Effects of ionic liquids on membrane fusion and lipid aggregation of egg-PC liposomes

https://doi.org/10.1016/j.colsurfb.2014.11.021Get rights and content

Highlights

  • Effects of ionic liquids on membranes were investigated using pyrene fluorescence.

  • Vibronic bands of pyrene have been used to assess the effects of ionic liquids.

  • Ionic liquids with short alkyl chains were more effective than polyethoxylated ones.

  • 1-Dodecyl-3-methylimidazolium bromide was the most effective one.

Abstract

In this study we have explored the effects of different groups of ionic liquids (ILs) on membrane fusion. The ILs used contain different head groups: N-methylimidazolium, 3-methylpyridinium and N-methylpyrrolidinium; short alkyl or ether functionalized side chains (with one or two ethoxy functionalities), paired with chloride anion. These ILs have been compared with 1-dodecyl-3-methylimidazolium bromide as example of a highly lipophilic IL. The effect of ILs on membrane fusion was investigated through pyrene steady state fluorescence probing, using the IE factor and excimer/monomer ratio (IE/IM) as parameters. The ratio between the vibronic bands of pyrene (I1/I3 ratio) has been used to monitor the effect of ILs on the aggregation properties of egg-PC liposomes. The effect of different ILs’ families was evident; the pyridinium ILs induced a greater extent of fusion than pyrrolidinium and imidazolium ILs having the same side chain. Marginal effect could be attributed to different anions. ILs with short alkyl chains were usually more effective than ether functionalized ones. The aggregation behaviors of ILs having dioxygenated chains have been measured in buffer solution.

Introduction

Ionic liquids (ILs) have received considerable interest because of their unique physico-chemical properties such as very low volatility, wide electrochemical window, non-flammability, high thermal stability and wide liquid temperature range [1], [2]. ILs have been explored as alternative media for chemical reactions [3] and recently the applications on small industrial scale gained the first successful developments in pilot plants [4]. ILs have been claimed to be green solvents, despite their wide chemical composition and diversity before reaching a deeper knowledge of their behavior in the environment. In the last decade many studies have been conducted dealing with ILs properties such as ecotoxicity [5], [6], [7], biodegradability [8], biological effects on microorganisms [9], lipophilicity [10], physicochemical properties [11] and interaction with sediments [12], [13]. Due to their amphiphilic nature, some ILs have been also used as solvents for self-assembling structures such as micelles [14], [15], reverse micelles, as phase transfer catalysts [16] and amphiphilic self-assembling media [17]. In the literature there is plenty of data about the measurements of the micellar properties of ILs with long alkyl chains [18], [19], [20], but little is known about the behavior of ILs with short-chains or with chains containing oxygen atoms. Moreover, poor attention has been paid to the effects that ILs have on cell membrane properties, either using living cells or membrane models. Studies on changes in ILs structures pointed out the attention on their effect on model membranes [21], [22]. Thus a detailed knowledge of ILs’ effects and mode of action toward membranes, that are the interface between the cell and the environment, is needed for a deep evaluation of ILs biological effects.

Initial investigations on membrane effects of ILs with different alkyl chain lengths [22] highlighted that imidazolium ILs with alkyl chains longer than six carbon atoms deeply alter a membrane bilayer, inducing leakage of content and completely disrupting the model membrane at concentrations near 100 mM. ILs with shorter alkyl chains (four carbon atoms), even at concentrations up to 500 mM, induce a slow and little leakage of content that is not indicative of membrane disruption. Similar results have been recently reached in another study employing model membranes as biomimetic system and the microorganism Shewanella oneidensis [23]. In two recent studies a small group of ILs, 1-octyl-3-methylimidazolium chloride, 1-buytl-3-methylimidazolium tetrafluoroborate and 1-benzyl-3-methylimidazolium tetrafluoroborate have been evaluated on dipalmytoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) liposomes [24], [25]. The authors found that a long chain IL (1-octyl-3-methylimidazolium chloride) reduces the phase transition temperature of lipids and high molar ratio causes fusion of solid gel and liquid crystalline phases of liposomes. A short chain IL does not appreciably influence the phase transition temperature in the studied molar ratio.

ILs with different cationic head group and chain length have been recently studied and exhibit distinct mechanisms of membrane interaction, insertion and disruption that could be correlated with their biological activities, depending on the lipid nature and membrane lipid domains [26]. The results indicate, in particular, that both the ILs side chain composition (number and presence of oxygen atoms) and the ILs head-groups are important elements for membrane activity and cell toxicity.

In this paper we investigate the effects that ILs with different cationic head groups, anions and side-chain length have on membrane fusion using pyrene (Py) as fluorescence probe, considering also the presence of ethoxy units in the side chain of the cation moiety. Membrane fusion is commonly defined as a process that transforms two lipid bilayers from topologically distinct compartments into one connected compartment [27].

Liposomes of l-α-phosphatidylcholine (Egg-PC) were used as models for cell membranes [28]. The ILs used are composed by N-methylimidazolium 1, 3-methylpyridinium 2 or N-methylpyrrolidinium 3 head groups, functionalized with short alkyl chains (butyl a, ether side chains (2-methoxyethyl b, 2-(2-methoxyethoxy)ethyl c)) and with chloride, tetrafluoroborate or dicyanamide as counter anions. The head group structures and one of the three ILs families are depicted in Chart 1. For comparison we have also tested an IL with a long alkyl chain, 1-dodecyl-3-methylimidazolium bromide (1f), whose aggregation properties are well known and described in the literature [29], [30], [31].

The ether-functionalized imidazolium-based ILs 1b and 1c are a promising class of alternative solvents with some interesting properties, such as high solubility for carbohydrates [32], suitable features as reaction media for some biocatalytic processes [33] and for catalytic asymmetric reactions [34], capability to be exploited in dye-sensitized photoelectrochemical solar cells [35] and nanoparticles stabilizing properties [36]. In previous papers we have demonstrated that the introduction of one oxygen atom into the lateral chain of imidazolium-based ILs decreases their toxicity with respect to the alkyl counterparts toward the crustacean Daphnia magna and the bacterium Vibrio fischeri [37] although it also decreases their biodegradability in soils [38]. Herein a further goal was to verify if the previously observed trend of reducing toxicity by increasing the polarity of the cation [21] can be related to membrane fusion.

Section snippets

Materials

All reagents and solvents used were purchased from Aldrich; 1-methylimidazole, 3-methylpyridine, 1-methylpyrrolidine, 1-chlorobutane and 2-chloroethyl methyl ether were re-distilled before use to avoid the formation of colored impurities in ILs (see Supplementary Material for purification details). Pyrene was purchased from Fluka, Milan, Italy. Milli-RO water (resistivity 18.2  cm at 25 °C; filtered through a 0.22 μm membrane) was used for sample preparation.

Synthetic and purification procedures

Results and discussion

In a liposomal environment, Py forms excited state dimers (excimers) in a diffusion-limited reaction which depends upon membrane composition, temperature and Py concentration [44]. Furthermore, Py molecules are capable of rapid lateral diffusion, from which the formation of excimers depends. It has been recently reported that Py is predominantly localized in the interfacial region of the membrane [45].

Typical features of the emission spectra of Py are the maxima at 375, 385, and 395 nm due to

Conclusions

All tested ILs are able to induce fusion of egg-PC liposomes and alter lipid packing. However, as clearly indicated by the IE factor, the IM/IE ratio and the I1/I3 ratio values, the ILs tested cannot disrupt the bilayer at concentration up to 0.5 M with the exception of 1f, which acts as a surfactant and disrupts the egg-PC liposomes determining a great extent of membrane fusion also at low concentration. ILs that bear dioxygenated side chains do not aggregate at the tested concentrations in

Acknowledgements

We acknowledge the Ministero dell'Istruzione dell'Università e della Ricerca (MiUR) and the University of Bologna (Campus di Ravenna) for funding. We thank Regione Emilia Romagna (POR-FESR grant, Progetto Tecnopoli) for fellowship to D. Malferrari and C. Samorì. We acknowledge Giulia Balestrelli and Prof. Giuseppe Falini for the support in DLS measurements.

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