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Wang et al. Microstructures 2023;3:2023042 https://dx.doi.org/10.20517/microstructures.2023.46 Page 9 of 16

Figure 5. Anticancer effect of free Fe O NPs. Live/dead staining of MDA-MB-231-Luc cells cultured without or with free Fe O NPs
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before and after AMF irradiation (live cell: green fluorescence, dead cells: red fluorescence) (A). Quantified cell viability during culture
without or with free Fe O NPs before and after AMF irradiation (B). Cell viability was normalized to that cultured with PBS without free
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Fe O NPs before AMF irradiation. Data are the mean ± SD (n = 3). Significant difference: ***P < 0.001. N.S. : no significant difference.
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almost all the cells were alive [Figure 5A]. After AMF irradiation, the cells cultured without Fe O NPs were
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still alive, while almost all the cells cultured with Fe O -5, Fe O -10, and Fe O -20 were dead. The results
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indicated that the cells cultured with free Fe O NPs at a concentration of 5, 10, and 20 mg mL were
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completely killed after AMF irradiation. Some of the dead cells cultured with 20 mg mL L free Fe O NPs
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detached from the culture wells.
The WST-1 assay showed that the cells cultured without or with free Fe O NPs had the same high viability
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before AMF irradiation [Figure 5B]. After AMF irradiation, the viability of cells cultured without free Fe O
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NPs did not change significantly, while the viability of cells cultured with free Fe O NPs significantly
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decreased after AMF irradiation. Viability of cells cultured with 5, 10, and 20 mg mL free Fe O NPs
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decreased to 10.3% ± 6.9%, 3.1% ± 5.8%, and 2.7% ± 5.2%, respectively. All the live/dead staining and WST-1
assay results indicated that the breast cancer cells could be killed by the free Fe O NPs under AMF
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irradiation. A higher concentration of free Fe O NPs resulted in a higher killing effect. The killing effect of
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free Fe O NPs should be due to the high temperature generated by free Fe O NPs during AMF irradiation
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[Figure 4]. A higher concentration of the Fe O NPs could generate higher temperatures and enhance the
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killing efficiency.
Anticancer effect of agarose/Fe O hydrogels
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The interaction between cells and agarose/Fe O hydrogels should be different from that between cells and
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free Fe O NPs. The cells could be near the hydrogels without adhesion to the hydrogels. The cells could also
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