Photodynamic Therapy with Hexa(sulfo-n-butyl)[60]Fullerene Against Sarcoma In Vitro and In Vivo

Abstract

The hydrophilic molecular micellar hexa(sulfo-n-butyl)[60]fullerene (FC₄S), first synthesized in 1998 as a photosensitizer (PS) has been reported to exhibit high efficacy for singlet oxygen generation and antimicrobial photodynamic inactivation. The purpose of this study was to investigate the effects of photoactivated FC₄S for free radical generation and to mediate photodynamic therapy (PDT) of cancer in vitro and in vivo. The results demonstrated that following light irradiation, FC4S produced singlet oxygen, but after addition of electron donors such as ferrocytochrome c or NADH, FC4S also produced superoxide. The combination of FC4S with light irradiation was able to induce cytotoxicity to human fibrosarcoma cells and murine sarcoma 180 cells in vitro. Cell-killing was proportional to fluence as well as FC4S concentration. Photoirradiation by argon-ion laser after intraperitoneal injection of FC4S also resulted in inhibition of S180 tumor growth in vivo (up to 80% reduction of tumor volume). Hematological and blood biochemistry parameters of the cancer-bearing mice were improved by PDT. Based on these findings, we conclude that FC₄S has a great potential as a nanomedicine in PDT for cancer.

Figure 1

(A) Synthetic scheme and the chemical structure of molecular micellar hexa(sulfo-n-butyl)fullerene (FC₄S) in a form of sodium salt. Time-resolved ¹O₂ luminescence produced upon laser irradiation of FC₄S at (B) 512 nm and (C) 600 nm excitation in different solvents: (a) DMF, (b) DMSO, (c) H₂O, and (d) phosphate buffer solution (PBS).

Figure 2

(A) Dose-dependent superoxide radical production by FC₄S at concentrations: (a) none, (b) 12.5 μM, (c) 25.0 μM, and (d) 37.5 μM under fluorescence light excitation (27 W) in the presence of cytochrome c. (B) Time-resolved ¹O₂ luminescence emission from FC₄S (10 μM) in D₂O−H₂O (3:1) in the presence of an increasing concentration of NADH (molar ratio of FC₄S:NADH from 1:0 to 1:1000), under photoexcitation using a crystal pulse laser at 532 nm. (C) Plot of k_obs (1/lifetime of singlet oxygen, s⁻¹ versus NADH concentration.

Figure 3

Cell morphological changes with (b, d) and without (a, c) the addition of FC₄S (5 μM) followed by broad-band light irradiation (8 mW/cm² for a period of 40 min. (a), (b) are human fibrosarcoma cells and (c), (d) are murine sarcoma 180 cells. (a), (b) Giemsa stain × 400; (c), (d) Feulgen stain × 400. Killing of human fibrosarcoma cells (e) and murine sarcoma 180 cells (f) by PDT, with the addition of FC₄S (0–10.0 μM) and fluorescence light irradiation for 0–60 min (0–30 J/cm². Each data point represents mean ± SE of 4 replicates.

Figure 4

Representative pictures of ICR mice with S180 tumors growing for 30 days after being (A) untreated; (B) treated with intraperitoneal injection of FC₄S (5.0 mg/kg) combined with laser (100 J/cm² irradiation. (C) Tumor size growth curves of: tumor untreated control, 100 J/cm² laser alone, 15 mg/kg FC₄S (i.p.) dark control, 5.0 mg/kg FC₄S (i.p.) + 100 J/cm² laser, 10 mg/kg FC₄S (i.p.) 100 J/cm² laser, and 15 mg/kg FC₄S (i.p.) + 100 J/cm² laser irradiation. Each data represents mean SE ± of 10 mice. *p < 0 05; **p < 0 01; ***p <+0 001 PDT groups versus control groups.