4324Fast-field cycling magnetic resonance detection of intracellular iron in the nanomolar range - A pre-requisite for in-vivo study of inflammation

Abstract

Abstract Background Ultra-small superparamagnetic iron oxide (USPIO) nanoparticles are phagocytosed by macrophages and when subjected to magnetic resonance imaging (MRI) detect inflammation. We describe a novel MR technique where the magnetic field is rapidly cycled (Fast field-cycling MR, FFC-MR), assessing T1 dispersion over a range of low (100μT-0.2T) fields and offering superior T1-based iron quantification. AIM To quantify iron using the FFC-MR R1 (1/T1) dispersion profile and flow cytometry features of USPIO-laden cells, in comparison to colorimetric assays. Methods Murine J774 macrophage-like cells were incubated with 0–200μg/ml Fe as USPIO (ferumoxytol) for 16 hours, washed and suspended in 500μl PBS/2mM EDTA. Prussian blue staining confirmed USPIO phagocytosis. Relaxation was measured using a clinical, in-house built prototype 0.2T FFC-MR system with a custom test tube coil. R1 dispersion was derived from a saturation recovery sequence (Fig. 1A). R1 (s–1) values were calculated with a monoexponential curve fitting algorithm, R2 of fits were ≥0.999. R1 dispersion profiles were generated plotting R1 against the magnetic field (T). Quantification of side scatter (SCC) intensity and the USPIO-occupied fraction of total cell area was performed with imaging flow cytometry. A colorimetric assay provided validation of cell iron content. All data are mean±SEM, analysed with t-tests, Pearson correlation and linear regression; statistical significance set at p<0.05. Results Table 1 shows quantitative data derived by all 3 modalities with increasing USPIO exposure. FFC-MR detection of intracellular iron was excellent (p≤0.001 vs. control for all), with separation of average R1 dispersion profiles (Fig. 1B), strong correlation with colorimetry (r=0.993 p<0.001) and good fit on linear regression model (R2=0.9222, Fig 1C). Flow cytometry quantification of SCC was comparable (p≤0.001 for all), whereas USPIO-occupied area was only sensitive at exposures ≥10μg/ml USPIO (r=0.967 p<0.001 & r=0.983 p<0.001 vs. colorimetric respectively). FFC-MR detected iron at ≥1.12ng/μg protein. USPIO exposure (μg/ml medium) 0 5 10 40 80 100 200 FFC-MR Mean R1 1x106 cell suspension (s–1) 0.308±0.014 0.356±0.013** 0.432±0.016** 0.706±0.021** 1.174±0.031** 1.239±0.033** 1.599±0.041** Flow cytometry USPIO area/cell area 0.034±0.001 0.036±0.001 0.037±0.001* 0.069±0.001** 0.085±0.001** 0.090±0.001** 0.097±0.001** Flow cytometry SSC intensity 26860 32815** 39573** 69285** 80967** 82693** 86373** Colorimetric assay Iron concentration 1x106 cells (ng/μg protein) 0.115±0.118 1.121±0.045** 2.074±0.084** 5.496±0.134** 8.421±0.269** 9.771±0.100** 12.398±0.233** SSC = side-scattered light; *p<0.05 vs. control; **p<0.001 vs. control. Conclusion Field-cycling MR is capable of highly accurate intracellular USPIO quantification, which has potential to non-invasively detect clinically relevant amounts of iron in inflammatory cardiovascular diseases. Acknowledgement/Funding NHS Grampian Endowment Fund