In the past few years extracellular vesicles called exosomes have gained huge interest of scientific community since they show a great potential for human diagnostic and therapeutic applications. However, an ongoing challenge is accurate size characterization and quantification of exosomes because of the lack of reliable characterization techniques. In this work, the emphasis was focused on a method development to size-separate, characterize, and quantify small amounts of exosomes by asymmetrical-flow field-flow fractionation (AF4) technique coupled to a multidetection system (UV and MALS). Batch DLS (dynamic light-scattering) and NTA (nanoparticle tracking analysis) analyses of unfractionated exosomes were also conducted to evaluate their shape and internal structure, as well as their number density. The results show significant influence of cross-flow conditions and channel thickness on fractionation quality of exosomes, whereas the focusing time has less impact. The AF4/UV-MALS and DLS results display the presence of two particles subpopulations, that is, the larger exosomes and the smaller vesicle-like particles, which coeluted in AF4 together with impurities in early eluting peak. Compared to DLS and AF4-MALS results, NTA somewhat overestimates the size and the number density for larger exosome population, but it discriminates the smaller particle population.
COBISS.SI-ID: 5754138
Perforin (PFN) is one of the most important protein effectors of the immune system. It is produced by cytotoxic T lymphocytes and natural killer cells and helps with the clearance of virus-infected and tumor cells. PFN is a pore-forming protein that readily binds to the lipid membranes of target cells, oligomerizes at the cell surface and forms transmembrane pores that allow passage of ions and other larger molecules. Its characterization was hindered in the past by a lack of efficient and reliable expression systems that would result in pure and functional product. In this paper we present optimization of PFN expression in a baculovirus expression system. We optimized several parameters of murine PFN (mPFN) expression and purification and showed that the expressed product is pure and hemolytically active and that it forms pores in the plasma membranes of K562 cells. We could also observe circular pores formed on liposome membranes by cryo-electron microscopy (cryo-EM). Our protocol opens the door for further biochemical and biophysical assessment of PFN properties and interactions with small ligands and lipid membranes.
COBISS.SI-ID: 28754215
High internal phase emulsions of water in cyclooctene stabilised by sorbitan monooleate (Span 80) were cured by ring-openingmetathesis polymerisation to release fully open macroporous polymer foams wherein the surfactant was covalently incorporated into the poly(cyclooctene) strands via chain transfer reactions.
COBISS.SI-ID: 37876997