CHANGESINELASTIC ANDMORPHOLOGICAL PROPERTIES OF ERYTHROCYTE CELLS DUETOPORPHYRIN-MEDIATED PHOTO-OXIDATION

In this project, we are employing a micropipette manipulation technique coupled with optical microscopy to study changes in the shape and elasticity of red blood cells (erythrocytes) when subjected to oxidative stress caused by photosensitizer molecules, which generate reactive oxygen species upon light exposure. Previously, Prof. Rosangela Itri's group conducted studies on oxidative stress a photosensitizer, cisporphyrin CisDiMPyP, which is hydrophobic. Now, we are interested in studying the effect of more water-soluble molecules. This is significant in the field of photosensitization because altering the distance between reactive species generators and their targets also changes the oxidation mechanism. There are two main known mechanisms: Type I involves direct electron transfer to the substrate, while Type II uses singlet oxygen to oxidize membranes. This research will allow us to compare results obtained previously by the group and deepen our understanding of the mechanisms at play in this system. Red blood cells are excellent model membranes for this study as they lack a nucleus and organelles, facilitating the transition of knowledge from model membranes synthesized by the group to membranes of animal cells. Thus, we are able to measure the shear modulus (resistance of cells to cutting forces) at different levels of oxidative stress that cells undergo until they rupture (hemolysis). Additionally, we conduct control assays to ensure the validity of results: testing cells without photosensitizers and light irradiation, cells under light irradiation without photosensitizers, and cells with photosensitizers but without light irradiation. For cell manipulation, we employ laboratory-manufactured glass micropipettes coupled with a micromanipulator and filled with a PBS buffer solution. To control the pressure exerted on cells, we use a transducer and a hydraulic system that adjusts the height of water reservoirs. Furthermore, to assess cytoskeletal integrity, we employ atomic force microscopy to evaluate the surface of erythrocyte ghosts, which depending on the structure obtained for the cytoskeleton, will provide us with information on the denaturation of filaments forming these proteins.