Transgene-free remote magnetothermal regulation of adrenal hormones

Abstract

The field of bioelectronic medicines seeks to modulate electrical signaling within peripheral organs, providing temporally precise control of physiological functions. This is usually accomplished with implantable devices, which are often unsuitable for interfacing with soft and highly vascularized organs. Here, we demonstrate an alternative strategy for modulating peripheral organ function, which relies on the endogenous expression of a heat-sensitive cation channel, transient receptor potential vanilloid family member 1 (TRPV1), and heat dissipation by magnetic nanoparticles (MNPs) in remotely applied alternating magnetic fields. We use this approach to wirelessly control adrenal hormone secretion in genetically intact rats. TRPV1-dependent calcium influx into the cells of adrenal cortex and medulla is sufficient to drive rapid release of corticosterone and (nor)epinephrine. As altered levels of these hormones have been correlated with mental conditions such as posttraumatic stress disorder and major depression, our approach may facilitate the investigation of physiological and psychological impacts of stress.

Fig. 1. TRPV1 expression in the adrenal gland.

(A) Adrenal gland anatomy: The chromaffin cells of the medulla secrete epinephrine and norepinephrine. The ZF cells of the adrenal cortex secrete corticosterone. Both processes are Ca²⁺ dependent. (B) An illustration of Ca²⁺ influx through TRPV1 channels in response to heat or CAP. (C) TRPV1 expression in the rat adrenal gland visualized via immunofluorescence across the different zones. Scale bar, 500 μm. The zona reticularis (ZR) in rats functions similarly to the ZF. The red and yellow squares indicate locations of immunoanalysis of TRPV1 expression in the medulla (E and F) and ZF (G and H), respectively. (D) Quantification of TRPV1 and SYN expression and in the adrenal medulla and cortex [n = 4 to 6, means ± standard deviation (std.)] (E and F) Immunofluorescence images of TRPV1 (E) (red) and SYN (F) (red) in the medulla. (G and H) Immunofluorescence images of TRPV1 (green) in ZF. (H) Higher magnification of (G). (E to H) The nuclear stain DAPI is shown in blue. Scale bars, 50 μm.

Fig. 2. MNP and WNP synthesis and characterization.

(A) Organometallic synthesis of MNPs and WNPs in 1-octadecene (ODE) and dibenzyl ether (DBE) mixture or ODE alone, respectively. The NPs are rendered hydrophilic via surface intercalation of PMAO-PEG into the oleic acid coating. (B) Transmission electron microscope (TEM) images of MNPs (top) and WNPs (bottom). The bright surface layer corresponds to the PMAO-PEG coating (~2-nm thickness as shown in the inset. Inset scale bar, 10 nm). Scale bars, 50 nm. (C) Room temperature magnetization curves and the coercive field measured for MNPs and WNPs. (D) Field-cooled SQUID magnetization curve measured at 4 K for MNPs and WNPs. (E) Powder x-ray diffraction spectra of MNPs and WNPs with wüstite (blue) and magnetite (orange) references. a.u., arbitrary units. (F) Temperature change in MNP and WNP solutions (4 mg/ml) in response to AMF (H₀ = 15 kA/m and f = 515 kHz). Blue rectangle denotes AMF exposure. Inset shows measured SLP values for MNPs and WNPs under these AMF conditions.

Fig. 3. Magnetothermal stimulation of adrenal cells in vitro.

(A) Magnetothermal triggering of TRPV1 recorded as a Ca²⁺-dependent increase in Fluo-4 fluorescence. (B) A bright-field micrograph of a primary mixed adrenal cell culture. Scale bar, 50 μm. (C and D) Expression of TRPV1 (C) (green) and CgA (D) (red) in adrenal cell culture. The nuclear stain DAPI is shown in blue. Scale bars, 100 μm. (E to M) Ca²⁺ influx in a mixed adrenal cell culture in response to 10 μM CAP (E to G), AMF in the presence of MNPs (H to J), or AMF in the presence of WNPs (K to M). (E, H, and K) Fluo-4 fluorescence images of the mixed adrenal culture before and after CAP (E) or AMF (H and K). Scale bars, 20 μm. (F, I, and L) Normalized Fluo-4 fluorescence intensity for 140 cells versus time. AMF conditions: H₀ = 15 kA/m and f = 515 kHz; duration, 40 s. (G, J, and M) Average fluorescence change ΔF normalized to the average fluorescence F₀ during first 20 s (ΔF/F₀) of recording for cells exposed to CAP with and without CZP (G), cells responding to AMF stimulation in the presence of MNPs without and with CZP (J), and cells responding to AMF in the presence of WNPs (M). Solid line, mean; shaded area, SEM. Blue rectangle denotes AMF exposure.

Fig. 4. MNP delivery into adrenal gland.

(A) FEM of heat distribution within an adrenal gland injected with MNPs (40 mg/ml) in one (1 μl), two (0.8 μl each), or three (0.5 μl each) locations and exposed to AMF for 40 s. The adrenal gland (white dashed line) is surrounded by a layer of adipose tissue. Scale bar, 1 mm. (B) Surgical procedure for direct injection of MNPs into the rat adrenal glands 1 week before AMF stimulation. (C) Example of tracked positions inside the open field for a rat that underwent an adrenal injection surgery and a control naïve rat. Scale bar, 5 cm. (D and E) Total distance (means ± std.) traveled by the rats (D) and the average velocity (means ± std.) in the open field during the 10-min test. Control rats (n = 3, blue) and postsurgery rats (red, n = 4). No significant difference was observed between the two groups (two-sided Student’s t test). n.s., not significant. (F) H&E staining of rat adrenal glands injected with MNPs and WNPs and extracted 1 to 2 months following surgery. Black arrows indicate MNP and WNP locations. Scale bars, 500 μm.

Fig. 5. Magnetothermal stimulation of the adrenal gland in vivo.

(A) Distribution of NP locations and coverage areas in rats injected in one to three locations (n = 12). Scale bar, 1 mm. (B) Percent of injection sites out of total examined location sites over 12 examined adrenal gland slices with one to three injection sites in each. (C) A photograph of an anesthetized rat in the whole-body AMF stimulation apparatus. (D) A Finite Element method magnetic (FEMM) stimulation of anticipated AMF amplitude within the coil under the chosen driving parameters. A white oval indicates an approximate location of the adrenal glands during the stimulation experiments. The AMF amplitude values are consistent with those necessary to achieve high SLPs. (E and F) Confocal images of the c-fos immunofluorescence in the adrenal gland following AMF stimulation. (E) A mosaic of an entire slice of an adrenal gland injected with MNPs in two locations (white squares). Scale bar, 300 μm. (F) Confocal images in the presence of MNPs (top) or WNPs (bottom). Scale bars, 100 μm. (G) Percentage of the c-fos–expressing cells in the region of interest (ROI) following AMF stimulation in rats injected with MNPs or WNPs. Error bars represent means ± SEM (number of injection sites per group: MNPs, n = 7 and WNPs, n = 5. Two-sided Student’s t test with threshold **P < 0.01, P = 0.006). Photo credit: Dekel Rosenfeld, Massachusetts Institute of Technology.

Fig. 6. Magnetothermal regulation of adrenal hormones in vivo.

(A) Timeline for injection of MNPs into the adrenal glands and blood sampling through a jugular vein catheter. Heart rate recordings are performed on a separate day. (B and C) Normalized relative change in epinephrine (B) and corticosterone (C) concentrations averaged across the three time points following AMF stimulation as compared to the baseline level for each rat before AMF [*P < 0.05, one-way analysis of variance (ANOVA) followed by two-sided Student’s t test with threshold; epinephrine, P = 0.029; corticosterone, P = 0.038]. (D and E) Normalized relative change in epinephrine and corticosterone across four time points. Solid lines and shaded areas represent means and SEM, respectively [(B) to (E): n = 6 in WNP/MNP with AMF and n = 4 WNP/MNP without AMF]. (F) Ratio of the poststimulation (post) to prestimulation (pre) heart rates (*P < 0.05, two-sided Student’s t test with threshold, means ± SEM, n = 3, P = 0.018). (G) Average of relative heart rate slope [beats per minute per second (bpm/s)] from pre-stimulation baseline (pre stim) to post stimulation (post stim) values (*P < 0.05, one-sided Student’s t test with threshold, means ± SEM, n = 3, P = 0.033). (H) Mean change in heart rate measured before, during, and after exposure to AMF. (I) Heart rate slope (bpm/s) as measured before, during, and after exposure to AMF. (H and I) Solid lines and shaded areas represent means ± SEM, n = 3 per group. Blue rectangles denote AMF exposure.