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.

Keywords: cell–cell communication; microparticles; microvesicles; nanoparticles; nanovesicles.

Figure 1

Scanning electron micrograph of an isolate from peripheral blood of a healthy human donor (male, 28 years). A mass of microparticles and numerous residual erythrocytes can be seen. The image was taken using a Quanta TM 250 FEG (FEI, Hillsboro, OR) scanning electron microscope at FEI Quanta, Eindhoven, The Netherlands, by applying 1.5 kV.

Figure 2

Scanning electron micrograph of chosen regions of an isolate from peripheral blood of a healthy human donor (male, 28 years). In addition to numerous nanoparticles which are present in all the pictures, erythrocytes (A – black arrow, B), activated platelets (A – white arrow), tubules (C), tori (E – white arrows), starfish (F – black arrow) and a deformed erythrocyte exhibiting protrusion with a bulbous end (F – white arrow) were observed. (A – D) images taken using a LEO Gemini 1530 (LEO, Oberkochen, Germany) scanning electron microscope by applying 8 kV (A, C, and D) and 2.7 kV (B), at Åbo Akademi University, Åbo/Turku. Images E and F taken by Quanta TM 250 FEG (FEI, Hillsboro, Oregon, OR) scanning electron microscope at FEI Quanta, Eindhoven, The Netherlands, by applying 1.5 kV.

Figure 3

Representative characteristic shapes of nanoparticles found in an isolate from the blood of a patient with pancreatic cancer (female, 60 years). Shapes include shizocytes (A and B), dumbbell (C), submicron discocyte (D), and the corresponding shapes calculated by minimization of the membrane free energy (E and F). The shape in panel E was obtained for a relative volume v = 36 π V^2/3/A^3/2 = 0.65, where V is the volume of the vesicle, A is the surface area of the vesicle and for the relative average mean curvature <h> = 1/2A ∫(C₁ + C₂)dA = 1.32, where C₁ and C₂ are the two principal curvatures at a chosen point of the membrane surface and integration is performed over the entire surface of the vesicle A. For the shape in panel F, v = 0.55 and <h> = 1.055. Intrinsic principal curvatures were equal to 0 for both shapes. The images were taken using a LEO Gemini 1530 (LEO, Oberkochen, Germany) scanning electron microscope by applying 8 kV at Åbo Akademi University, Åbo/Turku, Finland.

Figure 4

Deformation of cell-derived material obtained by isolation procedure. Deformed cells from the blood of a healthy mare (aged five years) exhibit protrusions connected by thin necks, which were torn eventually to yield membrane-enclosed cell fragments (A). Close to the tube wall, the shear forces in the centrifuge are high and therefore the cell fragments in the isolate from the blood of a healthy human donor (male, 28 years) are elongated and exhibit preferential orientation (B). The images were taken using a LEO Gemini 1530 (LEO, Oberkochen, Germany) scanning electron microscope by applying 8 kV at Åbo Akademi University, Åbo/Turku, Finland.

Figure 5

Platelets and nanoparticles at different temperatures. Nanoparticles were isolated from platelet-rich plasma of a healthy human donor (female, 28 years) at different temperatures (A: 4°C, B: 20°C, C: 37°C), platelets from platelet-rich plasma of a mare (D: 4°C, E: 20°C, F: 37°C), nanoparticles isolated from blood of the mare (G: 4°C, H: 20°C, I: 37°C). The images were taken using a LEO Gemini 1530 (LEO, Oberkochen, Germany) scanning electron microscope by applying 8 kV at Åbo Akademi University, Åbo/Turku, Finland.

Figure 6

Budding of membranes. (A) Budding of erythrocytes induced by adding detergent to the suspension of erythrocytes (scanning electron microscopy performed at Åbo Akademi University, Åbo/Turku, Finland). (B) A budding erythrocyte found in an isolate from human blood with numerous nanoparticles (scanning electron microscopy performed at FEI Quanta, Eindhoven, The Netherlands). (C) Nanoparticles isolated from suspension of erythrocytes with added detergent. (D) Precursors of nanoparticles appearing at the top of the echinocyte spicules. (A, C, and D images taken using a LEO Gemini 1530 (LEO, Oberkochen, Germany) scanning electron microscope by applying 8 kV, at Åbo Akademi University, Åbo/Turku, Finland. Image (C) taken by Quanta TM 250 FEG (FEI, Hillsboro, OR) scanning electron microscope at FEI Quanta, Eindhoven, The Netherlands, by applying 1.5 kV). Image in Panel B was taken from Šuštar et al.