In vitro magnetothermal activation of TRPV1-expressing cells
To precisely evaluate the efficacy of FVIO-mediated magnetothermal activation of the TRPV1 channel, monoclonal HEK293T cell lines were generated that stably expressed 6× His-labeled TRPV1 channels. TRPV1 was labeled with the red fluorescent protein mCherry, which was separated by the posttranscriptional cleavage linker p2A. The western blotting showed that TRPV1 fusion protein was expressed on HEK293T cells (Figure S5, Supporting Information ). Figure 2a presents a schematic of the FVIO-mediated magnetothermal activation of TRPV1 and Ca2+ influx into cells. The fluorescent Ca2+ indicator Fluo-4 was loaded into transfected HEK293T cells to monitor the changes in the intracellular Ca2+ concentration in response to magnetothermal stimulation. After application of the anti-His-FVIOs targeting the TRPV1 channel, the change in green fluorescence intensity during magnetothermal stimulation was recorded in real time using confocal laser scanning microscopy (CLSM) equipped with an AMF generator (Figure S6, Supporting Information ).
Before performing the magnetothermal activation experiments, we measured the local change in temperature around the FVIOs under an AMF (20 mT, 200 kHz) to confirm that the local heating offered by the FVIOs was sufficient to activate the TRPV1 channel. FVIOs were modified with fluoresceinamine (FL; emits green fluorescence) for use as a molecular-scale thermometer. First, the fluorescence intensity and bulk temperature increase of the FVIOs-FL dispersion were recorded during AMF exposure. The percent change in fluorescence intensity decreased linearly with increasing temperature[8], reaching –1.97 ± 0.04%/°C for FL (Figure S7, Supporting Information ). On this basis, FVIOs-FL were used to target the TRPV1 channels expressed on cultured HEK293T cells (Figure 2b; Figure S8, Supporting Information ), and the change in FL fluorescence intensity was measured upon AMF exposure. The local temperature changes near the FVIOs were calculated according to the linear temperature-fluorescence intensity curve. After 30 s of AMF exposure, the local temperature in the immediate vicinity of TRPV1 on the cell membrane increased from 36 to 43 °C and then decreased rapidly to 36 °C in the absence of an AMF (Figure 2c). A local temperature increase to more than 43 °C near the FVIOs is sufficient to activate the TRPV1 channel by inducing a conformational change in the protein. In addition, the simulation results revealed that the local temperature decreases as the distance between the surface of a single FVIO and water increases (Figure S9a, Supporting Information ). No heat was generated in the cell culture media according to the infrared thermal images (Figure S9b, Supporting Information ).
FVIO-mediated magnetothermal activation of TRPV1 was systematically investigated through in situ Ca2+ imaging in cultured 293T cells, where Ca2+ influx indicates the opening of the TRPV1 channel and increased cellular activity. The fold changes in the intracellular fluorescence intensity (Ft/F0) of the Ca2+indicator Fluo-4 were recorded under different experimental conditions. Intense green fluorescence corresponding to the intracellular Ca2+ signal was observed upon AMF exposure. After treatment with anti-His-FVIOs, the Ft/F0of the TRPV1-expressing HEK293T cells increased with prolonged AMF exposure, which was attributed to successful heat-triggered Ca2+ influx, as shown in Fig. 2 (d and e). This increase in fluorescence induced by Ca2+ influx was observed only in TRPV1-expressing cells that were activated by anti-His-FVIOs-mediated magnetothermal stimulation and in the group treated by agonist capsaicin (CAP, 10 μM). The cells in the negative control group, were cultured in media containing the antagonist capsazepine (CZP, 5 μM), and no significant difference in fluorescence intensity was observed during anti-His-FVIO-mediated magnetothermal stimulation. Moreover, the control groups, including the TRPV1 no expressing group, the FVIO-treated group and the AMF-treated group, exhibited negligibleCa2+-dependent changes in fluorescence intensity .