Dextran-coated SPIONs (SPIONDEX) were synthesized in a cold gelation process, as described previously.26 In brief, an aqueous solution containing 8.8% (w/w) dextran, 2.4% (w/w) FeCl3·6H2O, and 0.9% (w/w) FeCl2·4H2O was prepared and filtered through a 0.22-µm membrane into an ice-cooled three-neck round-bottomed flask. Under an argon atmosphere, the temperature was adjusted to 0°C–4°C. NH3 was added to the solution, which resulted in a green-brownish suspension. After heating the solution at 75°C, the formed particles were cooled and dialyzed against water (MWCO 10 kDa) to remove excess salts. Redundant dextran was removed afterward by ultrafiltration in a centrifuge 5430R (Eppendorf, Hamburg, Germany) with Vivaspin20 filter units at 6,300× g for 10 minutes for multiple runs. The dextran molecules of the SPION coating were crosslinked with ECH. In this reaction, the pH of the particles was adjusted to basic conditions with 5 M NaOH and then ECH was added to a final concentration of 15% (v/v). The suspension was stirred for 24 hours and then it was again dialyzed against water for 24 hours, followed by ultrafiltration and sterile filtration through 0.22 µm filters.
The dextran coating of the particles was functionalized with carboxylic acid groups according to Huynh et al.23 Briefly, the pH of the suspension was adjusted to 12–13 with 5 M NaOH. After cooling the suspension, monochloroacetic acid was added and then it was heated to 60°C for 90 minutes, followed by neutralization with acetic acid. In the end, the particles were purified by dialysis against water and ultrafiltration. In the following, they are referred to as SPIONCMD.
The determination of the particles’ iron content was performed photometrically as described by Dokuzovic.27