Fig. 3 In vitro magnetothermal induction of Ca2+ influx in TRPV1-expressing 293T cells. (a, b) Fold changes in fluorescence intensity (Ft/F0 values) of Fluo-4 in 293T cells during magnetothermal treatment with anti-His-FVIOs at Fe concentrations ranging from 51 to 498 μg/mL, and with anti-His-SPIOs at Fe concentrations of 498, 816, and 1095 μg/mL. AMF conditions: H = 20 mT and f = 290 kHz. (c) SIM images of TRPV1-expressing cortical neurons (red) bound to anti-His-FVIO-FL (green). (d) Fold changes in fluorescence intensity (Ft/F0 values) of Fluo-4 as a function of time for TRPV1-expressing cortical neurons treated with FVIOs (324 μg/mL) or SPIOs (1095 μg/mL). AMF conditions: H = 20 mT and f = 290 kHz. The data are presented as the mean ± SEM; the solid lines indicate the means, and the shaded areas indicate the SEMs.
We further examined Ca2+ influx in cultured TRPV1-expressing cortical neurons using the nanoheaters anti-His-FVIOs. FL-labeled anti-His-FVIOs successfully targeted TRPV1 on cortical neurons (Figure 3c). Moreover, the cells were loaded with Fluo-4 to monitor Ca2+ influx (Figure S10, movie S2, Supporting Information). As shown in Fig. 3d, upon magnetothermal stimulation with anti-His-FVIOs at an Fe concentration of 324 μg/mL, Ca2+ fluorescence in cortical neurons rapidly increased from 50 to 200 s. In contrast, the Ca2+ fluorescence intensity in neurons increased slowly in response to magnetothermal stimulation by the anti-His-SPIOs at an Fe concentration of 1095 μg/mL. Therefore, the anti-His-FVIO nanoheater is believed to enable efficient magnetothermal neural activation at low Fe concentrations.