Cold Atmospheric Plasma Induces ATP-Dependent Endocytosis of Nanoparticles and Synergistic U373MG Cancer Cell Death

Abstract

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.

Low dose Cold Atmospheric Plasma induces membrane oxidation, stimulates endocytosis and enhances uptake of nanomaterials in Glioblastoma multiforme cells

“…To further elaborate on this hypothesis, the uptake of AuNPs into cells was tracked using Our previous work demonstrated that AuNPs accumulate in lysosomes, with significantly more accumulation in the case of CAP-treated cells, after 24 h incubation using confocal imaging and 3D-image construction 11 . Supplementary Figure S6 demonstrates the colocalisation of AuNPs with early and late endosome respectively, which shows a tendency of AuNPs uptake to enter the periphery region of the cells through endocytosis, then gather at the central zone of the cells via endosome trafficking into lysosomes.…”

“…Lysosomes (green) were also counterstained. We have previously demonstrated that AuNPs accumulate in lysosomes 24 hours after 30 s, 75 kV CAP treatment using confocal imaging and 3D-image construction 11 . We demonstrate here that that AuNPs enter U373MG cells mainly through endocytosis, which can be identified first in early and late endosomes after CAP treatment and eventually accumulate in lysosomes (Supplementary Figure S6).…”

“…Therefore, they have emerged as a promising reagent, combined with CAP, for anti-cancer therapy in recent studies 4,9,10 . In our previous study, we explored the potential of a combination treatment of CAP with gold nanoparticles, which showed promising synergistic cytotoxicity to U373MG Glioblastoma multiforme (GBM) cells 11 . The accelerated uptake and accumulation of AuNPs in U373MG cells induced by CAP can enhance the efficiency of pharmaceuticals delivery for tumour treatment and diagnosis.…”

“…The following study of CAP-accelerated AuNPs uptake was carried out using a high voltage dielectric barrier discharge (DBD) contained reactor which has been previously described and characterised 11 . U373MG cells were treated with a low dose of CAP treatment previously demonstrated to be non-lethal, at a voltage output of 75 kV for 30 s 33 .…”

“…We employ various clathrin and caveolin specific inhibitors and clathrin silencing to further determine that the CAP-induced endocytosis of AuNP and membrane damage response is clathrin-dependent. The accumulation of AuNPs inside U373MG cells was monitored using atomic absorption spectroscopy and the dose response curve of AuNPs with or without CAP treatment has been presented in previous study 11 . We further analysed the data according to a simulated uptake model to better understand the possible mechanism of CAP-stimulated AuNP uptake.…”