Plasma-activated saline solutions and method of making plasma activated saline solutions

Abstract

A method for manufacturing plasma-activated saline for treatment of cancer cells. The method comprises the steps of immersing a cathode in saline solution in a container, positioning an anode at a fixed distance from a surface of said saline solution in said container and applying electrical energy to said anode for a fixed period of time, wherein said fixed distance and said fixed period of time are selected to cause a plasma self-organized pattern at a surface of said saline solution with an atmospheric discharge between said anode and said cathode.

Claims

1. A method for manufacturing plasma-activated saline for treatment of cancer cells comprising: immersing a cathode in saline solution in a container; positioning an anode at a fixed distance from a surface of said saline solution in said container; and applying electrical energy to said anode for a fixed period of time; wherein said fixed distance and said fixed period of time are selected to cause a plasma self-organized pattern at a surface of said saline solution with an atmospheric discharge between said anode and said cathode and where in said plasma self-organized pattern comprises a double ring structure.

2. The method for manufacturing plasma-activated saline for treatment of cancer cells according to claim 1 wherein said fixed distance is 4-6 mm.

3. The method for manufacturing plasma-activated saline for treatment of cancer cells according to claim 1 wherein said fixed time is 40 seconds.

4. The method for manufacturing plasma-activated saline for treatment of cancer cells according to claim 1 wherein said double ring structure comprises a solid inner ring surrounded by a continuous outer ring.

5. The method for manufacturing plasma-activated saline for treatment of cancer cells according to claim 1 wherein said double ring structure comprises a solid inner ring surrounded by a discontinuous outer ring.

6. The method for manufacturing plasma-activated saline for treatment of cancer cells according to claim 1 wherein said double ring structure comprises a continuous inner ring surrounded by a continuous outer ring.

7. The method for manufacturing plasma-activated saline for treatment of cancer cells according to claim 1 wherein said double ring structure comprises a continuous inner ring surrounded by a discontinuous outer ring.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) For a more complete understanding of the present invention and the advantages thereof, reference is now made to the following description and the accompanying drawings, in which:

(2) FIG. 1 shows a schematic representation of the SOP plasma discharge setup capable of producing well-defined self-organized interface patterns at the surface of the liquid/plasma interface. Different air gap distances between the cathode and surface of liquid accommodated plasma. (d is the distance of air gap).

(3) FIG. 2A illustrate a current-voltage dependence for different air gap lengths with optical photographs of the self-organized stratified interface patterns.

(4) FIG. 2B is a diagram of a double ring self-organized pattern with a solid inner ring and a continuous outer ring in accordance with a preferred embodiment of the present invention.

(5) FIG. 2C is a diagram of a double ring self-organized pattern with a solid inner ring and a discontinuous outer ring in accordance with another preferred embodiment of the present invention.

(6) FIGS. 3A-3E show the Optical emission spectrum by the SOP plasma discharge above saline solution with different air gap length taken using UV-visible-NIR in the 200-850 nm wavelength range: (a) 2 mm, (b) 4 mm, (c) 6 mm, (d) 8 mm, and (e) 10 mm.

(7) FIGS. 4A and 4B H.sub.2O.sub.2 and NO.sub.2.sup. concentrations in saline solution treated by plasma with self-organized pattern plasma with different air gap length (Each air gap length treated by SOP plasma for 40 seconds): (a) H.sub.2O.sub.2 concentration and (b) NO.sub.2.sup. concentration. Student's t-test was performed, and the significance compared to the 2 mm is indicated as *p<0.005, **p<0.01, ***p<0.001. (n=3)

(8) FIGS. 5A and 5B show effects of seven media: RPMI/KSFM, saline solution (SS), and five plasma-activated media (saline solution activated by plasma with SOP at 2, 4, 6, 8, and 10 mm distance for 40 seconds' treatment) on viability of the BxPC-3 human pancreas cancer cells d the H6c7 human pancreas normal cells after 24 (a) and 48 (b) hours' incubation, respectively. The percentages of surviving cells for each cell line were calculated relative to controls (RPMI/KSFM).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

(9) The preferred embodiments of the invention and the experiments will be described with reference to the drawings.

(10) FIG. 1A shows a schematic representation of a self-organized pattern plasma discharge setup capable of producing well-defined self-organized interface patterns at the surface of a liquid/plasma interface. An anode 120 (a thin copper plate, thickness d=0.2 mm, =22 mm) is placed at the bottom of a glass-made well 110. A saline solution 200 is placed in the glass well. A tungsten cathode 130 of =2 mm is then installed above the saline solution surface. A ballast resistor 140 (90 K) is connected between the cathode 130 and a direct current (DC) power supply unit 150 (Power Design, Model 1570A, 1-3012V, 40 mA). A voltage is applied between the cathode 130 and the liquid-immersed anode 120, and a plasma 160 is formed in a small (2-10 mm) gap between the cathode and liquid surface accommodated. As shown in FIG. 2A, voltage of 300-1500 v were applied with currents of approximately 10-25 mA.

(11) In a series of experiments, saline solution was treated by discharge with a 2, 4, 6, 8, and 10 mm air gap length d between the cathode 130 and the surface of the plasma 200 to obtain plasma-activated solutions for treating cancer cells.

(12) A. Cell Cultures for the Experiments

(13) The human pancreas adenocarcinoma cancer cell line (BxPC-3) was acquired from American Type Culture Collection (ATCC). Cell lines were cultured in RPMI-1640 Medium (ATCC 30-2001) supplemented with 10% (v/v) fetal bovine serum (Atlantic Biologicals) and 1% (v/v) penicillin and streptomycin (Life Technologies). The human pancreatic duct epithelial normal cell line (H6c7, Kerafast) was cultured in Keratinocyte SFM (KSFM, Gibco) supplemented with prequalified human recombinant Epidermal Growth Factor 1-53 (EGF 1-53, Gibco), Bovine Pituitary Extract (BPE, Gibco), and 1% (v/v) penicillin and streptomycin (Life Technologies). Cultures were maintained at 37 C. in a humidified incubator containing 5% (v/v) CO.sub.2. Cultures were maintained at 37 C. in a humidified incubator containing 5% (v/v) CO.sub.2.

(14) B. Evaluation of Hydrogen Peroxide (H.sub.2O.sub.2) Concentration

(15) Fluorimetric Hydrogen Peroxide Assay Kit (Sigma-Aldrich) was used for measuring the amount of H.sub.2O.sub.2 in saline solution. A detailed protocol can be found on the Sigma-Aldrich website. Briefly, we added 50 l of standard curves samples, controls, and experimental samples (saline solution treated by SOP plasma with 2, 4, 6, 8, and 10 mm air gap) to the 96-well flat-bottom black plates, and then added 50 l of Master Mix (including Red Peroxidase Substrate Stock, 20 units/mL Peroxidase Stock, and Assay Buffer) to each of wells. We incubated the plates for 20 min at room temperature protected from light on and measured fluorescence by Synergy H1 Hybrid Multi-Mode Microplate Reader at Ex/Em: 540/590 nm.

(16) C. Evaluation of Nitrite (NO.sub.2.sup.) Concentration

(17) Nitrite level were determined by using the Griess Reagent System, including 50 ml Sulfanilamide Solution, 50 ml NED solution, and 1 ml Nitrite Standard, (Promega Corporation) according to the instructions provided by the manufacturer. Briefly, we added 50 l of standard curves samples, controls, and experimental samples to the 96-well flat-bottom plates. Then dispense 50 l of the Sulfanilamide Solution to all samples and incubate 5-10 minutes at room temperature. Finally, dispense 50 l of the NED solution to all wells and incubate at room temperature 5-10 minutes. The absorbance was measured at 540 nm by Synergy H1 Hybrid Multi-Mode Microplate Reader.

(18) D. Measurement of Cell Viability

(19) The cells were plated in 96-well flat-bottom microplates at a density of 3000 cells per well in 70 L of complete culture medium. Cells were incubated for 24 hours to ensure proper cell adherence and stability. Confluence of each well was confirmed to be at 40%. 30 l of RPMI, saline solution, and plasma-activated saline solutions were added to the corresponding cells. Cells were further incubated at 37 C. for 24 and 48 hours. The viability of the pancreas normal and cancer cells was measured with Cell Counting Kit 8 assay (Dojindo Molecular Technologies, MD). The original culture medium was aspirated and 10 L of CCK 8 reagent was added per well. The plates were incubated for 3 hours at 37 C. The absorbance was measured at 450 nm by Synergy H1 Hybrid Multi-Mode Microplate Reader. We normalized data according to control group (RPMI for BxPC-3, and KSFM for H6c7). We calculated the mean and standard deviation independently.

(20) E. Optical Emission Spectra Measurement

(21) UV-visible-NIR, a range of wavelength 200-850 nm, was investigated on plasma to detect various RNS and ROS (nitrogen [N.sub.2], nitric oxide [NO], nitrogen cation [N.sup.+2], atomic oxygen [O], and hydroxyl radical [OH]). The spectrometer and the detection probe were purchased from Stellar Net Inc. The optical probe was placed 2 cm in front of the plasma beam. Integration time of the collecting data was set to 100 ms.

(22) F. Statistical Analysis

(23) All results were presented as meanstandard deviation plotted using Origin 8. Student's t-test was applied to check the statistical significance (*p<0.05, **p<0.01, ***p<0.001).

(24) Results

(25) A. Current-Voltage Characteristics of Discharge

(26) FIG. 2A shows the current-voltage characteristics of the discharge with air gap at distance of 2-10 mm. With gap increasing, the discharge current decreases while discharge voltage increases. Similar features of the discharge voltage increasing with air gap length are found in the case of electrolyte anode/cathode discharge. See, Bruggeman, P. et al. DC excited glow discharges in atmospheric pressure air in pin-to-water electrode systems. Journal of Physics D: Applied Physics 41, 215201 (2008). The self-organized pattern appears at the plasma-liquid interface and the discharge is stabilized when the air gap length is about 6 mm. At 2 mm gap, the discharge voltage is low while discharge current is high, and the discharge pattern represents a single filament. As the air gap length increases from 2 mm to 4 mm, the anode spot changes to a double ring-like structure. At an air gap length of 4 mm, the double ring structure is a solid inner circle surrounded by a continuous outer circle. At an air gap length of 6 mm, the double ring structure is a solid inner circle surrounded by a discontinuous continuous outer circle formed of a plurality of circular dots. At air gap length of 8 mm, various types of self-organized patterns are formed above the liquid media surface as shown in FIG. 2A. When the air gap is 2 to 8 mm, the plasma discharge is stable. When the air gap is 10 mm however, the plasma discharge becomes unstable. If the air gap is larger than 10 mm, the plasma discharge cannot be sustained.

(27) B. Optical Spectrum of SOP Plasma

(28) We have measured spectra of plasma from the plasma-liquid interface. Typical optical emission spectra are shown in FIG. 3. One can see that with air gap length increasing, the emission intensity decreases. The identification of the emission bands was performed according to the reference. See, Pearse, R. W. B. & Gaydon, A. G. Identification of molecular spectra. (Chapman and Hall, 1976). In the 250-300 nm wavelength range, weak emission bands (258, 267, and 284) were detected as NO lines. See, Walsh, J. L. & Kong, M. G. Contrasting characteristics of linear-field and cross-field atmospheric plasma jets. Applied Physics Letters 93, 111501 (2008).

(29) Species at wavelengths of 337 and 358 nm were defined as N.sub.2.sup.3 or NO .sup.2 (denoted as N.sub.2/NO), because both species have possible optical emission at these wavelengths. See, Pearse, R. W. B. & Gaydon, A. G. Identification of molecular spectra. (Chapman and Hall, 1976). The emission bands between 300 and 500 nm have still not been clearly identified in the literature. See, Chen, W. et al. Treatment of Enterococcus faecalis bacteria by a helium atmospheric cold plasma brush with oxygen addition. Journal of Applied Physics 112, 013304 (2012). However, we anticipated that OH was present at 309 nm, the wavelength of 375 nm could be indicative of N.sub.2.sup.+/N.sub.2, and atomic oxygen (O) was denoted at the wavelength of 777 nm. Atomic oxygen (ground/excited states) is believed to have a significant effect on cells and therefore a broad biomedical application. See, Cheng, X. et al. The effect of tuning cold plasma composition on glioblastoma cell viability. PloS one 9, e98652 (2014). The dominant species of the spectra in these experiments are NO or N2 lines (258, 267, 337, and 357 nm), OH (309 nm), N.sub.2.sup.+ (391 nm), and O (777 nm).

(30) C. H.sub.2O.sub.2 and NO.sub.2.sup. Concentration

(31) Plasma species penetrate through the plasma-liquid interface and can produce chemically reactive species in the saline solution. Complex chemistry is associated with plasma produced species in liquid. See, Chen, Z., Cheng, X., Lin, L. & Keidar, M. Cold atmospheric plasma discharged in water and its potential use in cancer therapy. Journal of Physics D: Applied Physics 50, 015208 (2017). These reactions lead to the formation of short- and long-lived species. H.sub.2O.sub.2 and NO.sub.2.sup. are relatively long-lived species in the plasma-activated saline solution. The air gap length dependencies of the H.sub.2O.sub.2 and NO.sub.2.sup. concentrations in the plasma-activated saline solution with gap distance as a parameter are shown in FIG. 4. The concentration of H.sub.2O.sub.2 increased initially with air gap up to 4 mm then decreased except 10 mm as shown in FIG. 4a. The concentration of NO.sub.2.sup. increases with air gap from 2 mm to 8 mm, then decreases at 10 mm.

(32) D. Cell Viability of H6c7 and BxPC-3

(33) To investigate the potential of plasma-activated saline solution, we treated BxPC-3 human pancreas cancer cells and H6c7 human normal cells with them. RPMI, KSFM, and untreated saline solution were used as controls. FIG. 5 shows the cell viability of BxPC-3 human pancreas cancer cells and H6c7 human pancreas normal cells exposed to RPMI/KSFM, saline solution, and plasma-activated saline solutions for 24 h and 48 h. We can see that plasma-activated saline solutions have stronger effect on the cancer cells than that on the normal cells. For BxPC-3 cancer cells, when incubated for 24 h and 48 h, cell viability decreased firstly then increased with air gap length increasing. The minimum cell viability appears at 4 mm air gap. For H6c7 normal cells, when incubated for 24 h and 48 h, plasma with SOP at 6 mm air gap has the most significant effect of plasma-activated saline solutions.

(34) In the past it was found that under some conditions cold atmospheric plasma can be directly applied to cancer cells without influencing the healthy tissues. Keidar, M. et al. Cold plasma selectivity and the possibility of a paradigm shift in cancer therapy. British journal of cancer 105, 1295-1301 (2011); Keidar, M. Plasma for cancer treatment. Plasma Sources Science and Technology 24, 033001 (2015); Yan, D., Sherman, J. H. & Keidar, M. Cold atmospheric plasma, a novel promising anti-cancer treatment modality. Oncotarget 8, 15977-15995 (2017); Karki, S. B., Thapa Gupta, T., Yildirim-Ayan, E., Eisenmann, K. M. & Ayan, H. Investigation of nonthermal plasma effects on lung cancer cells within 3D collagen matrices. Journal of Physics D Applied Physics 50 (2017); and Karki, S. B., Yildirim-Ayan, E., Eisenmann, K. M. & Ayan, H. Miniature Dielectric Barrier Discharge Nonthermal Plasma Induces Apoptosis in Lung Cancer Cells and Inhibits Cell Migration. BioMed research international 2017 (2017)

(35) At the same time plasma-activated media have been explored and found to have a cytotoxic effect in oncology. In the above experiments, saline solutions were treated by plasma with various SOPs to be applied to human pancreatic cancer and normal cells. Discharge is formed between pin and liquid electrode and result in SOP formation dependent on discharge gap as shown in FIG. 2A.

(36) Transport of ROS/RNS across the plasma/liquid interface is affected by SOP. As such modification of saline solution by discharge is affected and controlled by SOP at the plasma-liquid interface. Typical optical emission spectra of such plasmas at different air gap were shown in FIG. 3 indicating that plasma at each air gap length contains ROS and RNS in the gas phase. ROS and RNS were also formed in plasma-activated saline solution. RNS are well known to induce cell death via DNA double-strand breaks and apoptosis, where ROS are capable of inducing the apoptosis and necrosis. See, Boehm, D., Heslin, C., Cullen, P. J. & Bourke, P. Cytotoxic and mutagenic potential of solutions exposed to cold atmospheric plasma. Scientific reports 6, 21464 (2016) and Kim, S. J. & Chung, T. Cold atmospheric plasma jet-generated RONS and their selective effects on normal and carcinoma cells. Scientific reports 6, 20332 (2016). Our results in FIG. 4 show that the H.sub.2O.sub.2 concentration is highest at 4 mm air gap distance while NO.sub.2.sup. concentration is highest at 8 mm air gap distance. Possible reactions illustrating the routes of H.sub.2O.sub.2 and NO.sub.2.sup. formation in liquid and plasma have been reported in our previous articles.sup.32,40,41. See, Chen, Z., Cheng, X., Lin, L. & Keidar, M. Cold atmospheric plasma discharged in water and its potential use in cancer therapy. Journal of Physics D: Applied Physics 50, 015208 (2017); Chen, Z., Lin, L., Cheng, X., Gjika, E. & Keidar, M. Treatment of gastric cancer cells with nonthermal atmospheric plasma generated in water. Biointerphases 11, 031010 (2016); and Chen, Z., Lin, L., Cheng, X., Gjika, E. & Keidar, M. Effects of cold atmospheric plasma generated in deionized water in cell cancer therapy. Plasma Processes and Polymers 13, 1151-1156 (2016). From FIG. 2 Awe can see that plasma average discharge power is growing with increasing air gap, which results in the temperature of plasma-activated saline solutions going up (except 10 mm). Since H.sub.2O.sub.2 is thermodynamically unstable, its rate of decomposition increases with rising temperature. See, Goss, D. J. & Petrucci, R. H. General Chemistry Principles & Modern Applications, Petrucci, Harwood, Herring, Madura: Study Guide. (Pearson/Prentice Hall, 2007). It should be pointed out that the plasma discharge becomes unstable at the air gap length of about 10 mm. The discharge must be re-ignited. As such, the discharge instability at 10 mm gap might lead to a low concentration of nitrite. FIG. 5 shows that plasma-activated saline solution affects cancer and normal pancreatic cells in a selective manner. Plasma with SOP activating saline solutions have more effect on cancer cells. The trend of pancreatic normal and cancer cells can be attributed to the trend of ROS and RNS concentration with different air gap distances. On the other hand, H.sub.2O.sub.2 reacts with NO.sub.2.sup. to form peroxynitrite OONO.sup. and H.sub.2O. See, Tian, W. & Kushner, M. J. Atmospheric pressure dielectric barrier discharges interacting with liquid covered tissue. Journal of Physics D: Applied Physics 47, 165201 (2014). ONOO.sup. is a powerful oxidant and nitrating agent that is known to be a much more damaging to cancer cells.sup.44. See, Beckman, J. S. & Koppenol, W. H. Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and ugly. American Journal of Physiology-Cell Physiology 271, C1424-C1437 (1996). Therefore, a synergistic effect of ROS and RNS is suspected to play a key role in the apoptosis of the plasma solutions. For BxPC-3 cancer cells, intracellular ROS-mediated up-regulation of DR5 can leads to apoptosis (procaspase-8 is a direct downstream target of DR5). See, Kong, R. et al. Dihydroartemisinin enhances Apo2L/TRAIL-mediated apoptosis in pancreatic cancer cells via ROS-mediated up-regulation of death receptor 5. PLoS One 7, e37222 (2012). On the other hand, intracellular generation of ROS induces increasing protein expression of Bax, disruption of the mitochondrial membrane potential and release of cytochrome c and AIF into the cytosol resulting in to the activation of caspase9/3 cascade. See, Zhang, R., Humphreys, I., Sahu, R. P., Shi, Y. & Srivastava, S. K., In vitro and in vivo induction of apoptosis by capsaicin in pancreatic cancer cells is mediated through ROS generation and mitochondrial death pathway, Apoptosis 13, 1465-1478 (2008). Therefore, plasma with SOP-induced intracellular generation of ROS induced apoptosis in BcPC-3 cancer cells might be orchestrated by the synergistic effects of both extrinsic and intrinsic pathways. The results indicate the cytotoxicity of plasma-activated saline solution is specific to pancreatic adenocarcinoma cancer cells. The plasma-activated saline solution at 4 mm air gap distance had the most significant affect in inducing cell death in pancreatic cancer cells. This is related to certain amounts of ROS and RNS generated by double ring-like structure plasma with SOPs

CONCLUSION

(37) The above experiments demonstrate that self-organized pattern plasma-activated saline solutions applied to both BxPC-3 human pancreatic cancer and H6c7 human pancreatic normal cells exhibit selective manners. The air gap at a distance between 2 and 10 mm results into various shapes of self-organized patterns (SOPs) on saline solution anode. A synergistic effect of RNS and ROS present in the plasma solution is suspected to play a key role in the cell death. The SOP plasma-activated saline solution at 4 mm air gap distance had the most significant affect in inducing cell death in both pancreatic normal and cancer cells. The SOP plasma-activated saline solutions have more serious effect on BxPC-3 human pancreatic adenocarcinoma cancer cells than H6c7 human pancreatic epithelial normal cells. These results suggest that SOP plasma-activated saline solutions can be used with anti-tumor effect for clinical applications.

(38) The foregoing description of the preferred embodiment of the invention has been presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form disclosed, and modifications and variations are possible in light of the above teachings or may be acquired from practice of the invention. The embodiment was chosen and described in order to explain the principles of the invention and its practical application to enable one skilled in the art to utilize the invention in various embodiments as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims appended hereto, and their equivalents. The entirety of each of the aforementioned documents is incorporated by reference herein.