Characterization of radical types, penetration profile and distribution pattern of the topically applied photosensitizer THPTS in porcine skin ex vivo

•The main penetration of the photosensitizer THPTS into the skin occurs up to 10 min incubation time.•A penetration time >10 min promotes the loss of functionality of THPTS.•An irradiation between 400 and 700 nm is needed to activate THPTS in order to develop its effect as photosensitizer.•During PDT treatment mainly reactive oxygen species (ROS) and lipid oxygen species (LOS) are produced.•At low dose always ROS are prominent, at high dose LOS increases, independent of previous skin treatment as well as used wavelength. The topical photodynamic therapy (PDT) is mainly used in the treatment of dermato-oncological diseases. The distribution and functionality of the photosensitizer Tetrahydroporphyrin-Tetratosylat (THPTS) was investigated using microscopic and spectroscopic methods after topical application to excised porcine skin followed by irradiation. The distribution of THPTS was determined by two-photon tomography combined with fluorescence lifetime imaging (TPT/FLIM) and confocal Raman microspectroscopy (CRM). The radicals were quantified and characterized by electron paramagnetic resonance (EPR) spectroscopy. Results show a penetration depth of THPTS into the skin down to around 12 ± 5 µm. A penetration of THPTS through the stratum corneum was not clearly observable after 1 h penetration time, but cannot be excluded. The irradiation within the phototherapeutic window (spectral range of visible and near infrared light in the range ≈ 650–850 nm) is needed to activate THPTS. An incubation time of 10 min showed the highest radical production. A longer incubation time affected the functionality of THPTS, whereby significant less radicals were detectable. During PDT mainly reactive oxygen species (ROS) and lipid oxygen species (LOS) are produced. Overall, the irradiation dose per se influences the radical types formed in skin. While ROS are always prominent at low doses, LOS increase at high doses, independent of previous skin treatment and the irradiation wavelength used.