Clin Hemorheol Microcirc. 2015;61(2):259-77. doi: 10.3233/CH-151998.
Shell matters: Magnetic targeting of SPIONs and in vitro effects on endothelial and monocytic cell function.
- 1Section of Experimental Oncology und Nanomedicine (SEON), ENT-Department, Erlangen, Germany.
- 2Laboratory of Molecular Cardiology, Department of Cardiology and Angiology, University Hospital Erlangen, Germany.
Superparamagnetic iron oxide nanoparticles (SPIONs) are versatile and easily functionalized agents with high potential for diagnostic and therapeutic intravascular applications. In this study, we analyzed the responses of endothelial (ECs) and monocytic cells to three different types of SPIONs, in order to assess the influence of physico-chemical properties on the biological reactions to SPIONs. The following formulations were used: (1) Lauric acid-coated and BSA-stabilized SPION-1,(2) Lauric acid/BSA-coated SPION-2 and (3) dextran-coated SPION-3. SPION-1 were strongly internalized by ECs and reduced their viability in static conditions. Additionally, they had a dose-dependent inhibitory effect on monocytic cell chemotaxis to MCP-1, but did not affect monocytic cell recruitment by ECs. SPION-2 uptake was less pronounced, both in ECs and monocytic cells, and these particles were better tolerated by the vascular cells. Not being internalized by endothelial or monocytic cells, SPION-3 did not induce relevant effects on cell viability, motility or endothelial-monocytic cell interactions.Taken together, localized accumulation of circulating SPION under physiologic-like flow conditions and their cellular uptake depends on the physicochemical characteristics. Our findings suggest that SPION-2 are suitable for magnetic targeting of atherosclerotic plaques. Due to their excellent biocompatibility and low internalization, SPION-3 may represent a suitable imaging agent for intravascular applications.
KEYWORDS: Atherosclerosis; SPION uptake; endothelial migration; endothelial-monocytic cell interactions; live-cell analysis; magnetic nanoparticles; monocytic cell chemotaxis
Supplement: Magnetic nanoparticles for cardiovascular applications – in vitro safety and utility evaluation
Superparamagnetic iron oxide nanoparticles (SPIONs) and ultra-small SPIOs consist of an iron oxide core, which is often coated with a shell of organic materials e.g. polysaccharides, or polymers to improve the particle stability (1-3). In the past, SPION/USPIOs were utilized for multiple clinical diagnostic applications, including magnetic resonance imaging (MRI) of lymph nodes, liver, intestines, and the cardiovascular system (4). As shown by the group of JH Gillard, iron oxide nanoparticles improve atherosclerotic plaque detection and characterization, but also provide prognostic information (5). These particles also represent a useful tool both for monitoring the efficacy of treatment with statins (6) and for the prediction of the risk of future cardiovascular events in asymptomatic patients with carotid atherosclerosis (7). Moreover, SPION-labeling allows visualization of the cells in vivo in order to monitor of cell therapies and track inflammatory cells by MRI (8). Concerning disease treatment, a promising experimental method to overcome the disadvantages of systemic drug therapies is magnetic drug targeting (9-12). By loading SPIONs with antiatherosclerotic/antiinflammatory drugs and their magnetic accumulation at the site of atherosclerotic lesion, the efficacy of pharmacological agents could be dramatically increased, contributing to the improved outcomes (13,14).
In spite of the promising results of the pilot studies in humans, the marketing of iron oxide-based contrast agents is currently at the still-stand. Addressing the substantial research and clinical interest in the use of SPIONs for MRI and for cell-labeling, we aim at the development of effective and safe SPIONs for intravascular applications. To predict in vivo responses to these particles, we performed extensive in vitro analyses, as the cellular effects of different SPIONs may differ depending on their size, charge, and coating (15).
We developed and tested three types of SPIONs: (A) SPION-1, lauric acid-coated iron oxide nanoparticles synthesized using a coprecipitation method (16), as described by Tietze et al. (11); (B) SPION-2, lauric acid/BSA-coated iron oxide nanoparticles synthesized by coprecipitation, subsequent in situ coating with lauric acid, and formation of an artificial albumin corona as described by Zaloga et al. (17); and (C) SPION-3, dextran-coated iron oxide nanoparticles synthesized according to the method described by Unterweger et al. (18).
We first investigated the effects of different SPION formulations on endothelial viability in static cell culture conditions using real-time cell analysis. In the parallel samples, we performed live cell microscopy, which allowed the observation of cell morphology and the measurement of confluence.
To assess the effects of circulating SPIONs on endothelial viability, we grew primary human endothelial cells (ECs) in the bifurcating slides, and perfused them with SPION-containing medium at arterial shear stress. To investigate the magnetic accumulation of circulating SPIONs, a magnet was positioned directly at the outer wall of the bifurcation and afterwards, accumulated particles were stained with Prussian blue. We furthermore analysed the TNF-α-induced endothelial recruitment of circulating monocytic cells following the exposure to flow with or without magnetic accumulation of SPIONs. EC migration was assessed in a modified barrier assay using silicone cell culture inserts and monocytic cell chemotaxis was analysed in a modified Boyden chamber assay. To quantify the cellular uptake of SPIONS, we measured the iron concentration per cell with microwave plasma atomic emission spectroscopy.
SPION-1 particles had hydrodynamic diameter of 126 nm and were negatively charged (ζ-potential of -34.6 mV). We have seen that SPION-1 particles were very strongly internalized by ECs under static conditions, which corresponded to their relatively high toxicity. At 24h post-application, cell viability relative of the control (untreated) samples was reduced by 50% in ECs treated with 100 µg/mL SPION-1. However, under physiologic-like flow conditions, ECs exposed to SPION-1 at 100µg/mL for 18h remained viable. In terms of magnetic accumulation, SPION-1 performed best of all tested particles, which was likely related to their larger size and the tendency to agglomerate. These particles had no effect on inflammatory cell adhesion to EC under non uniform shear stress, or on endothelial migration. Upon 2h incubation of THP-1 monocytic cells with SPION-1, we detected a dose-dependent decrease in THP-1 chemotaxis towards monocyte chemoattractant protein-1 (MCP-1), which inversely correlated with SPION-1 uptake as estimated by increase in cell granularity.
SPION-2 particles were smaller (79 nm), negatively charged (ζ-potential of -37.3 mV) and were characterized by moderate internalization by ECs under static conditions. Correspondingly, they were less toxic and better tolerated than SPION-1. We observed decreased cell numbers compared to untreated controls only at prolonged incubation times. As compared with SPION-1, smaller amounts of circulating SPION-2 were accumulated in the region of interest under external magnetic field. When incubating THP-1 monocytic cells with SPION-1 for 2 hours, we detected a 40% decrease in THP-1 chemotaxis towards MCP-1, but this effect was independent of SPION-2 concentration. Preincubation with these particles had no major effect on inflammatory cell adhesion to EC under non uniform shear stress, or on endothelial migration.
SPION-3 particles were similar in size as SPION 2, but had nearly neutral charge (ζ-potential of +0.1 mV). These particles were characterized by extremely low internalization by ECs and THP-1 cells and were most biocompatible or bio-inert, showing no effectr on EC viability even upon prolonged incubation times. Moreover, these particles had no effect on the analysed functional parameters (EC migration, monocytic cell recruitment, THP-1 chemotaxis) at any of the tested concentration. In contrast to SPION-1 and SPION-2, we detected no accumulation or localized uptake of circulating SPION-3 under external magnetic field using Prussian blue stain.
Our study indicates that an extensive analysis of cellular responses to SPIONs can facilitate the development of stable nanoparticles with improved biocompatibility. We have seen that despite good magnetic properties, SPION-1 present a relatively poor safety profile which precludes their use for clinically relevant applications. SPION-2 are characterized by improved biocompatibility and sufficient magnetic properties, which in our opinion makes them suitable carriers for magnetic drug targeting. Being internalized by various cells without inducing major cytotoxicity, SPION-2 can further serve for magnetic cell-labelling and cell-tracking. Based on our studies, SPION-3 constitute a candidate formulation with superb characteristics for imaging purposes, due to their excellent biocompatibility and low internalization. These findings are summarized in Figure 1.
Fig. 1: Potential applications of different types of SPIONs produced and investigated in our study.
This work was supported by the DFG (CI 162/2-1) and the EU (“NanoAthero” project FP7-NMP-2012-LARGE-6-309820).
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