2), KB (epidermal carcinoma ATCC® CCL-17™) and MSU-1.1 (nor-mal human fibroblasts, kindly provided by Prof. C. Kieda - CBM, CNRS,Orléans, France) cell lines. The cells were cultured in a complete medi-um composed of the basal medium (DMEM - Dulbecco's ModifiedEagle's Medium, Sigma-Aldrich - for HeLa and MSU-1.1 cells; EMEM -Eagle's Minimum Essential Medium, Sigma-Aldrich – for KB cells), 10%foetal bovine serum (FBS, Sigma-Aldrich) and 1% antibiotics (penicillin100 μl/ml, streptomycin 100 μg/ml, Sigma-Aldrich). The cells weremaintained under sterile conditions and incubated at 37 °C in a humid-ified atmosphere supplemented with 5% CO2. The cell morphology andconfluence were evaluated using an inverted microscope (Leica DMILLED).

2.8. Cytotoxicity assays

To assess cell viability in response to sample B (ZnO@Gd2O3), sampleD (ZnO@Gd2O3@OA-polySi@FA), sample E (ZnO@Gd2O3@OA-polySi@Doxo) and sample F (ZnO@Gd2O3@OA-polySi@FA@Doxo) nanoparticles,the cytotoxicity in various cell types was determined by theWST-1 CellProliferation Assay (Clontech). This test is based on the enzymatic cleav-age of the tetrazolium salt WST-1 to a water-soluble formazan dye,which may be quantified by absorbance (wavelength of λ ~ 420–480 nm) by living cells. When the cell cultures reached the appropriateconfluence, the cells were trypsinized and seeded in microtiter plates(tissue culture grade, 96 wells, flat bottom). All cell lines were seededat a density of 2.5 × 104 cells per well. After 24 h, the cell culture medi-um was removed, fresh media containing the increased concentrationsof samples B, D, E, Fwere added to each well (1, 6, 8, 16, 32, 100, 300 μg/ml in complete medium), and then the cells were incubated for another24 and 48 h. After incubation, 10 μl of the WST-1 Cell Proliferation Re-agentwas added to eachwell and incubated for 2–4 h. Then, absorbanceat wavelength of λ ~ 450 nm (the reference λ ~ 620 nm) was recordedagainst the background control with the use of a multiwell plate reader(Zenyth, Biochrom). All experiments were carried out in duplicate. Foreach experiment, the untreated cells served as a negative control, andthe cells incubated with 10% dimethyl sulfoxide – DMSO (Sigma-

Aldrich) served as a positive control. Data are presented as the mean± standard deviation (SD).

2.9. Confocal bioimaging

The cellular distribution, targeting of the nanoparticles and thedoxorubicin release in the cells were all performed using fluorescentstaining with a cell membrane dye concanavalin A (Molecular Probes,100 μg/ml), the luminescence of core/shell nanoparticles and doxorubi-cin autofluorescence. All the cell lines were seeded at a density of 10-5 cells/ml in a Lab-Tek™ 8 Chamber Slide. After one day, samples B, D,E, F (concentration of 300 μg/ml) were added. After 24, 48 and 72 h ofincubation, the medium with nanoparticles was discarded and thecells were washed, fixed (4% paraformaldehyde, 0.2% Triton X-100,Sigma-Aldrich) and stained with concanavalin A. This dye shows theemission maximum at a wavelength of λ ~ 668 nm, whereas the nano-particle luminescence/fluorescence and the doxorubicin autofluores-cence show the emission at wavelength of λ ~ 520 nm and λ~ 560 nm, respectively. Confocal imaging was performed with a LeicaTCS SP5 system.

2.10. Toxicity in vivo

The in vivo analyses were performed on the embryonic zebrafish(Danio rerio) animal model. For the provision of embryos, the adultzebrafish (the ABxTL wild type strain) were kept in aquaria with alight/dark cycle of 14 h/10 h at the International Institute of Molecularand Cell Biology in Warsaw. The fish were allowed to breed naturallyand the eggs were collected in plastic egg chambers, approximately1 h post fertilization (hpf). Any unfertilized embryos were removed,the eggs were then cleaned, sorted after the microscopic evaluationand distributed (20 embryos per dish) into 3.5 cm culture dishesusing a plastic pipette. At all stages, the developing embryosweremain-tained at 28 °C in an aqueous E3 medium. The zebrafish embryos werethen exposed for 96 h to three different concentrations (10, 50, 100μg/ml) of the nanoparticle samples being analysed (B, D, E). Duringthe exposure period (after 24 h and 48 h), photographs of the embryoswere captured under a stereomicroscope in order to estimate the per-centage of surviving embryos, as well as their morphological changes.The embryos were also observed to estimate the hatching rate every24 h.

3. Results and discussion

3.1. Morphology and structure characterization

ZnO@Gd2O3 (non-functionalized) core/shell nanoparticles were ob-tained and characterized earlier by Babayevska et al. [31]. Previously ob-tained results showed that the core, as-obtained ZnONPs (sample A) arehighly crystalline and have a hexagonal wurtzite ZnO structure. Follow-ing ZnO surface modification by the Gd2O3 shell (sample B), the diffrac-tion pattern consists of two sets of lines – the hexagonal wurtzite ZnOstructure and new peaks corresponding to (222) and (400) planes ofthe cubic Gd2O3 phase (Fig. S1). ZnO NPs have a uniform sphericalshape, are highly crystalline and monodispersed with a mean size ofabout 7 nm. After covering ZnO NPs with a Gd2O3 layer, the resultingZnO@Gd2O3 NPs are polydispersed with the core/shell structure andhave a mean size range of 30–100 nm. It was shown that the Gd2O3shell thickness of these nanoparticles (with the core size of approx.70 nm) is about 4 nm (sample B) [31]. These ZnO@Gd2O3 core/shellNPs are too large for in vitro/in vivomeasurements. Therefore for any fu-ture functionalization, the core/shell NPs were fractionated bycentrifugation.

The luminescent characteristics of these ZnO@Gd2O3 core/shell NPswere studied earlier by Babayevska et al. [31]. ZnO NPs (core) have in-tensive green luminescence. After covering the ZnO NPs surface with a

Fig. 3. FT-IR spectra of sample D (ZnO@Gd2O3@OA-polySi@FA), sample E (ZnO@Gd2O3@OA-polySi@Doxo) and sample F (ZnO@Gd2O3@OA-polySi@FA@Doxo).

Table 1The size of nanoparticles estimated using SAXS.

Sample Sample A Sample B Sample C Sample D Sample E Sample F

Diameter(nm)

10.4 ±1.1

40.2 ±3.4

43.0 ±3.5

46.4 ±4.2

46.6 ±3.2

42.8 ±4.3

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Gd2O3 shell, luminescence intensity decreases. The relative quantumyield (QY) was calculated according to Sun et al. [35]. The QY of theas-obtained ZnO NPs is about 20%, after Gd2O3 ZnO (NPs surface modi-fication 96 h) QY in green emissions is about 8%. The organic shell doesnot affect the luminescent efficiency of the whole system.

Despite the fact that the QY is not high, it is enough to use ZnONPs asfluorescent markers and we can see this in our experiments, presentedbelow.

The average sizes of the ZnO NPs; ZnO NPs, modified by Gd2O3; ZnONPs, modified by Gd2O3 and capped by OA and polysiloxanes; ZnO NPs,modified by Gd2O3 and capped by OA, polysiloxanes and FA; ZnO NPs,modified by Gd2O3 and capped by OA, polysiloxanes and Doxo; ZnONPs, modified by Gd2O3 and capped by OA, polysiloxanes, FA andDoxo, based on small angle X-ray scattering (SAXS), are given in Table1. From these results it is clear that the particle size increased upon coat-ing with (poly)aminosiloxanes, oleic and folic acids.

3.2. Functionalization and biofunctionalization of ZnO@Gd2O3nanoparticles

To provide water stability and to follow drug targeting, ZnO@Gd2O3nanoparticles were firstly functionalized by oleic acid andaminosiloxanes, and then biofunctionalized by folic acid and doxorubi-cin. Confirmation of the particle surface modification was attained viaFT-IR. Fig. 2 presents the FT-IR spectra of samples A, B, C.

In Fig. 2, the broad band centred at about 3315 cm−1 is assigned tothe O\\H stretching mode of the surface hydroxyl groups for all thesamples studied. These peaks may also be attributed to the HO-ZnO hy-droxyl species formed via the dissociative adsorption of water with ZnONPs, and probably to the water molecules adsorbed on the surface ofnanoparticles. The IR absorption peaks found at 457 cm−1 and680 cm−1 are attributed to Zn\\O. Two sharp peaks are located at1585 cm−1 and 1400 cm−1, which are characteristic of the COO andCOO-Zn stretching vibrations (samples A, B).

The new small additional peak at 547 cm−1 corresponds to a typicalGd_O vibration from the Gd2O3 layer (sample B). The Zn\\O stretchingmode vibration shifted to a lower frequency (peak at 420 cm−1), whichindicates the existence of the Gd2O3 shell on ZnO NPs' surface.

Following ZnO@Gd2O3 NPs surface modification by oleic acid and(poly)aminosiloxanes – TMAH and AEAPS (sample C), the spectrum at

Fig. 2. FT-IR spectra of sample A (ZnO NPs), sample B (ZnO@Gd2O3) and sample C (ZnO@Gd2O3@OA@polySi).

3702–3047 cm−1 range is broader than that presented on the graphfor samples A, B. This broad adsorption band besides HO-ZnO andadsorbed H2O may consist of the N\\H stretching vibration of aminogroups presented in the ligand. The two peaks located at 2932 and2877 cm−1 are due to the symmetric and asymmetric C\\H stretchingvibrations of the CH2 groups from oleic acid. Two small peaks, locatedat 1585 cm−1 and 1400 cm−1 and corresponding to the COO andCOO-Zn stretching vibrations, are smaller. The sharp peak at1483 cm−1 is also attributed to the N\\H stretching vibration fromthe amino groups. It was also found that the FT-IR spectra of ZnO exhibitabsorptions at 1007 cm−1 and 948, which could be ascribed respective-ly to the characteristic peaks of the Si\\O\\Si and Zn\\O\\Si symmetri-cal stretching vibrations. These peaks indicate that ZnO is bonded to theaminosiloxanes ligands through covalent chemical bonds.

Following ZnO@Gd2O3@OA@polySi NPs surfacemodification by folicacid, the white powder of ZnO changes colour to yellow. In Fig. 3 (sam-ple D), there are small peaks at 1616 cm−1 and 1520 cm−1 correspond-ing to the C_O groups from folic acid. The Doxomodified nanoparticleswere also confirmed by FT-IR spectroscopy (Fig. 4). Despite the fact thatthe nanoparticles were intensely purple following functionalization bydoxorubicin, the FT-IR peaks are weak. The bands at 1724 cm−1,1611 cm−1, and a small peak at 761 cm−1 are referred to as thestretching vibration of two carbonyl groups of the anthracene ring ofdoxorubicin. Following double functionalization by FA and Doxo, wemay see peaks from the COO− and COO-Zn stretching vibrations.

TheDoxo loading is due to electrostatic forces. In order to investigatethe nature of these electrostatic interactions, zeta potential measure-ments were performed (Table 2). The zeta potential measurements forpure Doxo at pH around 7 were also performed to analyse the influenceof electrostatic interactions between Doxo molecules and ZnO@Gd2O3@

Fig. 4.Adsorption of FA (sample D) and Doxo (sample E) on ZnO@Gd2O3@OA@polySi core/shell NPs surface at RT.

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Table 2Zeta potential of nanoparticles.

Sample Zeta potential (mV)

Sample A 33.9 ± 1.4Sample B 18.5 ± 0.8Sample C 25.7 ± 1.2Sample D 28,7 ± 1.6Sample E −40.7 ± 2.3Sample F −12.7 ± 0.82

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OA-polySi nanoparticles during the adsorption process. The electroki-netic potential of Doxo at neutral pH is close to isoelectric points(−1.99 mV), which means that doxorubicin molecules have a slightnegative charge on the surface. The mechanism of doxorubicin adsorp-tion is not mainly attributed to the electrostatic interactions. However,positively charged nanoparticles can involve hydrogen bonding and fa-cilitate the adhesion on the surface [36,37].

The stability of nanoparticles is one of the critical aspects in ensuringsafety of nanomaterials. It should be pointed out that formation of ag-gregates with a size larger than 5 μm may lead to capillary blockadeand embolism [38]. Thus, the particle size, size distribution and electro-kinetic potential of nanomaterials should be carefully monitored toevaluate the physical stability of nanoparticles. The core/shell ZnO@Gd2O3, ZnO@Gd2O3@OA-polySi@FA, ZnO@Gd2O3@OA-polySi@Doxoand ZnO@Gd2O3@OA-polySi@FA@Doxo nanoparticles were character-ized by DLS in terms of zeta potential, particles size and polydispersityindex (PDI). The nanoparticles dimensions monitored by DLS overtime showed that all core/shell nanoparticles are in the nanoscalerange in the first 24 h (Fig. 5a). A significant aggregation of unmodifiedZnO@Gd2O3 NPs, ZnO@Gd2O3@OA-polySi and ZnO@Gd2O3@OA-polySi@FA@Doxo NPs began after 24 h incubation, leading to an in-crease in particle size to 500–600 nm and PDI. After 72 h, all nanostruc-tures formed large clusters with particle size reachingmicrometer scale,probably due to their lower value of zeta potential. Fig. 5b shows thezeta potential profile of nanoparticles versus time. Except for ZnO@Gd2O3@OA-polySi@FA@DoxoNPs, the zeta potential of all nanoparticlesdecreased with the time of experiments, resulting in a high sticking ef-ficiency between nanosurfaces (aggregation). This is in agreement withthe general literature that a decrease in zeta potential leads to a de-crease in the physical stability of nanomaterials [39]. From this resultit can be seen that the highest stability of nanoparticles was observedin the first 24 h. It could be concluded that the dispersion stability ofloaded by drug nanoparticles remains a very challenging issue. It de-pends on various parameters, such as surface properties ofnanomaterials, their concentration in aqueous media, pH and adsorp-tion of molecules on their surface.

Fig. 5. The average size (a) and the absolute value of zeta potential (b)

The UV–Vis spectroscopy was used as an additional method whichconfirms surface functionalization and determines the amount of FAandDoxo adsorbedon the core/shell nanosurface. Fig. 4 presents the ad-sorption of FA and Doxo on the ZnO@Gd2O3@OA@polySi NPs surface atRT.

As can be seen after approximately 15 min of the functionalizationprocess, N16% (1.62 mg on 10 mg NPs) of FA was adsorbed. In thecase of Doxo adsorption, the adsorption efficiency was around 42%(74.1 mg on 10 mg NPs) after 15 min. of the functionalization. Theadsorbed amount of FA and Doxo during the 24 h period was virtuallyunchanged. The adsorption of folic acid and doxorubicine on ZnO@Gd2O3@OA@polySi NPs surface is related to the conjugation of the car-boxylic groups of FA and Doxo to the amine groups on the nanoparticlesurface modified by (poly)aminosiloxanes.

3.3. Magnetic Resonance Imaging

To validate our assumption about the efficiency of our particles asMRI contrast agents, magnetic resonance images were collected. Thesix samples of different nanoparticles concentration in water were lo-cated: 0 mM – tube 1, 0.35 mM – tube 2, 0.17 mM – tube 3, 0.08 mM– tube 4, 0.04mM – tube 5, 0.02mM– tube 6. The suspension in the cen-tral tube (1) is brighter in all T2-weighted images (TE = 20 ms, TE =180ms, TE = 320 ms) than in the remaining tubes (2–6). The NMR sig-nal disappears faster in the tubes with a higher concentration of nano-particles (Video Still 1). This property corresponds with the T2 timevalue, which is shortest for tube 6. These results confirm the efficiencyof the nanoparticles which were prepared in enhancing the negativecontrast in the Magnetic Resonance Imaging experiments (Fig. 6).

3.4. In vitro cytotoxicity

The cellular toxicity analysis was based on theWST-1 assay dedicat-ed to assessing the viability of the cells being examined. ObtainedOD re-sults at wavelength of λ ~ 450 nmmay be evaluated as a percentage ofthe cell viability compared to the control [40]. To assess the effect ofnanoparticles on cell viability, a wide spectrum of particle concentra-tions was examined at two time intervals.

After 24 h of HeLa cell incubation with sample B, the average cell vi-ability was concentration-dependent with the lowest value at 300 μg/ml. The incubation with sample D resulted in an average survival rateof 64% (Fig. S2a). Cell viability after incubationwith sample Ewas stable,but decreased dramatically at the highest concentration (300 μg/ml –1.6%). During incubationwith sample F, the cells expressed an increasedcytotoxic effect in the three highest concentrations (64, 100 and 300 μg/ml) (Fig. 7a). The HeLa cells showed a very high viability profile after

of nanoparticles in water as function of time during stability study.

Fig. 6. MRI T2-weighted images in tubes 1–6 with different concentrations of sample B (ZnO@Gd2O3) in water.

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48 h of incubation with samples B and D, except for the highest concen-tration (300 μg/ml) (Fig. S2b). A weak ability to reduce WST-1 salt wasobserved after incubation with sample E in concentrations of 100 and300 μg/ml. After incubation with sample F, cytotoxicity was observedat a dose as low as 32 μg/ml (Fig. 7b).

The KB cells after 24 h showed an average viability 107% after sampleB exposure. During incubation with sample D, the cells expressed an in-creased cytotoxic effect in the two highest concentrations (100 and 300μg/ml) (Fig. S3a). After 48 h incubation, the same cellular reaction wasdetected (Fig. S3b). The lowest viability was 2.89% and was observedas a result of the incubation with 300 μg/ml of sample E. A similar re-sponse was observed after incubation with sample F. The trend for thecell viability described above was also detected after 48 h of incubationwith the nanoparticles analysed (Fig. 8a, b).

During the 24 h incubation of samples B, D (Fig. S4a), and E, F (Fig.9a), in the case of the MSU-1.1 cells, the viability was stable with in-creasing cytotoxicity in a concentration of 300 μg/ml. A lethal effect atthis dose was observed after 48 h (Figs. S4b, 9b).

The in vitro study showed the cytotoxic profile of the B, D, E, F sam-ples. To assess the cytotoxicity of the new materials, it is important tostart with the basic ZnO@Gd2O3 core/shell NPs. This in vitro study con-firmed that these nanoparticles (10–50 nm) are highly biocompatiblein the range of concentrations (1–300 μg/ml) analysed, and thus maybe used as a basic nanostructure for multimodal nanocarriers. Our in-vestigations are in agreement with the observations of other authorsthat Gd-doped ZnO nanoparticles used for labelling HeLa cells have nocytotoxic effect, even in concentrations as high as 1 mM [41].

The ZnO@Gd2O3 core/shell structure was biofunctionalized withfolic acid in order to target the tested nanoparticles to the cancer cells

Fig. 7. HeLa cells viability after (a) 24 h and (b) 48 h of incubation with sample E (ZnO

analysed. HeLa and KB cells overexpress the folic acid receptor at avery high level, but an enhanced nanoparticle cellular uptake causedno reduction in viability within the 1–100 μg/ml range of concentra-tions. Other data also suggest that folate-coated ZnO nanoparticlesdoped with rare metals (i.e. Gd, Yb) have no effect on cell viability [42].

A correlation analysis of the absorption and the amount of doxorubi-cin showed that concentrations of 100 and 300 μg/ml should be themost efficient, since they contain an effective dose of doxorubicin atthe in vitro level. Its effect is reflected in the cellular response observed(Figs. 7, 8). These data correspond to our previous results concerning cy-totoxicity and the drug-loading profile of magnetite@polydopamine@doxorubicin NPs [43]. The differences between the cellular responsesof cancer cellswere connectedwith their different sensitivity to the che-motherapeutic agent [44].

Themost visible effect of the improved cellular uptakewas observedafter the incubation of sample Fwith theHeLa cells. The reduction in cellviability was four times greater compared with the effect of sample E.These study results indicate that the nanoparticles tested are efficientin the targeted drug delivery system.

3.5. Confocal bioimaging

The incubation of the cellswith sample B resulted in thefluorescenceof the nanoparticles being observed within the cell interior. The micro-scopic study of sample D confirmed the targeting process in these cells,observed with a very high intensity in the cancer HeLa and KB cells, dueto the preferential binding of folic acid to the overexpressed surface re-ceptors (Fig. S5). The lowest nanoparticle specific fluorescence signals

@Gd2O3@OA-polySi@Doxo) and sample F (ZnO@Gd2O3@OA-polySi@FA@Doxo).

Fig. 8. KB cell viability after (a) 24 h and (b) 48 h incubation with sample E (ZnO@Gd2O3@OA-polySi@Doxo) and sample F (ZnO@Gd2O3@OA-polySi@FA@Doxo).

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were detected in the case of sample D following incubation with theMSU-1.1 cells.

Confocal observations of sample E established an effective drug de-livery using nanoparticles. The nucleus/cytoplasm doxorubicin releaseprocess was observed as early as the first day. Microscopic analyses ofsample F confirmed these results (Fig. 10). Please note, that on Fig. 10band c the red autofluorescence of doxorubicin and artificial green colourof membrane 647 nm dye are presented. This approach has allowed usto show indirect evidence for samples E and F cellular distribution. It wasnot our intention to distinguish florescence signal from the NPs and cellmembrane.

The Z-stack mode performed in all cases confirmed the internaliza-tion process (Video Still 2).

The literature on the subject includes many scientific reportsconcerning cytotoxicity and biological imaging based on ZnOnanocrystals doped with Co, Cu, Ni, Au, Gd, the Fe3O4 core/shell, ZnOnanowires targeted to integrin αvβ3, ZnO QDs coated with folate-con-jugated chitosan loaded with doxorubicin, ZnO@Gd@Doxo and folate-coated ZnO doped with rare metals [41,42,45–50]. This report is as-sumed to be the first describing the cytotoxicity profile and cellular im-aging of core/shell ZnO@Gd2O3, ZnO@Gd2O3@OA-polySi@FA, ZnO@

Fig. 9.MSU-1.1 cells viability after (a) 24 h and (b) 48 h incubation with sample E (Zn

Gd2O3@OA-polySi@Doxo and ZnO@Gd2O3@OA-polySi@FA@Doxo NPstoward HeLa, KB, MSU-1.1 cells.

3.6. In vivo toxicity

At present, the zebrafish appears to be a promising in vivo vertebratemodel for nanotoxicological studies. Themost important characteristicsof this laboratory animal include its small size, rapid development, shortlife cycle, optical transparency, high level of genetic homology tohumans and low culture cost [51]. A wide spectrum of nanomaterials(metallic, magnetic, polymeric, carbon and QD) has been examined interms of in vivo toxicity using theDanio reriomodel [52–57]. Our studiesincluded both embryo survival and hatching rates, aswell as themalfor-mation analyses of zebrafish embryos after incubationwith samples B,Dand E. The embryoswere not exposed to full range of concentrations (1–300 μg/ml). A close study of literature revealed that nanoparticles con-centrations of 50 and 100 mg/l were the lethal doses for zebrafish em-bryos [58] and we didn't want to exceed these values. Based on theresults from the in vitro study, we have shortlisted 3 samples (B, D, E)and used these in in vivo tests. Samples E and Fwere similar in their ac-tion on cancer cells but sample Ewas slightly more toxic to normal cells

O@Gd2O3@OA-polySi@Doxo) and sample F (ZnO@Gd2O3@OA-polySi@FA@Doxo).

Fig. 10. Image of HeLa cells after 24 h incubationwith (a) - sample B (ZnO@Gd2O3), violet colour (concanavalin A) - cellmembrane, green colour – nanoparticle luminescence; (b) - sampleE (ZnO@Gd2O3@OA-polySi@Doxo), green colour (concanavalin A) - cell membrane, red colour – doxorubicin; (c) - sample F (ZnO@Gd2O3@OA-polySi@FA@Doxo), green colour(concanavalin A) - cell membrane, red colour – doxorubicin. Magnification 63×, confocal microscope Leica TCS SP5. (For interpretation of the references to colour in this figure, thereader is referred to the web version of this article.)

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than sample F. In order to cover full range with least number of sampleswe have decided to omit sample F from further analysis. Although toxic-ity testing on zebrafish younger than 120 hpf is not considered as an an-imal testing (Directive 2010/63/UE and Polish ACT of 15 January 2015on protection of animals used for scientific or educational purposes)we feel obliged to limit the number of embryos/larvae that need to besacrificed in experiments.

3.6.1. Survival and hatching rateThe embryo survival rates were evaluated after 24 and 48 h of incu-

bation with the nanoparticles being tested. Microscopic observationsshowed embryo survival rates approx. 90–100% after incubation withsamples B, D and E in concentrations of 10, 50, 100 μg/ml and at time in-tervals (24, 48 h) (Fig. 11a). This may be connectedwith the function ofchorion, i.e. the three-layered outer membrane surrounding and

Fig. 11. Embryo survival (a) and hatching rates (b) after incubation with sample B (ZnO@Gd2O

protecting the embryo [59]. On the other hand, some research papersdescribe the ability of very small Ag nanoparticles (11.3 nm) to passthrough the chorion pores [60]. Bai et al. described the toxicity of ZnOnanoparticles in concentrations of 50 and 100 mg/L as the lethal dose[58]. For Fe2O3 the survival rate was dose- and time-dependent [61].

The nanoparticles affected the hatching rate of the zebrafish embry-os. The hatching time for the control began between 48 and 72 hpf. Ex-posure of embryos to sample B at 100 μg/ml resulted in no hatchingbeing observed even at 96 hpf. The same type of nanoparticles at 50μg/ml reduced the hatching rate at 96 hpf, whereas at a lower concen-tration (10 μg/ml), the hatching occurred earlier than in the control.During incubation with sample D, the dose of 10 μg/ml has no negativeeffect on the hatching ratio, whereas the concentration of 50 μg/ml re-duced the rate after 72 and 96 hpf. The concentration of 100 μg/mlafter 72 and 96 hpf inhibited totally the hatching process. Sample E

3), sample D (ZnO@Gd2O3@OA-polySi@FA) and sample E (ZnO@Gd2O3@OA-polySi@Doxo).

Fig. 12. Malformations of zebrafish embryos exposed for 48 h to (a) sample B (ZnO@Gd2O3), (b) sample D (ZnO@Gd2O3@OA-polySi@FA), (c) sample E (ZnO@Gd2O3@OA-polySi@Doxo).

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reduced the hatching after 96 h for the concentrations of 50 and 100 μg/ml (Fig. 11b) and as was the case with sample B, stimulated hatching at10 μg/ml. Retarded embryo hatching after exposure to ZnO, Ag, Au, Pt,TiO2 and Si nanoparticles was also described by other authors [60,62,63]. On the other hand, Samaee et al. showed the premature hatchingtime and decrease in time required for normal hatching after incubationwith TiO2NPs in a dose-dependentmanner [64]. Zhou et al. investigatedthe effect of surface chemical properties on the toxicity of 17 types ofZnO nanoparticles to the embryonic zebrafish and declared that it mayincrease the toxic impact [65].

3.6.2. Developmental abnormalitiesAs shown in Figs. 12 and 13, as a result of 48 h exposure to samples B,

D and E, embryos failed to develop the normal morphology. The mosttypical malformations at 48 hpf initiated after exposure to sample Bwere a bent trunk and tail malformation. Contact of the embryos withsample D resulted in yolk sac oedema andpericardial oedema. Pericardi-al oedema, yolk sac oedema and bent trunkwere the most frequent ab-normalities of embryos following incubation with sample E. Toxicactions of sample E were expected due to the interactions with theDNA double helix and confirmed other authors' findings concerningthe cardiotoxicity of drug analysed [66,67]. All types of malformationswere dose-dependent. No correlation between the premature hatchingobserved for samples B, D and E at a low concentration andmalformations was detected.

The results of the present study remain in agreementwith the obser-vations reported by other authors on the effect of bare nanoparticles onthe malformation development. The data concerning the toxicity ofSiNPs described such abnormalities as pericardial oedema, yolk sac oe-dema, as well as tail and head malformations [68]. The toxicity assess-ments of the near-infrared up conversion luminescent LaF3:Yb,Erinjected through the chorion into the early developed zebrafish embry-os showed some abnormalities, including a non-depleted or malformedyolk, spinal and tail malformations, pericardial sac or yolk formations,

Fig. 13.Malformations of zebrafish embryos exposed for 48h to (a) sample E (ZnO@Gd2O3@OA-E3 salt.

stunted body or eye growth, as well as pericardial or yolk sac oedema[51].

The results obtained in this paper indicate that all three types ofnanoparticles tested have a slight effect on the survival rates, and influ-ence the hatching rate, as well as being the cause of the embryomalformations. Literature sources describe three mechanisms for thetoxic effect of different nanoparticle types (Fe2O3, carbon nanotubes,TiO2, ZnO). The first mechanism involved aggregation and sedimenta-tion on the chorion surface and changes in the mechanical propertiesof the membrane, as well as the effect on chorionic hatching enzymes[69]. The second approach concerned interference with the nutrient ex-change between the embryos and the environment [70]. The third strat-egy was related to the toxic effect of the metal ions released from thenanoparticles and the induction of DNA damage, as well as the genera-tion of reactive oxidative species ROS [71,72].

As far as the authors of the present study are aware, there are nodataconcerning the influence of the core/shell ZnO@Gd2O3, ZnO@Gd2O3@OA-polySi@FA, ZnO@Gd2O3@OA-polySi@Doxo nanoparticles on thezebrafish embryo development. In vivo nanotoxicity studies providedthe survival and hatching rates, aswell as the developmental abnormal-ities. These data are regarded as highly informative, since zebrafishshare many physiological characteristics with higher vertebrates; thus,the toxicological results obtained in this study may be compared withthose of studies on the developmental toxicity in mammals [61]. Thein vitro-in vivo correlation of the recorded results is moderate. Due tothe muchmore complex animal response (biodistribution, degradabili-ty), in vivo nanotoxicity cannot be properly addressed using in vitro ex-perimental systems [64].

4. Conclusions

Physicochemical analyses showed the excellent potential applicabil-ity of the newly produced core/shell ZnO@Gd2O3 nanoparticles as effec-tive and efficient novel double-function T2 contrast agents for the

polySi@Doxo) (50 μg/ml), (b) sample B (ZnO@Gd2O3) (100 μg/ml), (c) - aqueous solution of

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magnetic resonance, as well as for fluorescent imaging. Biological stud-ies based on the in vitro cytotoxicity profile lead to the conclusion thatsamples B and D are biocompatible within a particular concentrationranges. ZnO@Gd2O3 nanoparticles with biofunctionalized agents (folicacid, doxorubicin) exhibit sufficient biological activity and are effectivenanocarriers for targeted drug delivery systems. In vivo results of thisstudy, will broaden the current knowledge on the potential toxicologi-cal effects of ZnO@Gd2O3 based nanoparticles and support sustainableexpansion of nanotechnology.

In summary, physicochemical and biological analyses indicate thatZnO@Gd2O3 core/shell nanoparticles are promising options for multi-modal imaging and targeted drug delivery. The results meet modernmedicine expectations. The presented solution may result in early dis-ease diagnostics, monitoring of therapy results, while reducing drugdoses, thus providing very important health, social and economiceffects.

The future lies in optimizing the results obtained andmoving towardthe development of personalized therapy. Using molecular biology andgenetic diagnostics tools, it is possible to establish the overexpression ofsurface protein which is characteristic and specific only to a given pa-tient, thus creating some targeted and personalized therapy.

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.msec.2017.07.009.

Conflict of interest

The authors declare that there is no conflict of interest regarding thepublication of this paper.

Funding sources

This work was supported by the National Centre for Research andDevelopment under research grants: “Nanomaterials and their applica-tion to biomedicine” (PBS1/A9/13/2012), and “Development of an inno-vative technology using transgenic porcine tissues for biomedicalpurposes” (INNOMED/I/17/NCBR/2014).

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Anna Woźniak - Ph.D. in Biology, University of MedicalScience in Poznan (2006), Manager of Biological Laboratoryin NanoBioMedical Centre, Adam Mickiewicz University inPoznań, Poland. Research interests: cell cultures; cytotoxicitystudy of metallic, magnetic, polymer, carbon nanoparticles;toxicity study of metallic, magnetic nanoparticles in vitro(zebrafish): hatching ratio, viability, developmentalmalformations assays; nanoparticle-based delivery study:nucleic acids, drugs, fluorescent dyes, targeted drug delivery,multimodal NPs; cancer cell lines/tumours implantation intomice, basic medical procedures, planning and providing pro-cedures and experiments, providing procedures, handling(Certificate of Polish Society of Laboratory Animal Science –

PolLASAno2066/2015). Foundermember of Zebrafish PolishAssociation.

Bartosz F Grześkowiak - Ph.D., Agricultural sciences –Biotechnology, Poznan University of Life Sciences,NanoBioMedical Centre, Adam Mickiewicz University inPoznan (2014), specialist in Biological Laboratory inNanoBioMedical Centre, Adam Mickiewicz University inPoznań, Poland. Research interests: synthesis, characteriza-tion and biofunctionalization of nanoparticles; developmentof nanosystems for nucleic acid delivery into cells; investiga-tion of nanoparticles:DNA complexes internalization intocells; cytogenetic and physical gene mapping, gene expres-sion analysis.

Nataliya Babayevska - Ph.D. in Technical Sciences, Speciali-zation: Materials Science, Institute for Single Crystals NASof Ukraine, (2010); specialist in Chemical Laboratory inNanoBioMedical Centre, AdamMickiewicz University in Poz-nań, Poland. Research interests: nanoparticles and films ofrare earth oxides and semiconductor oxides; synthesis andstudy ofmorphology, structure and luminescence properties.Patents/patent applications: Patent of Ukraine “Red EmissionPhosphor”, N.V. Babayevska, O.N. Bezkrovna, S.S. Olіynik,Yu.N. Savvіn, A.V. Tolmachov. Patent of Ukraine № 89,32811 2010 Dire p.

Tomasz Zalewski - Ph.D. in Physics, Adam MickiewiczUniversity in Poznan, Poland, (2007), Manager of MRI Spec-troscopy Laboratory in NanoBioMedical Centre, AdamMickiewicz University in Poznań, Poland. Scientific disci-pline: physics, biophysics, medical physics. Research inter-ests: bioimaging using MRI, NMR spectroscopy; moleculardynamics in biological systems; contrast agent for MRI andFMT.

Monika Drobna -Master Degree in Biotechnology at PoznańUniversity of Life Sciences (2016), PhD student in Depart-ment of Molecular and Clinical Genetics, Institute of HumanGenetics, Polish Academy of Science, Poznań, Poland. Re-search interests: analysis of miRNA expression profile inchildhood T-cell acute lymphoblastic leukaemia (T-ALL)and functional studies of influence of inhibition andmimicryof chosen miRNA on leukaemia cells cultures.

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and

Marta Woźniak-Budych - PhD in chemistry, Faculty ofChemical Technology, Poznan University of Technology(2015), specialist in Chemical Laboratory in NanoBioMedicalCentre, Adam Mickiewicz University in Poznań, Poland. Re-search interests: physical chemistry; membrane techniques;separation of lowmolecular weight organic compounds pro-duced by fermentation process.

A. Woźniak et al. / Materials Science

Małgorzata Wiweger - PhD, Head of the Zebrafish Core Fa-cility in International Institute of Molecular and Cell Biology,Warsaw, Poland. Specialties: cell and molecular biology; tis-sue culture; microscopy (light, fluorescent and confocal);zebrafish; drug screens; histology; developmental biology;cartialge, bone; proteoglycans; multiple osteochondromas;plant biology. Member of Polish Laboratory Animal ScienceAssociation and founder member of Zebrafish Polish Associ-ation.

Ryszard Słomski - Habilitation inmedicine, Full professor ofmedicine. Head of the Department of Biochemistry and Bio-technology, Poznań University of Life Sciences, Professor atInstitute of Human Genetics, Polish Academy of Sciences inPoznan. Research interests: preparation of gene constructscontaining human genes, nanodelivery to bacterial and ani-mal cells, including large animals and preparation of othergene constructs for prokaryotic and eukaryotic systems;characterization of novel human, animal and plant genes;paternity and relationship testing based onDNA studies;mo-lecular diagnostics of human diseases; bioimaging.

615Engineering C 80 (2017) 603–615

Stefan Jurga - Director of the NanoBioMedical Centre, Poz-nań, Poland. Research interests: nanotechnology in materialscience and biomedical research; structure and dynamics insoft matter and solid state studied by MRI, NMR, X-ray scat-tering; optical, electron and atomic microscopies; NMR re-laxation and diffusion; Raman and FTIR spectroscopies;nanotechnology fabrication techniques. Prof. Jurga has helda wide range of responsible functions in the Polish academicsystem: from 2005 to 2007 he was appointed as Poland'sVice- Minister of Science and Higher Education of Poland;as Vice Dean (1987–1990), Vice Rector (1990–1996) Rectorof the Adam Mickiewicz University (1996–2002) in Poznań.

ZnO@Gd2O3 core/shell nanoparticles for biomedical applications: Physicochemical, in vitro and in vivo characterization1. Introduction2. Materials and methods2.1. Materials2.2. Synthesis of core/shell ZnO@Gd2O3 core/shell nanoparticles2.3. Functionalization of ZnO@Gd2O3 core/shell nanoparticles by oleic acid and aminosiloxanes2.4. Biofunctionalization of ZnO@Gd2O3@OA-polySi core/shell nanoparticles by folic acid and doxorubicin2.5. Physicochemical characterization2.6. Magnetic Resonance Imaging2.7. Cell culture2.8. Cytotoxicity assays2.9. Confocal bioimaging2.10. Toxicity in vivo

3. Results and discussion3.1. Morphology and structure characterization3.2. Functionalization and biofunctionalization of ZnO@Gd2O3 nanoparticles3.3. Magnetic Resonance Imaging3.4. In vitro cytotoxicity3.5. Confocal bioimaging3.6. In vivo toxicity3.6.1. Survival and hatching rate3.6.2. Developmental abnormalities

4. ConclusionsConflict of interestFunding sourcesReferences