Introduction
Magnetothermal neurostimulation is emerging as a powerful technique for
gaining insight into intricate brain circuits and performing therapeutic
investigations of neurological disorders given its distinct advantages,
such as minimal invasiveness, tether-free operation, deep brain
accessibility, and high spatial resolution[1-2].
In this technique, biocompatible magnetic nanoparticles are utilized as
nanoheaters to generate heat locally through the hysteresis loss process
under alternating magnetic fields (AMFs)[3]. The
increase in local temperature can activate the thermosensitive ion
channels that are overexpressed on the cell membrane and trigger an
influx of calcium (Ca2+) into neurons, thereby
modulating neural activity[4]. To date,
magnetothermal neurostimulation has been demonstrated to bidirectionally
modulate neuronal activity[5-8], successfully
regulate blood glucose[9-10] and adrenal hormone
levels in vivo[11], and control the motor
behaviors of worms[8, 12],
flies[13] and freely moving
mice[14]. However, current techniques still suffer
from low activation efficiency and high required dosages of nanoheaters,
leading to an unexpectedly long stimulus-response time and potential
safety concerns[13].
According to the principles of magnetothermal neurostimulation, the
precisely timed activation of a single neuron depends on both the
expression level of thermosensitive ion channels and the heating
efficiency of the nanoparticles under an AMF[11,
15-16]. Transient receptor potential cation channel subfamily V
member 1 (TRPV1), the thermosensitive ion channel most commonly used in
neuromodulation, can be activated by local heat (> 43
°C)-induced conformational changes in the channel
pore[17-20]. Moreover, the rate of TRPV1 opening
increases exponentially with increasing surrounding
temperature[19, 21]. Because an increase in TRPV1
expression in vivo requires viral vector-mediated gene transfection,
which comes with high safety risks, the key to quickly and safely
activating TRPV1 lies in boosting the heat-generating performance of
magnetic nanoparticles[3-4, 11]. Unfortunately,
the superparamagnetic iron oxides (SPIOs) and ferritin protein widely
used in magnetothermal neurostimulation show poor thermal conversion
efficiency due to their superparamagnetic nature and minimal hysteresis
loss[22-25]. In addition, an AMF with a high
amplitude (H) and frequency (f ) is frequently needed to improve
the heat-generating ability of the
nanoheater[26-28]. Nevertheless, less improvement
has been achieved because of the biologically acceptable limit (H ×f ≤ 5 × 109A/(m·s))[28-30]. Recognizing the above
limitations, developing a high-performance magnetic
nanoparticle-mediated stimulation technique is a valuable approach for
realizing efficient and safe magnetothermal
neuromodulation[31-33].
Significant efforts have been made to synthesize various magnetic
nanoparticles with enhanced heat-generating performance given their wide
application in magnetic hyperthermia, controlled drug delivery, cell
signal transduction activation and magnetothermal
neurostimulation[30, 34-39]. The thermal
conversion efficiency of nanoparticles is commonly evaluated using the
specific absorption rate (SAR), which mainly depends on the intrinsic
magnetic properties of the nanoparticles[40-42].
Despite tuning their composition, shape, size and surface chemistry of
various magnetic nanoparticles, most exhibit relatively low SAR values
ranging from 250 W/g to 1000 W/g[30, 43-45]. As a
result, these nanoparticles take more time to reach the threshold
temperature and activate TRPV1, leading to long latency times
(approximately 14.7 s) for behavior onset[13, 18,
46]. Applying a high dose of magnetic nanoparticles is an alternative
method to reduce the heating time[26].
Nevertheless, using excessive amounts of nanoheaters increases the risk
of nonnegligible safety issues in the brain, such as dopamine neuron
damage, homeostatic disruption and inflammatory
responses[47]. We previously reported that novel
biocompatible ferromagnetic vortex-domain iron oxide nanorings (FVIOs)
exhibit an ultrahigh SAR of greater than 3000 W/g because of their
unique vortex-to-onion magnetization reversal phenomenon upon AMF
exposure[48]. Notably, as our simulation results
revealed, only 8.70 × 10-6 ng of FVIOs could
effectively increase the local temperature from 37 °C to 43 °C, whereas
33.6 × 10-6 ng of SPIOs was needed under the same AMF
conditions (Figure S1, Supporting Information ). In addition,
relatively large size and biocompatible FVIOs are more stable in the
brain, which is favorable for long-term and repeated magnetothermal
neurostimulation in vivo[48]. As such, FVIOs are
likely to be high-performance nanoheaters that facilitate quick
activation of TRPV1, which might provide an opportunity to establish a
highly efficient and safe magnetothermal neurostimulation technique.
In the present study, we comprehensively investigated FVIO-facilitated
magnetothermal neurostimulation in vitro and in vivo, and compared the
results with those of widely used SPIO-based approaches. The
newly designed FVIOs with an anti-His antibody coating triggered
Ca2+ influx in TRPV1-expressing HEK293T cells
and cortical neurons with a minimum Fe concentration of 54 μg/mL, which
was 20.27-fold lower than that of the SPIOs under the same AMF
treatment. In vivo magnetothermal stimulation in the central nucleus of
the amygdala (CeA) further demonstrated that FVIO treatment quickly
evoked fear behaviors in mice, exhibiting a response time that was
2.3-fold faster than that in the SPIO-treated group. Overall, the lowest
effective dose of FVIOs was 0.05 μg in vivo, 16.7 fold less than the
effective dose of SPIOs. More importantly, the SPIO ( 0.8
μg)-treated mice exhibited significant histopathological changes
(such as vacuolar degeneration) and upregulated proinflammatory cytokine
(such as interleukin-6 (IL-6)) expression in the CeA. In contrast, the
FVIO ( 0.05 μg)-treated mice showed negligible histopathological
alterations and changes in proinflammatory cytokine expression. In
addition, in the FVIO-treated groups, fear behaviors were successfully
induced in the mice on the 60th day after injection.
However, in the SPIO-treated group, fear behaviors could not be induced
on the 30th day. Notably, only 0.28 μg of
FVIOs activated the CeA endogenously expressing TRPV1 and elicited fear
behaviors in transgene-free mice. The aim of this work was to establish
an efficient and safe FVIO-facilitated magnetothermal neuromodulation
technique, which is expected to be a powerful tool for future
neuroscience investigations and therapeutic applications to neurological
disorders.