Vid Šuštar

Isolated microvesicles from peripheral blood and body fluids as observed by scanning electron microscope

Microvesicles are sub-micron structures shed from the cell membrane in a final step of the budding process. After being released into the microenvironment they are free to move and carry signaling molecules to distant cells, thereby they represent a communication system within the body. Since all cells shed microvesicles, it can be expected that they will be found in different body fluids. The potential diagnostic value of microvesicles has been suggested, however, a standardized protocol for isolation has not yet been agreed upon. It is unclear what is the content of the isolates and whether the isolated microvesicles were present in vivo or-have they been created within the isolation procedure. To present evidence in this direction, in this work we focus on the visualization of the material obtained by the microvesicle isolation procedure. We present scanning electronic microscope images of microvesicles isolated from blood, ascites, pleural fluid, cerebrospinal fluid, postoperative drainage fluid and chyloid fluid acquired from human and animal patients. Vesicular structures sized from 1microm downto 50nm are present in isolates of all considered body fluids, however, the populations differ in size and shape reflecting also the composition of the corresponding sediments. Isolates of microvesicles contain numerous cells which indicates that methods of isolation and determination of the number of microvesicles in the peripheral blood are to be elaborated and improved.

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

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.

Visualization of internalization of functionalized cobalt ferrite nanoparticles and their intracellular fate.

In recent years, nanoparticles (NPs) and related applications have become an intensive area of research, especially in the biotechnological and biomedical fields, with magnetic NPs being one of the promising tools for tumor treatment and as MRI-contrast enhancers. Several internalization and cytotoxicity studies have been performed, but there are still many unanswered questions concerning NP interactions with cells and NP stability. In this study, we prepared functionalized magnetic NPs coated with polyacrylic acid, which were stable in physiological conditions and which were also nontoxic short-term. Using fluorescence, scanning, and transmission electron microscopy, we were able to observe and determine the internalization pathways of polyacrylic acid–coated NPs in Chinese hamster ovary cells. With scanning electron microscopy we captured what might be the first step of NPs internalization – an endocytic vesicle in the process of formation enclosing NPs bound to the membrane. With fluorescence microscopy we observed that NP aggregates were rapidly internalized, in a time-dependent manner, via macropinocytosis and clathrin-mediated endocytosis. Inside the cytoplasm, aggregated NPs were found enclosed in acidified vesicles accumulated in the perinuclear region 1 hour after exposure, where they stayed for up to 24 hours. High intracellular loading of NPs in the Chinese hamster ovary cells was obtained after 24 hours, with no observable toxic effects. Thus polyacrylic acid–coated NPs have potential for use in biotechnological and biomedical applications.

Mechanisms for the formation of membranous nanostructures in cell-to-cell communication

Cells interact by exchanging material and information. Two methods of cell-to-cell communication are by means of microvesicles and by means of nanotubes. Both microvesicles and nanotubes derive from the cell membrane and are able to transport the contents of the inner solution. In this review, we describe two physical mechanisms involved in the formation of microvesicles and nanotubes: curvature-mediated lateral redistribution of membrane components with the formation of membrane nanodomains; and plasmamediated attractive forces between membranes. These mechanisms are clinically relevant since they can be affected by drugs. In particular, the underlying mechanism of heparin’s role as an anticoagulant and tumor suppressor is the suppression of microvesicluation due to plasma-mediated attractive interaction between membranes.

Number of microvesicles in peripheral blood and ability of plasma to induce adhesion between phospholipid membranes in 19 patients with gastrointestinal diseases

It was recently shown that the plasma protein-mediated attractive interaction between phospholipid membranes could in the budding process cause adhesion of the bud to the mother membrane [J. Urbanija, N. Tomsic, M. Lokar, A. Ambrozic, S. Cucnik, M. Kanduser, B. Rozman, A. Iglic, V. Kralj-Iglic, Coalescence of phospholipid membranes as a possible origin of anticoagulant effect of serum proteins, Chem. Phys. Lipids 150 (2007) 49-57]. Since in the in vivo conditions the budding of cell membranes leads to the release of microvesicles into the circulation, a hypothesis was put forward that the ability of plasma to cause adhesion between membranes supresses the microvesiculation process. In the present work, this hypothesis was tested in a population of 19 patients with gastrointestinal diseases. The number of microvesicles in peripheral blood of patients was determined by flow cytometry while the ability of plasma to cause adhesion between membranes was determined by adding patients plasma to the suspension of giant phospholipid vesicles created by electroformation method, and measuring the average effective angle of contact between the adhered vesicles. Statistically significant negative correlations between the number of microvesicles and the average effective angle of contact (Pearson coefficient -0.50, p=0.031) and between the number of microvesicles per number of platelets and the average effective angle of contact (Pearson coefficient -0.64, p=0.003) were found, which is in favor of the above hypothesis. Patients with gastrointestinal cancer had larger number of microvesicles (difference 140%, statistical significance 0.033) and smaller average effective angle of contact (difference 20%, statistical significance 0.013) compared to patients with other gastrointestinal diseases.

Effect of engineered TiO2 and ZnO nanoparticles on erythrocytes, platelet-rich plasma and giant unilamelar phospholipid vesicles

BackgroundMassive industrial production of engineered nanoparticles poses questions about health risks to living beings. In order to understand the underlying mechanisms, we studied the effects of TiO2 and ZnO agglomerated engineered nanoparticles (EPs) on erythrocytes, platelet-rich plasma and on suspensions of giant unilamelar phospholipid vesicles.ResultsWashed erythrocytes, platelet-rich plasma and suspensions of giant unilamelar phospholipid vesicles were incubated with samples of EPs. These samples were observed by different microscopic techniques. We found that TiO2 and ZnO EPs adhered to the membrane of washed human and canine erythrocytes. TiO2 and ZnO EPs induced coalescence of human erythrocytes. Addition of TiO2 and ZnO EPs to platelet-rich plasma caused activation of human platelets after 24 hours and 3 hours, respectively, while in canine erythrocytes, activation of platelets due to ZnO EPs occurred already after 1 hour. To assess the effect of EPs on a representative sample of giant unilamelar phospholipid vesicles, analysis of the recorded populations was improved by applying the principles of statistical physics. TiO2 EPs did not induce any notable effect on giant unilamelar phospholipid vesicles within 50 minutes of incubation, while ZnO EPs induced a decrease in the number of giant unilamelar phospholipid vesicles that was statistically significant (p < 0,001) already after 20 minutes of incubation.ConclusionsThese results indicate that TiO2 and ZnO EPs cause erythrocyte aggregation and could be potentially prothrombogenic, while ZnO could also cause membrane rupture.

Suppression of membrane microvesiculation--a possible anticoagulant and anti-tumor progression effect of heparin.

Heparins (unfractionated and low molecular weight (LMWH) heparins) primarily used as anticoagulants, were found to be effective also in slowing down the development of some types of cancer. On the other hand, the number of microvesicles in the peripheral blood originating from the budding of cell membranes (mostly platelets) is increased in hypercoagulabile states as well as in cancer, indicating a possible common underlying mechanism. It was hypothesized that by mediating an attractive interaction between phospholipid membranes heparin suppresses microvesiculation and thereby acts as an anticoagulant and anti-tumor agent. In this work, the effect of LMWH nadroparin on phospholipid membranes was tested in vitro in a system of giant phospholipid vesicles (GPVs) created by electroformation and observed under the phase contrast microscope. Plasma of different blood donors containing different concentrations of nadroparin was added to the suspension of GPVs to induce adhesion between GPVs. The attractive interaction between membranes was assessed by measuring the average effective angle of contact between the adhered GPVs. It was found in healthy donors, in a donor with gastrointestinal cancer and in a donor with rheumatoid arthritis that adding therapeutic doses of nadroparin to the plasma samples enhanced adhesion of phospholipid membranes in a dose and time-dependent manner while nadroparin alone had no effect within the therapeutic concentration range. The results are in favor of the hypothesis that suppression of microvesiculation underlies both, the anticoagulant and the anti-tumor progression effect of heparin.

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.

Post - prandial rise of microvesicles in peripheral blood of healthy human donors

BackgroundMicrovesicles isolated from body fluids are membrane - enclosed fragments of cell interior which carry information on the status of the organism. It is yet unclear how metabolism affects the number and composition of microvesicles in isolates from the peripheral blood.AimTo study the post - prandial effect on microvesicles in isolates from the peripheral blood of 21 healthy donors, in relation to blood cholesterol and blood glucose concentrations.ResultsThe average number of microvesicles in the isolates increased 5 hours post - prandially by 52%; the increase was statistically significant (p = 0.01) with the power P = 0.68, while the average total blood cholesterol concentration, average low density lipoprotein cholesterol concentration (LDL-C) and average high density lipoprotein cholesterol concentration (HDL-C) all remained within 2% of their fasting values. We found an 11% increase in triglycerides (p = 0.12) and a 6% decrease in blood glucose (p < 0.01, P = 0.74). The post - prandial number of microvesicles negatively correlated with the post - fasting total cholesterol concentration (r = - 0.46, p = 0.035) while the difference in the number of microvesicles in the isolates between post - prandial and post - fasting states negatively correlated with the respective difference in blood glucose concentration (r = - 0.39, p = 0.05).ConclusionsIn a population of healthy human subjects the number of microvesicles in isolates from peripheral blood increased in the post - prandial state. The increase in the number of microvesicles was affected by the fasting concentration of cholesterol and correlated with the decrease in blood glucose.

Morphology, biophysical properties and protein-mediated fusion of archaeosomes.

As variance from standard phospholipids of eubacteria and eukaryotes, archaebacterial diether phospholipids contain branched alcohol chains (phytanol) linked to glycerol exclusively with ether bonds. Giant vesicles (GVs) constituted of different species of archaebacterial diether phospholipids and glycolipids (archaeosomes) were prepared by electroformation and observed under a phase contrast and/or fluorescence microscope. Archaebacterial lipids and different mixtures of archaebacterial and standard lipids formed GVs which were analysed for size, yield and ability to adhere to each other due to the mediating effects of certain plasma proteins. GVs constituted of different proportions of archaeal or standard phosphatidylcholine were compared. In nonarchaebacterial GVs (in form of multilamellar lipid vesicles, MLVs) the main transition was detected at Tm = 34. 2°C with an enthalpy of ΔH = 0.68 kcal/mol, whereas in archaebacterial GVs (MLVs) we did not observe the main phase transition in the range between 10 and 70°C. GVs constituted of archaebacterial lipids were subject to attractive interaction mediated by beta 2 glycoprotein I and by heparin. The adhesion constant of beta 2 glycoprotein I – mediated adhesion determined from adhesion angle between adhered GVs was in the range of 10−8 J/m2. In the course of protein mediated adhesion, lateral segregation of the membrane components and presence of thin tubular membranous structures were observed. The ability of archaebacterial diether lipids to combine with standard lipids in bilayers and their compatibility with adhesion-mediating molecules offer further evidence that archaebacterial lipids are appropriate for the design of drug carriers.