MANISH ADHIKARI

Cold atmospheric plasma (CAP), a near room temperature ionized gas, has shown potential application in many branches of medicine, particularly in cancer treatment. In previous studies, the biological effect of CAP on cancer cells and other mammalian cells has been based solely on the chemical factors in CAP, particularly the reactive species. Therefore, plasma medicine has been regarded as a reactive species-based medicine, and the physical factors in CAP such as the thermal effect, ultraviolet irradiation, and electromagnetic effect have been regarded as ignorable factors. In this study, we investigated the effect of a physical CAP treatment on glioblastoma cells. For the first time, we demonstrated that the physical factors in CAP could reinstate the positive selectivity on CAPtreated astrocytes. The positive selectivity was a result of necrosis, a new cell death in glioblastoma cells characterized by the leak of bulk water from the cell membrane. The physically-based CAP treatment overcomed a large limitation of the traditional chemically based CAP treatment, which had complete dependence on the sensitivity of cells to reactive species. The physically-based CAP treatment is a potential non-invasive anti-tumor tool, which may have wide application for tumors located in deeper tissues. Glioblastoma multiforme (GBM) is characterized as a highly invasive, aggressive brain tumor 1. Individuals with GBM face a poor prognosis, with few surviving past the 2-year mark 1,2. A combination of chemotherapy, surgical resection, and radiotherapy is the gold standard for glioblastoma therapy, however, each component has its own drawbacks 1,3,4. Glioblastoma tumors generally originate deep in the brain and a new treatment option, particularly a non-invasive method, is needed to enhance the anti-cancer efficacy and decrease damage to normal tissues. CAP is a cocktail containing different reactive oxygen species (ROS), reactive nitrogen species (RNS), other charged particles, neutral particles, and electrons as well as physical factors, such as thermal effect, ultraviolet (UV), and electromagnetic (EM) waves 5-7. CAP has wide application in many areas, ranging from plasma chemistry, surface modification, decomposition of gaseous pollutants, medical sterilization, and microbial decontamination 8-12. CAP also shows a wide application in cancer treatment 13-16. CAP treatment has demonstrated strong and selective anti-cancer capacity in many cancer cell lines, including breast cancer, colorectal cancer, cervical cancer, skin cancer, and brain cancer 15. CAP also effectively inhibits the growth of subcutaneous xenograft tumors as well as melanoma by a transdermal treatment above the skin of the tumor site 17. In addition, some recent clinical trials have started to show the promising anti-tumor effect of CAP 18,19. To date, all reported anti-cancer effects of CAP treatment, both in vitro and in vivo have generally been regarded as the cellular responses to the chemical factors, particularly the reactive species 20-22. Experiments using CAP-activated medium further support this conclusion 23-27. H 2 O 2 has been regarded as a key player resulting in plasma medicine being referred to as H 2 O 2-medicine, but is also denoted as NO 2-medicine and other reactive species-based medicine in some cases 27-29 .

Cold atmospheric plasma (CAP) generated at temperature below 40°C is an emerging field in biomedical applications. CAP affects living cells by elucidating the physical and biochemical mechanisms. CAP-mediated drug delivery is one of the most promising methods used nowadays by generating mostly in volume or surface dielectric barrier discharge or by CAP jet. The CAP produced various kinds of free radicals according to the gas used, UV radiation, and heat along with change in surface pH which eventually aid in transdermal drug delivery. The lipophobic drugs or higher-molecular-weight drugs are unable to penetrate upper layer of the skin. The CAP alone can change the lipophilicity of the stratum corneum (upper layer of skin) according to the treatment time and temperature. Combination of CAP with antibodies, nanoparticles, and quantum dots also helps in efficient drug delivery. This chapter revealed this multidisciplinary field of research.

BACKGROUND: Many patients with CNS tumors express interest in initiating a ketogenic diet (KD). Following a discussion with the physician regarding the limited clinical evidence of KD for CNS tumors, interested patients were referred for evaluation by an oncology-certified dietitian. METHODS: We performed a single institution retrospective chart review of patients with CNS tumors on a KD. Demographics, clinical characteristics, and diet information were extracted from patient charts. Descriptive statistics were conducted to summarize patterns of adherence to KD across patient characteristics. RESULTS:

Seed germination and vegetative growth are two important plant growing stages that are vulnerable to physical and biological stress. Improvement in crop germination potential and seedling growth rate generally leads to high crop productivity. Cold plasma is a promising technology used to improve seed germination and growth. Structural changes on tomato seed surface exposed with cold air plasma jet for a different time period (1 min, 5 min, 10 min) was examined by SEM. For in-depth study, different physiological parameter such as seed germination and seedling growth, biochemical parameter such as reactive species status, antioxidants and phytohormone, and molecular analysis of various gene expression was also evaluated. Drought stress tolerance potential of cold plasma primed tomato seedling was also examined under 30% PEG stress. Cold plasma seed priming modulates tomato seed coat and improves the germination efficiency. It also induces growth, antioxidants, phytohormone, defense gene expression, and drought stress tolerance potential of tomato seedling. Cold plasma seeds priming augment the reactive species at a molecular level within seedlings, which changes the biochemistry and physiological parameters of plants by inducing different cellular signaling cascades.

Background: Autophagy is reported as a survival or death-promoting pathway that is highly debatable in different kinds of cancer. Here, we examined the co-effect of cold atmospheric plasma (CAP) and silymarin nanoemulsion (SN) treatment on G-361 human melanoma cells via
autophagy induction. Methods: The temperature and pH of the media, along with the cell number, were evaluated. The intracellular glucose level and PI3K/mTOR and EGFR downstream pathways were assessed. Autophagy-related genes, related transcriptional factors, and autophagy induction were estimated using confocal microscopy, flow cytometry, and ELISA. Results: CAP treatment increased the temperature and pH of the media, while its combination with SN resulted in a decrease in intracellular ATP with the downregulation of PI3K/AKT/mTOR survival and RAS/MEK transcriptional pathways. Co-treatment blocked downstream paths of survival pathways and reduced PI3K (2 times), mTOR (10 times), EGFR (5 times), HRAS (5 times), and MEK (10 times). CAP and SN co-treated treatment modulates transcriptional factor expressions (ZKSCAN3, TFEB, FOXO1, CRTC2, and CREBBP) and specific genes (BECN-1, AMBRA-1, MAP1LC3A, and SQSTM) related to autophagy induction. Conclusion: CAP and SN together activate autophagy in G-361 cells by activating PI3K/mTOR and EGFR pathways, expressing autophagy-related transcription factors and genes.

Over the past few decades, microwave (MW) radiation has been widely used, and its biological effects have been extensively investigated. However, the effect of MW radiation on human skin biology is not well understood. We study the effects of pulsed high-power microwaves (HPMs) on melanoma (G361 and SK-Mel-31) and normal human dermal
fibroblast (NHDF) cells. A pulsed power generator (Chundoong) was used to generate pulsed HPMs (dominant frequency: 3.5 GHz). For treatment 1, 5, 15, and 45 shots are given to cells in which the electromagnetic energy of 0.6 J was delivered to the cells at each trigger shot. Cell viability, proliferation rate, apoptosis, cell death, metabolic activity, and oxygen-free radical regulation were evaluated after the MW exposure at low and high doses. MW exposure increased the viabilities and proliferation rates of both melanoma cell lines in a dose-dependent manner, while no significant effects on the fibroblast cells were observed.

Plants are very vulnerable to pathogen attacks and environmental stress as they are exposed to harsh environments in natural conditions. However, they have evolved a self-defense system whereby reactive oxygen and nitrogen species (RONS) act as double-edged swords by imposing (at higher concentration) and mitigating (at lower concentration) environmental stress. Cold plasma is emerging as a feasible option to produce a variety of RONS in a controlled manner when amalgamate with water. Cold plasma activated/treated water (PAW) contains a variety of RONS at concentrations, which may help to activate the plant’s defense system components. In the present study, we examine the effect of cold atmospheric-air jet plasma exposure (15 min, 30 min, and 60 min) on the water’s RONS level, as well as the impact of PAW irrigation, (assigned as 15PAW, 30PAW, and 60PAW) on tomato seedlings growth and defense response. We found that PAW irrigation (priming) upregulate seedlings growth, endogenous RONS, defense hormone (salicylic acid and jasmonic acid), and expression of key pathogenesis related (PR) gene. 30 min PAW contains RONS at concentrations which can induce nontoxic signaling. The present study suggests that PAW irrigation can be beneficial for agriculture as it
modulates plant growth as well as immune response components.

Non-thermal atmospheric pressure plasma sources operated in ambient environments are known to generate a variety of reactive oxygen and nitrogen species which could be applied for various biomedical applications. Herein, we fabricate a micro-dielectric barrier discharge plasma device by using screen-printing technology and apply it for studying immuno-stimulatory effects. We demonstrate a tumor-suppressive role for plasma-stimulated macrophages in metastatic solid cancers that directly elicit proliferation and are responsible for tumor relapse mediated by mesenchymal shift. Using microarray analysis, we observed that cold plasma stimulates and differentiates monocyte cells into macrophages as demonstrated by expression of several cytokine/chemokine markers. Moreover, plasma treatment stimulates the differentiation of pro-inflammatory (M1) macrophages to a greater extent. These stimulated macrophages favor anti-tumorigenic immune responses against metastasis acquisition and cancer stem cell maintenance in solid cancers in vitro. Differentiation of monocytes into anticancer macrophages could improve the efficacy of plasma treatment, especially in modifying pro-tumor inflammatory microenvironment through effecting highly resistant immunosuppressive tumor cells associated with tumor relapse.

With the rising incidences of cancer cases, the quest for new metal based anticancer drugs has led to extensive research in cancer biology. Zinc complexes of amino acid residue side chains are well recognized for hydrolysis of phosphodiester bond in DNA at faster rate. In the presented work, a Zn(II) complex of cyclen substituted with two L-tryptophan units, Zn(II)-Cyclen-(Trp) 2 has been synthesized and evaluated for antiproliferative activity. Zn(II)-Cyclen-(Trp) 2 was synthesized in $70% yield and its DNA binding potential was evaluated through QM/MM study which suggested good binding (G = À9.426) with B-DNA. The decrease in intensity of the positive and negative bands of CT-DNA at 278 nm and 240 nm, respectively demonstrated an effective unwinding of the DNA helix with loss of helicity. The complex was identified as an antiproliferative agent against U-87 MG cells with 5 fold increase in apoptosis with respect to control (2 h post incubation, IC 50 25 mM). Electrophoresis and comet assay studies exhibited an increase in DNA breakage after treatment with complex while caspase-3/b-actin cleavage established a caspase-3 dependent apoptosis pathway in U-87 MG cells after triggering DNA damage. In vivo tumor specificity of the developed ligand was validated after radiocomplexation with 99m Tc (>98% radiochemical yield and specific activity of 2.56 GBq/mmol). Avid tumor/muscle ratio of >6 was depicted in biodistribution and SPECT imaging studies in U-87 MG xenograft model nude mice.

Ionizing radiation induced perturbations in cellular redox homeostasis are manifested as cell cycle arrest, inflammatory response, and apoptosis. Present study was focused on determination of radioprotective efficacy of a secondary metabolite N-acetyl-L-tryptophan glucoside (NATG) isolated
from radioresistant bacterium Bacillus sp. INM-1. Macrophage J774A.1 cells were treated with NATG (0.025–200 mg/ml) before g-irradiation (10-50 Gy) and radioprotective efficacy in terms of cellular metabolic activity was assessed using MTT assay. Radiation-induced intracellular ROS generation and its inhibition by NATG (0.25 mg/ml) pretreatment was evaluated using 2’,7’-dichlorodihydrofluorescein diacetate (H2DCFDA) probe. Effects of NATG pretreatment with or without g-irradiation (20 Gy) on cell cycle perturbations and apoptosis regulation was evaluated using flowcytometry. Results of the study displayed 0.25 and 50 mg/ml as effective dose (ED) and lethal dose (LD50) dose of NATG. A significant (p<0.05) decrease in DCFDA fluorescence corresponding to decreased ROS
levels was observed in NATG pretreated irradiated cells as compared to irradiated alone cells. Cell cycle progression analysis demonstrated decrease (10%) in G1, G2, and S phase cells at 24 h post-treatment time interval in NATG pretreated cells as compared to control group. However, radiation induced
or NATG-induced treatment did not cause any alterations in G2/M arrest. APO-BrDU analysis demonstrated significant (p<0.05) reduction in apoptosis level at 24 h time interval in
NATG pretreated irradiated cells as compared to alone irradiated cells. Conclusively, present study suggests that NATG offers radioprotection by apoptosis inhibition mechanism without altering cell cycle progression in J774A.1 cells. Further studies to evaluate detailed molecular mechanisms of radioprotection offered by NATG are ongoing.

Radiation can produce biological damage, mainly oxidative stress, via production of free radicals, including reactive oxygen species (ROS). Nanoparticles are of interest as radioprotective agents, particularly due to their high solubility and bioavailability. Silymarin is a hepatoprotective agent but has poor oral bioavailability. Silymarin was formulated as a nanoemulsion with the aim of improving its bioavailability and therapeutic efficacy. In the present study, we evaluated self-nanoemulsifying drug delivery systems (SNEDDS) formulated with surfactants and co-surfactants. Nano-silymarin was characterized by estimating % transmittance, globule size, and polydispersity index, and by transmission electron microscopy (TEM). The nano-silymarin obtained was in the range of 3–8 nm diameter. With regard to DNA damage,measured by a plasmid relaxation assay, maximum protection was obtained at 10 µg/mL. Cytotoxicity of nano-silymarin to human embryonic kidney (HEK) cells was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Protective efficacy against -radiation was assessed by reduction in micronucleus frequency and ROS generation, using the 2,7-dichlorodihydrofluoresceindiacetate (H2DCFDA) assay. Radiation-induced apoptosis was estimated by microscopic analysis and cell-cycle estimation. Nano-silymarin was radioprotective, supporting the possibility of developing new approaches to radiation protection via nanotechnology.

Purpose: To check the comparative efficacy of DNA protection by silymarin and its nanoformulation as an effective radiation countermeasure agent in ameliorating γ-radiation-induced Genotoxicity.
Methods: The study performed suggests the efficacy of silymarin and its nanoformulation specifically in ameliorating γ-radiation-induced genotoxic effects at cellular, plasmid DNA levels etc.
Results: The retention of super-coiled DNA following treatment of DNA with various concentrations of silymarin (parent compound) was found to be maximum at 25μg/ml, whereas better retention was seen at 10μg/ml in case of silymarin nanoformulation. Micronuclei count also reduced maximally at 10μg/ml when treated with silymarin nanoformulation as compared to 25μg/ml using parent compound.
Summary : Silymarin and its nanoformulation showed no toxic effects on DNA. The nanoformulation demonstrated better results in terms of protection of genetic material against γ-radiation due to increase in surface area and hence improved bioavailability. The nanoformulation can be of use in mitigating the deleterious effects of radiation and plausible biothreat agents.

Purpose: - Estimation of Antioxidant and radioprotective efficacy of silymarin: An in vitro study
Methods: - The study shows the efficacy of silymarin as an antioxidant by calculating ABTS, DPPH,
EPR studies and radioprotective efficacy was performed by analysis of Micronuclei count.
Results: - The value of free radical scavenging activities of silymarin against DPPH (>65%) and ABTS
(22%) were evaluated. Based on the calculated g values, the strong EPR signal stability shows the
antioxidant activity of silymarin. Reduction in micronucleus count was seen at 25μg/ml with 2Gy γ-
irradiation in HEK cells.
Conclusions: - Silymarin has the capacity of minimizing the deleterious of radiation. Due to its non-toxic
nature and safety in humans, silymarin could be used as a radiation countermeasure agent (RCM) agent
during nuclear/radiological emergencies.

The recent innovation of micro-dielectric barrier discharge (µ-DBD) cold atmospheric plasma technology developed by PBRC paved a new way of approaches in the treatment of melanoma. In this study we used novel drug delivery method of topical administering of a nanoemulsion of flavonolignan (silymarin) and called as small nano emulsifying drug delivery system (SNEDDS) [1-3] to achieve a therapeutic effect against melanoma cells. In our previous report, combination of cold atmospheric plasma and nanoparticle has synergistically presented its potential in cancer therapy [4]. Now we have developed a strategy for plasma assisted nano drug delivery system, in which SNEDDS with µ-DBD plasma is likely contributed to enhancing selective permeability in the topical region through the induction of membrane disruption by plasma species leading to facilitated intracellular diffusion of SNEDDS towards melanoma sites.
(i) SNEDDS was developed by incorporate the silymarin into oil-in-water (o/w) based nanocarrier to increase its bioavailability. The size and morphology along with increase in cellular uptake of SNEDDS in melanoma cells using cold plasma were also checked by using transmission electron microscopy.
(ii) Cellular viability and cell death were assessed and safe dosage estimated by using MTT and propidium iodide respectively. Growth inhibition, apoptosis in melanoma cells also calculated using PI alone and annexin-V PI apoptosis kit. Reactive oxygen species and reactive nitrogen species were assessed by flow cytometry using DCFDA and DAFFM dyes.
(iii) Melanoma specific enzymes i.e., NAMPT and DNA methyltransferase also showed drastic reduction in its activity which suggested confirmation of melanoma reduction.
(iv) Molecular events to apoptosis include reduction in PI3K, Akt and gradually increase in p53 strongly confirms decline in melanoma. Inhibition of epithelial mesenchymal transition (EMT) and melanoma stem cells were also observed.
References:
(1) Parveen R et al. Arch Pharm Res. 2011;34(5):767-774.
(2) Parveen R et al. Int J Pharm. 2011 413(1-2):245-253.
(3) Adhikari M et al. Mutat Res Genet Toxicol Environ Mutagen. 2015 792:1-11.
(4) Kaushik N et al. Biomaterials. 2016; 87: 118-130.

Melanin is primarily designated as animal cutaneous pigment and considered separately from similar fungal or bacterial pigments. Pigmentation disorders comprise various kinds of diverse conditions that are usually categorized by altered melanocyte embryological development, melanin concentration, defects in melanogenesis, flaws in biogenesis of melanosomes, problems in Melanosomes transport and its survival or both, and result in altered pigmentation of the skin. Many of the disorders by pigmentation are extremely common like melasma and vitiligo whereas some are very rare. Many gene mutations related to pigmentation disorder already described, but the function of their final product protein and their implication in melanogenesis are only partially understood, brings new clues in the understanding of the pigmentation process. In recent years, several reports have demonstrated the wide range of nonthermal atmospheric pressure plasma applications on melanogenesis. This chapter serves as a comprehensive update on the current understanding of the pigmentation production, disease associated with interaction of skin pigmentation with UV rays, hypermelanogenesis and hypomelanogenesis, as well as treatments for this common, yet therapeutically challenging conditions using surgical, hormonal, immunological, antioxidative and cold atmospheric plasma treatment compounds.