Targeted T1 Magnetic Resonance Imaging Contrast Enhancement with Extraordinarily Small CoFe2O4 Nanoparticles.

Extraordinarily small (2.4 nm) cobalt ferrite nanoparticles (ESCIoNs) were synthesized by a one-pot thermal decomposition approach to study their potential as magnetic resonance imaging (MRI) contrast agents. Fine size control was achieved using oleylamine alone, and annular dark-field scanning transmission electron microscopy revealed highly crystalline cubic spinel particles with atomic resolution. Ligand exchange with dimercaptosuccinic acid rendered the particles stable in physiological conditions with a hydrodynamic diameter of 12 nm. The particles displayed superparamagnetic properties and a low r2/ r1 ratio suitable for a T1 contrast agent. The particles were functionalized with bile acid, which improved biocompatibility by significant reduction of reactive oxygen species generation and is a first step toward liver-targeted T1 MRI. Our study demonstrates the potential of ESCIoNs as T1 MRI contrast agents.


Chemicals
Sodium hydroxide (NaOH) (≥99%), dichloromethane (DCM) (≥99.5%, p.a., ACS, ISO    S-4 Figure S4: ESIoN size as a function of the heating rate. Varying the heating rate from 12.6 to 24.7 °C/min -1 had no effect on the particle size. Figure S5: ESIoNs synthesised with 0.85 mmol of Fe(acac)3 and 60 mmol of oleylamine at different reaction times. There was no difference in particle size when a reaction time of 10, 30 or 60 minutes was used.

Synthesis of cobalt-ferrite nanoparticles with Fe(acac)3:Co(acac)2 molar
ratio of 1.5:1  Figure S6: Cobalt-ferrite nanoparticle size as a function of precursor amount when synthesised with Fe:Co molar ratio of 1.5:1. The particle size tends to decrease with a decrease in precursor, thus increase in precursor-to-OLA ratio, however the size distribution is wide. Control over particle size with oleylamine is less effective than when a Fe:Co molar ratio of 2:1 was used.
S-6 Figure S7: TEM micrographs and corresponding size distribution graphs of cobalt-ferrite nanoparticles synthesised with 20 ml oleylamine and 1.4-0.03 mmol precursors at a Fe:Co ratio of 1.5:1 (A-F). Scale bar is 100 nm.

X-ray diffraction (XRD) of OLA-FexOy and OLA-CoFe2O4 nanoparticles
XRD powder samples were investigated in Theta-2Theta geometry using a Bruker AXS Advance D8 diffractometer operating with copper K-alpha line and equipped with a Lynxeye detector.
S-7   (Table S3). We hypothesised that the aggregation was due to excess oleylamine hindering exchange with DMSA. To remove excess oleylamine, a pre-transfer dialysis step was introduced: 10 mg of particles in 10 ml of cyclohexane were added to Snakeskin® dialysis tubing (3.5 kDa molecular weight cut-off, 22 mm x 35 feet diameter, Thermo Scientific). The tubing was placed into a 500 ml glass beaker and 300 ml of chloroform was added. Following stirring for 1.5 hours, the chloroform was discarded, and 300 ml of fresh chloroform was added.
Finally, after stirring for a further 1.5 hours, the chloroform was discarded, and the nanoparticles were extracted from the tubing. Dynamic light scattering measurements revealed a significant decrease in Dh from 87 ± 2 nm to 11 ± 1 nm after particle dialysis ( Figure S10 The table below shows how the calculated particle concentration relates to the elemental concentration measured by ICP-OES. As nanoparticle induced toxic effects have been associated with the available surface area 6 , the total number of particles is also shown below. The particles were diluted accordingly to 100 µg/ml.

Cytotoxicity and oxidative stress assay interference testing
Additional cytotoxicity and oxidative stress assays were performed in the absence of cells (unless otherwise stated) to test for nanoparticle interference. Fluorescence values were normalised to the negative control (cell medium, water or PBS as indicated in the figure caption) and are an average of three measurements. Fluorescence measurements of DMSA and bile-acid derivative functionalised nanoparticles in cell culture medium alone were conducted to assess for optical interference. Figure S13(A,B) shows a decrease in signal as particle concentration increases. Both iron oxide and cobalt-ferrite particles caused a concentration-dependent reduction in fluorescence.
Metal oxide particle interference at higher concentrations has been widely reported for other assays such as MTT, LDH and DCF. 7 8 To check the particles for interference with a positive signal, positive results were generated by incubating cells with 1% triton X-100 for 24 hours at 37°C, S-13 following which nanoparticles were added. Figure S13(E) shows that particles did not cause an increase in signal when incubated with the positive control, thus false positive results from particle interference are ruled out. However, fluorescence decreased as particle concentration increased as they had in the particles only experiments ( Figure S13(A,B)). Thus, false negative results are possible due to the minor decrease in fluorescence caused by particle optical interference. To counteract particle interference, the cytotoxicity and ROS values were corrected for the factor of interference to the range of the absolute values in the particles-only experiment ( Figure S13(A,B)).
The potential for particles to cause conversion of the reactive oxygen species dye (2′,7′dichlorodihydrofluorescein (H2DCF)) was then assessed. Particles were incubated with H2DCF, which was prepared according to the protocol of Ivask et al. (2015). 9 SIN-1 was used as the positive control as it can process H2DCF in a cell-free environment. 10 Neither DMSA nor bile-acid functionalised ESCIoNs and ESIoNs caused conversion of H2DCF ( Figure S13(C,D)). Finally, we checked for particle interference with the already converted ROS dye after incubation with SIN-1. Particles were incubated with 10 µM of SIN-1 for 1 hour at room temperature in the dark. As shown in Figure S13(F), particles did not cause a reduction or increase in ROS signal.

Nuclear magnetic resonance (NMR) of bile-acid ligand synthesis
NMR spectroscopy was carried out to verify conversion of cholic acid to amino cholate. NMR spectra were recorded using a Bruker Avance II multi-core liquid NMR spectrometer with a proton resonance frequency of 300 MHz. Chemical shifts (δ) are listed in parts per million (ppm) and are

Ninhydrin test for bile-acid coupling to DMSA nanoparticles
The ninhydrin-based Kaiser test is a fast and simple method to detect the presence or absence of primary amines. 11 We employed a Kaiser test to qualitatively indicate conjugation of amino   S-21