D16 - Magnetic Iron Oxide Nanoparticles Enhance Exosome Production by Upregulating Exosome Transport and Secretion Pathways

Introduction: : Exosomes are lipid-coated extracellular vesicles (30 – 150 nm) that play an important role in intercellular communication by transferring bioactive molecules such as cytokines and microRNAs among different cell populations. Exosomes often exhibit specific tissue or cell tropism and have good biocompatibility and low toxicity. Growing evidence indicates that exosomes are intricately involved in various physiological and pathological processes, including immune response, tissue regeneration, and tumor progression. The ability to modulate exosome production is of significant interest for both basic research and therapeutic applications.
Although the mechanisms governing exosome production are not yet fully understood, numerous studies have shown that exosome production is influenced by various intra- and extracellular signals that affect their biogenesis, transport, and secretion. Magnetic iron oxide nanoparticles (MNPs) along with applied magnet can exert direct forces on structural proteins, cell membranes, or subcellular organelles, inducing a variety of cellular responses. Previous studies proved that MNPs can modulate macrophage polarization and stiffness sensing. Therefore, there is significant overlap among the cellular uptake of MNPs, MNP-mediated cellular responses, and the exosome production pathway.
In this study, we investigated the potential effects of oscillating magnetic force on exosome production in two types of cancer cells: 4T1 cells (epithelial origin) and RAW264.7 cells (myeloid origin). Both cell types are known to respond to mechanical cues such as membrane stretch. We examined force-induced changes in cell morphology, actin cytoskeleton remodeling, and gene expression in pathways relevant to exosome production.

Materials and

Methods: : MNPs were synthesized by themodecomposition, coated with a mixture of DSPE-PEG1K and DSPE-PEG2K, and dispersed in Millipore water. The size and composition of iron oxide nanocrystals were analyzed by TEM, XRD, and DLS.
For magnetic stimulation, cells were incubated in the culture medium containing 20 μg/mL MNPs in T75 flasks for 24 hours. Next, cells were washed with PBS and incubated in FBS-free medium under an oscillating magnetic field (OMF) for 24 hours. The OMF was generated with a magnet array assembled on a rotating plate with an orbiting radius of 19 mm and a frequency of 0.5 Hz. The distance between the cells and the magnets was set at 3 mm. The magnetic force exerted on intracellular MNPs was simulated with a MATLAB program developed in house. The cells in the control group were seeded at the same density, incubated in the medium without MNPs for 24 hours, and incubated in FBS-free medium for another 24 hours.
The cellular uptake of MNPs was quantified using a Ferrozine assay. The changed in cell morphology and cytoskeleton were examined by fluorescence microscopy. 30 genes involved in 7 major signaling pathways relevant to exosome production were evaluated by RT-qPCR. The 2−ΔΔCt was used to calculate relative gene expression. Z-score of each gene was used to compare relative gene expression from different treatment groups in the heatmap.
Exosomes were harvested through sequential ultracentrifugation. The morphology, surface markers, and quantity of the exosomes were examined using TEM, Western blotting, and NTA according to MISEV2023.

Results, Conclusions, and Discussions:: As-synthesized nanocrystals have a uniform size of 15 ± 2 nm, as measured by TEM (Figure 1A). The XRD pattern of the nanocrystals reveals the characteristic cubic spinel structure of magnetite (Figure 1B). Both types of cells exhibited dose- and time dependent uptake of MNPs (Figures 1C-D).
Our study found that loading with MNPs alone did not change the cell morphology (Figure 2A-B). However, when subjected to a magnetic field, the cells became more spread and exhibited an increase in the actin staining. After incubated under a static magnetic field, both cells exhibited polarization along the force direction. Interestingly, cells treated with the OMF did not exhibit polarization along the force, presumably because the cell response is relatively slow compared to the rapid change of OMF. Yet OMF still promoted the formation of F-actin in filopodia in RAW264.7 cells.
We analyzed 30 genes involved in 7 major signaling pathways relevant to exosome production, including autophagy, PI3K-AKT, apoptosis, Notch, mechanosensing, exosome secretion, and regulation of actin cytoskeleton. There is extensive crosstalk among the 7 pathways. A simplified diagram is shown in Figure 2C. Surprisingly, the OMF enhanced the expression of most genes examined in both RAW264.7 and 4T1 cells (Figure 2D).
In both cell types, the vesicles from the unstimulated and OMF-treated groups exhibited similar morphology (Figure 3A), with size range of 30−200 nm (Figure 3C−F). Western blotting confirmed CD81, CD9, and CD63 expression in these vesicles (Figure 3B). NTA revealed that the vesicles in both the untreated (control) and the OMF-treated groups had similar size distributions. OMF stimulation significantly increased exosome production by 60 ± 12% more exosomes in RAW264.7 cells, and 46 ± 29% more in 4T1 cells (Figure 3G-H).
Our findings reveal that magnetic force applied to intracellular MNPs induces vesicle relocation, promotes the formation of actin stress fibers, and upregulates genes related to exosome transport, secretion, and exosome biogenesis. Notably, these forces substantially enhance exosome production, particularly MNP-containing exosomes. In summary, our study offers valuable insights into MNP-driven exosome production and presents potential strategies for enhancing cell communication and modulating nanoparticle distribution in nanomedicine.

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