This is the first study showing that singlet oxygen kinetics of topically applied photosensitizers coincides with the microarchitecture of skin, e.g., fissures and hair follicles. The kinetics indicate a chemical interaction of singlet oxygen with the skin, which allows differentiating between residual crème, e.g., in the follicular orifice, and photosensitizer penetrated into the skin. We show the feasibility of an easy-to-use fiber optic application providing the opportunity for in situ investigation, as well as a setup with focused optics for high-resolution two-dimensional scanning of singlet oxygen luminescence kinetics in skin samples. The results show that time-resolved singlet oxygen luminescence detection in tissue is a desirable tool for medical therapy, diagnostics, and evaluation of singlet oxygen interaction with biological environments.
LEDs have more and more influence on daily life as well as on scientific equipment. In this paper we want to report
results gained with the first LED powered setup for time resolved detection of the singlet oxygen luminescence in
solution as well as in cell suspension. The results show, that this setup can compete with the best laser powered setups
worldwide. The high sensitivity comes along with a superior long term stability and wavelength versatility. Setups based
on LED excitation can simplify the technical part of such measurement very much and reduce the costs, making this
technology available for a wider scientific community.
Singlet oxygen plays a crucial role in photo-dermatology and photodynamic therapy (PDT) of cancer. Its direct observation by measuring the phosphorescence at 1270 nm, however, is still challenging due to the very low emission probability. It is especially challenging for the time-resolved detection of singlet oxygen kinetics in vivo which is of special interest for biomedical applications. Photosensitized generation of singlet oxygen, in pig ear skin as model for human skin, is investigated here. Two photosensitizers (PS) were topically applied to the pig ear skin and examined in a comparative study, which include the amphiphilic pheophorbide-a and the highly hydrophobic perfluoroalkylated zinc phthalocyanine (F64PcZn). Fluorescence microscopy indicates the exclusive accumulation of pheophorbide-a in the stratum corneum, while F64PcZn can also accumulate in deeper layers of the epidermis of the pig ear skin. The kinetics obtained with phosphorescence measurements show the singlet oxygen interaction with the PS microenvironment. Different generation sites of singlet oxygen correlate with the luminescence kinetics. The results show that singlet oxygen luminescence detection can be used as a diagnostic tool, not only for research, but also during treatment. The detection methodology is suitable for the monitoring of chemical quenchers' oxidation as well as O2 saturation at singlet oxygen concentration levels relevant to PDT treatment protocols.
For the fist time worldwide we report high amplitude NIR-Luminescence signals, measured in pig skin. The measurements are achieved with a recently developed setup that was shown to provide superior performance for 1O2- luminescence detection in vitro. This setup has been adapted to allow now the detection of singlet oxygen signals in vivo. Pig ears were used for first measurements as a widely accepted in vivo model for human skin.
The consumption of singlet oxygen quenchers during illumination of cells incubated with photosensitizers is a serious
issue for the determination of singlet oxygen kinetics in cells. Using LNCaP cells incubated with pheophorbide a it will
be shown, that already an illumination of 100 nJ/cell dramatically changes the value of the observables. This finding has
consequences for the spatial resolution of any time-resolved singlet oxygen measurement, since the radiative rate
constant of singlet oxygen in aqueous solutions is very small.
KEYWORDS: Luminescence, Molecules, Quantum efficiency, Oxygen, Fullerenes, Dendrimers, Chromophores, Absorption, Energy transfer, Molecular energy transfer
The photophysical properties of DAB-dendrimers from 1st to 4th generation as well as Diaminohexane - all
substituted with the in maximum achievable quantity of pheophorbide a (Pheo) molecules were studied in comparison
with a novel hexapyropheophorbide a - fullerene hexaadduct (FHP6) and a fullerene [6:0]-hexaadduct which carries
twelve pyropheophorbide a units (FHP12) using both steady-state and time-resolved spectroscopic methods. It was
found that neighboring dye molecules covalently linked to one DAB- or fullerene moiety due to the length and high
flexibility of carbon chains could stack with each other. This structural property is the reason for the possibility of
formation different types of energy traps, which were resolved experimentally. The dipole-dipole resonance F&diaero;rster
energy transfer between the dye molecules coupled to one complex caused a very fast and efficient delivery of the
excitation to a trap. As result the fluorescence as well as the singlet oxygen quantum yields of the different complexes
were reduced with increasing number of dye molecules per complex. Nevertheless in every case the singlet oxygen
generation was less influenced then the fluorescence quantum yield, exposing the complex to a non-negligible amount of
excited oxygen in the singlet state. While the fullerene complexes turned out to be stable under these conditions, the
DAB-dendrimer-backbones were completely fragmented to small rudiments carrying just one or a small number of dye
molecules.
Various tetrapyrrolic compounds are well-known photosensitizers in PDT. One of the important unsolved problems in PDT research is the selective accumulation of such compounds in tumor tissues. In this context different carrier systems for efficacious and directed transport of the dyes to the tumor tissue are still under investigation. From these investigations new problems have arisen like change of the sensitizer's photoactivity and photostability. As a result the relation between energy and electron transfer processes (that means the relative efficacy of sensitization via type I and II) during illumination can change. The photophysical properties of Pheoporbide a in different sensitizer-carrier systems (antibodies and liposomes), are investigated.
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