Fig 1.
Simulation of electric field distribution and temperature measurements in culture media.
(a) Schematic of the experimental setup showing a cuvette partially filled with a 500 µL media and two aluminum plate electrodes separated by a 4-mm gap. (b) Numerical simulations of electric field distributions at applied voltages of 400 V (1), 1,600 V (2), and 2,000 V (3). Color bar indicates electric field intensity distribution in kV/cm. (c) Temperature measurement during IRE procedures. (1) Experimental setup showing the position of a cc-type thermocouple within the media. (2) Measured temperature profiles under different IRE conditions.
Table 1.
Summary of electric pulse parameters and energy indices used in high- and low-electric-field conditions at 1 Hz.
Table 2.
Gene sequence for polymerase chain reaction.
Fig 2.
Cell Viability and apoptosis under various IREs in A549 cells.
(1) Cell viability after various electric field intensity treatment with pulse counts of 10, 20, and 40 pulses fixed at 20μs. (2) Cell viability at a fixed electric field of 4000 V/cm and 20 pulses, with pulse durations of 10, 20, and 40 μs. (3) Comparison of cell viability between 1000 V/cm and 4000 V/cm. (4) Apoptosis across increasing electric field strengths at a fixed pulse duration of 10 μs and pulse number of 20. (5) Apoptosis at electric field strength of 4,000 V/cm and 20 pulses with varying pulse durations of 10, 20, and 40 μs. (6) Comparison of apoptosis between 1000 V/cm and 4000 V/cm. Data are represented as mean ± SEM (n = 3). Multiple-group comparisons were analyzed using one-way ANOVA followed by Tukey’s HSD post hoc test, with significance indicated as: ###p < 0.001. Two-group tests were analyzed using unpaired two-tailed t-tests, with significance indicated as: *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 3.
The electrophysiological responses of A549 cells.
(1) Change in electrical resistance (Ω) in response to different electric field strengths. (2) Cytomembrane potential (CMP). (3) Cytosolic calcium concentration. Data is represented as mean ± SEM (n = 3). Multiple-group comparisons were analyzed using one-way ANOVA followed by Tukey’s HSD post hoc test, with statistical significance indicated as: ###p < 0.001; N.S, not significant. Two-group tests were analyzed using unpaired two-tailed t-tests, with significance indicated as: *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 4.
Impact of variable IRE parameters on mitochondrial dysfunction and oxidative stress.
(1) Mitochondrial membrane potential (MMP). (2) H2O2 production. (3) NO production. Data is represented as mean ± SEM (n = 3). Multiple-group comparisons were analyzed using one-way ANOVA followed by Tukey’s HSD post hoc test, with statistical significance indicated as: ###p < 0.001; N.S, not significant. Two-group tests were analyzed using unpaired two-tailed t-tests, with significance indicated as: *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 5.
Evaluation of total DNA damage in A549 cells.
Total DNA damage was quantified by the combined percentage of cells positive for phosphorylated ATM, and H2A.X. Data represent mean ± SEM (n = 3). Multiple-group comparisons were analyzed using one-way ANOVA followed by Tukey’s HSD post hoc test, with statistical significance indicated as: ###p < 0.001. Two-group tests were analyzed using unpaired two-tailed t-tests with significance indicated as: **p < 0.01, ***p < 0.001.
Fig 6.
Differential gene expression analysis in A549 cells.
Real-time PCR showing the relative mRNA expression levels normalized to the reference gene ClAO1, involved in DNA damage sensing (ATM (1) and ATR (2)), stress response coordination (CHK1 (3), CHK2 (4)), DNA repair (PARP1 (5), and BRCA1 (6), and apoptosis (p53 (7) and BAX (8)). Data are presented as mean ± SEM (n = 3). Multiple-group comparisons were analyzed using one-way ANOVA followed by Tukey’s HSD post hoc test, with statistical significance indicated as: #p < 0.05, ##p < 0.01, ###p < 0.001; N.S, not significant. Two-group tests were analyzed using unpaired two-tailed t-tests, with significance indicated as: *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 7.
in vitro ultrastructural analysis via transmission electron microscopy.
(1 & 2) Control cells showed intact plasma membranes, nuclear membranes, and mitochondria. (3 & 4) HEF caused severe plasma membrane rupture, substantial nuclear membrane inflation, and extensive mitochondrial damage. (5 & 6) LEF induced moderate plasma membrane disruption, mild nuclear membrane stress, and partial mitochondrial damage. Arrows indicate structures: (→) plasma membrane, (→) nuclear membrane, and (>) mitochondria. Scale bars represent magnification as indicated.
Fig 8.
in vivo ultrastructural analysis via transmission electron microscopy.
(1 & 2) Control tissues showed intact plasma membranes, nuclear membranes, and mitochondria. (3 & 4) HEF caused severe plasma membrane rupture, pronounced nuclear membrane inflation, and mitochondrial damage. (5 & 6) LEF caused substantial plasma membrane rupture and mitochondrial damage. Arrows indicate structures: (→) plasma membrane, (→) nuclear membrane, and (>) mitochondria. Scale bars represent magnification as indicated.