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Various complex biological effects occur when ultrasonic compression waves travel through biological material. The myriad of biological outcomes instigated by ultrasound are evident when viewed through the lens of the hallmarks of cancer. Herein, we summarise the therapeutic potential of ultrasound, enhanced by microbubbles, for the treatment of cancer.

Supplementary Data from Release of HMGB1 in Response to Proapoptotic Glioma Killing Strategies: Efficacy and Neurotoxicity

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumour in adults. Treatments include surgical resection, radiotherapy, and chemotherapy. Despite this, the prognosis remains poor, with an impacted quality of life during treatment coupled with brain tumour recurrence; thus, new treatments are desperately needed. In this review, we focus on recent advances in G-protein-coupled receptor (GPCR) targets. To date, the most promising targets are the chemokine, cannabinoid, and dopamine receptors, but future work should further examine the melanocortin receptor-4 (MC4R), adhesion, lysophosphatidic acid (LPA) and smoothened (Smo) receptors to initiate new drug-screening strategies and targeted delivery of safe and effective GBM therapies.

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 H2O2plays a partial role in CAP‐induced cytotoxicity in tumour-spheres. We conclude that ROS‐dependent cytotoxicity is induced uniformly throughout glioblastoma and epidermoid tumourspheres by direct CAP treatment.

This review focuses on recent advances in 3D culture systems that promise more accurate therapeutic models of the glioblastoma multiforme (GBM) tumor microenvironment (TME), such as the unique anatomical, cellular, and molecular features evident in human GBM. The key components of a GBM TME are outlined, including microbiomes, vasculature, extracellular matrix (ECM), infiltrating parenchymal and peripheral immune cells and molecules, and chemical gradients. 3D culture systems are evaluated against 2D culture systems and in vivo animal models. The main 3D culture techniques available are compared, with an emphasis on identifying key gaps in knowledge for the development of suitable platforms to accurately model the intricate components of the GBM TME.

Ultrasound is a sound wave with frequencies ranging between 20 kHz and 20 MHz. Ultrasound is able to temporarily and repeatedly open the BBB safely and enhance chemotherapeutic delivery without adverse effects.(Deprez et al., 2021). This novel technique in drug delivery benefits from the powerful ability of ultrasound to produce cavitation activity. Cavitation is the generation and activity of gas-filled bubbles in a medium exposed to ultrasound. As the pressure wave passes through the media, gas bubbles expand at low pressure and contract at high pressure. This leads to oscillation which produces a circulating fluid flow known as microstreaming around the bubble with velocities and shear rates proportional to the amplitude of the oscillation. At high amplitudes the associated shear forces can cut open liposomes (Wanigasekara et al., 2021; Deprez et al., 2021). Vesicles denser than the surrounding liquid are drawn into the shear field surrounding an oscillating bubble. If the shear stress is greater than the strength of the vesicle, it will burst and spill its contents. In a liposome, the vesicle will reform, often at a smaller size than before meeting the shear field. Hence, some interior liquid must be released during the break down. (Pitt et al., 2004) This protocol describes the use of an ultrasound probe to trigger the release of liposomes in glioblastoma cells. This method uses an ultrasound device which is set to the following parameters: Time = 3 min, Pulse = 59 /01, Amplitude = 20%. The ultrasound technique is an easy and reliable technique making it useful in the study of a variety of areas such as oncology. When applied to an ultrasonic transducer, the Pulser part of the instrument generates short, large amplitude electric pulses of controlled energy, which are transformed into short ultrasonic pulses. The VCX 750 is the ultrasonic liquid processor used for this experiment. It is powerful and versatile and can process a wide range of sample types and volumes for many different applications.

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.

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 IC50 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.

Plasma can be described as the fourth state of matter, and as a neutral ionised gas composed of neutral and charged particles, along with electrons, UV-radiation, radicals and electric fields. CAP is generated at temperatures below 40 ℃ and at near atmospheric pressures rendering it appropriate for use in biomedical applications such as an anti-cancer tool, that has already shown its cytotoxicity effects, selectivity potential and efficacy as a Glioblastoma (GBM) therapy. How CAP interacts with cells depends on many chemical and physical factors, with CAP activity varying with differing plasma discharges, cell types and culture conditions. Physical factors such as UV and heat have been shown to have had little effect on DNA integrity. Chemical factors are reported to have a more substantial effect due to the production of reactive oxygen species during CAP treatment. Characterising a new device is important as different plasma discharge modes arising from plasma-self adaptation leads to different interactions between cells and plasma. The novel device seen here in Figure 1, is a large Pin-to-Plate non-thermal atmospheric plasma generator. It consists of 88 pin electrodes which are powered by AC Supply (Leap100, PlasmaLeap Technologies, Dublin, Ireland) and has a resonance frequency of 30 to 125 kHz, a discharge frequency of 50 to 3000 Hz with a power range of 50 to 400 W and a discharge gap of a maximum of 55 mm. The electrodes/samples work area is encased in a fitted container to minimize the escape of Cold Atmospheric Plasma generated reactive species into the environment. The position of pins and a plate electrode acts as a ground, reduces power consumption, increases stability and diffuses the discharge across the entire surface. The discharge has also been seen to diffuse to a larger area than just the pin tip meaning more interactions between reactive species and cells can occur. This protocol describes the steps involved in assessing the cytotoxic effects of a novel large Pin-to-Plate non-thermal atmospheric plasma on mammalian cells. PURPOSE: Step-by-step instructions for the use of the Pin-to-Plate device, non-thermal atmospheric plasma, for the treatment of mammalian cells. SCOPE: To provide a standardized protocol for using the Pin-to-Plate device to generate Cold Atmospheric Plasma to treat mammalian cells for use by any laboratory using the Leap100 system device.

Silver nanoparticles (AgNP) have a multitude of uses, which range from anti-microbial to anti-cancerous etc. They are sometimes preferred to other metal-based nanoparticles due to their attractive physicochemical properties. This protocol is a standardized method of producing and characterising PVA coated AgNPs which range from 10 to 20 nm in diameter. These particles are suited to lab based in-vivo and in-vitro assays. The addition of PVA to AgNPs is to increase the stability of the nanoparticles.

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.

The exponential growth of emerging multidrug-resistant microorganisms, including foodborne pathogens affecting the shelf-life and quality of foods, has recently increased the needs of the food industry to search for novel, natural and eco-friendly antimicrobial agents. Macroalgae are a bio-diverse group distributed worldwide, known to produce multiple compounds of diverse chemical nature, different to those produced by terrestrial plants. These novel compounds have shown promising health benefits when incorporated into foods, including antimicrobial properties. This review aims to provide an overview of the general methods and novel compounds with antimicrobial properties recently isolated and characterized from macroalgae, emphasizing the molecular pathways of their antimicrobial mechanisms of action. The current scientific evidence on the use of macroalgae or macroalgal extracts to increase the shelf-life of foods and prevent the development of foodborne pathogens in real food products and their influence on the sensory attributes of multiple foods (i.e., meat, dairy, beverages, fish and bakery products) will also be discussed, together with the main challenges and future trends of the use of marine natural products as antimicrobials.

Platinum nanoparticles (PtNPs) have been investigated for their antioxidant abilities in a range of biological and other applications. The ability to reduce off-target 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 human 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 both cancerous U-251 MG cells and non-cancerous HEK293 cells against CAP induced cytotoxicity. PtNPs may be useful as a catalytic antioxidant for healthy tissue and for protecting against CAP-induced tissue damage.Graphical AbstractPtNPs are potent catalase and superoxide dismutase mimetics which makes them strong antioxidant candidates for the protection of cells against oxidative stress. CAP was generated using a Dielectric Barrier Device (DBD) system with a voltage output of 75 kV at a frequency of 50 Hz. A range of concentrations of 3nm uncoated PtNPs combined with CAP were examined in human U-251 MG Glioblastoma (GBM) cells and non-cancerous human embryonic kidney HEK293 cells. The protective effects of PtNPs against CAP were explored using several biochemical indicators of oxidative stress and cytotoxicity.

This is a protocol for citrate reduction methods to synthesize gold nanoparticles. Gold nanoparticles (GNP or AuNP) are sub-micrometre particles with various sizes ranging from 1nm to 8 µm created from gold. Besides, they also have different shapes, including spherical, sub-octahedral, octahedral, decahedral, nano triangles, etc. Due to their properties, GNPs have been used in many areas such as electronics, nanotechnology and biomedicine. In biomedical application, spherical and rod shapes are mainly used. Spherical usually is synthesized using a chemical reduction method, while Rod-shape uses a seed-mediated synthesis method. Different shapes and sizes of GNP could give various optical properties and cellular uptakes; by this, GNPs have broader use in modern biomedical applications both in vivo and in vitro. The citrate reduction method was initially introduced by Turkevich, where we use trisodium citrate as a reducing agent. The method widely studied in 1951, a substantial amount of important information regarding this system has been deposited in literature. It was demonstrated that GNP's size variation could readily be realized by controlling the concentration of sodium citrate. Absorbance, zeta potential and size of gold nanoparticles (GNPs) will be determined by using UV/Vis, Zetasizer and Scanning Transmission Electron Microscopy (STEM), respectively.

The objective of this study was to investigate the antimicrobial and anticancer properties of a fucoidan extract and subsequent fractions isolated from the macroalgae Fucus vesiculosus. The fractions obtained (>300 kDa, 300 kDa fraction presented the lowest IC50 values (0.052% - 24 h; 0.032% - 6 days). The potential bioactivity of fucoidan as an antimicrobial and anticancer agent was demonstrated in this study. Hence, the related mechanisms of action should be explored in a near future. European Commission Horizon 2020 Science Foundation Ireland Spanish Ministry of Science, Innovation and Universities

Plasma, the fourth state of matter, is an ionized gas that can directly interact with cells and tissues. A non-thermal plasma with weak collisions between electrons and heavy particles that is applied under atmospheric conditions is known as cold atmospheric plasma (CAP) (Conway et al., 2019). CAP generates short and long-lived reactive oxygen species (ROS), reactive nitrogen species (RNS), neutral particles, molecules, electrons an electromagnetic field, weak ultraviolet radiation and weak heating affect (Yan et al., 2018) that has lead to its' application in wound healing, sterilization and cancer treatment (von Woedtke et al., 2020). Di-electric barrier discharge devices are one of the most common CAP devices that generate short but wide plasma between an anode and a cathode using atmospheric air, and can be used to intensively treat a large sample area (Yan et al., 2016). The purpose of this protocol is to outline the steps involved in setting-up a di-electric barrier discharge cold atmospheric plasma (DBD-CAP) device for the generation of reactive oxygen species on cell cultures. This protocol provides procedures for direct treatment with and without post treatment storage and also indirect treatment using DBD-CAP on cell cultures.

Pyrazolopyrimidinone is a fused nitrogen-containing heterocyclic system, which acts 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. We have tested the synergistic anti-cancer effects of 15 pyrazolopyrimidinones, synthesised in a two-step process, combined with cold atmospheric plasma (CAP), a novel innovation generating reactive species with other unique chemical and physical effects. We identify two pyrazolopyrimidinones that act as prodrugs and display enhanced reactive-species dependent cytotoxicity when used in combination with cold atmospheric plasma. Synergistic activation was evident for both direct CAP treatment on prodrug loaded tumour cells and indirect CAP treatment of prodrug in media prior to adding to tumour cells. Our results demonstrate the potential of CAP combined with pyrazolopyrimidinones as a programmable cytotoxic therapy against cancer. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/429831v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@56c8aeorg.highwire.dtl.DTLVardef@1913ea9org.highwire.dtl.DTLVardef@a8f522org.highwire.dtl.DTLVardef@75a529_HPS_FORMAT_FIGEXP M_FIG C_FIG

This study presents the covalent grafting of a hyaluronic acid-lysozyme (HA-LZ) composite onto corrosion-resistant silane-coated AZ31 Mg alloy via EDC-NHS coupling reactions. The HA-LZ composite coatings created a smooth and hydrophilic surface with the increased concentration of functional lysozyme complexed to the hyaluronic acid. This was confirmed by the measurement of AFM, water contact angle, and quantification of hyaluronic acid and lysozyme. The colonization of S.aureus on HA-LZ composite-coated substrates was significantly reduced as compared to the hyaluronic acid, lysozyme coated and uncoated AZ31 controls. Such activity is due to the enhanced antibacterial activity of lysozyme component as observed from the spread plate assay, propidium iodide staining, and scanning electron microscopy. Furthermore, morphology of the osteoblast cells, alkaline phosphatase activity and DNA quantification studies demonstrated the improved biocompatibility and osteoinductive properties of HA-LZ-coated substrates....

The quality of patient care has increased dramatically in recent years because of the development of lightweight orthopedic metal implants. The success of these orthopedic implants may be compromised by impaired cytocompatibility and osteointegration. Biomimetic surface engineering of metal implants using biomacromolecules including hyaluronic acid (HA) has been used an effective approach to provide conditions favorable for the growth of bone forming cells. To date, there have been limited studies on osteoblasts functions in response to metal substrates modified with the hyaluronic acid of different molecular weight for orthopedic applications. In this study, we evaluated the osteoblasts functions such as adhesion, proliferation, and differentiation in response to high- and lowmolecular-weight HA (denoted as h-HA and l-HA, respectively) functionalized on Ti (h-HA-Ti and l-HA-Ti substrates, respectively) and corrosion-resistant silane coated-AZ31 Mg alloys (hHA-AZ31 and l-HA-AZ31). The DNA quantification study showed that adhesion and proliferation of osteoblasts were significantly decreased by h-HA immobilized on Ti or AZ31 substrates when compared to low-molecular-weight counterpart over a period of 14 days. On the contrary, h-HA significantly increased the osteogenic differentiation of osteoblast over l-HA, as confirmed by the enhanced expression of ALP, total collagen, and mineralization of extracellular matrix. In particular, the h-HAAZ31 substrates greatly enhanced the osteoblast differentiation among tested samples (l-HA-AZ31, l-HA-Ti, h-HA-Ti, and Ti alone), which is ascribed to the osteoinductive activity of h-HA, relatively up-regulated intracellular Ca2+ ([Ca2+]i ) and Mg2+ ([Mg2+]i ) concentrations as well as the alkalization of the cell culture medium. This study suggesting that HA of appropriate molecular weight can be successfully used to modify the surface of metal implants for orthopedic applications

Glioblastoma multiforme (GBM) is the most common and biologically aggressive brain tumour. The current standard therapy for GBM consists in surgical resection, followed by radiotherapy and chemotherapy. Yet, the treatment is limited due to the area for the surgical resection and for the inability of some drugs to cross the brain blood barrier, leading to a general prognostic of no more than a year. Cold atmospheric plasma (CAP) is a new approach in the treatment of this challenging disease. CAP interaction with cells is dependent on physical and chemical factors, with different plasma discharges, cell type, and culture conditions leading to different CAP activity. Considering the plasma self-adaptation that different plasma discharge modes can undergo, which leads to different interaction plasma/cells, the characterization of a new device is essential. In this study we analysed the effect of a novel large pin-to-plate non-thermal atmospheric plasma on U-251 MG cells under different conditions. The analysis of reactive oxygen and nitrogen species (RONS) on plasma, media and cells were also assessed. We were able to demonstrate that the pin-to-plate device is cytotoxic to GBM cells in a dose, time and ROS dependent manner. The measurements of RONS on plasma/media also give us an insight on the chemical effect of this novelty device, and the possibility to better understand the use of this device as a promising GBM therapy.

Biofilms are complex microbial communities that present serious contamination risks to our environment and health. In this study, atmospheric air plasma and airborne acoustic ultrasound technology were applied to inactivate Escherichia coli and Listeria innocua biofilms. Both technologies were efficient in controlling, or completely inactivating, the target bacterial biofilms. Viability and metabolic assays, along with microscopy analysis, revealed that atmospheric air plasma and airborne acoustic ultrasound damaged both the bacterial biofilm cells and its structural integrity. Scanning electron microscopy images highlighted the disruption of the biofilms and pore formation in bacterial cells exposed to both the plasma and acoustic treatments. Elevated reactive oxygen and nitrogen species in bacterial cells treated with atmospheric air plasma, demonstrated their primary role in the observed bacterial inactivation process. Our findings provide potential antimicrobial strategies to combat bacterial biofilms in the food and healthcare sectors.

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.

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), however, 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 demonstrated that UA induces a caspase independent, JNK dependent, mechanism of cell death. Migration studies established that UA inhibits GBM cell migration in a time dependent manner that is independent of the JNK signalling pathway. The cytotoxic insult induced by UA resulted in the formation of acidic vesicle organelles (AVOs), speculating activation of autophagy. However, inhibitor and spectrophotometric analysis demonstrated that autophagy was not responsible for the formation of the AVOs and confocal microscopy identified the AVO’s as lysosomes. Further investigation using isosurface visualisation of confocal imaging determined co-localisation of lysosomes with the previously identified acidic vesicles, thus providing evidence that lysosomes are likely to be playing a role in UA induced cell death.Collectively, our data identifies that UA rapidly induces a lysosomal associated mechanism of cell death in addition to UA acting as an inhibitor of GBM cell migration.Abstract Figure

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.

Cold atmospheric plasma (CAP) has demonstrated synergistic cytotoxic effects with nanoparticles, especially promoting the uptake and accumulation of nanoparticles inside cells. However, the mechanisms driving the effects need to be explored. 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 (30 s, 75 kV). CAP treatment induces cellular membrane damage, mainly via lipid peroxidation as a result of reactive oxygen species (ROS) generation. Membranes rich in peroxidated 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. Our data 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 which will be useful for developing more efficient deliveries of nanoparticles and pharmaceuticals into cancer cells for tumour therapy and diagnosis. This mechanism of RONS-induced endocytosis will also be of relevance to other cancer therapies that induce an increase in extracellular RONS.

Lateral flow immunochromatographic assays are a powerful diagnostic tool for point-of-care tests, based on their simplicity, specificity, and sensitivity. In this study, a rapid and sensitive gold nanoparticle (AuNP) immunochromatographic strip is produced for detecting aflatoxin B1 (AFB1) in suspicious fungi-contaminated food samples. The 10 nm AuNPs were encompassed by bovine serum albumin (BSA) and AFB1 antibody. Thin-layer chromatography, gel electrophoresis and nuclear magnetic resonance spectroscopy were employed for analysing the chemical complexes. Various concentrations of AFB1 antigen (0&ndash, 16 ng/mL) were tested with AFB1 antibody&ndash, BSA&ndash, AuNPs (conjugated AuNPs) and then analysed by scanning electron microscopy, ultraviolet&ndash, visible spectroscopy, and Zetasizer. The results showed that the AFB1 antibody was coupled to BSA by the N-hydroxysuccinimide ester method. The AuNPs application has the potential to contribute to AFB1 detection by monitoring a visible colour change from red to purple-blue, with a detection limit of 2 ng/mL in a 96-well plate. The lateral flow immunochromatographic strip tests are rapid, taking less than 10 min., and they have a detection capacity of 10 ng/g. The smartphone analysis of strips provided the results in 3 s, with a detection limit of 0.3 ng/g for AFB1 when the concentration was below 10 ng/g. Excellent agreement was found with AFB1 determination by high-performance liquid chromatography in the determination of AFB1 among 20 samples of peanuts, corn, rice, and bread.

Pharmaceuticals, and more recently biopharmaceuticals, have become the mainstay for antineoplastic treatments in combination with surgical interventions and radiation therapy. In recent years, advances have been made in the development of nanotechnological interventions for the treatment of cancer alone or in combination with existing therapeutic modalities. Nanotechnology used for therapeutic drug delivery and sensitization of photodynamic, sonodynamic, and radiotherapy are now being tested in preclinical and clinical trials for the treatment of cancer. This article reviews the current state of the art for nanotechnology therapies with an emphasis on targeted drug delivery and the observed and likely benefits when used in combination with existing therapeutic approaches.

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.

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 as well as a factor increase of up to 2.86 for levels of reactive nitrogen species. At higher frequencies, reactive oxygen species are more dominant than reactive nitrogen species 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 co-factors such as pro-drugs and nano particles for a large volume of samples and conditions due to the use of well plates.

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.

The macroalgal hydrocolloid industry is a rapidly growing industry with an annual growth in the global production of 2–3 %. Hydrocolloids from macroalgae are mainly in the form of polysaccharides with other nutrients, such as vitamins, minerals, and proteins. Due to its potential industrial applications, macroalgae have been used as raw materials for hydrocolloids extraction. Compared to the conventional extraction methods, emerging innovative assisted extraction technologies (e.g., Ultrasound/Microwave assisted extraction) have been developed to maximise the extraction yields, efficiency and thereby, maintaining sustainability along the process. These novel techniques are considered as clean and green strategies, with a potential for a large-scale production; thus, avoiding or reducing the use of chemicals. However, more research is required to establish their mechanism of action in order to finally implement them at industrial level. This paper reviews the most relevant strategies and technologies involved in the production of hydrocolloids from macroalgae. European Commission Horizon 2020

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 62.5mA, duty cycle of 7.5kVA and input frequency of 50-60Hz) and 10nm 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 (IC50of 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 cytotoxicityin vitro.

The possibility of releasing gold nanoparticles (GNP) into the environment has been rapidly increasing with the wide spread and flourishing application of gold nanoparticles (GNPs) in a wide range of areas. Consequently, environmental effects of GNP, 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 shape and size of GNPs on 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 circular and star/flower-shaped GNPs sized from 0.7 nm to large aggregates of 400 nm have been synthesised. After mixed with GNPs and reacting agents of GNPs accordingly and incubated for 48 hours, the relative survival rates of each kind of fungus was calculated and compared. The results indicated that with similar sizes, star/flower-shaped GNPs are more toxic to fungi than circular-shaped GNPs; the toxicity of star/flower-shaped GNPs increases with smaller sizes. The results also showed that different species of fungus reacts differently to same GNPs, and Penicillium chrysogenum was relatively more sensitive under the exposure to GNPs.

This paper reports the corrosion resistant and cytocompatible properties of the hyaluronic acid-silane coating on AZ31 Mg alloy. In this study, the osteoinductive properties of high molecular weight hyaluronic acid (HA, 1–4 MDa) and the corrosion protection of silane coatings were incorporated as a composite coating on biodegradable AZ31 Mg alloy for orthopaedic applications. The multi-step fabrication of coatings first involved dip coating of a passivated AZ31 Mg alloy with a methyltriethoxysilane-tetraethoxysilane sol-gel to deposit a dense, cross-linked and corrosion resistant silane coating (AZ31-MT). The second step was to create an amine-functionalised surface by treating coated alloy with 3-aminopropyl-triethoxy silane (AZ31-MT-A) which facilitated the immobilisation of HA via EDC-NHS coupling reactions at two different concentrations i.e 1 mg.ml−1 (AZ31-MT-A-HA1) and 2 mg.ml−1 (AZ31-MT-A-HA2). These coatings were characterised by Fourier transform infrared spectroscopy, atomic force microscopy and static contact angle measurements which confirmed the successful assembly of the full coatings onto AZ31 Mg alloy. The influence of HA-silane coating on the corrosion of Mg alloy was investigated by electrical impedance spectroscopy and long-term immersion studies measurements in HEPES buffered DMEM. The results showed an enhanced corrosion resistance of HA functionalised silane coated AZ31 substrate over the uncoated equivalent alloy. Furthermore, the cytocompatibility of MC3T3-E1 osteoblasts was evaluated on HA-coated AZ31-MT-A substrates by live-dead staining, quantification of total cellular DNA content, scanning electron microscope and alkaline phosphatase activity. The results showed HA concentration-dependent improvement of osteoblast cellular response in terms of enhanced cell adhesion, proliferation and differentiation. These findings hold great promise in employing such biomimetic multifunctional coatings to improve the corrosion resistance and cytocompatibility of biodegradable Mg-based alloy for orthopaedic applications. [Figure not available: see fulltext.]. © 2018, Springer Science+Business Media, LLC, part of Springer Nature.

Fungi, which are common in the environment, can cause a multitude of diseases. Warm, humid conditions allow fungi to grow and infect humans via the respiratory, digestive and reproductive tracts, genital area and other bodily interfaces. Fungi can be detected directly by microscopy, using the potassium hydroxide test, which is the gold standard and most popular method for fungal screening. However, this test requires trained personnel operating specialist equipment, including a fluorescent microscope and culture facilities. As most acutely infected patients seek medical attention within the first few days of symptoms, the optimal diagnostic test would be rapid and self-diagnostic simplifying and improving the therapeutic outcome. In suspensions of gold nanoparticles, Aspergillus niger can cause a colour change from red to blue within 2 min, as a result of changes in nanoparticle shape. A similar colour change was observed in the supernatant of samples of human toenails dispersed in water. Scanning electron microscopy, UV/Vis and Raman spectroscopy were employed to monitor the changes in morphology and surface plasmon resonance of the nanoparticles. The correlation of colour change with the fungal infection was analysed using the absorbance ratio at 520 nm/620 nm. We found a decrease in the ratio when the fungi concentration increased from 1 to 16 CFU/mL, with a detection limit of 10 CFU/mL. The test had an 80% sensitivity and a 95% specificity value for the diagnosis of athlete's foot in human patients. This plasmonic gold nanoparticle-based system for detection of fungal infections measures the change in shape of gold nanoparticles and generates coloured solutions with distinct tonality. Our application has the potential to contribute to self-diagnosis and hygiene control in laboratories/hospitals with fewer resources, just using the naked eye. Graphical abstract Colorimetric method for fungi detection with gold nano particles.

Background: The use of plasma-activated liquids such as PBS, medium or simply plasma-activated water (PAW) has been receiving increasing attention for applications in cancer treatments. Amongst the reactive species contained in these solutions, hydrogen peroxide appears to play a pivotal role in causing cytotoxic effects. H2O2 concentrations can be correlated with reduced cell viability and growth and used as an indicator of the potential efficacy of a plasma-activated liquid. Objective: To investigate the cytotoxic mediators generated in water specific to high-voltage DBD-ACP. Method: Using a high-voltage dielectric barrier atmospheric cold plasma (DBD-ACP) system, we examined PAW-mediated cytotoxic effects on different mammalian cell lines employing a set-up where short-lived reactive species can be discounted and activated liquids with long-term stability are generated. Results: The PAW potency could be modulated using voltage level, treatment time and post-treatment storage time and target-related characteristics such as surface to volume ratio. All of these parameters effected cell viability in a hydrogen peroxide concentration correlated manner. The susceptibility of two cancer cell lines to PAW was similar to that observed for two non-cancer cell lines and the toxicity of plasma-activated water exceeded that of the corresponding hydrogen peroxide concentrations. Conclusion: In cytotoxic plasma activated water an essential role for H2O2 has been demonstrated multi-fold, yet further contributing factors are apparent and remain to be identified.

The application of plasmonic-enhanced Raman imaging of cancer cells and drug delivery is gaining momentum. Here, we propose a new theranostic strategy based on an efficient plasmonic-tunable Raman/Fourier transform infrared (FTIR) spectroscopy imaging, to simultaneously evaluate the anticancer drug scattering cellular imaging and the Raman scattering molecular vibration signals in living cells. This technique allows to monitoring the drug release throughout the cell cycle and in vivo biodistribution and biocompatibility with low dose drug therapy (200 μg/mL) and low toxicity effect. This system can directly track in real-time the delivery and release of an anticancer drug (mitoxantrone, MTX) from gold nanostars in single living cells and in mice (healthy and lung cancer mice models), revealing a strong accumulation in the heart of healthy mice 5 min after administration and infiltration in the tumor site of lung cancer mice 5 h after systemic injection. This in vivo SERS detection method holds a great promise for application in image-guided cancer chemotherapy or as a nonspecific anti-inflammatory therapy for patients with cardiovascular diseases or chronic heart failure.

Background: Non-thermal atmospheric plasma (NTAP) is an ionised gas produced under high voltage that can generate short-lived chemically active species and induce a cytotoxic insult in cancer cells. Cell-specific resistance to NTAP-mediated cytotoxicity has been reported in the literature. The aim of this study was to determine whether resistance against NTAP could be overcome using the human glioma cell line U373MG. Methods: Non-thermal atmospheric plasma was generated using a Dielectric Barrier Device (DBD) system with a maximum voltage output of 120 kV at 50 Hz. The viability of U373MG GBM cells and HeLa cervical carcinoma cells was determined using morphology, flow cytometry and cytotoxicity assays. Fluorescent probes and inhibitors were used to determine the mechanisms of cytotoxicity of cells exposed to the plasma field. Combinational therapy with temozolomide (TMZ) and multi-dose treatments were explored as mechanisms to overcome resistance to NTAP. Results: Non-thermal atmospheric plasma decreased cell viability in a dose (time)-dependent manner. U373MG cells were shown to be resistant to NTAP treatment when compared with HeLa cells, and the levels of intracellular reactive oxygen species (ROS) quantified in U373MG cells were much lower than in HeLa cells following exposure to the plasma field. Reactive oxygen species inhibitor N-acetyl cysteine (NAC) only alleviated the cytotoxic effects in HeLa cells and not in the relatively NTAP-resistant cell line U373MG. Longer exposures to NTAP induced a cell death independent of ROS, JNK and caspases in U373MG. The relative resistance of U373MG cells to NTAP could be overcome when used in combination with low concentrations of the GBM chemotherapy TMZ or exposure to multiple doses. Conclusions: For the very first time, we report that NTAP induces an ROS-, JNK- and caspase-independent mechanism of cell death in the U373MG GBM cell line that can be greatly enhanced when used in combination with low doses of TMZ. Further refinement of the technology may facilitate localised activation of cytotoxicity against GBM when used in combination with new and existing chemotherapeutic regimens.

To investigate the influence of starvation on the biochemical response of Aspergillus niger. The biochemical impact of starvation was determined by morphological observation, immunofluorescent analysis, High-performance liquid chromatography (HPLC) and western blot over 8 days. Results showed that starvation can inhibit fungi survival rate in a time-dependent manner. A. niger exhibited active responses to starvation such as secretion of some 40 kDa proteins to manage changes in water balance. Conidiophores disintegrated from lack of nutrient. The immunofluorescent analysis demonstrated elevated ROS accumulation in starved cells (PA. niger growth by inducing cell apoptosis.