STRUCTURAL INVESTIGATION OF EXTRACELLULAR VESICLES AND THEIR INTERACTION WITH BIOMIMETIC INTERFACES

Over the last decade, research about extracellular vesicles (EVs), the nanosized membrane bound vesicles, secreted by all kinds of cells to deliver a physiological cargo and their influence on the cell-mediated communication has been rising as a hot topic. Different EVs, have different characteristics and activities, so their assessment will allow for their engineering and development for medical purposes. According to that, researchers have been applying mainly biological and biochemical approaches for studying EVs’ properties, including their common biomarkers, protein, and lipid profiles, to exploit them for therapeutic or diagnostic purposes. Here, an alternative novel physical approach to study EVs’ properties is carried by inspecting the EVs’ physical characteristics and connecting them to their activity. In the current study, multiple complementary physical methods are applied to study the characteristics of a new isolation of EVs from neuronal cells (N2a cells) after being treated with a promising Parkinson’s Disease drug molecule, namely the oligosaccharide OligoGM1 (hence, named as Oligo EVs). Those Oligo EVs (that are EVs purified from cells treated with OligoGM1) were studied in comparison with the Ctrl EVs (isolated from the untreated cells). Dynamic light scattering (DLS), nanoparticle tracking analysis (NTA) and atomic force microscopy (AFM) were applied for studying the difference between the EVs’ hydrodynamic and geometric diameters. Additionally, the EVs’ spectroscopic molecular profiles and protein/lipid ratios were investigated by attenuated-total-reflection Fourier-transform infrared spectroscopy (ATR-FTIR), in addition to performing a structural study of their surrounding membrane, using small-angle X-ray scattering (SAXS). Each applied technique has provided particular information of the subtle differences between the characteristics of both types of EVs under study. Then, an investigation of the EVs uptake mechanisms was carried with bottom-up approaches. In this contest, there is an urging need to design different membrane models with different complexities and geometries, to be used as biomimetic interfaces for mimicking the interaction mechanisms of incoming macromolecules such as the EVs. Indeed, the design of such model membranes can be considered as technique-specific. The requirements of each applied physical approach entail specific parameters to be tuned in the designed model membrane, such as its transition temperature, deuteration degree, bilayer thickness, transverse, and lateral structure. Therefore, in parallel, an original study was conducted on a novel model membrane made of the phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), sphingomyelin (SM) and cholesterol (Chol), according to the molar ratio of DMPC:SM:Chol; 2:1:0.15, to mimic the structure of the lipid rafts (i.e. phase separated highly ordered lipid domains within a bulk fluid phase lipid), to be used as a biomimetic interface for studying the uptake mechanisms of the EVs. DMPC was used as the main matrix for this model in contrast to other common lipids of lower transition temperatures (Tm) as 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), due to its better accessibility by a wider range of physical techniques such as differential scanning calorimetry (DSC) because of its higher Tm, and neutron reflectometry (NR) by benefiting from its commercially available deuterated forms. Hence, after building-up the lipid raft model, DSC, NR and AFM, were used in a complementary fashion to fully characterize that model membrane thermotropically, as well as its transverse/lateral structure at different temperatures following the organisation and formation of its phase separated domains upon lipid melting. After setting up the different model membranes to be used, the interaction and uptake mechanisms of the EVs were thoroughly studied where many details were unveiled. The presented results include studying the EVs’ preference to a specific lipid phase as observed by AFM and the progression of their interaction sites with time. The thermotropic behaviour of the target model membranes after interacting with the EVs was also addressed by DSC, while the difference between the lipidic exchange and the protein insertions from the EVs into the target membranes were investigated through ATR-FTIR and NR. Moreover, complementary neutron and X-ray small and wide-angle scattering (SANS, SAXS and WAXS) measurements were performed to study the structural alterations in the target membrane bilayer and the form factor of the mixed systems as well as the correlation distance of the acyl chains (CH2) groups of the target membranes upon the EVs uptake. A comprehensive understanding of the obtained results is introduced in the current study, connecting the significance of the outcomes from each technique in order to acquire a holistic knowledge of the EVs uptake mechanisms and their functionality as a result of their physical parameters, while distinguishing the intricate differences imposed in the Oligo EVs as a result of treating the N2a cells with OligoGM1.