Magnetothermal Multiplexing for Selective Remote Control of Cell Signaling

Abstract

Magnetic nanoparticles have garnered sustained research interest for their promise in biomedical applications including diagnostic imaging, triggered drug release, cancer hyperthermia, and neural stimulation. Many of these applications make use of heat dissipation by ferrite nanoparticles under alternating magnetic fields, with these fields acting as an externally administered stimulus that is either present or absent, toggling heat dissipation on and off. Here, we motivate and demonstrate an extension of this concept, magnetothermal multiplexing, in which exposure to alternating magnetic fields of differing amplitude and frequency can result in selective and independent heating of magnetic nanoparticle ensembles. The differing magnetic coercivity of these particles, empirically characterized by a custom high amplitude alternating current magnetometer, informs the systematic selection of a multiplexed material system. This work culminates in a demonstration of magnetothermal multiplexing for selective remote control of cellular signaling in vitro.

Keywords: AC magnetometer; cellular signaling control; magnetic nanoparticle; multiplexed magnetothermal control; selective nanoparticle heating.

Figure 1.

Design of multiplexed control of cell signaling using selective magnetothermal stimulation. (a) and (b) Doping of cobalt into Fe₃O₄ results in higher magnetic anisotropy, making cobalt ferrite MNPs magnetically harder compared to Fe₃O₄. (c) and (d) Schematic representation of multiplexed magnetothermal control of cell signaling using two different MNP ensembles that respond selectively to paired AMF conditions. (c) A high-amplitude, low-frequency AMF is sufficient to access major hysteresis loops for both MNPs, with the major hysteresis loops of Co_xFe_3-xO₄ MNPs inherently larger, causing them to heat preferentially. (d) An AMF with low amplitude and high frequency results in major hysteresis loops only for less coercive MNPs (Fe₃O₄), and minor loops for Co_xFe_3-xO_4. The MNPs only dissipate heat effectively when they are exposed to their respectively paired AMF conditions, triggering the opening of the heat sensitive TRPV1 ion channels exogenously expressed in the HEK cells. Calcium ions flow into the cells through the activated TRPV1 channels and bind to GCaMP6s indicators, producing an increase in green fluorescence.

Figure 2.

Characterization of magnetic nanoparticles. (a-h), TEM of Fe₃O₄ (a-d) and Co_xFe_3-xO₄ (e-h) magnetic nanoparticles and their size distributions from TEM (inset histograms). (i) Powder XRD of Fe₃O₄ and Co_xFe_3-xO₄ nanoparticles indicates single-crystal structure (inverse spinel). (j) Magnetization data were collected with a SQUID magnetometer to confirm the difference between hard and soft MNPs. To exclude physical rotation of suspended magnetic particles in water, the measurement was performed at 260K. (k) Incorporated cobalt concentration in each ensemble was analyzed by ICP-AES. Each line shows relative cobalt intensity to iron intensity at 234.830nm.

Figure 3.

Measurement of dynamic magnetization of MNP ensembles using custom high amplitude AC-magnetometer. (a) Design, photograph, and circuit diagram of the custom AC-magnetometer (ACM) used to capture dynamic magnetization under AMF. (Scale bar = 1 cm). (b) 2D coil design to detect dynamic magnetization. The spiral design of the sense coil results in a voltage induced by the changing magnetization of the sample. The compensation coil wound in the opposite direction cancels the majority of the voltage induced by the driving AMF. Ferrofluid is loaded into the 3D printed hollow spherical chamber of the sample holder. (Scale bar 1 = mm) (Blue – raw sample signal, Green – water control sample signal, Red – net sample signal). (Scale bars: voltage = 4V, time = 50 μs). (c, d) Dynamic hysteresis loops for ferrofluids of 16.2 nm Fe₃O₄ (c) and 16.3 nm Co_0.24Fe_2.76O₄ (d) were collected at room temperature under AMFs with ƒ = 75 kHz and amplitudes ranging 0–120 mT. (Black line represents VSM data, jet color lines correspond to ACM data). (e-h), Specific loss power (SLP) of MNP ensembles measured empirically via ACM (e, g – solid lines, closed circle markers, measured at 75 kHz and linearly scaled by frequency to 101.2 kHz) and calorimetry (e, g - dashed lines, open diamond markers, shadowed areas represent standard deviation, measured at 101.2 kHz), and calculated via dynamic hysteresis model (f, h). (i-l) Coercive fields H_c for MNP ensembles calculated from ACM measurements (i, k) and simulated via the dynamic hysteresis model (j, l).

Figure 4.

Optimization of field conditions and thermographic verification for multiplexed thermal control (a) Multiplexing factor as a function of AMF amplitudes H₁ and H₂, for frequencies f₁=522 kHz and f₂=50 kHz. The grey area represents excluded conditions H₁>H₂; a pair of AMFs in which both the amplitude and frequency of one condition is higher than the other is unsuitable for magnetothermal multiplexing. (b) Temperature profiles and (c) Thermographic images of two 10 μl MNP solution droplets (Fe₃O₄ 16.3 nm - red and Co_0.24Fe_2.76O₄ 18.6nm - blue) exposed sequentially to two distinct AMFs for 20 s (70 kA/m, 50 kHz and 10 kA/m, 522 kHz), at each time frame. Dashed lines in temperature profiles (b) represent the times of the thermographic frames in (c). (Scale bar = 5 mm)

Figure 5.

Cellular responses selectively triggered by multiplexed magnetothermal control. (a-c) Confocal images of HEK293T cells co-transfected with both TRPV1-p2A-mCherry (a, c) cytoplasmic mCherry expression) and GCaMP6s (b, c). (Scale bar = 25 μm). (d-g) Cellular responses to multiplexed magnetothermal heating. Temperature profiles (upper plots) of the ferrofluids and the normalized GCaMP6s fluorescence ΔF/F₀ (lower heat maps) of 100 randomly automatically chosen HEK239T cells plotted within the same time frame (0–70 s). Side panels indicate normalized GCaMP6s fluorescence images of the cells at different time-points during the experiments. (Scale bars = 40 μm). For each ferrofluid, the non-pairing AMF was applied at first. Afterward the same field of view (FOV) was imaged when cells were exposed to the pairing AMF. Each ΔF/F₀ profile is from same FOV for each cover slip. AMFs were applied for 20 s (color boxes in temperature profiles and white lines in heat maps: from 20 s to 40 s). (d) Fe₃O₄ at 10 kA/m 522 kHz AMF. (e) Fe₃O₄ with 70 kA/m 50 kHz AMF. (f) Co_0.24Fe_2.76O₄ with 10 kA/m 522 kHz AMF. (g) Co_0.24Fe_2.76O₄ with 70 kA/m 50 kHz AMF.