The synthesis and characterization of the probes were previously reported by Ahmed et al.25 (link) In brief, Para-isothiocyanatobenzyl-deferoxamine (p-NCS-Bz-DFO; CheMatech, Dijon, France) or fluorescein isothiocyanate (FITC; Sigma-Aldrich, St. Louis, MO, USA) were conjugated to human serum albumin (HSA; Sigma-Aldrich) by adding p-NCS-Bz-DFO or FITC to HSA and stirring at room temperature (RT) and stable pH. The products were purified on a desalting column, and the product was freeze-dried. To conjugate the maleic anhydride (MalA), solid MalA was added while stirring at RT to a solution of HSA-DFO or HSA-FITC at pH 9. The resulting Mal-HSA-DFO was purified by dialysis and the Mal-HSA-FITC was purified on a desalting column. Solutions of the products were sterile filtered and freeze-dried. The approximate average numbers of conjugated FITC, DFO, and Mal groups could be determined by comparisons of the average molecular masses of the different HSA modifications with the molecular mass of unconjugated HSA. The respective masses were analyzed by mass spectrometry (MS) (Bruker ultrafleXtreme MALDI-TOF/TOF with a Smartbeam-II laser (Bruker Daltonik, Bremen, Germany)).