Intra- and intercellular fate of iron oxide nanoparticles in platelets
K. Aurich, J. Wesche, R. Koch, J. Fuhrmann, A. Greinacher (Greifswald, Germany)
Platelets - Physiology and Disorders of platelet number and function
Time: 17:15 - 18:15
Objective: Platelet concentrates (PC) are transfused to prevent or treat bleeding in patients with severe thrombocytopenia or platelet function defects. To distinguish between platelet populations in platelet survival studies cell labeling is often required. We established under GMP conditions a non-radioactive platelet labeling method with iron oxide nanoparticles (NP) contained in Resovist® (Bayer AG, Germany). In vitro platelet function is not influenced by the labeling method, in addition survival studies in NOD/SCID mice indicate similar survival behaviour compared to non labeled platelets. Here we report on particle internalization into platelets and the stability of magnetic labeling in vitro and ex vivo.
Methods: Platelets from PC and freshly isolated from whole blood were incubated with fluorescent iron oxide nanoparticles (NanoscreenMAG-CMX-R, Chemicell, Germany) with similar structure to Resovist® for 1 hour at 37 °C. After purifying cells from excess nanoparticles, cells were analyzed by electron microscopy and fluorescence microscopy for detailed localization of NP. Labeling rate was determined by flow cytometry, mean intracellular iron content by atomic absorption spectroscopy. Labeling stability was analyzed over 24 hours by flow cytometry. Furthermore magnetic labeled platelets were added to whole blood samples in order to determine labeling stability ex vivo. Samples were taken at certain time points within 24 hours and analyzed by flow cytometry.
Results: Platelets internalize nanoparticles mainly in the open canalicular system (OCS), some are found in the alpha-granula. Only a small percentage of nanoparticles is found on the outer cell membranes. Labeling rates differ between platelets from PC (44.2%±21.2%) and platelets freshly prepared from whole blood (78.8%±9.1%). The mean cellular iron content per platelet is 0.33 pg±0.02 pg for PC and 0.51 pg±0.06 pg for platelets from whole blood. After spiking magnetic labeled platelets into whole blood, the labeling rate of platelets remained stable. We observed no exchange of NP between the labeled platelets and non-labeled platelets in whole blood.
Conclusion: We demonstrate that magnetic platelet labeling rate differs between platelet types, but remains stable over 24 hours without any measurable exchange of NP between platelets.