Contribution of headgroup and chain length of glycerophospholipids to thermal stability and permeability of liposomes loaded with calcein.

Biological membranes are complex systems that are composed of lipids, proteins and carbohydrates. They are difficult to study, so it is established practice to use lipid vesicles that consist of closed 'shells' of phospholipid bilayers as model systems to study various functional and structural aspects of lipid organisation. To define the effects of the structural properties of lipid vesicles on their phase behaviour, we investigated their headgroup and chain length, and the chemical bonds by which their acyl chains are attached to the glycerol moiety of glycerophospholipid species, in terms of phase transition temperature, enthalpy change and calcein permeability. We used differential scanning calorimetry to measure the temperature and enthalpy changes of phase transition, and fluorescence to follow calcein release through the bilayer structure. Our data show that longer acyl chains increase the stability of the lipid bilayers, whereas higher salt concentrations decrease the thermal stability and widen the phase transitions of these lipid bilayers. We discuss the possible reasons for the observed phase transition behaviour.

Structural properties of archaeal lipid bilayers: small-angle X-ray scattering and molecular dynamics simulation study.

Aeropyrum pernix is an aerobic hyperthermophilic archaeon that grows in harsh environmental conditions and as such possesses unique structural and metabolic features. Its membrane interfaces with the extreme environment and is the first line of defense from external factors. Therefore, lipids composing this membrane have special moieties that increase its stability. The membrane of A. pernix is composed predominantly of two polar lipids 2,3-di-O-sesterterpanyl-sn-glicerol-1-phospho-1'(2'-O-α-D-glucosyl)-myo-inositol (AGI) and 2,3-di-O-sesterterpanyl-sn-glicerol-1-phospho-myo-inositol (AI). Both have methyl branches in their lipid tails and ether linkages and carbohydrates in their headgroup. These moieties significantly affect the structure and dynamics of the bilayer. To provide a molecular level insight into these characteristics, we used here Molecular Dynamics (MD) simulations of lipid bilayers of composition similar to those of the archaeal membranes. First, we show that the electron density profiles along the normal to the bilayers derived from the simulations are in good agreement with the profiles obtained by the small-angle X-ray scattering (SAXS) technique, which provides confidence in the force fields used. Analyses of the simulation data show that the archaeal lipid bilayers are less hydrated than conventional phosphatidylcholine (PC) lipids and that their structure is not affected by the salt present in the surrounding solution. Furthermore, the lateral pressure in their hydrophobic core, due to the presence of the branched tails, is much higher than that at PC-based lipid bilayers. Both the methyl branched tails and the special headgroup moieties contribute to slow drastically the lateral diffusion of the lipids. Furthermore, we found that the lipid head groups associate via hydrogen bonding, which affects their reorientational dynamics. All together, our data provide links between the microscopic properties of these membranes and their overall stability in harsh environments.

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.

Electroporation of archaeal lipid membranes using MD simulations.

Molecular dynamics (MD) simulations were used to investigate the electroporation of archaeal lipid bilayers when subjected to high transmembrane voltages induced by a charge imbalance, mimicking therefore millisecond electric pulse experiments. The structural characteristics of the bilayer, a 9:91 mol% 2,3-di-O-sesterterpanyl-sn-glicerol-1-phospho-myo-inositol (AI) and 2,3-di-O-sesterterpanyl-sn-glicerol-1-phospho-1'(2'-O-α-D-glucosyl)-myo-inositol (AGI) were compared to small angle X-ray scattering data. A rather good agreement of the electron density profiles at temperatures of 298 and 343 K was found assessing therefore the validity of the protocols and force fields used in simulations. Compared to dipalmitoyl-phosphatidylcholine (DPPC), the electroporation threshold for the bilayer was found to increase from ~2 V to 4.3 V at 323 K, and to 5.2 V at 298 K. Comparing the electroporation thresholds of the archaeal lipids to those of simple diphytanoyl-phosphatidylcholine (DPhPC) bilayers (2.5 V at 323 K) allowed one to trace back the stability of the membranes to the structure of their lipid head groups. Addition of DPPC in amounts of 50 mol% to the archaeal lipid bilayers decreases their stability and lowers the electroporation thresholds to 3.8 V and 4.1 V at respectively 323 and 298 K. The present study therefore shows how membrane compositions can be selected to cover a wide range of responses to electric stimuli. This provides new routes for the design of liposomes that can be efficiently used as drug delivery carriers, as the selection of their composition allows one to tune in their electroporation threshold for subsequent release of their load.

Cell membrane integrity and internalization of ingested TiO(2) nanoparticles by digestive gland cells of a terrestrial isopod.

The present study was motivated by the paucity of reports on cellular internalization of ingested titanium dioxide (TiO(2)) nanoparticles (nano-TiO(2)). The model invertebrate (Porcellio scaber, Isopoda, Crustacea) was exposed to food dosed with nano-TiO(2) containing 100, 1,000, 3,000, or 5,000 µg nano-TiO(2) per gram of food. After 14 d of exposure, the amount of Ti in the entire body was analyzed by inductively coupled plasma-mass spectrometry, and elemental analyses of tissue cross sections were performed by particle induced X-ray emission. In addition, a series of toxicological markers including feeding parameters, weight change, and survival, as well as cytotoxic effects such as digestive gland cell membrane stability, were monitored. Internalization of ingested nano-TiO(2) by the isopod's digestive gland epithelial cells was shown to depend on cell membrane integrity. Cell membranes were found to be destabilized by TiO(2) particles, and at higher extracellular concentrations of nano-TiO(2), the nanoparticles were internalized.

The importance of a validated standard methodology to define in vitro toxicity of nano-TiO2.

Several in vitro studies on the potential toxicity of nano-TiO(2) have been published and recent reviews have summarised them. Most of these reports concluded that physicochemical properties of nanoparticles are fundamental to their toxicological effects. No published review has compared in vitro tests with similar test strategies in terms of exposure duration and measured endpoints and for this reason we have attempted to assess the degree of homogeneity among in vitro tests and to assess if they afford reliable data to support risk assessment. The responses in different in vitro tests appeared to be unrelated to primary particle size. The biologically effective concentrations in different tests can be seen to differ by as many as two orders of magnitude and such differences could be explained either by different sensitivities of cell lines to nanoparticles or by effect of the test media. Our review indicates that even when the in vitro tests measure the same biomarkers with the same exposure duration and known primary particle sizes, it is insufficient merely to use such data for risk assessment. In the future, validated standard methods should include a limited number of cell lines and an obligatory selection of biomarkers. For routine purposes, it is important that assays can be easily conducted, false negatives and false positives are excluded and unbiased interpretation of results is provided. Papers published to date provide an understanding of the mode on nano-TiO(2) action but are not suitable for assessment and management of risk.

Biological reactivity of TiO2 nanoparticles assessed by ex vivo testing.

Isolated digestive gland epithelium from a model invertebrate organism was used in an ex vivo system to assess the potential of nanoparticulate TiO(2) to disrupt cell membranes. Primary particle size, surface area, concentration of particles in a suspension, and duration of exposure to TiO(2) particles were all found to have effects, which are observed at concentrations of nano-TiO(2) as low as 1 µg mL(-1). The test system employed here can be used as a fast screening tool to assess biological potential of nanoparticles with similar chemical composition but different size, concentration, or duration of exposure. We discuss the potential of ex vivo tests to avoid some of the limitations of conventional in vitro tests.

Effect of ingested titanium dioxide nanoparticles on the digestive gland cell membrane of terrestrial isopods.

The aim of this study was to find out whether ingested titanium dioxide nanoparticles (nano-TiO(2)) cause cell membrane damage by direct contact or by lipid peroxidation. We assessed lipid peroxidation and digestive gland cell membrane stability of animals fed on food dosed with nano-TiO(2). Conventional toxicity measures were completed to determine if cellular effects are propagated to higher levels of biological complexity. An invertebrate model organism (Porcellio scaber, Isopoda, Crustacea) was fed with food containing nanosized TiO(2) and the result confirmed that at higher exposure concentrations after 3 d exposure, nano-TiO(2) destabilized cell membranes but lipid peroxidation was not detected. Oxidative stress as evidenced by lipid peroxidation was observed at longer exposure durations and high exposure doses. These data suggest that cell membranes are destabilized by direct interactions between nanoparticles and cell membrane, not solely via oxidative stress.

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.

Toxicity of abamectin to the terrestrial isopod Porcellio scaber (Isopoda, Crustacea).

To determine effects of the antiparasitic veterinary drug abamectin on the isopod Porcellio scaber, animals were exposed for 21 days to Lufa 2.2 soil spiked at concentrations of 3-300 mg/kg dry soil. After exposure, abamectin residues in the isopods were analysed using a novel analytical method. Toxicity was evaluated on different levels of biological organisation: biochemical, cellular and the individual organism. Measurements included glutathione S-transferase (GST) activity and stability of cell membranes in the digestive gland, animal mass gain or loss, food consumption, behaviour and mortality. LC50 for the effect of abamectin on survival of P. scaber was 71 mg/kg dry soil. The most obvious sublethal effects were reduced food consumption and decreased body mass (NOEC 3 mg/kg dry soil). Additionally, loss of digging activity and reduced GST activity (NOEC 30 mg/kg dry soil) and cell membrane destabilization (NOEC 10 mg/kg dry soil) were recorded. Abamectin only slightly accumulated in the isopods, with bioaccumulation factors always being <0.1. Based on these results and current information on environmental levels of abamectin, it is not likely that isopods will be affected by abamectin, but further studies with exposure through faeces are recommended.

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 (C(60)) or a reference chemical (ZnCl(2)). 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.

Hazardous potential of manufactured nanoparticles identified by in vivo assay.

New products of nanotechnologies, including nanoparticles, need to be assessed according to their biological reactivity and toxic potential. Given the large number of diverse nanomaterials, a tiered approach is favoured. The aim of our work presented here is to elaborate an in vivo assay with terrestrial invertebrates (Porcellio scaber), which could serve as a first step of hazard identification of nanoparticles. We adapted the widely used acridine orange/ethidium bromide (AO/EB) assay to be applicable for cell membrane stability assessment of entire organ where the animal was exposed in vivo. The digestive glands (hepatopancreas) of terrestrial isopods were taken as a model test system. The assay was validated with Cu(NO(3))(2) and surfactants. The results showed that all tested nanoparticles, i.e. nanosized TiO(2), nanosized ZnO and fullerenes (C(60)) have cell membrane destabilization potential. As expected, C(60) is the most biologically potent. The AO/EB in vivo assay proved to be fast because response is recorded after 30 min of exposure, relatively simple because digestive glands are inspected immediately after isolation from exposed animals and promising approach because different types of nanoparticles could be tested for their biological potential. This assay provides data for the identification of hazardous potential of nanoparticles before subsequent steps in a tiered approach are decided.

Lysosomal membrane stability in laboratory- and field-exposed terrestrial isopods Porcellio scaber (Isopoda, Crustacea).

Two established methods for assessment of the cytotoxicity of contaminants, the lysosomal latency (LL) assay and the neutral red retention (NRR) assay, were successfully applied to in toto digestive gland tubes (hepatopancreas) of the terrestrial isopod Porcellio scaber (Isopoda, Crustacea). In vitro exposure of isolated gland tubes to copper was used as a positive control to determine the performance of the two methods. Lysosomal latency and the NRR assay were then used on in vivo (via food) laboratory-exposed animals and on field populations. Arbitrarily selected criteria for determination of the fitness of P. scaber were set on the basis of lysosomal membrane stability (LMS) as assessed with in toto digestive gland tubes. Decreased LMS was detected in animals from all polluted sites, but cytotoxicity data were not in agreement with concentrations of pollutants. Lysosomal membrane stability in the digestive gland tubes of animals from an environment in Idrija, Slovenia that was highly polluted with mercury (260 microg/g dry wt food and 1,600 microg/g dry wt soil) was less affected than LMS in laboratory animals fed with 5 and 50 microg Hg/g dry weight for 3 d. This probably indicates tolerance of P. scaber to mercury in the mercury-polluted environment and/or lower bioavailability of environmental mercury. In animals from the vicinity of a thermal power plant with environmental mercury concentrations three to four orders of magnitude lower than those in Idrija, LMS was severely affected. In general, the LL assay was more sensitive than the NRR assay. The LMS assay conducted on digestive gland tubes of terrestrial isopods is highly recommended for integrated biomarker studies.