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


Introduction
Cold atmospheric plasma (CAP) is increasingly studied for applications across the food industry, medicine, energy storage and for driving catalytic reactions. Technological developments and preclinical studies have led to CAP testing in a growing number of clinical trials for cancer treatment 1,2 . Research is ongoing to explore the combination of CAP with other cancer therapies, including nanotechnology-based, radio-and chemo-therapy [3][4][5] .
Gold nanoparticles (AuNPs) are considered to be weakly or non-toxic to human cells and highly effective for delivery through the blood brain barrier, especially for ~20 nm diameter AuNPs [6][7][8] . Meanwhile, AuNPs are known to be readily manufactured and designed for targeting delivery of various compounds into cells. 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.
In general, the citrate-capped cationic AuNPs may absorb serum proteins onto their surface in cell culture medium and thereby stimulate receptor-mediated endocytosis pathways, including clathrin-mediated, caveolae-mediated and clathrin/caveolae independent endocytosis 8 . Without special surface functionalisation, AuNPs enter cells and are trapped in vesicles 8,12,13 or enter the nucleus, depending on their size/shape 14,15 . It has also been demonstrated that clathrin-mediated endocytosis is the specific mechanism of normal AuNPs cellular uptake 16 . Meanwhile, AuNPs with functionalised surface chemistries/ligands can directly penetrate the membrane and enter the cytoplasm 17 . However, the detailed mechanism whereby CAP and AuNPs have synergistic biological effects on cancer cells and the uptake of AuNPs affected by CAP needs to be further explored.
CAP generates a unique physical and chemical environment, including generating short-and long-lived reactive nitrogen species (RNS, e.g. excited N2, N2 + , ONOOand NO • , etc.) and reactive oxygen species (ROS, e.g. • OH, O, • O2and O3, etc.), photons as well as heat, pressure gradients, charged particles, and electrostatic and electromagnetic fields, many of which are known to induce biological effects [18][19][20] . Parallels to this can be found in phagocytes of the immune system. Enzymatic production of reactive oxygen and nitrogen species (RONS) along with various hypohalous acids, especially hypochlorites, play a significant role in respiratory bursts, also known as oxidative bursts, which are used in the clearance of tumour cells by phagocytic immune cells including neutrophils, macrophages and monocytes 21 . Anti-cancer cytotoxicity induced by respiratory bursts has been shown to induce spontaneous regression in mouse tumour models [22][23][24] . ROS has emerged as a double-edged sword to cancer cells.
Evidence shows that higher levels of ROS are generated in cancer cells by comparison with normal cells, which is attributed to the higher metabolic activities and more rapid proliferation of transformed cells 25 . Hence, the cellular antioxidant system works under a heavier load to protect tumour cells from oxidative stress, suggesting it may be possible to selectively overload and eliminate them with locally induced ROS production 26 .
Reactive species can induce a free radical chain reaction in the membrane lipids, known as lipid peroxidation/oxidation, which leads to oxidative degradation of the lipids and therefore a disruption of the membrane function and induced injury and disorder to cells. The peroxidated lipid products can induce further propagation of the free radical reactions 27 . It has been shown that several ROS and RNS generated by CAP as well as natural biological processes can induce cell injures via lipid peroxidation 28 . For example, as an oxidant prominent in air pollution, O3 has been proven to be responsible for the lipid peroxidation damage in lung cells [29][30][31] . Hydroxyl radicals ( • OH) react with various cellular components, including membrane lipid 32 . Superoxide ( • O2 -) can form peroxynitrite (ONOO -), which is able to initiate lipid peroxidation, after reacting with nitric oxide (NO) 32 . RNS, such as NO2 and ONOOH, also interact with lipids to form nitrated lipids, which have been demonstrated to play roles in vascular and inflammatory cellular signalling pathways 28 .
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 . Numerical modelling of the uptake of AuNPs, indicates that the CAP treatment stimulated a new uptake route, which can be clathrin-mediated membrane repairing rapid endocytosis according to experimentally observed behaviour. RONS generation by CAP is characterised using H2DCFDA, optical emission spectroscopy and quantitative colorimetric titration methods. We provide evidence, using the TBARS assay and a C11-BODIPY lipid peroxidation sensor, that CAP generated RONS induce lipid peroxidation, membrane damage and thereby activate the membrane reparation via rapid endocytosis. 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.

Results
Numerical modelling of the uptake of AuNPs by GBM cells 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. The uptake of nanoparticles by cell populations in vitro has previously been modelled according to a phenomenological rate equation approach 34-36 , and the approach can be extended to further investigate the role of CAP in AuNP uptake by U373MG Glioma Cells.
The rate of uptake of AuNPs into a cell can be described by the equation: where NAuNP is the number of internalised gold nanoparticles, D is the initial dose of AuNPs, (D-NAuNP) allows for the depletion of the applied AuNP dose, and kdoub is the doubling time of the cells. N1/R1, N2/R2and N3/R3 allow for three different principle uptake pathways, with 6 respective limiting capacities of N and rates R. The first two terms describe independent active and passive uptake mechanisms, respectively, with limiting cellular capacities N1(0) = N1max and N2(0) = N2max, such that: (3) Figure 1 (solid blue line) shows the simulated uptake of AuNPs, normalised to the maximum uptake observed for AuNP + CAP, for the case of R1 = 3 x 10 -3 hr -1 , R2 = 2.5 x 10 -5 hr -1 , R3 =0, which faithfully reproduces the experimentally observed behaviour. Quenching of the active uptake of AuNPs by NaN3 is best simulated by addition of a further term in equation 2, such that dN1dt= -N1*(D-NAuNP)*R1-N1*NaN3*R4 (4) where NaN3 is the effective dose of sodium azide, and R4 allows for the rapid depletion of the active uptake pathway. The experimentally observed uptake was well simulated (solid orange line) by a value of R4 = 3 x 10 -5 hr -1 , keeping all other rates as before.
In simulating the increased uptake of AuNPs upon CAP treatment, it was noted that the enhancement of a single pathway described by equations (2-4) by CAP treatment, by increasing a single uptake rate, did not faithfully reproduce the experimentally observed behaviour, as the uptakes were limited by the parameters N1max and N2max. Rather, faithful reproduction of the observed behaviour required the introduction of independent uptake mechanisms for untreated and CAP treated AuNP uptake, an observation which was critical to the interpretation of the effects of CAP treatment on the cells. Such a pathway can be represented by: dN3/dt = -N3*(D-NAuNP)*R3 (5) such that N3(0) = N3max. Upon the application of CAP, the enhanced uptake was well fitted (solid yellow line) by R3 = 2.5 x 10 -4 hr -1 , keeping all other rates as before.
The modelling process therefore indicates that CAP treatment increases the capacity of the U373MG cells to uptake AuNPs by introducing a new uptake channel. The modelling parameters employed are detailed in Table 1. Note that the parameters relating to the limiting cell uptake, Nnmax, were determined by the definition of the dose as 100 mg/mL. Furthermore, the process was one of simulation, rather than a mathematical fitting, so the parameters should be considered within ~10% confidence.  The electron energy distribution function (EEDF) of the plasma was also determined. As seen in Supplementary Figure S1, the EEDF remained close to a ratio of 7 during CAP treatment, which indicated that the electron energies were distributed more so on the lower end of the energy scale (11 -12 eV) than the higher energy levels (18.8 eV). The low variability of the EEDF indicated that the electric field was stable, and that the formation of the reactive species was in a steady state manner. Using Gastec ozone detector tubes, the concentrations of generated O3 in the extracted gas were measured post-discharge of CAP treatment (Figure 2b Having confirmed RONS generation in plasma, the concentration of hydrogen peroxide (H2O2), nitrite (NO -2) and nitrate (NO -3) in culture media was next measured. As seen in Figure 2c, the RONS generated in CAP-treated phenol red-free medium are presented after normalising to the values of untreated medium. By direct comparison previous results 38 , CAP treatment of culture media for 30 s generated very low amounts of H2O2 (~20 μM), NO 2-(~5 μM), and NO 3-(~30 μM). These concentrations are at least 15-fold and 200-fold lower than the IC50 values we measured previously for U373MG cells (315 µM, >1200 µM and >600 µM respectively) and therefore are essentially non-toxic 39 .
To further investigate CAP induced ROS generation in U373MG cells, H2DCFDA, a cell permeable ROS fluorescence sensor, was preloaded into cells before CAP treatment for 0.5 h.
After CAP treatment, cells were collected and the fluorescence of H2DCFDA was measured using flow cytometry. As seen in Figure 2d, CAP treatment induced increased intracellular H2DCFDA fluorescence, compared to the untreated group. The mean fluorescence was observed to significantly increase by 4-5 fold above untreated controls (Supplementary Figure   S1).

Tracking AuNPs in endosomes and lysosomes and effects of CAP treatment on endocytosis of AuNPs.
A membrane repair mechanism has been described for cells 44,45 . Through rapid endocytosis, cells can quickly remove damaged regions of membranes from the cell surface. These impaired membranes can be trafficked into endosomes and finally to lysosomes. In our case, the rapid endocytosis may contribute to the increased uptake of AuNPs or other compounds into cells following CAP treatment.   46 . In Supplementary Figure S6, the white arrows identify examples of co-localisation of AuNPs (red) with either early (left) or late (right) endosomes (orange).
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).
To monitor the immediate rate of change of endocytosis after CAP treatment, U373MG cells were treated with CAP for 30 s, 75 kV, then incubated with transferrin conjugated with Alexa Fluor™ 546, which is used as an early endosome marker, for 5 min, then fixed with 4% PFA.
Transferrin specifically binds to the Transferrin receptor on the cell membrane to deliver Fe 3+ atoms via receptor-mediated endocytosis. Iron-carrying transferrin then releases iron in the acidic environment of lysosome and will be recycled to the cell membrane. Therefore, early endosomes, including recycling endosomal pathways, were marked with red fluorescence within the confocal image ( Figure 4a). As seen from the imaging, within 5 mins after CAP treatment, the number of transferrin-containing endosomes was greater compared to the control group. To statistically analyse the increase of endosomes induced by CAP treatment, more than 50 cells in each group were analysed using the ImageJ software. Quantification of transferrin uptake confirmed a significant increase of endosomes 5 min after CAP treatment ( Figure 4b, p<0.0001).
Endocytosis is typically subdivided into four types, including clathrin-mediated endocytosis (CME), caveolae-mediated endocytosis, macropinocytosis and phagocytosis. As seen in Table   2, Pitstop, chlorpromazine (CPZ), Methyl-β-cyclodextrin (MβCD), filipin, genistein and amiloride are specific inhibitors suppress certain types of endocytosis and were used in this study to delineate the specific endocytic pathway activated by CAP.

13
To further confirm that clathrin-mediated endocytosis played the main role in CAPaccelerated cellular uptake, MISSION® Endoribonuclease-prepared siRNA (esiRNA) against human Clathrin heavy chain 1 (CLTC) was used to disrupting endocytosis mediated by clathrin coated pit formation. Cells were preincubated with MISSION® esiRNA (human CLTC) and MISSION® siRNA transfection reagent for 24 h, then treated with CAP for 0-30 s at 75 kV, as indicated in Figure 5, and then incubated with 100 μg/ml AuNPs in medium for 3 h and observed by confocal microscopy. As seen in Figure 5a-d, a significant decrease in AuNPs was observed in clathrin-silenced cells compared to the control groups. Following CAP treatment, we did not observe any increase in AuNP uptake in clathrin-silenced cells. To further quantify the uptake of AuNPs affected by clathrin-silencing and CAP treatment, more than 60 cells in each group were analysed using ImageJ software (Supplementary Table S4 for original data).
Figure 5e represents this data and demonstrates that clathrin silencing inhibited more than 50% of the baseline AuNP uptake; moreover, no increase of AuNP uptake was measured following CAP treatment in the clathrin-silenced group. Together, this confirms that clathrinmediated endocytosis played an important role in AuNP uptake, and accelerated endocytosis following CAP treatment was clathrin dependent.

Discussion
The cytoplasmic membrane separates and protects the cellular interior from the exterior environment and provides specific and efficient exchange channels for the remaining intercellular balance and cell viability. Therefore, the integrity of the membrane is vital for all cells. Mammalian cells have developed efficient membrane repair mechanisms that can recover and reseal an injured cytoplasm membrane quickly to retain cell viability. Although investigations of the precise membrane repairing mechanisms have been limited, four possible mechanisms, including patch, tension reduction and more recently exocytosis/endocytosis and budding repair mechanisms were recently proposed 45 . The study of cytoplasmic membrane repair usually employs bacterial pore-forming toxins, such as Streptolysin O, to create mechanical injuries on membranes 45 . Meanwhile, lipid peroxidation is a complex process that damages cellular membrane structure and function, which is CAP is known to generate reactive species and thereby cause lipid peroxidation of cells. In this research, we explored CAP-induced lipid peroxidation using low dose DBD CAP treatment and studied the possible mechanisms of accelerated cellular uptake of AuNPs following CAPinduced oxidative membrane damage.
As seen in Figure 1, the uptake and accumulation of AuNPs into U373MG cells has been modelled for further investigation. To faithfully reproduce the experimentally observed results, a new independent uptake rate was added in the model (equation 5). This numerical model indicates that CAP treatment may introduce a new uptake route, which has now been determined as an independent membrane repairing, clathrin-mediated, endocytosis pathway.
As seen in Figure  respectively, which can induce lipid peroxidation 50 . Furthermore, as seen in Figure 2b, the significant increase of O3 levels during low dose cold plasma treatment shows the ability to peroxidise membrane lipids. The generated RONS in the CAP-treated culture medium, including hydrogen peroxide, nitrite and nitrate, were also quantitatively measured (As seen in Figure 2c). By comparison with our previous study 38,42 , after the same low does CAP treatment (30 s, 75 kV), the levels of generated H2O2, NO -2, and NO -3 in culture medium are relatively low cytotoxic to U373MG cells, but still are able to induce lipid peroxidation.
From another perspective, the CAP treated group also showed significantly greater H2DCFDA fluorescence levels compared to the control group (Figure 2d), which confirmed the CAP- The TBARS assay has been widely applied in food and biomedical research for detecting lipid peroxidation, as it can precisely and specifically measure the cellular level of MDA 52 . Figure 2e directly confirmed the CAP-induced lipid peroxidation, as a significant increase of MDA after CAP treatment appeared compared to the control group.
It has been shown that CAP treatment can alter membrane structures, which may be partly due to the reactive species-caused lipid peroxidation 42, 51,53,54 . However, the cell membrane remains PI impermeable after exposing to 75 kV CAP for 30 s, which demonstrates that the oxidised membrane may remain mechanically intact and the U373MG cells retain viability after a low dose of CAP treatment (30 s, 75 kV) (Figure 2f), which aligns with the results indicating low concentration of RONS in the CAP-treated medium ( Figure 2c). As seen in Figure   3, there was a significant increase in the green fluorescence level of C11-BODIPY observed, which demonstrated that CAP treatment (30 s, 75 kV) induced significant peroxidation of BODIPY fatty acid, leading to a shift of the emission peak. There was also a significant increase of the vesicle structure marked by oxidised green C11-BODIPY tracked inside the U373MG cells, which may be the peroxidised membrane trafficked inside cells via endosomes ( Figure   3a). The increase of endosomes was also confirmed using transferrin conjugated with Alexa Fluor™ 546. As seen in Figure 4a, b, the number of transferrin-marked endosomes within cells was significantly increased just 5 min after CAP treatment (30 s, 75 kV).
Therefore, we propose that low dose CAP treatment can cause non-lethal cytoplasmic membrane damage which triggers a rapid membrane repair system. The increased endocytosis induced by membrane repair then accelerates the uptake of AuNPs into U373MG 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. Therefore, it further confirms that U373MG cells mainly accumulate AuNPs into lysosomes via uptake into early then late endosomes after CAP treatment (30 s, 75 kV).
Various endocytosis inhibitors, AuNPs tracking and transferrin staining were used to determine the specific endocytosis pathways activated by CAP treatment. Transferrin is an essential iron-binding protein that facilitate iron-uptake in cells via transferrin receptor and Clathrin-mediated endocytosis. Figure 4c shows that the formation of transferrin-trafficking endosomes was inhibited in both groups with 0 and 30 s CAP treatment, after exposure to specific/non-specific clathrin inhibitors, including pitstop, CPZ and MβCD. CAP treatment no longer enhanced endocytosis when clathrin was inhibited. Tracking the accumulation of AuNPs after 24 hours incubation demonstrates the long-term effects retained post CAP treatment and various inhibitors (Figure 4d), which suggests similarly that CAP treatment can specifically activate clathrin-mediated endocytosis to enhance cellular uptake of AuNPs.
Clathrin silencing combining CAP treatment further confirmed that the CAP-triggered endocytosis for membrane repairing is clathrin dependent ( Figure 5).
In summary, we report that the enhanced uptake of AuNPs induced by CAP can be as a result of ROS-caused lipid peroxidation, leading to rapid cytoplasma membrane repairing via clathrin-dependent endocytosis. This contributes to our understanding of the cellular effects induced by CAP, especially membrane damage and endocytosis activation, which can be employed for efficient uptake of nanomaterials and pharmaceuticals into cells when combining CAP with cancer therapies. This mechanism of RONS-induced endocytosis will also be of relevance to researchers optimizing other cancer therapies that induce an increase in extracellular RONS.

Methods
Cell Culture and Gold Nanoparticle Treatment. U373MG-CD14, human brain glioblastoma cancer cells (Obtained from Dr Michael Carty, Trinity College Dublin) were cultured in DMEMhigh glucose medium (Merck) supplemented with 10% FBS (Merck) and maintained in a 37 ℃ incubator within a humidified 5% (v/v) CO2 atmosphere. Gold nanoparticles were synthesised by trisodium citrate reduction of auric acid. 20nm sphere citrate-capped AuNPs were used to treat cells whose properties were determined in a previous study 11 . The gold colloid was concentrated to 2500 μg/ml then diluted in culture medium to 100 μg/ml which is non-toxic to U373MG cells. The culture medium containing 100 μg/ml AuNPs was then used to treat cells as indicated in the relevant figures.
CAP Configuration and Treatment. The current research uses an experimental atmospheric dielectric barrier discharge (DBD) plasma reactor, which has been described and characterised in detail 33,55 . All U373MG cells were treated within containers, which were placed in between two electrodes, at a voltage level of 75 kV for 30 s. Prior to CAP treatment, the culture medium was removed, and fresh culture medium was added into the cell culture container at 5% of the well working volume to prevent drying during treatment. Afterwards, fresh culture medium containing 100 μg/ml AuNPs or inhibitors as indicated was added to reach the well final working volume and incubated with cells at 37 ℃ for the indicated time.

H2DCFDA Assay and Optical Emission Spectroscopy (OES) and Ozone measurement.
H2DCFDA (Thermo Fisher Scientific) was used to detect ROS induced by CAP treatment.
U373MG cells were seeded into the TC dish 35 standard (35x10mm, Sarstedt) at a density of 2×10 5 cells/ml and incubated overnight to allow adherence. After washing twice with PBS, cells were incubated with 25 μM H2DCFDA in serum-free medium for 30 min at 37 ℃. Cells were then washed with PBS twice, culture medium once and then treated with CAP at 75 kV for 30s. The fluorescence of H2DCFDA was then measured using flow cytometry.
Optical emission spectroscopy was carried out using an Edmund Optics CCD spectrometer with a spectral resolution of between 0.6 nm to 1.8 nm. The spectra were measured using BWSpec TM software with a spectral range between 200 and 850 nm and were acquired every 7.5 s with an integration time of 1500 ms. Total relative intensity of each emission line was calculated using the integral of the area under each peak. EEDF was calculated using a line ratio method (N2 at 337 nm and N2 + at 391 nm) 56 . O3 was sampled using a standard Gastec sampling pump in conjunction with a Gastec detection tubes immediately after plasma discharge had ceased. For Propidium iodide (PI) staining, U373MG cells were seeded into TC Dish 35 at a density of 2×10 5 cells/ml and incubated overnight to allow adherence. Cells were then exposed to CAP 75kV for 30s. Afterwards, cells were incubated at 37 ℃ for 30 minutes, and collected by trypsinisation, resuspended into 1ml PBS. Resuspended cells were stained with 1µg/ml PI for 5 minutes. The fluorescence of PI was then measured using BD Accuri™ C6 Plus flow cytometry at FL2 (585/40nm) standard filter.