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

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.

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.

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.

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.