Introduction: Strong coupling between nanotechnology and cell/molecular biology leaded to a breakthrough in medicine in the last decade due to the exiting opportunities in designing and developing a tailored approach in response to different disease.
Magnetic nanoparticles (NPs) have attracted the attention of scientific community for biological and medical purposes as promising materials in drug or gene delivery, DNA/biomolecules separation, hypothermal treatment of tumours, contrast agents for imaging[1], and recently in tissue engineering and theranostic applications[2],[3]. Here novel biomimetic, fully biodegradable and cytocompatible NPs fabricated by doping hydroxyapatite (HA) with Fe ions (FeHA), avoiding the presence of poorly tolerated magnetic secondary phases and any coating, were biologically analysed in vitro. In detail a live monitoring of intracellular fate of FeHA NPs were deeply investigated, and the biodistribution was investigated in vivo, in a pilot study.
Materials and Methods: FeHA NPs were prepared by a neutralization process using FeCl2 and FeCl3 as a source of Fe2+ and Fe3+ doping ions[4]; HA NPs and commercial fluidMag NPs (Chemicell) were used as control groups. Mouse pre-osteoblast cells line (OBs), MC3T3-E1, and human Osteosarcoma cell line, MG63, were cultured with 100 μg/ml NPs up to 72 hours. The molecular pathways of cellular response (apoptosis/necrosis, ROS production and autophagy) to NPs were investigated. Moreover the mechanism of internalization by Caveolae-mediated endocytosis was studied. In a pilot in vivo experiment the biodistribution of different concentrations of NPs (ranging from 0.5 mg/kg up to 50 mg/kg) was evaluated.
Results and Discussion: The in vitro live monitoring showed that FeHA NPs were rapidly and easily internalized by both cell lines without producing significant cell damages and death (Fig.1). In detail the OBs uptake of FeHA NPs seems to be mediated by Caveolae-mediated endocytosis, while a different endocytic mechanism is required by MG63 cell line. Moreover, NPs seem to act as modulator of autophagy pathway, evaluated by the quantification of the processing of LC3β-I to LC3β-II necessary for autophagosome assembly. The in vivo study showed the absence of systemic toxicity even with the higher concentration.
Fig.1. Intracellular localization of FeHA NPs: A. FITC-NPs and endosome (red); B. TEM image of internalized NPs (*).
Conclusion: The data obtained on the cellular uptake of FeHA NPs lay the basis to clarify the intracellular fate of the FeHA NPs and open brilliant prospective for their use as innovative tools for nanomedicine. FeHA NPs could be injected and guided to a desired body site by an external magnetic field, avoiding any toxicity. Moreover, FeHA NPs could be easily functionalized with several biomolecules to direct cell fate in medical applications.
Eu Project SMILEY (NMP4-SL-2012-310637); Flagship Project NanoMAX (PNR-CNR 2011-2013)
References:
[1] M.I. Khan et al., Biomaterials 33 (2012) 1477-1488
[2] Amirfazli A. et al., Nature Nanotech 2(8), 467, 2007.
[3] Arruebo M. et al., Nano Today 2, 22, 2007
[4] Tampieri A. et al., Acta Biomater. 2012 Feb;8(2):843-51