Title Page 1

Abstract 7

Contents 10

List of abbreviations 19

List of symbols 22

Chapter 1. Introduction 23

1.1. Nonthermal atmospheric pressure plasma 24

1.2. Plasma in biomedical research 26

1.3. Plasma treated liquids 29

1.4. Cancer and immunogenic cell death 32

1.5. Bacterial pathogens 33

1.6. Objectives of this thesis 34

Chapter 2. Physiological solutions treated with argon gas plasma induce immunogenic cell death in lung cancer cells 35

2.1. Introduction 36

2.2. Material and methods 38

2.2.1. Material and reagents 38

2.2.2. Maintenance of Cell Culture 38

2.2.3. Macrophage-cancer cell co-culture 39

2.2.4. Details of plasma source and liquid treatment 39

2.2.5. Cell viability Analysis 40

2.2.6. Detection of NO species both intracellular and extracellular of cells 41

2.2.7. Intracellular ROS estimation 41

2.2.8. Flow cytometry 42

2.2.9. qRT-PCR 42

2.2.10. Immunoblotting 42

2.2.11. Live/dead cell staining 43

2.2.12. Statistical analysis 43

2.2.13. PTLs exhibit specific suppression of A549 and MDA-MB231 cell growth 45

2.2.14. Accumulation of intracellular (RONS) induced by PTLs 46

2.2.15. PTLs stimulate MDA-MB231 and A549 cells to emit DAMPs 47

2.2.16. Modification of macrophagic cellular organelles after co-culturing with PTL-treated cancer cells 49

2.2.17. Cancer cells immunogenicity enhanced by PTLs 50

2.3. Discussion 51

Chapter 3. Nitric oxide water induced Immunogenic cell death via sensitizing lung adenocarcinoma towards ferroptosis and autophagy 59

3.1. Introduction 60

3.2. Materials and methods 62

3.2.1. Set-up for Indirect co-culture experiment 62

3.2.2. Microwave torch plasma system and characterization of NOW 62

3.2.3. Production, and characterization of NOW 63

3.2.4. Cell viability 64

3.2.5. Colony forming assay 65

3.2.6. Intracellular NO/ROS analysis 65

3.2.7. Lipid peroxidation assay 65

3.2.8. Flow cytometry 66

3.2.9. qRT-PCR 66

3.2.10. Western blotting 66

3.2.11. Immunofluorescence 67

3.2.12. Bio-Transmission electron microscopy 67

3.2.13. Bioinformatics analysis 67

3.2.14. Statistical analysis 68

3.3. Results 68

3.3.1. Physicochemical properties of gaseous reactive species and NOW 68

3.3.2. Cancer cell growth inhibition via NOW 68

3.3.3. NOW instigates ICD via DAMP molecule release 71

3.3.4. NOW-induced mitochondrial dysfunction leading to autophagy 75

3.3.5. NOW instigates ferroptosis-signature genes via intracellular RONS 76

3.3.6. Macrophage organelles enhancement via NOW treatment 79

3.3.7. Suppression of immune blockade-related proteins via NOW 81

3.4. Discussion 82

Chapter 4. Decontaminating gastrointestinal infection causing bacteria with plasma-treated liquids 87

4.1. Introduction 88

4.2. Materials and methods 91

4.2.1. Bacterial species utilized 91

4.2.2. Characterization of plasma system 91

4.2.3. Characterization of plasma treated liquids 92

4.2.4. Inactivation of bacteria via PTLs 93

4.2.5. Live/dead cell staining of bacteria 93

4.2.6. Biofilm assay 94

4.2.7. Intracellular ROS detection 95

4.2.8. Lipid Peroxidation 95

4.2.9. Scanning electron microscopy 95

4.2.10. ATP assessment 96

4.2.11. PMAxxᵀᴹ- based qPCR 96

4.2.12. qRT-PCR analysis 96

4.2.13. Pathogenic analysis of S. enterica 97

4.2.14. Investigation of adhesion and invasion 97

4.2.15. Statistical analysis 98

4.3. Results and discussion 98

4.3.1. Physicochemical characterization 98

4.3.2. Bacterial inactivation via PTLs 103

4.3.3. Surface morphology analysis by SEM 107

4.3.4. Biofilm prevention 107

4.3.5. ROS stress mediated cell damage 109

4.3.6. Pathogenicity dysregulation of S. enterica 112

4.3.7. PTLs restrict VBNC 113

CHAPTER 5. Summary and future Scope 116

5.1. Summary 117

5.2. Future scope 119

References 124

Curriculum Vitae 155

List of Figures 14

Figure 1.1. Four fundamental states of matter 25

Figure 1.2. (A) An overview of plasmas and the vast array of microscopic properties that... 26

Figure 1.3. Timeline highlighting key milestones in the development of biomedical... 28

Figure 1.4. (A) Typical configurations of CAP for biomedical applications; jet, dielectric... 29

Figure 1.5. Schematic representation of RONS interaction in gaseous and liquid phase 30

Figure 2.1. The co-culture setup of THP-1 and A549 cells using trans-wells with 0.4 μm... 39

Figure 2.2. Details of plasma jet set-up. (A) A schematic representation of the experimental... 40

Figure 2.3. The co-culture setup of THP-1 and A549 cells using trans wells 41

Figure 2.4. Impact of plasma treatment on the physical characteristics of plasma-treated... 45

Figure 2.5. Plasma treated liquids induce cytotoxicity and intracellular (A) Cytotoxicity of... 46

Figure 2.6. Immunogenic cell death induction via PTLs. (A, B) Flow cytometry examines the... 48

Figure 2.7. Gene expression of DAMPs post PTL exposure; including CRT, HMGB-1, and... 49

Figure 2.8. PTLs exposed cancer cells triggered activation of mitochondria and lysosome... 50

Figure 2.9. Immunomodulation by PTLs (A) Levels of pro-inflammatory cytokine-related... 51

Figure 3.1. Schematic diagram and parameters of microwave plasma system for producing... 64

Figure 3.2. Physicochemical characteristics of nitric oxide water. (A) Gas phase FTIR... 70

Figure 3.3. Antiproliferative action of NOW on cells. (A) Cell viability of A549 (lung cancer... 71

Figure 3.4. Immunogenic cell death induction post NOW exposure on cells. (A, B) Flow... 73

Figure 3.5. Autophagy and mitochondrial damage caused by NOW. Histograms illustrating... 74

Figure 3.6. Transmission Electron Microscopy (TEM) images displaying mitochondrial... 75

Figure 3.7. TIMER plot representing the TP53/P53 expression in LUAD 76

Figure 3.8. TCGA analysis representing the co-expression of genes related to ferroptosis in... 77

Figure 3.9. Delivery of NO influenced intracellular ROS mediated cellular damage. (A)... 78

Figure 3.10. Activation of macrophage post co-culture with PTLs exposed cancer cells. (A)... 80

Figure 3.11. Inhibition of immunosuppressive protein expressions via NOW. Histograms and... 82

Figure 4.1. Plasma-treated liquids for inactivation of gastrointestinal and nosocomial... 90

Figure 4.2. Biofilm inhibition via PTLs experimental plan 94

Figure 4.3. Experimental set-up of Plasma device (A) Configuration of the experiment and... 99

Figure 4.4. Impact of plasma treatment on the physicochemical characteristics of PTLs. (A)... 101

Figure 4.5. Inhibition of bacterial colonies after PTLs treatment. (A) Images of the CFU of... 105

Figure 4.6. Bacterial cell death induction via PTLs. (A) Flow cytometric analysis... 106

Figure 4.7. Scanning Electron Microscopy (SEM) analysis for images of E. coli and S aureus... 107

Figure 4.8. PTLs sufficiently inhibit the formation of both E. coli and S. aureus.... 108

Figure 4.9. (A) Intracellular ROS detected via H₂DCFDA dye and (B) DCF intensity... 109

Figure 4.10. Antioxidants related gene modulation post PTLs (A) Expression of oxidative... 110

Figure 4.11. Pathogenicity of S. enterica via Type III secretion system pathway 112

Figure 4.12. Impaired pathogenicity of S. enterica via PTLs (A) Effect of cell viability direct... 113

Figure 4.13. Effect of PTLs on the VBNC formation. (A) Effect on cellular metabolism and... 115

Figure 5.1. Impact of plasma treated liquid on the fate of cells 118

Non-thermal atmospheric pressure plasma or cold atmospheric plasma (CAP) is characterized as a partially ionized gas. CAP is a cutting-edge and unique medical technology with numerous other applications, including the treatment of cancer microbial inactivation and wound healing. CAP can be generated by adding energy (heat or electromagnetic fields) to a neutral gas to make it electrically conductive. Physical components of plasma include visible light and electromagnetic radiation, as well as chemical constituents such as free electrons, reactive oxygen, and nitrogen species (RONS), free radicals, and positively and negatively charged ions. These reactive species have demonstrated a range of biological amenities, including anti-inflammatory, anti-microbial, anti-cancer, and tissue regeneration properties.

The first objective focused on the impact of cold atmospheric plasma-treated liquids (PTLs) to mediate immunogenic cell death. RONS in such liquids generate a cocktail that triggers specific toxicity targeting cancer cells. These reactive species exhibit greater persistence in the aqueous phase as compared to the gaseous phase. The use ofindirect plasma therapy to treat cancer has progressively increased attention in the field of plasma medicine. The purpose of this study was to treat cancer by inducing immunomodulation using phosphate-buffered saline (PT-PBS) and Ringer's lactate (PT-RL) solutions treated with plasma. PTLs reduced the proliferation of cancer cells and caused the least amount of cytotoxicity in non-tumor lung cells. The anti-cancer mechanism induced via PTLs in the induction of immunogenic cell death (ICD). Hallmark proteins of ICD known as damage-associated molecular patterns (DAMPs) enhancement were observed following the PTLs expression. Through the formation of pro-inflammatory cytokines, DAMPs, and decreased expression of the immunosuppressive protein; integrin-associated protein (IAP), or cluster of differentiation (CD47), we discovered that PTLs increase intracellular nitrogen oxide species buildup and elevate immunogenicity in cancer cells. Furthermore, PTLs instigated enhanced fluorescence of macrophage organelles (lysosomes and mitochondria) tagged dyes in A549 cells. This action can be co-related with the gain of functionality of macrophages. Collectively we have created a therapeutic strategy using PTLs to induce anti-cancerous immunity.

In the second study, the ICD induction and the impact on several cell death pathways by nitric oxide water (NOW) were explored. Non-small cell lung cancer (NSCLC), particularly lung adenocarcinoma (LUAD), significantly influences cancer-related mortality and is frequently considered poor therapeutic responses due to genetic alterations. Exploring regulated cell death (RCD) mechanisms offers promising avenues to augment immunotherapy by reshaping the tumor microenvironment (TME). Here, we investigate the prospective of microwave plasma-infused NO to initiate ICD while concurrently modulating autophagy and ferroptosis signaling in LUAD-associated A549 cells. Plasma treatment results in stable NO species nitrite/nitrate (NO2−/NO3-) in the water, altering its physiochemical properties. Analysis of ICD markers reveals increased expression of DAMPs at both protein and mRNA levels post-NOW exposure. Intracellular NO accumulation and mitochondrial dysfunction may have triggered autophagy induction. Flow-cytometry and western blotting confirm alterations in autophagy regulators Beclin-1 and SQSTM1. Furthermore, NOW treatment induces lipid peroxidation and upregulates ferroptosis-associated genes, as determined by qRT-PCR.

Transmission electron microscopy (TEM) imaging reveals autophagosome formation and loss of cristae structures, supporting the occurrence of autophagy and ferroptosis. These findings propose that NOW induced autophagic and ferroptosis cell death may enhance ICD, offering a promising strategy to modulate immunogenicity within the TME.

The intended purpose of the third study was to establish whether PTLs demonstrate an antibacterial effect on gastrointestinal pathogenic microorganisms. Nosocomial infections or hospital-acquired infections are now a serious threat and difficult to cure due to rising antibiotic resistance in biofilms, and pathogens. Although the antibacterial properties of CAP have been well investigated, its lack of portability is a disadvantage. The antibacterial potential of plasma has been observed frequently, however, the mechanism induced by the plasma-treated liquids has not been completely understood. Plasma treatment delivers reactive oxygen and nitrogen species (RONS) in the liquid samples, and among many RONS some of them are considered to have a longer lifespan. In the current study, Argon-jet plasma-treated physiological solutions, including plasma-treated Ringer's lactate (RL) phosphate-buffered saline (PBS), and physiological saline to investigate the bactericidal effect against Gram-negative Escherichia coli, Salmonella enterica, and Gram-positive Staphylococcus aureus bacteria. In addition, the effect of PTLs on the pathogenic efficiency of Salmonella enterica was also investigated via direct co-culture with colon cells and macrophages. We observed that these PTLs process long-lived RONS such as hydrogen peroxide and NO-species that have contributed to intracellular ROS accumulation which ultimately resulted in lipid peroxidation and eventually led to the inhibition of bacterial growth. Moreover, this study demonstrated that PTLs have efficiently dysregulated the pathogenicity of S. enterica via modulating T3SS-related effector genes. Also, in comparison to commercial disinfectants, PTLs cause a relatively less viable but not culturable (VBNC) state in bacteria demonstrating its biocompatible nature. In conclusion, this study provides new insights into the potential of cold atmospheric plasma-treated liquids-mediated cancer immunomodulation and microbial deactivation.