Publication
Reactive Oxygen Species (ROS) is a family of oxygen-based active molecules, which are deeply involved in numerous crucial processes of cellular metabolism, where the redox equilibrium is very important. An imbalance between ROS and the cell ability to neutralize them with antioxidants results in an Oxidative Stress (OS). This leads to cell alterations and impaired physiological functions. OS is involved in a growing list of human pathologies, from brain disorder, like Alzheimer and Parkinson's disease, to various forms of cancer (skin melanoma) and eye pathologies (cataract, macular disease), as well as in the aging process. Sometimes, inducing OS selectively in diseased cells can also act as a powerful therapy. This is the basis of PhotoDynamic Therapy (PDT), a newly developed medical application to cure cancer. It is based on ROS generation through photo-sensitive molecules (also referred to as PhotoSensitizers, or PS) under specific illumination. The goal of this work was to develop an efficient way to tune and characterize OS and related damage to biological systems. For this purpose, we synthesized a water-soluble derivative of fullerene-based PS, since the pristine molecule is known to generate ROS under visible illumination at very high yield. The efficiency of our custom-made PS in generating ROS was characterized by Electron Spin Resonance (ESR). Parallel studies were performed on a porphyrin-based PS and on TiO2 nanoparticles. The effects of oxidative stress provoked via PS were investigated for selected proteins and for a large variety of cells. The extent of OS was finely tuned by adjusting the experimental conditions such as PS concentration in the buffer. The changes upon OS were studied by three modern experimental techniques: ESR, Synchrotron Infrared MicroSpectroscopy (SIRMS) and Atomic Force Microscopy (AFM). We have obtained a promising set of data on the consequences of OS detected by these local techniques. During the OS certain chemical bonds are interrupted and others are created which manifest in the change of the lattice vibrations detected for entire cells using SIRMS. These stresses affect, first of all, the constituents of the cells. For example, OS on a protein induces conformational changes as measured by ESR using spin labeled proteins. We have shown that larger organizations of proteins like cytoskeletal filaments must also change under OS, since the cell stiffness decreases considerably. AFM measurements on various cell types (neurons, fibroblast, bladder cell, glioblastoma…) and on healthy and cancerous cells have clearly shown the progressive decrease of the Young's modulus as OS was switched on.