Development of nanotechnological methods for charaterization of clinical extracellular vesicles

Our research group has been investigating extracellular vesicles (EV) since 2014 and in the previous NFFA-410 project we focused analysis on EV from patients with traumatic brain injury (TBI). Generally, EV are nanosized, heterogeneous membrane structures, which are secreted by cells into body fluids during physiological and pathological processes. Along with the lipids and specific proteins in the bilayer membrane, EV can carry nucleic acids. Thus, EV posses a great diagnostic and prognostic value since they can be easily accessed from body fluids and can reveal condition of originating cells. During NFFA-410 we identified the following EV-specific issues in applying the advanced nanotechnology methods: 1) microscopy in air; 2) purity of EV for further characterization methods. Scaning electron and atomic force microscopy in air provided valuable details of EV morphology. However, we detected artefacts pointing to possible detrimental effect of vacuum on EV integrity and structure. In contrast to analysis of EV that we performed directly in body fluid in NFFA-410 and thus in presence of soluble proteins, in current proposal we plan to analyse purified EV. Purified EV are necessary for specific nanotechnolgies e.g. Raman spectroscopy. We have introduced in our laboratory gravity gel filtration (GGF) as method for total EV isolation and performed immunoblot detection on CD9 tetraspanin as EV marker on obtained GGF fractions.
In this project proposal our objectives are to characterize purified EV with the following nanotechnologies: 1) atomic force microscopy in liquidand scaning electron microscopy (SEM with STEM detector) will be used for EV visualisation; 2) optical tweezers, Raman spectroscopy and surface-enhanced Raman spectroscopy (SERS) will be applied to study content of EV on single-EV level. The proposed characterization of EV will be performed on samples from TBI patients and combined with our complementary detection methods such as immuno blot.

Structure and composition of extracellular vesicles from patients with severe traumatic brain injury

Severe traumatic brain injury (TBI) is a life-threatening intracranial injury caused by external force that leads to altered consciousness and coma. Pathophysiology of severe TBI is complex and involves initial injury as a direct consequence of trauma, followed by series of events leading to further neuronal damage and reduction in the number of synapses at places distant from the initial injury site. Recovery after severe TBI depends on plasticity response that is capable of restoring lost synaptic contacts. This requires multifaceted and highly regulated communication between neurons and glial cells. Despite extensive studies, underlying mechanisms of recovery are widely unknown. Extracellular vesicles (EVs) are nanosized particles and newly recognized mediators of intercellular communication. Studies of the EV composition and function in human central nervous system physiology and pathophysiology are in its infancy due to ethical and medical limitations to access clinical EV samples. We have collected cerebrospinal spinal fluids (CSF) from patients with severe TBI and healthy controls and performed initial EV analyses. Our unpublished data of nanoparticle tracking, western blot and ELISA analyses show that EVs are present in clinical CSF samples after severe TBI. The aim of this study is to extend those analyses by applying and optimizing new nanotechnologies for further characterization of EVs in clinical CSF and investigate correlation between EV features and patient injury and outcome. The following nanotechnologies are proposed: 1) atomic force and scanning transmission electron microscopy (EM) will be used for EV visualisation and concentration determination; 2) Raman spectroscopy will be applied to study EV content.