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LAMMP Seminar Video
Photosensitization with tetrapyrroles within the confined medium of a biological membrane
Benjamin Ehrenberg, PhD

Hydrophobic or amphiphilic tetrapyrrole sensitizers are taken up by cells and are usually located in cellular lipid membranes. Singlet oxygen is photogenerated by the sensitizer and it diffuses in the membrane and causes oxidative damage to membrane components. In this talk I shall describe two aspects of the localized photochemistry that is occurring in the confined membrane’s medium. Singlet oxygen’s intrinsic lifetime in the membrane is 15-35 µs, depending on the constituting lipids, while its lifetime in H2O is 3 µs. Therefore, any oxidative damage that singlet oxygen might cause to membrane proteins or lipids, must occur while it diffuses in the membrane, before escaping out of the lipid phase. Thus, the efficiency of photosensitization depends, among others, on the vertical location of the sensitizer in the membrane. We employed several classes of tetrapyrrole sensitizers (porphyrins, chlorins, phthalocyanines and porphyrazines), that were modified to alter their hydrophobicities and their insertion modes into membranes. We studied the consequential effect of the molecular structures and their membrane topography on the effective damage that is caused to membrane components. The uptake of the sensitizers by membranes, their intra-membrane locations and the kinetics of damage were determined by spectroscopic methodologies, including fluorescence quenching by iodide, the parallax method for fluorescence quenching and fluorescence resonance energy transfer (FRET). Depth effects were clearly observed in artificial membranes, but also in cells in vitro. The oxidative damage that is inflicted by the photosensitization process can occur to two classes of targets in the membrane: lipids and proteins. Depolarization of the Nernst electric potential on cells' membranes has been observed in cellular photosensitization, but it is not clear whether lipid oxidation is a relevant factor leading to abolishing the resting potential of cells' membranes and to their death. We studied the effect of the membrane’s lipid composition on the dissipation of the electric potential that is generated across the membrane. We employed electrochromic membrane probes that have a high fluorescence signal response to the potential. We found a correlation between the structure and unsaturation of lipids and the leakage of the membrane, following photosensitization. As the extent of non-conjugated unsaturation of the lipids is increased from 1 to 6 double bonds, the kinetics of depolarization become faster. When liposomes are composed of a mixture similar to that of natural membranes, and photosensitization is being carried out under usual PDT conditions, photodamage to the lipids is not likely to cause enhanced

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