Leading OpinionUniversal cell labelling with anionic magnetic nanoparticles☆
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], [2], [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], [11], [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], [14], [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 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], [25], [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], [31], [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 AMNP-labelled cells for MRI detection and for magnetic manipulations.
Section snippets
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 (γFe203)
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
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
Cell internalized AMNP
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], [48], [49], [50], [51]. Whatever the cell type, cell proliferation was not affected by AMNP labelling. Besides, iron load dilution scaled with cell
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
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
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
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,
Acknowledgments
We are grateful to C. Rivière, P. Smirnov and J.-P. Fortin for their respective contribution to this work.
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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.