A Highly Water-Soluble C60-Oligo-Lysine Conjugate as a Type I and Type II Photosensitizer with Enhanced ROS Generation and Photocytotoxicity

Abstract

C₆₀ has been regarded as a suitable photosensitizer for photodynamic therapy due to its excitation in the phototherapeutic window (650–900 nm), high quantum yields of ¹O₂ generation, and low dark toxicity. However, the use of this molecule in biomedical applications has been limited by its high aggregation tendency in polar solvents (e.g., water), resulting in quenching of its excited states. In this study, a C₆₀-peptide conjugate, C₆₀-oligo-Lys, with a lower aggregation tendency was investigated by chemical, physical, and photophysical methods in comparison to a previously reported water-soluble C₆₀-PEG conjugate. Photoinduced ¹O₂ generation was evaluated by both phosphorescence at 1274 nm and the electron spin resonance method in an aqueous solution, with comparison to the control C₆₀-PEG, revealing the superior capacity of the C₆₀-oligo-Lys conjugate. Importantly, the photoinduced type I electron transfer reaction is occurring in C60-oligo-Lys very efficiently, even in the absence of an e^– donor, presumably due to the partially unprotonated amines in the peptide, to form O₂^•– and ^•OH, which are generated in a further enhanced way by the addition of a physiological concentration of NADH. These species are more harmful to the target cells, including hypoxic tissues with limited oxygen concentration. Femtosecond transient absorption spectroscopy revealed different excited state dynamics for C₆₀-oligo-Lys and C₆₀-PEG at short time scales in water. By an in vitro cellular assay, significant cytotoxicity of C₆₀-oligo-Lys was observed (IC₅₀ < 1 μM) on HeLa cells under visible light irradiation (527, 630, and 660 nm), while very limited cytotoxicity was observed for C₆₀-PEG (IC₅₀ > 25 μM) under the same conditions. The strongly enhanced photocytotoxicity of C₆₀-oligo-Lys can be ascribed to the higher generation of both type I and type II ROS in addition to the potential affinity of the positively charged oligo-Lys moiety for the negatively charged cell membrane. The C₆₀-oligo-Lys conjugate reported in this study therefore shows high potential as a core photosensitizer for photodynamic therapy.