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.