The effects of magnetite (Fe₃O₄) nanoparticles on electroporation-induced inward currents in pituitary tumor (GH₃) cells and in RAW 264.7 macrophages

Abstract

Aims: Fe₃O₄ nanoparticles (NPs) have been known to provide a distinct image contrast effect for magnetic resonance imaging owing to their super paramagnetic properties on local magnetic fields. However, the possible effects of these NPs on membrane ion currents that concurrently induce local magnetic field perturbation remain unclear.

Methods: We evaluated whether amine surface-modified Fe₃O₄ NPs have any effect on ion currents in pituitary tumor (GH₃) cells via voltage clamp methods.

Results: The addition of Fe₃O₄ NPs decreases the amplitude of membrane electroporation-induced currents (I(MEP)) with a half-maximal inhibitory concentration at 45 μg/mL. Fe₃O₄ NPs at a concentration of 3 mg/mL produced a biphasic response in the amplitude of I(MEP), ie, an initial decrease followed by a sustained increase. A similar effect was also noted in RAW 264.7 macrophages.

Conclusion: The modulation of magnetic electroporation-induced currents by Fe₃O₄ NPs constitutes an important approach for cell tracking under various imaging modalities or facilitated drug delivery.

Keywords: free radical; ion current; iron oxide.

Figure 1

The effect of Fe₃O₄ NPs on I_MEP in GH₃ pituitary tumor cells. In these experiments, cells were bathed in Ca²⁺-free Tyrode’s solution containing 10 mM CsCl. The cell was held at −80 mV and hyperpolarizing pulses to −200 mV with a duration of 300 msec at a rate of 0.1 Hz were applied. (A) Superimposed current traces obtained in (a) the absence of Fe₃O₄ NPs and (b) the presence of 30 μg/mL Fe₃O₄ NPs and (c) 100 μg/mL Fe₃O₄ NPs. The upper part indicates the voltage protocol used. (B) The concentration–response curve for Fe₃O₄ NP-induced inhibition of I_MEP in these cells (mean ± standard error of the mean; n = 5–12 for each point). Current amplitudes obtained at the different concentrations (10 μg/mL–1 mg/mL) of Fe₃O₄ NPs were measured at the end of hyperpolarizing pulses (ie, −200 mV). The smooth blue line represents the best fit to the Hill equation as described under Materials and methods. Note: The IC₅₀ value, maximally inhibited percentage of I_MEP, and Hill coefficient for NP-induced inhibition of I_MEP were calculated to be 45 μg/mL, 23%, and 1.1, respectively. Abbreviation: Fe₃O₄ NPs, magnetite nanoparticles.

Figure 2

Effects of Fe₃O₄ NPs on I–V relation of I_MEP in GH₃ cells. In these experiments, cells were bathed in Ca²⁺-free Tyrode’s solution containing 10 mM CsCl, and I_MEP was elicited from −50 mV to different potentials ranging from −200 to 0 mV with 20 mV increments. The data (mean ± standard error of the mean; n = 8–11) were obtained in the absence (■) and presence (□) of Fe₃O₄ NPs (100 μg/mL). Notably, addition of the NPs reduced the slope of I_MEP at the voltage ranging between −100 and −200 mV, although no change in the threshold potential of this current was observed. Note: ^*Significantly different from controls measured at each voltage. Abbreviation: Fe₃O₄ NPs, magnetite nanoparticles.

Figure 3

The dual effect of Fe₃O₄ NPs on I_MEP in GH₃ cells. In these experiments, cells were bathed in Ca²⁺-free Tyrode’s solution containing 10 mM CsCl. The cell was held at −80 mV and hyperpolarizing steps to −200 mV with a duration of 300 msec at a rate of 0.1 Hz were then delivered. (A) Superimposed current traces obtained (a) in the absence of Fe₃O₄ NPs, (b) 1 minute, and (c) 3 minutes after the addition of NPs (3 mg/mL). (B) Bar graph showing summary of the effect of 3 mg/mL Fe₃O₄ NPs on I_MEP in GH₃ cells (mean ± standard error of the mean; n = 6 for each bar). Bar (a) is the control, and bars (b) and (c) were obtained 1 and 3 minutes after the addition of Fe₃O₄ NPs (3 mg/mL), respectively. Notes: ^*Significantly different from control. ^**Significantly different from those obtained 1 minute after the addition of NPs. Abbreviation: Fe₃O₄ NPs, magnetite nanoparticles.

Figure 4

Activity of membrane electroporation-induced channels in the absence (A) and presence (B) of Fe₃O₄ NPs in GH₃ cells. In these experiments, cells were bathed in Ca²⁺-free Tyrode’s solution containing 10 mM CsCl and 1 mM LaCl₃. Single-channel events were elicited by long-lasting ramp pulse ranging between −200 and +100 mV with a duration of 1.5 seconds at a rate of 0.05 Hz. Downward deflections indicate the opening events of the channel. The inset in (A) indicates the voltage protocol examined. The right-hand graph in (A) and (B) represents an amplified current trace corresponding to that appearing in the red dashed box in the left-hand graph. Notes: The straight blue line shown on the right side illustrates a linear I–V relation of membrane electroporation-elicited channels in (A) control and (B) during exposure to Fe₃O₄ NPs (100 μg/mL). Notably, no change in single-channel conductance was demonstrated in the presence of Fe₃O₄ NPs, although it decreased the probability of channel openings. Abbreviation: Fe₃O₄ NPs, magnetite nanoparticle.

Figure 5

Lack of effect of Fe₃O₄ NPs on delayed rectifier K⁺ current (I_K(DR)) in GH₃ cells. Cells, immersed in Ca²⁺-free Tyrode’s solution containing 1 μM tetrodotoxin and 0.5 mM CdCl₂, were held at −50 mV and depolarizing pulses ranging from −50 to +60 mV in 10 mV increments with a duration of 1 second were applied. Superimposed current traces shown in (A) are control, and those in (B) were recorded 2 minutes after the addition of 100 μg/mL Fe₃O₄ NPs. No discernible change in I_K(DR) kinetics and amplitude was demonstrated when cells were exposed to Fe₃O₄ NPs. Note: The uppermost part indicates the voltage protocol used. Abbreviation: Fe₃O₄ NPs, magnetite nanoparticles.

Figure 6

No effect of Fe₃O₄ NPs on I_K(erg) in GH₃ cells. In these experiments, cells were bathed in a high-K⁺, Ca²⁺-free solution. Each cell was held at −10 mV and a 1-second long hyperpolarizing pulse from −10 to −90 mV at a rate of 0.01 Hz was applied. (A) Superimposed I_K(erg) obtained in the absence (a) and presence (b) of 100 μg/mL Fe₃O₄ NPs. The inset indicates the voltage protocol used. (B) Bar graph showing summary of the effects of Fe₃O₄ NPs (100 μg/mL), methadone (10 μM), and single-walled nanotubes (30 μg/mL) on I_K(erg) (mean ± standard error of the mean; n = 5–7 for each bar). The peak amplitude of I_K(erg) in response to membrane hyperpolarization from −10 to −90 mV was measured in each cell. I_MEP amplitudes obtained in different concentrations of NPs were measured at −200 mV. Note: ^*Significantly different from control. Abbreviations: Fe₃O₄ NPs, magnetite nanoparticles; SWNT, single-walled nanotubes.

Figure 7

Effect of Fe₃O₄ NPs on I_MEP recorded from RAW 264.7 macrophages. The experiments were conducted in cells bathed in Ca²⁺-free Tyrode’s solution. (A) Original traces of I_MEP obtained in the absence and presence of Fe₃O₄ NPs. The current trace labeled (a) is control, and those labeled (b) and (c) were obtained in the presence of 30 μg/mL and 100 μg/mL Fe₃O₄ NPs, respectively. The inset indicates the voltage protocol used. (B) Bar graph showing summary of the effects of Fe₃O₄ NPs (30 μg/mL and 100 μg/mL) on I_MEP recorded from RAW 264.7 macrophages (mean ± standard error of the mean; n = 6–10 for each bar). Note: ^*Significantly different from control. Abbreviation: Fe₃O₄ NPs, magnetite nanoparticles.

Figure 8

The effect of Fe₃O₄ NPs on superoxide production in GH₃ cells. The experiments were conducted in cells bathed in Ca²⁺-free Tyrode’s solution. Bar graph showing summary of the effects of Fe₃O₄ NPs (100 μg/mL and 3 mg/mL) with or without GH₃ cells (mean ± standard error of the mean; n = 4–5 for each bar). Note: ^*Significantly different from control. Abbreviation: Fe₃O₄ NPs, magnetite nanoparticles.