Long-term magnetothermal neurostimulation in vivo
To evaluate the potential of long-term and repeated neuromodulation using FVIOs, fear behavior testing in mice was conducted over 60 days. As shown in Fig. 7a, the FVIOs retained the ability to magnetothermally elicit freezing behaviors in mice on the 60th day. However, no significant changes in percentage of freezing time were observed among the SPIO (0.8 μg)-treated mice, which may be due to SPIO biodegradation in the CeA. To confirm this, 7-Tesla magnetic resonance imaging (MRI) was employed to evaluate the changes in the concentrations of the magnetic nanoheaters in the CeA regions on the 1st, 30th, and 60th days using ultrashort echo time (UTE) pulse sequences. Linear correlations of the transverse relaxation rates (1/T2 values) of the FVIOs and SPIOs were first obtained (Figure S11, Supporting Information). Figure 7b presents MR images of mouse brains with which the concentrations of FVIOs and SPIOs that remained in the CeA regions could be determined (marked with yellow arrows) on the 1st, 30th, and 60th days. The relative signal intensity of the FVIOs measured in the CeA remained stable for 60 days (Figure 7c), suggesting that the amount of FVIOs remained unchanged at the injection site. However, the SPIO signal in the CeA notably decreased on the 60th day, indicating their disappearance. The disappearance of the SPIOs is mainly ascribed to their smaller size in comparison with the FVIOs, which makes their biodegradation and diffusion easier. In contrast, the FVIOs were highly stable under physiological conditions, giving them great potential for long-term and repeated neural stimulation and therapy[55].