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Published in final edited form as: IEEE Trans Plasma Sci IEEE Nucl Plasma Sci Soc. 2021 Sep 30;49(11):3317–3324. doi: 10.1109/tps.2021.3113260

Synergistic effects of an atmospheric pressure plasma jet and pulsed electric field on cells and skin

Chunqi Jiang 1, Edwin A Oshin 2, Siqi Guo 3, Megan Scott 4, Xi Li 5, Cathryn Mangiamele 6, Richard Heller 7
PMCID: PMC8653988  NIHMSID: NIHMS1757605  PMID: 34898731

Abstract

Nonthermal atmospheric pressure plasmas produce reactive plasma species including charged particles and reactive oxygen nitrogen species, which are known to induce oxidative stress in living cells in liquid or tissue. In the meantime, pulsed electric fields have been widely used in reversible or irreversible electropermeabilization for either the delivery of plasmid DNA or inactivation of cancer cells. This work discusses the synergistic effects of nanosecond pulsed plasma jets and pulsed electric field on inactivation of pancreatic cancer cells in vitro and enhancement of plasmid DNA delivery to guinea pig skin in vivo. Higher inactivation rates of the cancer cells in suspension were obtained with combined treatment of 300-ns 50 kV/cm pulsed electric field and a 1-min exposure of a nanosecond pulsed, 250-μm plasma jet. Increased efficiency of gene electrotransfer to skin was also observed after a 3-min treatment of a nanosecond pulsed, 1-mm plasma jet. Application of the plasma alone at the same dosage did not have significant effect on gene delivery. These findings signify the dosage-dependent cell-response to both the electric fields and plasma. Importantly, the use of cold plasma to increase the sensitization of the biological cells in response to pulsed electric fields could be an effective approach to enhance the desired effects in electroporation-based applications.

Index Terms—: Atmospheric-pressure plasma, plasma jet, nanosecond pulsed power, plasma medicine, gene delivery, cancer therapy, synergistic effect

I. Introduction

Nonthermal atmospheric-pressure plasma jets are known to induce oxidative and nitrosative stress in cells and tissues by producing reactive oxygen and nitrogen species (RONS) in liquid or material surfaces[1–3]. Depending on the dosage and application methods, the plasma-induced cellular effects range from proliferation[4–6], apoptosis[4, 7, 8], necrosis[9], or direct cell/microorganism ablation[10, 11]. Such oxidative stress in cell suspensions and in tissues can also be induced by electroporation, which uses pulsed electric fields to produce reversible or irreversible effects on cell membranes and structures[12, 13]. For example, high-intensity electric pulses of nanosecond duration have been employed to penetrate the plasma membrane and modify internal cell structures, resulting in necrotic or apoptotic cell death[14–16]. The method of cell ablation using nanosecond pulse electric field (nsPEF) has shown promise in cancer therapy in studies with murine models[17–19] and human studies[20]. However, local relapse due to incomplete ablation and clinical complications associated with the treatment protocol and the use of needle electrodes presents challenges in electroporation-based therapeutic applications[21–23]. In addition, electroporation has also been considered an effective means of delivering plasmid DNA to multiple tissues including skin and enhancing gene expression compared to injection alone[24–27]. Gene electrotransfer (GET) to the skin typically uses electrode-array applicators applying ms-duration pulsed electric fields of hundreds of volts per centimeter for the treatment[25, 27, 28]. Challenges in efficient GET to skin include targeting expression to deeper tissue layers with minimally invasive techniques as well as balancing the sensitization of the skin to pulses with the effectiveness of GET[24, 25, 27, 28]. Synergistic effect of electric field and nonthermal atmospheric-pressure plasmas was reported against Staphylococcus aureus in suspension[29], inactivating both the rat liver epithelial and tumorigenic cells[30], or permeabilizing phospholipid bilayer (with molecular dynamics simulation)[31]. Although as both electroporation and cold plasma were reported to generate extracellular and intracellular ROS[12, 13, 32], the reinforcement on oxidative stress on cells may not be the only mechanism that is associated with the synergistic effects[30]; electric current, field, charge[30, 33, 34], pressure[35] as well as gas temperature[24, 36–38] could also pay critical roles in cell stimulation or disruption, which could be achieved with different exercises of the two physical approaches.

In this study, synergistic effects of nanosecond pulsed, atmospheric-pressure plasma jets (ns-APPJ) and pulsed electric fields were evaluated for inactivation of pancreatic (Pan02) cancer cells in vitro and plasmid DNA delivery to skin in vivo. Two slightly different helium plasma jets, generated in ambient air, were used for the in vitro and in vivo studies, respectively: a 250-μm single-needle plasma jet for the cancer treatment study and a 1-mm needle-to-ring plasma jet for the gene transfer study. The Pan02 cancer cells in suspension were treated with nsPEF followed by the plasma exposure for different times. A viability assay was used to evaluate the inactivation effects on cells with different treatment conditions. The effect of the ns-APPJ on gene delivery to guinea pig skin were assessed by comparing the gene expression using plasma, GET and combined treatment.

II. Experimental Design and methods

A. Nanosecond pulsed plasma jets

Two ns-APPJs were used here for the in vitro and in vivo studies, respectively. The choice of the plasma jets and their associated electrode configurations was based on the specific requirements of the applications, the previous measurements of the reactive plasma agents for similar setups[32, 39], and the pilot testing results.

For the in vitro cancer cell inactivation study, a single needle microplasma jet was used. The same plasma jet for cancer cell inactivation [32] and details of the electrode configurations [40, 41] were reported previously. For the in vitro inactivation study, the ns-APPJ was applied to a 96-well plate, with a diameter of 6.8 mm for each well. A microplasma jet was hence better suited for this application, to allow the plasma-induced effect to be highly localized and ensure the minimum influence on the surrounding wells during the plasma exposure. A schematic of the experimental setup of the plasma impinging on the suspended cells in a 96-well plate is shown in Fig. 1a. The high voltage needle electrode was made of a stainless-steel tube having an outer diameter (OD) of 0.50 mm and an inner diameter (ID) of 0.26 mm. The gap distance between the needle electrode outlet and the liquid surface was kept constant at 5 mm. Ultrapure (>99.999%) helium was used as the working gas and flowed through the hollow needle electrode at a flow rate of 70 SCCM (standard cubic centimeter per minute), which was controlled by a calibrated rotameter. Similar to our previous work[32], the plasma was powered by 140 ns, 10 kV pulses at a pulse repetition rate of 2 kHz. The pulses were provided by a custom-built solid-state high voltage pulse generator capable of 10 kV maximal voltage at a repetition rate of 10 kHz and a constant pulse duration of 140 ns[32].

Fig. 1.

Fig. 1.

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Schematic of the experimental setup of (a) the 10-kV pulsed microplasma jet impinging on cell suspension in a 96-well plate for the cancer cell inactivation study; and (b) the 6-kV pulsed plasma jet impinging on skin for the gene transfer study

The plasma jet that was used for the in vivo gene delivery study had a larger diameter. For the gene delivery study, a larger surface coverage on the subject under treatment was needed. The electrode configuration was based on dielectric barrier discharges, similar as our previously used design[42–44] and others[45, 46]. It consisted of a center stainless steel tubing (ID = 1.16 mm, OD = 1.55 mm), a middle insulation ceramic tube (ID = 1.6 mm, OD = 3.2 mm), and an outer grounding ring. The grounding ring was made of 0.3 mm thick and 3 mm wide copper strip. The top edge of the grounding ring was spaced from the high voltage nozzle surface for 10 mm and the lower edge of the ring was 3 mm above the exit of the ceramic insulation tube. Details of the electrode configuration were reported previously[44]. The same pulsed power supply was used to provide 140 ns, 6 kV pulses at 1 kHz to the center tubular electrode. Up to 30 mm-long plasma jet was produced in ambient air when ultrapure helium was fed through the powered center tube at a flow rate of 985 SCCM. Fig. 1b shows the schematic of the experimental setup of the plasma jet impinging on the skin of a guinea pig during the in vivo plasma treatment. To obtain the conduction current that was produced between the electrode nozzle and the skin, additional experiments were conducted, where the guinea pig was replaced with a 15 mm-thick piece of pig skin. Protocol of the pig skin preparation was provided previously[39]. The gap distance between the electrode nozzle and the skin surface was kept at 10 mm for all the studies.

B. Inactivation of Pancreatic cancer cells in vitro

Cell culture.

Murine pancreatic adenocarcinoma (Pan02) cell lines were obtained from the Division of Cancer Treatment and Diagnosis (DCTD, NCI). The cells were maintained in RPMI-1640 (ATCC® 30–2001™) supplemented with 10% FBS (Atlantic Biological), 100 IU of penicillin and 100μg/ml streptomycin and cultured in a 37°C incubator supplied with 5% CO2.

nsPEF and plasma treatment.

Pan02 cells of 5×106 mL−1 in 0.1 mL complete medium were placed in a 1 mm-gap cuvette, and treated with 60 ns, 5 kV pulses for 20 s with a pulse repetition rate of 1 Hz (nsPEF). Under this pulse condition, the electric field between the gap of the cuvette can be assumed constant at 50 kV/cm during pulsing. After the nsPEF treatment, cells were resuspended and diluted in complete medium to have a concentration of 2 × 105 mL−1. The cell suspension was then transferred to 96-well plates with 0.1 mL per well for further treatment.

Total 12 treatment groups with n = 5 wells in each group were used in the study. Each well has 0.1 mL cell suspension at 2 × 105 mL−1, treated or untreated by nsPEF. Four control groups including two negative controls and two nsPEF treatment-only groups were used to evaluate the significance of the treatment outcome. The negative control includes one untreated group and the other that was treated with helium flow for 60 s. The nsPEF-control groups include one group that was only treated by the nsPEF protocol and the other that was treated by nsPEF plus helium flow (60 s). The 8 plasma treatment groups consist of 4 plasma treatment-only groups and 4 plasma plus nsPEF treatment groups. While the plasma was powered at the same pulse condition, different treatment time, 15, 30, 45 and 60 s were applied with and without nsPEF protocol. For all the plasma treatment groups, the nozzle-to-liquid gap distance was kept at 5 mm.

Cell viability assay.

WST-1 cell viability assay[26] was carried out in the in vitro study. Immediately after treatment, cells were incubated at 37°C and 5% of CO2 for 18 hours. After adding 10 μL WST-1 reagent to each well, cells were incubated for another 2 hours. Viability assessment was under-taken based on absorbance measurements using MultiSkan MCC/340 microplate reader (Fisher Scientific, Hampton, NH) with an excitation wavelength at 450 nm and a reference wavelength at 630 nm. The cell viability (%) was calculated using the formula: treated sample (OD450-OD630)/control (OD450-OD630) × 100%. Cells that were left untreated were used as control.

Statistical Analysis.

Results are expressed as mean ±SD (standard deviation) of at least 5 repeated experiments. Statistical analysis was performed using one-way ANOVA. P <0.05 was considered significant.

C. Delivery of plasmid DNA to skin in vivo

Animals.

Female Hartley guinea pigs weighing approximately 275–325 g were used for this study. All animals were housed in the Old Dominion University animal facility, which is AAALAC accredited. All procedures were approved by the Old Dominion Institutional Animal Care and Use Committee. All USDA regulations as well as NIH guidelines for the care and use of laboratory animals were followed. Prior to initiation of procedures, all animal subjects were quarantined and acclimated for a minimum of 7 days following arrival.

Plasmid DNA delivery to skin.

Endotoxin-free plasmid DNA encoding luciferase, gWizLuc (size, 6,732 bps), was purchased from Aldevron (Fargo, ND). Plasmid DNA was suspended in sterile saline at 2 mg/mL. To reduce the number of animals in the study, we utilized 6 treatment sites on each flank of the guinea pig. There was a 1–2 cm gap between treatment sites, which was maintained to avoid any treatment interference between sites. There was a total of 24 treatment sites which were assigned to six groups with n = 4 sites in each group. The six treatment groups consisted of the control (injection only), gene electrotransfer (GET) only, plasma treatment only for 1 min or 3 min, and combined treatment groups: plasma for 1 min or 3 min plus GET. Each treatment site received a 50 μL injection of gWizLuc at a concentration of 2 mg/mL. For the plasma treatment, the gap distance between the plasma nozzle and the skin surface was kept constant at 10 mm. For the GET treatment, a multi electrode array (MEA) containing 16 spring-loaded pins serving as non-penetrating electrodes, configured in a 4 × 4 array and equally spaced for a 2 mm – interelectrode distance[27, 28, 47]. Only 4 electrodes were active at a time. Each set of 4 electrodes delivered 4 pulses and nine sequences were used to sequentially activate all 16 electrodes (4 electrodes at a time)[27]. Total of 72 pulses were applied for each GET treatment. The electric pulse parameters were 150 ms-duration, 35 V pulses at a pulse repetition rate of 6.7 Hz (or 150 ms pulse-to-pulse interval). The electric pulses were generated using a high voltage power supply (Ultravolt HV Rack 1/4C24-P250, Ronkonkoma, NY) that was controlled by a customized program utilizing LabVIEW 8.2.1(National Instruments, Austin, TX). Sites that were assigned to the combined treatment groups were exposed to plasma for 1 min or 3 min, and immediately subjected to the pulsed GET treatment.

Bioluminescence imaging.

Animals were imaged for bioluminescence on days 1, 2, 9, and 14. During imaging, each animal was anesthetized and received subcutaneous injections of D-luciferin (Gold Biotechnology, Inc., St. Louis, MO) to the scruff of the neck. D-luciferin was administered at a concentration of 30 mg/ml and a dose of 150 mg/kg. An IVIS Spectrum in vivo Imaging System (PerkinElmer, Akron OH) was used to capture and quantitate bioluminescence signal.

III. Results

A. Voltage waveforms and power consumptions of the plasma sources

Voltage and current were measured at the load (i.e., the high voltage electrode) for both plasma jets using a high voltage probe (Tektronix 6015A) and a wideband current monitor (Pearson 2877), respectively. The jet current was obtained by subtracting the discharge current measured between the electrode nozzle and the substrate (cell suspension or skin) when the plasma was off (without helium flow) from when the plasma was on.

Figure 2 shows the voltage and current waveforms for the microplasma jet impinging on buffer solution in a 96-well plate (Fig. 2a) and the 1-mm plasma jet impinging on pig skin (Fig. 2b). For the microplasma jet, the full width at half maximum (FWHM) of the voltage pulse and the maximum voltage were measured to be 140 ns and 9.7 kV, respectively. Although the discharge current was dominated by the displacement component, its maximal value (169 mA) was comparable with the peak jet current (122 mA). The energy per pulse can be obtained by integrating the product of voltage and current over a sufficient period of time, e.g., 1200 ns, and it was 84 μJ for the condition used in this study. In addition, the electric charge delivered from the needle-anode toward the liquid can be calculated by integrating the jet current over time. The maximal positive charge was calculated to be 4 nC. To estimate the power density on the substrate surface, we assumed the surface area at the substrate to be the same as the nozzle cross section, 4.9 × 10−4 cm2. The average power density at the surface was obtained to be 342 W/cm2.

Fig. 2.

Fig. 2.

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Voltage, current and jet current waveforms of (a) the microplasma jet impinging on cell suspension in a 96-well plate and (b) the 1 mm-plasma jet impinging on skin

For the 1-mm plasma jet, the FWHM of the voltage pulse was the same and the maximal voltage was 6.1 kV, as shown in Fig. 2b. The maximal jet current was relatively small (45 mA), about 5% of the maximal pulsed current (850 mA). Since in this configuration the jet current was only the conduction component of the current that branched out from the total pulsed current, it was expected that the jet current was much smaller than the main discharge current. From the total voltage and current measured at the electrode, the energy per pulse that was deposited in the plasma system was calculated to be 1.6 mJ. The positive charge transferred from the electrode nozzle to the skin surface was calculated based on the jet current to be 1 nC. Considering nozzle cross section of 0.02 cm2, the average power density was calculated to be 80 W/cm2. Comparing with the microplasma jet, this 1-mm plasma jet had less power density and less electric charge delivery, both were about 4 of their corresponding quantities.

B. Inactivation of Pancreatic cancer cells in vitro

The viability of Pan02 cells in suspension in response to different treatment conditions using plasma and/or nsPEF is shown in Fig. 3. Exposing the cells to helium flow for 60 s or nsPEF-treatment alone did not show significant impact on the cell viability. This insignificant impact on cell viability was also observed when the helium flow was combined with the nsPEF treatment. A dosage dependence of the cell viability on plasma treatment time, from 15 s to 60 s with every 15 s increment, was observed: the longer the plasma treatment time, the less survival rate of Pan02 cells. When combining the nsPEF treatment with the plasma, the impact of the nsPEF was not significant on the cell inactivation until the plasma treatment time was increased to 60 s. This implies that the nsPEF treatment on cells may have increased the sensitization of the cells to plasma. However, this increased sensitization may be reversible, such that only when the dosage of the plasma treatment is sufficiently high, e.g. more than 60 s, the impact on cell inactivation could be enhanced significantly, e.g. by sixfold.

Fig. 3.

Fig. 3.

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Viability of Pan02 cancer cells under different treatment conditions: the lightly shaded are the negative control groups (untreated or helium flow); the diagonally striped are the nsPEF or nsPEF plus helium flow; the blue confetti-filled are the plasma treatment groups; and the purple densely dotted are the plasma and nsPEF combined groups. The plasma was powered by 140 ns, 10 kV pulses at 2 kHz for treatment time of 15, 30, 45 and 60 s. Treatment condition of the nsPEF was constant: 60 ns, 5 kV pulses for 20 s at a pulse repetition rate of 1 Hz (i.e. 20 pulses). ** P< 0.01 and *** P<0.001 by one-way ANOVA.

C. Delivery of plasmid DNA to skin in vivo

Following treatment, guinea pigs were followed for approximately 2 weeks with periodic imaging to determine level of luciferase expression. Comparing the control group (injection only) with the plasma treatment alone, the difference in expression was not significant. Delivery of gWizLuc with GET (150 ms, 35 V, 72 pulses) resulted in an increase in gene expression, implying that GET enhanced plasmid DNA delivery to skin, which agreed with our previous studies[25, 27, 28, 47]. Adding only 1-min plasma treatment to the GET protocol did not show significant difference from GET alone. However, increasing the plasma treatment time to 3 min showed higher gene expression than the GET alone, indicating the sensitization of the skin to GET might have been affected by the plasma treatment and the enhancement of gene delivery could be achieved with the combined plasma and GET approaches.

IV. Discussion

For the weakly-ionized ns-APPJs, the important plasma agents that contribute to biological effects may be heating, UV photons, RONS, and electric field.

The gas temperature of both plasma jets were previously determined to be 300 K by measuring the rotational temperature of the N2 second positive system[41] or the ground state atomic oxygen[48]. The temperature of the substrate directly under plasma treatment for similar setups have also been previously reported. Song et al. reported the water temperature in the immediate vicinity of plasma-water interface was 293 K (i.e. 20 °C) using a fiber optic probe (Neoptix T1S-2M) inserted in the plasma and on/above the substrate surface[40]. The use of pig skin as the substrate increased the substrate temperature slightly by 2 °C, comparing with water or buffer solution[39]. For in vivo treatment, Edelblute et al. reported the change of the skin surface temperature of BALB/c mice before and after 3-min plasma jet treatment was negligible[49]. These temperature measurements on similar plasma jets have shown that the effect of heating on the substrate can be negligible as the increase of the substrate temperature is less than 5 °C for the region directly under plasma exposure. Although any small temperature increase could in principle induce low-level of protein unfolding in the cells or tissue, the bio-effects are expected to be too low to cause any cellular response as the elevated temperature is below hyperthermia (40 – 46 °C)[24, 38, 50–52].

The role of UV photons from a similar plasma jet was assessed with measurements of UV emission intensity in the range of 0.05 – 0.1 mW/cm2 and deemed too low to have significant effect on cells[53].

Pulsed electric fields are known to generate ROS and induce stimulative or oxidative effects in cells[54–59]. Pakhomova et al. reported that nsPEF exposures induced ROS generation in cell suspensions, cell-free media and individual cells[55]. The associated oxidation both extracellularly and intracellularly could contribute to cell damage or increase the sensitization of the cells to electropermeabilization[55]. Nuccitelli et al. demonstrated that ROS generation was triggered by nsPEF treatment in human pancreatic cancer cells, resulting in apoptosis that was intracellular Ca2+ dependent[54]. More recently, nsPEFs with a low field strength (100 ns, 5 kV/cm, 10 pulses) were reported to induce a low concentration of intracellular ROS and to stimulate proliferation of endothelial cells[56]. For ns-APPJs, it was reported that reactive oxygen species (ROS) including O, OH and H2O2 were generated in the plasma-substrate interface[60–66]. Previous studies have shown that the amount of ROS produced in the liquid increased with the plasma exposure time[32]. In this study, we observed that the Pan02 survival rate decreased with increasing the plasma treatment time, which implied that the plasma-produced ROS played an important role in causing irreversible damage to the cancer cells, likely by oxidative stress. Adding a mild dosage of nsPEF treatment to the cells may have only altered the sensitization of the cells to oxidative stress[12, 13], which was activated when the level of plasma-induced oxidative stress increased sufficiently high with plasma treatment time of 1 min.

However, for the case of using skin as the substrate in gene transfer study, we need to reevaluate the roles of ROS. Humidity at the substrate surface was known to favor O and OH production in atmospheric-pressure plasma jets impinging on water or skin[39, 60, 61]. The use of skin as the substrate resulted in less OH or H2O2 production at the substrate comparing to water or cell media[39]. In this study, the use of plasma alone or applying the plasma treatment for 1 min did not facilitate the plasmid DNA delivery or improve the expression levels achieved with GET. After 3-min plasma exposure, however, expression levels achieved following delivery using GET was improved. Although moderate level of ROS at the skin surface could play an important role in inducing oxidative stress and increasing the sensitization of cells to electroporation, transient electric field at the surface may also have induced a similar effect[12].

To evaluate the effect of the transient electric field, induced by the streamers, on the cells or tissue, the substrate can be treated as an equivalent circuit that consists of a capacitor, whose capacitance is proportional to the dielectric constant, in parallel with a resistor, whose resistance is inversely proportional to the conductivity of the substrate. While the dielectric constant or relatively permittivity, εr, of water or cell media can be approximated to 80, εr of skin is typically on the order of 103 to 104 for a wave frequency of less than 10 MHz[67, 68]. The conductivities of cell suspensions and skin are 0.2 S/m[69, 70] and 0.2 – 0.4 mS/m[67] at room temperature, respectively. However, the muscle and fat tissue underneath the skin would increase the effective conductivity of the skin or guinea pig substrate to be in the range of 0.01 – 0.1 S/m[67]. The relaxation time, τ (= εrε0/σ), can then be estimated for cell suspension and skin to be ~4 ns and >90 ns, respectively. ε0 is the permittivity in vacuum. However, the jet current of the plasma jet impinging on cell suspension (Fig. 2a) showed the current decay time about 50 ns, much longer than the estimated time constant. This is possibly due to the higher resistivity at the gas-liquid interface, dominated by the weakly ionized plasma and void of charge carrying medium, compared with that in the liquid medium and hence has an increased relaxation time. For the case of the pig skin, the decay time of the jet current, ~100 ns, agreed well with the estimation (Fig. 2b). For both cases, the duration of the jet current indicated the approximate duration of the transient electric field in cell suspension or on the skin surface. A recent direct measurement of the electric field near the surface of a substrate (chicken breast or saline solution) showed that the electric field intensity of ~20 kV/cm was induced by a 1 μs-duration pulsed plasma jet[71]. This field intensity agreed well with the modeling findings where 23 kV/cm maximum field was calculated at the surface of the substrate that had a dielectric constant of 80[72]. The modeling study also showed that field intensity and its penetration depth (into the substrate) decreased with increasing the dielectric constant[72]. For both cell suspension and skin, the nanosecond-duration electric field induced by the streamer was only prominent at the substrate surface. The impact of the electric field on cell suspension is hence less important when compared with long-lived ROS, which is able to access cells deep in the medium. On the other hand, for assisting GET delivery of plasmid DNA to skin, the transient electric field could directly induce electroporation at the skin surface or indirectly do so by producing mild level of oxidative stress at the surface. It is hence worth noting that the plasma-induced electric field could play an important role in improving the GET efficiency and enable non-thermal plasma to be a safe and effective approach for enhancing GET procedures.

After all, more studies are needed to better understand the synergistic effects on cells and tissue induced from the plasma and pulsed electric field. Better defined quantitative measurements of the exogenous RONS dosage such as the equivalent total oxidation potential [73], intracellular ROS levels, membrane potentials and the associated bioeffects have equal importance to achieve better understanding and for optimizing the cold plasma and nsPEF-based or GET-based techniques for use in various applications.

V. Conclusion

This study evaluated the synergistic effects of nanosecond pulsed plasma jets and pulsed electric fields for inactivation of pancreatic (Pan02) cancer cells in vitro and plasmid DNA delivery to skin in vivo. Dosage dependent inactivation of cancer cells in suspension was observed with different plasma treatment times. Although the mild nsPEF treatment does not impact the cell viability, adding the nsPEF protocol prior to the plasma treatment changed the sensitization of the cells, which resulted in a sixfold-increase in the cell inactivation compared with plasma treatment alone. As nsPEF may induce low-level of oxidative stress, the RONS generated by the plasma was considered to play a primary role by increasing the level of stress and causing irreversible damage to cells. The application of a larger diameter plasma jet at a relatively low dosage (with reduced surface power density and charge delivery to the substrate) up to 3 min did not affect the plasmid DNA delivery to guinea pig skin. However, it was found that the same dosage of plasma treatment increased the expression levels achieved following delivery with GET. Both oxidative stress (due to either the plasma-produced RONS or the transient electric field at the surface) or plasma-induced electropermeabilization could play important roles here in increasing effective delivery. Further studies are needed to isolate these plasma agents in order to better understand the enhancement effects. Importantly, the use of nanosecond pulsed plasmas to increase the sensitization of the biological cells in response to pulsed electric fields could be an effective approach to enhance the desired effects in electroporation-based applications.

Fig. 4.

Fig. 4.

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Enhancement in gene expression due to combined treatment of plasma and GET (at 35 V). Note for the legend: IO, injection only; GET, gene electrotransfer; P1, Plasma treatment for 1 min; P3, plasma treatment for 3 min. There were 4 treatment sites per experimental group (n = 4). The plasma was powered by 140 ns, 6 kV pulses at 1 kHz for 1 min or 3 min. The GET was applied with 72 pulses of 150 ms, 35 V pulses at 6.7 Hz.

Acknowledgments

This work was supported in part by the National Institutes of Health under Grant No. NIBIB R01 EB023878 and in part by the US Air Force Office of Scientific Research under Grant No. FA9550-17-1-0257.

Biographies

Chunqi Jiang (M’01–SM’09) received her Ph.D. (2002) from Old Dominion University (ODU), USA. She worked in the pulsed power research group at the Department of Electrical Engineering – Electrophysics, University of Southern California (USC) first as a Postdoctoral Research Associate (2002 – 2005) then a Research Assistant Professor (2008 – 2012) and Research Associate Professor (2013). She is currently Associate Professor with the Frank Reidy Research Center for Bioelectrics and the Electrical and Computer Department, ODU. Her research interests include fundamental studies of atmospheric pressure nanosecond pulsed plasma sources and their applications for environmental and biomedical fields.

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Edwin Oshin received the B.S. in electrical and electronics engineering from Afe Babalola University, Ado-Ekiti (ABUAD), Nigeria in 2018, and a M.S. degree in Biomedical Engineering from Old Dominion University (ODU), Norfolk, VA, USA, in 2020. He is currently a Ph.D. student at ODU. His research interest includes fundamental studies and applications of nanosecond atmospheric pressure plasma for cancer therapy.

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Siqi Guo received the B. Med. degree in clinical medicine from the School of Medicine Zhejiang University in 1991, and the M.S. degree in oncology from the Academy of Military Medical Sciences (AMMS) China in 1997. He was a resident at the Department of Internal Medicine, Taizhou Hospital, Zhejiang, and the Department of Oncology, Affiliated Hospital of AMMS, Beijing, China, from 1991 to 1997. He worked at the Affiliated Hospital of AMMS from 1997 to 2004. He completed his three post-doctoral trainings at the Department of Pathology, University of Alabama at Birmingham, the Department of Microbiology and Immunology, Virginia Commonwealth University, and the Frank Reidy Research Center for Bioelectrics (FRRCBE), Old Dominion University, from 2005 to 2012. He joined the FRRCBE in 2013, where he is currently a Research Associate Professor. His research focuses are gene electrotransfer and its applications, and nanosecond pulsed electric field tumor ablation and induced immune response.

Biosketches and photos for Megan Scott and Cathryn Mangiamele are not available.

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Xi Li is currently a PhD candidate in Old Dominion University, Norfolk, VA, USA. She received the Bachelor of Science degree from Wuhan University, Wuhan, China, in 2011 and Master of Engineering degree from the graduate school of China Academy of Engineering Physics, Beijing, China, in 2014. Her research interests include pulsed power plasma, electron beam irradiation and their applications.

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Richard Heller is currently Professor in the Department of Medical Engineering at the University of South Florida Colleges of Medicine and Engineering. He previously was Executive Director of the Center for Bioelectrics at Old Dominion University (ODU) as well as Professor and Eminent Scholar in the School of Medical Diagnostics and Translational Sciences in the College of Health Sciences at ODU. Dr. Heller received his B.S. degree in Microbiology from Oregon State University, and a Ph.D. in Medical Sciences from the University of South Florida, College of Medicine. The major focus of his research is to develop in vivo delivery procedures for drug and non-viral gene therapy. His research group has developed new protocols or devices that are being tested for potential therapies for cancer, wound healing, and vascular diseases (peripheral and coronary ischemia) as well as vaccine and immunotherapy protocols.

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Contributor Information

Chunqi Jiang, Frank Reidy Research Center for Bioelectrics and the Department of Electrical and Computer Engineering, Old Dominion University, Norfolk, VA 23529 USA.

Edwin A. Oshin, Frank Reidy Research Center for Bioelectrics and the Department of Electrical and Computer Engineering, Old Dominion University, Norfolk, VA 23529 USA.

Siqi Guo, Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk, VA 23529 USA.

Megan Scott, Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk, VA 23529 USA.

Xi Li, Frank Reidy Research Center for Bioelectrics and the Department of Electrical and Computer Engineering, Old Dominion University, Norfolk, VA 23529 USA.

Cathryn Mangiamele, Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk, VA 23529 USA.

Richard Heller, Department of Medical Engineering, University of South Florida, Tampa, FL 33612 USA.

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