www.advhealthmat.de www.MaterialsViews.com FULL PAPER © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 1 wileyonlinelibrary.com Managing Magnetic Nanoparticle Aggregation and Cellular Uptake: a Precondition for Efficient Stem-Cell Differentiation and MRI Tracking DOI: 10.1002/adhm.201200294 Delphine Fayol, Nathalie Luciani, Lenaic Lartigue, Florence Gazeau, and Claire Wilhelm* The labeling of stem cells with iron oxide nanoparticles is increasingly used to enable MRI cell tracking and magnetic cell manipulation, stimulating the fields of tissue engineering and cell therapy. However, the impact of mag- netic labeling on stem-cell differentiation is still controversial. One compro- mising factor for successful differentiation may arise from early interactions of nanoparticles with cells during the labeling procedure. It is hypothesized that the lack of control over nanoparticle colloidal stability in biological media may lead to undesirable nanoparticle localization, overestimation of cellular uptake, misleading MRI cell tracking, and further impairment of differentia- tion. Herein a method is described for labeling mesenchymal stem cells (MSC), in which the physical state of citrate-coated nanoparticles (dispersed versus aggregated) can be kinetically tuned through electrostatic and mag- netic triggers, as monitored by diffusion light scattering in the extracellular medium and by optical and electronic microscopy in cells. A set of statistical cell-by-cell measurements (flow cytometry, single-cell magnetophoresis, and high-resolution MRI cellular detection) is used to independently quantify the nanoparticle cell uptake and the effects of nanoparticle aggregation. Such aggregation confounds MRI cell detection as well as global iron quantifica- tion and has adverse effects on chondrogenetic differentiation. Magnetic labeling conditions with perfectly stable nanoparticles—suitable for obtaining differentiation-capable magnetic stem cells for use in cell therapy—are subse- quently identified. Researchers in tissue engineering and cell therapy have recently used iron oxide nanoparticles coupled with MRI to follow the fate of therapeutic cells and cellular grafts in the organism. [2] They also harnessed magnetic forces to create replacement tissues in vitro [1a,1b,3] or potentiate cell therapy through in vivo cell manipulations. [1c,4] However, the incor- poration of magnetic nanomaterials into stem cells remains an issue with regard to their differentiation and therapeutic capa- bilities. Controversial effects of magnetic labeling on stem-cell differentiation have been reported, but the underlying mecha- nisms are still not understood. [5] A tight control over the conditions of cell labeling and the consequences for cell phenotype and functions is thus mandatory. Never- theless the interactions of nanoparticles with cells are dramatically influenced by the behavior of nanoparticles in biological media, containing or not containing pro- teins and eventually being supplemented with transfection agents. [6] The critical role of “cell vision” has been recently highlighted: what sort of nanomaterials would the cells actually see and process? [7] A crucial parameter that is most often neglected or poorly characterized in nanotoxicology studies is the physico-chemical state of the nanoparticles, i.e. their ten- dency to aggregate and eventually sediment on cells. The nano- material aggregation state may not only directly change the cellular responses, [8] but also profoundly modify and bias the means to monitor the biological effects or to quantify the dose of nanoparticles. Therefore we believe that a thorough control over the stabilization/destabilization of nanoparticles, together with improved and complementary methods for nanoparticles dosage and localization in the cellular environment, will help to interpret, replicate, and compare nanotoxicology studies. Conventional methods for measuring the cellular uptake of nanomaterials are based on global titration in a population composed of millions of cells, yielding an average cell load. Inductively coupled plasma (ICP) spectroscopy, [9] UV-visible spectrometry, [10] SQUID (superconducting quantum interfer- ence device) magnetization measurements, [11] MR relaxom- etry [12] and imaging, [13] or EPR quantification [14] are accurate Dr. D. Fayol, Dr. N. Luciani, Dr. L. Lartigue, Dr. F. Gazeau, Dr. C. Wilhelm Laboratoire Matière et Systèmes Complexes (MSC) UMR 7057 CNRS & University Paris Diderot, Paris, France E-mail: claire.wilhelm@univ-paris-diderot.fr 1. Introduction Magnetic nanoparticles have exciting potential uses in cell imaging and cell therapies. The nanoscopic size of these mate- rials enables them to enter the cells and import unique prop- erties related to their responsivity to magnetic fields. [1] For example, individual cells become visible by magnetic reso- nance imaging (MRI), can be remotely manipulated by means of external magnetic fields, or can be destroyed by magnetic hyperthermia. Adv. Healthcare Mater. 2012, DOI: 10.1002/adhm.201200294