C60-fullerenes: detection of intracellular photoluminescence and lack of cytotoxic effects

Abstract

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 microg/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.

Figure 1

Physical properties of methanol C₆₀. (A). Fullerenes suspended in water, methanol, and toluene. (B). UV/Vis absorption spectra of C₆₀ suspended in methanol at a concentration of 0.2 mg/ml. (C). Samples were excited with 488 nm and PL spectra were recorded. (D). Measurements of particle size distributions of C₆₀ in methanol (solid line) or in toluene (dashed line). (E) TEM micrograph of fullerene crystals in methanol drop-deposited onto a copper grid. Scale bar is 50 nm.

Figure 2

MALDI-TOF spectral analysis of C₆₀ preparations. C₆₀ was prepared in toluene (Panel A), in the water-soluble fullerene extracted from toluene (panel B) and in methanol (panel C). Representative aliquots of each preparation were analyzed by MALDI-TOF using α-cyano-4-hydroxycinnamic acid as the matrix. Spectra were acquired in the positive ion reflectron mode using the reflectron. The instrument was calibrated externally using a mixture of standard peptides (angiotensin II, 1046.54 Da; Substance P, 1347.736 Da; bombesin, 1619.823 Da; and ACTH clip 1–17, 2093.087 Da).

Figure 3

Cellular uptake of methanol C₆₀. (A). Phase contrast image of a MDA MB231 cell which has internalized a C₆₀ cluster. Intracellular C₆₀ retains its PL signature. Scale bar is 20 μm. (B). Confocal microscopy of internalized C₆₀ aggregates (red) identified with arrows. Methanol C₆₀-treated MCF10A cells were plated on collagen coated chamber slides, fixed, counterstained with FITC-phalloidin. A compiled 3-dimensional projection of optically sectioned z-stack is shown. Scale bar is 5 μm.

Figure 4

C₆₀ does not inhibit cell proliferation. MCF 10A and (Panel A) MDA MB 231 (Panel B) cell lines were cultured either in the absence or presence of methanol C₆₀ (0.2 mg/ml) and cell proliferation was assayed by crystal violet staining. ◆ Control, no C₆₀, ■ 10 μg C₆₀, ▲ 50 μg C₆₀, X 250 μg C₆₀. (Panel C). MDA MB 231 cells were simultaneously stained with calcein and ethidium using a live-dead assay kit. Lack of red-colored cells and the presence of cells stained in green indicate the lack of toxicity (Panel D). MDA MB 231 cells were either untreated (open box □) cultured with varying amounts 10 (gray ), 50 (patterned ) and 100 μg (filled ■) of C₆₀ for 48 h and analyzed for cell cycle progression by flow cytometry.

Figure 5

Water soluble toluene nano C₆₀ also does not block cell proliferation. Absorption spectra (A) and particle sizes (B) of water soluble nano C₆₀ from toluene are consistent with those reported in literature. The peak absorption wavelengths are indicated by arrows in A and the average particle size of the water soluble C₆₀ is 122 nm. MDA MB 231 (C) and HepG2 (D) cells were cultured with 2.7 μg (dotted line) or 27.4 μg (dashed line) of water soluble toluene nano C₆₀ or were untreated (solid line) and cell proliferation was assayed by crystal violet staining method.