Atmospheric Plasma Meets Cell: Plasma Tailoring by Living Cells

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Biological and Medical Applications of Materials and Interfaces

Atmospheric plasma meets cell: plasma tailoring by living cells Li Lin, Dayun Yan, Eda Gjika, Jonathan H. Sherman, and Michael Keidar ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b10620 • Publication Date (Web): 02 Aug 2019 Downloaded from pubs.acs.org on August 4, 2019

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ACS Applied Materials & Interfaces

Atmospheric plasma meets cell: plasma tailoring by living cells Li Lin*, Dayun Yan, Eda Gjika, Jonathan H. Sherman, and Michael Keidar * Corresponding author: [email protected] Department of Mechanical and Aerospace Engineering, The George Washington University, 800 22nd St NW, Washington, DC 20052, United States of America. Abstract The applications of the cold atmospheric plasma jet (CAPJ) in cancer treatment have been investigated for over a decade, focused on the effect that the CAPJ creates on cancer cells. Here we report for the first time on the impact that cells have on the CAPJ during treatment. To better understand these CAPJ-cell interactions, we analyzed the CAPJ behaviors in the presence of several normal and cancer cell lines and investigated the CAPJ selectivity. A more in-depth study of plasma self-organization patterns utilizing a model which contains a combination of normal and cancer cells, reveals that the cells’ capacitance can be an important predictor of plasma jet behavior. Cancer cells can direct the jet either toward or away from normal cells, which depends on the boundary condition behind the cell colony. Both experimental and theoretical results show that a grounded copper board beneath the cell-culture dish leads to opposite CPAJ behaviors comparing with a floating boundary condition. In conclusion, our findings indicate that plasma can be self-adaptive towards cancer cells, and such a feature can be manipulated. Therefore, using the permittivity difference among cell lines may help us focus plasmas upon cancer cells at the vicinity of normal tissues and maximize the selectivity of plasma treatments. Keywords plasma medicine, selective treatment, cold atmospheric plasma, cell feedback, self-organization pattern, capacitance imaging Significance Statement Cold plasma cancer therapy is currently one of the hottest topics in plasma engineering. The advantage of plasma treatment of cancer is its selectivity that may lead cancer cells to apoptosis with limited damage to normal cells (limited side effect). However, previous research focuses on comparing cell viabilities among different cell lines with the same plasma treatments. In this work, we describe a discovery that cells can affect the plasma. Based on our analysis of such cell feedback effects, future optimization of the plasma treatment can be achieved, and personalized treatment based on plasma adaptation can be envisioned. INTRODUCTION The Cold Atmospheric Plasma (CAP) has wide applications in modern medicine, material processing, environmental engineering, and agriculture 1-4. One of the most significant applications of the Cold Atmospheric Plasma Jet (CAPJ) is the selective treatment which causes cell death in cancer cells while having a limited effect on normal cells 5. In a previous study, we reported that human neuroblastoma cells show a more significant reduction in cell viability compared to healthy fibroblasts when undergoing the same CAPJ treatment condition 6. Similarly, after CAPJ treatment a more significant percentage of adherent lung cancer cells detached from the cell culture plate while the bronchial epithelial cells continue to remain attached to the substrate 7. This specific anti-cancer effect is related to the tumor suppressor gene p53 8. In a recent study, we compared ten cancer cell lines and revealed that the anti-cancer capacity of the CAPJ treatment was inversely proportional to the extracellular H2O2 consumption rate 9. However, for a better understanding of the interaction between the CAPJ and its biomedical targets, it is essential to understand how the CAPJ operates. A typical CAPJ is generated by feeding a noble gas through a pair of electrodes with couple kV sinusoidal waves or pulses at radio frequency 10. When the ACS Paragon Plus Environment

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discharge voltage is approaching its peak, the free electrons naturally existing in the gas are driven towards the anode 11. During their travels, some of these electrons collide with other neutral atoms and molecules. For those electrons with enough kinetic energy, electron-impact ionizations occur during the collisions and provide more electrons to ionize more particles. This process is referred to as “avalanche” 11. During the avalanche, not all electrons can ionize, but more electrons with lower energy and can excite other species. This makes the avalanche region luminous, which is thus referred to as the “plasma bullet” for its bullet-like appearance 12. On the other hand, during the avalanche, the newly generated electrons are accelerated to the anode while other species such as positive ions remain almost stationary since they are much heavier than electrons 11. Therefore, a positive ion cloud appears and turns into a new anode which attracts the free electrons at the front to start another avalanche 11. The ion cloud is also known as the “streamer head,” leaves a quasi-neutral plasma column behind connecting the streamer head and the anode 11. This is how the plasma bullet propagates forward. Such a process is also referred to as the “ionization wave” because it is an ionization process (plasma bullet) propagating forward 13. The entire process usually repeats in the same frequency as the discharge voltage waveform. Therefore, the “plasma bullet” cannot be observed by the naked eye but only through a high-speed camera, and the entire CAPJ appears as a continuous luminous beam for the naked eye. In addition, during the propagation, hundreds of plasma chemistry reactions generate Reactive Nitrogen Species (RNS) and Reactive Oxygen Species (ROS) along with the electromagnetic wave emission (from radio frequency to ultraviolet) as a result of particle collisions during the ionization wave propagation in ambient air 14, 15. During the propagation process of CAPJ, the gas flow rate, the discharge voltage, waveform, frequency, and power are some of the critical parameters that control CAPJ. These parameters can influence the chemical composition, its corresponding Optical Emission Spectrum (OES) 16-18, and the behavior of the plasma bullet 19, 20. The CAPJ is also affected by the environmental humidity, the impurity of the noble gas supply and the feeding gas type which can also be used to manipulate the electron temperature and chemical compositions of CAPJ 21-23. Another set of critical parameters are based on the properties of the target which is a boundary condition of CAPJ. The thickness of a dielectric target below the jet can affect the plasma bullet behavior and the species flux 24. Also, the distance from the target can influence the chemical composition of CAPJ 25. However, all previous biomedical studies of CAPJ focus on the treatment without taking into account the target feedback effect on CAPJ. During any CAPJ treatments, while target (i.e. cancer cells) can also alter the plasma as a boundary condition of both electromagnetism and plasma chemistry such as the release and deposition of species 26. In other words, two CAPJs with identical hardware settings are different when treating different type of cells. In this work, we discover the effect of living cells on CAPJ by analyzing its spatially averaged electron density, the capacitance distribution of target, and the temporally averaged brightness distribution at the CAPJ-target contact surface, which is also known as Self-Organization Pattern (SOP) 27, 28. Free electrons in CAPJ are the origin of ionizations and attachments which produce positive and negative ions. Electrons determine the electric field strength of the streamer head, and they are also involved in the generation of all reactive species, either directly or indirectly 29. Electron density is also a critical parameter for estimating other properties of CAPJ, such as electron temperature 19, 30. SOP indicates how the ionization wavefront distributes at the vicinity of the target, since the electrons which ionize species during collisions contribute to the high-energy tail of the Electron Energy Distribution Function (EEDF), while at the same location, more electrons can merely excite chemical species and provide OES 30. Capacitance imaging implies how electron density and SOP are related to the target permittivity. These results and conclusions from the in-vitro experiments are then supported by a theoretical analysis which also suggests how other CAPJ and target parameters can be used to manipulate their interactions. Based on the findings of a cell feedback signal in this work, future research studying self-adaptive plasma will assess the ability of the CAPJ to optimize itself and consequently fit a variety of applications 31-34. RESULTS Electron density ACS Paragon Plus Environment

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We first investigated how the electron density of CAPJ is altered by cells immersed in the medium, which is named as “cell-medium target” in this work. Figure 1A illustrates an example of the temporally resolved electron density captured by Rayleigh Microwave Scattering (RMS) system when the CAPJ is treating E6/E7/hTERT cells. The contour plot was depicted in a 2D time domain where the color bar represents the value of electron density. The horizontal “micro-time” axis represents the time in a discharge period, while the vertical “macro-time” axis represents the time after the power was switched on. For each discharge period, an electron density peak appeared at about 10 μs of the micro-time. From 0 to 50 seconds of the macro-time, a lower electron density peaks with later appearance indicated a warm-up time before the plasma jet reached its steady state. In such a steady state (from about 50 to 300 seconds of the macrotime), the electron density peaks had constant height and appearance time with limited disturbance. All electron densities discussed below in this paper refer to the peak values at steady states of the working CAPJ. Figure 1B summarizes the measured electron density peaks of CAPJ with different cell lines as targets. Among the different cell lines tested, cancer cells resulted in an overall lower electron density than normal cells. For instance, the electron densities of U87MG and B16F10 cancer cells were significantly lower than that of the healthy E6/E7/hTERT and HaCaT cells. An exception was the MDA-MB-231 breast cancer cells and the healthy primary fibroblast. The difference in electron density was not statistically significant for these two cell lines even though breast cancer cell had a lower average electron density. These results suggest that cancer cells, as a target, may weaken the plasma jet.

Figure 1. The resulting electron densities in the plasma jet during treatments. (A) An example of spatially averaged and temporally resolved electron density of the CAPJ during the treatment of E6/E7/hTERT cells. (B) A summary of all electron density peaks of the plasma jet at steady state for different cell lines. Error bars indicate the standard deviations of measurement repeats. Self-organization pattern The SOP is a luminous CAPJ-target contact region. According to the mechanism of streamer propagation, the luminous region is where the “avalanche” occurs. Therefore, the brightness distribution of SOP indicates where the CAPJ propagates. Our study relies on single and double cell experimental setups for investigating plasma self-organization patterns. For each single-cell test, the cell colony was located at the positive-x region on the right while the no-cell region locates at the negative-x region. CAPJ was aligned to shoot at the boundary of the colony (x = 0 mm). For each double-cell test, the normal cell colony was located at the negative side and the cancer cell colony was located at the positive side with their boundary was located at x = 0 mm for the CAPJ to aim at. An example of the single B16F10 test is shown in Figure 2A. There was an obvious bright dot at around x = -3 mm and y = 23 mm, while there was no such brightness at the positive x side. Note that the brightness at the top of this image was from the plasma jet ACS Paragon Plus Environment

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rather than the SOP. Therefore, the SOP of CAPJ shooting at the boundary of B16F10 colony was asymmetric. To strictly quantify the symmetry, the brightness value summations between y = 22.5 and 23.5 mm for all cases were summarized in Figure 2B. It is clear that B16F10 “pushes” the brightness away to the no-cell region at the negative-x side, while all other three cancer cells lines have no such significant asymmetry. Recall that in Figure 1B, B16F10 made the lowest electron density while other the cancer cells lines result in significant higher electron densities in CAPJ. Moreover, Figure 2B also shows the SOP of the double-cell test. When B16F10 was seeded on the right of HaCaT, more brightness had been observed on the HaCaT side. Similarly, when U87MG was seeded on the right of E6/E7/hTERT, more brightness was observed on the E6/E7/hTERT side. However, such effect was not strong when MDAMB-231 was on the right of fibroblast. Also recall that in Figure 1B, the difference of “B16F10 versus HaCaT” and “U87MG versus E6/E7/hTERT” were significant while the difference of “MDA-MB-231 versus fibroblast” was not significant. These trends imply that the cell line which makes a lower electron density in CAPJ can “push” the SOP away. Actually, the SOP results can be easily explained based on the electron densities shown in Figure 1B. The cell colony which could result in a lower electron density supposed to result in less excited species. In the case of cancer cell colony on the right of the normal cell one, more free electrons which are involved in the “avalanche” above the normal cell colony could make the streamer propagate to the normal cell side, and thus appeared as an asymmetric SOP. Therefore, the electron density is reflecting how cancer cells propel the CAPJ towards normal cells in the double-cell cases. Namely, the cell colonies direct the CAPJ in selecting where or which target to treat. However, such a phenomenon can be reversed when placing a ground electrode under the cell culture dish. Due to the blockage of the copper plate from the bottom and the CAPJ generator from the top, only the side view was provided for this extra experiment. Figure 2C shows a side-view example of the CAPJ directed at the boundary between E6/E7/hTERT and U87MG colonies covered by 20 mL DMEM medium. The contact spot appeared as a bright ellipse. Figure 2D shows the comparison between “E6/E7/hTERT versus U87MG” and pure DMEM, which were the average brightness taken from y = 0.15 mm to 0.35 mm. For comparison, the contacted spot brightness distribution for the pure DMEM case was symmetric. In contrast, the contacted spot was inclined to the right (positive x side) where the U87MG colony was seeded while E6/E7/hTERT colony located at the negative x side. The boundary of these two cell colonies was located at x = 0 mm. This result was opposite to the case without an electrode under the dish (Figure 2B). Consequently, we displayed that the target-triggered permittivity effect can be reversed by the grounding the cathode. A similar conclusion can be found in another simulation studies which shows that the electric field of streamer propagation is proportional to the target permittivity with a grounded cathode behind 35. This result might also explain the stronger killing effect of U87MG when compared with E6/E7/hTERT with a metal plate beneath the dish, which had been reported earlier 36, 37.

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Figure 2. The self-organization patterns when the plasma jet is directed to the boundary of cell colonies. (A) A bottom-view example of the no-cell region at the negative-x region and B16F10 at the positive-x region. (B) The brightness summary of the bottom-view self-organization patterns that taking average brightness from y = 22.5 mm to 23.5 mm for each case. (C) A side-view example of E6/E7/hTERT the negative-x region and U87MG at the positive-x region with a grounded copper plate beneath the dish. (D) The brightness summary of the side-view self-organization patterns that taking average brightness from y = 0.15 mm to y = 0.35 mm. Capacitance imaging During in-vitro treatments, the cell colonies with media were the targets and also the boundary conditions of CAPJ. For dielectric targets, it has been previously reported how their permittivity changes the plasma jet 35. To study the interactions of the electron density, SOP and such boundary conditions, the permittivity of cell-medium targets was analyzed. Figure 3A shows an example of the imaging that a 500 μL U87MG cell solution was located at the center of the dish, while Figure 3B shows the corresponding photo. This example indicates that a U87MG cell colony has a higher capacitance compared with the surrounding area of the dish. Figure 3C summarizes the average capacitances of five different cell colonies. Data were the average values taken in a square area from -0.1 cm to 0.1 cm for both x and y axis. The average capacitance of the B16F10 colony was the highest, which corresponded to the lowest electron density observed in the plasma jet (Figure 1B). In contrast, the capacitance of E6/E7/hTERT colony was the lowest, which corresponded to the highest electron density observed in the plasma jet. Since all imaging was performed using the same electrodes (same geometry), the highest capacitance of the B16F10 colony indicated the highest permittivity of B16F10 cells. Similarly, the E6/E7/hTERT colony displayed the lowest capacitance and permittivity. In short, we conclude that a higher permittivity of the cell colony can make a lower electron density in CAPJ. The permittivity of cells thus determined where CAPJ should come into contact with the target. This result might explain the strong resistance to CAPJ treatments of the B16F10 colony compared with all other cancer cell lines as reported in a previous study 38. ACS Paragon Plus Environment

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Figure 3. The capacitance imaging of the cell colonies at the centers of dishes. (A) An example of capacitance imaging of U87MG. (B) The photo of a 500 μL U87MG cell solution. (C) The comparison of average capacitance in the square region of x and y from -1.00 cm to 1.00 cm. Error bars represent the standard deviations of the cell colony capacitance. DISCUSSION To understand how the target permittivity could affect the plasma jet with and without a ground electrode, we derived a general mathematical model between the permittivity of the dielectric target and the electric field of the plasma bullet which is a space charge consisting of the streamer head and the target surface charge. The scenario is an electrostatic problem of the moment when the plasma bullet is approaching to the vicinity of the target surface. Considering a 1D model that the anode of CAPJ device locates at 0 cm; a positive ion cloud as the streamer head locates at Lp; the target front surface locates at Lp+LB; the target thickness is LT. Therefore, the back surface of the target locates at Lp+LB+LT. Between the anode and the streamer head, we defined an effective permittivity of the plasma column as 𝜎𝐿𝑃

𝜀𝑃 = 2𝜋𝑓𝐴𝜀

,

0

where σ is the conductivity of the plasma column, f is the discharge frequency, A is the cross-section area of the plasma column, and ε0 is the permittivity of vacuum. The plasma column conductivity in this model is based on the impedance of a capacitor: 𝑍 = 𝜎 ―1 = (2𝜋𝑓𝐶) ―1, where C is the capacitance that 𝐶 = 𝜀0𝜀𝑃 , and , we 𝐴/𝐿𝑃, where εP is the effective relative permittivity of plasma. Since have ACS Paragon Plus Environment

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,

(1)

where neP is the electron density in the plasma column, e is the unit charge, μe is the mobility of electron, me is the mass of an electron, and νcoll is the electron-neutral collision frequency. The permittivity between the streamer head and target surface is ε0, since it is a region not yet ionized, and the permittivity of the helium-air mixture is close to the one of vacuum 39, 40. The target permittivity is denoted as εT. The electric field of plasma bullet defined as the field located at the center point between the streamer head and target surface can be expressed as the superposition of background electric field (from the electrodes) and the field of charges: 𝐸𝐵 = 𝐸𝐵 ― 𝑏𝑔 +

𝑞 2𝜋𝜀0(𝐿𝐵/2)2

,

(2)

where the first term on the right-hand-side EB-bg is the electric field of the electrodes at the bullet position, while the second term is the Coulomb's law of the streamer head and target surface charge. They share the same charge amount of 𝑞 = 𝑛𝐵𝑒 to ensure the charge balance of the entire model, where nB is the ion density of the streamer head. To evaluate EB-bg, we consider the model of capacitors in series with the equivalent capacitance 1

1

1

𝐶𝑒𝑞 = (𝐶𝑃 + 𝐶𝐵 + 𝐶𝑇) where 𝐶𝑃 =

𝐴𝜀0𝜀𝑃 𝐿𝑃

―1

,

(3)

as the effective capacitance of plasma column, 𝐶𝐵 =

bullet region between the streamer head and target surface, and 𝐶𝑇 =

𝐴 𝜀0

as the capacitance of the plasma

𝐿𝐵 𝐴𝜀0𝜀𝑇 𝐿𝑇

as the capacitance of the target.

Based on the “voltage divider rule”, we have 𝐸𝐵 ― 𝑏𝑔 =

𝜑𝐹𝑆 ― 𝜑𝑆𝐻 𝐿𝐵

=

(𝜑0 ― 𝜑𝐵𝑆)𝐶𝑒𝑞

,

𝐿𝐵𝐶𝐵

(4)

where φFS, φSH, φ0, and φBS are the potential at the target front surface, streamer head, anode, and target back surface respectively. Therefore, Eq. (2) yields 𝐿𝑇

𝐿𝑃

𝐸𝐵 = (𝜑0 ― 𝜑𝐵𝑆)/(𝜀𝑇 + 𝜀𝑃 + 𝐿𝐵) +

𝑛𝐵𝑒

.

(5)

2𝜋𝜀0(𝐿𝐵/2)2

Combining with Eq. (1), we obtain a general mathematical expression of the plasma bullet electric field 𝐿𝑇

𝐸𝐵 = (𝜑0 ― 𝜑𝐵𝑆)/(𝜀𝑇 +

2𝜋𝑓𝐴𝜀0𝑚𝑒𝜐𝑐𝑜𝑙𝑙 𝐿𝑃𝑛𝑒𝑃𝑒

2

+ 𝐿𝐵) +

𝑛𝐵𝑒 2𝜋𝜀0(𝐿𝐵/2)2

.

(6)

When the target back surface is a ground electrode, 𝜑𝐵𝑆 = 0. However, when the target back surface is floating, the potential is determined by the plasma bullet charge which can be considered as a dipole. Thus, the target back surface potential locates at the negative charge side of the dipole and can be calculated as 𝜑𝐵𝑆 = ∆𝜑 ―

𝑛𝐵𝑒𝐿𝐵

,

(7)

4𝜋𝜀0𝜀𝑇(𝐿𝐵/2 + 𝐿𝑇)2

where Δφ is the potential drop along the plasma column which is proportional to neP-1. Therefore, Eq. (6) and Eq. (7) explains the entire mechanism behind the phenomena introduced in this work. When the target back surface is attached to a ground electrode, the plasma bullet electric field is proportional to the target permittivity which agrees with the phenomenon shown in Figure 1 and 3 assuming that a higher electric field creates a higher electron density in CAPJ. When the target back surface is floating, the target permittivity appears in both the numerator and the denominator of the first term in Eq. (6). Therefore, only when the electron density in the plasma column is low, the bullet electric field is inversely proportional to the target permittivity. This is because of the plasma column term 𝐿𝑇

contribution of 𝜀𝑇, while Δφ cannot limit the term 4𝜋𝜀 𝜀


0 𝑇(𝐿𝐵/2

+ 𝐿𝑇)2

2𝜋𝑓𝐴𝜀0𝑚𝑒𝜐𝑐𝑜𝑙𝑙 𝐿𝑃𝑛𝑒𝑃𝑒2

dominates and limits the

in the numerator since the potential drop

in the numerator can at most cancel out the φ0. In this work, as shown in Figure 1B, the electron density is mostly less than 1012 cm-3 with 8 kV discharge voltage which is under the “low electron density” situation, considering the same CAPJ generator can achieve an electron density higher than 2x1012 cm-3 with 5 kV and same gas flow rate when there is no target 30. Moreover, Eq. (6) suggests that a lower LB ACS Paragon Plus Environment

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can increase the term 4𝜋𝜀 𝜀


0 𝑇(𝐿𝐵/2

Page 8 of 21 𝐿𝑇

+ 𝐿𝑇

, and add more limit to the contribution of 𝜀𝑇 in the denominator. This ) 2

agrees with the common sense that the target can affect the plasma bullet more when the streamer is closer to the target. Eq. (6) and (7) also indicate additional CAPJ features which agree with other publications. For example, the electric field of a plasma bullet is usually stronger when the ground electrode appears 41. This is because of φBS in Eq. (7) is positive and implies that the potential drop along the plasma column is greater than the potential difference made by the streamer head electric field. In other words: 𝜑0 ― 0 > 𝜑0 ― 𝜑𝐵𝑆. However, if the electron density in the plasma column is high enough and results in a low Δ φ, the electric field of streamer head dominates. This explains why the streamer head potential can increase along the plasma jet when the discharge voltage is high, as reported in an old publication 42. In addition, using the same mathematical model, the electric field inside the target can be easily derived to 𝜀𝑇

𝑛𝐵𝑒

𝐸𝑇(𝑧) = (𝜑0 ― 𝜑𝐵𝑆)/(𝐿𝑇 + 𝜀𝑃𝐿𝑃 + 𝜀𝑇𝐿𝐵) ― 4𝜋𝜀 𝜀 𝑧2, 0 𝑇

(8)

where z is the location in the target from the target front surface. The right-hand-side is inversely proportional to the target permittivity. However, the first term is positive and the second one is negative. This equation suggests that the electric field in the target can be inversely proportional to the target permittivity, which agrees with the simulation results published previously 35. However, due to the negative second term, this equation also indicates that such feature occurs only when the electric field of streamer does not dominate. Please note that Eq (8) is less relevant to the main topic of this work but more discussion of it can be found in the Supplementary Materials. Other features of CAPJ implied by this mathematical model including the decay of plasma bullet electric field along the propagation distance, the weaken of target inner electric field for a further target position, and the strengthen of it when the streamer head is closer to the target. These trends all agree with common sense. Overall, these equations can be utilized to manipulate the CAPJ and they will be used in future for optimization and self-adaptive control. As an additional example, a finite element simulation of an electrostatic model using the software FEMM is discussed in the Supplementary Materials. In this study, we revealed how the permittivity of the cell colony could alter the direction of the CAPJ during the in-vitro treatment. We conclude that the electron density of the CAPJ can be affected by the permittivity of the targeted cell colony. When there is no electrode beneath the cells, a higher permittivity of the cell colony could result in a lower electron density for the plasma jet. This causes the plasma jet to lean more on one side by being directed at the boundary between two cell lines or the boundary of a singlecell colony. When a ground electrode is placed under the cell colony, such targeted permittivity effect can be reversed. Therefore, when there are two or even more cell lines facing the plasma jet, the cell permittivity can affect the CAPJ by determining which target the CAPJ will come into contact with. Based on the mathematical relationship we derived and supplemented with the FEMM simulation, we also revealed the mechanism of such a targeted permittivity effect. This study provides a strong basis for further understanding of selective cancer treatment, adaptive plasma control, and future possible clinical applications. MATERIALS AND METHODS Cell culture Four cancer cell lines were investigated in this study, including B16F10 (murine melanoma), MDA-MB231 (human breast adenocarcinoma), PA-TU-8988T (human pancreatic adenocarcinoma), and U87MG (human glioblastoma). In addition, three normal cell lines were also investigated, including E6/E7/hTERT (human astrocyte), HaCaT (human keratinocytes), and mouse primary fibroblasts. The U87MG cells were purchased from the American Type Culture Collection (ATCC) while the rest of the cells line were donated from other laboratories at the George Washington University. Cell lines U87MG, MDA-MB-231, PA-TU-8988T, E6/E7/hTERT, and the primary mouse fibroblasts were cultured in DMEM medium (Life Technologies, 11965-118) supplemented with 10% (v/v) fetal bovine serum (Atlanta Biologicals, S11150) and 1% (v/v) Penicillin/Streptomycin. The B16F10 cells were cultured in RPMI-1640 (ATCC, 30-2001) also supplemented with 10% (v/v) fetal bovine serum and 1% (v/v) Penicillin/Streptomycin, the HaCaT ACS Paragon Plus Environment

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cells were cultured in Keratinocyte-SFM medium (Gibco) with supplements (Gibco; 37000-15) and 0.5% (v/v) Penicillin/Streptomycin. Cells were seeded at a concentration of 5x105 cell/mL and the cell cultures were maintained under standard conditions at 37°C in a humidified incubator containing 5% CO2. Electron density measurement As shown in Figure 4, 500 μL cells in medium (5x105 cell/mL) was carefully seeded at the center of a 100 mm cell culture dish for each cell line. Cells were cultured for 6 hours under standard cell culture condition. During this period, the cells were able to attach to the dish, which is critical for establishing invitro cell growth. After the incubation was complete, 20 mL of DMEM medium was added before proceeding with measurements. A schematic of the CAPJ generator is illustrated in Figure 4 along with the hardware for measuring the electron density of the plasma jet: the Rayleigh Microwave Scattering (RMS) system. The CAPJ in this work was generated by a 10 LPM helium flow passing through a pair of electrodes powered by an 8 kV pk-pk sinusoidal wave at 12.5 kHz. In this experiment, the propagation of CAPJ was a cathode-directed streamer. Therefore, the streamer head was positively charged. For the temporally resolved electron density measurement, the cell-medium target was 3 cm away from the nozzle. A microwave of 12.6 GHz was generated and emitted from a horn antenna. The microwave beam was then scattered by the plasma jet which was considered as a dipole 43. The scattered wave carried the information of conductivity as a function of electron density which was recorded by Matlab in real-time through an oscilloscope. More details of the RMS diagnostic technology can be found in an old publication 43.


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Figure 4. A schematic representation of the in-vitro experimental setup and a Cold Atmospheric Plasma Jet with Rayleigh Microwave Scattering for performing electron density measurements. Observation of the plasma self-organization pattern The experiment of SOP includes two different setups: the single-cell setup and the double-cell setup. The procedure of the single-cell setup is shown in Figure 5A. As part of this setup, 500 μL of cells in the medium were carefully seeded at a 1 cm distance from the center of the 100 mm cell culture dish. In this manner, the boundary of each cell population aligned with the axis of the plasma jet. In other words, the plasma jet was applied at the boundary of the cell colony. Similarly, cells were incubated for 6 hours under standard experimental conditions. After the incubation was complete, 20 mL of medium was added, and the SOP for each cell line was recorded. The protocols of the double-cell test procedure are shown in Figure 5B. For this setup, we designed a plastic cell barrier to separate the two cell colonies during the incubation as shown in Figure 5C. The barrier was positioned at the center of the 100 mm cell culture dish. One normal cell line and another cancer cell line was seeded in the two wells of the barrier. In each well, 500 μL of cells in medium (5x105 cells/mL) was added. Cells were incubated for 6 hours. After the incubation, the barrier was removed creating a small gap between the two cell colonies as shown in Figure 5D. This gap represents the boundary between the two cell colonies. To quantify the SOP, a camera was fixed below the dish and recorded a bottom view of the SOP video during the CAPJ treatment in each test. Using the Matlab command “VideoReader”, each video frame was converted to a matrix of optical brightness distributions. The summation of all the test frames divided by the number of frames thus represented the average brightness distribution of the dynamic SOP video. For each single-cell line test, the center of the cell colony was located 1 cm from the center of the dish. This allowed the CAPJ nozzle to be pointed at the boundary of each colony and show how the presence of cells can make the SOP asymmetric. In each double-cell experiment, the difference between cell lines was also expected to reflect a certain asymmetry.


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Figure 5. In-vitro cell culture for observing the self-organization patterns of CAPJ. (A) The cell preparation procedure of one cell line. (B) The procedure of double-cell test. (C) The 3D model of the cell barrier. (D) The E6/E7/hTERT cell colony (left) and U87MG cell colony (right) in 20 mL of DMEM medium. Capacitance imaging For the capacitance imaging, the cell colony was located at the center of the dish (Figure 6). A sinusoidal wave (10 V pk-pk, 25 MHz) was applied on the series of a 0.47 pF reference capacitor and a pair of copper electrodes (5 mm x 1 cm each with 1 mm gap). The electrodes were attached to a linear translation stage for position control. The electrodes were 1 mm away from the surface of the medium and swept over the dish during each measurement for the imaging. Once the sample permittivity changed, the impedance between the electrodes changed accordingly along with the voltage applied to the reference capacitor. This voltage signal was sent to a 10x signal multiplier circuit with a core of LM7171 high-speed operational amplifier before it was finally sent to the oscilloscope and recorded in Matlab.

Figure 6. The schematic illustration of the capacitance imaging. SUPPORTING INFORMATION (attached) Figure S1. An electrostatic simulation model of a streamer propagation at the vicinity of a dielectric target. Figure S2. Average electric field of the streamer head space charge. Figure S3. Average electric field in the target. Figure S4. The axial potentials and electric fields of the target back surface with and without grounding the electrode. ACKNOWLEDGMENTS The authors would like to thank the following professors at the George Washington University: Dr. Lijie Grace Zhang for donating the MDA-MB-231 cell, Dr. Sotomayor for donating the B16F10 cells, Dr. Farid Murad for donating the PA-TU-8988T cells and Dr. Mary Ann Stepp for donating the HaCaT cells and the mouse primary fibroblasts. This research is supported by the National Science Foundation, grant 1465061 and 1747760.

AUTHOR CONTRIBUTIONS M.K. provides the initial idea that the cell colony capacitance may play a key role of the CAPJ-cell interactions. L.L. and D.Y. are responsible for the cell culture and preparation of B16F10, E6/E7/hTERT, MDA-MB-231, PA-TU-8988T and U87MG. E.G. is responsible for the cell culture and preparation of HaCaT and Fibroblast. L.L. also contributes to the measurement and data processing of CAPJ electron density, SOP, and capacitance imaging. L.L. developed the 1D mathematical model and simulated the 2D electrostatic scenario in FEMM. All authors are involved in the text editing. REFERENCE ACS Paragon Plus Environment

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Figure 1. The resulting electron densities in the plasma jet during treatments. (A) An example of spatially averaged and temporally resolved electron density of the CAPJ during the treatment of E6/E7/hTERT cells. (B) A summary of all electron density peaks of the plasma jet at steady state for different cell lines. Error bars indicate the standard deviations of measurement repeats.


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Figure 2. The self-organization patterns when the plasma jet is directed to the boundary of cell colonies. (A) A bottom-view example of the no-cell region at the negative-x region and B16F10 at the positive-x region. (B) The brightness summary of the bottom-view self-organization patterns that taking average brightness from y = 22.5 mm to 23.5 mm for each case. (C) A side-view example of E6/E7/hTERT the negative-x region and U87MG at the positive-x region with a grounded copper plate beneath the dish. (D) The brightness summary of the side-view self-organization patterns that taking average brightness from y = 0.15 mm to y = 0.35 mm.


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Figure 3. The capacitance imaging of the cell colonies at the centers of dishes. (A) An example of capacitance imaging of U87MG. (B) The photo of a 500 μL U87MG cell solution. (C) The comparison of average capacitance in the square region of x and y from -1.00 cm to 1.00 cm. Error bars represent the standard deviations of the cell colony capacitance.



Figure 4. A schematic representation of the in-vitro experimental setup and a Cold Atmospheric Plasma Jet with Rayleigh Microwave Scattering for performing electron density measurements.


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Figure 5. In-vitro cell culture for observing the self-organization patterns of CAPJ. (A) The cell preparation procedure of one cell line. (B) The procedure of double-cell test. (C) The 3D model of the cell barrier. (D) The E6/E7/hTERT cell colony (left) and U87MG cell colony (right) in 20 mL of DMEM medium.



Figure 6. The schematic illustration of the capacitance imaging.


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Atmospheric Plasma Meets Cell: Plasma Tailoring by Living Cells

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