Jernej Zupanc

Cellular Internalization of Dissolved Cobalt Ions from Ingested CoFe2O4 Nanoparticles: In Vivo Experimental Evidence

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 Co(2+), Fe(2+), and Fe(3+) 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. Co(2+) ions were found to be more toxic than nanoparticles. We confirmed that Co(2+) 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.

Effects of magnetic cobalt ferrite nanoparticles on biological and artificial lipid membranes

Background 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. Methods 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. Results 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. Conclusion 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.

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.

Biological reactivity of nanoparticles: mosaics from optical microscopy videos of giant lipid vesicles

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(2)O(4) nanoparticles on the population of 1-palmitoyl-2-oleoyl-sn-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.

A new approach to analyse effects of nanoparticles on lipid vesicles

Manufactured nanoparticles are potentially capable of inducing defects in lipid membranes. The effects of nanoparticles on cell membranes are one of the key issues in nanomedicine, nanotoxicology, food and pharmaceutical application of products of nanomaterials and others. Our aim is to demonstrate the nanoparticle - lipid vesicle interactions and to develop a controllable experimental setup for data acquisition. We studied interactions between nanoparticles (C60) and lipid vesicles (POPC), using ZnCl2 as a positive control. Light microscopy computer aided image segmentation was developed and population differences among vesicles incubated in different media were assessed. Data obtained by statistical image analysis methods revealed that nanoparticles (C60) caused changes in vesicle size distribution in the population of lipid vesicles as well as a burst of vesicles in time and in a concentration gradient. No significant changes in shape of vesicles were recorded. The advantage of the experimental set up presented here is that it employs statistical image analysis methods and direct microscopy observation of large populations of lipid vesicles. We discuss the applicability of this in vitro approach in analysing the effects of nanoparticles on simplified biological membranes.

FTIR microscopy reveals distinct biomolecular profile of crustacean digestive glands upon subtoxic exposure to ZnO nanoparticles

Abstract Biomolecular profiling with Fourier-Transform InfraRed Microscopy was performed to distinguish the Zn2+-mediated effects on the crustacean (Porcellio scaber) digestive glands from the ones elicited by the ZnO nanoparticles (NPs). The exposure to ZnO NPs or ZnCl2 (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 ZnCl2, and some only upon exposure to ZnO NPs. Both the ZnO NPs and the ZnCl2 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 ZnCl2, 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 Zn2+ are not the only inducers of the response to ZnO NPs. Low bioavailable fraction of Zn2+ 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.

Markov random field model for segmenting large populations of lipid vesicles from micrographs.

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.

Interactions of nanoparticles with lipid vesicles: A population based computer aided image analysis approach

Novel properties of nanoparticles have numerous potential technological applications but at the same time they underlie new kinds of biological effects. Uniqueness of nanoparticles and nanomaterials requires a new experimental methodology. Much evidence suggests that nanoparticles affect cell membrane stability and subsequently exert toxic effects. For this kind of research, lipid vesicles are of high value due to controllability and repeatability of experimental conditions. The aim of work presented here was to develop a computer aided analysis of lipid vesicles shape transformations. We studied a population of palmitoyloleoylphosphatidylcholine (POPC) lipid vesicles after exposure to nanoparticles (C60) or a reference chemical (ZnCl2). With the use of computer image analysis methods, we detected differences in size distributions of vesicles in different exposure groups. Though, at the present state, we are not able to precisely identify effects of nanoparticles on shape transformations of vesicles, those incubated with nanoparticles were in average larger than those in other groups. This population based approach holds many promises for future investigation of nanoparticles-lipid vesicles, or even nanoparticles-biological membranes interactions. However, in order to get reliable results, numerous images have to be analyzed which requires improved and highly automated image segmentation and analyses methods.

Analysis of lipid vesicle populations from microscopy video sequences

Conducting research on lipid vesicles is very convenient, since they provide a stable and controllable environment for in vitro observations. Their resemblance to biological cell membranes allows biologists to assess hazardous potential of nanoparticles by exposing the vesicles instead of live organisms. When considering behavior of vesicles during incubation with nanoparticles, majority of existing research focus on observing single vesicles only. Our approach provides an ability to observe thousands of lipid vesicles for more representative behavior estimation. We developed an efficient algorithm to transform video sequences acquired with video microscopy into quantitative data. This includes steps required to filter noise, use multiple frames for more precise content presentation, detection of regions of interest, and segmentation of circular and non-primitively shaped vesicles. Presented work is a crucial step towards the creation of an automated computer analysis for lipid vesicles behavior assessment.

Lipid Vesicle Shape Analysis from Populations Using Light Video Microscopy and Computer Vision

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.