Fig. 2 In vitro magnetothermal activation of TRPV1 triggers Ca2+ influx. (a) Schematic of FVIO-mediated magnetothermal activation of TRPV1 triggering of Ca2+influx. Fluo-4 was used as the Ca2+ indicator. (b) SIM images of TRPV1-expressing HEK293T cells (red) bound to FL labeled anti-His-FVIOs (green). (c) Local temperature increase near the FVIOs upon AMF exposure determined by measuring the change in green fluorescence intensity of anti-His-FVIOs- FL target the cell membrane. (d) Fold changes in fluorescence intensity (Ft/F0 values) and (e) color maps of Fluo-4 in HEK293T cells under different experimental conditions. TRPV1-expressing, TRPV1+; TRPV1-nonexpressing, TRPV1-; before AMF exposure, AMF OFF; during AMF exposure, AMF ON; with FVIOs treatment, FVIOs+; without FVIOs treatment, FVIOs-.; Ca2+ free group, TRPV1+-FVIOs+-AMF-Ca2+-free group. AMF conditions: H = 20 mT and f = 290 kHz. The solid lines and shaded areas represent the means and standard error of the mean (SEMs), respectively, the data presented as the mean ± SEM.
We further studied the influence of the Fe concentration on the stimulus-response time and fold changes in fluorescence intensity due to Ca2+ influx into cells. As shown in Fig. 3a, the response time of Ca2+ influx into cells decreased when the Fe concentration from the anti-His-FVIOs in the HEK293T cell culture media ranged from 51 to 498 μg/mL. HEK 293T cells treated with anti-His-FVIOs at an Fe concentration of 324 μg/mL showed the fastest response in terms of Ca2+ influx and the greatest fold change in fluorescence intensity (Figure S3a, movie S1 , Supporting Information ). Ca2+ influx into cells occurred only at an Fe concentration of 1095 μg/mL or greater in the SPIO-treated groups (Figure 3b). Notably, the minimum Fe concentration needed for TRPV1 activation was 51 μg/mL in the FVIO-treated group, which was 20.27-fold lower than that in the SPIO-treated group. This finding is consistence with our early simulation results showing that a single TRPV1 channel can be activated using a less FVIOs than SPIOs (Figure S1, Supporting Information ). Together, this experimental evidence suggests that FVIOs with superior heat-generating performance could enable efficient magnetothermal activation of thermosensitive TRPV1 expressed on cells.