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

Figure 1. Anticancer experimental scheme of free Fe O NPs (A), agarose/Fe O hydrogels (B-D), and gelatin/Fe O porous scaffolds
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(E-G). Three modes (sitting, transwell, and adhesion modes) were used to simulate the cells near or far away from or directly adhered
to the matrices.

MATERIALS AND METHODS
Materials
Iron (II) chloride tetrahydrate (FeCl ·4H O, ≥ 99%), iron (III) chloride hexahydrate (FeCl ·6H O, ≥ 97%),
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and sodium citrate tribasic dihydrate (≥ 99%) were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Diethylene glycol (DEG, 99%), sodium hydroxide (NaOH, 99.99%), acetic acid, ethanol, ethyl acetate,
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), and N-hydroxysuccinimide (NHS) were obtained
from Wako Pure Industries, Ltd. (Tokyo, Japan). N-methyldiethanolamine (NMDEA, 99%) was purchased
from Tokyo Kasei. Kogyo Co., Ltd, and gelatin (porcine) was obtained from Nitta Gelatin (Osaka, Japan).

Synthesis and Characterization of Fe O NPs
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Fe O NPs were synthesized using Fe (II) and Fe (III) in a mixture solution of DEG and NMDEA
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(1:1, v/v) [49,50] . First, 4 mmol FeCl ·4H O and 2 mmol FeCl ·z`6H O were added and dissolved in an 80 mL
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polyols mixture under a nitrogen atmosphere at room temperature. Next, 40 mL of 400 mM NaOH in a
polyol mixture was dropped into the iron salts solution and stirred for 3 h. Then, the mixed solution was
heated to 220 °C and reacted for 12 h under the protection of nitrogen. After the reaction, the black
precipitates were collected and washed with ethanol/ethyl acetate solvent (1:1, v/v) three times to obtain
bare Fe O NPs. The obtained bare Fe O NPs were redispersed in sodium citrate aqueous solution and
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reacted at 60 °C for 24 h to modify the Fe O NPs with citrate anions. After centrifugation, washing, and
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lyophilization, the citrate-modified Fe O NPs were obtained. The prepared Fe O NPs were redispersed in
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PBS to obtain the Fe O NPs colloidal solution at a concentration of 5 mg mL , 10 mg mL , and 20 mg mL ,
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which were named free Fe O -5 (5 mg mL ), free Fe O -10 (10 mg mL ), and free Fe O -20 (20 mg mL ),
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respectively. The structure and mean diameter of the citrate-modified Fe O NPs were analyzed by a
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transmission electron microscope (TEM, JEOL 2100F, Japan). The hydrodynamic size of the citrate-
modified Fe O NPs was analyzed by dynamic light scattering (DLS, Beckman Coulter, USA).
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