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The combined application of ultrasound (US) and plasma microbubble (PMB) is a promising technology against glioblastoma (GBM). Here, we determined the effects of US and PMB (alone and combined) on U‐251 MG GBM and A431 epidermoid carcinoma spheroids under different conditions. The synergistic treatment of US‐ and PMB‐induced tumor sphere cell death in a time‐, dose‐, treatment frequency‐, and reactive oxygen species dependent manner, while multiple treatments augmented this effect. Synergistic treatments significantly reduced three‐dimensional GBM spheroid regrowth and cell proliferation while inducing cytotoxicity, spheroid shrinkage, and damage to the tumor microenvironment. PMB‐induced cytotoxicity throughout the tumor sphere, likely via long‐lived reactive oxygen and nitrogen species (H2O2, NO2−, and NO3−) and other reactive species. [ABSTRACT FROM AUTHOR]

Three-dimensional (3D) cell culture models can help bridge the gap between in vitro cell cultures and in vivo responses by more accurately simulating the natural in vivo environment, shape, tissue stiffness, stressors, gradients and cellular response while avoiding the costs and ethical concerns associated with animal models. The inclusion of the third dimension in 3D cell culture influences the spatial organization of cell surface receptors that interact with other cells and imposes physical restrictions on cells in compared to Two-dimensional (2D) cell cultures. Spheroids' distinctive cyto-architecture mimics in vivo cellular structure, gene expression, metabolism, proliferation, oxygenation, nutrition absorption, waste excretion, and drug uptake while preserving cell–extracellular matrix (ECM) connections and communication, hence influencing molecular processes and cellular phenotypes. This protocol describes the in vitro generation of tumourspheroids using the low attachment plate, hanging drop plate, and cellusponge natural scaffold based methods. The expected results from these protocols confirmed the ability of all these methods to create uniform tumourspheres. [ABSTRACT FROM AUTHOR]

The aim of this study was to determine the effects of a pin‐to‐plate cold atmospheric plasma (CAP) on U‐251 MG three‐dimensional (3D) glioblastoma spheroids under different conditions. 3D tumorspheres showed higher resistance to the CAP treatment compared to 2D monolayer cells. A single CAP treatment was able to induce cytotoxicity, while multiple CAP treatments augmented this effect. CAP was also able to induce cytotoxicity throughout the tumoursphere, and we identified that reactive oxygen species (ROS) plays a major role, while H2O2 plays a partial role in CAP‐induced cytotoxicity in tumourspheres. We conclude that ROS‐dependent cytotoxicity is induced uniformly throughout glioblastoma and epidermoid tumourspheres by direct CAP treatment. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A large gap pin‐to‐plate, atmospheric‐pressure plasma reactor is demonstrated as means of in vitro study of plasma species interactions with cell cultures. By employing optical emission and optical absorption spectroscopy, we report that the pin‐to‐pate plasma array had an optimal discharge frequency for cell death of 1000 Hz in ambient air for the target cancer cell line, human glioblastoma multiform (U‐251MG). The detected plasma chemistry contained reactive oxygen and nitrogen species including OH, N2, N2+ and O3. We show that by varying the plasma discharge frequency, the plasma chemistry can be tailored to contain up to 8.85 times higher levels of reactive oxygen species (ROS) as well as a factor increase of up to 2.86 for levels of reactive nitrogen species (RNS). At higher frequencies, ROS are more dominant than RNS, which allows for a more dynamic and controlled environment for sample study without modifying the inducer gas conditions. When used for treatment of culture media and cell cultures, variation of the plasma discharge frequency over the range 1000–2500 Hz demonstrated a clear dependence of the responses, with the highest cytotoxic responses observed for 1000 Hz. We propose that the reactor offers a means of studying plasma–cell interactions and possible cofactors such as pro‐drugs and nanoparticles for a large volume of samples and conditions due to the use of well plates. [ABSTRACT FROM AUTHOR]

Cold atmospheric plasma (CAP) enhances uptake and accumulation of nanoparticles and promotes synergistic cytotoxicity against cancer cells. However, the mechanisms are not well understood. In this study, we investigate the enhanced uptake of theranostic nanomaterials by CAP. Numerical modelling of the uptake of gold nanoparticle into U373MG Glioblastoma multiforme (GBM) cells predicts that CAP may introduce a new uptake route. We demonstrate that cell membrane repair pathways play the main role in this stimulated new uptake route, following non-toxic doses of dielectric barrier discharge CAP. CAP treatment induces cellular membrane damage, mainly via lipid peroxidation as a result of reactive oxygen species (ROS) generation. Membranes rich in peroxidised lipids are then trafficked into cells via membrane repairing endocytosis. We confirm that the enhanced uptake of nanomaterials is clathrin-dependent using chemical inhibitors and silencing of gene expression. Therefore, CAP-stimulated membrane repair increases endocytosis and accelerates the uptake of gold nanoparticles into U373MG cells after CAP treatment. We demonstrate the utility of CAP to model membrane oxidative damage in cells and characterise a previously unreported mechanism of membrane repair to trigger nanomaterial uptake. This knowledge will underpin the development of new delivery strategies for theranostic nanoparticles into cancer cells. [ABSTRACT FROM AUTHOR]

Gold nanoparticles (AuNP) have potential as both diagnostic and therapeutic vehicles. However, selective targeting and uptake in cancer cells remains challenging. Cold atmospheric plasma (CAP) can be combined with AuNP to achieve synergistic anti-cancer cytotoxicity. To explore synergistic mechanisms, we demonstrate both rate of AuNP uptake and total amount accumulated in U373MG Glioblastoma multiforme (GBM) cells are significantly increased when exposed to 75 kV CAP generated by dielectric barrier discharge. No significant changes in the physical parameters of AuNP were caused by CAP but active transport mechanisms were stimulated in cells. Unlike many other biological effects of CAP, long-lived reactive species were not involved, and plasma-activated liquids did not replicate the effect. Chemical effects induced by direct and indirect exposure to CAP appears the dominant mediator of enhanced uptake. Transient physical alterations of membrane integrity played a minor role. 3D-reconstruction of deconvoluted confocal images confirmed AuNP accumulation in lysosomes and other acidic vesicles, which will be useful for future drug delivery and diagnostic strategies. Toxicity of AuNP significantly increased by 25-fold when combined with CAP. Our data indicate that direct exposure to CAP activates AuNP-dependent cytotoxicity by increasing AuNP endocytosis and trafficking to lysosomes in U373MG cells. [ABSTRACT FROM AUTHOR]

Pyrazolopyrimidinones are fused nitrogen-containing heterocyclic systems, which act as a core scaffold in many pharmaceutically relevant compounds. Pyrazolopyrimidinones have been demonstrated to be efficient in treating several diseases, including cystic fibrosis, obesity, viral infection and cancer. In this study using glioblastoma U-251MG cell line, we tested the cytotoxic effects of 15 pyrazolopyrimidinones, synthesised via a two-step process, in combination with cold atmospheric plasma (CAP). CAP is an adjustable source of reactive oxygen and nitrogen species as well as other unique chemical and physical effects which has been successfully tested as an innovative cancer therapy in clinical trials. Significantly variable cytotoxicity was observed with IC 50 values ranging from around 11 μM to negligible toxicity among tested compounds. Interestingly, two pyrazolopyrimidinones were identified that act in a prodrug fashion and display around 5–15 times enhanced reactive-species dependent cytotoxicity when combined with cold atmospheric plasma. Activation was evident for direct CAP treatment on U-251MG cells loaded with the pyrazolopyrimidinone and indirect CAP treatment of the pyrazolopyrimidinone in media before adding to cells. Our results demonstrated the potential of CAP combined with pyrazolopyrimidinones as a programmable cytotoxic therapy and provide screened scaffolds that can be used for further development of pyrazolopyrimidinone prodrug derivatives. [Display omitted] • Synthesis and screening of bioactive pyrazolopyrimidinones against tumour cells. • Discovery of two pyrimidone bicycle compounds as prodrugs activated by cold plasma. • Low cytotoxic reactive species activates prodrug against glioblastoma cells. [ABSTRACT FROM AUTHOR]

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 >100-fold greater when treated with both CAP and PVA-AgNP compared with either therapy alone (IC 50 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. • Combination of PVA-AgNP and CAP result in a synergistic effect of more than 100-fold in GBM cells. • Reactive species necessary for enhanced cytotoxicity. • CAP induced changes in PVA-AgNP size and surface charge distribution. • Novel darkfield spectral imaging determined enhanced uptake, aggregation and subcellular accumulation of PVA-AgNP by CAP. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]