Apolonija Bedina Zavec

Biological properties of extracellular vesicles and their physiological functions.

In the past decade, extracellular vesicles (EVs) have been recognized as potent vehicles of intercellular communication, both in prokaryotes and eukaryotes. This is due to their capacity to transfer proteins, lipids and nucleic acids, thereby influencing various physiological and pathological functions of both recipient and parent cells. While intensive investigation has targeted the role of EVs in different pathological processes, for example, in cancer and autoimmune diseases, the EV-mediated maintenance of homeostasis and the regulation of physiological functions have remained less explored. Here, we provide a comprehensive overview of the current understanding of the physiological roles of EVs, which has been written by crowd-sourcing, drawing on the unique EV expertise of academia-based scientists, clinicians and industry based in 27 European countries, the United States and Australia. This review is intended to be of relevance to both researchers already working on EV biology and to newcomers who will encounter this universal cell biological system. Therefore, here we address the molecular contents and functions of EVs in various tissues and body fluids from cell systems to organs. We also review the physiological mechanisms of EVs in bacteria, lower eukaryotes and plants to highlight the functional uniformity of this emerging communication system.

Evidence-Based Clinical Use of Nanoscale Extracellular Vesicles in Nanomedicine

Recent research has demonstrated that all body fluids assessed contain substantial amounts of vesicles that range in size from 30 to 1000 nm and that are surrounded by phospholipid membranes containing different membrane microdomains such as lipid rafts and caveolae. The most prominent representatives of these so-called extracellular vesicles (EVs) are nanosized exosomes (70-150 nm), which are derivatives of the endosomal system, and microvesicles (100-1000 nm), which are produced by outward budding of the plasma membrane. Nanosized EVs are released by almost all cell types and mediate targeted intercellular communication under physiological and pathophysiological conditions. Containing cell-type-specific signatures, EVs have been proposed as biomarkers in a variety of diseases. Furthermore, according to their physical functions, EVs of selected cell types have been used as therapeutic agents in immune therapy, vaccination trials, regenerative medicine, and drug delivery. Undoubtedly, the rapidly emerging field of basic and applied EV research will significantly influence the biomedicinal landscape in the future. In this Perspective, we, a network of European scientists from clinical, academic, and industry settings collaborating through the H2020 European Cooperation in Science and Technology (COST) program European Network on Microvesicles and Exosomes in Health and Disease (ME-HAD), demonstrate the high potential of nanosized EVs for both diagnostic and therapeutic (i.e., theranostic) areas of nanomedicine.

Inclusion bodies as potential vehicles for recombinant protein delivery into epithelial cells.

BackgroundWe present the potential of inclusion bodies (IBs) as a protein delivery method for polymeric filamentous proteins. We used as cell factory a strain of E. coli, a conventional host organism, and keratin 14 (K14) as an example of a complex protein. Keratins build the intermediate filament cytoskeleton of all epithelial cells. In order to build filaments, monomeric K14 needs first to dimerize with its binding partner (keratin 5, K5), which is then followed by heterodimer assembly into filaments.ResultsK14 IBs were electroporated into SW13 cells grown in culture together with a “reporter” plasmid containing EYFP labeled keratin 5 (K5) cDNA. As SW13 cells do not normally express keratins, and keratin filaments are built exclusively of keratin heterodimers (i.e. K5/K14), the short filamentous structures we obtained in this study can only be the result of: a) if both IBs and plasmid DNA are transfected simultaneously into the cell(s); b) once inside the cells, K14 protein is being released from IBs; c) released K14 is functional, able to form heterodimers with EYFP-K5.ConclusionsSoluble IBs may be also developed for complex cytoskeletal proteins and used as nanoparticles for their delivery into epithelial cells.

Blood and Synovial Microparticles as Revealed by Atomic Force and Scanning Electron Microscope

Microvesicles which are pinched off the cell membrane can be considered extracellular organelles which medi- ate interaction between distal cells. They were suggested to play an important role in many diseases including autoim- mune disorders, however, standard methods for their assessment have not yet been decided upon while their clinical rele- vance and the underlying mechanisms are yet unclear. We present a pilot study results involving atomic force microscope (AFM) and scanning electron microscope (SEM) images of the material isolated from peripheral blood of healthy donors and from synovial fluid of patients with psoriatic arthritis and rheumatoid arthritis, which is expected to contain microve- sicles. Micrographs reveal in the samples isolated from blood the presence of globular and tubular structures which are most probably microvesicles while the identity of grain-like structures isolated from synovial fluid remains obscure. To the best of our knowledge the AFM and SEM images of the material isolated from synovial fluid are presented for the first time.

Archaeosomes can efficiently deliver different types of cargo into epithelial cells grown in vitro

Archaeosomes are a type of liposomes prepared from the polar lipids of various Archaeobacteria. These have unique structural features that increase the lipid bilayers stability even under high temperatures, low or high pH, presence of phospholipases and bile salts. This makes them ideal as basis for the development of new drug, gene and vaccine delivery systems. In this study we prepared large unilamellar archaeosomes (400nm size) from Aeropyrum pernix K1 and demonstrated their potential as base for the development of an efficient and universal system for drug or therapy delivery to epithelial cells. Our archaeosomes may be used to deliver small fluorescent molecules (calcein), smaller proteins (60kDa listeriolysin), large protein aggregates (e.g. keratin 14) and plasmid DNA, into epithelial cells grown in culture. The delivery efficiency for small molecules is already quite high at this initial stage of development, around 40%. Our unilamellar archaeosomes are also not toxic to keratinocytes even at high doses (500μg/ml).

Effect of shear stress in the flow through the sampling needle on concentration of nanovesicles isolated from blood

&NA; During harvesting of nanovesicles (NVs) from blood, blood cells and other particles in blood are exposed to mechanical forces which may cause activation of platelets, changes of membrane properties, cell deformation and shedding of membrane fragments. We report on the effect of shear forces imposed upon blood samples during the harvesting process, on the concentration of membrane nanovesicles in isolates from blood. Mathematical models of blood flow through the needle during sampling with vacuumtubes and with free flow were constructed, starting from the Navier–Stokes formalism. Blood was modeled as a Newtonian fluid. Work of the shear stress was calculated. In experiments, nanovesicles were isolated by repeated centrifugation (up to 17,570 × g) and washing, and counted by flow cytometry. It was found that the concentration of nanovesicles in the isolates positively corresponded with the work by the shear forces in the flow of the sample through the needle. We have enhanced the effect of the shear forces by shaking the samples prior to isolation with glass beads. Imaging of isolates by scanning electron microscopy revealed closed globular structures of a similar size and shape as those obtained from unshaken plasma by repetitive centrifugation and washing. Furthermore, the sizes and shapes of NVs obtained by shaking erythrocytes corresponded to those isolated from shaken platelet‐rich plasma and from unshaken platelet rich plasma, and not to those induced in erythrocytes by exogenously added amphiphiles. These results are in favor of the hypothesis that a significant pool of nanovesicles in blood isolates is created during their harvesting. The identity, shape, size and composition of NVs in isolates strongly depend on the technology of their harvesting. Graphical Abstract Figure. No caption available.

Extracellular vesicles concentration is a promising and important parameter for industrial bioprocess monitoring

Extracellular vesicles (EVs) are membrane vesicles that are produced by cells to be released into their microenvironment. In this study, we present the EV concentration as a new factor for optimization of industrial bioprocess control. The release of EVs depends on many cell properties, including cell activation and stress status, and cell death. Therefore, the EV concentration might provide a readout for identification of the cell state and the conditions during a bioprocess. Our data show that the EV concentration increased during the bioprocess, which indicated deteriorating conditions in the bioreactor. This increase in EV concentration in the fermentation broth was the consequence of two different processes: cell activation, and cell death. However, the release of EVs from activated living cells had a much weaker impact on EV concentration in the bioreactor than those released during cell death. EVs and cells in the bioprocess environment were quantified by flow cytometry. The most accurate data were obtained directly from unprocessed samples, making the monitoring of the EV concentration a rapid, easy, and cheap method. These EV concentrations reflect the conditions in the bioreactor and provide new information regarding the state of the bioprocess. Therefore, we suggest EV concentration as a new and important parameter for the monitoring of industrial bioprocesses.