We present a method for giant lipid vesicle shape analysis that combines manually guided large-scale video microscopy and computer vision algorithms to enable analyzing vesicle populations. The ...method retains the benefits of light microscopy and enables non-destructive analysis of vesicles from suspensions containing up to several thousands of lipid vesicles (1-50 µm in diameter). For each sample, image analysis was employed to extract data on vesicle quantity and size distributions of their projected diameters and isoperimetric quotients (measure of contour roundness). This process enables a comparison of samples from the same population over time, or the comparison of a treated population to a control. Although vesicles in suspensions are heterogeneous in sizes and shapes and have distinctively non-homogeneous distribution throughout the suspension, this method allows for the capture and analysis of repeatable vesicle samples that are representative of the population inspected.
Massive 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.
Washed 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.
These results indicate that TiO2 and ZnO EPs cause erythrocyte aggregation and could be potentially prothrombogenic, while ZnO could also cause membrane rupture.
Emerging fields such as nanomedicine and nanotoxicology, demand new information on the effects of nanoparticles on biological membranes and lipid vesicles are suitable as an experimental model for ...bio-nano interaction studies. This paper describes image processing algorithms which stitch video sequences into mosaics and recording the shapes of thousands of lipid vesicles, which were used to assess the effect of CoFe
O
nanoparticles on the population of 1-palmitoyl-2-oleoyl-
-glycero-3-phosphatidylcholine lipid vesicles. The applicability of this methodology for assessing the potential of engineered nanoparticles to affect morphological properties of lipid membranes is discussed.
Abstract
Background
Massive 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 TiO
2
and ZnO agglomerated engineered nanoparticles (EPs) on erythrocytes, platelet-rich plasma and on suspensions of giant unilamelar phospholipid vesicles.
Results
Washed 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 TiO
2
and ZnO EPs adhered to the membrane of washed human and canine erythrocytes. TiO
2
and ZnO EPs induced coalescence of human erythrocytes. Addition of TiO
2
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. TiO
2
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.
Conclusions
These results indicate that TiO
2
and ZnO EPs cause erythrocyte aggregation and could be potentially prothrombogenic, while ZnO could also cause membrane rupture.
With a model invertebrate animal, we have assessed the fate of magnetic nanoparticles in biologically relevant media, i.e., digestive juices. The toxic potential and the internalization of such ...nanoparticles by nontarget cells were also examined. The aim of this study was to provide experimental evidence on the formation of Co2+, Fe2+, and Fe3+ ions from CoFe2O4 nanoparticles in the digestive juices of a model organism. Standard toxicological parameters were assessed. Cell membrane stability was tested with a modified method for measurement of its quality. Proton-induced X-ray emission and low energy synchrotron radiation X-ray fluorescence were used to study internalization and distribution of Co and Fe. Co2+ ions were found to be more toxic than nanoparticles. We confirmed that Co2+ ions accumulate in the hepatopancreas, but Fe n+ ions or CoFe2O4 nanoparticles are not retained in vivo. A model biological system with a terrestrial isopod is suited to studies of the potential dissolution of ions and other products from metal-containing nanoparticles in biologically complex media.
There are some molecular dynamic simulations but a paucity of experimental evidence of the effects of C-60 fullerene on lipid bilayers. The aim of this study is to assess the potential for disruption ...of the lipid bilayer by C-60 suspended in water. We selected a C-60 suspension that has previously been shown to provoke cell membrane destabilisation in vivo. Electromobility measurements show significant negative surface charge on the C-60 nanoparticles suspended in a glucose solution and a zeta potential of −26
mV. The prevalent C-60 clusters have hydrodynamic radii of approximately 2
nm. Phase contrast microscopy and computer aided image analysis results show that C-60 causes shape transformations and rupture of unilamellar phospholipid vesicles, indicative of changes in their average mean curvature. Small-angle X-ray scattering reveals that C-60 provokes disruptions of external membranes of multilamellar vesicles only after freeze and thaw cycles. Here, the liposomes undergo breakage and annealing steps which increase the probability for fullerenes to insert into the MLVs. Our experimental findings confirm the potential of C-60 to reconstruct lipids in biological membranes. This research enhances our understanding of the impact of engineered nanoparticles on cell membranes.
The purpose of this work is to provide experimental evidence on the interactions of suspended nanoparticles with artificial or biological membranes and to assess the possibility of suspended ...nanoparticles interacting with the lipid component of biological membranes.
1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid vesicles and human red blood cells were incubated in suspensions of magnetic bare cobalt ferrite (CoFe2O4) or citric acid (CA)-adsorbed CoFe2O4 nanoparticles dispersed in phosphate-buffered saline and glucose solution. The stability of POPC giant unilamellar vesicles after incubation in the tested nanoparticle suspensions was assessed by phase-contrast light microscopy and analyzed with computer-aided imaging. Structural changes in the POPC multilamellar vesicles were assessed by small angle X-ray scattering, and the shape transformation of red blood cells after incubation in tested suspensions of nanoparticles was observed using scanning electron microscopy and sedimentation, agglutination, and hemolysis assays.
Artificial lipid membranes were disturbed more by CA-adsorbed CoFe2O4 nanoparticle suspensions than by bare CoFe2O4 nanoparticle suspensions. CA-adsorbed CoFe2O4-CA nanoparticles caused more significant shape transformation in red blood cells than bare CoFe2O4 nanoparticles.
Consistent with their smaller sized agglomerates, CA-adsorbed CoFe2O4 nanoparticles demonstrate more pronounced effects on artificial and biological membranes. Larger agglomerates of nanoparticles were confirmed to be reactive against lipid membranes and thus not acceptable for use with red blood cells. This finding is significant with respect to the efficient and safe application of nanoparticles as medicinal agents.
Biomolecular profiling with Fourier-Transform InfraRed Microscopy was performed to distinguish the Zn
2+
-mediated effects on the crustacean (Porcellio scaber) digestive glands from the ones elicited ...by the ZnO nanoparticles (NPs). The exposure to ZnO NPs or ZnCl
2
(1500 and 4000 µg Zn/g of dry food) activated different types of metabolic pathways: some were found in the case of both substances, some only in the case of ZnCl
2
, and some only upon exposure to ZnO NPs. Both the ZnO NPs and the ZnCl
2
increased the protein (∼1312 cm
−1
; 1720-1485 cm
−1
/3000-2830 cm
−1
) and RNA concentration (∼1115 cm
−1
). At the highest exposure concentration of ZnCl
2
, where the effects occurred also at the organismal level, some additional changes were found that were not detected upon the ZnO NP exposure. These included changed carbohydrate (most likely glycogen) concentrations (∼1043 cm
−1
) and the desaturation of cell membrane lipids (∼3014 cm
−1
). The activation of novel metabolic pathways, as evidenced by changed proteins' structure (at 1274 cm
−1
), was found only in the case of ZnO NPs. This proves that Zn
2+
are not the only inducers of the response to ZnO NPs. Low bioavailable fraction of Zn
2+
in the digestive glands exposed to ZnO NPs further supports the role of particles in the ZnO NP-generated effects. This study provides the evidence that ZnO NPs induce their own metabolic responses in the subtoxic range.
Giant unilamellar lipid vesicles, artificial replacements for cell membranes, are a promising tool for in vitro assessment of interactions between products of nanotechnologies and biological ...membranes. However, the effect of nanoparticles can not be derived from observations on a single specimen, vesicle populations should be observed instead. We propose an adaptation of the Markov random field image segmentation model which allows detection and segmentation of numerous vesicles in micrographs. The reliability of this model with different lighting, blur, and noise characteristics of micrographs is examined and discussed. Moreover, the automatic segmentation is tested on micrographs with thousands of vesicles and the result is compared to that of manual segmentation. The segmentation step presented is part of a methodology we are developing for bio-nano interaction assessment studies on lipid vesicles.
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