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. 2006 Dec 14;4:14.
doi: 10.1186/1477-3155-4-14.

C60-fullerenes: Detection of Intracellular Photoluminescence and Lack of Cytotoxic Effects

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Free PMC article

C60-fullerenes: Detection of Intracellular Photoluminescence and Lack of Cytotoxic Effects

Nicole Levi et al. J Nanobiotechnology. .
Free PMC article

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.

Figures

Figure 1
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
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
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
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 formula image), 50 (patterned formula image) and 100 μg (filled ■) of C60 for 48 h and analyzed for cell cycle progression by flow cytometry.
Figure 5
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.

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