Photosensitization in lysosomal mimetic membranes to investigate the cell death mechanism related to autophagy.

Abstract

Generating reactive oxygen species (ROS) in specific intracellular targets is the best strategy to maximize the Photodynamic Therapy (PDT) efficiency, since the short lifetime and high reactivity limited the ROS distribution inside cells. During years literature indicated mitochondria as target-organelle for PDT, however reports more recents indicate lysosomes as organelle-key to enhanced PDT efficiency. Photosensitizers (PS) that photodamage lysosomes impair the autphagic function pro-survival resulting in cell death associated to autophagy and increase the PDT efficiency regardless to the interaction level with membranes (T.M. Tsubone, tese de doutoramento 2016). These fact break some paradigms accepted in literature (i) binding in membrane is directly associated to the phototoxicity, (ii) mitochondria is the key-organelle in terms of specificity for PDT and (iii) apoptose is the cell death mechanism suitable in terms of better cell-killing efficiency. Therefore, once the photodamage in lysosomes has large potential to oppose with paradigms in this field, our goal is unveil the lysosome role in the cell death associated to the autophagy. Then, this project will investigate the effects of photosensitization lipid bilayers representing lysosomal membranes, which it will be mimicked by giant unilamellar vesicles (GUVs) in an unprecedented way in the literature.Since pores formed by photosensitization allows leak enzymes and/or macromolecules related to a specific mechanism of cell death (apoptosis and/or autophagy), our intention is to evaluate the increase in permeability of the lysosomal membrane via determination of pore size. We will also investigate the formation of membrane domains by photo-oxidation and its relation with the lysosomal-autophagic pathway as a result of PDT.For this, we choose two photosensitizers that localizes in lysosomes: TPPS2a (porphyrin) and DMMB (phenotiazine) in two membrane model: i) GUV containing only unsaturated phospholipid (simple and well known system) and ii) GUV to mimetic lysosomes (intracellular mimetic system, unprecedented in the literature).Both pore size and domains generated by photosensitization will be investigated under fluorescence optical microscopy and atomic force microscopy techniques.