Labelling of granulocytes by phagocytic engulfment with 64Cu-labelled chitosan-coated magnetic nanoparticles.

Purpose: The aim of the present work was to perform the labelling of granulocytes by their engulfment with chitosan-coated magnetic (64)Cu nanoparticles (MNPs) in order to obtain a radiopharmaceutical suitable for dual imaging (PET-MRI) of inflammatory/infective diseases.

Procedures: Specimens of 5-20 mg MNPs were washed with saline-isotonic solution and recuperated by magnetic decantation; 15-58 μg Cu(2+) (CuCl(2)·H(2)O) in 50 μl of acidified (pH 5.5) saline solution was added to the MNPs re-suspended saline-isotonic solution; 10 mg MNPs was allowed to react with 16 μg (64)Cu [(64)Ni(p,n) at 12-9 MeV] followed by anion exchange chromatography with a specific activity of 56 MBq/μg. Pellets of granulocytes were obtained from peripheral blood; MNPs engulfment by granulocytes was obtained and granulocyte-engulfed viability was assessed by the trypan blue exclusion (TBE) test performed at 5 min, 2 h and 4 h; assessment of the release of (64)Cu from labelled granulocytes in plasma was performed by measuring the radioactivity of both the cellular pellet and the supernatant solution.

Results: Our data showed the binding capacity of chitosan-coated MNPs for cationic metal. The amount of Cu(+2) chelated captured per milligram of MNPs was constant and independent of the reagent concentrations. In all cases, more than 90% of the engulfed granulocytes were positive to the TBE test. The MNPs were localised within the cells.

Conclusion: In our in vitro model, MNPs are taken up by granulocytes through phagocytosis, whereas previously described methods were based on the use of a chelating agent that permit Cu to cross the cell membrane. Moreover, the (64)Cu-engulfed granulocytes showed a high stability of up to 80% of retained radioactivity after 24 h of incubation.