Discharge assessment assay of medium
A hundred µL of HeLa cell suspension at 6.0 × 104 − 1.0 × 105 cell/mL were prepared in wells of a 96-well plate. The cell suspension was incubated at 37 oC with 5% CO2 using a culture medium composed of a regular medium which consists of minimum essential medium with 10% fetal bovine serum and 2% penicillin-Streptomycin mixed solution. After 24 h incubation, the cells were adhered to the bottom surface and used for potential stimulation as shown in Fig. 2. We prepared cells in two conditions; one experienced exposure to potential stimulation and the other was placed in the grounded medium (control). The distance between a needle tip and a medium's surface was 1.5 mm. A pulsed discharge that propagates between the needle and the medium forms the potential in the liquid 24,25. The potential fluctuation of the medium at the direct discharges was transferred through a SUS 316 cylindrical wire to the medium including HeLa cells. To generate plasma on the medium of direct plasma a pulsed voltage with an amplitude of ± 7.5 kV, a rise time of 8 µs, width of 9 µs and 5 kpps of pulse repetition rate was applied for 30 or 60 min to the needle electrode. Figure 3 shows typical waveforms of the applied voltage to the needle electrode and current flowing through the needle and the ground. As shown in Fig. 3, current spikes, corresponding to discharge occurrence, appear with voltage increase and drop. The first discharge occurs at 1.3 kV. The pulse width of the discharge current spikes are 88 ns, indicating that once a discharge propagates to the other side then it stops. These results show that discharge mode did not shift to arcing from streamer. The typical streamer channel is < 30 Td with the electron temperature Te < 2.7 eV 26. Such electrons mainly vibrationally excite N2 and O2 in the air and generate NO in the atmosphere 27. Therefore, the media with cell were closed to prevent contamination of chemical species. If a discharge is generated in the medium with cell, the effect of potential stimulation on the cells cannot be extracted. When an electric discharge occurs, H2O2, relatively long-lived RONS, is generated in the medium by the chemical reaction through R1 and R2 28.
H2O + P → *OH + *H + P (R1)
*OH + *OH + M → H2O2 + M (R2)
where P is an energetic particle from the plasma (e.g., electron) and M is a collision partner (e.g., N2 or O2). Figure 4 shows H2O2 concentration in the medium below the needle electrode (direct plasma), that of potential stimulation, and that of control after 60 min-plasma generation. H2O2 concentration shows 0.67 mg/L for direct plasma. In contrast, H2O2 was not detected in the medium with and without potential stimulation.
Cell concentration assay
The effect of potential stimulation on cells was evaluated by changes in the cell activity. Figure 5 shows changes in the concentration of living cells in the medium with and without potential stimulation for 30 min. The cell concentrations were measured immediately after treatment (0 h) and after 24 hours of incubation since the treatment (24 h). Figure 5 (a) was obtained after replacing the medium with phosphate-buffered saline (PBS) followed by cell concentration assay. The concentration of treated cells decreases in both 0 h and 24 h. We evaluated %decrease due to potential stimulation by Eq. 1.
$$\%Decrease=\frac{{n}_{\text{s}} }{{n}_{\text{c}}}\times 100 \left[\%\right]$$
1
where ns is the concentration of living cell with potential stimulation (/mL) and nc is that without potential stimulation (/mL). In Fig. 5., %decrease is 85% at 0 h (p = 0.0055) and 86% at 24 h (p = 0.00099), respectively. HeLa cells adheres on the bottom using proteins such as extracellular matrix and adhesion molecules 29. Local electric fields created by potential fluctuations might affect their protein function by changing its conformation 30,31. Since the cell concentration assay was performed after replacing the medium with PBS, the cells detached by potential stimulation might be discarded leading cell concentration decrease. Figure 5 (b) shows the concentration of cells counted after stimulation without replacing the medium with PBS. Nevertheless, %decrease was 84% at 0 h (p = 0.012) and 84% at 24 h (p = 0.0033). This result is in good agreement with the result in Fig. 5 (a). Consequently, potential stimulation affects the cells but not just the inactivation of the adhesive protein.
To discuss the subsequent effects, a proliferation ratio Rp was obtained by Eq. 2.
$${R}_{\text{p}}=\frac{{n}_{24\text{h}} }{{n}_{0\text{h}}}$$
2
where n24h is the concentration of cells incubated for 24 h after potential stimulation and n0h is that without incubation after potential stimulation. Rp is 2.11 and 2.14 for control and stimulation in Fig. 5 (a), and 2.61 and 2.55 in Fig. 5 (b), respectively. Considering that the initial cell concentration was 6.0 × 104 cell/mL in Fig. 5 (a) and 1.0 × 105 cell/mL in Fig. 5 (b), it is natural that Fig. 5 (a) shows higher cell proliferation ratios. However, it should be noted that the ratio is maintained at the same level as the control group, even though potential stimulation reduces the concentration of living cells. The cells may recover from temporal damage due to the stimulation. Considering that the doubling time of HeLa cells is within 24 h 32,33, the cells may recover in a few hours after the stimulation. Alternatively, potential stimulation may shorten the cell cycle. The mechanism underlying how the cell proliferation rate is compensated (Fig. 5) should be clarified in the future. In this study, further experiments were conducted on the effects of potential fluctuations during plasma generation on cell membranes.
When a needle-liquid electric discharge occurs, an electric potential is formed in the liquid phase and requires more than several ten seconds to relax without ground 24. Once potential is formed in medium including cell, the capacitors such as cell membrane and nuclei are charged depending on the voltage duration 34–36. The voltage corresponds to the potential induced in a liquid medium by a pulsed discharge. A time difference between the first discharge and the subsequent discharge with a reverse polarity is 200 µs at maximum as shown in Fig. 3. Assuming that the relaxation time of the cell membrane potential in the HeLa cell is about the same millisecond as that of the nerve cell 29, the maximum potential is contentiously applied to inside and outside the cell for 200 µs. Considering that such a potential difference leads the transient mechanical compressive force due to Maxwell stress, perforation may occur on the cell membrane. To evaluate this, the influence of potential stimulation on the cell membrane was studied. We added membrane-impermeable fluorescent dye to the medium before and after the stimulation, and microscopically observed the fluorescence-stained cells. This approach allows us to estimate whether the perforations formed in the membrane are temporary or stationary.
Influence of potential stimulation on the cell membrane
Fluorescence microscopy observations revealed that potential stimulation causes temporal pores on the membrane through which fluorescent reagent transports into the living cells. Figure 6 shows the result of the microscopic fluorescent observation for the cells with and without stimulation. To elucidate the biological effect of plasma-induced potential stimulation, fluorescent regents MitoRed and SYTOX-Green in DMSO were added at (a) 0 h and (b) 24 h after potential stimulation for 30 min and (c) before the stimulation. In Fig. 6 (a), cells were stained with MitoRed but not stained with SYTOX-Green. To quantitively evaluate the effect, the ratio of the number of cells simultaneously stained by both MitoRed and SYTOX-Green divided by the number of cells stained by MitoRed, GR ratio, were calculated. GR ratio enables us to assess the number of surviving cells with a non-lethal damage on the membrane. GR ratio was 0.097 in control group and 0.28 in stimulation group (Fig. 6a; Table 1). After 24 h (Fig. 6b), GR ratio was 0.12 in control group and 0.043 in stimulation group (Table 1). The number of MitoRed-stained cells with and without stimulation increased at almost the same, owing to proliferation. These results are consistent with Fig. 5. Conversely, many cells were stained by SYTOX-Green when added at before stimulation, even they were also stained by MitoRed (Fig. 6c). GR ratio was 0.16 in control group but 0.96 in stimulation group, which is significant high (Table 1). This result indicates that potential stimulation enables transport of SYTOX-Green into cells. It also should be noted that the cells with and without stimulation in Figs. 6 (a) and (b) were not stained by SYTOX-Green when the reagent added at 0 h and 24 h later since stimulation. These results suggest that potential stimulation make temporal pores on the membrane of HeLa cells. Further experiment was conducted to evaluate the molecular transport out of the cell. LDH level of each medium after cell exclusion with and without potential stimulation for 30 min was measured. LDH elutes to the medium due to poration 37. The control showed high LDH levels at 1.115 ± 0.008 as shown in Fig. 7. This is because phenol red and LDH that is originally contained in fetal bovine serum of the medium gives a positive bias to absorbance for colorimetry 38. Nevertheless, potential stimulation shows slight but significant increase as 1.174 ± 0.045 than control (p = 0.021, n = 15). Above results show that potential stimulation induces molecular transport into and out of HeLa cell by temporal pores formation. This might be involved in the cell inactivation as shown in (Fig. 5).
Possible mechanism underlying temporal poration by potential stimulation
Electric field across the membrane by the first discharge (Fig. 3) was evaluated using LTspice XVII. When a streamer discharge occurs, the potential of the needle electrode is conducted to the culture medium. This allow us to propose an equivalent circuit composed of an upper membrane Cm1, a cytoplasm Rc, a lower membrane Cm2 of an adhered HeLa cell, bottom of polystyrene 96-well Cb, blank space of 96-well plate (skirt) Cs, and 200 mm space Ca as shown in Fig. 8 (a), where C is the capacitance and R is the resistance. These values are calculated by Eqs. (3–4) as follows.
$$C=\frac{{\epsilon }_{0}{\epsilon }_{\text{r}}}{d}$$
3
$$\rho =\frac{1}{\sigma }$$
5
where \({\epsilon }_{0}\) is electric constant as 8.854 × 10− 12 F/m, \({\epsilon }_{\text{r}}\) is relative permittivity, d is distance of the capacitor (m), ρ is resistivity (Ωm), derived by (3), l is the length (m), S is the cross-section area of the resister (m2), and σ is the conductivity (mS/cm). Cm1 and Cm2 are both 9.929 × 10− 15 F, Rc is 2.5 × 106 Ω, Cb is 1.705 × 10− 20 F, Cs is 6.542 × 10− 21 F, Ca is 5.560 × 10− 23 F based on the physical characteristics as follows. For HeLa cell, \({\epsilon }_{\text{r}}\) of cell membrane is 6.25, derived by averaging 5.7 for erythrocyte and 6.8 for lymphocyte, d is 7 nm, σ of cytoplasm is 3.2 mS/cm, and vertical l of cytoplasm of an adhered HeLa cell on the well bottom is 1 µm 39–41. Since the horizontal radius of the adhered HeLa cell is 40 µm 42, an equivalent circuit was constructed in the smallest unit by assuming that the other elements except voltage source were regarded as the same radius as well as the adhered HeLa cell. Voltage source was set as a pulse mode with 1.3 kV for 200 µs based on Fig. 3. For the 96-well plate, \({\epsilon }_{\text{r}}\) of polystyrene is 2.3 43, d of bottom is 1.5 mm according to the product manufacturer. \({\epsilon }_{\text{r}}\) of air is 1.0. Figure 8 (b) shows the simulation result. An applied voltage shows the potential of the medium with cell. Vm1 and Vm2 show the voltage across the upper and the lower membrane, respectively. The voltage applied to the membrane is divided depending on each capacitance. As shown in Fig. 8 (b) as the current charges the capacitors, Vm1 and Vm2 gradually increase and reach 53 V and 27 V at 200 µs. The electric field was calculated as 7.6 × 103 kV/mm for the upper membrane and 3.9 × 103 kV/mm for the lower membrane. It is obvious that the electric field intensity decreases with a comprehensive equivalent circuit including all the intracellular components. It should be noted that the simulation result also shows that a larger electric field is applied to the upper cell membrane, comparing to the lower cell membrane, i.e., perforation may occur in the upper cell membrane. Deng et al. applied 6.0 kV of pulsed voltage between parallel plates inserted into a cell suspension at a distance of 1 cm and observed cell morphology after 15 min 34. The cell morphology did not change as pulse width at 10 µs. In contrast, the cell membrane partially collapsed as that at 100 µs 34. For this study, the electric field is maintained up to 200 µs. It was experimentally and theoretically shown that the potential induced in the liquid phase during plasma irradiation has a reversible perforation effect on the HeLa cell membrane. Plasma irradiation could be used to efficiently transport the generated RONS and other target molecules into cells.