Intracellular uptake of anionic superparamagnetic nanoparticles as a function of their surface coating
Introduction
Cell labeling with magnetic nanoparticles is an increasingly common method for in vitro cell separation as well as for in vivo imaging owing to their signal amplification properties in magnetic resonance imaging (MRI). Magnetic cell labeling also raises very promising developments for therapy, by allowing magnetic intracellular hyperthermia [1], [2]. All these applications require that cells efficiently capture the magnetic nanoparticles either in vitro or in vivo. In vivo, requisites for cell targeting are to graft high affinity ligands on the nanoparticles surface in order to favor specific interactions [3] and, at the same time, to prevent the interactions with serum protein and subsequent capture by the reticuloendothelial system. In vitro, magnetic labeling only needs a high capture of the nanoparticles by the cells, following the endocytosis pathway. Beyond interesting developments in cell biology (to purify or to manipulate magnetically intracellular organelles) [4], [5], an efficient in vitro magnetic labeling offers promising new approaches in cell-based therapy. The cells of interest (for instance T lymphocytes [6] or stem cells [7]) are isolated and labeled in vitro before their transplantation in vivo [8], [9], [10]. It is then possible to track their migration in vivo (homing or recruitment for example) by high resolution MRI [7] thanks to the signal amplification due to the magnetic properties of the labeled cells. The most commonly used iron oxide nanoparticles are dextran coated [11] but do not present sufficient cellular uptake to enable cell tracking, probably because of a relatively inefficient fluid phase endocytosis pathway. However, significant improvements in the magnetic labeling efficiency and versatility were achieved by the attachment on the nanoparticles surface of a transfection agent or a small peptide, known to facilitate cell internalization [7], [12].
In this paper, we present a new class of iron oxide nanoparticles, anionic maghemite nanoparticles (AMNP). We demonstrate that it is a highly versatile system suitable either for a high efficiency non specific cellular uptake mediated by adsorptive endocytosis (using bare AMNP), either for specific cell recognition allowed by the nanoparticle surface modification and the binding of a specific ligand. We show that bare anionic maghemite nanoparticles, free of any dextran coating, exhibit a surprisingly high level of cell internalization that is comparable with nanoparticles modified with Tat peptide [12] or encapsulated into dendrimers [7]. They interact strongly and non specifically with the plasma membrane thanks to their surface negative charges. This adsorption step, which appears to be ubiquitous, precedes the internalization step and governs the overall cell uptake. Alternatively, to induce receptor mediated endocytosis pathway and cell specific magnetic labeling, it is necessary to reduce the non specific nanoparticles/membrane interactions and to force the recognition of the nanoparticles by the membrane receptors. We show that the non specific adsorptive endocytosis pathway can be inhibited by steric hindrance, due to the coating of the AMNP surface with albumin or with dextran. We also demonstrate that AMNP coated with albumin are good candidates for the binding on the nanoparticle surface of a cell membrane high affinity ligand, like an antibody.
In the present paper, the surface modifications and the colloidal stability of the anionic maghemite nanoparticles are characterized by a new method, magnetically induced birefringence measurement, that is sensitive to the hydrodynamic volume of the particles. Cell uptake assays are performed for comparison with AMNP, with BSA-coated AMNP and with dextran coated iron oxide nanoparticles. Quantification of particle uptake is obtained using new complementary magnetic assays, magnetophoresis (MP) and electron spin resonance (ESR).
Section snippets
Chemical synthesis
The iron oxide nanoparticles studied in this work are made of maghemite (γFe2O3), a ferrimagnetic crystal with an inverse spinel structure: where A and B stand, respectively, for tetraedric and octaedric sites, and Δ stands for lacuna. The ionic precursor is synthesized according to the Massart's method [13] by alkalizing an aqueous mixture of iron (II) chloride and iron (III) chloride. With use of Fe(NO3)3, the so obtained magnetite ()
Particle surface modification
We have shown in a previous paper [22] that the measurement of the magnetically induced birefringence relaxation, providing the distribution of hydrodynamic diameters of superparamagnetic nanoparticles, allowed to detect the binding reaction of a macromolecule on the nanoparticle surface and moreover the eventual onset of nanoparticle aggregation. The robustness of the method was proved by comparing nanoparticles fractionated in size by gel filtration. In the present paper, we use this method
Anionic nanoparticles versus dextran-coated nanoparticles
The capture of anionic nanoparticles by HeLa cells (Fig. 6b) exceeds the capture of dextran-coated iron oxide nanoparticles (Fig. 8b) by three orders of magnitude. For RAW macrophages, the difference is slightly less pronounced, since they capture a smaller amount of anionic particles than HeLa cells, whereas the inverse effect is observed for dextran-coated nanoparticles. Previous studies reported cellular uptake of dextran-coated nanoparticles varying from 0.011 to of iron per cells (1
Conclusion
In summary, anionic maghemite nanoparticles represent a new type of superparamagnetic label that shows a high affinity for cellular membrane mainly due to electrostatic interactions. Their non specific adsorption on virtually any mammalian cells and their subsequent internalization into endosomes offer the opportunity to label a wide variety of cells with comparable efficiency than other magnetic nanoparticles specially engineered to facilitate their entry into cell. It opens up new
Acknowledgements
We are grateful to B.de Crossa for preparing electron microscopy samples and to O. Clément and P.-Y. Brillet for their enlightening discussions and for providing dextran coated nanoparticles. This work was financially supported by the CNRS program Physique et Chimie du vivant and by the French research ministry (ACI “Technologies pour la santé”).
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