Intrinsic magnetism and hyperthermia in bioactive Fe-doped hydroxyapatite
Introduction
In recent years magnetic nanoparticles (MNPs) have received significant attention owing to their potential biomedical applications [1], [2], [3]. Indeed magnetic particles have been progressively incorporated as support materials for enzyme immobilization, and have been used as drug-delivery agents, contrast agents for magnetic resonance imaging (MRI) as well as heat mediators for hyperthermia-based anti-cancer treatments and many other exciting biotechnological applications [4], [5], [6], [7]. Nanoparticles are amorphous semicrystalline structures with at least one dimension ranging between 10 and 100 nm. A number of their characteristics, e.g. size uniformity, surface area, adsorption kinetics, superparamagnetism and magnetic moment, can be finely tuned during the production process for specific purposes [8]. Among the most popular MNPs used in medicine [9] and biotechnology [10], [11] are iron oxide-based phases (maghemite or magnetite) whose long-term effects in the human body are not yet fully assessed [12], [13]. Such materials are classified as “superparamagnetic”, indicating their ability to become magnetized upon exposure to a magnetic field without showing permanent magnetization (remanence) once the field is turned off. This ability is used in nanomedicine as an efficient tool to move nanoparticles into the body towards target organs. One of the most important criteria in using MNPs is the absence of any toxicity: to this end, over the last decade the surface of MNPs has been modified through the creation of biocompatible layers made of organic polymers, inorganic phases or metals deposited on the existing surface [14]. Due to the importance of having no- toxic MNPs for the above-mentioned applications, the present work is focused on the development of an innovative biocompatible and bioresorbable superparamagnetic-like phase by doping hydroxyapatite (HA) with Fe ions, avoiding the presence of poorly tolerated magnetic secondary phases. This new magnetic apatite could represent, by virtue of its bioactivity, a conceptually new type of scaffold for hard tissue regeneration. At present the use of magnetic stimulation or guidance in the field of regenerative medicine is coming up as one of the most attractive concepts [15], [16]. The use of magnetic fields influencing and addressing cell behavior has been already described [17], [18] and more recently it became the basic concept to design new magnetizable scaffolds able to be activated by an external magnetic field [19], [20]. Besides a direct stimulating influence on cells, the in vivo behavior of a magnetizable scaffold can be finely tuned by injecting MNPs as shuttles for active biomolecules (VEGF, BMP, etc.), driving signals towards the scaffolds [21], [22] that stimulate bone regeneration and vascular remodeling.
This new magnetic apatite could also be a valid bioactive, bioresorbable and non-toxic substitute for magnetite in magnetic-based therapies such as hyperthermia. Hyperthermia, which in this context refers to raising the local temperature for limited periods of time, is an effective treatment for different types of cancer. Tumoral cells can be killed by exposing MNPs, deliberately placed in proximity of the tumoral tissues, to an external magnetic field.
The use of Fe-HA particles in place of magnetite, which has completely dominated the MNP market but has also given rise to concerns about its long-term toxicity, can significantly extend the use of all kinds of magnetic-based therapies.
Although Fe is a vital element in the human body, its concentration within hard tissue is low and its presence into the body scarcely affects bone remodeling [23]. On the other hand, the biocompatibility and bioactivity of HA is already well established [24], [25], [26], [27] and in fact more than 60% of the currently available bone graft substitutes involve calcium phosphate-based materials [28]. In this view the design of a new Fe-HA phase endowed with superparamagnetic ability is very promising for application either as active scaffold for bone and osteochondral regeneration or as nontoxic biodegradable magnetic nanocarriers.
The inclusion of Fe ions in the apatite lattice has been already studied in a previous work: Ming Jiang et al. [29] studied only the local geometry and the distribution of Fe2+ and Fe3+ in the HA lattice, neglecting the investigation of any intrinsic magnetic properties. The authors found that Fe2+ and Fe3+ preferentially occupy specific crystal sites in the HA lattice, i.e. Fe3+ in the Ca(1) and Fe2+ in the Ca(2) position, which correspond to 4f and 6h sites having respectively No. 4 and No.6 calcium ions.
Other authors have reported a synthesis procedure for magnetic HA that involves introducing only Fe2+ ions during the neutralization process [30], [31], [32], [33]. In particular, Wu et al. [33] measured the magnetic properties of such synthesized powder but did not discriminate between the actual formation of a new Fe-HA and magnetite as secondary phase, which indeed represented the main contributor to the magnetization signal.
The present paper takes inspiration from the fact that, in principle, it is possible to introduce both Fe species into the HA lattice at different Ca sites with a specific coordination [29] in order to generate two different sublattices whose interaction could induce superparamagnetic behavior. With this purpose, we developed and optimized a synthetic procedure to obtain a magnetic (Fe2+,Fe3+)-lattice substituted HA, minimizing the formation of magnetite as secondary phase.
Section snippets
Synthesis methods
To prepare Fe-HA powder, a phosphoric acid (Aldrich, 85 wt.% pure, 44.40 g in 300 ml H2O) solution was added dropwise into a basic suspension of calcium hydroxide Ca(OH)2, (Aldrich, 95 wt.% pure, 50 g in 400 ml H2O) containing Fe ions, over a period of 2 h, under constant heating and stirring. The total amounts of Fe ions with respect to Ca ions were adjusted so as to obtain: Fe/Ca = 20 mol.%. To study the relationship between the synthesis parameters and physicochemical properties of the powders, the
Results and discussion
Fe-HA powders obtained by the three synthesis methods are characterized by primary particles in the range 5–20 nm agglomerated in larger grains of about 5–10 μm as revealed by SEM analysis.
ICP analysis confirms the presence of Fe in the powders at a level of 90% with respect to the one nominally introduced as reagent. For all the prepared Fe-HA samples, the molar ratio (Fe + Ca)/P ranges between 1.61 and 1.73 (Table 1, Table 2, Table 3), while Ca/P ratio is lower than the theoretical one: 1.31 < Ca/P <
Conclusions
A neutralization method has been employed to synthesize HA nanopowders in which Ca is partially substituted by Fe2+ and Fe3+. The simultaneous addition of both Fe species under controlled synthesis conditions leads to Fe-HA with a (Fe + Ca)/P ratio very close to the theoretical one (Ca/P = 1.67), Fe3+/Fe2+ ratio ∼3 and a very small content of magnetite as secondary phase. XRD, ICP and TEM analysis confirm that both Fe2+ and Fe3+ ions enter the HA lattice. The new Fe-HA exhibits very low
Acknowledgements
We are very grateful to Dr. G. Celotti (ISTEC-CNR) for valuable discussion on crystallographic aspects, and we kindly thank Dr. Y. Piñeiro-Redondo (University of Santiago de Compostela) for hyperthermia measurements. We would like to acknowledge European Project MAGISTER (NMP3-LA-2008-214685) for funding our research.
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