Fast field-cycling magnetic resonance detection of intracellular ultra-small iron oxide particles in vitro: Proof-of-concept

Abstract

Purpose: Inflammation is central in disease pathophysiology and accurate methods for its detection and quantification are increasingly required to guide diagnosis and therapy. Here we explored the ability of Fast Field-Cycling Magnetic Resonance (FFC-MR) in quantifying the signal of ultra-small superparamagnetic iron oxide particles (USPIO) phagocytosed by J774 macrophage-like cells as a proof-of-principle.

Methods: Relaxation rates were measured in suspensions of J774 macrophage-like cells loaded with USPIO (0-200 μg/ml Fe as ferumoxytol), using a 0.25 T FFC benchtop relaxometer and a human whole-body, in-house built 0.2 T FFC-MR prototype system with a custom test tube coil. Identical non-imaging, saturation recovery pulse sequence with 90° flip angle and 20 different evolution fields selected logarithmically between 80 μT and 0.2 T (3.4 kHz and 8.51 MHz proton Larmor frequency [PLF] respectively). Results were compared with imaging flow cytometry quantification of side scatter intensity and USPIO-occupied cell area. A reference colorimetric iron assay was used.

Results: The T₁ dispersion curves derived from FFC-MR were excellent in detecting USPIO at all concentrations examined (0-200 μg/ml Fe as ferumoxytol) vs. control cells, p ≤ 0.001. FFC-NMR was capable of reliably detecting cellular iron content as low as 1.12 ng/µg cell protein, validated using a colorimetric assay. FFC-MR was comparable to imaging flow cytometry quantification of side scatter intensity but superior to USPIO-occupied cell area, the latter being only sensitive at exposures ≥ 10 µg/ml USPIO.

Conclusions: We demonstrated for the first time that FFC-MR is capable of quantitative assessment of intra-cellular iron which will have important implications for the use of USPIO in a variety of biological applications, including the study of inflammation.

Keywords: Fast field-cycling magnetic resonance; Inflammation; Ultrasmall superparamagnetic iron oxide particles (USPIO).

Graphical abstract

Fig. 1

Outline of the workflow used to delineate area occupied by USPIO and cell granularity, based on calculated cellular features on the Amnis ImageStreamX Mark II. A: Single cells were selected by plotting area against aspect ratio assuming that most non-adherent macrophages were round in shape. Focused cells were selected by plotting gradient against contrast and selecting cells with highest values of both features. SSC was measured to assess granularity. B: Both custom-generated (green) and the ‘adaptive erode’ mask (no. 83) were used to identify USPIO-free intracellular area. USPIOs were identified using ‘intensity’ mask with the pixel intensity range (0–600). Intracellular USPIOs were identified by selecting overlapping cell- and USPIO-occupied areas. C: Images from two BF channels were included in the final calculation, given the slight difference between these images (white arrows). D: Calculation used to identify the relative areas of the cells occupied by USPIO. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

A: Iron quantification (from phagocytosed USPIO) using a 2,2′-Bipyridine colorimetric assay as a standard reference. Cell iron uptake appears non-linear, best described by the 3rd order polynomial y = 2E-06x3 − 0.0009x2 + 0.1616x + 0.3092; R² = 0.999. All points represent triplicate mean ± SEM. B: Prussian blue visualised in J774 macrophages exposed to 40 µg/ml ferumoxytol USPIO and Perl stain; ×400 magnification light microscopy. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

R₁ dispersion curves of non-fixed (A) and fixed (B) J774 cell suspensions exposed to 0–200 µg/ml USPIO. Each data point represents a mean of triplicates ± SEM.

Fig. 4

Effect of increasing macrophage USPIO exposure concentration on the average relaxivity of the USPIO compared to a reference NMRD profile in fresh (A) and fixed (B) cells. The average ratio between the NMRD profile and the reference profile shows that the relaxivity of internalised USPIO saturates with concentration, which is expected, but this effect is almost twice as important in fresh cells.

Fig. 5

Imaging flow cytometric analysis of USPIO-exposed non-fixed and fixed cells. A: Macrophage imaging and localisation of intracellular USPIO aggregates (highlighted in green). B: The relative USPIO-occupied cell area as calculated by custom-generated image masks, and SSC intensity reflecting cell granularity (expressed as mean ± SEM), both increasing with higher USPIO exposure, based on intracellular iron amounts quantified colorimetrically (µg iron/ µg protein). Regression lines shown for all three. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)