Nanoparticles isolated from blood: a reflection of vesiculability of blood cells during the isolation process

Abstract

Background: Shedding of nanoparticles from the cell membrane is a common process in all cells. These nanoparticles are present in body fluids and can be harvested by isolation. To collect circulating nanoparticles from blood, a standard procedure consisting of repeated centrifugation and washing is applied to the blood samples. Nanoparticles can also be shed from blood cells during the isolation process, so it is unclear whether nanoparticles found in the isolated material are present in blood at sampling or if are they created from the blood cells during the isolation process. We addressed this question by determination of the morphology and identity of nanoparticles harvested from blood. Methods: The isolates were visualized by scanning electron microscopy, analyzed by flow cytometry, and nanoparticle shapes were determined theoretically. Results: The average size of nanoparticles was about 300 nm, and numerous residual blood cells were found in the isolates. The shapes of nanoparticles corresponded to the theoretical shapes obtained by minimization of the membrane free energy, indicating that these nanoparticles can be identified as vesicles. The concentration and size of nanoparticles in blood isolates was sensitive to the temperature during isolation. We demonstrated that at lower temperatures, the nanoparticle concentration was higher, while the nanoparticles were on average smaller. Conclusion:These results indicate that a large pool of nanoparticles is produced after blood sampling. The shapes of deformed blood cells found in the isolates indicate how fragmentation of blood cells may take place. The results show that the contents of isolates reflect the properties of blood cells and their interaction with the surrounding solution (rather than representing only nanoparticles present in blood at sampling) which differ in different diseases and may therefore present a relevant clinical parameter.

Viscosity of Plasma as a Key Factor in Assessment of Extracellular Vesicles by Light Scattering

“…The concentration of EVs in ex-vivo samples of blood plasma was estimated at 10 10 particles/mL [3], but may be increased as a result of various pathological conditions [4,5]. Despite intensive investigations of these membrane particles in the last few decades, numerous issues remain unresolved, an important one being the effect of pre-analytical handling of samples on the results [5,6,7,8,9,10,11,12]. The dynamics of a vesicle membrane and its contents in ex-vivo samples is poorly understood.…”

“…A possibility of EV transformations due to the aggressive processes of isolation from body fluids should be taken into account. It was suggested [12] that EVs result from self-organization of the available physico-chemical components following their exposure to various external factors. Consequently, viewing EV isolates as composed of particles in the state as formed in the body is to some degree questionable.…”

The role of extracellular vesicles in phenotypic cancer transformation

“…It is considered that EVs are membrane-enclosed compartments, released into the surroundings of practically all cell types, both in vivo and in vitro 6. After separation from the mother membrane, vesicles with various types of cargo become mobile and may travel from the extracellular/intercellular space to blood (Figure 1).…”

“…An increased number of circulating EVs were found in blood isolates of patients with gastrointestinal cancer 31–33. It is, however, important to bear in mind that the EVs found in blood isolates are not necesarilly the native circulating vesicles but can also be formed during sampling and isolation procedures due to exposure of the cells to thermal and mechanical stress 6. Nevertheless, studying EVs isolated from blood and other body fluids of cancer patients is of special interest, not only because cancer cells are particularly prone to vesiculation, but also because of greater vulnerability and fragmentation of blood cells (platelets) in cancer patients, which could be reflected in a higher concentration of EVs in blood-isolates6 which could be used as a valuable diagnostic marker 2…”

Morphological alterations of T24 cells on flat and nanotubular TiO2 surfaces

“…The aggregation of negative electrical charges at highly curved edges (7), such as the nanotubular edges of TiO 2 nanotube surface, should be analyzed using positively charged fluorescent dyes. We hope that the study of shape transformation of malignant cancer cells on different nanostructured surface may set the ground for the development of the novel intervention strategies in cancer treatment (20,21). …”