Efficient internalization of silica-coated iron oxide nanoparticles of different sizes by primary human macrophages and dendritic cells
Introduction
Magnetic nanoparticles are a widely studied class of nanoparticles as they offer great possibilities in a number of diagnostic applications and therapies (Laurent et al., 2009, Veiseh et al., 2009). In recent years, a new generation of nanoparticles with complex structures has emerged, carrying different functionalities which allow for the achievement of multiple tasks simultaneously, e.g. combined imaging and delivery of drugs to specific target organs (Riehemann et al., 2009). The core–shell architecture offers an attractive platform for development of multifunctional nanoparticles. The visualization function (i.e. for MRI applications) is performed via the core while the protective shell can be used for surface modification e.g. for targeted drug delivery (Salgueiriño-Maceira and Correa-Duarte, 2007, Kunzmann et al., 2011).
The MRI technique without the use of contrast agents has a limited sensitivity as well as a low resolution giving insufficient pathological information. The use of superparamagnetic iron oxide nanoparticle (SPION) based MRI contrast agents is well established and a number of SPIONs are approved by the U.S. Food and Drug Administration (FDA) (Hamm et al., 1994, Reimer and Balzer, 2003). For biomedical applications it is crucial that SPIONs have a well-defined shape, are monodisperse, and exhibit a high magnetization value (Laurent et al., 2009). However, SPIONs cannot be used as prepared, the agglomeration at physiological pH (Lu et al., 2007) and the rapid clearance from the blood being important drawbacks. Coating with an additional biocompatible layer such as dextran (Dutz et al., 2007), polyethylene glycol (Barrera et al., 2009) or silica (Bumb et al., 2008) is one of the common strategies to avoid these problems. Silica offers an inert coating material, preventing the aggregation of the superparamagnetic core in liquid media and enhancing their chemical stability. Furthermore, silica is a versatile material for surface modification which opens to new possibilities for the synthesis of nanoparticles for combined diagnostics and therapy (Liong et al., 2008, Bumb et al., 2010).
Among various methods of coating the iron oxide nanoparticles with silica the most common routes are the Stöber method (Stöber et al., 1968, Lu et al., 2002, Deng et al., 2005, Im et al., 2005) and the microemulsion process (Santra et al., 2001, Lu et al., 2007). The Stöber method, mainly used for synthesis of silica particles, is also applied for obtaining core–shell nanoparticles (Rao et al., 2005). Even though this method is technically simple, the core–shell nanoparticles are multicore and highly polydisperse, which limits their use for biomedical applications. The water-in-oil microemulsion (w/o), or inverse microemulsion, is a preferred method for the synthesis of monodisperse silica shell–magnetic core nanoparticles (Yi et al., 2005, Yi et al., 2006, Lu et al., 2007, Zhang et al., 2008, Narita et al., 2009). Even if the initially synthesized particles are uniform in size and shape, due to the residual condensation reaction during the washing steps, the final particles are strongly necked, aggregated and often multicore nanoparticles. These characteristics will result in the increase of the overall size, decreased surface area and reduced colloidal stability, which will hinder their potential use for in vitro or in vivo applications that require small size, high monodispersity and non-aggregated nanoparticles.
To promote a safe use of nanomaterials in biomedical applications, the cellular interactions of nanoparticles have to be carefully addressed (Kunzmann et al., 2011, Shvedova et al., 2010). At the same time, potential consequences for the immune system are important to consider (Dobrovolskaia and McNeil, 2007). Macrophages and dendritic cells are key players in the immune system (Banchereau and Steinman, 1998, Gordon and Taylor, 2005), and the use of primary human monocyte derived macrophages (HMDM) and dendritic cells (MDDC) is more relevant in in vitro model systems compared to the transformed cell lines that are commonly used in toxicity studies. Furthermore, when nanoparticles are applied in the clinical setting, it has to be ensured that the macrophage function is not impaired (Witasp et al., 2009b) and that dendritic cells are not activated to induce adverse immune reactions (Vallhov et al., 2006).
In order to use SPIONs as MRI agent, they have to be efficiently internalized by target cells. Targeting of immune cells facilitates diagnostic imaging, and with combined features such as tumor antigen loading (de Vries et al., 2005), therapeutic effects could be enhanced. Nowadays, MRI agents are used for a number of different applications. Macrophages are mainly involved in nonspecific uptake of particles and cellular debris. Thus, besides the classical liver imaging, MRI agents offer the possibility to track macrophages in vivo, allowing e.g. the depiction of immune responses (Fleige et al., 2002) and further permit a deeper insight into the process of diseases or pathologies (Flogel et al., 2008). In addition, when SPIONs are internalized by dendritic cells, MRI serves as an ideal tool for detecting migration of dendritic cells, offering the possibility to achieve an optimal immunotherapy (de Vries et al., 2005, Kobukai et al., 2010). Labeling of immune-competent cells with SPIONs enables the visualization of specific tissues with enrichment of such cells, which is relevant in e.g. cancer therapy (Valable et al., 2008).
With the aim to generate nanomaterials with improved performance for imaging applications as MRI and cell tracking, we synthesized different sized superparamagnetic silica-coated iron oxide nanoparticles, referred to as core–shell nanoparticles (CSNPs). We compared these to dextran-coated iron oxide nanoparticles (nanomag®-D-spio) commercially available and similar to clinically used SPIONs. The CSNPs were shown to display superior relaxivity values compared to the commercial nanoparticles, and good biocompatibility when incubated in vitro with primary human immune-competent cells, although some dose-dependent toxicity of the smaller silica-coated nanoparticles was recorded for primary human dendritic cells. Cellular internalization of the particles was shown to be active (actin cytoskeleton-dependent), and the degree of particle internalization by primary human macrophages appeared to depend on the surface coating, with significantly higher uptake of CSNPs compared to nanomag®-D-spio.
Section snippets
Chemicals
Nanomag®-D-spio was purchased from Micromod Partikeltechnologie GmbH (Rostock-Warnemuende, Germany). Iron oxyhydroxide, FeO(OH) (30–50 mesh), Triton-X100 (analytical grade), cyclohexane (99.5%) and hexanol (98%) were purchased from Sigma Aldrich (St. Louis, MO) and oleic acid, tetraethyl orthosilicate (TEOS) (99.5%) and NH4OH (28%) from Fluka (Sigma Aldrich). All chemicals were used as received. Ethanol was of 99.9% purity and the water was MilliQ grade with a resistivity of 18 MΩ.
Synthesis of silica-coated iron oxide nanoparticles (CSNPs)
The synthesis
Characterization of the nanoparticles
TEM was used to characterize the size and morphology of the CSNPs and nanomag®-D-spio nanoparticles. Hereafter the CSNPs are referred to as CSNPs 30 nm, 50 nm, 70 nm, and 120 nm, an approximation from the mean diameter presented in Table 1. In Fig. 1, the spherical shape, the core–shell architecture and the non-agglomerated character of CSNPs 30 nm (A), 50 nm (B), 120 nm (C) and 70 nm (D) are displayed. The TEM micrographs of nanomag®-D-spio (Figs. 1E–F) exhibit a cluster-like morphology with a high
Discussion
In the present study, the magnetic properties, biocompatibility and efficiency of cellular internalization of novel silica-coated CSNPs compared to commercial dextran-coated nanomag®-D-spio were evaluated in primary human macrophages and dendritic cells. The CSNPs revealed superior magnetic properties, and displayed a higher efficiency of cellular uptake when compared to the nanomag®-D-spio. The CSNPs were non-toxic to macrophages at all sizes tested whereas the smaller nanoparticles (30 nm and
Conclusions
We have compared the cytotoxicity and cellular uptake by primary human immune-competent cells of two types of SPIONs: de novo synthesized silica-coated iron oxide nanoparticles of different sizes (ranging from 30 nm to 120 nm) and commercially available dextran-coated iron oxide nanoparticles (nanomag®-D-spio) with average cluster sizes of 20 nm and 50 nm. The degree of uptake by macrophages was shown to be greater for silica-coated particles than for the dextran-coated particles. The observed
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Acknowledgments
The authors are supported by the Seventh Framework Programme of the European Commission (EC-FP7-NANOMMUNE-Grant Agreement No. 214281), the ARC Program 05/10-335 of the French Community of Belgium, the Knut and Alice Wallenberg Foundation, the Swedish Research Council for Working Life and Social Research, and the Swedish Research Council. We thank Liliane Diener, Swiss Federal Laboratories for Materials Testing and Research, and Margareta Grandér and Brita Palm, Karolinska Institutet, for
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These authors have contributed equally.