By combining commonly used Protamine Sulfate (Pro) with superparamagnetic dextran coated iron oxide nanoparticle (SPIO) Ferumoxides (FE), a complex is formed that can be used to magnetically label stem cells and other mammalian cells. Cells take up FEPro complexes by macropinocytosis. Once internalized, iron oxide particles remain transiently in the endosomelysosome compartment and can be visualized on Prussian blue stain. Since it is possible that labeled cells may contain SPIO nanoparticles for long periods of time it is necessary to determine if the iron can be released from the endosomelysosome compartment and become available for metabolic pathways. We determined whether FEPro labeling altered expression of transferrin receptor (TfR1) and ferritin, proliferation, production of reactive oxygen species (ROS) in MSC and HeLa cells as part of the preclinical evaluation for investigative new drug (IND) submission to FDA. FEPro labeled MSCs and HeLa cells were cultured for a period of 1 to 28 days. Cells were evaluated for TfR1 and ferritin gene expression by Real TimePCR, protein levels by Western blotting, proliferation assay and ROS analysis. FEPro labeling of Hela and MSCs resulted in a decrease in TfR1 mRNA (day 17, in HeLa and day 114, in MSCs) and protein (day 37, in HeLa and MSCs). Ferritin mRNA was increased in labeled HeLa on day 7, and in labeled MSCs on day 14 and 28. Ferritin protein was significantly increased in HeLa for days 114 and in MSC day 128. FEPro labeling elicited changes in iron metabolism and storage but did not change in cell proliferation or ROS. Early detection of labeled cells in vivo by cellular MRI requires the development of novel pulse sequences or image processing to improve the sensitivity to low numbers of iron oxide labeled cells in tissues. A postprocessing positive contrast technique, Susceptibility Gradient Mapping (SGM), was investigated and compared with a gradient compensation based approach White Marker technique and an offresonance based approach Inversion Recovery ONresonance water suppress (IRON) technique with SPIO labeled tumor model in rats. The SGM, White Marker and IRON positive contrast images were acquired when the labeled tumors were approximately 5 mm (small), 10 mm (medium) and 20 mm (large) in diameter to evaluate their sensitivity to the dilution of the SPIO nanoparticles as the tumor cells proliferate. In vivo MRI has demonstrated that all three positive contrast techniques can increase the conspicuity of SPIO labeled cells by producing high signal intensities in surrounding areas. For small and medium tumors, SGM clearly delineated the regions with SPIO labeled cells and the Number of Positive Pixels (NPP) detected from SGM within the region of interest is significantly larger than that from the White Marker and IRON techniques: 1288 155 (p<0.01) vs. 330 130 from White Marker and 8552 from IRON for small tumors and 2584 177 (p<0.05) vs. 2047 378 from White Marker and 126 91 from IRON for medium tumors. For large tumors, SGM resulted in similar NPP as the White Marker technique: 5137 1633 (pNS) vs. 4514 1185 and IRON failed to generate positive contrast images with a 200Hz suppression band. These results indicate that post processing approaches can be useful in detecting magnetically labeled cells in vivo and have also revealed that hemorrhage that appears as hyperintensities on positive contrast images may interfere with the detection of SPIO labeled cells.
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