Development of in cells EPR protocols for a new service platform: spin labeled biological samples, interaction with metals and nanomaterials

Electron Paramagnetic Resonance (EPR) spectroscopy is a versatile and powerful analytical technique used for the direct detection and characterization of systems containing one or more unpaired electrons, referred to as paramagnetic systems. These include stable and transient free radicals, paramagnetic transition metals and diamagnetic systems marked with molecules called spin labels. EPR is considered the gold standard technique for studying oxidative stress. Over the last few decades, EPR spectroscopy of complex biological systems, particularly proteins, has developed considerably, due to the possibility to investigate the structure, dynamics and function of biological macromolecules and their interactions, in a non-destructive manner. Despite these advances, EPR is still mainly applied to solution systems, often at room or cryogenic temperatures and at relatively high sample concentrations. These conditions deviate from the living organisms’ conditions. This gap limits the relevance of EPR spectroscopy in studies of real biological phenomena. This thesis aims to address this gap by conducting continuous wave EPR (CW-EPR) studies on paramagnetic systems in cellular environments. This work has been developed as part of a wider initiative for the development and standardization of advanced methodologies for EPR spectroscopy within the Core Facilities and Advanced Instrumentation Service (FAST) at the Istituto Superiore di Sanità (ISS). This project aims to enhance the capabilities of the EPR platform, particularly in the context of biological and biomedical research and to establish EPR as a reliable and accessible complementary technique for the scientific community. Six systems were studied in the project: endogenous paramagnetic systems, such as Hydroxytyrosol -Cu(II) complexes, graphene oxide (GO) and its reduced forms (rGOs), diamagnetic systems spin labeled with conventional or novel spin labels, such as aggregating protein (salmon calcitonin (sCT) and human α-synuclein (hα-syn)), NarJ chaperon protein interacting with NarG peptide and Chinese hamster ovary (CHO) cells. The central focus that unifies the experiments presented in this thesis is the use of CW-EPR spectroscopy to study how biological systems (whether inherently paramagnetic or rendered paramagnetic through spin labeling) interact with cellular environments. The experiments explore a wide variety of molecules and materials (small molecules, membranes, nanomaterials, aggregating proteins, and protein–peptide complexes), but all share the common goal of adapting and optimizing consolidated EPR methodologies and developing innovative ones for biological applications, under conditions as close as possible to the cellular environments. The strategy adopted in each experiment involves an initial characterization of the systems at room and cryogenic temperatures, varying the pH and the concentration, if appropriate. This preliminary phase allows to understand the EPR signal under controlled conditions. Once characterized these systems were delivered into cells or, when this was not possible, studied in interaction with cells, with the aim of investigating how the EPR signal is affected by the cellular environment. Hydroxytyrosol (HDT)-copper(II) complexes were studied in TBNC cells: the study demonstrated that HDT's chelation of endogenous copper in cells was detectable and proportional to the HDT concentration. EPR study of spin-labeled CHO cell membranes, used to examine the effects of neodecanoic acid (NDA) revealed a time-dependent alteration in membrane fluidity. For graphene oxide (GO) and its reduced forms (rGOs), EPR enabled the detection of carbon-centered defects and metal impurities, while interaction studies with cells suggested membrane interaction without altering the overall membrane properties. Protein aggregation models, including salmon calcitonin (sCT) and mutant human α-synuclein (hα-syn), labeled with novel and conventional spin labels, were studied in contact with HT22 neuronal cells, and provided insights into aggregation behavior and label stability in reducing environments. Protein-peptide interactions, using spin-labeled chaperone protein NarJ and its interactor NarG peptide, delivered within E. coli. Dual labeling with 15N and 14N spin labels enabled to distinguish the two molecules in cell, albeit with limited resolution due to line broadening in cellular environments. The results confirm that CW-EPR can provide relevant biophysical and biochemical information even under challenging cellular conditions. However, the main limitation identified remains the relatively low sensitivity of CW-EPR under physiological conditions and low analyte concentrations, which makes rigorous system-specific optimization essential. The project has led to the successful validation of several sample preparation and labeling protocols and can contribute to the ongoing effort of integrating EPR spectroscopy into life science research. This work has provided the ISS EPR Facility with reliable and validated protocols and tools suitable to offer a wider portfolio of user-customized EPR-based analyses to support research on cellular mechanisms in health and disease such as neurodegenerative and tumor diseases. Protocols are being compiled and made available through the ISS platform to foster reproducibility and broader adoption.