Novel Therapeutic Approaches to Treat Brain Cancer Combining Cold Atmospheric Plasma, Therapeutic Prodrugs and Gold Nanoparticles

Glioblastoma Multiforme (GBM) makes up approximately 45% of all primary brain tumours. State of the art treatment at present involves concurrent and adjuvant temozolomide (TMZ) with radical radiotherapy which extends median survival from 12.1 months (radical radiotherapy alone) to 14.6 months according to the study of the European Organization for Research and Treatment of Cancer (EORTC) Brain Tumour and Radiotherapy Groups and the National Cancer Institute of Canada (NCIC) Clinical Trials Group. Meanwhile, National Cancer Registry Ireland presented that GBM represents over 40% of all malignant brain tumours and had the worst five-year net survival (4%) compared to overall malignant brain cancer (five-year net survival, 19%) in Ireland. Long term survival of patients with GBM has not been significantly improved in the last 20 years. GBM tumours also have presented high level of resistance to normal treatments. Therefore, novel therapies to treat GBM are urgently needed. This study aimed to investigate efficient therapeutic methods by combining novel interventions, including cold atmospheric plasma (CAP), gold nanoparticles (AuNPs) and specific chemotherapeutic compounds to overcome the barriers of GBM treatment. Over the past decade CAP has emerged as a novel approach in health care area, especially cancer therapy. CAP generates chemically active species such as reactive oxygen species (ROS) and reactive nitrogen species (RNS) and has been demonstrated to act in synergy with a selection of traditional chemotherapeutic compounds which could reduce the effective concentrations of drugs needed at the tumour and may allow for targeted toxicity at sites exposed to the plasma field. AuNPs, well known as biocompatible drug delivery and diagnosis agents for cancer therapy, have been demonstrated to have synergistic anti cancer effects in combination with CAP treatment. In this project, for the first time, we investigated and described the detailed mechanism iii behind the synergistic anti-cancer effects between AuNPs and CAP treatment. Chapter 2 and Chapter 3 demonstrated that low dose treatment of CAP treatment was capable of promoting the uptake of AuNPs into glioblastoma U373MG cells via stimulated membrane repair clathrin-dependent endocytosis. The intracellular accumulation of AuNPs was tracked using atomic absorbance spectrometry (AAS) and simulated with numerical modelling to identify the enhanced uptake routine. AuNPs were tracked into early endosomes, late endosomes and finally lysosomes using specific fluorescent probes and confocal microscope. The lipid oxidation of cancer cells induced by CAP treatment was confirmed by various methods, including confocal microscopy, Thiobarbituric Acid Reactive Substances (TBARS) assay and flow cytometry. Meanwhile, the related endocytosis pathway was determined to be clathrin dependent using multiple clathrin and caveola specific inhibitors and clathrin siRNA. In Chapter 4, we performed the screening of 47 prodrug candidates for their cytotoxicity against U373MG cells in combination with CAP treatment. The selection of chemotherapeutic compounds provided by collaborators have been tested to determine dose response curves with or without CAP treatment using Alamar Blue assay, thus, to characterise their synergistic potential in combination with CAP. Two leading candidates which showed significant cytotoxicity with CAP, have been identified from 47 compounds. Furthermore, the mechanism behind the synergistic cytotoxicity between one of the leading candidates, JW-04-061, and CAP treatment has been investigated. It has been demonstrated that reactive species, especially short-lived species, generated in culture medium may play a main role in the oxidation and activation of the prodrug during CAP treatment

Investigating the Role of Gold Nanoparticle Shape and Size in Their Toxicities to Fungi

Gold nanoparticles (GNPs) are increasingly being used in a wide range of applications, and such they are being released in greater quantities into the environment. Consequently, the environmental effects of GNPs, especially toxicities to living organisms, have drawn great attention. However, their toxicological characteristics still remain unclear. Fungi, as the decomposers of the ecosystem, interact directly with the environment and critically control the overall health of the biosphere. Thus, their sensitivity to GNP toxicity is particularly important. The aim of this study was to evaluate the role of GNP shape and size in their toxicities to fungi, which could help reveal the ecotoxicity of GNPs. Aspergillus niger, Mucor hiemalis, and Penicillium chrysogenum were chosen for toxicity assessment, and spherical and star/flower-shaped GNPs ranging in size from 0.7 nm to large aggregates of 400 nm were synthesised. After exposure to GNPs and their corresponding reaction agents and incubation for 48 h, the survival rates of each kind of fungus were calculated and compared. The results indicated that fungal species was the major determinant of the variation of survival rates, whereby A. niger was the most sensitive and M. himalis was the least sensitive to GNP exposure. Additionally, larger and non-spherical GNPs had relatively stronger toxicities

A Novel, Rapid, Seedless, in Situ Synthesis Method of Shape and Size Controllable Gold Nanoparticles using Phosphates

We hereby report a novel synthesis method of size and shape controllable gold nanoparticles that is rapid, in situ and seedless. Unlike most currently employed size and shape controllable synthesis methods, it takes place in a single step under room temperature within ~15 minutes. While mixtures of gold nanospheres around 70 nm and gold nanoplates with width ranging from 100 nm to 1000 nm can be synthesized in about 15 minutes by standard synthesis method using N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid (HEPES) to reduce Au(III), gold nanoflowers or mixtures of smaller gold nanospheres and nanoplates can be synthesized with the addition of disodium phosphate (Na2HPO4) or monosodium phosphate (NaH2PO4), respectively. Increasing the concentration of phosphate added significantly reduces the formation time of gold nanoparticles to seconds. By increasing the molar ratio of Na2HPO4 : HEPES and NaH2PO4 : HEPES, the size of gold nanoflowers and gold nanoparticle mixtures can be tuned from ~60 nm down to 1 nm and from ~70 nm to ~2.5 nm, respectively. The systematic structural changes are accompanied by similarly systematic colour changes associated with shifting of the surface plasmon resonance. The proposed mechanism of the synthesis process is also presented

Platinum Nanoparticles Inhibit Intracellular ROS Generation and Protect Against Cold Atmospheric Plasma-induced Cytotoxicity

Platinum nanoparticles (PtNPs) have been investigated for their antioxidant abilities in a range of biological and other applications. The ability to reduce off-target cold atmospheric plasma (CAP) cytotoxicity would be useful in Plasma Medicine; however, little has been published to date about the ability of PtNPs to reduce or inhibit the effects of CAP. Here we investigate whether PtNPs can protect against CAP-induced cytotoxicity in cancerous and non-cancerous cell lines. PtNPs were shown to dramatically reduce intracellular reactive species (RONS) production in U-251 MG cells. However, RONS generation was unaffected by PtNPs in medium without cells. PtNPs protect against CAP induced mitochondrial membrane depolarization, but not cell membrane permeabilization which is a CAP-induced RONS-independent event. PtNPs act as potent intracellular scavengers of reactive species and can protect against CAP induced cytotoxicity. PtNPs, showing no significant biocorrosion, may be useful as a catalytic antioxidant for healthy tissue and for protecting against CAP-induced tissue damage

Investigating the Role of Gold Nanoparticle Shape and Size in Their Toxicities to Fungi

Gold nanoparticles (GNPs) are increasingly being used in a wide range of applications, and such they are being released in greater quantities into the environment. Consequently, the environmental effects of GNPs, especially toxicities to living organisms, have drawn great attention. However, their toxicological characteristics still remain unclear. Fungi, as the decomposers of the ecosystem, interact directly with the environment and critically control the overall health of the biosphere. Thus, their sensitivity to GNP toxicity is particularly important. The aim of this study was to evaluate the role of GNP shape and size in their toxicities to fungi, which could help reveal the ecotoxicity of GNPs. Aspergillus niger, Mucor hiemalis, and Penicillium chrysogenum were chosen for toxicity assessment, and spherical and star/flower-shaped GNPs ranging in size from 0.7 nm to large aggregates of 400 nm were synthesised. After exposure to GNPs and their corresponding reaction agents and incubation for 48 h, the survival rates of each kind of fungus were calculated and compared. The results indicated that fungal species was the major determinant of the variation of survival rates, whereby A. niger was the most sensitive and M. himalis was the least sensitive to GNP exposure. Additionally, larger and non-spherical GNPs had relatively stronger toxicities

Cold Atmospheric Plasma Induces Silver Nanoparticle Uptake, Oxidative Dissolution and Enhanced Cytotoxicity in Glioblastoma Multiforme Cells

Silver nanoparticles (AgNP) emerged as a promising reagent for cancer therapy with oxidative stress implicated in the toxicity. Meanwhile, studies reported cold atmospheric plasma (CAP) generation of reactive oxygen and nitrogen species has selectivity towards cancer cells. Gold nanoparticles display synergistic cytotoxicity when combined with CAP against cancer cells but there is a paucity of information using AgNP, prompting to investigate the combined effects of CAP using dielectric barrier discharge system (voltage of 75 kV, current is 62.5 mA, duty cycle of 7.5kVA and input frequency of 50–60Hz) and 10 nm PVA-coated AgNP using U373MG Glioblastoma Multiforme cells. Cytotoxicity in U373MG cells was \u3e100-fold greater when treated with both CAP and PVA-AgNP compared with either therapy alone (IC50 of 4.30 μg/mL with PVA-AgNP alone compared with 0.07 μg/mL after 25s CAP and 0.01 μg/mL 40s CAP). Combined cytotoxicity was ROS-dependent and was prevented using N-Acetyl Cysteine. A novel darkfield spectral imaging method investigated and quantified AgNP uptake in cells determining significantly enhanced uptake, aggregation and subcellular accumulation following CAP treatment, which was confirmed and quantified using atomic absorption spectroscopy. The results indicate that CAP decreases nanoparticle size, decreases surface charge distribution of AgNP and induces uptake, aggregation and enhanced cytotoxicity in vitro

Ursolic Acid Inhibits Collective Cell Migration and Promotes JNK-Dependent Lysosomal Associated Cell Death in Gioblastoma Multiforme Cells

Ursolic acid (UA) is a bioactive compound which has demonstrated therapeutic efficacy in a variety of cancer cell lines. UA activates various signalling pathways in Glioblastoma multiforme (GBM) and offers a promising starting point in drug discovery; however, understanding the relationship between cell death and migration has yet to be elucidated. UA induces a dose dependent cytotoxic response demonstrated by flow cytometry and biochemical cytotoxicity assays. Inhibitor and fluorescent probe studies demonstrate that UA induces a caspase independent, JNK dependent, mechanism of cell death. Migration studies established that UA inhibits GBM collective cell migration in a time dependent manner that is independent of the JNK signalling pathway. Cytotoxicity induced by UA results in the formation of acidic vesicle organelles (AVOs), speculating the activation of autophagy. However, inhibitor and spectrophotometric analysis demonstrated that autophagy was not responsible for the formation of the AVOs. Confocal microscopy and isosurface visualisation determined co-localisation of lysosomes with the previously identified AVOs, thus providing evidence that lysosomes are likely to be playing a role in UA induced cell death. Collectively, our data identify that UA rapidly induces a lysosomal associated mechanism of cell death in addition to UA acting as an inhibitor of GBM collective cell migration

Cell Death Induced in Glioblastoma Cells by Plasma-Activated-Liquids (PAL) is Primarily Mediated by Membrane Lipid Peroxidation and not ROS Influx

Since first identified in 1879, plasma, the fourth state of matter, has been developed and utilised in many fields. Nonthermal atmospheric plasma, also known as cold plasma, can be applied to liquids, where plasma reactive species such as reactive Oxygen and Nitrogen species and their effects can be retained and mediated through plasma-activated liquids (PAL). In the medical field, PAL is considered promising for wound treatment, sterilisation and cancer therapy due to its rich and relatively long-lived reactive species components. This study sought to identify any potential antagonistic effect between antioxidative intracellularly accumulated platinum nanoparticles (PtNPs) and PAL. We found that PAL can significantly reduce the viability of glioblastoma U-251MG cells. This did not involve measurable ROS influx but instead lead to lipid damage on the plasma membrane of cells exposed to PAL. Although the intracellular antioxidative PtNPs showed no protective effect against PAL, this study contributes to further understanding of principle cell killing routes of PAL and discovery of potential PAL-related therapy and methods to inhibit side effects

Reactive oxygen species (ROS): utilizing injectable antioxidative hydrogels and ROS-producing therapies to manage the double-edged sword

Reactive oxygen species (ROS) are generated in cellular metabolism and are essential for cellular signalling networks and physiological functions. However, the functions of ROS are ‘double-edged swords’ to living systems that have a fragile redox balance between ROS generation and elimination. A modest increase of ROS leads to enhanced cell proliferation, survival and benign immune responses, whereas ROS stress that overwhelms the cellular antioxidant capacity can damage nucleic acids, proteins and lipids, resulting in oncogenic mutations and cell death. ROS are therefore involved in many pathological conditions. On the other hand, ROS present selective toxicity and have been utilised against cancer and pathogens, thus also acting as a double-edged sword in the healthcare field. Injectable antioxidative hydrogels are gel precursors that form hydrogel constructs in situ upon delivery in vivo to maintain an antioxidative capacity. These hydrogels have been developed to counter ROS-induced pathological conditions, with significant advantages of biocompatibility, excellent moldability, and minimally invasive delivery. The intrinsic, readily controllable ROS-scavenging ability of the functionalised hydrogels overcomes many drawbacks of small molecule antioxidants. This review summarises the roles of ROS under pathological conditions and describes the state-of-the-art of injectable antioxidative hydrogels. A particular emphasis is also given to current ROS-producing therapeutic interventions, enabling potential application of injectable antioxidant hydrogels to prevent the adverse effects of many cancer and infection treatments

Cold Atmospheric Plasma Induces Accumulation of Lysosomes and Caspase-independent Cell Death in U373MG Glioblastoma Multiforme Cells

Room temperature Cold Atmospheric Plasma (CAP) has shown promising efficacy for the treatment of cancer but the exact mechanisms of action remain unclear. Both apoptosis and necrosis have been implicated as the mode of cell death in various cancer cells. We have previously demonstrated a caspase-independent mechanism of cell death in p53-mutated glioblastoma multiforme (GBM) cells exposed to plasma. The purpose of this study was to elucidate the molecular mechanisms involved in caspase-independent cell death induced by plasma treatment. We demonstrate that plasma induces rapid cell death in GBM cells, independent of caspases. Accumulation of vesicles was observed in plasma treated cells that stained positive with acridine orange. Western immunoblotting confirmed that autophagy is not activated following plasma treatment. Acridine orange intensity correlates closely with the lysosomal marker Lyso TrackerTM Deep Red. Further investigation using isosurface visualisation of confocal imaging confirmed that lysosomal accumulation occurs in plasma treated cells. The accumulation of lysosomes was associated with concomitant cell death following plasma treatment. In conclusion, we observed rapid accumulation of acidic vesicles and cell death following CAP treatment in GBM cells. We found no evidence that either apoptosis or autophagy, however, determined that a rapid accumulation of late stage endosomes/lysosomes precedes membrane permeabilisation, mitochondrial membrane depolarisation and caspase independent cell death