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 .