Application of Cold Atmospheric Plasma in the Treatment of Cancer, Viruses, and Bacteria

Plasma medicine is an emerging interdisciplinary field that combines physics, biomedical, and clinical applications. Cold atmospheric plasma (CAP) technologies, such as Helium Plasma, have been widely used in the field called plasma medicine to inhibit cancer. The main effect of these plasmas is created through highly reactive chemical species. The chemical species generated from CAP are called reactive oxygen species (ROS) and reactive nitrogen species (RNS). CAP, a partly ionized gas formed at atmospheric pressure, has quasi-neutral charges composed of positive and negative charged ions, charged radicals, neutral atoms, and ultraviolet (UV) photons. CAP has gained considerable interest due to its extraordinary ability to influence biological processes and its operation at room temperature. Through the electric breakdown of gases such as helium (He) or argon (Ar) between electrodes driven by a specific voltage, plasma is generated. CAP contains a reactive combination of electrons, ions, excited atoms, molecules, reactive species (e.g., OH, O, H2O2, O3, NO, NO2, etc.), ultraviolet (UV) photons, and electromagnetic (EM) waves, collectively termed reactive agents (RAs). Since plasma generates a wide range of RAs, it has been used in many fields, including medical applications, dentistry, the biomedical sterilization of tools and dental instruments, wound healing, dermatology, and clinical oncology.In oncology, exposing cancerous cells to CAP leads to the generation of free radicals and RAs that are toxic to cancer cells and can induce apoptotic cell death in glioblastoma (GBM) cells. This dissertation offers insight into the applications of CAP in cancer treatment, specifically glioblastoma, detailing cell interactions with plasmas and genetic alterations at molecular levels using RNA sequencing. This study demonstrates how CAP treatment can play a significant role in programmed cell death (apoptosis) and how cells respond to stress from oxidation caused by the generation of ROS. Despite the promising potential of CAP in treating various cancers, its physical effects have not been completely explored. This study analyzed both the chemical and physical potential of CAP in vitro and in vivo on GBM. Glioblastoma multiforme (GBM) is one of the most common and aggressive forms of malignant brain cancer in adults, highly resistant to treatment due to its infiltrative pattern of invasion, rapid growth, and frequent relapses. Standard treatments include surgery, radiotherapy, and Temozolomide (TMZ), but the prognosis remains poor. CAP works by increasing RONS and targets tumor spread. TMZ is an oral, alkylating, chemotherapeutic agent approved by the FDA for GBM treatment, inducing apoptosis by adding a methyl group to the nitrogen-containing bases in DNA, causing breaks in the DNA. However, nearly half of GBM patients are resistant to TMZ due to the methyl guanine methyltransferase (MGMT) DNA repair system. Therefore, a combination therapy of CAP and TMZ is proposed as an essential treatment for GBM. In this study, two different types of human glioblastoma cell lines, TMZ-resistant and TMZ-sensitive, were investigated. The combined effect of CAP and TMZ treatment was analyzed in two ways: 1) CAP treatment followed by immediate replacement with TMZ solution, and 2) CAP treatment pre-applied to the cells before adding TMZ without replacing the media. These methods were performed to check the potential synergistic effect of the co-treatment and the selectivity of CAP in potentially sensitizing the cells post-treatment. The study examined lactate dehydrogenase (LDH) activity, indicating cytotoxicity, along with apoptosis and caspase-3 activity, which imply the anticancer nature of the combination. The findings suggest that CAP enhanced the effects of TMZ in GBM cells, both in sensitive and resistant cell lines, likely due to the reactive species generated by CAP. CAP sensitized both TMZ-resistant T98G and TMZ-sensitive A172 GBM cells to chemotherapy, with reactive species from CAP significantly increasing apoptosis and DNA damage. The combination therapy led to elevated caspase-3 activity and LDH levels, indicating enhanced apoptosis and cellular stress. These promising results indicate that CAP pre-treatment can improve TMZ responses, offering a non-invasive adjunct therapy for GBM that warrants further in vivo validation. Data from subcutaneous studies demonstrated the promising potential of CAP as a non-invasive treatment modality for GBM in a subcutaneous mouse model. Combining CAP with TMZ led to significant tumor inhibition, achieving a 49.84% reduction on day 7 and a significant 85.64% reduction on day 14 compared to control groups. Further investigation into CAP’s physical aspects revealed its ability to mediate cancer suppression through EM waves that can penetrate human bone fibula, leading to secondary ROS generation. This study also explored CAP's role in sensitizing glioblastoma cells to TMZ, even penetrating the bone/skull barrier in mouse models. A single 1-minute, non-invasive direct application of CAP on the skull of mice led to 79% tumor inhibition in the co-treatment. This non-invasive approach shows promise in overcoming GBM treatment challenges and warrants further clinical translation. The study also delved into the molecular mechanisms of CAP treatment, utilizing high throughput RNA-sequencing to explore genetic alterations and outcomes at the molecular level. CAP treatment induced ROS-mediated stress responses and DNA damage, upregulating genes involved in oxidative stress, DNA repair, and apoptosis, while downregulating histone genes and DNA damage markers. Transcriptomic analysis highlighted alterations in key pathways such as cell cycle regulation, P53, MAPK signaling, and DNA damage response. Homology modeling simulations revealed structural alterations in proteins post-treatment, suggesting potential therapeutic targets. The identification of key pathways such as P53 signaling, MAPK signaling, and stress response pathways provides potential targets for enhancing therapeutic efficacy. Next, a novel cold plasma source based on non-equilibrium plasma in a sealed glass tube, named radial cold Plasma Discharge Tube (PDT) or Cold Plasma Tube (CPT), was developed. This PDT demonstrated sensitization and enhancement of therapeutic efficacy of TMZ in combination treatment. In vivo studies using U87MG intracranial models showed that a single 7-minute PDT pretreatment with TMZ led to remarkable tumor inhibition, achieving a 96.25% reduction in tumor growth. PDT alone reduced tumor growth by 43.75%, demonstrating its skull-penetrating effects. The combination treatment also extended the mean survival of PDX tumor-bearing mice by over 100% (110+ days vs. 51 days). PDT's ability to enhance blood-brain barrier permeability highlights its potential in improving GBM treatment. The study also focused on the biomedical applications of CAP in treating viruses like influenza (H1N1) and SARS-CoV-2, the latter causing COVID-19. CAP effectively reduced influenza virus titers and showed significant promise in decontaminating SARS-CoV-2, including on personal protective equipment (PPE). CAP reduced influenza virus titers by approximately 3.77 log TCID50/mL in water and 2.83 log TCID50/mL in media after 120 seconds of exposure. Similarly, DBD devices achieved reductions of 2.11 to 3.11 log TCID50/mL. These findings highlight CAP's potential for decontaminating PPE and improving infection control. The dissertation next explored CAP's potential for decontaminating PPE. For Methicillin Resistant Staphylococcus aureus (MRSA) treatment, CAP demonstrated significant antimicrobial efficacy. Treatment on bacterial colonies showed zones of inhibition averaging 1.88 cm and 2.15 cm for 5 and 10 minutes of exposure, respectively. Colony formation assays demonstrated a 5-log reduction and a 99.999% reduction in bacterial count. Scanning Electron Microscopy (SEM) analysis revealed structural damage to MRSA cells, including membrane blebbing and disruption. These results are particularly significant given that MRSA infections contribute to approximately 80,461 severe illnesses and 11,285 deaths annually in the United States alone, according to FDA statistics. Characterization of a 5×1 array multiplasma jet system ensured consistent delivery of reactive species. Electron densities measured via Rayleigh microwave scattering (RMS) were 12.8 × 10^11 cm-3 at a discharge frequency of 17.5 kHz, 12.9 × 10^11 cm-3 at 16.5 kHz, and 13.4 × 10^11 cm-3 at 16.1 kHz. Throughout these studies, CAP's efficacy was attributed to its generation of RONS which induce oxidative stress, DNA damage, and apoptosis in cancer cells, viruses, and bacteria. The physical properties of CAP, including electromagnetic waves, were also found to contribute to its therapeutic effects, particularly in penetrating barriers such as the skull and blood-brain barrier in mouse models. In conclusion, this comprehensive study demonstrates the versatility and potential of CAP technology across various biomedical applications. It shows significant promise in treating cancer, decontaminating viruses like influenza and SARS-CoV-2, and combating antibiotic-resistant bacteria such as MRSA. CAP emerges as a promising, non-invasive, cost-effective, and adaptable approach for enhancing cancer treatments, virus decontamination, and bacterial infection control. These findings warrant further investigation and optimization for clinical translation, potentially revolutionizing treatment strategies in oncology, infection control, and beyond. Future research should focus on standardizing protocols, optimizing treatment parameters, and ensuring safety for widespread clinical adoption, aiming to enhance therapeutic outcomes and infection control.

Author

• Soni, Vikas

Language

• English

Keyword

• Molecular Biology
• Cancer
• Biomedical Engineering
• Plasma Medicine
• Bioinformatics
• Cell Biology

Date created

• 2024

Type of Work

• Dissertation

License

• All rights reserved

Rights statement

• In Copyright

GW Unit

• Mechanical & Aerospace Engineering

Degree

• Ph.D.

Advisor

• Keidar, Michael

Committee Member(s)

• Keir, Stephen T
• Crandall, Keith A
• Wood, Steven C
• Karunasena, Enusha
• Sherman, Jonathan H

Persistent URL

•  https://scholarspace.library.gwu.edu/etd/sn009z747