Cationic fullerenes are effective and selective antimicrobial photosensitizers

Abstract

Fullerenes are soccer ball-shaped molecules composed of carbon atoms, and, when derivatized with functional groups, they become soluble and can act as photosensitizers. Antimicrobial photodynamic therapy combines a nontoxic photosensitizer with harmless visible light to generate reactive oxygen species that kill microbial cells. We have compared the antimicrobial activity of six functionalized C(60) compounds with one, two, or three hydrophilic or cationic groups in combination with white light against gram-positive bacteria, gram-negative bacteria, and fungi. After a 10 min incubation, the bis- and tris-cationic fullerenes were highly active in killing all tested microbes (4-6 logs) under conditions in which mammalian cells were comparatively unharmed. These compounds performed significantly better than a widely used antimicrobial photosensitizer, toluidine blue O. The high selectivity and efficacy exhibited by these photosensitizers encourage further testing for antimicrobial applications.

Figure 1

Synthesis of BF1–3 (i) 200ºC, 45 min. (ii) Acetic anhydride, pyridine, 18 hr. (iii) CBr₄, DBU, acetone, toluene, 4.5 hr. (iv) K₂CO₃, methanol, H₂O. Et, ethyl; Ac, acetyl; DBU, 1,8-Diazabicyclo[5.4.0]undec-7-ene.

Figure 2

Synthesis of BF4–6

Figure 3

UV-Visible Absorption Spectra of BF4–6 and TBO at 10 μM in 1:9 DMSO:Water

Figure 4

PDI of Bacteria with BF1–3 (A) S. aureus (10⁸ cells per ml) were incubated for 10 min with BF1–3 at a 100 μM concentration in PBS, followed, or not, by a wash (centrifugation and resuspension) and illumination with 400–700 nm light at an irradiance of 200 mW/cm². Aliquots were removed from the suspensions after the various fluences of light had been delivered and the CFU had been determined. Values are means of six independent experiments, and bars are SEM. *p < 0.05; **p < 0.01; two-tailed unpaired t test. (B) E. coli (10⁸ cells per ml) was treated the same as S. aureus.

Figure 5

PDI of S. aureus with BF4 S. aureus at 10⁸ cells per ml was incubated with the specified concentrations of BF4 for 10 min, followed by a wash and illumination with white light. Values are means of six independent experiments, and bars are SEM.

Figure 6

PDI of Bacteria and Yeast with BF4–6 (A–D) (A) S. aureus was incubated with a 1 μM concentration of BF4–6, and (B) E. coli, (C) C. albicans, and (D) P. aeruginosa, all at 10⁸ cells per ml, were incubated with BF4–6 at 10 μM concentrations for 10 min, followed by a wash and illumination with white light. Values are means of six independent experiments, and bars are SEM. *p < 0.05, **p < 0.01, ***p < 0.001; two-tailed unpaired t test.

Figure 7

Comparison of PDI of E. coli and Fibroblasts with BF4–6 and TBO (A) E. coli was incubated with a 10 μM concentration of BF4–6 or TBO in the presence of 10% FBS for 10 min, followed by a wash and illumination with white or red light, respectively. (B) L929 murine fibroblasts at 80% confluence were incubated with BF4–6 or TBO, both at a 10 μM concentration, for 10 min in the presence of 10% FBS, followed by a wash and illumination with white or red light. The survival fraction was calculated by using the MMT viability assay. Values are means of six independent experiments, and bars are SEM.