Genetic manipulation of iron biomineralization enhances MR relaxivity in a ferritin-M6A chimeric complex

Abstract

Ferritin has gained significant attention as a potential reporter gene for in vivo imaging by magnetic resonance imaging (MRI). However, due to the ferritin ferrihydrite core, the relaxivity and sensitivity for detection of native ferritin is relatively low. We report here on a novel chimeric magneto-ferritin reporter gene – ferritin-M6A – in which the magnetite binding peptide from the magnetotactic bacteria magnetosome-associated Mms6 protein was fused to the C-terminal of murine h-ferritin. Biophysical experiments showed that purified ferritin-M6A assembled into a stable protein cage with the M6A protruding into the cage core, enabling magnetite biomineralisation. Ferritin-M6A-expressing C6-glioma cells showed enhanced (per iron) r2 relaxivity. MRI in vivo studies of ferritin-M6A-expressing tumour xenografts showed enhanced R2 relaxation rate in the central hypoxic region of the tumours. Such enhanced relaxivity would increase the sensitivity of ferritin as a reporter gene for non-invasive in vivo MRI-monitoring of cell delivery and differentiation in cellular or gene-based therapies.

2.7 Å cryo-EM structure of vitrified M. musculus H-chain apoferritin from 200 keV “screening microscope”

“…We show that basic settings for automated cryo-electron microscopy imaging, corresponding to what is widely considered as a "screening cryomicroscope", can be used to determine structures at high resolution. We use mouse H-chain apoferritin to produce an atomic model resolved at 2.7 Å (see Supplementary material for methodological details) and compare it to the corresponding 2.24 Å crystal structure (PDB ID: 3WNW [18]).…”

Ultraparamagnetic Cells Formed through Intracellular Oxidation and Chelation of Paramagnetic Iron

“…Such capabilities would facilitate, for example, the study of commensal and pathogenic microbes inside mammalian hosts and the development of magnetically engineered microbial diagnostic and therapeutic agents . However, because of the stringent pH, iron concentrations and redox potentials required for the synthesis of magnetosomal magnetite and other forms of superparamagnetic or ferromagnetic iron oxides, attempts to engineer the formation of these minerals in natively non‐magnetic species such as E. coli have had limited success …”

Biomolecular MRI reporters: Evolution of new mechanisms

“…However, magnetite/maghemite mineralization in mammalian cells or bacteria, other than magnetotactic bacteria or their close relatives [24], has not been reported, and is challenging due to the strict chemical conditions required for such mineralization. Recent attempts to engineer or evolve ferritin have resulted in particles with somewhat stronger MRI contrast, but no evidence of minerals other than ferrihydrite [25–27]. Other biomolecular T 2 reporters that may interest the reader include hemoglobin [28], tyrosinase [29], and mms6 [30].…”