K6 - Optimizing Iron Oxide Nanoparticle Uptake in Natural Killer Cells for Enhanced Magnetic Particle Imaging-Based Tracking

Introduction: : Natural killer (NK) cell immunotherapy is an exciting avenue currently being explored for cancer treatment. Similar to T cells, NK cells are a type of white blood cell capable of identifying and eliminating infected or malignant cells. However, unlike T cells, which primarily rely on antigen-specific, antibody-dependent mechanisms, NK cells induce cytotoxicity through antigen-independent pathways. While NK cell-based therapies offer distinct advantages, their clinical development is hindered by a lack of effective tools for tracking cell migration and persistence within the tumor microenvironment. Magnetic nanoparticles present a potential solution through magnetic particle imaging (MPI), a non-invasive imaging modality that enables real-time, three-dimensional visualization based on the nonlinear dynamic magnetization of iron oxide nanoparticles (IONPs). Nevertheless, efficient IONP labeling of NK cells remains challenging as they are suspension cells with low endocytic activity and limited cytoplasmic volume. This study aims to identify optimal culture conditions and nanoparticle characteristics that facilitate measurable IONP uptake in NK92MI cells, a clinically relevant NK cell line, while preserving cell viability and functional integrity.

Materials and

Methods: : We evaluated the uptake of four commonly used IONPs (Fig 1A) at 100 μg/mL by NK92MI cells (1.2x10^6 cells/mL) in three media conditions (serum-free, reduced serum, and serum-containing) at 4 hours and 24 hours. After incubation, NK92MI cells were washed, fixed, and the iron content was measured via MPI using high-sensitivity 2D scans, followed by Prussian blue staining to visualize iron uptake within the cells.

Results, Conclusions, and Discussions:: NK92MI cell viability was maintained >90% across all media conditions following incubation with each IONP formulation. Among the IONP formulations tested, VivoTrax, VivoTrax Plus, and Perimag IONPs demonstrated detectable MPI signal following incubation with NK92 cells (Fig 1B). This MPI signal was observed across all media conditions tested at both 4-hour and 24-hour timepoints. However, although their signal was detectable, the Perimag IONPs exhibited a weak signal, whereas the VivoTrax, VivoTrax Plus, and poly(maleic anhydride-alt-1-octadecene) (PMAO) IONP formulations demonstrated significant nanoparticle aggregation, both at the macroscopic and microscopic levels (Fig 1C-1E). Prussian blue staining showed that many of the IONPs appeared bound to the cell surface rather than internalized intracellularly. Because NK92MI cells are suspension cells, separating the nanoparticle aggregates from the cells is difficult, complicating accurate quantification of cellular uptake and highlighting a need for improved nanoparticle formulations. To remove nanoparticle aggregates, a Ficoll-Paque density gradient centrifugation step was applied to separate the cells from the aggregates. While this method reduced non-cell-associated particles, it also resulted in substantial loss of cell mass, rendering the protocol impractical for downstream applications that require large cell numbers (Fig 1F). The ability to non-invasively track adoptively transferred NK cells in vivo remains a major unmet need for NK cell immunotherapy. Our findings indicate that VivoTrax, VivoTrax Plus, and Perimag could produce an MPI-detectable signal in the measured culture conditions. However, this apparent success was tempered by weak signal or substantial nanoparticle aggregation and challenges in discriminating between cellular uptake and extracellular particle association. The observed aggregation not only complicated signal interpretation but also posed a significant barrier to downstream applications, such as clinical translation or longitudinal tracking in animal models. Although Ficoll density separation reduced extracellular debris, it did so at the expense of considerable NK cell loss. Given these challenges, future efforts are focused on developing optimized nanoparticle formulations, via flash nanoprecipitation, that are stable in media and are specifically engineered for uptake by NK cells. Ultimately, we aim to create a labeling platform that combines high signal detectability with minimal toxicity, ensuring compatibility with therapeutic use and in vivo imaging applications.

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