C60-Fullerenes: detection of intracellular photoluminescence and lack of cytotoxic effects
© Levi et al; licensee BioMed Central Ltd. 2006
Received: 11 September 2006
Accepted: 14 December 2006
Published: 14 December 2006
We have developed a new method of application of C60 to cultured cells that does not require water-solubilization techniques. Normal and malignant cells take-up C60 and the inherent photoluminescence of C60 is detected within multiple cell lines. Treatment of cells with up to 200 μg/ml (200 ppm) of C60 does not alter morphology, cytoskeletal organization, cell cycle dynamics nor does it inhibit cell proliferation. Our work shows that pristine C60 is non-toxic to the cells, and suggests that fullerene-based nanocarriers may be used for biomedical applications.
Recent advances in materials science have fueled tremendous interest in numerous potential biomedical applications of various nanomaterials. For example, fullerene C60 molecules are unique for their multi-functional uses in materials science and optics [1–4], and are considered for a variety of biological applications (reviewed in ), such as imaging probes , antioxidants [7–9] and drug carriers (taxol) . Our laboratory is interested in exploring whether novel multifunctional nanoparticles can be designed for cancer therapy and diagnosis. Realization of such a goal requires a better understanding of the interactions between nanoparticles and cells and it is important to determine whether or not the particles by themselves impact cell growth and differentiation. We have chosen C60 for initial studies because the established chemistries afford us the flexibility to couple various biologically interesting and relevant molecules.
However, some undesirable properties of C60 present specific challenges. For example, due to its inherent hydrophobicity, C60 is poorly soluble and naturally forms large micron-sized clusters in aqueous media. Therefore, organic solvents are routinely used for solubilization of C60  Consequently, cell biological studies with pristine C60 have been limited.
Whereas chemical conjugation of C60 to various water soluble molecules improves the overall aqueous compatibility, pristine C60 is routinely dissolved in toluene [12, 13], tetrahydrofuran (THF)  or other organic solvents, and then exchanged into water by extracting the organic phase with water. The resultant preparation is often referred to 'water soluble C60' which is typically of light yellow color and is estimated to contain a few hundred micrograms of C60/ml . It has been suggested that the aqueous C60 is toxic to cultured cells and the toxic effects are due to peroxidation of lipids in cell membranes [16–19]. Various groups have reported that C60 (prepared using different methods) is not toxic [20–24] and some have attributed the toxicity of C60 to the side chains present on the functionalized C60 . Possible mechanisms that might contribute to the observed toxicity of nano C60, include the solvent effects like atmospheric exposure of solvents such as THF (according to the manufacturer). Additionally, acquisition of ionogenic groups upon C60 crystal formation in aqueous media via THF solvent exchange have been reported to contribute to the potential biological consequences . In support of these possibilities, a recent study suggests that toxicity of THF-derived water soluble nano C60 is abolished by removing THF by γ-irradiation. .
The conflicting data on cytotoxic effects of C60 merits attention and requires a resolution if these materials are to become biologically useful. The following simple hypothesis may reconcile with the mutually contradictory data on the cytotoxic effects of pristine fullerenes. C60 undergoes modifications during the preparation of water soluble C60, and such changes are responsible for the cytotoxic effects. Whereas the precise nature of such modifications is unknown at present, the hypothesis can be tested and the effects of C60 can be unequivocally examined if C60 can be applied to cells in such a way that obviates the need of preparing water soluble C60.
Studies presented in this manuscript examine the key issue of observed cytotoxic effects of C60 in cultured normal and malignant breast epithelial cells. We have developed a new, yet simple, method to directly apply C60 to cultured cells by modifying an established cell biological technique used in anoikis studies [28, 29].
Although several key properties of fullerenes, such as the characteristic photoluminescence (PL) of C60 are well characterized in solutions  and polymer complexes , few have examined such properties in cellular environment. Photoluminescence of crystalline C60 occurs due to coupling of the vibrational modes of the lattice with electronic transitions and the PL signature of fullerene crystals may be useful to track the presence of C60. Results presented in this work demonstrate that unmodified C60 crystals are taken up by cells and intracellular C60 retains its optical properties, as determined by measurements of PL. Significantly, our studies reveal that C60 prepared by a variety of methods up to 200 μg/ml is not toxic to a number of cell types.
Results and discussion
To eliminate the use of toxic organic solvents for applying C60 to cells, we have adapted methods routinely used in cell culture studies involving polymer coating of tissue culture dishes following solvent evaporation [28, 29, 32]. Colloidal suspensions of C60 in methanol (0.2 mg/ml) were prepared by sonication as described in Materials and Methods and applied to tissue culture dishes as an uniform coating. The organic phase is allowed to evaporate in a tissue culture hood, which leaves behind a coating of C60 on the dish. Cells are plated on to these dishes of C60. The C60 plated using this technique requires minimal manipulation and does not contain harsh organic solvents in cell culture. We refer to this preparation of C60 as 'methanol C60.'
1) Properties of methanol C60
C60 exhibits a characteristic reddish orange PL signature in the solid state with a peak at 735 nm [31, 36, 37]. Methanol C60 retained this key property that is dependent on the interstitial spacing between C60 molecules in the crystalline structure with a broad peak around 750 nm (Figure 1C). These spectral findings are consistent with the established behavior of C60, which exhibits slight shifts in the absorption and PL peaks dependent upon the temperature  and the solvent used to disperse C60 . Consistent with the properties described above, methanol C60 suspensions, when applied to tissue culture substrata, exhibited readily detectable crystal sizes and marked PL when visualized by light microscopy (discussed in the next section). Together, these data suggest that C60 remains adequately suspended in methanol and that the spectral characteristics are similar to those prepared in other organic solvents.
Particle size measurements confirm the stability of methanol- C60 suspensions. Dynamic laser light scattering measurements show that toluene C60, used as a reference (Figure 1D), yields uniformly sized particles with a mean size of 32.7 nm, consistent with published data [18, 38]. Parallel measurements with methanol C60 reveals two peaks at 106 nm and 342 nm size, which indicates heterogeneity in the particle size (Figure 1D).
Transmission electron microscopy (TEM) was used to verify cluster sizes of fullerenes dried from methanol (Figure 1E). Methanol C60 clusters were observed in a wide range of sizes including large clusters in the micron range although many clusters smaller than 10 nm were observed. TEM micrographs corroborate particle size data obtained by dynamic light scattering which indicates the presence of a heterogenous mixture of variably sized clusters. Furthermore, following evaporation of methanol, the majority of fullerene clusters do not reaggregate, and have a range of sizes of tens of nanometers, although some larger clusters also exist. TEM data differ from that of the dynamic light scattering results in this regard since the light scattering apparatus accounts for the average of all sizes of fullerene clusters in solution.
Prolonged sonication of C60 in various organic solvents is routinely employed to prepare solutions of C60 [13, 39]. As an additional measure to ascertain that suspension and sonication of C60 in methanol has not introduced any modifications into the fullerene, we analyzed each preparation by matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry.
2) Growth of cells in presence of methanol C60
Previous studies have suggested that water soluble nano-C60 compromises the integrity plasma membrane, possibly due to lipid peroxidation . To determine whether C60 applied to cells by a different method would produce a similar toxic effect, we have tested the effects of methanol C60 on cultured cells. First, we have examined whether C60 crystals are taken up by cells.
The presence of intracellular C60 crystals was verified via examination through multiple focal planes using confocal microscopy. Normal breast epithelial cells (MCF10A) cultured overnight on methanol C60 were trypsinized, replated on collagen I, fixed in paraformaldehyde, extracted with 0.1% Triton X-100 and stained with FITC-labeled phalloidin for counterstaining. C60 crystals were readily evident by their characteristic reddish orange PL signature (Figure 3B). Multiple crystals of C60 of varying sizes were present in different focal planes, indicating their intracellular localization. Initial examination shows that intracellular C60 does not interfere with cell spreading on ECM or alter microfilament reorganization following attachment to ECM. Untreated (control) cells, processed in parallel, on the other hand, do not exhibit orange PL. Similar results were obtained with MDA MB 231 and MDA MB 435 breast cancer cells (data not shown). Since cytoskeletal reorganization following integrin activation involves a series of complex signaling events beginning with integrin activation and orchestrated activation of Rho GTPases , our results suggest that treatment of C60 is unlikely to interfere with the events following cell- ECM interactions.
3) Cell survival in presence of pristine C60
As discussed in the Introduction, there is a lack of consensus on the effects of C60 on cell growth, and we have hypothesized that the apparent cytotoxic effects of the nanoparticle are due to the methods of preparation and application of C60 to cells. Therefore, we have reassessed the effects of C60 on cell proliferation using methanol C60 and water soluble nano-C60 prepared from toluene.
Culturing of MCF10A and HepG2 cells with up to 27.4 μg/ml (27.4 ppm) of water soluble nano C60 derived from toluene had no effect on cell proliferation (Figures 5C & D). The lack of cytotoxic effects was confirmed by two different assays (crystal violet staining and live-dead cell assays) and cell cycle analyses. The amounts of C60 used in these experiments is comparable to those used in previous studies where extreme toxicity was reported with other water soluble nano C60 preparations [18, 19]. Thus, our findings with methanol C60 and water soluble nano C60 prepared from toluene demonstrate that cell proliferation is not inhibited by fullerenes and the nanoparticle does not exert toxic effects in cell culture.
Our efforts to increase the concentration of the nano C60 in cell culture studies is limited by the maximum concentration of C60 achievable in the water soluble preparation derived from toluene. Cell culture and proliferation in presence of other carbon nanomaterials, such as nanotubes, has also been successfully reported [42, 43] and such findings are consistent with our data that show cell growth in presence of pristine C60 is feasible. While several researchers (for example, see [44, 45]) report that nanotubes indeed are cytotoxic, a recent publication  attributes such toxicity to, at least, in part to technical issues. This is analogous to our hypothesis that methods of preparation of C60 accounts for the observed divergent cytotoxic effects of C60. Taken together, our data suggest that C60 particles can be utilized for the design and development of multi-functional nanoparticles and the core nanoparticle is unlikely to adversely affect cell physiology.
An important finding of this study is that C60, when applied as methanol suspension, is non-toxic to a variety of cell types and does not interfere with cell proliferation. This finding is supported by cell proliferation assays, cell cycle analyses and vital stains. Further, cells continuously cultured with C60 showed no defects in cell spreading and cytoskeletal organization, indicating the underlying cell-matrix interactions and signaling pathways are not adversely affected by C60. Our results are supported by other studies which show that C60, consistent with its well established electron acceptor properties, is a potent anti-oxidant [20, 47]. This key finding differs from several published reports [16, 18, 19, 41] which suggested that pristine nano C60 is toxic. To reconcile with the cell type differences, we have employed several normal and malignant epithelial cells and tested their proliferation in presence of toluene-derived water soluble nano C60. Some investigators have reported weak toxicity of a preparation of polyvinyl pyrrolidine (PVP) and C60 in cell culture and animal models compared to PVP alone [48, 49]. However, it should be noted that the amount of C60 used in those studies significantly exceeded that used in the present work and the method of preparation of C60 is different.
Whereas several studies have examined the effects of C60 on a variety of cells, few studies have examined whether fullerene crystals are taken up by the cells. Confocal microscopy of methanol C60-treated cells onto collagen matrices reveals intracellular C60 nanocrystals of varying sizes in normal and malignant breast cancer cells (Figure 3B). We believe that this is a first demonstration of intracellular pristine C60 crystals using the PL signature as the reporter. The data shown in Figure 3B suggests that internalized C60 retains its crystal structure as evident from its bright reddish orange PL. While we demonstrate of larger C60 crystals in cells by confocal microscopy, smaller crystals (≤ 200 nm) may not be detectable by this technique. Recent reports indicate the ability to detect fluorescence of carbon nanotubes in cellular systems [50–54]. These findings suggest the possibility of detecting intracellular C60 fluorescence, although the signal is generally weaker than the infrared signal of nanotubes. While other nanoparticles such as functionalized nanotubes [55, 56] and gold nanoparticles  are reported to be internalized through endosomal pathways, the route of internalization of pristine C60 is not known. Our data also suggest that the PL may be used as a reporting tool to estimate intracellular C60 levels, provided the yield from the smaller crystals can be quantitatively measured.
In summary, our work describes a simple and rapid method for application of C60 to cultured cells and to investigate the interactions of C60 with cells. We provide evidence that pristine C60 is taken up by normal and malignant cells and the intracellular C60 retains its PL signature. Finally, we demonstrate that continuous culture of cells with C60 is non-toxic and that cell adhesion, cytoskeletal reorganization following integrin activation and cell proliferation following treatment with C60 remain unaffected. The reported toxicity of pristine C60 is most likely due to incompletely understood solvent effects or to chemical modifications of the C60 that may occur during preparation. A key implication of our research is that fullerene-based nanoparticles could possibly be utilized for biomedical applications without negative consequences from the fullerenes themselves.
C60 fullerenes are useful for several biological applications. Here we described a new and simple method of applying these materials to cells and shown that they are taken up by cells. Significantly, we demonstrate that unmodified C60 fullerenes are not toxic to cells. This finding should clarify the issue of perceived toxic effects of fullerenes and enhance developing novel biomedical applications using these nanoparticles.
Materials and methods
C60 fullerenes (Sigma Chemical Co) were sonicated in methanol at 0.2 mg/ml using a water bath sonicator (Branson) for 30 minutes to create a suspended fullerene solution which is referred to as methanol C60. 'Water- soluble' nano C60 suspensions were prepared from toluene using published procedures [12, 13]. To prepare a 'nano-C60' suspension from toluene 0.5 mg of C60 was added per ml of toluene. The suspension was sonicated for 10 minutes in a water bath (Branson) until a uniform purple solution was obtained and all C60 had been dissolved as determined by observation. Following sonication in toluene, an equal volume of deionized water was added to the toluene/C60 suspension and an organic/water phase separation was observed. This solution was sonicated in a water bath until all the toluene had evaporated (no more purple solution left), typically requiring about 2–6 hours depending on batch quantity.
Fullerene suspensions were characterized by UV/Vis absorption (Beckman DU7500 spectrometer) and fluorescence spectroscopy. Photoluminescence (PL) measurements were made using a Safire2 multifunctional monochromator based microplate reader (Tecan Instruments). Because methanol C60 suspensions settle rapidly, spectra were recorded within 10 minutes of sonication.
Size measurements of the colloidal fullerene suspensions prepared from methanol and toluene were carried out using a light scattering Zetasizer Nano-S light scattering instrument (Malvern Instruments, Southboro, MA). Sonicated methanol C60 suspensions were immediately measured to prevent settling of the particles. Recording of the spectra was routinely completed within 10 minutes of sample sonication.
Transmission electron microscopy
Transmission electron microscopy was done on fullerene clusters dried from methanol onto formvar grids. A Phillips TEM Transmission Electron Microscope (model 400, 120 keV) was used and a sample of C60 in methanol was dried onto a formvar grid for observation of the clusters.
An Esquire MALDI-TOF mass spectrometer (Bruker Daltonics Instruments, Billerica, MA) was used to measure the masses of molecular species present in the various C60 preparations. Solutions containing C60 were mixed with equal volumes of saturated matrix solution (10 mg α-cyano-4-hydroxycinnamic acid per mL of 0.05% trifluoroacetic acid and 25% CH3CN). Mass spectra were recorded in positive and negative ionization modes using the reflectron mode and calibrations were performed using a peptide mass calibration kit supplied by Bruker Daltonics.
Normal (MCF10A) and malignant (MDA MB 435 and MDA MB 231) human mammary epithelial cell lines, and human liver carcinoma cell line (HepG2) were obtained from the American Type Culture Collection (Manassas, VA) and cultured under standard conditions.
Methanol C60 suspensions were prepared and immediately applied to 12-well tissue culture dishes based on a protocol used for anoikis assays [29, 32]. Following application of the suspensions, methanol was allowed to evaporate from the culture dishes while standing open in a sterile hood. Cells were plated onto the coated dishes and cultured in regular growth media in a tissue culture incubator. Cell proliferation was measured using crystal violet assays . Culture dishes were rinsed with phosphate buffered saline (PBS) and stained in crystal violet stain (0.25% w/v in 50% methanol) for 10 minutes. Following rinsing of the dishes to remove excess stain, the dishes were air-dried, the protein-bound dye was solubilized in 50% methanol and the absorbance was recorded at 540 nm . Each sample was measured in triplicate and the experiments were repeated at least twice. For some experiments, cell proliferation was assessed with a live-dead cell assay kit (Molecular Probes) containing calcein AM and ethidium dyes. Fluorescence microscopy was used to determine cell viablity by examining ratios of green (viable) to red (dead) cells.
Cell cycle profiles were determined by flow cytometry using established protocols [29, 60]. Cells were trypsinized and fixed in 70% ethanol for at least 24 h at 4°C, stained with propidium iodide and subjected to flow cytometric analysis on a BD FACStar instrument. The DNA content of cells in various phases of cell cycle was determined by Modfit program.
Light and confocal microscopy
All cells lines were incubated with 200 μg of C60 from the methanol preparation for 24 hours at 370C. Following incubation, cells were extensively washed with PBS to remove adherent extracellular fullerene clusters, trypsinized, and replated on collagen I coated (5 μg/cm2) chamber slides . Samples were either directly viewed by phase contrast microscopy using an Olympus microscope or processed for confocal microscopy. Light microscopy images were recorded with a standard white light source without a UV filter. For confocal microscopy preparation, samples were fixed in 4% paraformaldehyde, extracted with 0.5% Triton X-100, incubated with FITC- labeled phalloidin (Molecular Probes) to visualize actin cytoskeletal filaments, and mounted with the anti-fade kit (Molecular Probes) [32, 60]. Samples were viewed on a Zeiss LSM 510 confocal microscope. Detection of C60 was accomplished by excitation at 458 nm and the use of a long pass filter for λ > 650 nm. Images were optically sectioned and the projections of the compiled z-stack were imported into Adobe Photoshop (version CS2).
This work was supported by the funds from the Department of General Surgery, Wake Forest University School of Medicine and Kulynych Family Funds for Medical Research (GLP). Mass spectrometry was performed in the Biomolecular Resource Laboratory of the Comprehensive Cancer Center of Wake Forest University, supported by grant 5 P30 CA12197-30 from the National Cancer Institute of the National Institutes of Health.
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