Abstract
The article presents the results of studying the physicochemical properties of exosome preparations obtained by ultrafiltration, which indicate a high degree of the composition and properties dependence of the obtained product on the material of the filters used. Quantitative determination of proteins and nucleic acids in exosome samples using UPN-50 filters allows us to conclude that the content of the main impurity compounds in the preparation is significantly reduced compared to dispersions obtained using filters with pore sizes of 220 and 450 nm. Analysis of flow cytometry data made it possible to demonstrate that when using the UPN-50 filter, an increase in the contribution to the dispersion of all types of fractions of non-exosomal size was observed, the appearance of which can result from fraction destruction associated with pore size or filter material properties. drying of the dispersion was observed in the studied exosome samples. Fraction sizes ranged from 40 to 450 nm (an average of about 200 nm). Exosomes from the entire variety of membrane vesicles are fractions that have the most suitable characteristics that allow them to be used as a nanoscale drug delivery vehicle while ensuring the necessary quality control of the drug at the sample preparation stage.
Highlights
From the moment of discovery until the beginning of the 21st century, biovesicles were not given much importance, but as soon as their direct participation in intercellular communication was proved, many scientists of the world showed a genuine interest in studying of exosomes.Exosomes, as a rule, include a fraction of membrane vesicles with a diameter of 40-100 nm, which are secreted into the intercellular space by various types of tissues and organs cells
Aliquots of 1.8 ml of blood serum were taken into 2 ml tubes, centrifuged using a MiniSpin microcentrifuge (Eppendorf, Germany) at 4000 x g rpm for 30 min. 1 ml of th00e obtained supernatant was taken with a medical syringe and filtered simultaneously through the following membranes: Millex-GV (Durapore) with a membrane diameter of 25 mm and a pore size of 0.22 μm; LCR, with a membrane diameter of 25 mm and a pore diameter of 0.45 μm, and on a Millipore installation (USA) with a vacuum pump using UPN-50 filters (50 kDa, Vladipor, Russia)
We previously suggested that when exosomes are obtained, the use of filters with a smaller pore diameter at the ultrafiltration stage can lead to a regular decrease in the concentration of proteins and nucleic acids
Summary
As a rule, include a fraction of membrane vesicles with a diameter of 40-100 nm, which are secreted into the intercellular space by various types of tissues and organs cells. It is known that the structure of the exosomal capsule is a double lipid membrane with an integrated layer of external transmembrane proteins. It has been established that exosomes are released by B- and T-lymphocytes, platelets, dendritic cells, mast cells, as well as epithelial cells.
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