Synthesis and Characterization of SPIONs

SPIONs were synthesized as described previously using an adjusted protocol by Elbialy et al. (23 (link), 32 (link)) The nanoparticles were sterilized by filtration through a 0.2 µm syringe filter (Sartorius, Goettingen, Germany). The hydrodynamic size, iron concentration, magnetic susceptibility, and zeta potential of the SPIONs were then characterized as described by Mühlberger et al. and Boosz et al. (21 (link), 23 (link)) The iron content was investigated at a dilution of 1:25 in dH₂O, dissolved in 65% nitric acid, using atomic emission spectroscopy with an Agilent 4200 MP-AES (Agilent Technologies, Santa Clara, CA, USA) with an iron solution of 1000 mg Fe/L as an external standard (Bernd Kraft, Duisburg, Germany). Measurements were performed in triplicates at a wavelength of 371.993 nm, which were then averaged. SPIONs were diluted with sterile dH₂O to the intended concentration for all experiments.