Synthesis of SPIO Nanoparticles

Large and small SPIO NWs (L-SPIO NWs and S-SPIO NWs, respectively) were synthesized from 15 to 25 kDa dextran, Fe(III) chloride, and Fe(II) chloride using a modified Molday precipitation method^{30 (link)} as described previously. ^{26 (link)} The ratio between dextran and iron salts determined the final size of the nanoparticles.^{26 (link)} Particles were resuspended in sterile phosphate-buffered saline (PBS), filtered through a 0.45 μm filter, and stored at 4 °C. Size and ζ potential were determined using a Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, UK). The intensity-weighted distribution was used to report the hydrodynamic diameter of nanoparticles and liposomes. For TEM analysis of S-SPIO NWs and L-SPIO NWs, the imaging of nonstained samples applied on carbon grid was performed using a FEI Tecnai Spirit BioTwin electron microscope at 100 keV. For TEM analysis of Feraheme, the imaging of nonstained particles was performed at Thermo Fisher Scientific (Americas Nanoport, Hillsboro, OR) using an FEI Apreo microscope. The imaging was performed in immersion mode with STEM 3+ detector and landing energy of 30 keV.