Article pubs.acs.org/IC
Fe-Doping-Induced Magnetism in Nano-Hydroxyapatites Vincenzo Iannotti,*,†,& Alessio Adamiano,*,‡,& Giovanni Ausanio,† Luciano Lanotte,† Giuliana Aquilanti,§ John Michael David Coey,∥ Marco Lantieri,⊥ Gabriele Spina,# Maria Fittipaldi,# George Margaris,∇ Kalliopi Trohidou,∇ Simone Sprio,‡ Monica Montesi,‡ Silvia Panseri,‡ Monica Sandri,‡ Michele Iafisco,‡ and Anna Tampieri‡ †
CNR-SPIN and Department of Physics “E. Pancini”, University of Naples “Federico II”, Piazzale V. Tecchio 80, I-80125 Napoli, Italy Institute of Science and Technology for Ceramics (ISTEC), National Research Council (CNR), Via Granarolo 64, 48018 Faenza, Italy § Elettra-SincrotoneTrieste S.C.p.A., s.s. 14, km 163.5, I-34149 Basovizza, Trieste, Italy ∥ School of Physics, Trinity College, Dublin 2, Ireland ⊥ ISC-CNR, via Madonna del Piano 10, 50019 Sesto Fiorentino (FI), Italy # Department of Physics, University of Florence, via Sansone 1, 50019 Sesto Fiorentino (FI), Italy ∇ Institute of Nanoscience & Nanotechnology, NCSR “Demokritos”, Aghia Paraskevi, 15310 Athens, Greece ‡
S Supporting Information *
ABSTRACT: Doping of biocompatible nanomaterials with magnetic phases is currently one of the most promising strategies for the development of advanced magnetic biomaterials. However, especially in the case of iron-doped magnetic hydroxyapatites, it is not clear if the magnetic features come merely from the magnetic phases/ions used as dopants or from complex mechanisms involving interactions at the nanoscale. Here, we report an extensive chemical−physical and magnetic investigation of three hydroxyapatite nanocrystals doped with different iron species and containing small or no amounts of maghemite as a secondary phase. The association of several investigation techniques such as X-ray absorption spectroscopy, Mössbauer, magnetometry, and TEM allowed us to determine that the unusual magnetic properties of Fe2+/3+-doped hydroxyapatites (FeHA) occur by a synergy of two different phenomena: i.e., (i) interacting superparamagnetism due to the interplay between iron-doped apatite and iron oxide nanoparticles as well as to the occurrence of dipolar interactions and (ii) interacting paramagnetism due to Fe3+ ions present in the superficial hydrated layer of the apatite nanophase and, to a lesser extent, paramagnetism due to isolated Fe3+ ions in the apatite lattice. We also show that a major player in the activation of the above phenomena is the oxidation of Fe2+ into Fe3+, as induced by the synthesis process, and their consequent specific positioning in the FeHA structure.
1. INTRODUCTION
hydroxyapatite (HA)one of the most important inorganic phases for biomaterials applicationsdoped with iron ions have been proposed.11,12 Most researchers have focused on the magnetic abilities conferred by the formation of iron oxide secondary phases (usually magnetite or maghemite) but have not clearly addressed the magnetic properties of iron-doped HA. Only a few works have tried to shed light on the mechanisms underlying the magnetic behavior of doped HAs, but they lack in-depth structural and magnetic characterizations.10,11 It was previously shown12−14 that the specific positioning of Fe2+/3+ ions into the HA structure generated magnetic properties not ascribable to the presence of a secondary iron
Superparamagnetic iron oxide nanoparticles (SPIONs) have been investigated for almost 20 years and are, together with those based on gadolinium oxide, the most important clinically approved magnetic nanoparticles (MNPs).1 The documented relationship between their use and the outbreak of acute adverse effects such as nephrogenic systemic fibrosis, formation of apoptotic bodies, inflammation, and other significant toxic effects justifies concerns about their administration.2−8 To circumvent these issues, in the past few years an increasing number of studies have focused on doping wellknown biocompatible and bioactive materials with magnetic ions (i.e., Fe, Ni, Co, etc.) to achieve magnetic biomaterials endowed with better biological features and higher degradability in physiological conditions in comparison to SPIONs.9,10 In this respect, several methods for the synthesis of © 2017 American Chemical Society
Received: January 3, 2017 Published: April 5, 2017 4446
DOI: 10.1021/acs.inorgchem.6b03143 Inorg. Chem. 2017, 56, 4446−4458
Article
Inorganic Chemistry
disposable electrophoretic cell (DTS1061, Malvern Ltd., Worcestershire, U.K.). Twenty runs of 3 s each were collected in each measurement. 2.3. XRD Measurements. The X-ray diffraction (XRD) patterns of the samples were recorded with a D8 Advance diffractometer (Bruker, Karlsruhe, Germany) equipped with a Lynx-eye positionsensitive detector using Cu Kα radiation (λ = 1.54178 Å) generated at 40 kV and 40 mA. XRD spectra were collected in the 2θ range from 20 to 60° with a step size (2θ) of 0.02° and a counting time of 0.5 s. Quantitative evaluation of phase compositions and cell parameters was performed by full-profile Rietveld analysis of the XRD spectrum (TOPAS v. 4.2, Bruker AXS, Karlsruhe, Germany). The crystallinity degree of the samples was calculated according to eq 1
oxide phase, associated with excellent biocompatibility and enhanced osteogenic ability in vitro, in comparison with undoped and randomly Fe-doped HA nanoparticles (NPs). In addition, these NPs were already reported to be suitable for cell internalization and guidance by magnetic fields, thus showing potential for cell therapy and gene delivery.15 Since new therapies based on magnetic activation of cells and biomaterials are some of the most promising concepts in nanomedicine, a deeper knowledge of the mechanisms acting at the atomic scale and determination of the magnetic properties would greatly help materials scientists to develop new biomaterials with adequate magnetic and biological features. In this regard, the aim of the present work was to highlight compositional and structural aspects related to the occurrence of magnetic properties in a previously reported iron-doped HA,12 thus confirming that a specific positioning of Fe2+/3+ ions is a key aspect with respect to the final magnetic properties. We report on the production and characterization of iron-doped HA NPs endowed with different magnetic properties, generated by a precise setting of Fe2+ and Fe3+ ions. Notably, an interacting superparamagnetic behavior with a saturation magnetization as high as 130 emu/(g of Fe) at room temperature, much higher than those found for SPIONs, was observed in Fe2+/3+-doped HA (FeHA). In addition, this work provides new insights into the important role of the oxidative reaction of Fe2+ to Fe3+ in their positioning in the NPs during their precipitation, as well as of the formation of small amounts of a secondary iron oxide phase (maghemite) characterized by an unusual disposition of iron atoms. Furthermore, we found that Fe2+ ions substituted for Ca2+ in the crystalline hub of HA, while Fe3+ substituted Ca2+ ions in the nonapatitic environment surrounding the NP crystalline core.
crystallinity (%) = 100
C A+C
(1)
where C is the sum of peaks area and A is the area between the peaks and the background in the diffraction pattern.16 2.4. Fourier-Transformed Infrared Analysis. The infrared spectra were recorded in the wavelength range from 4000 to 400 cm−1 with 2 cm−1 resolution using a Nicolet 380 FT-IR spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, US). A powdered sample (approximately 1 mg) was mixed with about 200 mg of anhydrous KBr. The mixture was pressed at 10 Torr pressure into 7 mm diameter disks. A pure KBr disk was used as a blank. The infrared splitting factor (IR-SF) was calculated by adding the measured intensities of the two ν4(PO4) vibration bands at 565 and 605 cm−1 in the absorbance mode and dividing their sum by the intensity of the valley between these absorption bands and the baseline after a baseline correction between 1200 and 250 cm−1.17,18 2.5. ICP Analysis. The quantification of Ca, Fe, and P was carried out with an ICP-OES spectrometer (ICPAES:Liberty 200, Varian, Clayton South, Australia). A 20 mg portion of powder was dissolved in 2 mL of HNO3 (Aldrich, 65 wt % pure), and the solution volume was increased to 100 mL with deionized water. The analytical emission wavelengths were as follows: Ca 422.673 nm, Fe 259.940 nm, and P 213.618 nm. The obtained values are expressed in terms of (Fe + Ca)/ P, Ca/P mol, Fe/Ca molar ratio, and Fe wt %. 2.6. Determination of Iron(II) and Iron(III). The amount of Fe2+ was measured by a colorimetric method based on the use of ophenanthroline (Merck 1,10-phenanthroline, ≥99% pure): ferrous ions in the presence of o-phenanthroline form the stable red-orange complex [(C12H8N2)3Fe]2+ in the pH range 4−5; this complex is detectable at 510 nm by UV−visible spectrophotometry (Lambda 35 UV/vis spectrometer; PerkinElmer Instruments, USA). A 20 mg portion of powder was dissolved in 0.8 mL of H2SO4 (Aldrich, 96 wt % pure) after it was verified that sulfuric acid did not affect the concentration of the Fe2+-complexed compound, at least in the time required to make the analysis. A 10 mL portion of sodium citrate buffer (0.1 M, pH 4) and 5 mL of a 1 wt % hydroxylamine solution were added to the solution containing sulfuric acid to set the pH at around 4−5 and to avoid Fe2+ oxidation; then an adequate volume of o-phenanthroline solution at 0.2 wt % was added to the solution to set the nominal Fe2+/o-phenanthroline molar ratio at 1/3. The volume of the final solution was increased to 50 mL using Millipore water. The amount of Fe3+ was calculated by the difference between the total amount of Fe (determined by ICP) and the amount of Fe2+ determined by UV−vis.12 2.7. Thermogravimetric Analysis. The carbonate content was evaluated on dried samples by thermogravimetric analysis (TGA) investigations using a Stanton STA 1500 (Stanton, London, U.K.) apparatus. About 10 mg of apatite was weighed into a platinum crucible and heated from room temperature to 1100 °C under nitrogen flow. The heating rate was 10 °C/min, and alumina was used as the reference standard. The CO32− content was evaluated according to the weight loss observed between 550 and 950 °C. 2.8. TEM Analysis. Transmission electron microscopy (TEM) observations were made using a FEI Tecnai F20 TEM instrument equipped with a Schottky emitter and operating at 120 and 200 keV. The instrument is equipped with a Fischione high angle annular dark
2. EXPERIMENTAL SECTION 2.1. Synthesis of Iron-Doped Hydroxyapatite. For the synthesis of hydroxyapatite doped with Fe(II) and Fe(III) (FeHA), a phosphoric acid solution (Aldrich, 85 wt % pure, 20.75 g in 300 mL of H2O) was added dropwise into a basic suspension of calcium hydroxide Ca(OH)2 (Aldrich, 95 wt % pure, 23.40 g in 400 mL of H2O). FeCl2·4H2O (Aldrich, 99 wt % pure, 6.03 g in 75 mL of H2O) and FeCl3·6H2O (Aldrich, 97 wt % pure, 8.28 g in 75 mL of H2O) were added together as sources of Fe2+ and Fe3+ ions during the neutralization process. The ratio between the two iron ions was set to 1, and their amounts with respect to Ca2+ ions were adjusted so as to obtain Fe/Ca ≈ 20 mol %. The synthesis was carried out in a heating mantle set at 45 °C, and the temperature was controlled by means of a thermometer placed in the reacting solution. Two more hydroxyapatites doped with a single iron ion were synthesized introducing only Fe(II) (FeHA2) or Fe(III) (FeHA3) respectively in the Ca(OH)2 suspension, using the same Ca/P molar ratio. The synthesis of stoichiometrically pure hydroxyapatite (Hap) was carried out by simple neutralization of the Ca(OH)2 without adding any iron precursor, under the same conditions used to synthesize iron-doped HAs (e.g. temperature, Ca/P ratio, etc.). Once the neutralization reactions were completed (pH 6.0 ± 0.5), the solutions were stirred at 45 °C for 3 h and then left to age for 24 h at room temperature without further stirring. Samples were removed from the mother liquor by centrifugation, repeatedly rinsed with water, and then freezedried. The obtained powders were manually ground in a mortar and sieved (
