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Page 10 of 16 Wang et al. Microstructures 2023;3:2023042 https://dx.doi.org/10.20517/microstructures.2023.46

be far away from the hydrogels or directly adhere to the hydrogels. To simulate these interactions between
the breast cancer cells and hydrogels, three culture modes were used to investigate the MH anticancer effect
of the agarose/Fe O hydrogels. When the MDA-MB-231-Luc cells were cultured near the agarose/Fe O 4
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hydrogel discs (sitting mode), most of the cells near the agarose/Fe O -5 hydrogel discs were dead, and cell
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viability decreased significantly after AMF irradiation [Figure 6A and B]. Almost all the cells near the
agarose/Fe O -10 and agarose/Fe O -20 were dead, and their viability further decreased to the lowest level.
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The cell viability near agarose/Fe O -5, agarose/Fe O -10, and agarose/Fe O -20 decreased to 11.3% ± 4.8%,
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3.9% ± 2.2%, and 3.6% ± 5.1%, respectively, after AMF irradiation [Figure 6B]. However, the cells near the
agarose hydrogel discs without Fe O NPs were alive, and their viability did not change before and after
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AMF irradiation.
When the breast cancer cells were cultured far away from the agarose/Fe O hydrogel discs (transwell
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mode), the agarose/Fe O hydrogels with a high concentration of Fe O NPs (agarose/Fe O -10 and
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agarose/Fe O -20) could kill almost all the cells and significantly decrease cell viability to very low level by
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AMF irradiation [Figure 6C and D]. However, only a small number of cells cultured with the
agarose/Fe O -5 were killed by AMF irradiation. After AMF irradiation, cell viability in the transwell of
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agarose/Fe O -5, agarose/Fe O -10, and agarose/Fe O -20 decreased to 85.7% ± 5.7%, 6.0% ± 3.5% and 2.8% ±
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5.9%, respectively [Figure 6D].
The breast cancer cells adhered on the agarose/Fe O hydrogel discs (adhesion mode) were most efficiently
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killed by AMF irradiation. Almost all the cells cultured with all the agarose/Fe O hydrogel discs were dead,
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and their viability was the lowest compared to the sitting and transwell modes [Figure 6E and F]. Viability
of breast cancer cells adhered on the agarose/Fe O -5, agarose/Fe O -10, and agarose/Fe O -20 decreased to
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7.1% ± 1.2%, 2.7% ± 4.6%, and 2.3% ± 6.5%, respectively, after AMF irradiation [Figure 6F]. This should be
due to the direct heating effect of the cells by the agarose/Fe O hydrogels when the cells adhered to the
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hydrogels.
Anticancer effect of gelatin/Fe O porous scaffolds
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The anticancer effect of gelatin/ Fe O porous scaffolds was investigated by using the same methods as those
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used for the agarose/Fe O hydrogels [Figure 7]. The cells cultured with the gelatin porous scaffold before
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and after AMF irradiation and with the gelatin/Fe O porous scaffolds before AMF irradiation were alive
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with high viability. However, after AMF irradiation, some dead cells were observed in the gelatin/Fe O -5
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and gelatin/Fe O -10, and almost all the cells were dead in the gelatin/Fe O -20 when the cells were cultured
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near the scaffolds [Figure 7A] or adhered in the scaffolds [Figure 7E]. When the cells were cultured far away
from the scaffolds, a small part of the cells cultured with the gelatin/Fe O -20 were killed [Figure 7C]. After
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AMF irradiation, the viability of breast cancer cells cultured with gelatin/Fe O -5, gelatin/Fe O -10, and
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gelatin/Fe O -20 was 94.2% ± 9.1%, 80.3% ± 7.8%, and 6.2% ± 5.2% in sitting modes [Figure 7B],
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99.8% ± 5.0%, 96.0% ± 5.1%, and 87.0% ± 3.4% in transwell modes [Figure 7D] and 90.8% ± 2.5%,
67.8% ± 3.2%, and 4.7% ± 3.4% in adhesion modes [Figure 7F], respectively.
The above results indicated that the breast cancer cells could be killed by either free Fe O NPs,
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agarose/Fe O hydrogels, or gelatin/Fe O porous scaffolds. However, the anticancer effect depended on the
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matrices and interactions. The free Fe O NPs showed the highest killing effect, while the gelatin/Fe O 4
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porous scaffolds showed the lowest. Matrices used to embed Fe O NPs showed an inhibitory influence on
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the killing effect of Fe O NPs. In particular, the porous scaffolds had a more inhibitory influence than the
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hydrogels. This should be due to the influence of matrices on the magnetic thermal property of the Fe O 4
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NPs. The interaction between breast cancer cells and Fe O NP-embedded hydrogels or porous scaffolds
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