Background

Several nanoparticle-based cancer medicines are in clinical use because they have demonstrated safety and efficacy for diagnosis, cancer drug delivery, and hyperthermia (Marchal et al., 2015; Wu et al., 2015). Yet, there remain critical gaps in our understanding of cancer nanomedicine, largely because details of interactions between nanoparticles and immune cells remain unknown. Others and we have identified nanoparticle-immune cell interactions as critical variables affecting in vivo nanoparticle fate and retention in tumors (Sheen et al., 2014; Zanganeh et al., 2016; Korangath et al., 2020). Flow cytometry enables quantitative analytical sorting of mixed cell populations by combining light scattering with detection of a fluorescent signal from appropriately labeled cells. To track nanoparticle uptake in cells, the nanoparticles are often labeled with a fluorescent dye to enable detection. The presence of dyes or other ‘excipient’ molecules on nanoparticles can change nanoparticle pharmacokinetics and biodistribution in unintended and unrealized ways, and do not provide sufficient analyte for quantitative validation of nanoparticle concentration. Here we demonstrate the use of the magnetic property of unlabeled iron oxide nanoparticles (no fluorescence labeling) taken up by the cells, when administered intravenously. Our developed assay provides quantitative and cell-specific data of unaltered nanoparticle uptake following systemic (i.e., intravenous) delivery. Our assay exploits the directional response of magnetic iron oxide nanoparticles (MIONs) to high-gradient magnetic fields, which causes cells containing the MIONs to sediment near the magnet, whereas cells devoid of MIONs remain in supernatant. In this manner, cells are ‘pre-sorted’ by magnetic separation to differentiate between the two populations before flow cytometry analysis. Following sorting, cells can be analyzed further by inductively coupled plasma mass spectrometry (ICP MS) or ferene-s assay (Hedayati et al., 2018) to validate concentration of the analyte (Fe) which is then related to intracellular nanoparticle content; and, by other gene- or protein-based array technologies to assess biological responses at the molecular level within cells (Figure 1).


Figure 1. Concept of magnet-assisted cell isolation for detailed downstream analysis. When injected into the tail vein, iron oxide nanoparticles accumulate in organs and tumor. After harvest, tissues can be dissociated into single cells and nanoparticle-associated cells can then be isolated by placing the entire mixture on a permanent rare earth magnet. Cells associated with the iron oxide nanoparticles (internal or membrane bound) will separate from the suspension and attach to the tube wall proximal to the magnet. Cells not associated with a significant concentration of nanoparticles will remain in suspension (supernatant). Magnetically sorted cells can then be further analyzed with a flow cytometer to distinguish among cell types or lineages. Absolute amount of iron in cells can be determined by inductively coupled plasma mass spectrometry (ICPMS), or by other quantitative iron assays. Nanoparticle-induced changes in cells at the molecular level can be identified by further analysis using gene- or protein-based array technologies. Comparisons with cells not associated with the nanoparticles is also possible by collecting and analyzing the supernatant.