Cytotoxic effects of plasma-irradiated fullerenol Daiki Kanno, Hiromasa Tanaka, Kenji Ishikawa*, Hiroshi Hashizume, and Masaru Hori Nagoya University, Furo-cho, Chikusa, Nagoya 464-8603, JAPAN *
E-mail: [email protected]
accepted in Journal of Physics D: Applied Physics (July 2018) DOI: 10.1088/1361-6463/aad510
Fullerenol dissolved in water was irradiated with a non-equilibrium atmospheric pressure plasma. Plasma irradiation altered the fullerenol solution by introducing carbonyl groups, ether bonds, and intercalated nitrate anions. The resulting plasma-irradiated fullerenol (PF) was added in cell culture medium. PF-supplemented medium exhibited a cytotoxic effect on HeLa cells. Notably, HeLa cells cultured in the PF-added cell culture medium generated intracellular reactive oxygen and nitrogen species (RONS), such that the endocytosis of PF induced the apoptotic cell death. The cytotoxic effect inducing apoptosis was found in the PF-supplemented medium.
1. Introduction Fullerenol has hydroxyl groups bonded to the skeletal structure of fullerene C60, rendering fullerenol highly water soluble. Numerous researchers have reported that this nanoparticle, though non-cytotoxic itself, exhibits high potential for the scavenging of radicals.1-12) Notably, fullerenol has attracted much attention for bio-applications as a scavenger of reactive oxygen and nitrogen species (RONS) such as superoxide anion radical O2−▪ and hydroxyl radical ▪OH, as reported by Saitoh et al.13,14) Fullerenol also has been shown to suppress lipid accumulation during differentiation.13,14) Moreover, fullerenol has antibacterial activity against Propionibacterium acnes and Staphylococcus epidermidis, as reported by Aoshima et al.15) However, the mechanism underlying these biomedical effects of fullerenol remains unclear. Nonetheless, these functional characteristics suggest that this nanoparticle may find use in multiple clinical applications. Recently, non-equilibrium atmospheric pressure plasma (NEAPP) has received much attention in biomedical applications. Iseki et al.16) has reported a selective killing effect of cancer cells without killing normal cells following direct irradiation by NEAPP of cultured cancer cells in petri dishes. Subsequent work employed cell culture medium that had been irradiated by NEAPP, a reagent designated plasma-activated medium (PAM). Culturing of glioblastomas (brain cancer cells) and astrocytes (normal cells) in PAM yielded selective killing of glioblastomas compared to astrocytes, as reported by Tanaka et al.17) PAM also was shown to exhibit in vivo antitumor activity, providing reduced tumor volumes when administered subcutaneously in a mouse xenograft model of ovarian cancer.18) Cell death in
RAS/MPKK-mediated cell proliferation signaling pathways were downregulated by PAM cultivation.19) Thus, PAM injection may provide a cancer therapeutic application that is less invasive and causes less inflammation in the living body. Intriguingly, it was recently reported that the plasma-activated Ringer's lactate solution also exhibits antitumor effects.20) Since PAM retains antitumor effects even after several hours of storage, irradiation-induced RONS with antitumor activity are presumably long-lived. The actual concentrations of hydrogen peroxide (H2O2), nitrite anion (NO2−), and nitrate anion (NO3−) generated in PAM were measured and a synergistic cell death effect of tens-of-μM-level H2O2 and mM-level of NO2− were reported by Kurake et al.21,22) Intracellular RONS such 2
as ONOO−, NO, and O2−▪ were observed by various fluorescent probes, and lipid peroxidation was detected in HeLa cells incubated in PAM.23,24) However, these RONS might be degraded by enzymes present in the living body (i.e., under in vivo conditions). Substances other than RONS, such as modified glucose and organic acids, may play an important role in antitumor effects, as proposed by Kurake et al.21) In vivo, the antitumor effect of PAM was decreased by catalases, a class of enzyme known to act as H2O2 scavengers. Catalase treatment decreased H2O2 generation when the medium was treated with plasma, and this enzyme enhanced cell viability of HeLa cells without inducing cytotoxicity, as reported by Satoh et al.25) These results partially explain why the anti-tumor effect of PAM is decreased when PAM is used in vivo although the same reagent is effective in vitro. Therefore, a method to deliver the RONS generated by plasma treatment is needed when the reagent is to be applied to cancer cells in vivo, to avoid enzymatic inactivation of antitumor agents. Here we focus on a method of storing radicals (such as oxygen atom radicals (▪O), ▪OH radicals, and ▪NO radicals) that are generated during NEAPP irradiation of liquid, yielding long-lived RONS. Nanoparticle such as fullerenol scavenges, for example, ▪OH with generating hydroxyl groups on the C−C and C=C bonds of fullerenol and destabilizes the fullerenol structure.26) If unstable fullerenol passes through cell membranes before decomposing to form other intracellular radicals, fullerenol might serve as a long-lived anti-tumor agent. This observation suggests that fullerenol is a candidate mediator of long-lived cytotoxicity. Namely, it would be a kind of nanoparticle-deliver or nanocarriers of drugs.27-36) Thus, our research motivates a combination of nanoparticles and plasma treatments, i.e., nanoparticle-supplemented plasma-activated medium. In the current study, we investigated the cytotoxic effects on cancer cells of a combination of both NEAPP and fullerenol. First, we observed the cytotoxicity of plasma-irradiated fullerenol (PF) using HeLa cells. To understand the mechanisms of cell killing by PF, we separately assessed the effects of modification of an aqueous solution of fullerenol by NEAPP irradiation, which yields structural changes in fullerenol itself. The endocytosis of fullerenol in HeLa cells was evaluated using PF added into the culture medium. We propose a cytotoxic mechanism of PF on the basis of physicochemical analyses of PF modification. 3
2. Experimental Fullerenol (C60(OH)n・mH2O (n>40, m>8), Cat. No. 793248; Sigma-Aldrich, St. Louis, MO) was used in this study. Fullerenol was dissolved at 1600 µM in 1 mL of Mill-Q water with mixing by vortex; the resulting solution was transferred to a 30-mm glass petri dish. The fullerenol solution was positioned at a distance of 3 mm from the exit of the NEAPP source. Figure 1 shows a photograph of the experimental set-up for NEAPP irradiation of the liquid. The plasma source was operated by applying a 60-Hz-ac power source (9 kV0-p) to a stream of argon gas flowing at 2 slm.37) The plasma source had an exit with a size of 1×20 mm2. Under these conditions, an otherwise-unobstructed plasma plume extended for a distance of about 7 mm into free space. The NEAPP delivered 2×1016 cm-3 of electrons and 4×1014 cm-3 of oxygen atoms, measured by the vacuum ultraviolet absorption spectroscopy (VUVAS).37,38) The NEAPP was placed 3 mm from the liquid surface and irradiation was allowed to proceed for 3 min. The 30-mm-diameter petri dish was rotated while held at a tilt angle of 10° during the irradiation to provide uniform irradiation of the liquid. After irradiation, the solution was completely dried for 1 h by evaporation using a hotplate at 70°C. The plasma-irradiated fullerenol was then collected from the bottom of the dish. HeLa cells (a human cervical cancer cell line) were used. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM: Cat. No. 5796; Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS) and penicillin (100 U/mL)-streptomycin (100 µg/mL) (P/S) (“DMEM+FBS+P/S”) in a humidified incubator containing 5.0% CO2 at 37°C. Cell lines were subcultured by diluting to yield 70% confluence. For cell viability tests, HeLa cells were suspended at 25000 cells/mL in DMEM+FBS+P/S and then distributed at 200 µL (5000 cells) per well in a 96-well plate. After 24 h, spent medium was replaced with 200 µL of experimental or control medium; samples were tested in triplicate. After cultivation for another 24 h, the sample was replaced
5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) reagent 4
(Promega Corp., Madison, WI). After cultivation for 1 h, the absorbance was measured at 490 nm to determine viable cell numbers. For the evaluation of apoptotic death, HeLa cells were suspended at 25000 cells/mL in DMEM+FBS+P/S and distributed at 1 mL per 35-mm glass bottom dish. After 24 h, the medium was replaced with 1 mL of sample. After cultivation for another 24 h, 1 mL of DMEM containing 0.50 µM Cell Event Caspase 3/7 detection reagent (Invitrogen, Carlsbad, CA) and 2.7 µg/mL 4’,6-diamidino-2-phenylindole (DAPI; DOJINDO, Kumamoto, JPN) was added to each sample, and culturing was allowed to proceed for another 30 min. Cells were observed with fluorescence and differential interference contrast (DIC) microscopy (OLYMPUS, Tokyo, Japan). As a reference, plasma activated medium (PAM) was prepared. DMEM (6 mL) was placed in a 35-mm dish and irradiated with NEAPP for 3 min with constant stirring at 300 rpm. For use in cultivation, PAM was diluted 1:15 in DMEM. For assessment of the effect of scavenging H2O2, the PAM was supplemented with catalase (Wako, Osaka, Japan) to a final concentration of 0.25 mg/mL and incubated for 1 h at 37°C in a 5.0% CO2 environment. For assessment of the endocytosis of fullerenol, HeLa cells were suspended at 25000 cells/mL in DMEM+FBS+P/S and distributed at 1 mL per 35-mm glass bottom dish. After 24 h, the medium was replaced with 1 mL of sample. After cultivation for another 24 h, 1 mL of DMEM containing 2.7 µg/mL DAPI was added to each sample, and culturing was allowed to process for another 15 min. Cells were observed by fluorescence microscopy. For assessment of intracellular RONS, HeLa cells were suspended at 25000 cells/mL in DMEM+FBS+P/S and distributed at 1 mL per 35-mm glass bottom dish. After 24 h, the medium was replaced with 1 mL of sample. After cultivation for another 24 h, 1 mL of DMEM containing 2.7 µg/mL DAPI and 10 µM CM-H2DCFDA (Invitrogen, Carlsbad, CA) was added to each sample, and culturing was allowed to proceed for another 5 h. Cells were observed by fluorescence microscopy. For the analysis of the chemical composition of fullerenol, Fourier-transform infrared spectroscopy (FT-IR; is50, Thermo Scientific, Waltham, MA) measurements were performed on a pellet of 0.2 µmol of PF and 30 mg of KBr (Wako, Osaka, JPN); this pellet 5
was formed by pressure treatment at 9800 N for 10 min. All assays were performed in duplicate and the empirical standard deviations were calculated. Empirical standard deviations demonstrate the reproducibility of parallel assays but do not allow statistical analysis of variance. The chi-square test was used for the statistical determination of significances (p < 0.05 = significant; p < 0.01 = high significant). Asterisks signifying statistical significance are shown in the figures.
3. Results and discussion 3-1. Cytotoxic effects on HeLa cells of plasma-irradiated fullerenol (PF) Cytotoxic effects of PF on HeLa cells were evaluated. After a 24-h cultivation, cell viabilities were measured by the MTS assay, allowing comparison among cultures grown in DMEM+FBS+P/S (control sample), medium added with 50 µM fullerenol, and medium added with 50 µM PF, as shown in Fig. 2. No change in cell viability was observed between the control and the fullerenol-added cultures. In contrast, the PF-added medium yielded an approximately 30% loss of cell viability. This result indicated that fullerenol is not itself cytotoxic, but NEAPP irradiation renders a solution of fullerenol cytotoxic to HeLa cells. Meanwhile, the activation of caspase 3/7 as an apoptotic marker was observed by fluorescent staining. Figure 3 shows the DIC, caspase-activation and DAPI staining fluorescent images after the 24-h cultivation with and without fullerenol and PF. In the DIC images, cell morphology for the PF-added culture samples shows apparently shrinkage and rounded cell-shapes, indicating characteristics of apoptosis but not necrosis. Green fluorescence, which arises from caspase activation, indicated that PF-supplemented medium induces the apoptotic cell death of HeLa cells. It should be noted that weak fluorescence was unable to determine the activation, so that fullerenol has substantially weak fluorescence. The DAPI staining fluorescence represents dead cells resulting from loss of cell viability during culturing in the PF-supplemented medium. Thus, exposure to PF induces apoptosis in HeLa cells. Figure 4 shows cell viabilities after 24-h-cultivation in the PF-added medium with and without catalase. The PF-added medium killed 30% of HeLa cells even without H2O2 (i.e., with catalase) As a reference, the viability of cell grown in PAM was 40% and 100% 6
without and with catalase, respectively. Thus, the cytotoxic activity of PAM was sensitive to catalase addition. No antitumor effect was observed for standard medium containing catalase, indicating that the antitumor effects of PAM reflected H2O2 generated (directly or indirectly) by irradiation. In contrast, the cytotoxic effect of PF-supplemented medium was retained after catalase treatment. This result indicated that the cytotoxic effect of PAM is related to H2O2, while the effect of the PF-added medium appears be arise from the PF itself.
3-2. Endocytosis of fullerenol and intracellular RONS PF's cell-killing effects were investigated to assess whether the PF acted from within or without the cells. Since it is known that the fullerenol is a fluorescent molecule,39) the endocytosis of fullerenol or PF was observed by fluorescence microscopy. Figure 5(a) shows fluorescent images after 24-h cultivation of HeLa cells in DMEM supplemented with 800 µM PF. Green fluorescence indicates endocytosed PF; blue fluorescence indicates DAPI staining of dead cells, in which the dye can penetrate and stain nuclear DNA. The merged images clearly show overlap of the dead cells and those that have endocytosed PF. This result indicated that the death of the HeLa cells is induced by the endocytosis of PF. A fluorescent probe method was used to assess the presence of intracellular RONS in HeLa cells cultured in DMEM containing 50 µM PF. Figure 5(b) shows fluorescent images after 24-h cultivation. Green fluorescence due to intracellular RONS and blue fluorescence due to DAPI staining of dead cells were observed together only in the cells grown in PF-added-DMEM. Thus, intracellular RONS appear to result from cultivation in PF added-DMEM.
3-3. Structural change of fullerenol by NEAPP irradiation The chemical structure of PF was measured by FT-IR of PF in combination with KBr. Figure 6 shows FT-IR spectra for fullerenol (unirradiated, i.e., 0 min) and PF; in this case, PF was prepared by 1.5 and 3.0 min irradiation with NEAPP. As the NEAPP irradiation interval rose, increased absorbance was observed for peaks assignable to carbonyl (C=O) groups (1700 cm−1), hydroxyl (−OH) groups (1400 cm−1), and ether 7
(C−O−C) bonds (1000 and 1200 cm−1). In contrast, a peak for C=C bonds (at 1600 cm−1) remained almost constant in intensity. Major peaks were assignable to the literature values; for instance, computed infrared and Raman spectra of fullerenol were previously reported by Dawid et al.40) Experimental infrared spectra are assigned by hydroxyl (-OH) absorption bending mode at 1430 to 1440 cm−1 and fullerene skeleton stretching mode at 1600 to 1650 cm−1.41) Notably, the spectrum for PF exhibited novel carbonyl and ether bond absorptions. Interestingly, a peak for nitrate ion (NO3−) at 1400 cm−1 was detected only in the plasma-irradiated samples. Hamwi et al. has reported intercalation of NO3− between two fullerenol molecules.42) Separate work has indicated that, when a plasma flare comes in contact with the surface of water at atmospheric pressure, the water's pH falls (to approximately 5 or 6) and NO3− is generated in the water.43,44) We postulate that PF with intercalated nitrate anions plays an important role in the generation of intracellular RONS, resulting in the enhanced cytotoxicity of PF. However, no cytotoxic effect was observed when cells were grown in DMEM supplemented with 2000 µM NO3−.45) Thus, nitrate itself clearly does not contribute directly to the observed cytotoxic effect of PF. Plasma-generated RONS such as OH▪ and NO3− may synergistically generate a chemically active functionalization of fullerenol. Similarly, organic molecules have been reported to be synergistically functionalized by RONS such as peroxides and nitrites, as reported by Kurake et al.46) We propose that the RONS-functionalized fullerenol can be regarded as an antitumor drug carrier. Further clarifications of this hypothesis will be needed. Fullerenol has hydroxyl (-OH) groups that yield peaks at 3600 and 1700 cm−1. Since the C=C absorption was still observed in PF, the generated functional groups might be formed by replacement or functionalization of the OH groups. Ultraviolet (UV) or vacuum ultraviolet (VUV) emissions are known to dissociate chemical bonds under plasma irradiation, when the affected bond energies are smaller than those of the UV and VUV emissions. Irradiation with highly energetic photons may excite the fullerene skeleton to the single-excited state, resulting in intersystem crossing to the triplet-excited state, as argued by Markovic and Trajkovic.47) The triplet state can be efficiently quenched by molecular oxygen to generate large amounts of singlet oxygen, leading to electron transfer and forming the superoxide anion radical.47) Thus, the fullerene skeleton can yield reactive 8
oxygen intermediates by photochemical reactions. In water, fullerenol is a direct absorber of penetrating UV radiation and also can react with short-lived radicals of H▪ and OH▪ that are generated by photodissociation of water. In addition, long-lived RONS such as NO2− and NO3− may mediate the functionalization of fullerenol via its hydroxyl groups. Carboxylic acid-substituted fullerene derivatives (C60(C(COOH)2)2) also are potent ROS scavengers.48) However, the C60(C(COOH)2)2 scavenging effect was significantly less than half of that of fullerenol, as reported by Yin et al.48) The large electron affinity of the carboxylic group apparently facilitates the binding of O2−▪, leading to the suppression of secondary generation of O2−▪. As a result, the carboxylic acid moieties also facilitate the production of H2O2. The functionalization of the fullerenol skeleton is expected to contribute to the loss of cytotoxic chemical potential. Fullerenol induces ion permeability on the basis of pore formation in the lipid membrane, a process that is enhanced under acidic conditions, as reported by Rokitskaya et al.49) We note that the observed modifications (such as those from CO to COH and from OH− to NO3−) can occur chemically or electrochemically without any physical change in the fullerene skeleton. It is also noteworthy that the antitumor effects of PF may arise from plasma-activated functionalization of the skeleton. Ether bonds on fullerenol act as a reaction center with hydrogen ions (H+), as reported by Wu et al.50) DMEM is neutral or weakly basic, exhibiting pHs ranging from 7 to 9. Intracellular pH becomes weakly acidic to neutral in medium of pH 6.9 to 7.4. Endocytosis of PF containing ether bonds (C−O−C) presumably results in the formation of carbonyl groups (>C=O), as observed in the FT-IR measurements, due to the relatively higher intracellular H+ concentration. Changes in the conformation of fullerenol may induce defective C−C sites on this molecule. We suppose that destabilization of the fullerenol structure may induce the generation of secondary ▪OH radicals and O atoms by decomposition or subsequent generation of H2O2. This hypothesis is consistent with the results of our catalase addition experiments. The secondary OH effect on cytotoxicity has not been proven yet. Although RONS such as NO3− are actually generated by plasma irradiation, individual RONS do not appear to exhibit cytotoxicity; however, it should be considered whether a synergism between OH and NO3 plays a role in the antitumor effect. It has been suggested 9
that proendocytotic responses of nanoparticles may lead to oxidative stress and oxidative injury.33,34) The intracellularly proendocytotic oxidative responses were actually observed in these experiments, and the NO3-decorated fullerenol may however lead to produce cytotoxic oxidants such as ONOO‒. The remarkable functionalization of fullerenol (by plasma irradiation) and the resulting molecule’s endocytosis must be a trigger for the antitumor effect, even though purified fullerenol has no cytotoxicity itself. PF-induced intercellular RONS are presumed to be cytotoxic agents, leading to the killing of HeLa cells. The results obtained in the present study may find widespread application in medicine, and therefore deserve further study.
4. Conclusions The cytotoxic effects on cancer cells of plasma-irradiated fullerenol (PF) were investigated. Although no cytotoxicity was observed for fullerenol itself, PF demonstrated killing of HeLa cells. PF cytotoxicity was preserved regardless of catalase addition. PF harbors intercalated nitrate anions, as demonstrated by infrared spectroscopy. Cultivation in PF-added cell culture medium resulted in PF endocytosis and the generation of intracellular reactive oxygen and nitrogen species (RONS), yielding the induction of apoptotic cell death. Non-equilibrium atmospheric pressure plasma (NEAPP) irradiation of fullerenol enhanced the presence of functional groups such as carbonyl groups and ether bonds on the fullerenol skeleton, and created chemical bonds involving nitrate. Given that PF is known to penetrate weakly acidic cells, we hypothesize that endocytosed PF reacts with intracellular hydrogen ions, leading to the formation of ether and C-C bonds on the fullerenol skeleton. These reactions are expected to result in modification of the PF functional groups, yielding upregulation of intracellular RONS and inducing cell death, thus explaining the observed cytotoxicity of PF.
Acknowledgment This work was supported in part by JSPS-KAKENHI Grant Number 24108002.
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Figure Captions Fig. 1. (online color) Experimental setup of NEAPP irradiation to fullerenol in water.
Fig. 2. (online color) Cell viabilities after the cultivation in fullerenol-added DMEM and PF-added DMEM for 24 h.
Fig. 3. (online color) Fluorescent images of activation of caspase 3/7 after cultivation in PF-added DMEM for 24 h.
Fig. 4. (online color) Cell viabilities after cultivation in PAM and PF-added DMEM in existence of catalase for 24 h.
Fig. 5. (online color) (a) Fluorescent images of PF after cultivation in PF-added DMEM for 24 h. (b) Fluorescent images of intracellular RONS after cultivation in PF-added DMEM for 24 h.
Fig. 6. (online color) FT-IR spectra for fullerenol and PF for different irradiation time for 1.5 and 3.0 min.
Plasma (NEAPP source) 60 Hz, 9 kV0-p Ar 2 slm, 3 min 70°C 1h
... . .. .... ..... .
10° Fullerenol in 1 ml H2O (1600 µM)
PF added DMEM (50 µM)
PF-added DMEM Without catalase PAM (NEAPP 3min)
For viability tests; MTS assay
DMEM 25000 of HeLa cells (37 ºC 5% CO2)
For fluorescence detection; Caspase 3/7, DAPI, and H2DCFDA
Fig. 1. (online color)
* : p