A combined targeted/untargeted LC-MS/MS-based screening approach for mammalian cell lines treated with ionic liquids : Toxicity correlates with metabolic profile

This work presents the development and validation of a quantitative HILIC UHPLC-ESI-QTOF-MS/MS method for amino acids combined with untargeted metabolic profiling of human corneal epithelial (HCE) cells after treatment with ionic liquids. The work included a preliminary metabotoxicity screening of 14 different ionic liquids, of which 9 carefully selected ionic liquids were chosen for a metabolomics study. This study is focused on the correlation between the toxicity of the ionic liquids and their metabolic profiles. The method development included the comparison of different MS/MS acquisition modes. A sequential window acquisition of all theoretical fragment ion mass spectra (SWATH) method with variable Q1 window widths and narrow Q1 target windows of 5 Da for most of the amino acids was selected as the optimal acquisition mode. Due to the absence of a true blank matrix, C-13,N-15-isotopically labelled amino acids were utilized as surrogate calibrants, instead of proteinogenic amino acids. Partial least squares (PLS) analysis of the median effective concentrations (EC50) of 9 selected ionic liquids showed a correlation with their metabolic profile measured by the untargeted screening.Peer reviewe

Determination of the Main Phase Transition Temperature of Phospholipids by Nanoplasmonic Sensing

Our study demonstrates that nanoplasmonic sensing (NPS) can be utilized for the determination of the phase transition temperature (Tm) of phospholipids. During the phase transition, the lipid bilayer undergoes a conformational change. Therefore, it is presumed that the Tm of phospholipids can be determined by detecting conformational changes in liposomes. The studied lipids included 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). Liposomes in gel phase are immobilized onto silicon dioxide sensors and the sensor cell temperature is increased until passing the Tm of the lipid. The results show that, when the system temperature approaches the Tm, a drop of the NPS signal is observed. The breakpoints in the temperatures are 22.5 °C, 41.0 °C, and 55.5 °C for DMPC, DPPC, and DSPC, respectively. These values are very close to the theoretical Tm values, i.e., 24 °C, 41.4 °C, and 55 °C for DMPC, DPPC, and DSPC, respectively. Our studies prove that the NPS methodology is a simple and valuable tool for the determination of the Tm of phospholipids.Peer reviewe

Ionic liquids affect the adsorption of liposomes onto cationic polyelectrolyte coated silica evidenced by quartz crystal microbalance

The worldwide use of ionic liquids (ILs) is steadily increasing, and even though they are often referred to as "green solvents" they have been reported to be toxic, especially toward aquatic organisms. In this work, we thoroughly study two phosphonium ILs; octyltributylphosphonium chloride ([P-8444]CI)and tributyl(tetradecyl)phosphonium chloride ([P-14444]CI). Firstly, the critical micelle concentrations (CMCs) of the ILs were determined with fluorescence spectroscopy and the optical pendant drop method in order to gain an understanding of the aggregation behavior of the ILs. Secondly, a biomimicking system of negatively charged unilamellar liposomes was used in order to study the effect of the ILs on biomembranes. Changes in the mechanical properties of adsorbed liposomes were determined by quartz crystal microbalance (QCM) measurements with silica coated quartz crystal sensors featuring a polycation layer. The results confirmed that both ILs were able to incorporate and alter the biomembrane structure. The membrane disrupting effect was emphasized with an increasing concentration and alkyl chain length of the ILs. In the extreme case, the phospholipid membrane integrity was completely compromised. (C) 2015 Elsevier B.V. All rights reserved.Peer reviewe

Impact of Surface-Active Guanidinium-, Tetramethylguanidinium-, and Cholinium-Based Ionic Liquids on Vibrio Fischeri Cells and Dipalmitoylphosphatidylcholine Liposomes

We investigated the toxicological effect of seven novel cholinium, guanidinium, and tetramethylguanidinium carboxylate ionic liquids (ILs) from an ecotoxicological point of view. The emphasis was on the potential structure-toxicity dependency of these surface-active ILs in aqueous environment. The median effective concentrations (EC50) were defined for each IL using Vibrio (Aliivibrio) fischeri marine bacteria. Dipalmitoylphosphatidylcholine (DPPC) liposomes were used as biomimetic lipid membranes to study the interactions between the surface-active ILs and the liposomes. The interactions were investigated by following the change in the DPPC phase transition behaviour using differential scanning calorimetry (DSC). Critical micelle concentrations for the ILs were determined to clarify the analysis of the toxicity and the interaction results. Increasing anion alkyl chain length increased the toxicity, whereas branching of the chain decreased the toxicity of the ILs. The toxicity of the ILs in this study was mainly determined by the surface-active anions, while cations induced a minor impact on the toxicity. In the DSC experiments the same trend was observed for all the studied anions, whereas the cations seemed to induce more variable impact on the phase transition behaviour. Toxicity measurements combined with liposome interaction studies can provide a valuable tool for assessing the mechanism of toxicity.Peer reviewe

Effects of phosphonium-based ionic liquids on phospholipid membranes studied by small-angle X-ray scattering

The effects of ionic liquids on model phospholipid membranes were studied by small-angle X-ray scattering, dynamic light scattering (DLS) and zeta potential measurements. Multilamellar 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine liposomes and large unilamellar vesicles composed of L-alpha-phosphatidylcholine (eggPC) and L-alpha-phosphatidylglycerol (eggPG) (80:20 mol%) or eggPC, eggPG, and cholesterol (60:20:20 mol%) were used as biomimicking membrane models. The effects of the phosphonium-based ionic liquids: tributylmethylphosphonium acetate, trioctylmethylphosphonium acetate, tributyl(tetradecyl)-phosphonium acetate, and tributyl(tetradecyl)-phosphonium chloride, were compared to those of 1-ethyl-3-methyl-imidazolium acetate. With multilamellar vesicles, the ionic liquids that did not disrupt liposomes decreased the lamellar spacing as a function of concentration. The magnitude of the effect depended on concentration for all studied ionic liquids. Using large unilamellar vesicles, first a slight decrease in the vesicle size, then aggregation of vesicles was observed by DLS for increasing ionic liquid concentrations. At concentrations just below those that caused aggregation of liposomes, large unilamellar vesicles were coated by ionic liquid cations, evidenced by a change in their zeta potential. The ability of phosphonium-based ionic liquids to affect liposomes is related to the length of the hydrocarbon chains in the cation. Generally, the ability of ionic liquids to disrupt liposomes goes hand in hand with inducing disorder in the phospholipid membrane. However, trioctylmethylphosphonium acetate selectively extracted and induced a well-ordered lamellar structure in phospholipids from disrupted cholesterol-containing large unilamellar vesicles. This kind of effect was not seen with any other combination of ionic liquids and liposomes. (C) 2016 Elsevier Ireland Ltd. All rights reserved.Peer reviewe

Correlation between Ionic Liquid Cytotoxicity and Liposome-Ionic Liquid Interactions

This study aims at extending the understanding of the toxicity mechanism of ionic liquids (ILs) using various analytical methods and cytotoxicity assays. The cytotoxicity of eight ILs and one zwitterionic compound was determined using mammalian and bacterial cells. The time dependency of the IL toxicity was assessed using human corneal epithelial cells. Hemolysis was performed using human red blood cells and the results were compared with destabilization data of synthetic liposomes upon addition of ILs. The effect of the ILs on the size and zeta potential of liposomes revealed information on changes in the lipid bilayer. Differential scanning calorimetry was used to study the penetration of the ILs into the lipid bilayer. Pulsed field gradient nuclear magnetic resonance spectroscopy was used to determine whether the ILs occurred as unimers, micelles, or if they were bound to liposomes. The results show that the investigated ILs can be divided into three groups based on the cytotoxicity mechanism: cell wall disrupting ILs, ILs exerting toxicity through both cell wall penetration and metabolic alteration, and ILs affecting solely on cell metabolism.Peer reviewe

Assessing toxicity of ionic liquids utilizing zebrafish, cells, and liposomes

Ionic liquids are molten salts, with melting points usually below 100 °C. Because the physical and chemical properties of ionic liquids are easily tunable by combining different ionic liquid cations and anions, has their use become more popular in many pharmaceutical and industrial applications. Despite their “green reputation”, due to their low vapor pressures and growing use, there is only little information on the toxicity of ionic liquids, effects on the environment, and long-term impacts. Many ionic liquids are water-soluble and lipophilic, thus they can bioaccumulate in aqueous organisms and furthermore in animals at higher trophic levels and eventually even to humans. This doctoral thesis describes how a group of phosphonium-, imidazolium-, and guanidinium based ionic liquids interact with zebrafish, various cell lines, and biomimicking phospholipid vesicles (liposomes). The ionic liquids in this study are potential biomass dissolvers. Further, the ionic liquids were chosen to have different chain lengths or core atoms. The aim of the study was to determine how the selection of a target organism, cation chain length, and especially anion chain length of phosphonium-based ionic liquids affect the ionic liquid toxicity. In addition, a long-term exposure to potential biomass dissolving ionic liquids was assessed using adult zebrafish. These results were utilized to find novel methodologies to evaluate ionic liquid toxicity without the use of living organisms, using solely cell membrane mimicking liposomes. To investigate the influence of ionic liquids on cell rupturing, hemolysis and a real-time cytotoxicity assay were performed and the results were compared with liposome integrity information. Differential scanning calorimetry was used to study the penetration of the ionic liquid into the lipid bilayer. The effect of ionic liquids on the size and the zeta potential of negatively charged liposomes, were assessed in order to obtain information on the changes in the diameter and surface charge of the liposomes. In addition, nuclear magnetic resonance spectroscopy, more specifically diffusion ordered spectroscopy was utilized for studying whether the ionic liquids occur as unimers or aggregates and if they are attached to the lipid bilayer. By comparing the toxicity results obtained from the zebrafish and cytotoxicity assays to the lipid bilayer rupturing information, it was possible to predict the mechanism of toxicity of ionic liquids. Thus, the ionic liquids could be divided into three groups based on their toxicity mechanism: 1) plasma membrane rupturing ionic liquids, 2) ionic liquids that have an effect on both the cell membrane integrity and the cell metabolism, and 3) ionic liquids affecting solely on the cell metabolism.Ioniset nesteet ovat sulia suoloja, joiden sulamispisteet ovat yleensä alle 100 °C. Koska ionisten nesteiden fyysiset ja kemialliset ominaisuudet ovat helposti muokattavissa, yhdistämällä erilaisia kationeja ja anioneja, on niiden käyttö yleistynyt monissa lääketieteellisissä ja teollisissa sovelluksissa. Ionisten nesteiden myrkyllisyydestä, vaikutuksista ympäristöön ja pitkäaikaiskäytöstä on vain vähän tietoa huolimatta niiden alhaisten höyrynpaineiden aiheuttamasta ”vihreästä maineesta” ja niiden kasvavasta käytöstä. Monet ioniset nesteet ovat vesiliukoisia ja lipofiilisia ja voivat siten bioakkumuloitua vesistöjen organismeihin, ravintoketjun korkeammilla tasoilla oleviin eläimiin sekä lopulta jopa ihmisiin. Tämä väitöskirja kuvaa miten ryhmä fosfonium-, imidatsolium-, ja guanidiniumperäisiä ionisia nesteitä vuorovaikuttaa seeprakalojen, erilaisten solujen ja biomallintavien fosfolipidivesikkelien (liposomi) kanssa. Tässä työssä käytetyt ioniset nesteet ovat potentiaalisia biomassan liuottajia, joiden atomien ytimet sekä ketjujen pituudet vaihtelevat. Tutkimuksen tarkoituksena oli selvittää miten kohdeorganismin valinta, fosfoniumperäisten ionisten nesteiden kationin ketjun pituus, sekä varsinkin anionin ketjun pituus vaikuttaa ionisten nesteiden myrkyllisyyteen. Lisäksi, ionisten nesteiden pitkäaikaisvaikutuksia arvioitiin käyttämällä aikuisia seeprakaloja. Näitä tuloksia hyödynnettiin, jotta löydettäisiin uusia menetelmiä, joissa elävien organismien sijaan voitiin käyttää yksinomaan solukalvoja mallintavia liposomeja. Ionisten nesteiden vaikutusta solujen hajoamiseen tutkittiin hemolyysin ja reaaliaikaisen sytotoksisuusanalyysin avulla ja näitä tuloksia verrattiin liposomien eheystuloksiin. Differentiaalista pyyhkäisykalorimetria käytettiin selvittämään miten ioniset nesteet tunkeutuvat lipidikaksoiskerrokseen. Ionisten nesteiden vaikutusta negatiivisesti kokonaisvarautuneiden liposomien kokoon ja zetapotentiaaliin tutkittiin kuvaamaan miten ioniset nesteet vaikuttavat liposomien halkaisijaan sekä pintavaraukseen. Lisäksi, ydinmagneettista resonanssispektroskopiaa, tarkemmin diffuusio-erotteista spektroskopiaa, hyödynnettiin selvittämään ilmenevätkö ioniset nesteet unimeereina vai aggregaatteina ja ovatko ne kiinnittyneet lipidikaksoiskerroksen pintaan. Yhdistämällä seeprakaloista ja sytotoksisuusanalyyseista saadut tulokset lipidikaksoiskerroksen hajoamistuloksiin, oli mahdollista ennustaa ionisten nesteiden toksisuusmekanismi. Näin ollen ioniset nesteet voitiin jakaa kolmeen ryhmään: 1) plasmamembraanin hajottavat ioniset nesteet, 2) ioniset nesteet, jotka vaikuttavat sekä solun eheyteen että solun aineenvaihduntaan ja 3) ioniset nesteet, jotka vaikuttavat vain solun aineenvaihduntaan

Unraveling Interactions between Ionic Liquids and Phospholipid Vesicles Using Nanoplasmonic Sensing

Owing to their unique properties and unlimited structural combinations, the ubiquitous use of ionic liquids (ILs) is steadily increasing. The objective of the present work is to shed light onto the effects of amidinium- and phosphonium-based ILs on phospholipid vesicles using a nanoplasmonic sensing measurement technique. A new and relatively simple method was developed for the immobilization of large unilamellar vesicles on two different hydrophilic surfaces composed of titanium dioxide and silicon nitride nanolayers. Among the pretreatment conditions studied, vesicle attachment on both substrate materials was achieved with HEPES buffer in the presence of sodium hydroxide and calcium chloride. To get an understanding of how ILs interact with intact vesicles or with supported lipid bilayers, the ILs 1,5-diazabicyclo(4.3.0)non-5-enium acetate ([DBNH][OAc]), tributyl(tetradecyl)phosphonium acetate ([P14444][OAc]), and tributylmethylphosphonium acetate ([P4441][OAc]) were introduced into the biomimetic system, and the characteristics of their interactions with the immobilized vesicles were determined. Depending on the IL, in situ real-time IL binding and/or phospholipid removal processes were observed. Although [DBNH][OAc] did not have any significant effect on the phospholipid vesicles, the strongest and the most significant effect was observed with [P14444][OAc]. The latter caused clear changes in the phospholipid bilayer: the ILs interacted with the bilayers, resulting in deformation of the vesicles most probably due to the formation of vesicle–IL aggregates. Only a mild effect was observed when [P4441][OAc], at a very high concentration, was exposed to the intact vesicles. In general, these results led to new insights into the effects of ILs on phospholipid vesicles, which are of great importance to the overall understanding of the harmfulness of ILs on biomembranes and biomimicking systems. In addition, the present work highlights the pivotal role of this highly surface-sensitive indirect biosensing technique in scrutinizing and dissecting the integrity and architecture of phospholipid vesicles in the nanoscale range