Universal cell labelling with anionic magnetic nanoparticles

Biomaterials 29 (2008) 3161–3174

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Biomaterials journal homepage: www.elsevier.com/locate/biomaterials

Leading Opinion

Universal cell labelling with anionic magnetic nanoparticlesq Claire Wilhelm*, Florence Gazeau** Laboratoire Matie`re et Syste`mes Complexes (MSC), UMR 7057 CNRS et Universite´ Paris-Diderot, Paris, France

a r t i c l e i n f o

a b s t r a c t

Article history: Received 7 February 2008 Accepted 1 April 2008 Available online 1 May 2008

Magnetic labelling of living cells creates opportunities for numerous biomedical applications, from individual cell manipulation to MRI tracking. Here we describe a non-specific labelling method based on anionic magnetic nanoparticles (AMNPs). These particles first adsorb electrostatically to the outer membrane before being internalized within endosomes. We compared the labelling mechanism, uptake efficiency and biocompatibility with 14 different cell types, including adult cells, progenitor cells, immune cells and tumour cells. A single model was found to describe cell/nanoparticle interactions and to predict uptake efficiency by all the cell types. The potential impact of the AMNP label on cell functions, in vitro and in vivo, is discussed according to cellular specificities. We also show that the same label provides sufficient magnetization for MRI detection and distal manipulation. Published by Elsevier Ltd.

Keywords: Iron oxide nanoparticles Magnetism MRI Biocompatibility Cell therapy Cell manipulation

1. Introduction Magnetic labelling of cells raised up increasing interest due to the various biological or medical applications involving magnetism in living organisms. Magnetic forces are widely used to separate cells in vitro [1–3], but also to manipulate or attract cells by an external stimulus with applicability for basic study of cell migration [4,5], for tissue engineering [6,7] or for cell therapy [8,9]. However, the most developed applications concern the use of magnetic resonance contrast agent to identify and track the migration of magnetically labelled cells following infusion or transplantation in vivo [10–12]. In this field, different techniques have been developed to label non-phagocytic cells in culture using magnetic nanoparticles. The main requirement is to supply cells with sufficient magnetization to be detectable by MRI (or manipulated by magnetic forces), while maintaining cell viability and functionalities. Dextran-coated iron oxide nanoparticles (Ultrasmall Superparamagnetic Iron Oxide USPIO) approved for clinical MRI protocols were first experimented for in vitro cell labelling, but showed poor intracellular uptake, especially for cells that lack substantial phagocytic capacity [13– q Editor’s Note: This paper is one of a newly instituted series of scientific articles that provide evidence-based scientific opinions on topical and important issues in biomaterials science. They have some features of an invited editorial but are based on scientific facts, and some features of a review paper, without attempting to be comprehensive. These papers have been commissioned by the Editor-in-Chief and reviewed for factual, scientific content by referees. * Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (C. Wilhelm), fl[email protected] (F. Gazeau). 0142-9612/$ – see front matter Published by Elsevier Ltd. doi:10.1016/j.biomaterials.2008.04.016

15]. To facilitate cell labelling, different strategies have been developed. The first class of strategies is based on a receptor mediated approach. Immunoglobulins were covalently linked to the dextran polysaccharide coat of the iron oxide in order to induce specific recognition with receptors at the surface of targeted cell and then trigger receptor mediated endocytosis: monoclonal antibody (mab) to the mouse transferrin receptor OX26 [16], human transferrin [17], and anti CD-11 mab for dendritic cells [18]. This strategy is similar to labelling techniques used in magnetic cell sorting applications, although the labelling procedure is modified to promote endocytosis of the magnetic tag. It is species-specific and may suffer from an insufficient number of receptors at the surface. Coupling the particle surface to a translocation agent which is not dependent on a receptor, as the HIV tat peptide [19], has been shown to improve the cell labelling efficiency [20] with a cell uptake increasing with the tat peptide/particles ratio [21]. The second class of labelling techniques, currently chosen in most of cell imaging assays, involves the use of a transfection agent helping the internalization of the magnetic nanoparticles. This method has applications in the labelling of a wide variety of cells since its mechanism is non-specific. Highly charged macromolecules form large complexes with dextran-coated nanoparticles, adsorb to the cell membrane via electrostatic interactions and induce membrane bending [22] that triggers endocytosis. This strategy is similar to the one used to transfect oligonucleotides into cells. Transfection agents (TAs) include cationic peptides, lipids, polyamines, and dendrimers. It can be directly engineered on the particle surface, as for magnetodendrimers [23], a highly branched regular 3D carboxylated structure on the iron oxide core. More widely, TA is simply added for a given time to dextran-coated SPIO

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suspension to form complexes, whose size, zeta potential, stability in culture medium as well as MR relaxivities and interactions with cells are finely tuned by the nature of the TA and particles/TA ratio [24–26]. Hence, despite its simplicity of use, the control of the complexes formed by TA and nanoparticles and their subsequent properties are not easily achievable. Nevertheless, different TAs, each of them complexed with USPIO ferrumoxides, have been successfully used for efficient magnetic labelling of various cell types with incubation time of at least 6–12 h [27,28]. However, inhibition of the chondrogenic differentiation of mesenchymal stem cells labelled with poly-L-lysine and ferrumoxides was observed [29]. Recently the clinically approved polycationic peptide – protamine sulfate – was proposed as a highly efficient TA to label mature [25] as well as stem cells [30–32] without any effect on their differentiation capacity in vitro or in vivo. The low molecular weight of protamine sulfate leads to smaller and better controlled complexes as compared with PLL. However, recent studies pointed out the possible precipitation of the TA-nanoparticles complexes and adsorption of these complexes on the plasma membrane of cells rather than internalization [26,33]. Also involving electrostatic interactions with cell, another class of efficient magnetic label has emerged in the last few years. It consists of dextran-free iron oxide nanoparticles coated with charged monomers. They are characterized by the absence of polymers, a small size (hydrodynamic diameter < 50 nm), a negative zeta potential and an electrostatic stabilization in colloidal suspension. The anionic citrate-coated USPIO (VSOP-C125 developed by Ferropharm, Germany) was shown to be incorporated by macrophages much faster and with a better efficiency than their carboxy-dextran counterparts [34]. Similar citrate-coated nanoparticles (VSOP-C184) with a very small size (iron core of 4 nm) are now under phase 2 clinical development [35]. At the same time, we demonstrated that anionic nanoparticles coated with dimercaptosuccinic acid (DMSA) were internalized by macrophages and Hela cells [36,37] in much higher amounts than classical dextran-coated nanoparticles. Surface coating was pointed out as a key factor to allow for non-specific interactions with plasma membrane. Since, a wide variety of cells have been labelled after short incubation with anionic monomer-coated nanoparticles without impairment of the cell viability and functionality. The aim of this paper is to review and document the use of anionic monomer-coated maghemite nanoparticles (AMNPs) for cell labelling. This labelling method, which leads to endosomal internalization of

the particles, is very simple (no modification of nanoparticles surface, no addition of transfection agent), rapid (20 min to 2 h), efficient and applicable for every kind of cell. Since its mechanism has been fully characterized, the labelling procedure is reliable and uptake efficiency is predictable knowing cell size, incubation time and extracellular iron concentration. Here different aspects are developed concerning the mechanism of cell uptake, the intracellular pathway and biocompatibility of AMNP and the use of AMNPlabelled cells for MRI detection and for magnetic manipulations. 2. Anionic monomer-coated nanoparticles (AMNPs): synthesis, characterization and cell labelling protocol The stability of magnetic nanoparticles in colloidal aqueous suspension (ferrofluid) requires repulsive interactions to counterbalance the globally attractive Van der Waals and dipole–dipole interactions. Electrostatic interactions between charged nanoparticles have been proposed by Massart [38] as an alternative to the steric repulsions between polymer coated nanoparticles, which are classically used in commercial ferrofluids. The nanoparticles used in this study are maghemite (gFe203) nanoparticles synthesized by alkaline coprecipitation of iron(III) and iron(II) salts. Adsorption of citrate anions to the ferric oxide surface confers to the particles a net negative charge due to carboxylic groups (zeta potential ¼ 30 mV) and ensures the colloidal stability in the range of pH from 3 to 11 and for ionic strengths lower than 0.35 mol/L. Alternatively, the particles can be coated with dimercaptosuccinic acid, which possesses thiol groups in addition to carboxylic groups [39]. The mean size of magnetic core is about 8 nm and the size distribution is described by a log-normal distribution with a polydispersity of 0.35. The hydrodynamic size, determined by dynamic light scattering, is about 30 nm. Cell labelling was performed in culture medium (RPMI) without addition of serum, but supplemented with 5 mM sodium citrate to ensure equilibrium between free and particle-bound citrate ions. Iron concentration varied from 0.05 to 20 mM and incubation time from 10 min to 8 h at 37  C. Unless otherwise stated, the incubation with nanoparticles was followed by two washing steps and by a chase at 37  C in particlefree culture medium to achieve the complete internalization. To demonstrate the non-specific nature of labelling with AMNP, this labelling procedure was performed for a wide variety of mammalian cells (see Table 1), including phagocytic and nonphagocytic cells, different species (rat, mouse, human), different

Table 1 Cell types that have been labelled with AMNP Cell types (origin)

d (mm)

K (mM)1

Immune cells Raw macrophages (mouse) Hybridomas (mouse) Dendritic cells (human) OT-1 lymphocytes (mouse) EL4-B lymphocytes (human)

11.7  0.8 12 12.2  1.6 8.4  0.6 9.2  1.5

17  4 44  31 29  8 43  26 30  10

Tumour cells HeLa ovarian carcinoma (human) PC3 prostatic carcinoma (human) HuH7 hepatic carcinoma (human)

20.2  2.6 14.6  2.3 11.6  1.7

Therapeutic adult cells Hepatocytes (mouse) Gingival fibroblasts (human) Smooth muscle cells (rat) Therapeutic stem cells or progenitor cells Myogenic precursor cells (pig) Endothelial progenitor cells (human)

si (h)

Fo

mp (pg)

Ref.

6  0.2 2.4  0.2 6.6  0.4 1.3  0.1 4.1  0.2

1.3  0.2 0.4  0.1 0.9  0.1 2  0.8 1.4  0.2

25  5 1.5  0.3 1.8  0.5 1.1  0.5 1.3  0.4

33 5.5 15.2 2.2 8

[37] [50] [42] [51]

17  5 16  5 27  11

18  0.3 8.1  0.2 3.4  0.2

1  0.1 0.4  0.1 1.2  0.1

2.5  0.2 1.8  0.3 1.5  0.2

37.5 13.4 8.3

[36] [66,67]

20  4 17  3 14  1

30  13 24  8 29  8

21.3  0.8 13.5  0.6 5.8  0.2

1.4  0.2 1.6  0.5 1.5  0.1

1.9  0.3 1.6  1 1.9  3

49 28 12.4

[68] [49] [47]

14  2.5 13.8  2.1

22  11 39  12

3.5  0.2 8.9  0.3

1.8  0.3 1.4  0.2

1.1  0.3 1.9  0.3

4.8 20.5

[55] [48]

mo (pg)

The different parameters describing the particle uptake of each cell type are indicated. d is the cell diameter, K is the affinity constant of AMNP for the cell membrane, m0 is the binding capacity on plasma membrane (in mass of attached particles), si is the characteristic time for internalization, f0 is the maximal fraction of internalized membrane, mp is the predicted mass of iron per cell for a labelling condition of [Fe] ¼ 20 mM for 2 h at 37  C.

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cell types (adult cells, progenitor or stem cells, immune cells and commonly used tumour cells), different cell size and culture properties (growth in confluence or in suspension, primary cultures or cell lineages). 3. Labelling cells with AMNP: a generic internalization pathway The pathway of AMNPs in mammalian cells was directly highlighted by TEM investigations. To distinguish between fluid-phase endocytosis and adsorptive endocytosis, cell/AMNP interactions were investigated both at 37  C and at 4  C. Membrane trafficking and internalization process are known to be inhibited at 4  C, so that only adhesion on cell plasma membrane can occur, if existing. By contrast, incubation at 37  C permits different routes of internalization. It was observed that, both at 4  C and 37  C (Fig. 1), anionic nanoparticles were attached on the plasma membrane in the form of clusters. It is a marked difference with dextran-coated nanoparticles which do not adsorb on cell membrane [37]. Cells were then washed and incubated at 37  C to follow the internalization process, which show the hallmarks of endocytosis pathway (Fig. 1). Soon after 10 min chase, invaginations of cell membrane with bound AMNPs and clathrin coated pits containing particles’ clusters were both observed. At 20 min, nanoparticles were densely packed into early endosomes, which were subsequently conveyed in the cytoplasm to fuse with late endosomes. After 60 min chase, all the nanoparticles were confined within late endosomes or lysosomes dispersed throughout the cytoplasm. It is noteworthy that the same uptake pathway was observed for all types of mammalian cells tested (Fig. 1). Thus AMNPs occupy successively different compartments of the endocytosis pathway (early endosome, late endosome, lysosome), which could be individually purified by magnetic separation and biochemically characterized [40]. Organizations of nanoparticles on the plasma membrane and within intracellular vesicles were fully characterized by TEM, small angle neutron scattering and dynamic magneto-optical birefringence for Hela cells [41]. AMNPs do not behave as single particles, but are clustered into aggregates decorating the endosomal membrane and/or homogeneously dispersed in the endosome. From magnetic measurements on purified endosomes – namely, magnetization curve and magnetophoretic mobility – the number of particles per endosome was estimated in the range of 1000 to 5 00 00 depending on the labelling conditions. Due to their high content in magnetic particles, these submicronic intracellular vesicles acquired a large magnetic moment (up to 5  1015 A m2), when submitted to a magnetic field. As a consequence, magnetic endosomes attract each other due to dipole–dipole interactions and form chains of several endosomes (typically 2–8) aligned in the direction of the magnetic field (Fig. 2a). These chains colocalized with the lysosomal marker, LAMP1, reinforcing the view of late endosomal nature of intracellular magnetic vesicles (Fig. 2b). 4. Efficiency of AMNP uptake: a single model to quantify labelling in every cell type Nanoparticles uptake was measured quantitatively by two complementary methods, single cell magnetophoresis and electron spin resonance (ESR) [42]. Single cell magnetophoresis consists of measuring the velocity of cells in suspension, when they are submitted to a magnetic field gradient. This measurement provides the distribution of iron load per cell for the whole cell population. By contrast, ESR gives access to a mean value of iron load per cell. Uptake kinetics were evaluated both at 4  C and 37  C in the case of Hela cells and mouse macrophages (Raw) [36]. Uptake

Fig. 1. Intracellular pathway of anionic magnetic nanoparticles observed by TEM. Cells were incubated at 4  C and 37  C with AMNP ([Fe] ¼ 2 mM) for different times. At 4  C and for short times at 37  C, nanoparticles adsorb on plasma membrane. At 37  C, the membrane-adsorbed particles are progressively internalized following the endocytosis pathway. (Top) HeLa cells after 1 h incubation at 4  C and a consecutive incubation at 37  C for 10 min, 20 min and 1 h. (Middle) EL4-B lymphocytes after 10 min and 30 min incubation at 37  C. (Bottom) endothelial progenitor cells after 15 min and 1 h incubation at 37  C.

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Fig. 2. Endosomal localization of AMNP within the cell. After nanoparticles internalization ([Fe] ¼ 2 mM for 1 h at 37  C) Hela cells were submitted to a uniform magnetic field (0.1 T). (a) A TEM picture of the cells fixed under magnetic field shows the chaining of nanoparticles loaded intracellular compartments in the direction of the field. (b) Chains of intracellular compartments colocalize with LAMP1, which is a marker of late endosomes (from left to right: bright light/fluorescence microscopy/ merge of the two pictures).

efficiencies were compared for dextran-coated nanoparticles (SineremÒ, Guerbet Laboratories), bare AMNP and albumin-coated AMNP [37]. As shown in Fig. 3a, AMNP cell uptake was saturable both at 4  C and at 37  C and as function of incubation time or extracellular iron concentration. In the same range of incubation time (<10 h), the uptake of dextran-coated nanoparticles or BSAcoated AMNP at 37  C did not saturate and was, respectively, about 1000-fold and 100-fold less efficient than for AMNP. Moreover the uptake of albumin-coated AMNP decreased drastically when the ratio of albumin to AMNP was increased [37]. Despite a negative surface charge beared by the BSA, it was found to strongly adsorb on the anionic nanoparticles, so that steric interactions due to polymer shell were presumably hampering the adsorption process mediated by electrostatic interactions between AMNPs and cell membrane. These observations suggest two different mechanisms for cell uptake: electrostatic adsorptive endocytosis for AMNP and fluid-phase endocytosis for dextran or BSA-coated nanoparticles. It emphasizes that the non-specific affinity of AMNP for cell membrane is the key point to ensure high labelling efficiency. In an attempt to rationalize labelling procedure of every cell type, a formalism based on a mass action kinetics was tentatively developed for modelling the mechanism of AMNP uptake [36]. It assumes that AMNP cellular uptake consists of two processes occurring concomitantly: binding of AMNP on reactive sites on the cell membrane and internalization of the reactive sites by endocytosis pathway. The binding step is modelled by a Langmuir adsorption, with a finite number of binding sites (cationic sites on the cell membrane), leading to a maximum mass m0 of attached particles (binding capacity). Adsorption rate of nanoparticles is proportional to their extracellular concentration C and to the number of vacant binding sites. Desorption rate is proportional to the number of occupied sites. The constant rates for adsorption ka (in M1s 1) and desorption kd (s1) are defined. K ¼ ka/kd (in M1) measures the affinity of particles for cell membrane. If internalization occurs concomitantly, the binding capacity m0 is

Fig. 3. Quantification of nanoparticles uptake by different cells. (a) Comparative uptake of AMNP at 4  C and 37  C of BSA-coated AMNP and of dextran-coated SPIO at 37  C as a function of incubation time for HeLa cells. Extracellular iron concentration is [Fe] ¼ 15 mM. (b) Comparative uptake of AMNP by hepatocytes, gingival fibroblasts, endothelial progenitor cells, smooth muscle cells, HuH7 tumour cells and B lymphocytes as a function of extracellular iron concentration. Incubation times are indicated in the inset. Theoretical adjustments using the model detailed in the text are plotted as solid lines. Parameters deduced from the fit are indicated in Table 1.

assumed to be constant, which means that binding cationic sites are continuously regenerated. The internalization step is described as a first order kinetic saturation law, with F0 being the maximum fraction of surface that can be internalized (internalization capacity) and si is the characteristic time for internalization. This simple model was initially developed to adjust experimental uptake in Hela cells and Raw macrophages both at 4  C and 37  C [36]. Here it was tested to model uptake kinetics for a large variety of cell types. Fig. 3b shows the uptake curves for different mammalian cells, together with the theoretical adjustments. The parameters used to fit experimental data are summarized in Table 1. It is remarkable that this model was able to fit experimental uptake kinetics (as function of incubation time or extracellular concentration) for all tested cells. The robustness of the model is illustrated by Fig. 4, where the binding capacity m0 and the affinity K for cell membrane are plotted as a function of cell diameter. This graph highlights the non-specific nature of the AMNP interactions with plasma membrane. The affinity K is almost constant whatever the cell type and cell size, in the range of 1.6  107–4.4  107 M1. It is lower than reported values for the binding of charged liposomes

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5. Cell internalized AMNP

Fig. 4. Binding capacity m0 (in pg of iron) of AMNP on the plasma membrane and affinity constant K (in M1) as a function of the cell diameter d. The binding capacity scales with the surface of the cell: the best fit corresponding to m0(d) ¼ 0.0426  d2 is presented as a dotted line. Affinity constant of Fab fragment to its cell surface receptor and of anionic, cationic and Fab grafted liposomes to the cell surface are indicated for comparison.

on plasma membrane [43–45] and comparable with the affinity of a free Fab’ fragment (from 4D5 antibody) to its cell surface receptor [46]. The binding capacity scales with the square of cell diameter, which means that it is roughly proportional to the surface of the plasma membrane. The binding capacity per unit surface is 0.027 pg/mm2, corresponding to about 2.3  104 nanoparticles per mm2, independently of the cell type. It follows from these two findings that the efficiency of the adsorption step is predictable for all cell types, depending only on their external surface. Parameters characterizing the internalization process – i.e. the internalization time si and internalization capacity F0 – are also quite homogeneous for the different cell types, ranging from 0.4 to 1.8 h and from 1.5 to 2.5, respectively. However, macrophages show a unique internalization capacity of 25, in keeping with their specificity as professional phagocytes. In conclusion, the model is valid to predict labelling efficiency using AMNP. A maximum mass of iron, which may be internalized, can be predicted for all cell types (see Table 1). For example, small sized OT-1 lymphocytes will internalize 2.2 pg of iron at maximum, whereas large hepatocytes will uptake up to 49 pg. For cells having a low or moderate endocytosis capacity, the labelling efficiency is primarily governed by the adsorption process. Thus differences in cell uptake are principally related to the variation of cell surface area for one cell type to the other, as emphasized in Fig. 3b. Modelization also helps to optimize the labelling protocol. Increasing concentration of nanoparticles in the extracellular medium decreases the characteristic time of Langmuir adsorption at the cell surface (as 1/(kaC þ kd)). Hence particles bound on the cell surface, available for internalization at short time, are much more numerous and more rapidly regenerated for high extracellular concentration. As a consequence, the choice of high extracellular iron concentration combined with short incubation time is well suited to achieve efficient magnetic labelling. It is a huge difference with the other labelling procedure, which required long incubation times (usually about 24 h) to optimize the particles’ internalization and low concentration of transfection agent to avoid toxicity. Incubation time with AMNP can be limited to short period (typically 15 min to 2 h) and followed by a period of chase to permit the complete internalization of the particles (1–3 h).

During cell division, the nanoparticles load is equally shared by the daughter cells. Qualitatively, magnetic endosomes are distributed on both sides of the mitotic spindle (Fig. 5a), leading to the sharing of the endosomes. Quantitative detection of nanoparticles’ contents was performed up to 14 days after labelling for different cell types [47–51]. Whatever the cell type, cell proliferation was not affected by AMNP labelling. Besides, iron load dilution scaled with cell division rate (Fig. 5b) and the histograms of cell uptake conserved their relative width during division, showing fair inheritance of the nanoparticles from the parent cells. TEM observation [47,51] showed that the nanoparticle-loaded organelles become scarcer in the descendant cells, but also larger with more densely packed nanoparticles (Fig. 5c). Note that with mammalian cells, exocytosis of the nanoparticles was never observed. The long term becoming of AMNP in non-dividing cell was not investigated so far. More generally, the metabolic pathway for the degradation of iron oxide nanoparticles in lysosomes, which may represent their terminal compartments, is not fully understood. The low pH lysosomal environment is believed to allow long term degradation of the nanoparticles and metabolisation into assimilable ferric iron species. It was shown outside the cell that low pH and presence of metallic chelates like citrate were sufficient to dissolve dextran-coated nanoparticles [52]. Therefore it could be interesting to compare the metabolic pathway of cell internalized citrate-coated nanoparticles as compared to their dextran-coated counterparts. Moreover, it appears necessary to investigate expression of transferrin receptor and ferritin, following AMNP internalization, both of them being involved in cellular iron homeostasis [53]. 6. Biocompatibility of AMNP labelling: in vitro and in vivo cell functionalities The main requisite for a cell labelling technique is to preserve the normal cell behaviour. Biocompatibility of AMNP was verified at different levels in line with the cell specificity. In vitro viability and proliferation capacity were not affected by AMNP uptake (see Fig. 5b). Cell phenotype was not significantly affected or transiently affected, showing a reversible stress response to magnetic labelling. Human gingival fibroblasts (HGF) are phenotypically unique cells in adults since they contribute to the rapid healing of oral wounds. Transcription and secretion of connective tissue remodelling molecules by human gingival fibroblasts were quantified after labelling with AMNP (15 pg of iron per cell) [49]. Secretion of interleukins 1b, matrix metalloproteinases (MMP-1, -2 and -3) and tissue inhibitor of metalloproteinases TIMP-2 increased at day 1 after labelling, whereas TIMP-1 did not. The increase of interleukin 4 secretion began at day 3 while MMPs and TIMPs secretion decreased. Despite this transitory inflammatory reaction, HGF phenotype was stabilized 3 days after labelling. Vascular smooth muscle cells (VSMCs) have been proposed for cell therapy of aortic aneurysms or myocardial ischemia. The balance between MMPs and TIMPs was investigated after magnetic labelling (4 pg of iron per cell) [54]. There were no significant differences with control cells in the mRNA levels of MMP-2, MMP9, TIMP-1 and TIMP-3 both at 6 h and 24 h after labelling. Moreover the loading of VSMCs by AMNP did not modify their expression of alpha-actin. Endothelial progenitor cells (EPC) have potential for vascular repair at sites of ischemia and are also involved in vascularization of tumours. The effect of increasing uptake of AMNP (up to 12 pg of iron per cell) was quantified for different membrane proteins specific to the endothelial phenotype [48]. Slight differences with nonlabelled cells were observed at day 1 after labelling in expression of

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Fig. 5. Magnetic labelling of dividing cells. (a) Confocal microscopy of an AMNP-labelled Hela cells ([Fe] ¼ 10 mM for 1 h) during mitosis and under a 0.1 T magnetic field. Microtubules and F-actin are stained for immuno-fluorescence using, respectively, antitubulin green antibody and red phalloı¨din. Chains of magnetic endosomes can be seen on each sides of the mitotic spindle. (b) (Left) proliferation of magnetically labelled cells versus control cells. N is the number of cells as a function of days after labelling (N0 the initial number). Bars represent the variations of proliferation between non-labelled cells and cells labelled with different labelling conditions (EPC: [Fe] ¼ (2–5–10) mM for 30 min and 2 h; fibroblasts: (1–5–10–15–20) mM for 20 min; HuH7: 5 mM for 1 h; MPC: 10–25 mM for 1 h; SMC: 1–5 mM for 2 h). Cell number increases as N ¼ N0  2ðD=D0 Þ (solid lines), where D0 is the doubling time in days ((1.17  0.06) for EPC; (1.83  0.31) for fibroblasts, (1.33  0.07) for HuH7; (2.24  0.3) for MPC; (2.56  0.25) for SMC; (1.2  0.08) for lymphocytes). (Right) decrease of the mass of iron per cell (m) during proliferation. mi is the initial mass after labelling (EPC: 5 mM for 1 h; fibroblast: 10 mM for 20 min; SMC: 1 mM for 2 h; lymphocytes: 4 mM for 15 min). m decreases as m ¼ mi =2ðD=D0 Þ (dotted lines) in agreement with a redistribution of internalized particles among descendant cells (no exocytosis). Inset shows the histograms of iron mass per cell for different days after labelling of EPC. (c) TEM pictures of magnetically labelled cells at different days after labelling. (Top) OT-1 lymphocytes the day of labelling (4 mM for 15 min) (left) and 3 days later (right). (Bottom) smooth muscle cells the day of labelling (1 mM for 1 h) (left) and 7 days later (right). Magnetic endosomes become scarcer with cell division, but also bigger and more loaded with particles.

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E-selectin CD62E and of two tyrosine kinase receptors (VEGF-R2 and angiopoietin-1 receptor TIE2), but were normalized at day 2. Expression of PECAM-1 adhesion protein (CD31) rose slightly on day 2 and VE-cadherin (CD144) expression was stable at days 1 and 2. Hence despite a transient non-significant early response, labelling with AMNP preserved the expression of major proteins involved in neovascularization. The issue of differentiation capacity of labelled stem cells has been recently discussed given that inhibition of chondrogenesis was observed after labelling of mesenchymal stem cells (MSC) using the association of superparamagnetic iron oxide nanoparticles (FeridexÒ, AMAG Pharmaceuticals) and poly-L-lysine [29]. So far, there is no published data on the differentiation of multipotent stem cells after labelling with anionic nanoparticles, but rather on committed cells already engaged in a differentiation way. AMNPlabelled endothelial progenitors cells (EPC) isolated from human cord blood samples were able to differentiate in vitro and to selforganize to form tubular structure in matrigel regardless of their iron load or time after labelling (Fig. 6a) [48]. Myogenic precursor cells (MPC) produced by pig skeletal muscle explants were labelled with AMNP [55] and showed a non-altered capacity to proliferate, to differentiate into myotubes and form regenerated myofibers. Innocuity of AMNP uptake was also assessed by the behaviour of labelled cells in animal models of cell therapy. In vivo studies involving local or intravenous injection of AMNP-labelled cells and MRI monitoring are summarized in Table 2. To assess cell therapy consecutive to a local graft, labelled vascular smooth muscle cells were seeded endovascularly in already formed, expanding abdominal aortic aneurysm in rat [54]. AMNPlabelled VSMCs were actually able to stabilize the diameter of aneurysms by contributing to the aortic wall reconstruction (Fig. 6a). The majority of iron-loaded cells remained up to 1 month in the regenerating intimal tissue, as first detected by 1.5 T MRI and confirmed by histology and high resolution 9.4 T ex vivo MRI. Moreover magnetic cells were alpha-actin positive, thus participating to aortic regeneration. In another study, myofibers isolated from skeletal muscle were magnetically labelled with AMNP and implanted into the urethra of pigs to regenerate muscular function [55]. In this cell-based therapy of muscle disorder, myofibers act as a reservoir of myogenic precursor cells (MPC) which can escape the implant to differentiate and form new myotubes in injured areas. Interestingly, magnetically labelled myofibers implanted in vivo were still able to produce MPC capable of fusion and formation of new myotubes. Some of these MPC had internalized nanoparticles before migrating out of the implant to achieve their therapeutic function. In the last two studies, MRI associated with magnetic labelling of therapeutic cells allowed the immediate localization of cell seeding or tissue graft as well as the long term monitoring of cell becoming and tissue regeneration (Fig. 6b). AMNPs operate as an innocuous tracer both for non-invasive in vivo imaging and for ex vivo histology. These studies considered the engraftment or migration of labelled cells proximal to their sites of injection. Another cell therapy approach consists of the systemic transfer of cells which will be recruited and activated in some organs which are distant from the infusion site. In a first study [50], hybridomas (fusion between B lymphocytes and myeloma cells) labelled with 2.5 pg of iron were injected intraperitoneally to nude mice. MRI revealed the homing of the labelled cells to the spleen soon 24 h after infusion. Magnetic cells in the spleen were recovered, numbered and their iron load was quantified. This quantitative study established that the labelled hybridomas have been able both to migrate in their homing site and to proliferate in vivo. In a mouse model of autoimmune diabetes [56], T-cell homing to the pancreas was tracked by use of in vivo MRI and AMNP-

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labelled T-lymphocytes (1.6 pg of iron per cell). Since 11 days after their intravenous transfer (106 cells), labelled T-cells were found in the pancreas, in direct contact with necrotic b-cells. Hence autoreactive T-cell toxicity was preserved by labelling as also demonstrated by the similar rate of diabetes mellitus onset in mice transferred with unlabelled or labelled T cells at 30 days. Immune cells can also be used as cell-based therapeutic tools for the treatment of cancer. Cell imaging techniques are being developed to track cell redistribution in vivo and assess treatment efficacy. In this context, ovalbumin-specific splenocytes labelled with AMNP were adoptively transferred into mice with growing ovalbumin-expressing tumours [51]. MRI cell tracking at 7 T showed that the intravenously infused lymphocytes initially homed to the spleen (at 24 h) and migrated to the tumour in a second time, at 48 h and 72 h after transfer. Quantitative magnetic sorting of total splenocytes confirmed in vivo results and showed that transferred cells underwent divisions within the spleen before being recruited by the tumour. Moreover tumour expressing ovalbumin antigen regressed 6 days after cell transfer, whereas control tumour not expressing ovalbumin progressed. This again proved that both cell specificity and toxicity were not altered by labelling procedure. These different studies exemplified the absence of in vivo function alterations due to AMNP cell uptake. In the first series of studies, cells were able to migrate towards proximal sites after local injection, to differentiate if necessary and to induce tissue regeneration. In the second series, immune-competent cells infused through the systemic route were able to home towards secondary lymphoid organs, to proliferate and eventually to activate, before migrating towards targeted organs where they exerted their cytotoxic functions. 7. MRI cell detectability Magnetic labelling of cells has been mainly developed with the aim to monitor cell trafficking in vivo by non-invasive MRI [10,11]. Indeed superparamagnetic nanoparticles create local magnetic fields which modify the dynamics of magnetization of surrounding protons. It results in contrast enhancement that extends further than the dimensions of the particles or even the cells themselves [15,57,58]. MRI cell detection using AMNP as cellular contrast agent has been discussed using different spatial resolutions. At low resolution, the global signal enhancement depends on the concentration of labelled cells in a given area [47,50,59]. By contrast, high resolution MRI allows the detection of single individual cells [60]. Central to the detection of labelled cells is the fact that properties of contrast agent are modified following cell internalization [59]. The efficiency of the contrast agent is evaluated by its longitudinal (r1) and transverse (r2) relaxivities, which determine the magnetization relaxation rates as function of contrast agent concentration. When dispersed in colloidal suspension, AMNP have relaxivities of r1 ¼10.2 s1 mM1 and r2 ¼ 357 s1 mM1. In contrast, once internalized into intracellular cell endosomes, AMNP relaxivities change to r1 ¼1.1 s1 mM1 and r2 ¼ 248 s1 mM1. The drastic decrease of longitudinal relaxation is due both to the slowering of water diffusion inside the cell and to the saturation of relaxing effect of nanoparticles, once they are strongly confined into micrometric endosomes. The confinement of nanoparticles inside the cell directly influences their relaxing properties. At low resolution, the relaxation rate 1/T2 of a region of interest is directly proportional to the global iron concentration or to the cell density (Fig. 7a and b). 1/T2 decreases when cells divide (at constant number of cells), but remains constant with time for cell proliferating in a constant volume (no change in the global iron concentration) [47]. Moreover, due to the very high ratio r2/r1, the

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Fig. 6. Functionality of magnetically labelled cells. (a) AMNP-labelled endothelial progenitor cells ([Fe] ¼ 10 mM for 30 min) self-organize to form capillary network on matrigel. Successive pictures were acquired at 1 h, 6 h and 11 h after deposition on matrigel. Kinetics of formation and morphology of the network were similar to non-labelled cells. (b) Endovascular cell therapy using AMNP-labelled vascular smooth muscle cells ([Fe] ¼ 1 mM for 2 h) [54]. (Top) in vivo 1.5 T MR imaging of aortic aneurysm before (left) and after (right) local delivery of labelled VSMC. Gradient echo image evidenced hyposignals area on the part of aortic wall, where the cells were delivered. (Bottom) high resolution 9.4 T MR gradient echo image of the aortic section 28 days after delivery of the labelled VSMC (left). Corresponding histologic examination with Perls staining (right). Iron-loaded cells were present in the deep portion of the aortic wall, with a good correlation between MRI and histological localizations. (c) Cell-based anticancer therapy using tumour-infiltrating lymphocytes [51]. AMNP-labelled ovalbumin-specific lymphocytes (4 mM for 15 min) were infused intravenously to mouse with growing ovalbumin-expressing tumors. In vivo 7 T MRI (gradient echo sequences) reveals the pathway of lymphocytes in the body. Negative enhancement and increased volume of the spleen are observed 24 h after cell transfer, whereas no modification appeared in the tumour. By contrast, 72 h after cell transfer, the tumour showed hyposignal areas, whereas the spleen retrieved its normal aspect: lymphocytes first accumulate in the spleen, where they divide before migrating in a second time to the tumour.

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Table 2 Different in vivo models using AMNP as cell label Animal model

Cell types

Potential for cell therapy

Iron mass per cell (pg)/ labelling conditions

Number of injected cells/injection mode

MRI tracking

In vivo cell functionality

Nude mice [50]

Hybridomas

Trafficking of immune cell

(2.6  0.4)/ (8 mM, 20 min)

20  106/intraperitoneal

1.5 T in vivo 24 h post-injection

C57BL/6 mice bearing ovalbuminexpressing tumour [51]

Ovalbuminspecific splenocytes (OT-1)

Cell-based anticancer therapy

(1.3  0.1)/ (4 mM, 15 min)

3  106/ intravenous

7 T in vivo (24, 48, 72 h post-injection), 9.4 T ex vivo

Irradiated NOD mice [56]

Cytotoxic CD3þT-cells

Mechanism of autoimmune diabetes

(1.6)/ (2 mM, 1 h)

5  106/ intravenous

7 T in vivo up to 20 days

Mice [68]

Syngeneic hepatocytes

Cell therapy of liver diseases

(20  4)/ (5 mM, 15 min)

8  105/ intraportal infusion

1.5 T in vivo up to 32 days

- Homing to spleen - Cell proliferation within spleen - Initial homing to spleen (24 h) and cell division - Migration to the tumour (72 h) - Tumour regression - Homing to the pancreas - Contact with necrotic b-cells - Onset of diabetes mellitus - Implantation in the liver

Rat with expanding abdominal aortic aneurysm [54]

Syngeneic vascular smooth muscle cell

Endovascular cell therapy of aortic aneurysms

(3.9  0.9)/ (1 mM, 2 h)

106/local injection in aneurysm lumen

1.5 T in vivo up to 28 days 9.4 T ex vivo

Pig [55]

Myogenic precursor cells

Muscle therapy using sliced muscle autograft

(1.4  109 pg)/g of dried implant/ (25 mM, 2 h)

Autograft of labelled myofibers implants into the urethra

0.3 T in vivo up to 1 month

presence of labelled cells results, for spin echo sequences, in signal loss increasing exponentially with echo time. Spin echo sequences are thus well suited for quantification of labelled cells at low resolution in a given area, as, for example, in the spleen [50]. Gradient echo sequences are more sensitive to the presence of labelled cells, which act as susceptibility anomalies: r*2 relaxivity is one order of magnitude larger that r2. It is thus a sequence of choice for accurate detection of labelled cells. However, the quantitation of magnetically labelled cells on the basis of low resolution 1=T2* measurements is tricky because the signal will strongly depend on the distribution of labelled cell in the region of interest. By contrast, at high resolution, gradient echo sequence is the best suited sequence for the detection of small aggregates of cells or of single cells (Fig. 8a and b). A labelled cell appears as a signal void due to the default of proton magnetization created in its surrounding [60]. Indeed a magnetically labelled cell loaded with 5 pg of iron acquires a magnetic moment of typically 5  1013 A m2 when it is submitted to the MRI magnetic field. Due to its size and high magnetic moment, a labelled cell is seen by the surrounding protons as a static susceptibility artefact, creating a dipolar magnetic field inhomogeneity (Fig. 8c). Typically the field increment is 104 T at the surface of the cell and 107 T at 50 mm from its centre (with a decrease as 1/r3). Thus the more the proton is close to the cell, the faster is the Larmor precession of its magnetic moment. Protons nearby the cell become rapidly out of phase compared to protons at distance. This dephasing effect propagates spatially with time and the subsequent magnetization loss and signal void spread over a larger volume. As a consequence, the water volume affected by the presence of the labelled cell strongly exceeds the actual cell size and increases with the time before MR detection (echo time) (Fig. 8c). By this process, single AMNP-labelled cells have been detected in agarose gel for iron load in the range of 0.2–20 pg. The spatial

- Stabilization of aneurysm’s diameter - Aortic wall reconstruction - Regeneration of new functional myofibers in the urethra

resolution is the key point to determine the detection threshold, since protons magnetization will be added over the voxel volume. A resolution of at least 100 mm in the three directions is needed for accurate detection of single cell. For AMNP-labelled cells, it has been achieved using a 9.4 T MRI [60] or alternatively a 1.5 T MRI coupled with a superconductive low noise detection coil [61]. In the first case, the signal is increased proportionally to the field strength, whereas in the second case the detection noise is reduced. In both cases, the increase of signal/noise ratio allows to attain very high resolution (up to 20 mm). Cell distribution can be assessed in three dimensions, with a very good correspondence with optical microscopy as shown in Fig. 8b. Thus high resolution MRI combined with magnetic labelling may be complementary to histology (Fig. 6b) as a non-destructive three-dimensional imaging technique for ex vivo samples, with a very high sensitivity to the magnetic label (0.2 pg of iron for a voxel of 30 mm3). In vivo, the detection of labelled cells is complicated by the intrinsic variations of susceptibility in tissue, whose relaxing effects are amplified for high magnetic field and high echo time. Thus the use of short echo times associated to the highest possible resolution must be recommended for in vivo monitoring of cell distribution. In the model of cancer cell therapy previously described [60], it has been possible to detect single AMNP-labelled lymphocytes infiltrating the targeted tumour, 2 days after their intravenous injection [62]. Injected lymphocytes were initially loaded with 1.3 pg of iron and underwent division in the spleen, leading to iron load of 0.23 pg per cell at 48 h. The tumour was imaged in vivo at 48 h after lymphocytes injection in a 1.5 T MRI using a superconducting low noise surface coil [61]. A resolution of 59 mm was achieved allowing the detection of punctual signal voids dispersed in the tumour volume. These signal voids were attributed to the presence of labelled lymphocytes with an iron content as low as 0.2 pg. This finding was

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Fig. 7. Quantitative detection of labelled cells with low resolution MRI. (a) Longitudinal (1/T1) and transverse (1/T2) relaxation rates of protons magnetization at 1.5 T in presence of dispersed AMNP (empty circles) and of cell internalized AMNP (plain circle) at equivalent iron concentrations. Relaxivities of AMNP are modified following cell internalization [59]. (b) Transverse relaxation rate (1/T2) increases linearly with the number of labelled cells in agarose phantoms (0.5 ml, smooth muscle cells labelled with [Fe] ¼ 1 mM for 2 h) and decreases, for a constant number of cells in the phantom (106), when cells underwent division as a function of the number of days in culture. MR images (1.5 T) of the phantoms were obtained with a T2-weighted spin echo sequence (TR ¼ 2 s, TE ¼ 120 ms).

confirmed by histology, showing dispersed Prussian Blue-stained cells in the tumour tissue. Hence even under in vivo conditions, single AMNP-labelled can be monitored by MRI with a sensitivity allowing the detection of labelled cells after several divisions. 8. Magnetic manipulation of cells Beyond MRI detection, magnetic labelling of cells offers the opportunity to manipulate biological entities at a distance. Magnetically driven manipulation may involve either intracellular labelled organelles or the cell as a whole magnetic object. Due to their high content in magnetic particles, endosomes are sensitive to external fields. Under uniform field, magnetic endosomes elongate in the direction of the field and attract each other due to dipole–dipole interaction (Fig. 9a). Deformation of endosomes is limited by bending energy and thermal fluctuations of their membrane, so that the mechanical membrane properties can be deduced from the analysis of their deformation under magnetic field [63]. Beside, chains of intracellular endosomes can be rotated by external magnetic field (Fig. 9b): their dynamic response to this magnetic constraint provides unique information on the local mechanical properties of cell’s internal architecture [64]. Thus magnetic organelles serve as probe of their intimate environment. Cells tagged with AMNP can also be manipulated as a whole by external magnetic field gradients. The magnetic force experienced by each cell is Fm (in N) ¼ Mcell  grad B, where Mcell (in A m2) is the magnetic moment of the cell in the field B and grad B (in T/m) is the field gradient. For iron load from 1 to 20 pg, cell magnetic moments range from 1013 to 2  1012 A m2 at saturation. It is possible to

generate magnetic field gradients in the range of 10–50 T/m (over a distance up to 1 cm) using permanent magnets [3,42] and in the range of 500–1500 T/m (over tenth of mm) using thin magnetized tips [5], corresponding to forces on labelled cells in between 1 and 100 pN and up to a few nN. By these means, two applications could be developed involving the control of cell transport. On one hand, the ability to separate living magnetic cells on a microfluidic platform has been demonstrated [3]: cells pass through a miniaturised chamber and are deflected from the direction of the flow proportionally to their magnetic content (Fig. 9c). Such a continuous flow separation of magnetic cells offers the unique advantages, compared to bulk cell separations, to considerably reduce the volume of required material and to allow separations in sub-populations of different magnetizations. Besides, as both magnetic labelling and magnetic isolation are mild technologies, cells are not damaged after sorting and retain their biological activities. On the other hand, in a more basic point of view, the use of magnetic forces in the nN range allowed unravelling the role of mechanical forces during cell migration on their substrate. By applying external magnetic forces, it has been possible to control the cell movement during the early stage of the social amoeba Dictyostelium morphogenesis and to determine the force threshold to counterbalance chemotaxis during cell aggregation, by magnetically induced mechanotaxis (magnetotaxis) [5]. In a like manner, it has been showed that the different phases of capillary formation by endothelial progenitor cells could be piloted by a magnetic field gradient [48]. A capillary network could form along the lines of magnetic field gradient due to magnetic forces on

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Fig. 8. Single cell detection with high resolution MRI. (a) Gradient echo MR images (9.4 T) of agarose phantoms (0.3 ml) containing 105 (left) and 103 cells (right) (iron mass per cell ¼ 2.5  0.4 pg). In plane resolution is 23.5  23.5 mm2. Slice thickness is 125 mm. Numbers of cells per slice are, respectively, 817 and 8.17 [60]. (b) Optical microscopy (left) and 9.4 T gradient echo 3D images (right) of a cell monolayer deposited on an agarose gel. 3D cell distribution reveals the meniscus formed by the underlying gel. (c) (Top) local magnetic field created by a 5 mm single cell with an iron load of 5 pg magnetized at saturation. The local field modulus is represented as a colour code (in mT), together with the field lines in the top-right field map (200 mm  200 mm). The difference in proton’s Larmor frequency is proportional to the component of the local field in the direction of the static magnetic field. This component is represented by a colour code in the top-left field map. Arrows indicate the relative directions of protons magnetization 50 ms after a refocusing pulse. (For commodity, proton precession is represented in the plane of the image, but occurs in the perpendicular plane.) (Middle) sketches of protons magnetization as a function of time after the refocusing pulse: the phase perturbation propagates spatially with time. (Bottom) MR images (9.4 T) of a single cell (5.6 pg of iron) acquired with a gradient echo sequences for different echo times (TE) [60]. The voxel size is 40 mm3. The apparent size of the cell is much larger than its actual size and increases with the echo time.

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Fig. 9. Magnetic manipulations of cells. (a) TEM pictures of HeLa cells ([Fe] ¼ 10 mM for 1 h) fixed under a 0.1 T magnetic field. Intracellular endosomes loaded with AMNP are deformed in presence of a magnetic field. Statistical analysis of these deformations allows evaluating the bending rigidity and internal tension of endosomal membranes [63]. (b) Optical microscopy of living macrophages ([Fe] ¼ 2 mM for 1 h). Intracellular magnetic endosomes attract each other under magnetic field and form long cohesive chains distributed throughout the cytoplasm. These chains can be rotated inside the cell by simply changing the direction of the external field. The analysis of chains’ rotation in response to the magnetic torque gives access to the mechanical properties of their close environment. Cartography of intracellular viscoelasticity can be achieved using this unique method of intracellular microrheology [64]. (c) Magnetic cell sorting in a microfluidic platform [3]. AMNP-labelled cells injected through one microcanal are deflected according to their magnetization (or iron load), when they are submitted to a magnetic field gradient. They can be sorted with respect to their magnetic content and recovered in different exit microcanals. (d) Endothelial progenitor cells ([Fe] ¼ 5 mM for 2 h) forming a capillary network on matrigel can be attracted with a magnetic tip. The figure shows successive images of EPC near the tip. Some pseudopodes extend in the direction of the tip driven by a magnetic endosome. The magnetic force required to split cells interacting in the network is a measure of between-cells interaction force.

individual cells and conversely a preliminary formed network could be deformed due to magnetic forces. More generally, tissue engineering as well as cell-based therapy could benefit of magnetic properties of labelled cells for cell

targeting. In accordance with these outlooks, magnetic labelling has been recently used to capture and retain magnetically labelled endothelial cells on magnetized intravascular stent in the presence of blood flow [8,65].

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9. Conclusion MRI combined with magnetic cell labelling is currently becoming the method of choice to monitor cell migration in cell therapy assays. Beyond imaging, the concept of ‘‘magnetic cells’’ opens new possibilities for cell manipulation by non-contact constraints. Magnetic forces at a distance can be used to control the movement of flowing cells (with application in cell sorting), but also to influence the organization and the migration of cells on an engineered substrate or in a tissue. Therefore, tissue engineering as well as cell-based therapy could benefit of magnetically induced cell targeting or cell control. In that context, cell labelling with anionic magnetic nanoparticles shows the advantages of simplicity, rapidity, universality and predictable efficiency. Apart of in vitro innocuousness of AMNP labelling, the preservation of cell functionalities has been proven in vivo for different cell therapy assays. Besides, the value of AMNP labelling for MRI cell detection and monitoring of cell migration has been demonstrated. Finally, original methods of intracellular and cellular manipulations have been developed using this label. Acknowledgments We are grateful to C. Rivie`re, P. Smirnov and J.-P. Fortin for their respective contribution to this work. References [1] Miltenyi S, Muller W, Weichel W, Radbruch A. High gradient magnetic cell separation with MACS. Cytometry 1990;11(2):231–8. [2] Thiel A, Scheffold A, Radbruch A. Immunomagnetic cell sorting–pushing the limits. Immunotechnology 1998;4(2):89–96. [3] Pamme N, Wilhelm C. Continuous sorting of magnetic cells via on-chip freeflow magnetophoresis. Lab Chip 2006;6(8):974–80. [4] Riviere C, Marion S, Guillen N, Bacri JC, Gazeau F, Wilhelm C. Signaling through the phosphatidylinositol 3-kinase regulates mechanotaxis induced by local low magnetic forces in Entamoeba histolytica. J Biomech 2007;40(1):64–77. [5] Wilhelm C, Riviere C, Biais N. Magnetic control of Dictyostelium aggregation. Phys Rev E Stat Nonlin Soft Matter Phys 2007;75(4 Pt 1):041906. [6] Ito A, Hibino E, Kobayashi C, Terasaki H, Kagami H, Ueda M, et al. Construction and delivery of tissue-engineered human retinal pigment epithelial cell sheets, using magnetite nanoparticles and magnetic force. Tissue Eng 2005; 11(3–4):489–96. [7] Ito A, Takizawa Y, Honda H, Hata K, Kagami H, Ueda M, et al. Tissue engineering using magnetite nanoparticles and magnetic force: heterotypic layers of cocultured hepatocytes and endothelial cells. Tissue Eng 2004;10(5–6): 833–40. [8] Pislaru SV, Harbuzariu A, Gulati R, Witt T, Sandhu NP, Simari RD, et al. Magnetically targeted endothelial cell localization in stented vessels. J Am Coll Cardiol 2006;48(9):1839–45. [9] Arbab AS, Jordan EK, Wilson LB, Yocum GT, Lewis BK, Frank JA. In vivo trafficking and targeted delivery of magnetically labeled stem cells. Hum Gene Ther 2004;15(4):351–60. [10] Bulte JW, Kraitchman DL. Iron oxide MR contrast agents for molecular and cellular imaging. NMR Biomed 2004;17(7):484–99. [11] Arbab AS, Liu W, Frank JA. Cellular magnetic resonance imaging: current status and future prospects. Expert Rev Med Devices 2006;3(4):427–39. [12] Corot C, Robert P, Idee JM, Port M. Recent advances in iron oxide nanocrystal technology for medical imaging. Adv Drug Deliv Rev 2006;58(14):1471–504. [13] Yeh TC, Zhang W, Ildstad ST, Ho C. Intracellular labeling of T-cells with superparamagnetic contrast agents. Magn Reson Med 1993;30(5):617–25. [14] Weissleder R, Cheng HC, Bogdanova A, Bogdanov Jr A. Magnetically labeled cells can be detected by MR imaging. J Magn Reson Imaging 1997;7(1):258–63. [15] Dodd SJ, Williams M, Suhan JP, Williams DS, Koretsky AP, Ho C. Detection of single mammalian cells by high-resolution magnetic resonance imaging. Biophys J 1999;76(1 Pt 1):103–9. [16] Bulte JW, Zhang S, van Gelderen P, Herynek V, Jordan EK, Duncan ID, et al. Neurotransplantation of magnetically labeled oligodendrocyte progenitors: magnetic resonance tracking of cell migration and myelination. Proc Natl Acad Sci U S A 1999;96(26):15256–61. [17] Daldrup-Link HE, Rudelius M, Oostendorp RA, Settles M, Piontek G, Metz S, et al. Targeting of hematopoietic progenitor cells with MR contrast agents. Radiology 2003;228(3):760–7. [18] Ahrens ET, Feili-Hariri M, Xu H, Genove G, Morel PA. Receptor-mediated endocytosis of iron-oxide particles provides efficient labeling of dendritic cells for in vivo MR imaging. Magn Reson Med 2003;49(6):1006–13.

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Clinically applicable labeling of mammalian and stem cells by combining superparamagnetic iron oxides and transfection agents. Radiology 2003;228(2):480–7. [29] Kostura L, Kraitchman DL, Mackay AM, Pittenger MF, Bulte JW. Feridex labeling of mesenchymal stem cells inhibits chondrogenesis but not adipogenesis or osteogenesis. NMR Biomed 2004;17(7):513–7. [30] Arbab AS, Pandit SD, Anderson SA, Yocum GT, Bur M, Frenkel V, et al. Magnetic resonance imaging and confocal microscopy studies of magnetically labeled endothelial progenitor cells trafficking to sites of tumor angiogenesis. Stem Cells 2006;24(3):671–8. [31] Arbab AS, Yocum GT, Rad AM, Khakoo AY, Fellowes V, Read EJ, et al. Labeling of cells with ferumoxides-protamine sulfate complexes does not inhibit function or differentiation capacity of hematopoietic or mesenchymal stem cells. NMR Biomed 2005;18(8):553–9. [32] Guzman R, Uchida N, Bliss TM, He D, Christopherson KK, Stellwagen D, et al. Long-term monitoring of transplanted human neural stem cells in developmental and pathological contexts with MRI. Proc Natl Acad Sci U S A 2007;104(24):10211–6. [33] Schafer R, Kehlbach R, Wiskirchen J, Bantleon R, Pintaske J, Brehm BR, et al. Transferrin receptor upregulation: in vitro labeling of rat mesenchymal stem cells with superparamagnetic iron oxide. Radiology 2007;244(2):514–23. [34] Fleige G, Seeberger F, Laux D, Kresse M, Taupitz M, Pilgrimm H, et al. In vitro characterization of two different ultrasmall iron oxide particles for magnetic resonance cell tracking. Invest Radiol 2002;37(9):482–8. [35] Taupitz M, Wagner S, Schnorr J, Kravec I, Pilgrimm H, Bergmann-Fritsch H, et al. Phase I clinical evaluation of citrate-coated monocrystalline very small superparamagnetic iron oxide particles as a new contrast medium for magnetic resonance imaging. Invest Radiol 2004;39(7):394–405. [36] Wilhelm C, Gazeau F, Roger J, Pons JN, Bacri JC. 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Magnetophoresis and ferromagnetic resonance of magnetically labeled cells. Eur Biophys J 2002;31(2):118–25. [43] Lee KD, Nir S, Papahadjopoulos D. Quantitative analysis of liposome–cell interactions in vitro: rate constants of binding and endocytosis with suspension and adherent J774 cells and human monocytes. Biochemistry 1993;32(3):889–99. [44] Miller CR, Bondurant B, McLean SD, McGovern KA, O’Brien DF. Liposome–cell interactions in vitro: effect of liposome surface charge on the binding and endocytosis of conventional and sterically stabilized liposomes. Biochemistry 1998;37(37):12875–83. [45] Chenevier P, Veyret B, Roux D, Henry-Toulme N. Interaction of cationic colloids at the surface of J774 cells: a kinetic analysis. Biophys J 2000;79(3): 1298–309. [46] Sarup JC, Johnson RM, King KL, Fendly BM, Lipari MT, Napier MA, et al. Characterization of an anti-p185HER2 monoclonal antibody that stimulates

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[47]

[48]

[49]

[50]

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