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 clathrindependent 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. Cold atmospheric plasma (CAP) is increasingly studied in a growing number of clinical trials for cancer treatment 1,2 and research is ongoing to explore the combination of CAP with other therapies, including nanoparticles, radiotherapy and chemotherapy 3-5. Gold nanoparticles (AuNPs) are known to be weakly-toxic to human cells and be readily manufactured and designed for targeting delivery of various therapeutic compounds into cells. Citrate-capped cationic AuNPs may adsorb serum proteins onto their surface and thereby stimulate receptor-mediated endocytosis 6. Without special surface functionalisation, AuNPs enter cells and become trapped in vesicles 6-8 or enter the nucleus, depending on their size/shape 9,10. Meanwhile, AuNPs with functionalised surface chemistries/ligands can directly penetrate the membrane and enter the cytoplasm 11 .

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It has been shown that plasma generated in contact with liquid can be tailored to tune the composition of plasma functionalized liquids. For biomedical applications, it is necessary to understand the generation of the plasma treated liquids to modulate the composition and thus the biological response. In this work, two distinct discharge compositions were realized by modifying the location of the ground electrode in a pin-to-liquid plasma system. Through this simple modification to the configurations, the spatiotemporal characteristics of the discharge were significantly affected which, in turn, affected the composition of the generated plasma activated water (PAW). Colorimetric testing of the PAW generated from each system revealed that only one configuration was able to generate PAW with a high concentration of H2O2. Using time-, space-, and wavelength-resolved imaging of excited plasma species [OH, N2 (SPS), N2+ (FNS), and atomic O], the differences in PAW composition were linked to the differences observed in the discharge dynamics between the two configurations.

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.

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Collagen coatings were applied onto polystyrene microplates using a cold atmospheric pressure plasma process. The coatings were compared to standard wet chemical collagen thin films using microscopy, surface energy, infra-red spectroscopy, electrophoresis, and cell culture techniques. Thin films were also deposited on gold electrodes using both coating methods and their structural and barrier properties probed using cyclic voltammetry. While the wet chemical technique produced a thicker deposit, both films appear equivalent in terms of coverage, porosity, structure, and chemistry. Significantly, the cold plasma method preserves both the primary and secondary structure of the protein and this results in high biocompatibility and cell activity that is at least equivalent to the standard wet chemical technique. The significance of these results is discussed in relation to the benefits of a single step plasma coating in comparison to the traditional multi-step aseptic coating technique.

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.