(G-270) Stimulating Raw 264.7 Cell-Derived Exosomes Using Nanomagnets

Introduction:: Exosomes are lipid-coated extracellular vesicles (30 – 200 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. There is extensive interest in developing exosome-based agents for disease treatment. Of particular interest to cancer treatment are the macrophage-derived exosomes. Macrophages are one of the main cellular components in solid tumors. Importantly, several studies showed that macrophage-derived exosomes had an excellent tumor-targeting capability. A major challenge in using exosomes for therapeutic purposes is the low exosome production rate. It is known that exosome production relies on vesicle transport regulated by cytoskeletons such as myosin and actin filaments. Our previous study showed that magnetic nanoparticles (MNPs) internalized by the cells could induce the formation of actin filaments by exerting an intracellular force. Here we examine the effects of intracellular force on exosome production by macrophages, aiming to provide a new leverage for controlling exosome production in vitro and in vivo.

Materials and Methods:: MNPs with a magnetite nanocrystal core were synthesized using a published method. RAW264.7 cells (a mouse macrophage cell line) were cultured in DMEM supplemented with 10% FBS. For exosome production, RAW264.7 cells were seeded in T75 flasks and cultured overnight. The cells were incubated in a medium containing MNPs for 24 hours. After incubation, the cells were washed with PBS and incubated in fresh DMEM without FBS and MNPs. For magnetic stimulation, the flask was incubated above a magnetic plate rotating at 1 Hz. After incubation for 24h, the medium was collected and exosomes in the medium were collected using a centrifugation method. In brief, the medium was centrifuged once at 2000g for 10min and twice at 14000g for 30min and the pellets were discarded after each round of centrifugation. At the last step, the medium was centrifuged at 100,000g for 2 hours. The supernatant was discarded, and the pellets were dispersed in 1 mL PBS.


Results, Conclusions, and Discussions:: We synthesized magnetite nanocrystals of 15 nm in diameter (Fig. 1A). After 24-hour incubation in the medium containing MNPs, each cell contained 1.2 ± 0.2 × 10⁵ MNPs. Intracellular MNPs accumulated in the vesicles around the cell nuclei. In addition, MNPs had negligible cytotoxicity on RAW264.7 cells. We designed a magnetic plate consisting of arrays of Neodymium rare earth disc magnets. Numerical simulation showed that the magnitude of the oscillating magnetic force exerted on each cell was approximately 3 pN. The size distribution of the exosomes produced by all cells were similar. However, compared with untreated cells, the cells stimulated by intracellular magnetic force produced twice as many exosomes (Fig. 1D).


Acknowledgements (Optional): : This work was performed in part at the U.K. TEM, a member of the Nanotechnology Coordinated Infrastructure (NNCI), which is supported by the National Science Foundation (NNCI-2025075). This work is also supported by NIH/NIBIB R01 (R01EB026893).


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