Functionalized fullerenes mediate photodynamic killing of cancer cells: Type I versus Type II photochemical mechanism

Abstract

Photodynamic therapy (PDT) employs the combination of nontoxic photosensitizers (PS) and harmless visible light to generate reactive oxygen species (ROS) and kill cells. Most clinically studied PS are based on the tetrapyrrole structure of porphyrins, chlorines, and related molecules, but new nontetrapyrrole PS are being sought. Fullerenes are soccer-ball shaped molecules composed of 60 or 70 carbon atoms and have attracted interest in connection with the search for biomedical applications of nanotechnology. Fullerenes are biologically inert unless derivatized with functional groups, whereupon they become soluble and can act as PS. We have compared the photodynamic activity of six functionalized fullerenes with 1, 2, or 3 hydrophilic or 1, 2, or 3 cationic groups. The octanol-water partition coefficients were determined and the relative contributions of Type I photochemistry (photogeneration of superoxide in the presence of NADH) and Type II photochemistry (photogeneration of singlet oxygen) were studied by measurement of oxygen consumption, 1270-nm luminescence and EPR spin trapping of the superoxide product. We studied three mouse cancer cell lines: (J774, LLC, and CT26) incubated for 24 h with fullerenes and illuminated with white light. The order of effectiveness as PS was inversely proportional to the degree of substitution of the fullerene nucleus for both the neutral and the cationic series. The monopyrrolidinium fullerene was the most active PS against all cell lines and induced apoptosis 4-6 h after illumination. It produced diffuse intracellular fluorescence when dichlorodihydrofluorescein was added as an ROS probe, suggesting a Type I mechanism for phototoxicity. We conclude that certain functionalized fullerenes have potential as novel PDT agents and phototoxicity may be mediated both by superoxide and by singlet oxygen.

Figure 1

Structures of (A) BF1–BF3, (B) BF4–BF6. (C) UV-visible absorption spectra of BF1–BF3 and (D) BF4–BF6 in DMSO:water 1:9.

Figure 2

Survival curves of (A) LLC; (B) J774; and (C) CT26 cells after 24 h incubation with 2 μM BF1–BF6 followed by a wash and illumination with white light. A MTT assay was carried out after 24 h incubation. Values are means of 9 separate wells and bars are SD. Experiments were repeated at least twice.

Figure 2

Survival curves of (A) LLC; (B) J774; and (C) CT26 cells after 24 h incubation with 2 μM BF1–BF6 followed by a wash and illumination with white light. A MTT assay was carried out after 24 h incubation. Values are means of 9 separate wells and bars are SD. Experiments were repeated at least twice.

Figure 2

Survival curves of (A) LLC; (B) J774; and (C) CT26 cells after 24 h incubation with 2 μM BF1–BF6 followed by a wash and illumination with white light. A MTT assay was carried out after 24 h incubation. Values are means of 9 separate wells and bars are SD. Experiments were repeated at least twice.

Figure 3

Time course of apoptosis as measured by a fluorescent caspase assay in CT26 cells receiving BF4-PDT (80% lethal dose) or BF6-PDT 60% lethal dose).

Figure 4

Fluorescence micrographs of J774 cells that had been incubated with intracellular ROS probe H₂DCFDA, illuminated with 5 J/cm² 405 nm laser and imaged after 5 min. (A) H₂DCFDA without fullerene; (B) BF4 for 24 hours + H₂DCFDA. Scale bar is 100 μum.

Figure 5

Time decay curves of 1270-nm luminescence from singlet oxygen produced when BF4 (49 μM), BF6 (52 μM) or riboflavin (RBFL, 17 μM) were excited with a 5-ns 449-nm laser pulse. (A) deuterated methanol; (B) deuterated PBS; (C) compare BF6 in air or in nitrogen.

Figure 6

(A) Increase with illumination time (broad band white light) in ESR signal from superoxide-specific spin trap (DMPO-OOH) and BF4 or BF6 (35 μM) in presence of 1 mM NADH or 2 mM histidine in 1:3 H₂O:DMSO. (B) Oxygen consumption rates for BF4 or BF6 (35 μM) in presence of 1 mM NADH or 2 mM histidine with or without 5-mM sodium azide in 1:3 H₂O:DMSO determined by ESR oximetry.