Magnetic oxide particles with gold nanoshells: Synthesis, properties and cytotoxic effects

Accepted Manuscript Title: Magnetic oxide particles with gold nanoshells: synthesis, properties and cytotoxic effects Authors: Jakub Koktan, Karel Kr´alovec, Radim Havelek, ˇ Jarmila Kuliˇckov´a, Pavel Rezanka, Ondˇrej Kaman PII: DOI: Reference:

S0927-7757(17)30186-3 http://dx.doi.org/doi:10.1016/j.colsurfa.2017.02.052 COLSUA 21407

To appear in:

Colloids and Surfaces A: Physicochem. Eng. Aspects

Received date: Revised date: Accepted date:

17-1-2017 20-2-2017 20-2-2017

Please cite this article as: Jakub Koktan, Karel Kr´alovec, Radim Havelek, ˇ Jarmila Kuliˇckov´a, Pavel Rezanka, Ondˇrej Kaman, Magnetic oxide particles with gold nanoshells: synthesis, properties and cytotoxic effects, Colloids and Surfaces A: Physicochemical and Engineering Aspects http://dx.doi.org/10.1016/j.colsurfa.2017.02.052 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Magnetic oxide particles with gold nanoshells: synthesis, properties and cytotoxic effects

Jakub Koktana,b*, Karel Královecc, Radim Havelekc, Jarmila Kuličkováb, Pavel Řezankaa and Ondřej Kamanb

a

University of Chemistry and Technology Prague, Department of Analytical Chemistry,

Technická 5, 166 28 Prague 6, Czech Republic. b

Institute of Physics, Academy of Sciences of the Czech Republic, Cukrovarnická 10/112,

162 00 Prague 6, Czech Republic. c

University of Pardubice, Department of Biological and Biochemical Sciences, Studentská 95

532 10 Pardubice, Czech Republic. *

Corresponding author. Tel.: +420 220 318 417; fax: +420 233 343 184. E-mail address:

[email protected]

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GRAPHICAL ABSTRACT

HIGHLIGHTS 

Gold nanoshells are formed around superparamagnetic Mn-Zn ferrite with silica



The product shows low cytotoxic effects and efficient internalization into cells



The nanoparticles show typical optical properties and high magnetization

2

Abstract In this paper, a successful synthesis and detailed study of complex Mn-Zn ferrite core-shell nanoparticles, whose shell is composed of primary silica layer and secondary gold coating, are presented. Magnetic cores with chemical composition Mn0.61Zn0.42Fe1.98O4 and high specific magnetization are synthesized as a single-phase product by hydrothermal procedure. The cores are embedded in amorphous silica by a modified Stöber process, and gold shell is formed on their surface by a seed-and-growth synthesis. Analysis of intermediates and the final product by transmission electron microscopy reveals the course of the multistep synthesis. Energy dispersive X-ray spectroscopy, ICP-MS, and magnetic measurements provide coherent results on the chemical composition of the prepared particles. Colloidal stability of the complex particles in water is evidenced by dynamic light scattering and explained on the basis of zeta-potential measurements. In order to demonstrate the possibility of further functionalization, 4-mercaptobenzoic acid and 7-mercapto-4-methylcoumarin are covalently attached to the surface of the particles. Both the molecules provide strong SERS signals, and the nanoparticles modified with the coumarin derivate constitute an efficient label for fluorescence microscopy. Finally, biological study on two different cell lines is carried out, dealing with the viability and real-time monitoring of proliferation and adhesion of cells. The results indicate only weak cytotoxic effects, and cell viabilities remain generally higher than 90 %. Moreover, fluorescence microscopy demonstrates an extensive internalization of the nanoparticles.

Keywords Cytotoxicity;

Core-shell nanoparticles; Mn-Zn ferrite; Gold nanoshells; Magnetic

nanoparticles; Surface Plasmon Resonance

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1. Introduction Recently, a significant effort has been expended on the development of theranostic nanoparticles, that combine a diagnostic platform with therapeutically active agents [1]. Typical theranostic particles could serve as contrast agents for imaging techniques, mediators for therapeutic heating induced magnetically or by near-infrared (NIR) radiation, and advanced drug delivery systems with active targeting and controlled release of drugs at the same time. Specifically, nanoparticles with magnetic cores enable visualization of labeled structures by magnetic resonance imaging (MRI), localized heating (magnetic fluid hyperthermia), and magnetically targeted delivery through blood vessels. Gold nanoshells offer fundamentally different modalities, such as photothermal therapy and detection methods based on their plasmonic properties. However, the development of theranostic nanoparticles and a fortiori their deployment in clinical use are hindered owing to complex synthesis, problems related to their long-term stability, and other important issues like toxicity of magnetic cores and biological fate of the whole particles [2]. Traditionally, iron oxide nanoparticles based on magnetite, maghemite, and their mixtures were employed as magnetic cores, but their magnetic properties are somewhat restrictive in terms of modern applications. Higher MRI contrast effect (and thus lower dose of material administered) can be achieved by ferrites or doped magnetite phases owing to higher magnetization. The requirement for highly magnetic cores is particularly important as regards theranostic nanoparticles, that usually constitute larger architectures with relatively low content of the magnetic phase. Moreover, certain ferrites and other complex oxides whose Curie temperature can be adjusted slightly above the body temperature were suggested as promising mediators for the self-regulated magnetic fluid hyperthermia [3]. Mn-Zn ferrites represent a clear example of a magnetic phase which offers better performance in many applications than purely iron oxides, including the magnetite Fe3O4. The ratio of manganese and zinc ions, together with their distribution, determine the balance between antiparallel magnetic moments of tetrahedral and octahedral sublattices and therefore affects the behavior in external magnetic fields. For example, Mn0.6Zn0.4 Fe2O4 nanoparticles proved to possess the highest magnetization, Ms = 108 Am2/kg, in a series of Mn1-xZnxFe2O4 samples prepared by the thermal decomposition method, whereas lower value of Ms = 82 Am2/kg was observed for the Fe3O4 counterpart [4]. Also the MRI properties and hyperthermia

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performance were significantly enhanced for the composition with x = 0.4 compared to the magnetite. Several routes were proposed for the synthesis of Mn1-xZnxFe2O4 nanoparticles, such as coprecipitation, sol-gel processes, thermal decomposition, and hydrothermal procedure, each of them providing somewhat different material as regards the size distribution of particles, their morphology, crystallinity, and surface properties. The presently used hydrothermal synthesis is particularly attractive way because it is a facile, low-cost method that utilizes simple inorganic salt precursors, e.g., compared to the modern thermal decomposition procedure. The preparation of complex nanoparticles with magnetic cores and gold nanoshells is motivated by the combination of magnetic behavior and optical properties like Raman scattering enhancement [5] and absorption of light due to the surface plasmon resonance (SPR) [6]. Moreover, gold nanostructures possess exceptional chemical stability [7], and gold is generally considered as a biocompatible material suitable for medical applications [8,9]. Both these features are important when transitive metal oxides are involved as magnetic cores. Such cores have to be isolated by a durable and biologically inert barrier. Although gold nanoshells can be deposited directly on the surface of various magnetic particles, dielectric interface is essential for SPR to occur in the desired NIR region as well as for precise tuning of the resonance absorption [10]. Further, the encapsulation of magnetic cores into silica provides an intermediate structure with surface properties independent of the core material, which allows diverse functionalization routes and more robust procedures for the deposition of gold. The synthesis of gold nanoshells on silica or silica-coated particles is carried out mostly by a two-step procedure called the seed-and-growth method. In the first step, small gold nanoparticles are attached to the silica substrate, and in the second step, the immobilized seeds grow by reduction of an appropriate Au(III) precursor dissolved in a solution. Before the seeding step, it is necessary to introduce a suitable linker or layer that provides an efficient attachment of gold seeds to the silica surface. The most common linkers employed so far are 3-aminopropyltrialkoxysilanes [7,11], but excellent results were obtained also through the adhesion of polyelectrolytes [12]. As the growing step is concerned, several reducing agents were reported, namely formaldehyde [13], glucose [14], L-ascorbic acid [15], hydroxylamine

5

hydrochloride [16], etc. The differences among these agents are mainly related to the reaction kinetics and surface properties of the resulting gold nanoshells [7]. 2. Experimental 2.1. Chemicals and cell cultures Poly(sodium

4-styrenesulfonate)

30

wt%

in

H2O

with

Mr

≈

70 000

(PSS),

poly(diallyldimethylammonium chloride) 35 wt% in H2O with Mr ≈ 100 000 (PDADMAC), tetraethyl orthosilicate ≥ 99.0 % (TEOS), tetrakis(hydroxymethyl)phosphonium chloride 80 wt% solution in water (THCP), poly(vinylpyrrolidone) K 90 with Mr ≈ 360 000 (PVP), all Sigma–Aldrich. The human ovarian carcinoma cell line A2780 and the human lung carcinoma line A549 (both p53 wild-type) were obtained from the European Collection of Cell Cultures (ECACC). A2780 cells were propagated in RPMI 1640 medium supplemented with foetal bovine serum 10 %, L-glutamine 2 mmol/L, pyruvate 1 mmol/L, HEPES 10 mmol/L, MEM Non-Essential Amino Acids 10 µL/mL, penicillin 50 µg/mL, and streptomycin 50 µg/mL (all reagents from Life Technologies). A549 cells were cultured in Minimum Essential Medium Eagle with Lglutamine and sodium bicarbonate (Sigma-Aldrich) in the presence of foetal calf serum 10 %, pyruvate 1 mmol/L, HEPES 10 mmol/L, penicillin 50 µg/mL, and streptomycin 50 µg/mL (all supplements from Life Technologies). All cell lines were maintained and grown at 37 °C, 95 % humidity, 5 % CO2. A2780 and A549 cells in the maximum range of 20 passages and in an exponential growth phase were used for this study. 2.2. Synthesis of Mn0.61Zn0.42Fe1.98O4 ferrite nanoparticles (MZF) MZF particles were prepared by hydrothermal technique as follows. Aqueous solutions of Fe(III), Mn(II), and Zn(II) nitrates with concentrations determined by titration were mixed in ratio Mn : Zn : Fe = 0.6 : 0.4 : 1.80, forming the total of 5.6 mmol of metals, and concentrated in vacuo. Further procedure was performed under inert atmosphere in a glove bag. While stirring, a solution of NaOH was slowly added to reach pH = 10.0 at the final volume of 25 mL. During alkalization, brown precipitates were formed, and the so-obtained mixture was transferred into a Berghof DAB-2 pressure vessel with a 50 mL teflon insert and subjected to the hydrothermal treatment under autogenous pressure at 180 °C for 12 h. Thereafter, the

6

product was decanted, washed several times with water and transferred into ethanol. The typical amount of material prepared in a single batch was 430 mg. 2.3. Encapsulation of ferrite cores into silica (MZF@si) Approximately 115 mg of MZF dispersed in 12 mL of ethanol were added into 300 mL 12 % PVP aqueous solution and sonicated for 12 h. The particles were separated by centrifugation, washed once with 20 mL of ethanol, and the residue was redispersed in 190 mL of ethanol by ultrasound sonication for 2 h. The mixture was transferred into a round-bottom flask equipped with mechanical stirrer and placed in an ultrasound bath tempered to 25 °C by an external thermal regulator. During mechanical stirring and ultrasound irradiating, 0.6 mL of TEOS was added, followed by 32 mL of ammonia hydroxide solution 29 %. After one minute, the ultrasound was turned off, and the mixture was further agitated only by mechanical stirring. The procedure was terminated after 12 h. Raw MZF@si product was separated, washed four times with ethanol, three times with water and redispersed in 240 mL of water. The heavy fractions were removed from the product by centrifugation at 260 RCF for 15 min, supernatant was collected and concentrated to the volume of 20 mL. The typical yield of encapsulation was about 25 % with respect to magnetic cores. 2.4. Preparation of gold nanoshells with silica-coated ferrite cores Gold nanoshell formation was performed by seed-and-growth procedure. First, small gold nanoparticles (GNP), possessing approximately 2 nm in diameter, were prepared by Duff’s method [17]. Diluted solutions of THPC (2 mL, 67 µmol/L) and HAuCl4 (4 mL, 25 mmol/L) were added to a round-bottom flask containing aqueous solution of NaOH (93 mL, 6.5 mmol/L) under magnetic stirring. Immediately, a brownish suspension of GNP was formed. After 24 h the product was purified from mother liquor as follows. The suspension of nanoparticles was concentrated in vacuo to 20 mL and destabilized with 60 mL of acetone. The mixture was centrifuged at 7450 RCF for 1 h, and supernatant was removed. Nanoparticles were redispersed in water, and the residual acetone was evaporated in vacuo. The final volume of GNP suspension was adjusted to 100 mL. GNP nanoparticles were attached to the surface of MZF@si by electrostatic modification of silica. Suspension of MZF@si (2 mL, 0.20 mg(Mn)/g) was added to 1 mL aqueous solution of NaCl (3 mol/L) with PDADMAC (5 µL). The mixture was placed in an ultrasound bath and sonicated for 30 min. Nanoparticles were isolated by centrifugation and redispersed in pure 7

water (2 mL). This treatment was repeated in exactly the same way with PSS and again with PDADMAC. After the deposition of polyelectrolytes, nanoparticles were washed three times with water. The particles were added dropwise to a round-bottom flask with GNP (30 mL, 200 mg(Au)/L) in an ultrasound bath tempered to 25 °C. The mixture was treated by ultrasound overnight and subjected to centrifugation at 4200 RCF for 30 min. Silica-coated ferrite particles seeded with GNP (MZF@si@GNP) were sedimented on the bottom of the tube, whereas excess GNP nanoparticles, un-attached to silica, remained in the suspension. The product was washed six times with water to remove any residual GNP nanoparticles, and the final volume was adjusted to 20 mL (concentration approximately 0.02 mg(Mn)/g). Deposition of gold shell on the silica surface seeded with GNP was carried out through reduction of HAuCl4 by L-ascorbic acid. First, the so-called K-gold solution was prepared. HAuCl4 ·3H2O (0.25 mmol) was dissolved in water (100 mL), and then K2 CO3 (1.2 mmol) was added to alkalize the solution. The mixture was stored in dark and cold conditions overnight, which was accompanied by transition from pale yellow to colourless. The soobtained K-gold solution was diluted 20 times for further use. Diluted MZF@si@GNP particles (10 mL, concentration approximately 1 g(Mn)/g) were mixed with L-ascorbic acid (6 µmol) in a two-neck round-bottom flask. K-gold solution (20 mL) was added slowly dropwise (1 mL/min) while mechanical stirring and ultrasound irradiation was applied. The mixture changed its color from pale red, through purple to deep blue-green, indicating gradual growth and fusion of gold nanoparticles attached to the surface of silica. The raw product was separated by centrifugation and washed once with water. The heavy fraction was removed via centrifugation at 120 RCF for 5 min, and the corresponding supernatant was separated as the final product. The typical yield of gold nanoshell synthesis was about 40 % with respect to magnetic cores. 2.5. Functionalization of nanoshells with reporter and fluorescent molecules Potential application of MZF@si@Au for surface-enhanced Raman scattering (SERS) was investigated after functionalization with 4-mercaptobenzoic acid (MBA) and 7-mercapto-4methylcoumarin (MMC) as reporter molecules. Specifically, MZF@si@Au nanoparticles (100 µL, 4 mmol(Au)/L) were separated by centrifugation and redispersed either in acetonitrile solution of MMC (0.5 mL, 50 mmol/L) or in ethanol solution of MBA (0.5 mL, 50 mmol/L). Mixtures were agitated overnight. MZF@si@Au modified with MMC or MBA (denoted as MZF@si@Au-MMC and -MBA) were separated by centrifugation and washed 8

three times with either acetonitrile or ethanol, respectively, and once with water. The modified particles were redispersed in 0.5 mL of water, and the suspensions obtained in this way were directly analyzed. Moreover, MZF@si@Au-MMC nanoparticles were used as fluorescent probes for optical microscopy. 2.6. Physical and chemical characterization Chemical analysis Chemical analysis of solutions employed for hydrothermal synthesis was performed by chelatometric titration of Fe3+, Zn2+, and Mn2+. The precise chemical composition of the MZF product was determined by X-ray fluorescence spectroscopy (XRF, Panalytical Axios). The concentration of MZF@si nanoparticles was accurately determined by atomic absorption spectroscopy (AAS, GBC Scientific Equipment SensAA) with flame atomization. For this purpose, samples were prepared by wet chemical digestion in an open system with hydrofluoric acid and nitric acid. Hydrogen peroxide was added to reduce all manganese to Mn(II). Digestion was performed in triplicates, and the standard addition method with fourpoint calibration was used to eliminate any matrix effects. The measurement was carried out in an acetylene-air flame and the concentration was determined by the measurement of manganese content at λ = 279.5 nm. In contrast, MZF@si@Au concentration was determined by inductively-coupled plasma mass spectrometry (ICP-MS, Perkin-Elmer Elan 6000) measurements of gold and manganese content. The decomposition was carried out similarly to AAS but the sample was treated with mixture of nitric and hydrochloric acid to dissolve gold shell, and no hydrogen peroxide was used. X-ray diffraction and magnetometry The crystal structure, phase composition, and mean size of crystallites (dXRD) were determined by X-ray powder diffraction (XRD) in the Bragg-Brentano geometry (Bruker D8 diffractometer). The crystal phases were identified according to the ICSD database, and the program FULLPROF was used to analyze diffraction patterns by means of the Rietveld method. The Thompson-Cox-Hastings pseudo-Voigt profile was utilized to evaluate size and strain contributions to the peak broadening. A strain-free tungsten powder with particles size of 9.4 µm was utilized to determine the instrumental profile.

9

Magnetic properties of prepared nanoparticles were probed by SQUID magnetometry on wellcompacted powder samples by using an MPMS XL measuring system (Quantum Design). Silica and gold-coated samples were measured after defined drying at 105 °C for 10 h. Transmission electron microscopy The morphology and chemical composition of core-shell nanoparticles were studied by transmission electron microscopy (TEM, Philips CM 120) combined with energy dispersive X-ray spectroscopy (EDS, Edax silicon drift detector Apollo XLTW). Samples were prepared on carbon-coated copper grids by air-drying of aqueous suspension. Image analysis of 200 particles, whose cross-sections were measured by ImageTool software, was applied to evaluate the size distribution of MZF cores, MZF@si, and MZF@si@Au particles. The size of a particle, d, was estimated from the area of the observed cross-section, S, according to the spherical approximation: 𝑑 = 2 ∙ √𝑆/𝜋. The EDS analysis was applied for MZF@si@Au particles, and their composition was quantified by virtue of TEAM™ EDS software by using the MThin method with corrections on absorption and fluorescence. Weight fraction of elements was normalized on the sum of Au, Si, Zn, Mn, and Fe. Emission lines of K series were used to quantify the content of Si, Zn, Mn, and Fe, whereas Au content was quantified according to L emission lines. Hydrodynamic size and zeta-potential measurements The hydrodynamic size and colloidal stability of particles in aqueous suspension were investigated via dynamic light scattering (DLS, Malvern Instruments Ltd. Zetasizer) measurements. Hydrodynamic diameter was described by Z-average value, and the width of distribution was expressed by polydispersity index (PdI) [18]. In addition, the colloidal stability of nanoparticles under different pH was studied through analysis of zeta-potential, that was measured by laser Doppler electrophoresis carried on the same instrument. Optical properties The SPR effects were studied on dilute suspensions of particles in water by measurement of absorption spectra on a Cary 300 UV-Vis spectrophotometer (Agilent Technologies). SERS spectra were obtained by Raman NIR Advantage spectrometer (DeltaNu) with laser excitation line 785 nm, power 100 mW in the range of 200–2000 cm-1 with spectral resolution 4 cm-1. 2.7. Biological study 10

Evaluation of cytotoxicity by XTT assay The effects of MZF@si@Au nanoparticles on the proliferation and viability of A2780 and A549 cells were quantified by means of the XTT assay, i.e., a colorimetric assay for the determination of activity of mitochondrial dehydrogenases, which correlates with the number of living cells. In addition, the same experiments were performed with cisplatin (cis-Pt) as a standard to compare the cytotoxic effect of particles. The cells were seeded at optimal density, determined in preceding experiments, in a 96-well plate and were allowed to settle overnight. Thereafter, the cells were treated with MZF@si@Au nanoparticles at the concentration of 0.025–0.2 mmol(Au)/L and cis-Pt at 5 or 10 µmol/L for 48 h. After the given incubation, the cell viability was determined by using Cell Proliferation Kit II (XTT, Roche) according to manufacturer's instructions. The assay was carried out with 200 μL of cell suspension and 100 μL of XTT-labelling mixture (containing the 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)2H-tetrazolium-5-carboxanilide reagent). Absorbance was measured at 480 nm by a 96multiwell microplate reader Tecan Infinite M200 (Tecan Group Ltd.). The viability was quantified as described in Ref. [19] according to the following formula: (%) viability = (Asample - Ablank) / (Acontrol - Ablank) x 100, where A is the absorbance of the employed XTT formazan measured at 480 nm. All experiments were carried out as four independent replicates. The viability of the treated cells was normalized to the viability of cells treated with sterile deionized water (Lonza) as a vehiculum control. Cells treated with 5 % DMSO were used for positive control. Real-time monitoring of cells by xCELLigence system The RTCA SP xCELLigence system (Roche and ACEA Biosciences) was used to monitor cell adhesion and proliferation of A2780 and A549 cells treated with MZF@si@Au nanoparticles. The system had been tested by a Resistor Plate before the RTCA Single Plate station was placed inside the incubator at 37 °C with 5 % CO2. First, the optimal seeding concentration for experiments was optimized for each cell line. Background measurements were taken by adding 100 µL of an appropriate medium to the wells of an E-Plate 96. Cell suspension (90 µL) was added to each well of the E-plate 96 at the cell density of 15 000 cells per well for A2780 and 7 000 cells per well for A549. The cells were monitored every 30 minutes by the xCELLigence system. Approximately 24 h later, when the cells were in the log growth phase, the cells were exposed in tetraplicates to 10 µL of sterile deionized water with MZF@si@Au particles to obtain final concentrations of 0.2–0.025 mmol(Au)/L. Negative 11

controls received sterile deionized water for cell cultures (Lonza), whereas cells treated with 5 % DMSO were used as positive controls. Monitoring of cells was under way for 48 h. Evaluation of data was performed using xCELLigence 1.2.1 software, and the growth curves were normalized to the time point of the treatment. The presented results were obtained by averaging of at least four independent replicates under the same conditions. Fluorescence microscopy For each experiment, 350 000 of A549 cells in 2 mL of complete culture medium per well were seeded in 2-well chamber slides SPL (SPL Life Sciences, Korea). After seeding (usually 24 h later), the medium was replaced with fresh one, and particles were added to reach the concentration of 0.05 mmol(Au)/L. Both the MZF@si@Au-MMC and MZF@si@Au were tested to confirm the origin of fluorescence. Further, cells treated with a respective volume of sterile deionized water for cell cultures (Lonza) were used as a vehicle control. Following 3-h treatment, the cells were washed 3 times with phosphate-buffered saline (PBS), fixed with freshly prepared paraformaldehyde (4 %, 10 min, room temperature), and again washed 3 times with PBS. Then, the slides were covered with 2 mL of the complete culture medium containing 2 µL (100 mg/mL) of propidium iodide (PI) solution (Sigma-Aldrich) to counterstain cells nuclei. After 5 min of staining, three washing cycles in PBS followed, the slides were mounted with an antifading ProLong® Gold mounting medium (Life Technologies). The samples were inspected with a Nikon epifluorescence microscope system Eclipse 80i; the exposure time and dynamic range of the camera in all channels were adjusted to the same values for all of the slides to portray quantitatively comparable images. Band pass filter cube designed for DAPI was used to capture fluorescence of MZF@si@Au-MMC, whereas PI fluorescence was captured by using a TRITC band pass filter cube. Samples were visualized under oil-immersion at 1000 × magnification. Images were further processed and merged using NIS-Elements Advanced Research 4.13 (instrument, filters and software from Nikon). 2.8. Statistical analysis The results of image analysis, EDS measurements, and evaluation of cytotoxicity are expressed as arithmetic means. The respective standard deviations (sd) are calculated as uncorrected sample standard deviation according to formula 𝑠𝑑 = √

∑(𝑥−𝑥̅ )2 (𝑛−1)

. The significant

differences between the groups were analyzed using the Student's t-test. 12

3. Result and discussion 3.1. Chemical composition and crystal structure of ferrite cores The chemical composition of the prepared MZF nanoparticles was determined by XRF to be Mn0.61Zn0.42Fe1.98O4. The XRD analysis of the sample is illustrated in Fig. 1. Diffraction pattern showed a single-phase product of the spinel structure with symmetry Fd3̅m. The cubic lattice parameter a was equal to 8.4576(4) Å, and the mean size of crystallites was dXRD = 11 nm. The lattice constant is in good agreement with values reported for comparable samples, e.g., the hydrothermal treatment of a mixture of Mn2+ : Zn2+ : Fe3+ = 0.65 : 0.35 : 2 that was precipitated at pH = 10 provided Mn-Zn ferrite particles with a = 8.453 Å [20], whereas the bulk sample of Mn0.60Zn0.40Fe2O4 phase was characterized by somewhat larger cell volume with a = 8.4975 Å [21]. The minor difference might be attributed either to slightly different oxygen stoichiometries of nanocrystalline and bulk samples or to the presence of compressive strain in nanoparticles. Some difference in the oxygen stoichiometry can be expected taking into account the markedly distinct temperatures employed for the hydrothermal preparation of nanoparticles and for classical sintering of bulk samples. Moreover, it was evidenced that the actual oxygen stoichiometry of nanoparticles of certain metal oxides is higher in consequence of the saturation of their surface with oxygen [22]. Interestingly, such chemisorption of oxygen on La1-xSrxMnO3 particles is compensated by an increase in the oxidation state of manganese in the uppermost layers of the particles, which even induces a compressive lattice strain [22,23]. It is worthwhile to highlight that the composition of the product almost reflected the initial molar ratio of Mn and Zn nitrates in the starting solution. The amount of Fe(NO3)3 employed in the synthesis was intentionally substoichiometric (Mn+Zn) : Fe = 1.80 because lower molar ratio of Fe to the other metals guarantees that the obtained product is pure and does not contain hematite, α-Fe2O3. At the same time, the precipitation of the precursor before the hydrothermal treatment has to be performed under inert atmosphere. Otherwise, the presence of oxygen leads to partial oxidation of Mn(II), and thus -Fe2O3 and hausmannite phases, Mn3-xZnxO4, occur as admixtures (see also the diffraction patterns in Fig. S1 of Supplementary materials for illustration) [2]. 3.2. Silica coating and gold shell formation 13

The morphology and size of MZF nanoparticles, silica-coated particles (MZF@si), intermediate product with gold seeds (MZF@si@GNP), and final gold nanoshells (MZF@si@Au) is illustrated by representative transmission electron micrographs in Fig. 2 (for further images see Fig. S2 in Supplementary materials). The MZF@si particles are characterized by uninterrupted silica coating with smooth surface. Although the shell thickness slightly varies within each coated particle on account of very complex shape of the magnetic cores, its average thickness is very uniform in the whole sample. The magnetic cores of MZF@si are formed by small clusters of nanoparticles rather than individual ferrite nanoparticles. In details, the extensive image analysis of MZF@si nanoparticles revealed that the mean diameter of cores was approximately ̅̅̅ 𝑑i = 22 nm with standard deviation of 5 nm, and the mean diameter of silica-coated nanoparticles was 𝑑̅j = 38 nm (sd = 5 nm). Considering the difference between 𝑑̅i and 𝑑̅j , the mean thickness of silica coating was 8 nm (sd = 3 nm). Detailed histograms of di and dj are presented in Fig. 3. As regards the intermediate product, gold seeds are homogeneously immobilized on its surface in high density. The seeds are present exclusively on the silica surface, whereas unattached gold particles are not observed. Actually, no free gold seeds were recorded even after thorough washing with water, sonication, and long storage of the sample within several weeks, which indicates high stability and long-term disposability of the intermediate product. It is supposed that the small gold clusters are attached to the silica surface via electrostatic adhesion [12] since the negatively charged silica was modified to positively charged surface by adsorption of the polyelectrolytes (consecutive adsorption of PDADMAC, PSS, and PDADMAC, respectively) prior to the deposition of gold seeds. Polyelectrolytes adhesion on silica surface was discussed elsewhere [24] but in short terms, several layers of polymers and higher ionic strength of the solution used for the deposition (addition of NaCl) provide a thicker polyelectrolyte film, that increases the degree of coverage. Preliminary optimization of the gold shell formation suggests that the concentration of the Kgold solution and the content of MZF@si@GNP particles in the mixture are the crucial parameters. Too high K-gold concentration or too low MZF@si@GNP concentration resulted in nucleation of gold, whereby free gold nanoparticles were formed in the reaction mixture. Therefore, the K-gold solution was added dropwise to the seeded particles to keep its instantaneous concentration low and to prevent formation of free gold. Other parameters, 14

including the concentration of L-ascorbic acid and the temperature during reduction, did not seem to be comparably significant. According to TEM (see Fig. 2), the optimized gold reduction on the surface of MZF@si@GNP particles resulted in a rather rough gold layer. The gold shell, having been formed by gradual growth of gold seeds and their coalescence, is almost continuous but the individuality of the original gold particles as well as some minor imperfections (varying thickness and potentially even porosity) are observable. ̅̅̅𝑘 ) according to the image analysis of TEM The mean diameter of MZF@si@Au particles (𝑑 micrographs was estimated to 100 nm with standard deviation 14 nm (for histogram see Fig. 3). The average gold shell thickness was evaluated to 31 nm (sd = 7 nm) based on the difference between 𝑑̅𝑗 and ̅̅̅ 𝑑𝑘 . ̅̅̅̅̅̅ DLS measurements of dilute aqueous suspensions showed that Z-average diameters (𝑑 𝐷𝐿𝑆 ) of MZF@si and MZF@si@Au particles were 134 nm and 155 nm, respectively (see Fig. 4). The difference in the hydrodynamic radii does not correspond to the gold nanoshell thickness estimated from the TEM analysis. However, the hydrodynamic size includes also the hydration layer around particles and reflects their aggregation. Therefore, the smaller apparent thickness of gold nanoshell obtained from DLS can be explained by a more robust hydration layer bound to silica surface compared to gold surface on the assumption that the silica-coated particles do not aggregate more than the gold-coated ones. It worth mentioning that the silica is characterized by high surface density of both hydrogen bond donors and acceptors and is generally hydrophilic [25], which contributes to stronger hydration. Surprisingly, the intermediate MZF@si@GNP particles exhibited large hydrodynamic size of ̅̅̅̅̅̅ 𝑑𝐷𝐿𝑆 = 156 nm. This finding can be interpreted based either on higher aggregation of the intermediate product or based on a rather loose conformation of the adsorbed polyelectrolytes, that can form some type of corona around particles [26]. The dependence of the zeta-potential on pH is shown for silica-coated particles and final gold nanoshells in Fig. 5. From this point of view, silica-coated particles and gold nanoshells prepared by the above described procedure (i.e., by means of the reduction with L-ascorbic acid) possess similar surface properties. In both cases, the colloidal stability of their aqueous suspensions is given by strong coulombic repulsion that occurs for pH > 5. The observed increase of the MZF@si zeta-potential in suspension with pH > 9 is related to alkaline

15

hydrolysis of the silica layer. In contrast, the zeta-potential of MZF@si@Au nanoparticles remains essentially constant in the alkaline region, which suggests that the gold shell efficiently isolates the primary silica layer and prevents the hydrolysis. The zeta-potential determined for MZF@si@Au under neutral pH is comparable to the values published earlier for citrate-stabilized gold nanoparticles with size of 100 nm, that showed zeta-potential of 41 mV under pH = 7.4 [27]. The EDS analysis of MZF@si@Au was performed to evaluate the homogeneity of coating and chemical composition of the product. For this purpose, a larger agglomerate of particles was randomly selected and analyzed at five different spots. Average EDS spectrum is presented in Fig. 6, and the quantitative data are summarized in Table 1. The analysis proved that the composition of the sample is homogenous, at least, as the variation among whole coated particles is considered. Further, the results from EDS analysis can be compared with the concentration of Mn and Au in the suspension MZF@si@Au analyzed by ICP-MS. The ratio of Au and Mn according to ICP-MS expressed as the relative weight fraction 𝑤𝑡 (Mn) = 𝑚Mn ⁄(𝑚Au + 𝑚Mn ) was 0.009 compared to wt (Mn) = 0.006 determined by EDS measurements. Such difference is negligible taking into account the standard uncertainties of both methods and difficulties related to quantification in EDS analysis. 3.3. Magnetic properties The magnetic measurements of bare MZF cores confirmed the ferrimagnetic nature of the sample and high saturation magnetization (the hysteresis loops at 5 K and 300 K are shown in Fig. S4 of Supplementary materials, and the low temperature curve is also depicted in the inset of Fig. 7A). The specific magnetization of the cores in the field of H = 1500 kA/m was M1500 kA/m = 110 Am2/kg and 56 Am2/kg at 5 K and 300 K, respectively. The hysteresis loop of the MZF cores at 5 K reveal clear hysteresis with the coercive field of Hc = 21 kA/m, whereas practically negligible coercive field, Hc = 0.2 kA/m, is observed at room temperature. In order to get a better insight into the temperature dependence of magnetic behavior, the zero-field cooled (ZFC) and field cooled (FC) susceptibilities, χZFC and χFC, were measured. The temperature derivative of their difference, depicted in Fig. 7B, describes the distribution of blocking temperatures and indicates that most of the particles are in superparamagnetic state at room temperature, and only a negligible component of larger particles in blocked state occurs. 16

Similar response to external fields is observed also for the coated samples, MZF@si and MZF@si@Au, but their magnetizations are considerably lower due to the presence of diamagnetic silica and gold coating, that dilute the magnetic cores (see Fig. 7A). The low temperature data can be used to roughly estimate the weight ratio of the ferrimagnetic phase to the shell components, which leads to the relative weight fraction wt (Mn) = 0.009 (wt (Au) = 0.991, respectively). These values further confirm the aforementioned results of chemical analysis by both EDS and ICP-MS. Further, the ZFC-FC study of MZF@si@Au particles (see Fig. 7B) showed that the distribution of blocking temperatures was shifted to lower temperatures compared to MZF. This finding can be attributed to the suppression of interparticle dipolar interactions by diamagnetic coatings (see, e.g., the effect of silica coatings on different clusters of magnetic particles in [28]).

3.4. Optical properties The attachment of gold nanoparticles and gold shell formation were also investigated by UVVis absorption spectroscopy, and the spectra of all samples are depicted in Fig. 8. GNP used for the seeding step of synthesis do not possess a clear absorption band because of their small size of ≈2 nm, and only a small shoulder is visible at 500 nm. This anomalous behavior of ultrafine gold nanoparticles is usually discussed in relation to their altered electronic structure accompanied by dramatic broadening of the SPR band [29]. The same absorption feature is observed also in the spectrum of the intermediate product MZF@si@GNP with gold seeds attached to the silica surface. In contrast, the final gold nanoshells are characterized by a red-shifted absorption band with the maximum at 650 nm, whose considerable part extends to NIR, which is important for possible use in photothermal applications. The larger width of the SPR band can be explained in relation to the broad size distribution of the gold nanoshells (see also Fig. 3). Considering the geometry of an average nanoshell (the diameter of the inner sphere ≈40 nm, the thickness of the shell ≈30 nm), the localization of the band is in accordance with the dependences described elsewhere [30]. For example, 20 nm thick gold nanoshell deposited on 60 nm silica particles exhibited a maximum of plasmon resonance at 750 nm, whereas reduction of gold nanoshell thickness down to 5 nm was accompanied by a red shift of the maximum over 1000 nm.

17

SERS properties of the prepared gold nanoshells were studied after functionalization with suitable reporter molecules, namely 7-mercapto-4-methylcoumarin and 4-mercaptobenzoic acid [31,32]. Obtained spectra are depicted in Fig. 9. Only two strong vibrations, manifested at 1585 cm-1 and 1070 cm-1, were observed in the spectrum of MZF@si@Au-MBA (see ii and v in Fig. 9). These bands are assigned to aromatic ring vibrations [33]. On contrary, MZF@si@Au-MMC particles possessed more complex SERS spectrum with several highly intense Raman signals characteristic for the coumarin dye [31,34]. Vibrations at 1594 cm-1 and 1534 cm-1 can be assigned to in-plane C=C stretching of lactone ring and modes of benzene ring. The intense peak at 1170 cm-1 is probably associated with both the aryl ring breathing vibrations and the in-plane deformations of the group C(O)–C. Doublet present in the region 1100–1000 cm-1 can be ascribed to bending vibration of C–H and deformation vibration of C–O (see i and iv in Fig. 9) [35].

3.5. Biological tests The analysis of the activity of mitochondrial dehydrogenases for A2780 and A549 cells treated with MZF@si@Au nanoparticles provided quantitative data on cytotoxic effects of the sample at concentrations of 0.025–0.2 mmol(Au)/L (see Fig. 10). The results are supplemented by parallel experiments performed with cisplatin, however at lower concentrations of 5 and 10 µmol/L, to compare the determined viabilities to the cytotoxic effect of a standard anti-cancer drug. The viabilities of both types of cells incubated with nanoparticles at 0.025 mmol(Au)/L for 48 h did not differ significantly from the negative control. Only weak effects of the particles were observed at higher concentrations but the viabilities of the treated cells were still higher than 90 %. In contrast, the experiments with cis-Pt showed that the A2780 cells were strongly affected by even lower concentration of the compared agent. A549 cells seemed to be less susceptible, but the viability at 10 µmol/L still decreased to 77 %. These findings point to the very low toxicity of MZF@si@Au nanoparticles compared to conventional chemotherapy agents, which is not trivial taking into account the complex architecture of these particles and presence of transitive metal oxide in their cores. More detailed insight into the cytotoxic effects of MZF@si@Au sample is provided by the growth kinetics of cells incubated with the nanoparticles at different concentrations. Fig. 11 shows the time course of normalized cell indices (CI) of A2780 and A549 cells based on the 18

measurements by the xCELLigence system. For both the cell lines, the dependences are essentially identical and comparable with the vehiculum control, i.e., the adherent properties and proliferation of cells were not influenced, not even at concentration as high as 0.2 mmol(Au)/L. Finally, the fluorescence microscopy of A549 cells treated with MMC-tagged nanoparticles confirmed that an efficient internalization of the whole gold-coated nanoparticles occurred within 3 hours (Fig. 12). In spite of the low concentration applied, 0.05 mmol(Au)/L, an intense fluorescence signal with perinuclear localization was observed. The supplemental experiment with MZF@si@Au sample (see Fig. S5 in Supplementary material) proved that the observed fluorescence is given solely by the coumarin dye, whereas neither the gold nanoshells on its own nor autofluorescence contributed to the signal. 4. Conclusions Magnetic

nanoparticles

of

the

composition

Mn0.61Zn0.42Fe1.98O4,

possessing

high

magnetization M1500 kA/m = 110 Am2/kg at 5 K, were prepared via hydrothermal procedure. The particles were encapsulated into silica by a modified Stöber process (silica layer of 5 nm thickness) and further covered with gold nanoshells by a two-step seed-and-growth method. The detailed TEM analysis showed a rather rough, but almost continuous gold layer with mean thickness of approximately 30 nm. Importantly, surfactant-free suspensions of the prepared nanoparticles were colloidally stable in the pH range from 4 to 12. The chemical composition of the particles was analyzed by three independent methods, specifically ICPMS, EDS analysis, and SQUID magnetometry combined with XRF, all of which provided coherent results. Optical properties of the prepared particles were characterized by a significant SPR absorption band with a maximum at 650 nm. Moreover, they showed promising properties as regards possible SERS applications, which was demonstrated after their functionalization with two reporter molecules, namely 7-mercapto-4-methylcoumarin and 4-mercaptobenzoic acid. The functionalization with the former molecule provided the particles also with an efficient fluorescent label suitable for fluorescence microscopy. The toxicity was studied in vitro on two different cell lines (A2780 and A549 cells) by means of the quantification of the activity of mitochondrial dehydrogenases and real-time monitoring of their growth kinetics. The nanoparticles exhibited either insignificant or only very weak 19

toxic effect at concentrations of 0.025 to 0.2 mmol(Au)/L and did not interfere with proliferation of cells. Finally, the fluorescence microscopy of cells treated with the fluorescently tagged particles evidenced an efficient cellular uptake. All the properties suggest that the silica coated Mn-Zn ferrite particles with gold nanoshells represent suitable agents for biological and medical applications. Their cores possess high magnetization and their shells show plasmonic properties as SPR and SERS. An efficient fluorescence can be achieved by a facile functionalization and the negligible cytotoxicity is prospective for in vivo applications.

Acknowledgement This work was supported by the Czech Science Foundation [grant number 16-04340S] and by the Ministry of Education, Youth and Sports of the Czech Republic [MSMT No 20SVV/2016]. Further, we would like to thank also to our colleagues Dr. Karel Závěta and Dr. Zdeněk Jirák for careful reading and helpful comments.

20

5. References [1]

S.M. Janib, A.S. Moses, J.A. MacKay, Imaging and drug delivery using theranostic nanoparticles,

Adv.

Drug

Deliv.

Rev.

62

(2010)

1052–1063.

doi:10.1016/j.addr.2010.08.004. [2]

O. Kaman, T. Dědourková, J. Koktan, J. Kuličková, M. Maryško, P. Veverka, R. Havelek, K. Královec, K. Turnovcová, P. Jendelová, A. Schröfel, L. Svoboda, Silicacoated manganite and Mn-based ferrite nanoparticles: a comparative study focused on cytotoxicity, J. Nanoparticle Res. 18 (2016) 100. doi:10.1007/s11051-016-3402-5.

[3]

E. Pollert, P. Veverka, M. Veverka, O. Kaman, K. Závěta, S. Vasseur, R. Epherre, G. Goglio, E. Duguet, Search of new core materials for magnetic fluid hyperthermia: Preliminary chemical and physical issues, Prog. Solid State Chem. 37 (2009) 1–14. doi:10.1016/j.progsolidstchem.2009.02.001.

[4]

J.T. Jang, H. Nah, J.H. Lee, S.H. Moon, M.G. Kim, J. Cheon, Critical enhancements of MRI contrast and hyperthermic effects by dopant-controlled magnetic nanoparticles, Angew. Chemie - Int. Ed. 48 (2009) 1234–1238. doi:10.1002/anie.200805149.

[5]

J.H. Kim, P.P. Pompa, H.G. Baek, B.H. Chung, Nanoembossed gold nanoshell with a fluorescence-like strong SERS signal,

Nanotechnology.

27 (2016)

175704.

doi:10.1088/0957-4484/27/17/175704. [6]

P.K. Jain, K.S. Lee, I.H. El-Sayed, M.A. El-Sayed, Calculated absorption and scattering properties of gold nanoparticles of different size, shape, and composition: Applications in biological imaging and biomedicine, J. Phys. Chem. B. 110 (2006)

21

7238–7248. doi:10.1021/jp057170o. [7]

B.J. Jankiewicz, D. Jamiola, J. Choma, M. Jaroniec, Silica-metal core-shell nanostructures,

Adv.

Colloid

Interface

Sci.

170

(2012)

28–47.

doi:10.1016/j.cis.2011.11.002. [8]

S.C. Gad, K.L. Sharp, C. Montgomery, J.D. Payne, G.P. Goodrich, Evaluation of the Toxicity of Intravenous Delivery of Auroshell Particles (Gold-Silica Nanoshells), Int. J. Toxicol. 31 (2012) 584–594. doi:10.1177/1091581812465969.

[9]

J.M. Stern, V. V. Kibanov Solomonov, E. Sazykina, J.A. Schwartz, S.C. Gad, G.P. Goodrich, Initial Evaluation of the Safety of Nanoshell-Directed Photothermal Therapy in the Treatment of Prostate Disease, Int. J. Toxicol. 35 (2016) 38–46. doi:10.1177/1091581815600170.

[10] X. Ji, R. Shao, A.M. Elliott, R.J. Stafford, E. Esparza-Coss, G. Liang, Z.-P. Luo, K. Park, J.T. Markert, C. Li, Bifunctional Gold Nanoshells with a Superparamagnetic Iron Oxide-Silica Core Suitable for Both MR Imaging and Photothermal Therapy, J. Phys. Chem. C. Nanomater. Interfaces. 111 (2007) 6245. doi:10.1021/jp0702245. [11] S. Watanabe, T. Hiratsuka, Y. Asahi, A. Tanaka, K. Mae, M.T. Miyahara, Flow Synthesis of Plasmonic Gold Nanoshells via a Microreactor, Part. Part. Syst. Charact. 32 (2015) 234–242. doi:10.1002/ppsc.201400126. [12] R. Ashayer, S.H. Mannan, S. Sajjadi, Synthesis and characterization of gold nanoshells using poly(diallyldimethyl ammonium chloride), Colloids Surfaces A Physicochem. Eng. Asp. 329 (2008) 134–141. doi:10.1016/j.colsurfa.2008.07.004. [13] J. Choma, A. Dziura, D. Jamioła, P. Nyga, M. Jaroniec, Preparation and properties of

22

silica–gold core–shell particles, Colloids Surfaces A Physicochem. Eng. Asp. 373 (2011) 167–171. doi:10.1016/j.colsurfa.2010.10.046. [14] J. Tharion, J. Satija, S. Mukherji, Glucose mediated synthesis of gold nanoshells: A facile and eco-friendly approach conferring high colloidal stability, RSC Adv. 4 (2014) 3984. doi:10.1039/c3ra45815f. [15] B.L. Sanchez-Gaytan, S.-J. Park, Spiky Gold Nanoshells, Langmuir. 26 (2010) 19170– 19174. doi:10.1021/la1038969. [16] I.Y. Goon, L.M.H. Lai, M. Lim, P. Munroe, J.J. Gooding, R. Amal, Fabrication and Dispersion of Gold-Shell-Protected Magnetite Nanoparticles: Systematic Control Using Polyethyleneimine, Chem. Mater. 21 (2009) 673–681. doi:10.1021/cm8025329. [17] D.G. Duff, A. Baiker, P.P. Edwards, A new hydrosol of gold clusters. 1. Formation and particle size variation, Langmuir. 9 (1993) 2301–2309. doi:10.1021/la00033a010. [18] Zetasizer nano series user manual, (2013). [19] R. Havelek, P. Siman, J. Cmielova, A. Stoklasova, J. Vavrova, J. Vinklarek, J. Knizek, M. Rezacova, Differences in vanadocene dichloride and cisplatin effect on MOLT-4 leukemia and human peripheral blood mononuclear cells, Med. Chem. (Los. Angeles). 8 (2012) 615–21. [20] C. Rath, K.. Sahu, S. Anand, S.. Date, N.. Mishra, R.. Das, Preparation and characterization of nanosize Mn–Zn ferrite, J. Magn. Magn. Mater. 202 (1999) 77–84. doi:10.1016/S0304-8853(99)00217-6. [21] U. König, G. Chol, Röntgenbeugungs- und Neutronenbeugungsuntersuchungen an Ferriten der Reihe MnxZnl-xFe2O4, J. Apllied Crystallogr. 1 (1968) 124–126. 23

[22] P. Žvátora, M. Veverka, P. Veverka, K. Kn????ek, E. Pollert, V. Kr??l, G. Goglio, E. Duguet, K. Z??v??ta, O. Kaman, Influence of surface and finite size effects on the structural and magnetic properties of nanocrystalline lanthanum strontium perovskite manganites, J. Solid State Chem. 204 (2013) 373–379. doi:10.1016/jjssc.2013.06.006. [23] M. Kačenka, O. Kaman, Z. Jirák, M. Maryško, P. Veverka, M. Veverka, S. Vratislav, The magnetic and neutron diffraction studies of La1−xSrxMnO3 nanoparticles prepared via molten salt synthesis, J. Solid State Chem. 221 (2015) 364–372. doi:10.1016/j.jssc.2014.10.024. [24] S.T. Dubas, J.B. Schlenoff, Factors Controlling the Growth of Polyelectrolyte Multilayers, Macromolecules. 32 (1999) 8153–8160. doi:10.1021/ma981927a. [25] J. Trébosc, J.W. Wiench, S. Huh, V.S.-Y. Lin, M. Pruski, Solid-state NMR study of MCM-41-type mesoporous silica nanoparticles, J. Am. Chem. Soc. 127 (2005) 3057– 3068. doi:10.1021/ja043567e. [26] Y. Sun, Y.L. Sun, L. Wang, J. Ma, Y.W. Yang, H. Gao, Nanoassembles constructed from mesoporous silica nanoparticles and surface-coated multilayer polyelectrolytes for controlled drug delivery, Microporous Mesoporous Mater. 185 (2014) 245–253. doi:10.1016/j.micromeso.2013.11.020. [27] G. Sonavane, K. Tomoda, K. Makino, Biodistribution of colloidal gold nanoparticles after intravenous administration: Effect of particle size, Colloids Surfaces B Biointerfaces. 66 (2008) 274–280. doi:10.1016/j.colsurfb.2008.07.004. [28] T. Dedourkova, O. Kaman, P. Veverka, J. Koktan, M. Veverka, J. Kulickova, Z. Jirak, V. Herynek, Clusters of Magnetic Nanoparticles as Contrast Agents for MRI: Effect of Aggregation on Transverse Relaxivity, IEEE Trans. Magn. 51 (2015) 1–4. 24

doi:10.1109/TMAG.2015.2438112. [29] S. Link, M.A. El-Sayed, Shape and size dependence of radiative, non-radiative and photothermal properties of gold nanocrystals, Int. Rev. Phys. Chem. (2010) 409–453. doi:10.1080/01442350050034180. [30] L.R. Hirsch, A.M. Gobin, A.R. Lowery, F. Tam, R. a Drezek, N.J. Halas, J.L. West, Metal nanoshells, Ann. Biomed. Eng. 34 (2006) 15–22. doi:10.1007/s10439-005-90018. [31] B. Bassi, A. Taglietti, P. Galinetto, N. Marchesi, A. Pascale, E. Cabrini, P. Pallavicini, G. Dacarro, Tunable coating of gold nanostars: tailoring robust SERS labels for cell imaging, Nanotechnology. 27 (2016) 265302. doi:10.1088/0957-4484/27/26/265302. [32] X. Zhang, L. Guo, J. Luo, X. Zhao, T. Wang, Y. Li, Y. Fu, Metallic Nanoshells with Sub-10 nm Thickness and Their Performance as Surface-Enhanced Spectroscopy Substrate,

ACS

Appl.

Mater.

Interfaces.

8

(2016)

9889–9896.

doi:10.1021/acsami.6b01658. [33] A. Michota, J. Bukowska, Surface-enhanced Raman scattering (SERS) of 4mercaptobenzoic acid on silver and gold substrates, J. Raman Spectrosc. 34 (2003) 21– 25. doi:10.1002/jrs.928. [34] E. Vogel, A. Gbureck, W. Kiefer, Vibrational spectroscopic studies on the dyes cresyl violet and coumarin 152, J. Mol. Struct. 550–551 (2000) 177–190. doi:10.1016/S00222860(00)00385-9. [35] E. Vogel, W. Kiefer, Investigation of the metal adsorbate interface of the system silver coumarin and silver hydrocoumarin by means of surface enhanced Raman

25

spectroscopy,

Fresenius

J.

Anal.

Chem.

361

(1998)

628–630.

doi:10.1007/s002160050972.

26

Fig. 1. XRD pattern with Rietveld refinement of the structure for bare Mn0.61Zn0.42Fe1.98O4 magnetic nanoparticles. Vertical lines show the reflections of spinel structure with Fd3̅m symmetry.

27

Fig. 2. TEM micrographs: A) MZF nanoparticles, B) and C) MZF@si nanoparticles with different magnification, D) and E) MZF@si@GNP nanoparticles, F) and G) MZF@si@Au nanoparticles.

28

Fig. 3. Size distribution of particles: histograms for ferrite cores (d̅i = 22 nm, sd = 5 nm) and silica-coated particles (d̅j = 38 nm, sd = 5 nm) are based on the image analysis of MZF@si sample, whereas the histogram of gold-coated nanoparticles (̅̅̅ dk = 100 nm, sd = 14 nm) was evaluated from the final product. Histograms are fitted with LogNormal distribution function.

29

̅̅̅̅̅̅ Fig. 4. Distribution of hydrodynamic diameter for nanoparticles MZF@si (𝑑 𝐷𝐿𝑆 = 134 nm, PdI = 0.16),

MZF@si@GNP

̅̅̅̅̅̅ (𝑑 𝐷𝐿𝑆 = 156 nm,

PdI = 0.084),

and

MZF@si@Au

̅̅̅̅̅̅ (𝑑 𝐷𝐿𝑆 = 155 nm, PdI = 0.13) obtained by DLS. The presented distributions are average profiles of three measurements and were normalized to the same area.

30

Fig. 5. Zeta-potential of silica-coated particles and final gold nanoshells under various pH of their aqueous suspensions.

31

Fig. 6. EDS analysis of MZF@si@Au. Main peaks are designated by respective element and corresponding electron transitions. The presented spectrum is an average of five measurements performed at different spots of the agglomerate (see Fig. S3). The signal of copper originates from the TEM grid.

32

Fig. 7. Magnetic behavior of the studied particles. A) Magnetization curves: i) MZF@si@Au at 5 K and ii) MZF@si@Au at 300 K, the bottom-right inset is a detail of the same magnetization curves, and the upper-left inset displays iii) bare MZF cores at 5 K and iv) MZF@si at the temperature of 5 K. B) Temperature derivative of the difference of FC and ZFC susceptibilities measured in the field of H = 1.59 kA/m.

33

Fig. 8. UV-Vis absorption spectra of GNP, MZF@si, MZF@si@GNP, and MZF@si@Au in dilute aqueous suspensions.

34

Fig. 9. SERS spectra of i) MZF@si@Au-MMC, ii) MZF@si@Au-MBA, iii) MZF@si@Au, iv) MZF@si@Au substracted from MZF@si@Au-MMC and v) MZF@si@Au substracted from MZF@si@Au-MBA.

35

Fig. 10. Viability of A2780 and A549 cells incubated with MZF@si@Au nanoparticles at different concentrations (0.025–0.2 mmol(Au)/L) or with cis-Pt at concentrations as low as 5 and 10 µmol/L. The bar graphs represent mean values ± sd of four independent experiments. Asterisk indicates significant difference to control (t-test, p ≤ 0.05).

36

Fig. 11. Real-time monitoring of proliferation and cell adhesion by the xCELLigence system: (A) A2780 cells and (B) A549 cells treated with MZF@si@Au nanoparticles at concentrations of 0.025–0.2 mmol(Au)/L. Dash lines denote either the addition of MZF@si@Au suspension for analysis of cytotoxicity or addition of water or DMSO for negative and positive control, respectively.

37

Fig. 12. Fluorescence microscopy images of A549 cells incubated with nanoparticles and thoroughly washed with PBS: A) treatment with MZF@si@Au-MMC at 0.05 mmol(Au)/L for 3 h, B) treatment with MZF@si@Au under the same conditions. Merged photographs show both PI fluorescence (red) and MMC signal (blue). The scale bars are 10 µm.

38

Table 1. Chemical composition of MZF@si@Au according to EDS. Light elements and copper were excluded from the analysis. Element

Spot 1

Spot 2

Spot 3

Spot 4

Spot 5

Mean(sd)

Au [wt%] Si [wt%] Fe [wt%] Zn [wt%] Mn [wt%]

86.46 8.49 3.17 1.03 0.83

92.83 4.96 1.29 0.57 0.35

91.31 4.96 2.32 0.72 0.69

91.67 5.32 1.85 0.72 0.43

96.21 2.20 1.01 0.37 0.21

93(4) 5.2(1.5) 1.9(0.7) 0.7(0.2) 0.5(0.2)

39