Robust water-based synthesis of monodisperse ultrasmall SPIONs with tunable T1-T2 relaxometric behaviour as MRI contrast agents

Magnetite-based nanoparticles (MNPs) are widely investigated for biomedical applications including hyperthermia, drug delivery and magnetic resonance imaging (MRI). Precise control of their morphology is essential and typically achieved via thermal decomposition, though more scalable and energy-efficient approaches are needed, especially for ultrasmall (<5 nm) MNPs. Here, well-controlled MNPs were synthesized by optimizing a coprecipitation process conducted at low temperature and in air, without polymeric stabilizers or templates. The combined use of tetramethylammonium hydroxide (TMAOH) as a base, citric acid to quench growth, and controlled reaction temperature (from room temperature to 0 ◦C), enabled the reproducible formation of monodisperse, highly crystalline MNPs with core size tunable from 6.6 to 4.0 nm, as confirmed by (HR)TEM. The TMAOH could be readily replaced by citrate as biocompatible stabilizer, forming a 1 nm-thick shell (AFM) and ensuring long-term stability, even under magnetic fields. NMRD measurements (0.01-57 MHz) showed superparamagnetic behaviour and a size-dependent transition from T2-to T1-type relaxation, with r2/r1 ratio at 1.34 T decreasing from 3.3, 2.9, and 1.8 for 6.6, 5.3, and 4.0 nm particles, respectively. MRI at 3 T confirmed that the smaller MNPs exhibit significant T1 contrast. Finally, MNP@citrate were stable in cell culture medium, well tolerated at all tested concentrations (Cmax = 140 μg/mL) on Human Embryonic Kidney 293 (HEK) cells, and accumulated in the cytoplasm within 24 h incubation, as shown by reflectance confocal microscopy.