Jerome CANADY, et al.
CAP vs Cancer
August 19, 2019
Treat cancer with cold plasma? Purdue aerospace engineer helps bring first clinical trial
by Kayla Wiles

Cold atmospheric plasma technology, currently the only way to remove microscopic cancer tumors remaining from surgery, has been approved by the U.S. Food and Drug Administration for first-ever use in a clinical trial.

For solid tumor cancers such as those in the breast and lungs, standard treatment involves chemotherapy, radiation, surgery or all of the above. When these tumors aren’t fully removed, they can cause the cancer to come back. Approximately 20%-40% of women undergoing partial mastectomy in the U.S. each year, for example, return to surgery because of marginal tumors that the surgeon couldn’t see the first time around.

A multi-institute team, which included Purdue University aerospace engineer Alexey Shashurin, developed a pen-like electrosurgical scalpel that sprays a blue jet of cold plasma at any remaining cancerous tissue or cells for 2-7 minutes. The device targets only tumors, leaving surrounding tissue unharmed, as demonstrated in vitro, in vivo and in FDA-approved compassionate use cases prior to the clinical trial.

U.S. Medical Innovations LLC (USMI) and the Jerome Canady Research Institute for Advanced Biological and Technological Sciences (JCRI/ABTS) led the team and are sponsoring the clinical trial, with plans to recruit patients in September.

USMI developed and patented the first high-frequency electrosurgical generator with cold plasma for the selective treatment of cancer in 2014.

The technology, approved for an FDA phase I clinical trial in 20 patients, was developed by a team led by Canady, chief science officer of JCRI/ABTS, CEO of USMI and a research professor in the School of Engineering and Applied Sciences at The George Washington University; an engineering team led by Taisen Zhuang, the vice president of research and development at USMI; Michael Keidar, a professor in the School of Engineering and Applied Sciences and director of the Micropropulsion and Nanotechnology lab at The George Washington University; and Shashurin, an assistant professor in Purdue’s School of Aeronautics and Astronautics.

“Plasmas are very reactive, which can cause a variety of responses on the cellular level in biological tissue. But because they’re also extremely hot gases, there had been a push over the past 20 years to generate and test cold plasmas for biological applications,” Shashurin said.

In addition to developing cold plasma solutions for cancer treatment technology, Shashurin’s lab also conducts research on various topics of experimental plasma sciences. These include generation and diagnostics of miniature cold plasmas at atmospheric pressure, application of cold plasma for sterilization, laser-induced plasma for combustion diagnostics, advanced spacecraft propulsion and nanosecond repetitive plasma discharges for aerodynamic flow control.

In 2008, Keidar and Shashurin were among an early wave of researchers to develop a cold plasma generator and see that it produces responses from biological tissue. By 2011, the team had published a paper in the British Journal of Cancer showing that cold plasma selectively kills cancer cells in animal models.

Keidar and Shashurin began consulting with USMI in 2013 on creating an industrial-scale prototype of the cold plasma generator and its application for cancer treatment, based on a generator they had developed and patented. The goal was to integrate cold plasma with Canady Hybrid Plasma electrosurgical scalpels already used in operating rooms because these scalpels allow for bloodless surgery. This is due to their ability to cut and coagulate tissue at the same time, sealing off blood vessels.

This cold plasma technology selectively kills tumors through toxic molecules called reactive oxygen species, which damage targeted cancerous tissue but do not affect normal biological tissue. Lasers could also kill tissue, but the high heat would also bring permanent damage to surrounding tissue.

To bridge the advantages of electrosurgical scalpels with cold plasma, JCRI/ABTS and USMI converted standard high-frequency electrosurgical generators into ones that spray cold plasma.

“Cold plasma application is the fourth arm for the treatment of cancer, following chemotherapy, radiation and surgery. There’s no other ‘magic bullet’ out there for killing off residual tissue,” Canady said.

One of the clinical trial sites for this device will be Rush University in Chicago. Meanwhile, Shashurin’s lab at Purdue will continue to collaborate with USMI on further development of this technology.

The work aligns with Purdue's Giant Leaps celebration, acknowledging the university’s global advancements made in health, longevity and quality of life as part of Purdue’s 150th anniversary. This is one of the four themes of the yearlong celebration’s Ideas Festival, designed to showcase Purdue as an intellectual center solving real-world issues.


The effect of cold atmospheric plasma treatment on cancer stem cells
Barry Trink, Michael Keidar, Jerome Canady, Yeela Shamai, Maty Tzukerman

Intratumoral heterogeneity challenges existing paradigms for anticancer therapy. Accumulating evidence demonstrates that the model of cancer stem cells (CSCs) and the model of clonal evolution mutually contribute to intratumoral heterogeneity, as CSCs themselves undergo clonal evolution. The limitation of conventional anticancer therapies may lead to treatment failure and cancer recurrence, mainly due to drug resistance and self-renewal capacities of CSC. These two factors are responsible for resistance to standard oncology treatments. In this study, we examine cold atmospheric plasma (CAP) treatment of CSC in vitro. We demonstrate that two types of heterogeneous CSC populations derived from a single patient tumor are sensitive to the effects of plasma treatment. Surprisingly, the more aggressive CSC population (C13) was more sensitive to CAP treatment than the less aggressive type (C12).

Micro-sized cold atmospheric plasmasource for brain and breast cancer treatment
Zhitong Chen, Li Lin, Qinmin Zheng, Jonathan H.Sherman, Jerome Canady, Barry Trink, Michael Keidar

Micro-sized cold atmospheric plasma (μCAP) has been developedt o expand the applications of CAP in cancer therapy. In this paper, μCAP devices with different nozzle lengths were applied to investigate effects  on  both brain  (glioblastoma U87)  and  breast (MDA-MB-231)cancer  cells. Various diagnostic techniques were employed to evaluate the parameters of μCAP devices with different lengths such as potential distribution, electron density, and optical emission spectroscopy. The generation of short-and long-lived species (such as hydroxyl radical (OH), superoxide (O2-), hydrogen  peroxide  (H2O2),  nitrite  (NO2-),  et  al) were  studied.  These  data  revealed  that  μCAP treatment with a 20 mm length tube has a stronger effect than that of the 60 mm tube due to the synergetic  effects of reactive species and  free  radicals.  Reactive  species  generated  by  μCAP enhanced tumor cell death in a dose-dependent fashion and was not specific with regards to tumor cell type.
Scientific Reports volume 5, Article number: 18339 (2015)

Principles of using Cold Atmospheric Plasma Stimulated Media for Cancer Treatment
Dayun Yan, et al.
To date, the significant anti-cancer capacity of cold atmospheric plasma (CAP) on dozens of cancer cell lines has been demonstrated in vitro and in mice models. Conventionally, CAP was directly applied to irradiate cancer cells or tumor tissue. Over past three years, the CAP irradiated media was also found to kill cancer cells as effectively as the direct CAP treatment. As a novel strategy, using the CAP stimulated (CAPs) media has become a promising anti-cancer tool. In this study, we demonstrated several principles to optimize the anti-cancer capacity of the CAPs media on glioblastoma cells and breast cancer cells. Specifically, using larger wells on a multi-well plate, smaller gaps between the plasma source and the media and smaller media volume enabled us to obtain a stronger anti-cancer CAPs media composition without increasing the treatment time. Furthermore, cysteine was the main target of effective reactive species in the CAPs media. Glioblastoma cells were more resistant to the CAPs media than breast cancer cells. Glioblastoma cells consumed the effective reactive species faster than breast cancer cells did. In contrast to nitric oxide, hydrogen peroxide was more likely to be the effective reactive species.
A Novel Micro Cold Atmospheric Plasma Device for Glioblastoma Both In Vitro and In Vivo
by Zhitong Chen, et al.
Cold atmospheric plasma (CAP) treatment is a rapidly expanding and emerging technology for cancer treatment. Direct CAP jet irradiation is limited to the skin and it can also be invoked as a supplement therapy during surgery as it only causes cell death in the upper three to five cell layers. However, the current cannulas from which the plasma emanates are too large for intracranial applications. To enhance efficiency and expand the applicability of the CAP method for brain tumors and reduce the gas flow rate and size of the plasma jet, a novel micro-sized CAP device (µCAP) was developed and employed to target glioblastoma tumors in the murine brain. Various plasma diagnostic techniques were applied to evaluate the physics of helium µCAP such as electron density, discharge voltage, and optical emission spectroscopy (OES). The direct and indirect effects of µCAP on glioblastoma (U87MG-RedFluc) cancer cells were investigated in vitro. The results indicate that µCAP generates short- and long-lived species and radicals (i.e., hydroxyl radical (OH), hydrogen peroxide (H2O2), and nitrite (NO2−), etc.) with increasing tumor cell death in a dose-dependent manner. Translation of these findings to an in vivo setting demonstrates that intracranial µCAP is effective at preventing glioblastoma tumor growth in the mouse brain. The µCAP device can be safely used in mice, resulting in suppression of tumor growth. These initial observations establish the µCAP device as a potentially useful ablative therapy tool in the treatment of glioblastoma.
PLoS ONE 8(9):e73741 · September 2013

Cold Atmospheric Plasma for Selectively Ablating Metastatic Breast Cancer Cells
Mian Wang, et al.
Traditional breast cancer treatments such as surgery and radiotherapy contain many inherent limitations with regards to incomplete and nonselective tumor ablation. Cold atomospheric plasma (CAP) is an ionized gas where the ion temperature is close to room temperature. It contains electrons, charged particles, radicals, various excited molecules, UV photons and transient electric fields. These various compositional elements have the potential to either enhance and promote cellular activity, or disrupt and destroy them. In particular, based on this unique composition, CAP could offer a minimally-invasive surgical approach allowing for specific cancer cell or tumor tissue removal without influencing healthy cells. Thus, the objective of this research is to investigate a novel CAP-based therapy for selectively bone metastatic breast cancer treatment. For this purpose, human metastatic breast cancer (BrCa) cells and bone marrow derived human mesenchymal stem cells (MSCs) were separately treated with CAP, and behavioral changes were evaluated after 1, 3, and 5 days of culture. With different treatment times, different BrCa and MSC cell responses were observed. Our results showed that BrCa cells were more sensitive to these CAP treatments than MSCs under plasma dose conditions tested. It demonstrated that CAP can selectively ablate metastatic BrCa cells in vitro without damaging healthy MSCs at the metastatic bone site. In addition, our study showed that CAP treatment can significantly inhibit the migration and invasion of BrCa cells. The results suggest the great potential of CAP for breast cancer therapy.
Plasma 2018, 1(1), 218-228
Treatment of Triple-Negative Breast Cancer Cells with the Canady Cold Plasma Conversion System: Preliminary Results
by Xiaoqian Cheng, et al.
Triple-negative breast cancer is a phenotype of breast cancer where the expression level of estrogen, progesterone and human epidermal growth factor receptor 2 (HER2) receptors are low or absent. It is more frequently diagnosed in younger and premenopausal women, among which African and Hispanic have a higher rate. Cold atmospheric plasma has revealed its promising ant-cancer capacity over the past two decades. In this study, we report the first cold plasma jet delivered by the Canady Cold Plasma Conversion Unit and characterization of its electric and thermal parameters. The unit effectively reduced the viability of triple-negative breast cancer up to 80% without thermal damage, providing a starting point for future clinical trials.
Scientific Reports volume 8, Article number: 15418 (2018)

The Cell Activation Phenomena in the Cold Atmospheric Plasma Cancer Treatment
Dayun Yan, et al.

Cold Atmospheric Plasma (CAP) is an ionized gas with a near room temperature. CAP is a controllable source for reactive species, neutral particles, electromagnetic field and UV radiation. CAP showed the promising application in cancer treatment through the demonstration in vitro and in vivo. In this study, we first demonstrate the existence of an activation state on the CAP-treated cancer cells, which drastically decreases the threshold of cell vulnerability to the cytotoxicity of the CAP-originated reactive species such as H2O2 and NO2−. The cytotoxicity of CAP treatment is still dependent on the CAP-originated reactive species. The activation state of cancer cells will not cause noticeable cytotoxicity. This activation is an instantaneous process, started even just 2 s after the CAP treatment begins. The noticeable activation on the cancer cells starts 10–20 s during the CAP treatment. In contrast, the de-sensitization of activation takes 5 hours after the CAP treatment. The CAP-based cell activation explains the mechanism by which direct CAP treatment causes a much stronger cytotoxicity over the cancer cells compared with an indirect CAP treatment do, which is a key to understand what the effect of CAP on cancer cells.
October 2012
Cold Atmospheric Plasma in Cancer Therapy
Michael Keidar, et al.
Plasma is an ionized gas that is typically generated in high-temperature laboratory conditions. Recent progress in atmospheric plasmas led to the creation of cold plasmas with ion temperature close to room temperature. Areas of potential application of cold atmospheric plasmas (CAP) include dentistry, drug delivery, dermatology, cosmetics, wound healing, cellular modifications, and cancer treatment. Various diagnostic tools have been developed for characterization of CAP including intensified charge-coupled device cameras, optical emission spectroscopy and electrical measurements of the discharge propertied. Recently a new method for temporally resolved measurements of absolute values of plasma density in the plasma column of small-size atmospheric plasma jet utilizing Rayleigh microwave scattering was proposed [1,2]. In this talk we overview state of the art of CAP diagnostics and understanding of the mechanism of plasma action of biological objects. The efficacy of cold plasma in a pre-clinical model of various cancer types (long, bladder, and skin) was recently demonstrated [3]. Both in-vitro and in-vivo studies revealed that cold plasmas selectively kill cancer cells. We showed that: (a) cold plasma application selectively eradicates cancer cells in vitro without damaging normal cells. For instance a strong selective effect was observed; the resulting 60--70% of lung cancer cells were detached from the plate in the zone treated with plasma, whereas no detachment was observed in the treated zone for the normal lung cells under the same treatment conditions. (b) Significantly reduced tumor size in vivo. Cold plasma treatment led to tumor ablation with neighbouring tumors unaffected. These experiments were performed on more than 10 mice with the same outcome. We found that tumors of about 5mm in diameter were ablated after 2 min of single time plasma treatment. The two best known cold plasma effects, plasma-induced apoptosis and the decrease of cell migration velocity can have important implications in cancer treatment by localizing the affected area of the tissue and by decreasing metastasic development. In addition, cold plasma treatment has affected the cell cycle of cancer cells. In particular, cold plasma induces a 2-fold increase in cells at the G2/M-checkpoint in both papilloma and carcinoma cells at about 24 hours after treatment, while normal epithelial cells (WTK) did not show significant differences. It was shown that reactive oxygen species metabolism and oxidative stress responsive genes are deregulated. We investigated the production of reactive oxygen species (ROS) with cold plasma treatment as a potential mechanism for the tumor ablation observed.
Cancers 2019, 11(5), 671
Acidification is an Essential Process of Cold Atmospheric Plasma and Promotes the Anti-Cancer Effect on Malignant Melanoma Cells
by Christin Schneider, et al.
(1) Background: Cold atmospheric plasma (CAP) is ionized gas near room temperature. The anti-cancer effects of CAP were confirmed for several cancer types and were attributed to CAP-induced reactive species. However, the mode of action of CAP is still not well understood. (2) Methods: Changes in cytoplasmic Ca2+ level after CAP treatment of malignant melanoma cells were analyzed via the intracellular Ca2+ indicator fura-2 AM. CAP-produced reactive species were determined by fluorescence spectroscopic and protein nitration by Western Blot analysis. (3) Results: CAP caused a strong acidification of water and solutions that were buffered with the so-called Good buffers, while phosphate-buffered solutions with higher buffer capacity showed minor pH reductions. The CAP-induced Ca2+ influx in melanoma cells was stronger in acidic pH than in physiological conditions. NO formation that is induced by CAP was dose- and pH-dependent and CAP-treated solutions only caused protein nitration in cells under acidic conditions. (4) Conclusions: We describe the impact of CAP-induced acidification on the anti-cancer effects of CAP. A synergistic effect of CAP-induced ROS, RNS, and acidic conditions affected the intracellular Ca2+ level of melanoma cells. As the microenvironment of tumors is often acidic, further acidification might be one reason for the specific anti-cancer effects of CAP.

System and Method for Treating Cancer Through DNA Damage With Cold Atmospheric Plasma With Self-organized Patterns
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Field of the Invention

[0004] The present invention relates to systems and methods for using Cold Atmospheric Plasma (“CAP”) to treat cancer.

Brief Description of the Related Art

[0005] Breast cancer is one of the most common cancers diagnosed among American women (excluding skin cancers), which is the second leading cause of cancer death among women after lung cancer. See, C. E. DeSantis, J. Ma, A. Goding Sauer, L. A. Newman, A. Jemal, Breast cancer statistics, 2017, racial disparity in mortality by state, CA: a cancer journal for clinicians 67(6) (2017) 439-448. The global burden of breast cancer exceeds all other cancers and the incidence rates of breast cancer are increasing. A. Jemal, R. Siegel, J. Xu, E. Ward, Cancer statistics, 2010, CA: a cancer journal for clinicians 60(5) (2010) 277-300. Different treatment methods including surgical techniques, medication drugs, and radiation-based approaches are routinely being used for breast cancer. L. Hutchinson, Breast cancer: Challenges, controversies, breakthroughs, Nature Reviews Clinical Oncology 7 (2010) 669-670. However, additional treatment modalities need to be developed to minimize the morbidity and mortality associated with this disease. Breast cancer represent a multitude of different diseases with intratumoral and intertumoral genetic and epigenetic alterations.

[0006] Plasma medicine is emerging as an innovative field for cancer therapy, which combines biology, chemistry, plasma, and medicine. See, G. Fridman, G. Friedman, A. Gutsol, A. B. Shekhter, V. N. Vasilets, A. Fridman, Applied plasma medicine, Plasma Processes and Polymers 5(6) (2008) 503-533 and M. Keidar, Plasma for cancer treatment, Plasma Sources Science and Technology 24(3) (2015) 033001. Plasma is one of the four fundamental states of matter, and is a fully or partially ionized gas. See, M. Keidar, A. Shashurin, O. Volotskova, M. Ann Stepp, P. Srinivasan, A. Sandler, B. Trink, Cold atmospheric plasma in cancer therapy, Physics of Plasmas 20(5) (2013) 057101. Historically, plasma could be generated only at high temperatures or in vacuum, while more recent studies have reported on plasma generated at atmospheric pressure and at room temperature (cold atmospheric plasma, CAP). See, E. Stoffels, Y. Sakiyama, D. B. Graves, Cold atmospheric plasma: charged species and their interactions with cells and tissues, IEEE Transactions on Plasma Science 36(4) (2008) 1441-1457; S. B. Karki, T. T. Gupta, E. Yildirim-Ayan, K. M. Eisenmann, H. Ayan, Investigation of non-thermal plasma effects on lung cancer cells within 3D collagen matrices, Journal of Physics D: Applied Physics 50(31) (2017) 315401; and S. B. Karki, E. Yildirim-Ayan, K. M. Eisenmann, H. Ayan, Miniature dielectric barrier discharge nonthermal plasma induces apoptosis in lung cancer cells and inhibits cell migration, BioMed research international 2017 (2017).

[0007] CAP has attracted a lot of attentions because of its remarkable potential to affect biological processes. Yan, D.; Sherman, J. H.; Cheng, X.; Ratovitski, E.; Canady, J.; Keidar, M. Controlling plasma stimulated media in cancer treatment application. Appl. Phys. Lett. 2014, 105, 224101. The potential of CAP in diverse bio-medical applications has been explored, including wound treatments, blood coagulation, disinfection, control of inflammation, regenerative medicine, and cancer therapy. Z. Chen, H. Simonyan, X. Cheng, E. Gjika, L. Lin, J. Canady, J. H. Sherman, C. Young, M. Keidar, A novel micro cold atmospheric plasma device for glioblastoma both in vitro and in vivo, Cancers 9(6) (2017) 61. The efficacy of CAP in the proposed applications relies on the synergistic action of the reactive oxygen species (ROS), reactive nitrogen species (RNS), free radicals, ultraviolet (UV) photons, charged particles, and electric fields. See, S. B. Karki, T. T. Gupta, E. Yildirim-Ayan, K. M. Eisenmann, H. Ayan, Investigation of non-thermal plasma effects on lung cancer cells within 3D collagen matrices, Journal of Physics D: Applied Physics 50(31) (2017) 315401 and O. Volotskova, T. S. Hawley, M. A. Stepp, M. Keidar, Targeting the cancer cell cycle by cold atmospheric plasma, Scientific reports 2 (2012) 636.

[0008] ROS and RNS, combined or independently, are known to promote cell proliferation as well as cell death, additionally, extreme amounts of ROS and RNS may lead to the damage of proteins, lipids, senescence and induce apoptosis. P. Attri, T. Sarinont, M. Kim, T. Amano, K. Koga, A. E. Cho, E. H. Choi, M. Shiratani, Influence of ionic liquid and ionic salt on protein against the reactive species generated using dielectric barrier discharge plasma, Scientific reports 5 (2015) 17781 and Z. Chen, L. Lin, X. Cheng, E. Gjika, M. Keidar, Treatment of gastric cancer cells with nonthermal atmospheric plasma generated in water, Biointerphases 11(3) (2016) 031010. Many studies of CAP for cancer treatment have shown that CAP dose not harm normal tissues when applied at the appropriate dosages. See, A. Shashurin, M. Keidar, S. Bronnikov, R. Jujus, M. Stepp, Living tissue under treatment of cold plasma atmospheric jet, Applied Physics Letters 93(18) (2008) 181501 and S. N. Zucker, J. Zirnheld, A. Bagati, T. M. DiSanto, B. Des Soye, J. A. Wawrzyniak, K. Etemadi, M. Nikiforov, R. Berezney, Preferential induction of apoptotic cell death in melanoma cells as compared with normal keratinocytes using a non-thermal plasma torch, Cancer biology & therapy 13(13) (2012) 1299-1306. Taken together, CAP therapy has been introduced as a cost effective, rapid and selective treatment modality for killing cancer cells. In addition, CAP with self-organized patterns has recently attracted significant attentions on cancer therapy. Z. Chen, L. Lin, E. Gjika, X. Cheng, J. Canady, M. Keidar, Selective treatment of pancreatic cancer cells by plasma-activated saline solutions, IEEE Transactions on Radiation and Plasma Medical Sciences (2017) and Z. Chen, S. Zhang, I. Levchenko, I. I. Beilis, M. Keidar, In vitro Demonstration of Cancer Inhibiting Properties from Stratified Self-Organized Plasma-Liquid Interface, Scientific reports 7(1) (2017) 12163.

[0009] Self-organization is generally referred to as a process of spontaneous transition from a homogeneous stable state to a regular pattern in a spatially extended system. See, Radehaus, C., Dirksmeyer, T., Willebrand, H. & Purwins, H.-G. Pattern formation in gas discharge systems with high impedance electrodes. Physics Letters A 125, 92-94 (1987) and Jäger, D., Baumann, H. & Symanczyk, R. Experimental observation of spatial structures due to current filament formation in silicon pin diodes. Physics Letters A 117, 141-144 (1986). Self-organization is a complex and fascinating phenomenon commonly observed in both natural and technological contexts within diverse varieties of physics, chemistry and biology. Raizer, Y. P. & Mokrov, M. Physical mechanisms of self-organization and formation of current patterns in gas discharges of the Townsend and glow types. Physics of Plasmas 20, 101604 (2013) and Trelles, J. P. Formation of self-organized anode patterns in arc discharge simulations. Plasma Sources Science and Technology 22, 025017 (2013). Different types of self-organization phenomena have been reported in a wide range of plasmas, such as dielectric barrier discharge (see, Kogelschatz, U. Filamentary, patterned, and diffuse barrier discharges. IEEE Transactions on plasma science 30, 1400-1408 (2002)), high frequency discharge (see, Shi, J., Liu, D. & Kong, M. G. Plasma stability control using dielectric barriers in radio-frequency atmospheric pressure glow discharges. Applied physics letters 89, 081502 (2006)), gas flow stabilized discharges (see, Akishev, Y. et al. The influence of electrode geometry and gas flow on corona-to-glow and glow-to-spark threshold currents in air. Journal of Physics D: Applied Physics 34, 2875 (2001) and Shirai, N., Ibuka, S. & Ishii, S. Atmospheric DC glow discharge observed in intersecting miniature gas flows. IEEE Transactions on Plasma Science 36, 960-961 (2008)), resistively stabilized discharged (see, Laroussi, M., Alexeff, I., Richardson, J. P. & Dyer, F. F. The resistive barrier discharge. IEEE Transactions on Plasma Science 30, 158-159 (2002)), and discharges with liquid electrodes (see, Laroussi, M., Lu, X. & Malott, C. M. A non-equilibrium diffuse discharge in atmospheric pressure air. Plasma Sources Science and Technology 12, 53 (2003), André, P. et al. Experimental study of discharge with liquid non-metallic (tap-water) electrodes in air at atmospheric pressure. Journal of Physics D: Applied Physics 34, 3456 (2001) and Chen, Z., Zhang, S., Levchenko, I., Beilis, I. I. & Keidar, M. In vitro Demonstration of Cancer Inhibiting Properties from Stratified Self-Organized Micro-Discharge Plasma-Liquid Interface. arXiv preprint arXiv: 1701. 01655 (2017)). The self-organization phenomena associated with the formation of electrode patterns are significantly different among these discharges, which typically occur in the anode or cathode layer. Benilov, M. Bifurcations of current transfer through a collisional sheath with ionization and self-organization on glow cathodes. Physical Review E 77, 036408 (2008) and Schoenbach, K. H., Moselhy, M. & Shi, W. Self-organization in cathode boundary layer microdischarges. Plasma Sources Science and Technology 13, 177 (2004). Self-organization patterns (SOPs) of plasma include square-textures, square-lattices, square/hexagonal superlattices, hollow-hexagonal, multi-armed spirals, rotating-wheels patterns, etc. Dong, L., Fan, W., He, Y. & Liu, F. Self-organized gas-discharge patterns in a dielectric-barrier discharge system. IEEE Transactions on Plasma Science 36, 1356-1357 (2008) and Dong, L. et al. Collective vibration of discharge current filaments in a self-organized pattern within a dielectric barrier discharge. Physical Review E 85, 066403 (2012). The formation of these patterns depends on various parameters such as driving current, electrolyte conductivity, gap length, gas species, and so on. See, Shirai, N., Uchida, S. & Tochikubo, F. Influence of oxygen gas on characteristics of self-organized luminous pattern formation observed in an atmospheric dc glow discharge using a liquid electrode. Plasma Sources Science and Technology 23, 054010 (2014), Shirai, N., Ibuka, S. & Ishii, S. Self-organization pattern in the anode spot of an atmospheric glow microdischarge using an electrolyte anode and axial miniature helium flow. Applied Physics Express 2, 036001 (2009) and Zheng, P. et al. Self-organized pattern formation of an atmospheric-pressure, ac glow discharge with an electrolyte electrode. Plasma Sources Science and Technology 24, 015010 (2014). Recently, plasma discharges with the liquid electrode have been studied referring to applications ranging from water decontamination and activation (see, Locke, B., Sato, M., Sunka, P., Hoffmann, M. & Chang, J.-S. Electrohydraulic discharge and nonthermal plasma for water treatment. Industrial & engineering chemistry research 45, 882-905 (2006) and Ostrikov, K. K., Cvelbar, U. & Murphy, A. B. Plasma nanoscience: setting directions, tackling grand challenges. Journal of Physics D: Applied Physics 44, 174001 (2011)), to nanoparticle and materials synthesis (Ostrikov, K. K., Cvelbar, U. & Murphy, A. B. Plasma nanoscience: setting directions, tackling grand challenges. Journal of Physics D: Applied Physics 44, 174001 (2011) and Richmonds, C. & Sankaran, R. M. Plasma-liquid electrochemistry: rapid synthesis of colloidal metal nanoparticles by microplasma reduction of aqueous cations. Applied Physics Letters 93, 131501 (2008)), and medicine (see, Kong, M. G. et al. Plasma medicine: an introductory review. New Journal of Physics 11, 115012 (2009)). Therefore, self-organization in plasma interacting with surfaces is interest not only from a fundamental point of view as intrinsic and fascinating characteristics of nature, but also from practical standpoint in current and emerging technological applications.


[0010] The present invention creates plasma with different self-organization patterns (SOPs) to activate saline solution. The plasma activated saline solutions have anti-tumor effects on human cancer cells.

[0011] Plasma interacting with the liquid generates reactive oxygen species (ROS) and reactive nitrogen species (RNS) that act as key intermediate for cancer therapy. See, Boehm, D., Heslin, C., Cullen, P. J. & Bourke, P. Cytotoxic and mutagenic potential of solutions exposed to cold atmospheric plasma. Scientific reports 6 (2016); Chen, Z. et al. A Novel Micro Cold Atmospheric Plasma Device for Glioblastoma Both In Vitro and In Vivo. Cancers 9, 61 (2017). The present invention creates plasma with different self-organization patterns (SOPs) to activate a media such as saline solution. The plasma activated medias have anti-tumor effects on human normal and cancer cells. A camera was used to characterize the patterns of plasma with SOP. The spectra of plasma with SOPs were determined by UV-visible-NIR optical emission spectroscopy OES). The concentration of hydrogen peroxide (H2 O2 ) and nitrite (NO2 − ) was measured by using a Fluorimetric hydrogen peroxide assay kit, and the Griess reagent system, respectively. The cell viability of H6c7 and BxPC-3 was measured via Cell Counting KIT 8 Assay. Typically, saline solution is used to treat dehydration by injection into a vein, and it is also used to dilute medications to be given by injection. Based on the results, one can suggest that SOP plasma-activated saline solutions (plasma solutions) has the potential to be utilized as an oral medicine or drug injected into tumors.

[0012] In a preferred embodiment, the present invention is a method for manufacturing plasma-activated media for treatment of cancer cells. The method comprises immersing a first electrode in a media in a container, positioning a second electrode at a fixed distance from a surface of the media in the container, and applying electrical energy to the second electrode for a fixed period of time, wherein the fixed distance and the fixed period of time are selected to cause a plasma self-organized pattern at a surface of the media with an atmospheric discharge between the second electrode and the first electrode. The fixed distance preferably is 4-6 mm. The fixed time may be, for example, 40 seconds...

Method for making and Using Cold Atmospheric Plasma Stimulated Media for Cancer Treatment
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A method for preparing cold atmospheric plasma stimulated cell culture media with a cold atmospheric plasma system having a delivery port out of which an inert gas flows. The inert gas may be helium. The method comprises the steps of placing a cell culture media in a first well, the first well having a bottom and having a diameter greater than 20 mm; wherein the cell culture media placed in the first well has a volume of 4 ml or less, treating the cell culture media in the first well with cold atmospheric plasma, wherein the treating is performed with a gap between the delivery port and the bottom of the first well is between 2.5 cm and 3.5 cm, and transferring a portion of the treated media to cultured cancer cells in a second well. The cold atmospheric plasma may be applied for 0.5 minutes to 2 minutes.