2.2. SPION Synthesis and APTES Modification

The SPIONs were synthesized and modified with APTES (SPION@APTES) in a one-step co-precipitation method. In a typical synthetic procedure, 0.75 g of FeCl₃∙6H₂O and 0.375 g of FeCl₂∙4H₂O were dissolved in 50 mL de-ionized water (dH₂O) in a 100 mL three-necked round bottom flask. The solution was refluxed at 80 °C and stirred for 10 min under nitrogen atmosphere. Then, 10 mL of NH₄OH (28–30% aqueous solution) was injected dropwise, via a syringe to the solution. After one hour, 15 mL of EtOH, followed by a previously prepared solution of 2 mL of APTES in 25 mL EtOH, was added to the reaction mixture and stirred for further 3 h at 80 °C. Prior to the purification, the suspension was allowed to cool to room temperature (RT). Then, as-synthesized nanoparticles were retrieved from the mixture by the addition of acetone (5:1 v/v acetone/supernatant) as precipitant, followed by centrifugation for 10 min at 8500 rpm, resuspended in 20 mL dH₂O, and dialyzed against 2 L dH₂O for 24 h. The resulting magnetic nanoparticle concentration of 26.67 mg/mL was determined gravimetrically.