Size, charge and concentration dependent uptake of iron oxide particles by non-phagocytic cells

Abstract

A promising new direction for contrast-enhanced magnetic resonance (MR) imaging involves tracking the migration and biodistribution of superparamagnetic iron oxide (SPIO)-labeled cells in vivo. Despite the large number of cell labeling studies that have been performed with SPIO particles of differing size and surface charge, it remains unclear which SPIO configuration provides optimal contrast in non-phagocytic cells. This is largely because contradictory findings have stemmed from the variability and imprecise control over surface charge, the general need and complexity of transfection and/or targeting agents, and the limited number of particle configurations examined in any given study. In the present study, we systematically evaluated the cellular uptake of SPIO in non-phagocytic T cells over a continuum of particle sizes ranging from 33nm to nearly 1.5microm, with precisely controlled surface properties, and without the need for transfection agents. SPIO labeling of T cells was analyzed by flow cytometry and contrast enhancement was determined by relaxometry. SPIO uptake was dose-dependent and exhibited sigmoidal charge dependence, which was shown to saturate at different levels of functionalization. Efficient labeling of cells was observed for particles up to 300nm, however, micron-sized particle uptake was limited. Our results show that an unconventional highly cationic particle configuration at 107nm maximized MR contrast of T cells, outperforming the widely utilized USPIO (<50nm).

Figure 1

Hydrodynamic diameter of SPIO. The hydrodynamic diameter of SPIO particles was determined by DLS. Intensity measurements are reported and the peak intensity is provided for each distribution.

Figure 2

TEM of SPIO Cores. High magnification transmission electron microscopy images of the iron oxide particles were obtained with a JEOL 2010 operating at 200 kV. Structure analysis revealed the multiple core nature of the (A) 33.4 nm, (B) 53.5 nm and (C) 107 nm dextran-coated SPIO. Larger particles were composed of single cores; (D) 207 nm, (E) 289 nm and (F) 1430 nm. All scale bars are 50 nm, excluding (F) 1 μm.

Figure 3

Size distribution of SPIO core diameters. TEM measurements of the SPIO core diameter for (A) 33.4 nm, (B) 53.5 nm, (C) 107 nm and (D) all cores. The cores diameters were analyzed assuming that they were spherical and the frequency and cumulative distributions are plotted. Particle size appears to be determined by the number of cores per particle rather than the size of those constituent cores.

Figure 4

T₁ Relaxivity (R₁) measurements of SPIO. SPIO of various size were diluted in PBS to iron concentrations between (A) 0.1 mM and 2 mM or (B) 1 mM and 6 mM. T₁ values were then obtained using the minimum time sequence required to get reproducible values, because of precipitation issues. The inverse of the T₁ time, in seconds, was linearly fit against concentration to yield the particle R₁.

Figure 5

T₂ Relaxivity (R₂) measurements of SPIO. SPIO of various size were diluted in PBS to iron concentrations between (A) 0.1 mM and 2 mM or (B) 0.01 mM and 0.5 mM. The T₂ values were then obtained using a monoexponential curve fit. The inverse of these values, plotted against concentration, gives the R₂. Precipitation of the 1430 nm particles resulted in nonlinearity.

Figure 6

Dependence of SPIO loading on particle concentration. Fluorescently-labeled SPIO of various size and across a range of concentrations were incubated with 2×10⁶ T cells/mL at 37° C for 4 hours (excluding the 107 nm particle as indicated). SPIO uptake was then measured by flow cytometry. Each experiment was conducted in triplicate on at least two separate occasions and each data point represents the average value for the mean fluorescent intensity (MFI). Note the difference in x- and y-axes for (A) and (B).

Figure 7

Dependence of SPIO loading on surface charge. T cell uptake of fluorescently-labeled SPIO as a function of surface charge was examined by modulating the number of amines per particle for the (A) 33.4 nm, (B) 53.5 nm and (C) 107 nm particles. A gradient in the degree of functionalization was produced by glycidol blocking of amines. SPIO were incubated with T cells at saturating concentrations, 50 μg/mL, under identical conditions. Flow cytometry was then performed to assess the relative uptake of each SPIO. Each data point represents the mean fluorescent intensity (MFI). The loading of SPIO was rapid; (D) shows the representative uptake of fully-aminated 107 nm particles.

Figure 8

Viability of T cells incubated with SPIO. SPIO were incubated with T cells at various iron concentrations: 10 μg/mL [black], 50 μg/mL [white], and 100 μg/mL [grey]. After 4 hours (unless otherwise noted), viability was measured and normalized to cells grown in the absence of any particles (blank). All SPIO exhibited negligible impact on cell survival after 4 hours, excluding the 107 nm diameter particles. Reducing incubation time of these particles to 1 hour eliminated adverse effects at both low and saturating concentrations.

Figure 9

T2 Relaxation times of T cells labeled with SPIO. T cells were labeled with SPIO of various size and across a range of concentrations. The T2 relaxivity of 0.5×10⁶ SPIO-loaded T cells/mL in 300 μL was measured on a Bruker mq60 MR relaxometer operating at 1.41 T (60 MHz). The signal decrease observed following internalization of SPIO is dose dependent and saturation correlates well with values determined by flow cytometry. The 107 nm SSPIO produced maximum signal decrease.