Simplified preparation and characterization of magnetic hydroxyapatite-based nanocomposites

Accepted Manuscript Simplified preparation and characterization hydroxyapatite-based nanocomposites

of

magnetic

Stefania Scialla, Barbara Palazzo, Amilcare Barca, Luigi Carbone, Angela Fiore, Anna Grazia Monteduro, Giuseppe Maruccio, Alessandro Sannino, Francesca Gervaso PII: DOI: Reference:

S0928-4931(16)32142-7 doi: 10.1016/j.msec.2017.03.060 MSC 7568

To appear in:

Materials Science & Engineering C

Received date: Revised date: Accepted date:

10 November 2016 22 December 2016 9 March 2017

Please cite this article as: Stefania Scialla, Barbara Palazzo, Amilcare Barca, Luigi Carbone, Angela Fiore, Anna Grazia Monteduro, Giuseppe Maruccio, Alessandro Sannino, Francesca Gervaso , Simplified preparation and characterization of magnetic hydroxyapatite-based nanocomposites. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Msc(2017), doi: 10.1016/j.msec.2017.03.060

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ACCEPTED MANUSCRIPT Simplified preparation and characterization of magnetic hydroxyapatite-based nanocomposites Stefania Sciallaa,*,**, Barbara Palazzoa,b,**, Amilcare Barcac, Luigi Carboned, Angela Fiored,e, Anna Grazia Montedurod,e, Giuseppe Marucciod,e, Alessandro Sanninoa, Francesca Gervasoa a

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Department of Engineering for Innovation, University of Salento, Lecce, Italy b Ghimas S.p.A., c/o Dhitech S.c.a.r.l., Campus Ecotekne, Lecce, Italy c General Physiology Laboratory, DiSTeBA Department, University of Salento, Lecce, Italy d CNR NANOTEC-Institute of Nanotechnology c/o Campus Ecotekne, University of Salento, Lecce, Italy e Department of Mathematics and Physics, University of Salento, Via per Arnesano, 73100, Lecce, Italy * corresponding author:

Stefania Scialla, Department of Engineering for Innovation, University of Salento, Via Monteroni s.n., 73100

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Lecce, Italy, [email protected]

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** These authors contributed equally to this work

Keywords

superparamagnetic nanocomposites; dextran-grafted iron oxide nanoparticles; magnetic hydroxyapatite

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green-friendly coprecipitation; vibrating sample magnetometry; biomimetic materials

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Abstract

Authors aimed to provide a magnetic responsiveness to bone-mimicking nano-hydroxyapatite (n-HA). For

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this purpose, dextran-grafted iron oxide nanoarchitectures (DM) were synthesized by a green friendly and scalable alkaline co-precipitation method at room temperature and used to functionalize n-HA crystals. Different amounts of DM hybrid structures were added into the nanocomposites (DM/n-HA 1:1, 2:1 and 3:1weight ratio) which were investigated through extensive physicochemical (XRD, ICP, TGA and Zetapotential), microstructural (TEM and DLS), magnetic (VSM) and biological analyses (MTT proliferation

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assay). X-ray diffraction patterns have confirmed the n-HA formation in the presence of DM as a co-reagent. Furthermore, the addition of DM during the synthesis does not affect the primary crystallite domains of DM/n-HA nanocomposites. DM/n-HAs have shown a rising of the magnetic moment values by increasing DM content up to 2:1 ratio. However, the magnetic moment value recorded in the DM/n-HA 3:1 do not further increases showing a saturation behavior. The cytocompatibility of the DM/n-HA was evaluated with respect to the MG63 osteoblast-like cell line. Proliferation assays revealed that viability, carried out in the absence of external magnetic field, was not affected by the amount of DM employed. Interestingly, assays also suggested that the DM/n-HA nanocomposites exhibit a possible shielding effect with respect to the antiproliferative activity induced by the DM particles alone.

ACCEPTED MANUSCRIPT 1. Introduction The scientific interest in magnetic nanoparticles (MNPs) as one of the most versatile nanomaterials for bone tissue engineering is constantly increasing [1,2]. In the last 10 years, MNPs have been proposed for several applications such as MRI contrast agents [3], magnetic drug delivery [4], hyperthermic therapies [5]; currently, due to their nanoscale-sized interactions MNPs are also studied for their ability to induce cell differentiation by exerting magnetic stimuli directly on the mechanosensitive receptors of the cell membranes [6,7]. MNPs are usually produced as core-shell-like structures, consisting of: (i) a magnetic core,

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(ii) a protective polymeric and/or inorganic coating, and (iii) an optional surface functionalization, in order to promote both their stability and the interaction with cells [8,9]. On the other hand, the use of naked MNPs is not actually feasible, due to biofouling and aggregation processes into the bloodstream and consequent rapid

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segregation by macrophages [10]. Among MNPs, magnetite (Fe3O4) or maghemite (γ-Fe2O3) nanoparticles receive considerable attention [11,12] for their rather low toxicity [13] and their strong magnetic properties, enabling the targeting to specific tissue districts under the action of external magnetic fields [14]. In

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particular, maghemite revealed to have better chemical stability than magnetite, which is not stable in oxygen-abundant applications and it is disposed to oxidize to ferric oxi-hydroxides [15]. However, both

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show similar superparamagnetic properties [14]. In bone tissue engineering applications, magnetic fields not only promote bone fracture healing, spinal fusion and bone ingrowths, but are also able to regulate the orientation of matrix proteins and cells, as shown by in vitro as well as in vivo investigations [16-18]. Being the bone a mechano-sensitive tissue, mechanical stimuli from external magnetic fields have been

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successfully used to promote bone regeneration [19]. In this respect, single MNPs could provide ‘micromotions’ to membranes of osteogenic cells directly affecting the mechanotransduction receptors and

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triggering specific pathways of osteoblast differentiation [20]; moreover, the forces required for mechanotransduction via cell membrane deformation are in the order of magnitude (picoNewton) of those potentially triggered by MNPs [21,22].

Among bone substitution materials, hydroxyapatite (HA) is one of the most eligible. However, up

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today only few studies have been focusing on the enhancement of nano-hydroxyapatite (n-HA particles by MNPs. Biocompatibility, bioactivity and osseointegration properties of n-HA are strictly related to its nanosize, chemical composition and crystallinity [23, 24]. Numerous synthetic strategies exist for producing nHA and ion substituted n-HA crystals; such strategies include wet precipitation, hydrothermal and ultrasonic nebulization methods, electrodeposition, sol-gel and solid-state synthesis [25-30], and usually allow obtaining highly crystalline n-HA. Neverthless, to the best of our knowledge, poorly crystalline n-HA shows shorter terms of bioresorbability with respect to highly crystalline n-HA [31], although maintaining more favorable biological properties (e.g. absence of toxicity and/or inflammatory/immune responses). The HA crystal structure and adsorbing nature offer a wide variety of substitution [32], doping [33] and surface adsorption options, which allow specific tailoring of the material properties [34]. To date, several attempts to synthesize magnetic-HA composites via co-precipitation of HA in the presence of a ferro-fluid suspensions [34, 35] or to combine it with other route like spray-drying [36] have been suggested. In

ACCEPTED MANUSCRIPT addition, other mechano-chemical approaches [37] or in situ methods involving the only introduction of Fe2+ ions during the neutralization phase [38-42] have been developed. For instance, Mir and colleagues synthesized superparamagnetic iron oxide-HA nanocomposites by crystallization of HA in PVA-stabilized aqueous ferro-fluid nanotemplates [35]. They obtained the incorporation of the iron oxide nanoparticles in the interstitial spaces of HAp without disrupting its lattice structure. Moreover, Donadel and collaborators reported the use of n-HA for coating iron oxide nanoparticles by an efficient and relatively inexpensive method based on spray drying [36]; interestingly, the method produces super-paramagnetic n-HA core/shell

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structures consisting of a single HA phase and a double iron oxide phase (magnetite/maghemite). As well, the inclusion of Fe ions in the HA lattice has been studied by Jiang and coworkers [38]. They specifically investigated the distribution of Fe2+ and Fe3+ in the HA lattice, without focusing on magnetic properties. Otherwise, other authors suggested synthesis approaches for magnetic HA simply based on the introduction

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of Fe2+ or Fe3+ ions during the neutralization phase [39-41]; on this basis, Ansar and collaborators carried out the synthesis of iron oxide-HA by co-precipitation of iron salts and calcium phosphate precursors, in alkaline medium and N2-conditioned atmosphere [42]. They obtained iron oxide nanocrystallites embedded in a HA

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matrix with a plate-like structure with size ranging from 70 to 100 nm. Although the above-cited methods produced superparamagnetic nanoparticles, they require labor-intensive and extreme conditions (high

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temperature/pressure, organic solvents, high polymer content) and/or complex systems. Herein, we describe the improvement of the bone-mimicking features of n-HA by switching on some alternative properties. We aimed at providing magnetic responsiveness to our n-HA, while maintaining its

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high biocompatibility in the absence of a magnetic field. We produced magnetic nanocomposites based on low crystalline n-HA decorated by dextran-grafted iron oxide (DM) hybrid structures. To this aim, we

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adopted a green-friendly and scalable two-step process, carried out in aqueous solution, at room temperature and atmospheric pressure. In the first step we obtained dextran-grafted maghemite nano-architectures by a microwave-assisted alkaline co-precipitation route. In the second stage, the DM/n-HA nanocomposites were formed by precipitation in an aqueous system containing different amounts of colloidal DM and calciumphosphate precursors. Finally, the effect of DM/n-HA ratios on the nanocomposites features were

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extensively analyzed by physicochemical, structural and magnetic characterization, and biological assays.

2. Materials and methods

2.1 Chemicals Iron (II) chloride tetrahydrate (FeCl2•4H2O, 99%), iron (III) chloride hexahydrate (FeCl3•6H2O, 98%) and Dextran from Leuconostoc Mesenteroids (Mr 6000 Da) were purchased by Alfa Aesar. Sodium hydroxide anhydrous pellets (97%), hydrochloric acid (37%), phosphoric acid (85%), ammonium hydroxide (30%), and calcium acetate hydrate (99%) were purchased from Aldrich. 18,2 MΩ/cm water has been used. Eagle's

Minimum

Essential

Medium

(EMEM),

fetal

bovine

serum

(FBS),

L-glutammine,

penicillin/streptomycin antibiotic mix, Dulbecco’s phosphate buffer saline (D-PBS), paraformaldehyde

ACCEPTED MANUSCRIPT (PFA), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (Milan, Italy). All reagents and medium supplements were supplied as cell-culture tested.

2.2 Synthesis of dextran-grafted iron oxide nanoparticles (DM) DM hybrid structures were prepared by combining a dextran carboxylation and maghemite precipitation reactions modifying a method previously published by Walsh et al. [43]. Briefly, 3.8 g of FeCl3•6H2O, 1.4 g of FeCl2•4H2O and 8 g of dextran were added to 75 ml of water and vigorously stirred.

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Then, 80 ml of NaOH 1 M were quickly added and the obtained mixture vigorously stirred for 20 min and finally microwaved for 4 min (600 W) at 80°C. Afterwards, the mixture was cooled into an ice bath and contemporarily HCl 6 N was added until pH 7. After about 1 hour of cooling, the mixture was centrifuged at 4000 rpm for 40 min. The supernatant was purified by dialysis tubing cellulose membrane overnight

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(molecular weight cut-off 14 kDa). A colloidal magnetic suspension (ferrofluid) was thus obtained and then dried at 50°C overnight.

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2.3 Synthesis of DM/n-HA nanocomposites

Calcium-deficient hydroxyapatite nanocrystals (Ca/P=1.5 n-HA) were synthesized in the presence of

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the DM nanoarchitectures. 5 mM ammonium phosphate solution was prepared by dilution of phosphoric acid (85% w/w) with water, adjusting pH to 10 with NH4OH. After that, 10ml of three different aqueous colloidal DM suspensions (either 0.5, or 1.0 or 1.5mg/mL, respectively), were alternatively added to the ammonium

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phosphate solutions, making up the volume with water. An equal volume of a 7,5 mM (CH3COO)2Ca⋅H2O was slowly dropped into the vigorously stirred (at a rate of 350 rpm) ammonium phosphate/DM suspension.

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DM was introduced in the reaction at the weight ratios 1:1, 2:1 or 3:1 with respect to the amount of n-HA precipitated in the same reaction performed in the absence of DM. The synthesis was carried out at room temperature and atmospheric pressure. Ammonia solution was used to buffer to 10 the solution pH throughout the entire synthesis procedure. The reaction mixture was aged at room temperature overnight whilst stirring. The recovered precipitate was washed with water by centrifugation at 4000rpm and dried at

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37°C overnight.

2.4 Characterization of DM and DM/n-HA nanocomposites

2.4.1 Transmission electron microscopy DM and DM/n-HA nanocomposites morphology was characterized by TEM operating at 100kV (Jeol JEM-1011) equipped with a CCD camera ORIUS 831. The powders were dispersed in aqueous ammonium hydroxide solution and deposited onto a carbon-coated copper grid. 2.4.2 Dynamic light scattering and Z-potential measurements The hydrodynamic size and Z-potential of DM were acquired by dynamic laser scattering with a Zetasizer Nano ZS (Malvern Instruments, Herrenberg, Germany) operating in backscattering mode with a

ACCEPTED MANUSCRIPT He-Ne laser beam (λ=532nm) at 25°C; the scattering angle was set at 175° and 12,8°, in order to acquire the hydrodynamic size and Z-potential respectively. In order to obtain a signal of 100-500 Kcps, the particles were dispersed in aqueous solution at 0.1mg/ml. The experiment was repeated five times, and the results were averaged.

2.4.3 X-ray diffraction analyses Phase indication in powder samples was confirmed by XRD (Rigaku D-Max/Ultima diffractometer),

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operated at 40kV with Cu Kα radiation (Kα1=1.54056Å and Kα2=1.54439Å) within scanning range of 5°-80° (2θ) at step size of 0.02°/step, and a scanning speed of 3 s/step. The average crystallite domain size (along the [220] and [311] directions for γ-Fe2O3, and along the [002] and [310] directions for n-HA) was estimated applying the Scherrer’s equation: 𝐷 = (𝑘𝜆⁄𝛽𝑐𝑜𝑠𝜃)

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(Eq.1)

where k is a dimensionless crystallite shape factor, λ is the X-ray wavelength (Cu Kα1), β is the line broadening at half the maximum intensity (FWHM), after subtracting the instrumental line broadening, in

was evaluated according to the formula:

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𝜒 = (𝑋⁄𝑌) ∙ 100

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radians, and θ is the Bragg’s diffraction angle for plane (hkl). The degree of nanocomposites crystallinity (χ)

(Eq.2)

where X=net area of diffracted peaks and Y=net area of diffracted peaks + background area.

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2.4.4 Compositional analysis

The Fe, Ca and P atomic contents in the nanocrystal powder solutions

were determined by

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inductively-coupled plasma-mass spectrometry (ICP-MS) measurements. Samples were digested in aqua regia (HCl/HNO3 = 3:1 v/v) solution for three days, and then made up to the desired concentration with ultrapure water.

2.4.5 Thermal analysis

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The thermogravimetric analysis (TGA) was carried out on the prepared samples using a SDTQ600, TA Instrument analyzer (New Castle, DE, USA). DM and DM/n-HA powders were analyzed carrying out the scansion in the temperature range 25÷1200°C at a heating rate of 10°C/min in a nitrogen atmosphere. Al2O3 was used as reference material. The weight amount of dextran (DDM (wt%)) in DM nanoarchitectures and in the DM/n-HA nanocomposites was calculated according to the following formula: 𝑊𝐿𝐷 (𝑤𝑡%): 100 = 𝑊𝐿𝐷𝑀 (𝑤𝑡%): 𝐷𝐷𝑀 (𝑤𝑡%)

(Eq.3)

where WLD (wt%) and WLDM (wt%) corresponded to the dextran weight loss in the range 25÷450°C for pure dextran and for DM nanoarchitectures respectively.

2.4.6 Vibrating sample magnetometry

ACCEPTED MANUSCRIPT The magnetic behavior of the DM nanoarchitectures and DM/n-HA nanocomposites synthesized with 1:1, 2:1 and 3:1 ratios was investigated by using a vibrating sample magnetometer (Cryogenic Ltd). The magnetization curves (M-H) were recorded at 300 K on dry samples packed in gelatin capsules over the field range -10 kOe ÷ 10 kOe and normalized to the sample mass in order to quantify the moment in term of emu/g. M-H measurements were also performed up to 30 kOe to investigate if the response of DM/n-HA nanocomposites saturates.

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2.5 In vitro cell-based assays For the in vitro cytocompatibility assays, the immortalized MG63 cell line (human osteoblast-like cells) was adopted as suitable cell model [44-46]. Cell cultures were maintained in sterile conditions, with EMEM supplemented with 10% (v/v) FBS, 2 mM L-glutamine, penicillin (100 U/ml), streptomycin (100

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ng/ml), in a water-saturated atmosphere of 5% CO2 and 95% air at 37 °C. For propagation, cells were washed with D-PBS, detached with a 0.3% (w/v) trypsin solution and harvested by centrifugation; then, cell pellets were re-suspended and transferred to new flasks once reaching 70-90% confluence (every 2-3 days).

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All experiments were performed between passage 3 and 10 of propagation. For the treatments with n-HA and DM nanocomposites, MG63 cells were seeded in multi-well plates, and the day after were incubated for 24,

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48 and 72 hours with n-HA or DM dissolved in E-MEM medium at different concentrations (500, 50, 5 and 0.5 µg/µL serial dilutions). The same protocol was performed by incubating cell cultures with DM/n-HA nanocomposites at different ratios (1:1, 2:1, 3:1). Prior to the proliferation assays, n-HA, DM and DM/n-HA

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powders were sterilized by UV irradiation for 2 hours.

Viability of cultured cells was assessed by standard protocols of the 3-[4,5-dimethylthiazol-2-yl]-2,5-

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diphenyltetrazolium bromide (MTT) proliferation assay. Briefly, the MTT solution (in D-PBS, pH 7.4) was added to each culture well after treatments at the final concentration of 0.5 mg/mL, and plates were incubated at 37 °C for 4 h. Then, after medium removal the dark-blue formazan crystals produced by viable cells were solubilized by cell lysis with a 2-propanol/1N HCl solution; absorbance values from the lysates were measured at 550 nm. Absorbance data were normalized, and mean values (n = 6 for each experimental

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condition) were reported (± S.E.M.) as percent of the untreated control (100% proliferation) in graphics. For cell morphology analysis, the MG63 cells were seeded on sterile coverslips; after 24 h (50% ≤ confluence ≤ 75%), cells were incubated with DM/n-HA according to the described times and concentrations. After fixation by 4% (w/v) paraphormaldeyde (PFA) in D-PBS, samples were processed by a standard haematoxylin-eosin staining protocol. Stained samples on coverslips were dehydrated by 2-minute passages in the presence of increasing concentrations of ethanol (50, 70, 95 and 100% EtOH in distilled water) and final xylene incubation. Lastly, coverslips were mounted on microscope slides with the Eukitt® acrylic resin mix, and left to dry for 12-18 h before observations by light microscopy.

2.6 Statistical analyses Determinations of n-HA and γ-Fe2O3 crystallite domain size, bulk Ca/P, Fe/Ca, and Fe/P ratios,

ACCEPTED MANUSCRIPT TGA, particle size and Z potential were carried out 5 times on the same synthesis product. Cytotoxic experiments were performed in triplicate. Results are reported as mean ± standard error of the mean (S.E.M.). Data obtained from the experiments were compared by a two-tailed t-test. Differences were considered statistically significant at a significance level of 95%.

3. Results and Discussions

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3.1 DM nanoarchitectures characterization 3.1.1 Morphological and structural characterization

Hybrid nanostructures made of dextran-foils decorated by iron oxide cores have been produced through an alkaline co-precipitation method. The nanoarchitectures have been obtained from a solution

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containing chloride-based iron precursors and dextran (Fig.1 (d)). Dynamic light scattering analyses revealed a DM hydrodynamic diameter of 52±4nm. Iron oxide cores dimension resulted in the range of 3.0-5.0 nm as assessed by TEM observation (Fig. 1 (a)). Z-potential measurements displayed a negative charge of -

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26±1mV reasonably attributable to the carboxylate terminal groups of the dextran molecules. The crystal structures of iron oxide nanocrystals and DM nanoarchitectures have been investigated

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through XRD analyses (Fig. 1(b)). XRD patterns of naked iron oxide nanocrystals and DM nanoarchitectures revealed a cubic spinel crystalline structure in accordance to the standard data card of pure maghemite, γFe2O3 (JCPDS card no. 39-1346). Despite the similarity between X-ray diffraction patterns of maghemite

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and magnetite phases, the oxidative condition of the synthesized iron oxide cores and nanoarchitectures, together with their derived magnetic properties, confirmed the formation of a γ-Fe2O3 phase [47,48]. This hypothesis has been corroborated by comparing the relative d-spacing values of the as-synthesized samples

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with the JCPDS data of Fe3O4 (card no. 19-629) and γ-Fe2O3 (Table S1) [49]. A broad band resulted clearly distinguishable in the DM diffractogram at 2θ≈18° corresponding to the glycoside rings [50] of the sugar molecules possibly over-coating the magnetic cores. Furthermore, the widening of iron oxide reflections in the DM diffractogram suggest a decrease in the crystallite dimensions

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of the DM iron oxide cores. Consistently, the dimensions of the crystalline domains along the [311] direction (2θ≈36°) resulted 10 ± 2nm for the iron oxide synthesized in the absence of dextran, and 3 ± 0.5nm for the DM nanoarchitectures respectively. Furthermore, the widening of iron oxide reflections can be attributed to the drastic crystallinity reduction, which lower itself of 85 % with respect to the naked iron oxide one. This effect, together with the crystallite dimension decrease can be attributed to the interaction between dextran and iron oxide cores during the DM synthesis. It is worth noting that the values of the DM crystallite domain sizes are in good agreement with the dimensions estimated by TEM images, revealing that we are in the presence of a monocrystalline powder.

3.1.2 Thermal characterization and elemental analysis Thermal properties of DM nanoarchitectures and of each single component, namely dextran and γ-

ACCEPTED MANUSCRIPT Fe2O3 nanocrystals, were also studied through non-isothermal thermogravimetric analysis, by evaluating the variations of residual mass percentage of each sample as the temperature increases (Fig. 1(c)). In the thermograms, the γ-Fe2O3 phase exhibited a total weight loss of nearly 4%wt in the temperature range 25÷400°C, due to the removal of physically and chemically adsorbed water [51]. Dextran thermogram shows two distinct weight loss stages: the first one between 20 and 150°C (TDTG ~ 63°C) is nearly 5%wt and represents the waterfall; the second one between 150 and 450°C (TDTG ~ 310°C) with mass loss of 77%wt corresponds to the organic breakdown of polysaccharide dextran chains.

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The dextran residual mass at 1200°C was about 14%wt, caused by its incomplete combustion under an anaerobic atmosphere [52,53].

Contrarily to the thermograms of pure γ-Fe2O3 nanocrystals and dextran, TGA curve of DM presented diverse temperature-promoted weight losses. The first of these was related to the water

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displacement occurring in the range 25÷150°C C (TDTG ~ 54°C) with almost 6%wt of loss; the other two stages corresponded to the organic breakdown of polysaccharide dextran chains (range of temperature 150÷300°C, (TDTG = ~ 245°C) and 300÷500°C, (TDTG = ~ 400°C) with losses of 30%wt and 13%wt,

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respectively) (Fig. S1 (a)) [52,53]. Interestingly, the presence of maghemite nanocrystals in the DM nanoarchitectures resulted to catalyze the thermal decomposition of the adsorbed dextran molecules as

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displayed by the lowering of the decomposition temperature (TDTG ~ 245°C) when compared with the free dextran molecules (TDTG ~ 310°C) [52,53]. The last stage of decomposition between 500 and 1200°C shows two further weight losses (about 9% and 4% wt, respectively). The first one (TDTG ~ 614°C) is probably

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associated to the phase transition of γ-Fe2O3 to nonstoichiometric iron oxide that is also due to the interaction of the sample with the gases produced during heating, the second one (TDTG ~ 665°C) resulted correlated to

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the deoxidation of the iron oxide coherently with the fact that TGA analysis was performed under N2 atmosphere [54,55]. The different thermal behavior registered in N2 atmosphere between dextran alone and DM nanoarchitectures could be justified by the bonding of the dextran to the iron oxide core (e.g. through hydrogen bond and/or carboxylated-groups adsorbed on the surface) as also reported by Carp 2010. TGA quantitative analysis carried out on DM samples according to Eq.(3), provided a dextran-carboxylate content

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as large as 66%wt and an iron oxide nanocrystals content of 34%wt. ICP-MS measurements gave an iron content in the maghemite nanocrystals of 21%wt.

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Fig. 1. (a) TEM image of DM hybrid structures. (b) XRD patterns of DM and of naked maghemite (γ-Fe2O3) compared to JCPDS card no. 39-1346. (c) TGA curves of naked γ-Fe2O3, dextran and DM expressed as weight loss percentage as a function of the temperature (heating rate 10°C/min, N 2 atmosphere). (d) Picture of DM in powder form under the effect of a magnet.

3.2 DM/n-HA nanocomposite characterization

3.2.1 Morphological and structural characterization

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DM/n-HA magnetic nanocomposites have been obtained by precipitating calcium phosphate in the presence of different amounts of maghemite-decorated dextran hybrid architectures. The DM-to-n-HA ratio refers to the amount of colloidal DM used to perform the reaction with respect to the amount of HA precipitated in a reaction performed in the absence of DM.

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TEM images reveals the presence of DM nanoarchitectures on the n-HA crystals surface; however, they do not allow to discriminate an increase in the DM density in the three different DM/n-HA ratios. In Fig. 2 (a,b) TEM images of pure n-HA and nanocomposite DM/n-HA 2:1 are shown as an example. In particular, in nanocomposites pictures it is possible to observe plate-like n-HA crystals surface-decorated by DM hybrid structures (Fig. 2(b)). The morphology of DM/n-HA nanocomposites does not seem to be dependent on the amount of the magnetic complex introduced during the synthesis. Powder X-ray diffraction patterns (Fig. 2(c)) of both n-HA and magnetic nanocomposites show the characteristic diffraction maxima of hydroxyapatite single phase (JCPDS 9-432). The not well-defined diffraction maxima of both n-HA and magnetic nanocomposites indicate a relatively low degree of crystallinity. Furthermore, both n-HA and all the DM/n-HA nanocomposites, produced by loading different weight amounts of DM, exhibited X–ray diffraction patterns very similar to the deproteinated bone apatite

ACCEPTED MANUSCRIPT one (Fig. 2(c)), confirming the production of a not stoichiometric defective apatite. Unfortunately the n-HA [301] reflex overlapped the characteristic peak at 2θ ≈ 36° corresponding to the plane [311] of γ-Fe2O3, that therefore cannot be detected. The primary domain size of n-HA and nanocomposites along the c-axis ([002] ~ 25.9°) and its transversal direction ([310] ~ 39.9°), and their crystallinity degree are summarized in Table 1. The primary domain sizes do not change as a function of the DM/n-HA ratio and are equal to about 15nm and 6nm respectively.

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Table 1 Primary domain size along the c axis and its transversal directions for n-HA synthesized in the presence of different amount of dextran-maghemite. DM/n-HA ratio refers to the amount of DM used to perform the reaction respect to the amount of n-HA precipitated in a reaction performed in the absence of DM. All data are expressed as mean value ± S.E.M.

D002 (nm)

D310 (nm)

Cristallinity (%)

n-HA

15 ± 1.0

5.6 ± 0.6

43 ± 1

DM/n-HA 1:1

13.6 ± 1.8

4.8 ± 0.6

27 ± 2

DM/n-HA 2:1

14.1 ± 0.8

5.3 ± 0.6

23 ± 3

DM/n-HA 3:1

14.4 ± 1.2

6.3 ± 0.1

16 ± 5

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Samples

The crystallinity degree of the synthesized pure n-HA resulted equal to about 43 % (Table 1), consistently with the mild, biomimetic synthesis conditions and, particularly, with the non-stoichiometric Ca/P molar

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ratio employed. Moreover, the experimental data showed that the DM/n-HA crystallinity decreased down to 27%, 23% and 16% by increasing the DM content in the composite (Fig. 2(c) and Table 1). This trend has been attributed to the possible steric impediment operated by DM nanoarchitectures during the n-HA

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nucleation [56]. Therefore, the inclusion of DM does not induce a significant change in the dimensions of the primary crystallites, but rather does effect the development of the amorphous-to-crystalline phase ratio in the nanocomposite. The adsorption of DM nano-architectures onto the calcium phosphate nuclei can probably prevent the ripening of the amorphous towards crystalline phase. Furthermore, a less ordered surface of the

2(a) and (b)).

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nano-composites crystals with respect to the pure n-HA can also be observed through the TEM images (Fig.

3.2.2 Thermal characterization and elemental analysis Thermal decomposition curves of n-HA and DM/n-HA nanocomposites, the latter achieved at three different loading amounts of DM, namely 1:1, 2:1 and 3:1, have been reported in Fig. 2(d). For the n-HA, three weight losses have been observed. The first endothermic region from 25 to 300°C (TDTG~65°C) corresponds to the dehydration of the precipitated complex and specifically to the loss of physically adsorbed water molecules (~4%wt). Then, the additional heating from 300 to 1200°C caused two further weak losses (about 1,3%wt at about 485°C and 1,2%wt at 750°C respectively), which are the result of carbonate loss and gradual dehydroxylation of n-HA powder.

ACCEPTED MANUSCRIPT Thermal analysis of DM/n-HA nanocomposites obtained at different DM-to-HA weight ratios, showed three distinct stages of weight losses. A first stage, appearing in the range 25÷150°C (T DTG ~ 62 – 72 – 61°C for 1:1, 2:1 and 3:1 ratios, respectively, Fig. SI 1(b-c-d)) related to the water-evolving fall, provided a weight loss of about 8%. Then, in the temperature range 150÷500°C (TDTG ~ 252 – 262 – 258°C for 1:1, 2:1 and 3:1 ratios, respectively, Fig. SI 1(b-c-d)), the TGA curves showed a rapid weight loss, slightly higher than 10%wt, for each DM/n-HA sample. This loss can be attributed to the thermal decomposition of the dextran-phase in DM hybrid structures. This decomposition extends in the weak third weight loss stage

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visible between 500°C and 1200°C (TDTG ~ 758 – 731 – 755°C for 1:1, 2:1 and 3:1 ratios, respectively, Fig. SI 1(b-c-d)), without ever reaching the complete dextran degradation because of the anaerobic conditions. TGA quantitative analysis carried out on DM/n-HA samples according to Eq.(3), provided a dextrancarboxylate content in the range between 23 and 26 % wt. On the other hand, the theoretical dextran content

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of the DM/n-HA composite was expected between 33 and 44 % wt, as a function of DM/n-HA ratio. Considering the above, we hypothesized that during the nanocomposites synthesis the polymer bonded to the iron oxides cores in the DM nano-architectures was partially desorbed in favor of n-HA

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crystals surface sites. In other words it is conceivable that, if the interaction of nude iron oxide with n-HA is favored in respect to iron oxide cores with dextran, the capping molecules of iron oxide nanoparticles were

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partially replaced by surface n-HA anchoring sites.

ACCEPTED MANUSCRIPT Fig. 2. (a) TEM image of n-HA. (b) TEM image of DM/n-HA 2:1. (c) X–Ray diffraction patterns of the synthesized Cadeficient hydroxyapatite (n-HA) and DM/n-HA nanocomposites at 1:1, 2:1 and 3:1 weight ratios. At the bottom of the picture, the XRD reference of bulk hydroxyapatite is reported (JCPDS card no. 09-0432). (d) TGA curves of n-HA and DM/n-HA at different weight ratio 1:1, 2:1 and 3:1 expressed as weight loss percentage as function of the temperature (heating rate 10°C/min, N2 atmosphere).

In Table 2 mean values ± S.E.M of Fe/P and Fe/Ca ratios calculated by ICP-MS analysis are reported. It is possible to observe that the atomic Fe/P and Fe/Ca ratios increase by increasing the DM/n-HA ratio to 2:1

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then remains more or less constant, suggesting a saturation of adsorbed sites on the surface of n-HA crystals.

Table 2 Evaluation of Fe content of DM/n-HA nanocomposites expressed as mean value of Fe/P and Fe/Ca ratio ± S.E.M.

Fe/P

Fe/Ca

DM/n-HA 1:1

0,7±0,04

0,23 ±0,02

DM/n-HA 2:1

1,3±0,01

0,44 ±0,004

DM/n-HA 3:1

1,2±0,08

0,43 ± 0,04

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Samples

3.3 Magnetic properties

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Fig. 3 shows the magnetization as a function of the applied magnetic field for the DM nanoparticles and DM/n-HA complexes synthesized by varying the DM/n-HA ratios. The magnetization curves have been recorded at 300 K and normalized dividing by the sample mass. DM nanoarchitectures show a non-saturated

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magnetization behavior with a magnetization of about 4 emu/g measured at 30kOe (not shown), the maximum value of field applied in this study. This value of magnetization is much lower than that of bulk γ-

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Fe2O3 (80 emu/g) [13] due to the small size of the magnetic core of DM (of about 4nm), which makes these nanoparticles very sensitive to surface anomalies (e.g defects and spin canting) responsible of a high magnetic frustration. The magnetic moment of DM/n-HA nanocomposites increases by increasing the DM content up to a 2:1 ratio. However, the 3:1 ratio presents a response similar to the one at 2:1 ratio, which can be ascribed to the saturation of absorption sites on surface of the plate-like n-HA crystals, in agreement with

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the Fe/Ca and Fe/P results.

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Fig. 3. Magnetization curves of the DM nanoarchitectures and DM/n-HA nanocomposites as a function of DM/n-HA ratio recorded at 300 K.

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3.4 Evaluation of the DM, n-HA and DM/n-HA nanoparticles cytocompatibility The basic cytocompatibility of the DM, n-HA and DM/n-HA was assessed by evaluating

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proliferation and morphology of the MG63 human-derived osteoblast-like cells. MG63 have been grown in the presence of the nanoparticles added to the growth medium, in the absence of an external magnetic field. When the behavior of the DM alone were evaluated, no effects on cell viability were detected under the

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lowest tested concentration, i.e. 0.5 µg/mL DM, whereas a significant inhibition of cell proliferation was detected after 48 h treatment by 5 µg/mL DM, and the reduced proliferation was detected up to 72 hours post DM administration. This effect was induced also by 50 and 500 µg/mL DM concentrations; anyway, a dose-

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dependent increase was not detected, interestingly (Figure 4 (a)). Overall, assays on the DM-treated cells indicated that 5 µg/mL DM was the minimal tested concentration triggering cell responsiveness, and suggested that the exceeding concentrations should be ineffective in terms of bioactivity, under our experimental conditions. In parallel, when the n-HA nanoparticles alone were evaluated, viability of MG63

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cells was not significantly affected by the presence of serial dilutions (500, 50, 5 and 0.5 µg/mL) of n-HA added to the culture medium, up to 72 h of treatment (data not shown).

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Fig. 4. Cell viability analysis on MG63 cells. MTT proliferation assays were performed on cells grown in the presence of (a) serial dilutions (500, 50, 5 and 0,5 µg/ml) of the synthesized dextran-grafted maghemite nanoparticles (DM); (b) DM/n-HA nanocomposites at the 1:1 and (c) 2:1 and 3:1 ratios (5 µg/ml of concentration), after treatments for 24, 48 and 72 hours. Data are the mean values from n = 6 biological replicates, expressed as % proliferation (± S.E.M.) with respect to the untreated control cells. Qualitative evaluation of cell morphology was assessed by haematoxylin-eosin staining of MG63 cells grown in the presence of DM/n-HA composites: (d) untreated control; (e) cells grown in the presence of the 2:1 ratio of DM/n-HA nanocomposites, pointed by black arrowheads (2:1 pictures are representative, since no significant morphological variations occurred among the three DM/n-HA ratios), (f) MG63 cells with a 20-fold concentration of 2:1 DM/n-HA (100 µg/ml) [Magnif. 10X].

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Consequently, viability of cells was investigated in the presence of DM/n-HA nanocomposites synthesized with the 1:1, 2:1, and 3:1 DM/n-HA ratios. In the 1:1 ratio, the amounts of both DM and n-HA

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were considered to be equal, and treatments were conducted by 5 µg/mL of DM/n-HA. As shown in Fig. 4(b), MTT assays revealed no different proliferation between cells treated by DM/n-HA and the untreated control cells, up to 72 h. Interestingly, this analysis could suggest that the cytotoxicity previously observed for the DM nanoparticles alone is no more retained, once the DM is embedded in the DM/n-HA nanocomposites. Intriguingly, this masking effect of DM cytotoxicity by the HA component gives hints of a

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modular biomimicry of the synthesized nanocomposites. Then, both the DM/n-HA 2:1 and 3:1 ratios were also tested, keeping constant the concentration of HA present in the 1:1 ratio. Interestingly, for both the DM/n-HA 2:1 and 3:1 nano-architectures an increase of proliferation (~25%) has been observed at 48 h. Anyway, this trend turned back to control levels up to 72 hours of treatments (Fig. 4(c)). Assuming that this possible proliferative effect at intermediate times must be furtherly elucidated, also the DM/n-HA 2:1 and 3:1 ratios did not retain the DM-related toxicity, as previously shown for the 1:1 ratio. The biocompatible impact of the DM/n-HA on cells was also qualitatively evaluated by light microscopy analysis. As represented in Fig. 4(e), no evident changes in the plain cell morphology were observed by treating cells with DM/n-HA up to 72 h at each synthesis ratio, as well as no inhibition of migration and/or proliferation could be detected, compared to the untreated cells (Fig. 4(d)). No alterations of migration and adhesion processes could be detected even increasing the amount (20-fold) of the nanoparticles, thus potentially interfering with

ACCEPTED MANUSCRIPT cell proliferation. (Fig. 4(f)). 4. Conclusions An easy, aqueous synthesis enabling the development of magnetic HA nanoarchitectures has been presented in this study. n-HA crystals with an iron oxide surface functionality have been obtained. It was found that the introduction of dextran maghemite as co-reagent during the synthesis does not affect the n-HA primary crystalline domain; but on the other hand, it induces a decrease in the n-HA crystallinity. The

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magnetic behavior of this apatite-based material and the lack of hysteresis allowed establishing that all properties connected to magnetization can be switched on-off by external stimuli. In addition, the in vitro studies have shown favorable properties in terms of biocompatibility of the DM/n-HA nanocomposites in the absence of magnetic field, giving hints of high modulability with respect to the proliferation dynamics of

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osteoblast-like cells. The strong biomimetic properties of apatite nanocrystals together with the magnetic responsiveness of DMs could represent an optimized combination of structural and functional features to

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synthesize novel switchable nanocomposites. Furthermore, the co-precipitation method shows some advantages with respect to other methods for future application of our nanocomposites. In particular, the mild experimental conditions make possible the “in situ” functionalization of the nanocomposites with

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bioactive molecules. The obtained hetero-structures could be used for the fabrication of bio-devices intended to act as both bone filler and mechano-transductor agents.

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Acknowledgment

The authors wish to thank Mr. Cannoletta Donato (University of Salento) for the performed XRD

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analysis. The authors acknowledge the Regione Puglia NABIDIT - NANOBIOTECNOLOGIE e SVILUPPO PER TERAPIE INNOVATIVE Project (F31D08000050007) and Regione Puglia - FSC 2007-2013

References

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Intervento "FutureInResearch".

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Hydroxyapatite nanocomposites were co-precipitated with dextran-grafted maghemite

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Dextran-maghemite does not affect the crystalline domain of nano-hydroxyapatite

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Magnetic behavior of nanocomposites show a lacks of hysteresis

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Nanocomposites resulted biocompatible in the absence of magnetic field

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