MICRO-COLD ATMOSPHERIC PLASMA DEVICE

Abstract

A micro-sized CAP (CAP) employed to target glioblastoma tumors in the murine brain. Various plasma diagnostic techniques were applied to evaluate the physics of helium CAP such as electron density, discharge voltage, and optical emission spectroscopy. The direct and indirect effects of CAP on glioblastoma (U87MG-RedFluc) cancer cells in vitro were investigated and indicate that CAP-generated short- and long-lived species and radicals [i.e. hydroxyl radical (OH), hydrogen peroxide (H2O2), nitrite (NO2-), et al] with increasing tumor cell death in a dose-dependent fashion. Translation of these findings to an in vivo setting demonstrates that intracranial CAP is effective at preventing glioblastoma tumor growth in the mouse brain. The CAP device can be safely used in mice, resulting in suppression of tumor growth. These initial observations establish the CAP device as a potentially useful ablative therapy tool in the treatment of glioblastoma.

Claims

1. A system for treatment of an area having cancerous cells, comprising: a cold atmospheric plasma delivery device comprising: a housing having a channel within said housing, said channel having an entry port for connecting said channel to a source of gas and an exit port for gas flowing through said channel; a first electrode within said channel; a second electrode outside of said channel; and a capillary tube connected to said exit port.

2. A system for treatment of an area having cancerous cells according to claim 1, further comprising: a controller coupled adapted to control a flow rate of gas flowing from a gas source into said channel and to control a size of a plasma jet exiting said capillary tube.

3. A system for treatment of an area having cancerous cells, comprising: a source of inert gas; a source of electrical energy; a cold atmospheric plasma delivery device comprising: a housing having a channel within said housing connected to said source of inert gas, said channel having an entry port for connecting said channel to a source of gas and an exit port for gas flowing through said channel and said channel having a diameter greater than 1 mm; a first electrode within said channel, said first electrode being connected to said source of electrical energy, wherein electrical energy applied to said first electrode plasmatizes gas within said channel; a second electrode outside of said channel; and a tube connected to said exit port, wherein said tube has a diameter less than 500 m, and said tube directs plasmatized gas flowing out of said exit port onto a target.

4. A system for treatment of an area having cancerous cells according to claim 3, further comprising: a controller coupled adapted to control a flow rate of gas flowing from said gas source into said channel and to control a size of a plasma jet exiting said tube.

5. A system for treatment of an area having cancerous cells according to claim 3, wherein said inert gas comprises helium.

6. A system for treatment of an area having cancerous cells according to claim 3, wherein said target comprises cancerous cells.

7. A system for treatment of an area having cancerous cells according to claim 3, wherein said target comprises a medium.

8. A method of eradicating cancerous cells in an area, the method comprising: generating a cold atmospheric plasma jet directed at the area having cancerous cells via a capillary tube; and controlling the flow rate and size of the plasma jet to the cancerous cells.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] 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:

[0017] FIG. 1A is a schematic representation of the micro-sized cold atmospheric plasma setup.

[0018] FIG. 1B is an illustration of CAP direct treatment: CAP was directly applied to cells in DMEM (100 L).

[0019] FIG. 1C is an illustration of CAP indirect treatment: DI water was treated with CAP and then applied to cells in DMEM (70 L DMEM+30 L treated DI water).

[0020] FIG. 1D is an optical emission spectrum detected from the He CAP using UV-visible-NIR, in the 250-850 nm wavelength range.

[0021] FIG. 2A is a schematic of RMS experimental setup.

[0022] FIG. 2B is a graph of electron number and discharge voltage of He.

[0023] FIGS. 3A and 3B are RNS concentration in He CAP treated DI water (3A) and DMEM (3B). Student t-test was performed, and the statistical significance compared to CAP 5 second treatment is indicated as *p<0.05, **p<0.01, ***p<0.001. (n=3).

[0024] FIGS. 4A and 4B show relative methylene blue (MB) concentration for assessing the concentration of hydroxyl free radicals in He CAP treated DI water (4A) and DMEM (4B). Student t-test was performed, and the statistical significance compared to MB without CAP treatment (0 second) is indicated as *p<0.05, **p<0.01, ***p<0.001. (n=3).

[0025] FIGS. 5A and 5B show H.sub.2O.sub.2 concentration in CAP treated DI water (5A) and DMEM (5B). Student t-test was performed, and the statistical significance compared to CAP 5 second treatment is indicated as *p<0.05, **p<0.01, ***p<0.001. (n=3).

[0026] FIGS. 6A and 6B show cell viability of U87MG after 24 and 48 hours' incubation with He CAP indirect treatment (6A) and direct treatment (6B) for 0, 5, 10, 30, 60, and 120 seconds. The ratios of surviving cells for each cell line were calculated relative to controls (DMEM). Student t-test was performed, and the statistical significance compared to cells present in DMEM (0 s) is indicated as *p<0.05, **p<0.01, ***p<0.001. (n=3).

[0027] FIG. 7A is a photograph of a new CAP device for plasma delivery through an intracranial endoscopic tube to target glioblastoma tumors in the mouse brain for in vivo targeting of glioblastoma tumor with He CAP.

[0028] FIG. 7B illustrates Representative in vivo bioluminescence images illustrating glioblastoma tumor volume (i.e. light emission or radiance) at baseline and 2 days following He CAP or vehicle (helium) treatment. Areas of high photon emission are shown in red and low are shown in blue.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] FIG. 1A illustrates the CAP setup used in this work. At the end of a quartz tube, a 254 m inner radius capillary tube was attached and insulated by epoxy. The feeding gas for this study was industrial purity He which was injected into the quartz tube with a 0.1 L/min gas flow rate. FIG. 1B illustrates CAP direct treatment in which the CAP was used to treat culture medium containing glioblastoma cancer cells. FIG. 1C shows CAP indirect treatment in which the CAP was used to first treat DI water. After treatment, the DI water is transferred to culture medium containing cells (70 L DMEM (containing cells)+30 L treated DI water, total 100 L). The time for both direct and indirect treatment was 5, 10, 30, 60, and 120 seconds. FIG. 1D shows the spectrum of the He CAP with a flow rate of 0.1 L/min. The identification of the emission line and bands was performed mainly according to reference. See, Pearse, R. W. B.; Gaydon, A. G.; Pearse, R. W. B.; Gaydon, A. G., The identification of molecular spectra, Chapman and Hall London: 1976; Vol. 29. In the 250-300 nm wavelength range, weak emission bands were detected as NO lines. Species at the wavelength of 316, 337, 358, and 381 nm could be defined as N.sub.2 .sup.3 or NO .sup.2, as both of species have possible optical emission at these wavelengths. The helium bands were assigned between 500 and 750 nm in FIG. 1D. Species at a wavelength of 309 nm could be defines as OH. Both He bands between 250 and 425 nm could be defined as ROS/RNS.

[0030] The experimental RMS is shown in FIG. 2A. The dependence of the output RMS signal on parameters of the scattering channel can be expressed as U=AV, where A is the proportionality coefficient (A=263.8V/cm.sup.2) and V is the plasma volume. The volume of the plasma column was determined from Intensified Charged-Coupled Device (ICCD) images. is plasma conductivity by using the following expression: =2.8210.sup.4n.sub.e+.sub.m/(.sup.2+.sub.m.sup.2), .sup.1 cm.sup.1, where .sub.m is the frequency of the electron-neutral collisions, n.sub.e is the plasma density, and is the angular frequency. See, Shashurin, A.; Shneider, M.; Dogariu, A.; Miles, R.; Keidar, M., Temporary-resolved measurement of electron density in small atmospheric plasmas. Applied Physics Letters 2010, 96, (17), 171502; Lin, L.; Keidar, M., Cold atmospheric plasma jet in an axial DC electric field. Physics of Plasmas (1994-present) 2016, 23, (8), 083529. Combining these equations, yields n.sub.e=((.sup.2+.sub.m.sup.2)U)/((2.8210.sup.4A.sub.m)V). Therefore, we can calculate the total electron number in the plasma as N.sub.e=n.sub.eV=U(.sup.2+.sub.m.sup.2)/(2.8210.sup.4A.sub.m). The electron number of He CAP is presented in FIG. 2B, and the total electron number for one discharge period is 2.410.sup.9.

[0031] CAP treatment of DI water and DMEM were performed to induce changes in the concentration of ROS and RNS as a function of the treatment time. Indeed, as shown in FIG. 3, NO.sub.2.sup. concentration in He CAP treated DI water and DMEM increase with the duration of treatment time. The NO.sub.2.sup. mainly originates as NO (N.sub.2+e.fwdarw.2N+e, N+O.sub.2.fwdarw.+O, 4NO+O.sub.2+2H.sub.2O.fwdarw.4NO.sub.2.sup.+4H.sup.+), while most of NO is formed in the gas phase during the afterglow a few million seconds after the discharge pulse. The NO.sub.2.sup. concentration in DMEM treated with He CAP is higher than that in DI water.

[0032] CAP treatment of DI (de-ionized) water and DMEM (Dulbecco's Modified Eagle's medium) were performed to induce changes in the concentration of reactive oxygen species (ROS) and reactive nitrogen species (RNS) as a function of the treatment time. Indeed, as shown in FIG. 3, NO.sub.2.sup. concentration in He CAP treated DI water and DMEM increase with the duration of treatment time. The NO.sub.2.sup. mainly originates as NO (N.sub.2+e.fwdarw.2N+e, N+O.sub.2.fwdarw.NO+O, 4NO+O.sub.2+2H.sub.2O.fwdarw.4NO.sub.2.sup.+4H.sup.+)[36-38], while most of NO is formed in the gas phase during the afterglow a few million seconds after the discharge pulse. The NO.sub.2.sup. concentration in DMEM treated with He CAP is higher than that in DI water.

[0033] MB concentration was used to assess the concentration of .OH. It is known that MB reacts with .OH aqueous solutions leading to a visible color change. Satoh, A. Y.; Trosko, J. E.; Masten, S. J., Methylene blue dye test for rapid qualitative detection of hydroxyl radicals formed in a Fenton's reaction aqueous solution, Environmental science & technology 2007, 41, (8), 2881-2887.

[0034] As shown in FIG. 4, the relative MB concentration decreases with treatment time of He CAP. The relative MB concentration decreases by 9.3% after He CAP treated DI water for 120 seconds. While this change is large, He CAP treated DMEM showed a stronger degradation action (14.7%), suggesting that more short-living reactive species are generated in DMEM. Overall, these findings demonstrate that there is an increase in the concentration of OH as a function of CAP treatment time.

[0035] FIG. 5 shows the ROS concentration dependence on treatment time in He CAP treated DI water and DMEM. H.sub.2O.sub.2 generation might be attributed to the high electron density and energy of the plasma (He.fwdarw.He.sup.++e, He.sup.++H.sub.2O.fwdarw.H.sub.2O.sup.++He, H.sub.2O.sup.++H.sub.2O.fwdarw.H.sub.3O.sup.++.OH; He+e.fwdarw.He*+e, He*+H.sub.2O.fwdarw.He+.OH+H.; H.sub.2O+e.fwdarw.H.sub.2O*+e, H.sub.2O*.fwdarw..OH+H.; .OH+OH.fwdarw.H.sub.2O.sub.2). The H.sub.2O.sub.2 concentration increases with the duration of plasma treatment time in both DI water and DMEM, although the concentration of H.sub.2O.sub.2 generated in DI water is higher than DMEM.

[0036] The H.sub.2O.sub.2 concentration increases with the duration of plasma treatment time in both DI water and DMEM, although the concentration of H.sub.2O.sub.2 generated in DI water is higher than DMEM.

[0037] FIG. 6A shows the cell viability of the glioblastoma cancer cells treated with He CAP indirect treatment after 24 and 48 hours. The cell viability after 24 hours incubation (normalized by DMEM control) decreased by approximately 14.2%, 19.9%, 25.4%, 29.0%, 34.7%, and 39.2%, respectively, according to a 0, 5, 10, 30, 60, and 120 second treatment duration. After 48 hours incubation, the cell viability decreased by approximately 18.3%, 35.6%, 34.8%, 41.1%, 54.7%, and 51.4%, respectively, for a 0, 5, 10, 30, 60, and 120 second treatment duration. FIG. 6B shows the cell viability of glioblastoma cancer cells after 24 and 48 hours incubation with He CAP direct treatment (treat 100 L media containing cells) for 0, 5, 10, 30, 60, and 120 seconds duration. The cell viability of the glioblastoma cancer cells after 24 hours incubation (normalized by control) decreased by approximately 15.4%, 27.1%, 28.9%, 44.1%, and 65.1%, respectively, according to 5, 10, 30, 60, and 120 second treatment duration. Similar dose-dependent (i.e. increase treatment time) decreases in cell viability were found with 48 hour incubation.

[0038] In order to determine the effect of the novel plasma device effect on glioblastoma in vivo, we directly applied He CAP for 15 seconds to glioblastoma tumors in the brain of living mice via an implanted endoscopic tube as shown in FIG. 7A. Using in vivo bioluminescence imaging (FIG. 7B), the tumor volume in a control animal (helium only) increased nearly 400% over the course of two days, whereas He CAP treated tumor volume decreased approximately 50% compared with baseline levels. These striking findings demonstrate the potential of CAP to inhibit glioblastoma tumor growth in vivo.

[0039] CAP has received considerable attention for its potential biomedical applications. Emerging fields of application of CAP include wound healing, sterilization of infected tissue, inactivation of microorganisms, tooth bleaching, blood coagulation, skin regeneration, and cancer therapy. This has been collectively termed plasma medicine. Both direct and indirect applications of CAP have been shown to be effective for treating various cancer cells in vitro and in vivo. However, as discussed above, the treatment of tumors deep within the body has been hampered by the limitations of CAP deliver tools. Thus, we aimed to develop and study how application of a novel He CAP device could induce high cell death both in vitro and in vivo.

[0040] Treating culture media containing cells (direct treatment) was compared to treating DI water and removing it to culture media containing cells (indirect treatment). Our findings suggests that both He CAP direct/indirect treatment could induce high cell death in glioblastoma cancer cells (FIG. 6), although, in general direct treatment was more effective than that of indirect. Plasma contains the energies ions, free radicals, reactive species, UV radiation, and the transient electric fields inherent with plasma delivery, which interact with the cells and other living organisms. In many cases, it has been reported that plasma induced apoptosis in cancer cells had no adverse effect on the normal cells when administered at the comparable dosage provided to cancer cells. See, Mirpour, S.; Ghomi, H.; Piroozmand, S.; Nikkhah, M.; Tavassoli, S. H.; Azad, S. Z., The selective characterization of nonthermal atmospheric pressure plasma jet on treatment of human breast cancer and normal cells. IEEE Transactions on Plasma Science 2014, 42, (2), 315-322; Yan, D.; Talbot, A.; Nourmohammadi, N.; Cheng, X.; Canady, J.; Sherman, J.; Keidar, M., Principles of using cold atmospheric plasma stimulated media for cancer treatment. Scientific reports 2015, 5, 18339. As previously shown, the inactivation effect on bacteria by UV-radiation in mostly related to DNA/RNA damage at a UV wavelength of 200-280 nm. See., Chen, W.; Huang, J.; Du, N.; Liu, X. -D.; Wang, X. -Q.; Lv, G. -H.; Zhang, G. -P.; Guo, L. -H.; Yang, S. -Z., Treatment of enterococcus faecalis bacteria by a helium atmospheric cold plasma brush with oxygen addition. Journal of Applied Physics 2012, 112, (1), 013304. Thus, it can be argued that UV photons are not the major CAP species with our experimental setup (FIG. 1). The absorption cross-section of ROS in the UV spectral range is relatively small (Winter, J.; Tresp, H.; Hammer, M.; Iseni, S.; Kupsch, S.; Schmidt-Bleker, A.; Wende, K.; Dnnbier, M.; Masur, K.; Weltmann, K., Tracking plasma generated H2O2 from gas into liquid phase and revealing its dominant impact on human skin cells, Journal of Physics D: Applied Physics 2014, 47, (28), 285401), and the species lines between 300-350 are still not clearly determined. Radicals and electrons generated during plasma formation can be either short-lived or long-lived. These radicals or electrons reach a solution and form many complex reactions, which result in the formation of other short- and long-lived radicals or species. Short-lived radicals or species includes superoxide (O.sub.2.sup.), nitrite (NO), atomic oxygen (O), ozone (O.sub.3), hydroxyl radical (.OH), singlet delta oxygen (SOD, O.sub.2(.sup.1)), peroxynitrite (ONOO), and etc. See, Graves, D. B., The emerging role of reactive oxygen and nitrogen species in redox biology and some implications for plasma applications to medicine and biology. Journal of Physics D: Applied Physics 2012, 45, (26), 263001; Tian, W.; Kushner, M. J., Atmospheric pressure dielectric barrier discharges interacting with liquid covered tissue. Journal of Physics D: Applied Physics 2014, 47, (16), 165201. Long-lived species include hydrogen peroxide (H.sub.2O.sub.2) and nitrite (NO.sub.2).

[0041] In the case of CAP indirect treatment (treatment of DI water and transfer to cells in culture medium), one can argue that the primary effects are associated with long-lived species (H.sub.2O.sub.2 and NO.sub.2.sup.). RNS are known to induce cell death via DNA damage, while ROS can induce cell death by apoptosis and necrosis. S e e, Boehm, D.; Heslin, C.; Cullen, P. J.; Bourke, P., Cytotoxic and mutagenic potential of solutions exposed to cold atmospheric plasma. Scientific reports 2016, 6, 21464; Kim, S. J.; Chung, T., Cold atmospheric plasma jet-generated RONS and their selective effects on normal and carcinoma cells. Scientific reports 2016, 6, 20332. When He is used as a carrier gas, the concentration of H.sub.2O.sub.2 and NO.sub.2.sup. in DI water rises with time (FIG. 3 and FIG. 5). Analyzing cell viability, one can see that He CAP has a strong effect on glioblastoma (U87MG) cancer cells. The CAP-generated H.sub.2O.sub.2 and NO.sub.2 concentration increase with treatment time, in line with the linear increase in cancer cell killing efficiency. However, there might be additional antitumor pathways related to H.sub.2O.sub.2 and NO.sub.2.sup.. For example, H.sub.2O.sub.2 and NO.sub.2.sup. are believed to generate peroxynitrite (H.sub.2O.sub.2+2NO.sub.2.sup.2HONOO.sup.) that is known to be toxic to cells. HONOO.sup. is stable at basic pH (DI water <6) or otherwise it immediately decomposes into NO.sub.3.sup.. However, HONOO.sup. may be difficult to generate in media because media is buffered and only slightly basic (pH >7). See, Kurake, N.; Tanaka, H.; Ishikawa, K.; Kondo, T.; Sekine, M.; Nakamura, K.; Kajiyama, H.; Kikkawa, F.; Mizuno, M.; Hori, M., Cell survival of glioblastoma grown in medium containing hydrogen peroxide and/or nitrite, or in plasma-activated medium. Archives of biochemistry and biophysics 2016, 605, 102-108. On the other hand, aquaporins (AQPs) play a critical role to facilitate the passive diffusion of H.sub.2O.sub.2, especially AQP8. See, Bienert, G. P.; Chaumont, F., Aquaporin-facilitated transmembrane diffusion of hydrogen peroxide, Biochimica et Biophysica Acta (BBA)-General Subjects 2014, 1840, (5), 1596-1604; Ishibashi, K.; Hara, S.; Kondo, S., Aquaporin water channels in mammals, Clinical and experimental nephrology 2009, 13, (2), 107-117; Yan, D.; Talbot, A.; Nourmohammadi, N.; Sherman, J. H.; Cheng, X.; Keidar, M., Toward understanding the selective anticancer capacity of cold atmospheric plasmamodel based on aquaporins (Review), Biointerphases 2015, 10, (4), 040801. Not all AQPs can transport H.sub.2.sub.2, and AQPs show various transportation degrees of H.sub.2O.sub.2 in different types of human tumors. AQP1, 4, 8, and 9 are highly expressed in glioblastoma cell lines. Thus, the distinct expression pattern of AQP8 in glioblastoma might be responsible for cancer cells being sensitive to high H.sub.2O.sub.2 concentrations.

[0042] The effect of both short-lived and long-lived species or radicals is plausible when considering CAP direct treated glioblastoma cancer cells (in vitro, FIG. 6) and tumors in the mouse brain (in vivo, FIG. 7). Of note, DMEM comprises over 30 components such as inorganic salts, amino acids and vitamins and the effect of plasma irradiation on these compounds is still unknown. Similarly, the effect of plasma irradiation in murine brain is also unknown. Therefore, unknown reactive species or radicals could be generated during the irradiation of DMEM and during in vivo application. Short- and long-lived species or radicals in DMEM and mice brain containing .OH, NO, O.sub.2.sup., O, O.sub.3, O.sub.2(.sup.1), ONOO.sup., H.sub.2O.sub.2 and NO.sub.2.sup. are prominent components of antitumor plasma action. The concentration of .OH in DMEM treated by He CAP increases with treatment time (FIG. 4). .OH derived amino acid peroxides can contribute to cell injury because .OH itself and protein (amino acid) peroxides are able to react with DNA, thereby inducing various forms of damage. S e e, Gebicki, S.; Gebicki, J. M., Crosslinking of DNA and proteins induced by protein hydroperoxides, Biochemical Journal 1999, 338, (3), 629-636; Adachi, T.; Tanaka, H.; Nonomura, S.; Hara, H.; Kondo, S. -i.; Hori, M., Plasma-activated medium induces A549 cell injury via a spiral apoptotic cascade involving the mitochondrialnuclear network, Free Radical Biology and Medicine 2015, 79, 28-44.

[0043] When He CAP activates DMEM, the concentration of both H.sub.2O.sub.2 and NO.sub.2.sup. rises with treatment time (FIG. 3 and FIG. 5), and synergism of H.sub.2O.sub.2 and NO.sub.2.sup. mentioned above might be an important factor. CAP also produces significant amount of ozone (O.sub.3), which is known to have strongly aggressive effect on cells. See, Fridman, A.; Chirokov, A.; Gutsol, A., Non-thermal atmospheric pressure discharges. Journal of Physics D: Applied Physics 2005, 38, (2), R1-R24. Ozone has a role in the formation of biologically active.

[0044] ROS and RNS in aqueous media, which may be responsible for cell death. See, Takamatsu, T.; Uehara, K.; Sasaki, Y.; Miyahara, H.; Matsumura, Y.; Iwasawa, A.; Ito, N.; Azuma, T.; Kohno, M.; Okino, A., Investigation of reactive species using various gas plasmas. RSC Advances 2014, 4, (75), 39901-39905; Kaushik, N.; Uddin, N.; Sim, G. B.; Hong, Y. J.; Baik, K. Y.; Kim, C. H.; Lee, S. J.; Kaushik, N. K.; Choi, E. H., Responses of solid tumor cells in DMEM to reactive oxygen species generated by non-thermal plasma and chemically induced ROS systems. Scientific reports 2015, 5, 08587. Atomic oxygen (O) (including the ground state and all the excited states) is believed to have a significant effect on cells. Superoxide (O.sub.2.sup.) generated by plasma can activate mitochondrial-mediated apoptosis by changing mitochondrial membrane potential and simultaneously up-regulates pro-apoptotic genes and down-regulates anti-apoptotic genes for activation of caspases resulting in cell death. Riedl, S. J.; Shi, Y., Molecular mechanisms of caspase regulation during apoptosis, Nature reviews Molecular cell biology 2004, 5, (11), 897-907.

[0045] Singlet delta oxygen O.sub.2(.sup.1g) is another important ROS with the excitation energy of 0.98 eV. Highly reactive molecule O.sub.2(.sup.1g) not only produces oxidative damage in many biological targets but is also a primary active species in the selective killing of tumor cells in the emerging cancer therapy. Moreover, nitrite (NO) and superoxide (O.sub.2.sup.) can easily form ONOO.sup. once they collide. See, Pacher, P.; Beckman, J. S.; Liaudet, L., Nitric oxide and peroxynitrite in health and disease. Physiological reviews 2007, 87, (1), 315-424. ONOO.sup. is a powerful oxidant and nitrating agent, which is known to be highly damaging to tumor cells. See, Cheng, X.; Sherman, J.; Murphy, W.; Ratovitski, E.; Canady, J.; Keidar, M., The effect of tuning cold plasma composition on glioblastoma cell viability, PloS one 2014, 9, (5), e98652.

[0046] Overall, the above results and discussion indicate that both direct and indirect routes of delivering CAP might be useful and should be considered in a clinical medical application. A further understanding of the precise underlying mechanisms will allow for determination of the best combination when used as a treatment strategy.

[0047] The CAP setup consists of a 2 electrode assembly with a central powered electrode and a grounded outer electrode wrapped around the outside of quartz tube (FIG. 1A). At the end of the quartz tube, a 254 m inner radius capillary tube was attached and insulated by epoxy. The two electrodes were connected with a high voltage power supply. The feeding gas for this study was industrial purity helium (He) which was injected into the quartz tube with a 0.1 L/min gas flow rate. The voltage and current were measured by an oscilloscope with high voltage and current probes.

[0048] 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 radicals [OH]). The spectrometer and detection probe were purchased from Stellar Net Inc. The optical probe was placed at a distance of 1.0 cm in front of the plasma jet nozzle. Data were collected with an integration time of 100 ms.

[0049] The experimental RMS system is schematically presented in FIG. 2A. Two microwave horns were used for radiation and detection of microwave signal. Microwave radiation linearly polarized was scattered on the collinearly-oriented plasma channel and the scattered signal was then measured. The detection of the scattered signal was accomplished using a homodyne scheme by means of I/Q mixer, providing in-phase (I) and quadrature (Q) outputs. For the entire range of scattered signals, the amplifiers and mixer were operated in linear mode. The total amplitude of the scattered microwave signal was determined by: U={square root over (I.sup.2+Q.sup.2)}.

[0050] Human glioblastoma cancer cells (U87MG, Perkin Elmer) were cultured in Dulbecco's Modified Eagle Medium (DMEM, Life Technologies) supplemented with 10% (v/v) fetal bovine serum (Atlantic Biologicals) 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.

[0051] A fluorimetric hydrogen peroxide assay Kit (Sigma-Aldrich) was used for measuring the amount of H.sub.2O.sub.2, according to the manufacturer's protocol. Briefly, 50 l of standard curve, control, and experimental samples were added to 96-well flat-bottom black plates, and then 50 l of Master Mix was added to each of well. The plates were incubated for 20 min at room temperature protected from light and fluorescence was measured by a Synergy H1 Hybrid Multi-Mode Microplate Reader at Ex/Em: 540/590 nm.

[0052] RNS level were determined by using a Griess Reagent System (Promega Corporation) according to the instructions provided by the manufacturer. Briefly, 50 l of samples and 50 l of the provided Sulfanilamide Solution were added to 96-well flat-bottom plates and incubated for 5-10 minutes at room temperature. Subsequently, 50 l of the NED solution was added to each well and incubated at room temperature for 5-10 minutes. The absorbance was measured at 540 nm by Synergy H1 Hybrid Multi-Mode Microplate Reader.

[0053] An MB solution was prepared by dissolving MB podwer in DI water and DMEM. MB solutions (100 l per well, 0.01 g/L) in a 96-well flat-bottom black plate were treated by He CAP for 5, 10, 30, 60, and 120 seconds. The gap between the outlet of CAP and the surface of the samples was around 3 mm. As a control, two untreated MB solutions in triplicate were transferred to a 96-well flat-bottom black plate. .OH was measured as the absorbance at 664 nm by a Synergy H1 Hybrid Multi-Mode Microplate Reader.

[0054] U87 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. On day 2, 30 L of DI water was treated by He CAP for 0, 5, 10, 30, 60, and 120 seconds, and was added to cells. Cells were further incubated at 37 C. for 24 and 48 hours. The cell viability of the glioblastoma cancer cells was measured for each incubation time point with an MTT assay. 100 L of MTT solution (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Sigma-Aldrich) was added to each well followed by a 3-hour incubation. The MTT solution was discarded and 100 L per well of MTT solvent (0.4% (v/v) HCl in anhydrous isopropanol) was added to the wells. The absorbance of the purple solution was recorded at 570 nm with a Synergy H1 Hybrid Multi-Mode Microplate Reader.

[0055] U87 cells were plated in 96-well flat-bottom microplates at a density of 3000 cells per well in 100 L of complete culture medium. Cells were incubated for 24 hours to ensure proper cell adherence and stability. On day 2, the cells were treated by He CAP for 0, 5, 10, 30, 60, and 120 seconds. Cells were further incubated at 37 C. for 24 and 48 hours. An MTT assay was used to assess cell viability as described above.

[0056] All animal protocols were approved by the George Washington University Institutional Animal Care and Use Committee. 8 week old female athymic nude mice (Charles River, NU(NCr)-Foxn1.sup.nm) were anesthetized intraperitoneally (i.p.) [Ketamine (100 mg/kg) mixed with Xylazine (10 mg/kg)] and placed in a stereotaxic frame. The surface of the skull was visualized with a dissecting microscope and horizontally leveled between bregma and lambda. A small hole was drilled at the desired location. U87MG were resuspended in DMEM at a concentration of 510.sup.5 cell/L and injected into the frontal lobe at the following coordinates (relative to Bregma) using a Hamilton syringe: 2.2 mm ventral from the dorsal surface of the skull, 1.0 mm caudal, and 2.0 lateral. 510.sup.5 cells were injected at a depth of 2.2 mm and the syringe was then retracted to 1.8 mm and an additional 510.sup.5 cells were then administered. To allow for delivery of CAP, an endoscopic tube was then implanted at a depth of 2.2 mm and secured in place with dental cement. Mice were allowed to recover for 7 days and He CAP or vehicle control (He alone) was then administered. In brief, mice were anesthetized with isofluorane and CAP was applied via the implanted endoscopic tube at 5-second intervals, followed by 15 seconds off, for a total CAP treatment time of 15 seconds. Tumor size was estimated using in vivo bioluminescent imaging before and for up to 48 hours post CAP or vehicle treatment. For bioluminescent imaging, animals were anesthetized with isoflurane and the substrate luciferin was administered i.p. (150 mg/kg). The mice were then transferred to the light-sealed imaging cabinet of an IVIS Lumina K machine and positioned in a nose cone in order to maintain anesthesia. Bioluminescent images were acquired using a charge-coupled device camera cooled to 80 C. to achieve maximal sensitivity. Images were acquired at 10 minutes post substrate injection with an exposure time of 20 seconds, medium binning, F/Stop=1, and EM gain off.

[0057] Results were plotted using Origin 8 as meanstandard deviation. Student t-test was used to check the statistical significance (*p<0.05, **p<0.01, ***p<0.001).

[0058] In summary, the effect of a newly developed CAP on glioblastoma both in vitro and in vivo has been demonstrated. A variety of diagnostics tools were applied to the CAP including optical emission spectroscopy, microwave scattering, and potential measurement, which supplied evidence for reactions of plasma-generated species in media and the mouse brain. The CAP direct treatment has a stronger effect than the indirect treatment due to the synergetic effect of short- and long-lived species, while a strong effect of indirect CAP treatment on cancer cells is likely attributed to the action of long-lived species. The CAP is safe for mice and suppresses tumor growth in the mouse brain. These initial observations establish CAP as a potentially useful ablative therapy in glioblastoma.

[0059] 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.