Photophysical properties of a photocytotoxic fluorinated chlorin conjugated to four β-cyclodextrins

Abstract

A meso-tetrakis(pentafluorophenyl)-chlorin with the reduced pyrrole ring linked to an isoxazolidine ring (FC) has been conjugated to four β-cyclodextrins (CDFC). The CDFC exhibits excellent water solubility and is a potent photosensitizer towards proliferating NCTC 2544 human keratinocytes. The study by conventional steady state absorption and fluorescence spectroscopies and by time-resolved femto- and nanosecond laser flash spectroscopies suggests that in ethanol and pH 7 buffer the β-cyclodextrins embed the highly hydrophobic tetrakis(pentafluorophenyl)-chlorin macrocycle and strongly interact with the chlorin rings in the singlet and triplet manifolds. In these solvents, femtosecond spectroscopy suggests that the conjugate undergoes a rapid relaxation in the upper excited singlet states induced by photochemical and/or conformation change(s) at a rate of about 5 ps⁻¹ to fluorescent states whose lifetime is ∼8 ns. This interaction is destroyed upon addition of Triton X100 to buffer. Both FC and CDFC strongly fluoresce (Φ_F ∼ 0.5) in micelles. Similar behavior is observed at the triplet level. In ethanol and water, the initial transient triplet state absorbance decays within 1–3 μs yielding a longer lived triplet with spectral properties indistinguishable from that of original difference absorbance spectra. The determination of the molar absorbance in the 440–460 nm region (∼35 000 M⁻¹ cm⁻¹) leads to an estimate of ∼0.2 for the triplet formation quantum yield of FC in toluene and of FC and CDFC in Triton X100 micelles. Quenching of the CDFC triplets by dioxygen in buffer produces ¹O₂ in a good yield consistent with the effective photocytotoxicity of the chlorin–cyclodextrins conjugate towards cultured NCTC 2544 human keratinocytes. By contrast, FC which aggregates in buffer produces little if any ¹O₂.