Unraveling Interactions between Ionic Liquids and Phospholipid

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Unraveling interactions between ionic liquids and phospholipid vesicles using nanoplasmonic sensing Joanna Witos, Giacomo Russo, Suvi-Katriina Ruokonen, and Susanne Kristina Wiedmer Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b04359 • Publication Date (Web): 09 Jan 2017 Downloaded from http://pubs.acs.org on January 12, 2017

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Unraveling interactions between ionic liquids and phospholipid vesicles using nanoplasmonic sensing

Joanna Witos1, Giacomo Russo1, Suvi-Katriina Ruokonen1, Susanne K. Wiedmer1*

1

Department of Chemistry, P. O. Box 55, FIN-00014 University of Helsinki, Helsinki, Finland

KEYWORDS: [DBNH][OAc]; Ionic Liquids; Localized Surface Plasmon Resonance; Nanoplasmonic Sensing; [P14444][OAc]; [P4441][OAc]; Phospholipids

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ABSTRACT 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 into 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

ionic

liquids

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. While [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 for 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.


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INTRODUCTION Ionic liquids (ILs) are a class of organic salts exhibiting relatively low melting points (generally below 100 °C) increasingly attracting the interest of both academia and industry for their unique features.1, 2 Among those, their remarkable capability of solubilizing both organic and inorganic materials makes them perfect candidates as an environmental-benign source of alternative solvents.3 Furthermore, they exhibit low vapor pressure, severely limiting their release into the atmosphere, as well as very high degree of chemical and thermodynamical stability. For these reasons, they have been widely employed in a plethora of industrial applications,4, 5 such as in dye-sensitized solar cell manufacturing, in catalytic processes, and for biomass dissolution.

Albeit considered as greener solvents in comparison with the conventional volatile organic compounds they are gradually replacing, many ILs present instead noticeable toxicity,6,

7, 8

most of them being

water-soluble and having the tendency to rapidly accumulate in the soil and sediments, thus contaminating the water ecosystem. Even though many toxicity mechanisms for ILs have been hypothesized,9,

10

just a few of them are completely understood. Therefore, study on IL-cellular

membrane interactions, involving membrane binding, insertion, and rupture and/or membrane phospholipid removal, is a highly relevant research topic.11,

12

Many ILs can be classified as

conventional surfactants,13 which are well known as strong membrane disrupting agents. The surfactant properties are also highly linked to the aggregation of monomers, and some ILs aggregate (e.g. forming micelles) in aqueous solutions. Some authors have linked the toxicity of ILs to their n-octanol/water partitioning constants, i.e. lipophilicity,7 basically implying that the stronger their partitioning into the n-octanol phase the more they disrupt biological membranes, whereas others have related the toxicity of the ILs to the length of the alkyl chain of the cationic constituent.6 The latter effect has been suggested to be caused by dipolar interactions between the positively charged moieties of the ILs and 3 ACS Paragon Plus Environment

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the phosphate groups of the phospholipid head groups, allowing the insertion of ILs through the polar region of the membrane bilayer, resulting in membrane damage and, consequently, cell death. Other key factors affecting the toxicity of ILs are (a) the degree of functionalization of the side chain in the cationic part, (b) the anionic nature, (c) the cationic nature, and (d) the mutual influence of anion and cationic components in the IL.6

The toxicity of ILs can be realized by following their effects on the architecture of the lipid membrane of cells. The impact can be seen by structural rearrangements of membrane lipids, cell leakage, and/or through liposome rupture. For that reason, suitable biomimicking models can be utilized for probing interactions between ILs and liposomes in the nanoscale range. Reliable quantitative toxicity data can be achieved only by assaying the ILs on cell cultures; however, biomimicking technique can be used for shedding lights into the mechanisms behind the toxicity exerted by ILs. For this purpose, vesicles made of phospholipids (liposomes) were utilized in this work as biomimicking models of biological membranes. Phospholipid deposition and immobilization either as intact vesicles or as supported lipid bilayers (SLBs)14 on hydrophilic supports offer an interesting opportunity to model and examine the effect of ILs on the integrity and architecture of phospholipid bilayers. Phospholipid immobilization is generally achieved after exposure of small lipid vesicles to hydrophilic supports, most commonly silicon dioxide, silicon nitride, titanium dioxide, and aluminium oxide.15 Since 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) is one of the most abundant phospholipids present in eukaryotic cells,16 POPC liposomes have been considered as excellent models mirroring the natural membrane composition.

To date, several techniques are available for investigating biomembrane architecture on the nanoscale, including atomic force microscopy (AFM),17 quartz crystal microbalance-dissipation (QCM-D),17,

18


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and nanoplasmonic sensing (NPS).19,

20, 21

AFM is a scanning-probe microscopy technique that has

offered substantial assets in systematically exploring the cell membranes architecture taking advantage of high resolution, three-dimensional and in situ measurements, yielding valuable information about biomacromolecules under nearly physiological conditions.22 However, quantitative analysis of kinetic processes by AFM is not that straightforward.23 QCM-D is another surface sensitive technique widely employed to monitor the adsorption and desorption kinetics on nanomaterial surfaces in solution.24 In the technique changes in the resonance frequency and energy dissipation that are strictly related to the mass and structural viscoelastic properties of the absorbed compound, respectively, are monitored. NPS is a label-free optic technique allowing the study of surfaces and interfaces of metals that are due to light-induced electronic excitations called surface plasmons. For a metal nanosensor embedded in a dielectric spacer layer, the surface plasmons are due to oscillation of the electric field and polarization localized in space (a phenomenon known as localized surface plasmon resonance, LSPR). The position of the observed plasmonic signal is strictly dependent on the shape, size, and material of the studied nanomaterials, and by the refractive index (RI) of the medium in close proximity to them. To the best of our knowledge, NPS has been utilized for studying lipid vesicle adsorption onto various surface materials since 2008.19, 25, 26, 27 The dependence of the adsorption kinetics upon a plethora of conditions such as different sensor materials,20,

28

temperature,29 vesicle size,30 and the presence of divalent

cations31 have been thoroughly investigated. In general, Tris seems to be the buffer of choice and as to date there are no studies on the effect of the buffer component other than Tris on the formation of SLBs or intact adsorbed vesicles on different NPS sensor materials. Therefore one of the aims of this study was to investigate the role of HEPES buffer on the liposome adsorption process. Another important goal was to demonstrate the potential of NPS as sensitive methodology for gaining insights into compound-liposome interactions. To note is that NPS alone cannot provide detailed information on the nature of such interactions, but it can be successfully employed as a complimentary methodology. For 5 ACS Paragon Plus Environment

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instance, QCM-D and NPS techniques provide essentially complementary information as they have rather different surface sensitivity:32 while QCM-D measurements have a penetration depth of around 250 nm, NPS determinations are far more surface sensitive, reaching a penetration depth of around 10 nm, and as such they represent an ideal platform for probing the architecture of lipid vesicles adsorbed on hydrophilic supports.25

In the present work, we studied to the immobilization of POPC lipid vesicles on two different support surfaces, i.e. silicon nitride and titanium dioxide via NPS measurements and further we looked into the interactions between liposomes and three synthesized ILs: [DBNH][OAc] (1,5-diazabicyclo(4.3.0)non5-enium

acetate),

[P14444][OAc]

(tributyl(tetradecyl)phosphonium

acetate),

and

[P4441][OAc]

(tributylmethylphosphonium acetate) (Figure 1). The ILs chosen have potential biomass solubility properties and are therefore of great interest for the industry.33, 34 We have previously investigated the toxicity of [DBNH][OAc], [P14444][OAc], and [P4441][OAc] using various cell lines (human corneal epithelial cells, E. coli bacterial cells,35 and Chinese hamster ovary cells) and zebrafish.10 Furthermore, we have used liposomes as biomimicking membranes to gain more detailed information on ILmembrane interactions. The effect of [P14444][OAc] on eggPC/POPG liposome size and surface charge (zeta potential) was assessed using dynamic light scattering and zeta potential determinations.35 Small angle X-ray scattering was used to obtain information on the lamellar distance of POPC vesicles upon addition of [P14444][OAc] or [P4441][OAc] to MLVs.36 In addition, the effect of [P14444][OAc] on the zeta potential of eggPC/eggPG and eggPC/eggPG/cholesterol LUVs was assessed and the results demonstrate that the liposomes were coated with ILs at a concentration of 0.1 mM. The two studied sensor surfaces differ by their surface properties at the studied pH of 7.4; while titanium dioxide is essentially neutral at physiological pH,37 silicon nitride surfaces remain slightly negatively charged.38 Our recent studies10 have shown that [P14444][OAc] has a significant cytotoxicity 6 ACS Paragon Plus Environment

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towards Chinese hamster ovary cells (median effective concentration, i.e. EC50 of 5.5 µM), while the two other ILs, [DBNH][OAc] and [P4441][OAc], have EC50 values of 72.51 mM and 3.55 mM, respectively. Thus, they were classified as “harmless” and “practically harmless” on the same cultured cell monolayers. This study was carried out to get new insights into the effects that such ILs have on liposomes or SLBs, and to shed light into the mechanism behind membrane binding and structural rearrangement.

MATERIALS AND METHODS Chemicals POPC was purchased from Avanti Polar Lipids (Alabaster, AL, USA). N-(2-Hydroxyethyl)piperazineN′-(2-ethanesulfonic acid) (HEPES) and sodium dodecyl sulfate (SDS) were from Sigma (Darmstadt, Germany). Sodium hydroxide pellets were obtained from J.T. Baker Chemicals (Center Valley, PA, USA). Calcium chloride was purchased from VWR International Oy (Espoo, Finland). Ethanol (Etax A) was from Altia Oyj (Rajamäki, Finland). The pH solutions (7.0 and 10.0) used for calibrating the pH meter were purchased from Merck (Darmstadt, Germany). The pH meter was from Mettler Toledo (Columbus, Ohio, USA). Distilled water was purified with Millipore water purification system (Millipore, Molsheim, France). The ILs were synthesized in the Laboratory of Organic Chemistry (University of Helsinki, Finland). Detailed information on the synthesis and characterization of [DBNH][OAc], [P14444][OAc], and [P4441][OAc] can be found in the literature.10, 12, 39

Buffer and sample preparation The ionic strength of the buffer solution was 10 mM and its pH value was pH 7.4. Before use, the buffer solution was filtered through a 0.45-µm syringe filter (Gelman Sciences, Ann Arbor, MI, USA). 7 ACS Paragon Plus Environment

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Liposome stock solution (4.0 mM) was diluted in the appropriate buffer solution, either in HEPES or HEPES with 5.26 mM CaCl2, to obtain final concentration 0.15 mM. The ILs samples in water were prepared from their corresponding aqueous stock solutions (MilliQ water). All buffer and sample solutions were stored at +4 ºC. Vesicle preparation A 4.0 mM unilamellar lipid vesicles comprised of POPC were prepared by the extrusion method. Assupplied lipid chloroform stock solution was evaporated to dryness under a steam of pressurized air. The chloroform residues were removed by overnight evacuation under reduced pressure (8–100 mbar). The phospholipids were hydrated in MilliQ water for 60 min at 60 ºC and shaken to yield multilamellar vesicles (MLVs). The resulting dispersion was processed to large unilamellar vesicles (LUVs) at room temperature by extruding the dispersion 21 times through Millipore (Bedford, MA, USA) 100-nm pore size polycarbonate filters using a Liposo-Fast extruder. Routinely, determinations of the size of the liposomes were carried out by a Zetasizer Nano ZS instrument (Malvern Instruments, Malvern, Worcestershire, UK). The average diameter of LUVs and MLVs were 142.9 ± 17.5 nm and 732.1 ± 79.6 nm, respectively.

NPS measurements NPS measurements based on localized surface plasmon resonance were conducted on nanodisks in optical transmission mode using an Insplorion XNano instrument (Insplorion AB, Gothenburg, Sweden).30 Briefly, white light enters the measurement cell, passes the sensor chip, and exits through a quartz glass window. The transmitted light was collected by spectrophotometer and simultaneously recorded as a function of time. The frequency resolution of the NPS signal was set to 1 Hz.


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Silicon nitride and titanium dioxide sensor chip (Insplorion AB, Gothenburg, Sweden) were used as sensing surfaces. Prior to the experiments, the sensors were pretreated for 20 min with oxygen plasma using an UV ozone cleaner (UVC-1014 NanoBioAnalytics, Berlin, Germany). The sensors were reused up to 10 times and were cleaned with 1% SDS, MilliQ water, and 30% ethanol solution between the experiments. All experiments were performed two times under continuous flow (100.0 µL min-1) controlled by a Reglo-CPF Digital peristaltic pump (Ismatec, Wertheim, Germany) at ambient room temperature (22 °C). Measurement data was analyzed by the software Insplorer version 1.2 (Insplorion AB, Gothenburg, Sweden).

RESULTS AND DISCUSSION The increasing use of ILs in various industrial processes has resulted in a strong demand of achieving a better understanding of their toxicological impact. The toxicity of ILs is related to the lipophilicity and alkyl chain lengths of ILs. These properties affect the interaction between ILs and biological membranes6 and can enhance bioaccumulation, disruption and leakage of biomembranes, or even lead to premature cell death.40 However, there are only a few toxicity studies of amidinium- and phosphonium-based ILs and these concern enzymes, aquatic systems, and bacterial and mammalian cell lines.7, 41, 42, 43, 44, 45, 46, 47 The aim of this work was to demonstrate that NPS, being a highly surfacesensitive indirect sensing method, can be utilized as an innovative approach for studying the interactions between phospholipid vesicles and industrially relevant ILs. First the immobilization of phospholipid vesicles onto two different hydrophilic sensors (titanium dioxide and silicon nitride coatings) was optimized by varying the pretreatment conditions. Secondly, the selected ILs were introduced into the biomimicking nanosystems, enabling real-time monitoring of their interactions with phospholipid vesicles and SLBs. 9 ACS Paragon Plus Environment

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Phospholipid vesicle immobilization Phospholipid vesicles adsorb and self-assemble to form different adsorption patterns on sensors depending on their surface properties.25,

48

Phospholipid vesicles might remain as intact adsorbed

vesicles or they can rupture to form SLBs.48, 49 The vesicle adsorption process was studied on titanium dioxide- and silicon nitride-coated gold nanodisks. The rate of immobilization was followed by the tracking of the changes in the characteristic maximum-extinction wavelength that depends on nanoparticle properties like size, shape, interspacing, and local dielectric environment. When vesicles are adsorbed onto the substrate, an increase in the maximum-extinction wavelength is observed due to a higher RI of lipid vesicles than that of the surrounding buffer solution.

The effect of sensor preconditioning on the POPC vesicle adsorption process, using titanium dioxide and silicon nitride sensors, was studied. An organic HEPES buffer solution was chosen based on our earlier works, in which we have reported the importance of HEPES or other piperazine-based buffer components for the adsorption on phospholipids on fused-silica capillaries using capillary electrochromatography.50, 51 HEPES (and other piperazine-based buffers) forms a linkage between the phospholipid vesicles and the fused silica wall, whereas a highly stable semi-permanent phospholipid coating is obtained.

To elucidate the role of HEPES on the adsorption of POPC on different surfaces, four different pretreatment conditions were studied. In the first method, the sensor was only rinsed with HEPES buffer prior to liposome immobilization. In the second method, a fresh sensor was flushed with sodium hydroxide, water, and subsequently with HEPES buffer. In the third method, the sensor was flushed 10 ACS Paragon Plus Environment

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with HEPES buffer containing calcium ions (POPC:CaCl2 concentration ratio of 20) prior to immobilization of liposomes. In a fourth method, a fresh sensor was rinsed with sodium hydroxide, water, HEPES buffer, and subsequently with HEPES buffer containing calcium ions (POPC:CaCl2 concentration ratio of 20). After preconditioning, the liposome dispersion diluted in the appropriate buffer solution was introduced to the sensor surface by flushing the substrate until the signal remained stable for 10 min. Finally, the sensor was rinsed with the buffer solution to remove excess, unadsorbed, phospholipid vesicles. The immobilization procedures used for titanium dioxide and silicon nitride surfaces were identical.

Phospholipid immobilization performed on titanium dioxide sensors To determine whether or not the POPC vesicles adsorb on the titanium dioxide substrate, the change in the maximum-extinction wavelength (∆λ), the so-called “peak shift” was monitored as a function of time (Figure 2). As shown in Figure 2D, there was no interaction between POPC liposomes and the titanium dioxide surface when the surface was solely pretreated with HEPES buffer. Clearly, the linkage between the liposomes and the titanium dioxide surface cannot be formed simply by flushing the sensor with HEPES. To enhance vesicle attachment, a sodium hydroxide flush was used prior to HEPES flush and CaCl2 was mixed with the HEPES buffer. Titanium dioxide contains mixed covalent and ionic bonds and is reactive toward different inorganic and organic molecules and atoms.52 Titanium dioxide nanoparticles have been shown to contain various types of hydroxyl groups,53 which are reactive to e.g. atmospheric gases.54 Titanium dioxide sensors used in this work presumable contain free hydroxyl groups, which can be deprotonated by the 0.1 M sodium hydroxide making the surface reactive. Another plausible explanation for the reactivity caused by the sodium hydroxide flush, can be the breakage of ionic Ti-O-Ti bonds and formation of Ti-O-Na or Ti-OH groups and/or further unstable sodium titanate. Sodium hydroxide flush has been shown to improve the bioactivity of titanium dioxide 11 ACS Paragon Plus Environment

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nanotubes by creating nanofiber-like sodium titanate structure on the surface.55 However, in that study highly concentrated sodium hydroxide (5 M) was used at 60 °C and the layer was further heat-treated at 500 °C. Moreover Lee et al.56 have treated titanium dioxide surfaces on Ti alloys with sodium hydroxide gaining a porous sodium titanate hydrogel layer, which stabilized after dehydration at high temperature (600-800 °C).

Calcium chloride, on the other hand, acts as a strong fusogenic agent and has shown to enhance the adsorption of liposomes on fused silica capillary walls. In addition, it plays an important role for the formation of SLBs.50, 57, 58 As shown in Figures 2B and 2C, successful immobilization was obtained in both cases (NaOH flush and CaCl2 addition). Upon vesicle addition, we could initially observe a small decrease in the peak shift followed by continuous vesicle adsorption until reaching a saturation plateau. The initial small decrease is typical for adsorption of large unilamellar vesicles.30, 59 Clearly, sodium hydroxide and calcium ions aid the formation of linkage between the HEPES treated surface and the vesicles, enabling immobilization of phospholipids. The liposomes remained as intact vesicles on the surface under both conditions. No vesicle rupturing resulting in SLB formation was observed. Moreover, the POPC liposomes clearly interact less with the sodium hydroxide treated titanium dioxide surface (Figure 2C). The most efficient immobilization was achieved when calcium chloride was added to the HEPES buffer (Figure 2B). Our results are in good agreement with a recent biosensing study, in which the authors showed that divalent cations enhanced the immobilization of vesicles, with the most prominent effect attributed to calcium ions.32 The average final peak shift was 4.2 nm, and the time scale of the whole procedure was 35 min when CaCl2 was used with HEPES buffer. By comparison, the average final peak shift was only 2.5 nm when sodium hydroxide was used in the preconditioning before HEPES treatment. The time scale of vesicle attachment was similar in both cases. However, it has to be pointed out that the reproducibility of vesicle adsorption on titanium dioxide surface was only 12 ACS Paragon Plus Environment

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on a satisfactory level. Our results showed that the final peak shift of vesicle immobilization was in the range between 2.5-6.5 nm, suggesting that vesicles adsorbed on titanium dioxide surface in an unpredictable manner. To improve the reproducibility of phospholipid adsorption on titanium dioxide surface, a fresh sensor was treated with sodium hydroxide and buffer containing calcium ions before liposome immobilization. As can be seen in Figure 2A, phospholipids were successfully immobilized on the surface and remained as intact vesicles. The average final peak shift was 4.0 nm and the time scale of the whole procedure was again 35 min. Unfortunately, the reproducibility of the vesicles adsorption was still poor. For this reason, the titanium dioxide substrate was not utilized in our further studies.

Phospholipid immobilization performed on silicon nitride sensors For a comparison, the vesicle adsorption was performed on silicon nitride surface. Silicon nitride contains both covalent and ionic bonds, but predominantly covalent bonds.60 The chemical composition of silicon nitride changes in aqueous environment by formation of silanol groups, as a result of partial oxidation of the surface.38,

61

Furthermore, Kennedy et al. pointed out how oxynitride films under

certain conditions can be produced from silicon nitride dielectric films undergoing plasma anodization.62 Such formed dielectric films have a “three layers” sandwich structure, resulting in two outer “SiO2”-like layers and one inner “Si3N4” –like layer between them. Therefore, when the pH is above 2-3, the surface becomes negatively charged due to deprotonation of the silanol groups. As shown in Figure 3D, unsuccessful immobilization was again observed when solely HEPES buffer was used in the preconditioning. Clearly, HEPES alone was not enough for forming a link between the liposomes and the negatively charged surface. To improve phospholipids immobilization, sodium hydroxide was utilized in the sensor preconditioning. In view of earlier findings, specific ions such as sodium do not adsorb on the silicon nitride surface.38, 63 Our results show that when the silicon nitride 13 ACS Paragon Plus Environment

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surface was activated with sodium hydroxide, repeatable immobilization of phospholipid vesicles on the surface was obtained (Figure 3C). First, upon vesicle introduction a small decrease in the peak shift could be detected, as observed also with the titanium dioxide surface. Subsequently, a continuous increase in the NPS signal was observed. The attachment of vesicles was conducted until reaching a saturation coverage (a plateau value), with an average final peak shift of 3.5 nm. Consistent with the previous experiments performed on titanium dioxide surfaces, phospholipids remained also as intact vesicles on the silicon nitride substrate when sodium hydroxide and HEPES buffer were utilized in the preconditioning step. For comparison, the vesicle immobilization was performed in the presence of calcium chloride in the buffer solution. Additionally, we tested vesicle attachment after preconditioning the sensor with sodium hydroxide and a calcium-containing buffer. As seen in Figures 3A and 3B, we could initially observe a slight decrease in the NPS response followed by a continuous increase in the signal until an acceleration in the NPS response occurred, changing the slope. This acceleration is attributed to the movement of phospholipids toward the substrate surface caused by rupturing of liposomes into SLBs.32 The sensor-to-sensor stability and reproducibility in both cases were excellent with a final average peak shift of ~4.0 nm achieved within 10 min of immobilization. Our results complement the previous findings and show that calcium ions evidently promote supported bilayer formation on negatively charged surfaces.32,

50

The sensor pretreated only with HEPES buffer

containing calcium ions was used in further interaction studies concerning SLBs due to the shorter pretreatment time needed.

In order to further investigate phospholipid immobilization on the silicon nitride surface, the effect of the physical structure of the vesicle on the adsorption process was studied using also MLVs. The MLVs were prepared in the same manner as LUVs but without extrusion after hydration in water. Figure 4 shows the peak shift as a function of time for MLVs immobilization after different 14 ACS Paragon Plus Environment

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pretreatment procedures. The interaction of the MLVs with the silicon nitride sensor occurred only when calcium chloride was used during the vesicle attachment process (Figure 4A). Interestingly, the typical initial small decrease in the peak shift was not observed. This can be attributed to the wide size distribution of the heterogeneous vesicles containing also small vesicles. Most probably, smaller vesicles were filling the vacant areas of the coated gold nanodisks affecting the NPS response signal. The vesicles showed continuous adsorption until reaching a peak shift of 2.1 nm, where the slope changed and the adsorption accelerated indicating vesicle rupture and the beginning of SLB formation. The magnitude of the final peak shift was 4.0 nm obtained within 2 hours. The long duration of vesicle attachment was caused by the vesicle multilamellar structure: the greater the diversity and the broader the size distribution of vesicles, the longer is the immobilization process. In addition, the MLVs caused lower signal-to-noise-ratio than small unilamellar vesicles. Our finding is consistent with studies carried out by Jackman et al.30, who observed that vesicle attachment takes longer for bigger vesicles (160 nm diameter) due to the slower relaxation of the vesicle adlayer. As already pointed out, surprisingly, there was no vesicle adsorption when the silicon nitride substrate was activated with sodium hydroxide. These results are in contradiction with the studies carried out with LUVs, where the phospholipids were successfully immobilized and remained as intact vesicles. Evidently, the linkage between the MLVs and the silicon nitride surface cannot be formed by flushing the sensor with sodium hydroxide and calcium-containing buffer. The reasons for such unsuccessful immobilization might be attributed to the considerable differences in vesicle size and lamellarity between LUVs and MLVs. Since LUVs are composed of a single bilayer shell with a diameter of 100-1000 nm, while MLVs consisting of several concentric shells, are in the size range from 100 nm to 20 µm and possess a vesicle-within-vesicle structure,64 the LUVs are more prone than MLVs to undergo fusion onto the surface.65 Moreover, Gulcev et al.66 showed that the lamellarity of vesicle affects the adsorption rate


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onto the negatively charged fused silica capillary wall. The greater lamellarity, the smaller is the adsorption rate of phospholipids.

Our NPS measurements indicate that the most efficient, repeatable POPC-based coatings were achieved on the silicon nitride surface with excellent sensor-to-sensor stability and reproducibility. Moreover, by utilizing the sodium hydroxide in the preconditioning step or a HEPES buffer containing calcium chloride, phospholipids remained as intact, adsorbed vesicles or ruptured to form SLBs, respectively. Because of this, the silicon nitride substrate was employed in all further interaction studies.

Interactions between ILs and POPC liposomes To elucidate the impact of one amidinium- and two phosphonium-based ILs on phospholipid vesicles, POPC liposomes were immobilized on silica nitride sensor. Different pretreatment procedures were used resulting either in intact, adsorbed vesicles, or in the formation of SLBs. Each IL was exposed to the phospholipid vesicles either immobilized as intact supported vesicular layers (SVLs) or as SLBs in order to evaluate the suitability of both models in describing the IL-lipid vesicle interactions. The attachment of vesicles was confirmed by rinsing the POPC-based coating with appropriate buffer solution followed by a water rinse. ILs were further introduced at a concentration above the EC50 values, in order to ensure a possible effect of ILs.10 The EC50 value refers to the concentration causing a certain effect in 50% of the test group. As already stated in the Introduction, such values are generally assayed on cultured cells, as the NPS technique is unable to provide reliable quantitative information of analyte toxicity. However EC50 were here assumed as mere indications of effect of ILs on the biomembrane integrity and as a starting reference for the experimental design of the work. The rate of 16 ACS Paragon Plus Environment

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interaction was monitored by following the changes in the characteristic maximum-extinction wavelength of the immobilized phospholipid vesicles until the signal remained stable for 10 min. Finally, the sensor was rinsed with water to establish the effect of ILs on phospholipid vesicles. ILs were introduced to the sensor surface by the same procedure in order to gain references.

Interactions between [DBNH][OAc] and POPC liposomes To investigate whether the NPS technique could be exploited for studies on interactions between ILs and phospholipid vesicles, we first studied the effect of the amidinium-based IL [DBNH][OAc] on POPC. As already reported in Introduction, the cytotoxicity studies carried out on Chinese hamster ovary cells indicated an EC50 value equaling 72.51 mM for [DBNH][OAc].10 To ensure a possible effect, [DBNH][OAc] at concentration of 250 mM was introduced in the system. The effect of [DBNH][OAc] on silicon nitride as a function of time is shown in bottom of Figure 5A and 5B. The reference runs clearly indicate that [DBNH][OAc] did not interact with silicon nitride surface. The signal remained at the same level before the introduction of IL and after a water rinse, proving that [DBNH][OAc] did not adsorb on the sensor surface under the studied conditions. The observable changes in the characteristic maximum-extinction wavelength are only related to the differences in the RI values between IL and the surrounding water solution. The increase in peak shift indicates that [DBNH][OAc] has a higher RI than a water solution.

Subsequently, the effect of [DBNH][OAc] was studied on intact adsorbed vesicles (Figure 5A) and on SLBs (Figure 5B). In both cases, there were almost no changes in the peak shift values when flushing with HEPES buffer containing calcium chloride, HEPES buffer, and water solution, suggesting that the phospholipids were not flushed out and still remained on the sensor surface. Introduction of [DBNH][OAc] resulted in a fast increase of the NPS signal, while the following water rinse caused the 17 ACS Paragon Plus Environment

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opposite behavior i.e. a decrease in the signal. This is related to the differences in RI values between IL and the surrounding water solution as seen from the reference runs. Moreover, the equal changes in the characteristic maximum-extinction wavelength confirmed that phospholipids remained on the silicon nitride surface in the same form as before the introduction of the IL. This is a clear indication that [DBNH][OAc] does not induce any effect either on intact adsorbed vesicles or on SLBs. It was further confirmed in our previous works, where a long-term impact studies were performed using zebrafish

10

and the effect of [DBNH][OAc] on human corneal epithelial cells was assessed, that there were no significant impacts caused by [DBNH][OAc].12

Interactions between [P14444][OAc] and POPC liposomes We have previously shown that the phosphonium-based IL [P14444][OAc] shows high toxicity towards various types of cell lines.10,

12

To clarify the interactions between [P14444][OAc] and phospholipid

vesicles immobilized on a silicon nitride sensor, we first measured whether the IL adsorb on the sensor surface or not. After establishing a baseline in water solution, 1 mM [P14444][OAc] was introduced in the system and an adsorption was observed with a final peak shift of 0.3 nm (Figure 6A; reference curve). The following water rinse evidenced a rather weak interaction between the IL and the sensor surface. Therefore, we can conclude that the positively charged [P14444][OAc] is neither electrostatically nor hydrophobically adsorbed onto the negatively charged silicon nitride substrate.

In order to gain further insights into the [P14444][OAc] effects on phospholipid vesicles, [P14444][OAc], at a concentration of 1 mM, was introduced on the intact adsorbed phospholipid vesicles (Figure 6A) and on the SLB (Figure 6B). Addition of 1 mM [P14444][OAc] to the intact adsorbed vesicles led to an initial increase in the maximum-extinction wavelength (∆λ = 0.3 nm), which suggests that the IL is partitioning into the vesicle bilayer. Indeed, an increase in the maximum-extinction wavelength could 18 ACS Paragon Plus Environment

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also suggest a slight deformation of the phospholipid vesicles induced by the addition of the IL. In fact, the higher extent of adsorbed liposome deformation corresponds to a greater peak shift (∆λ) as a result of more lipid mass adsorbed near the substrate surface and thus within the LSPR evanescent field. Several authors measured the LSPR signal of adsorbed intact DPPC and DOPC lipid vesicles both on silicon nitride and titanium oxide sensor surfaces and have quantitatively described the extent of liposome deformation induced by temperature67 or by changes in the ionic strength,29 both at low and high surface coverages and for different lipid phase states. The following mild decrease in the peak shift might indicate that phospholipids are exchanged by IL molecules and that the liposomes are partially ripped off from the substrate surface. This sort of removal of lipids prior to liposome rupture has been shown by Lopez et al.68 using Triton X-100 surfactants. The plot suggests that the subsequent water rinse flushed away some lipid-IL aggregates from the sensor surface (∆λ = 0.3 nm). Nevertheless, it is clear that the integrity of the liposome architecture was not completely compromised and some phospholipids or their lipid-IL aggregates still remained on the silicon nitride substrate. Our results are consistent with recent studies, indicating that the phosphonium-based IL [P14444][OAc] has a remarkable effect on phospholipid vesicles10, 12, 24 by inducing structural rearrangement of the vesicles, followed by consequent lipid removal from the vesicles.

The effect of [P14444][OAc] was also assessed on SLBs. As a control experiment, 1 mM [P14444][OAc] was added to the silicon nitride substrate surface. An increase in the peak shift was observed until reaching a saturation coverage (Figure 6B; reference curve). The final peak shift was 0.8 nm, which corresponds to IL binding to the silicon nitride surface. To check whether the interactions are weak or strong, the substrate was rinsed with water. Our results demonstrate that most of the [P14444][OAc] molecules were flushed away, which is an indication of rather weak interactions between [P14444][OAc] and the silicon nitride surface. However, as shown in Figure 6B (reference curve), some of the IL 19 ACS Paragon Plus Environment

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aggregates were still attached to the surface. This may be attributed to the negative charge of the sensor or/and the fact that the HEPES buffer employed in the preconditioning step was enriched with calcium ions. When the procedure was carried out with calcium-free HEPES buffer, [P14444][OAc] was totally flushed away from the silicon nitride sensor surface (Figure 6A reference curve). We believe that such binding is mainly promoted by the presence of calcium chloride.

The plot describing the interaction between [P14444][OAc] at a concentration of 1 mM and SLBs as a function of time is presented in Figure 6B. As can be seen, initial addition of [P14444][OAc] led to a slight increase in the maximum-extinction wavelength followed by a decrease in the LSPR signal with a peak shift of 0.5 nm. This can be attributed to the partitioning of [P14444][OAc] into the SLBs and the consequent rearrangement of the lipid bilayer. Moreover, the shape of the curve corresponding to water rinse strongly suggests that a considerable amount of phospholipids were ripped off the sensor surface. Even though part of the lipid bilayer was still attached to the substrate, it is clear that the [P14444][OAc] exerts a damaging effect on the phospholipid bilayer.

Interactions between [P4441][OAc] and POPC liposomes Subsequently, we decided to look into the effect on phospholipid vesicle integrity of another phosphonium-based IL [P4441][OAc], categorized as ‘practically harmless’.10 Nevertheless, our preliminary data obtained by other techniques suggests that [P4441][OAc] has a minor effect on phospholipids. These findings motivated us to evaluate the impact of this IL on vesicles immobilized either as intact vesicles or as SLBs on the silicon nitride sensor surface. To ensure a possible effect, [P4441][OAc] at a concentration of 50 mM, which is much higher than its EC50 value,10 was introduced into the system. The reference runs clearly indicate that there was no adsorption of [P4441][OAc] on silicon nitride surface after a water flush (Figures 7A and 7B; reference curves). Initial fast increase in 20 ACS Paragon Plus Environment

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the maximum-extinction wavelength corresponds to the differences in the RI values between IL and the surrounding water solution. In the case of intact adsorbed phospholipids, addition of 50 mM [P4441][OAc] caused an immediate increase in the NPS response signal with a peak shift of 0.4 nm, followed by a small decrease in the signal (Figure 7A). Moreover, further introduction of [P4441][OAc] led to a slight increase in the signal. Changes in the maximum-extinction wavelength are affected by changes in the RI as well as in changes in the mass of the adsorbed vesicles on the sensor surface. Therefore, the variation in the adsorbed masses can be identified by a combined effect, i.e. small instant binding of [P4441][OAc] to phospholipid vesicles (increase in the NPS signal) in combination with lipid removal from the vesicles (decrease in the NPS signal) due to the formation of novel combined ionic liquid/lipid aggregates. The small decrease in the peak shift magnitude suggests that [P4441][OAc] affected only the surface layer of the liposome membrane. Moreover, subsequent water rinse flushed out some vesicles remaining on the sensor surface in the form of mixed phospholipid-IL aggregates. However, the response achieved after [P4441][OAc] introduction to the biomimetic system is rather different from that of [P14444][OAc]; in fact, the profile of the plot reported in Figure 6A suggests that [P14444][OAc], at a concentration of 1 mM, permeates deeper into the intact phospholipid vesicles. The stronger vesicle-IL interaction will result in a greater phospholipid removal

and structural

rearrangement of the immobilized liposomes. Conversely, the plot presented in Figure 7A supports binding and a more superficial interaction of [P4441][OAc] at a concentration 50 mM with the intact vesicles. In fact, a milder effect of [P4441][OAc] on the phospholipid vesicle architecture was observed. Furthermore, after the water rinse (Figure 7A), the maximum-extinction wavelength was only negligibly lower than that observed before IL introduction, thus suggesting that most of the intact liposomes were still immobilized on the sensor surface. This is presumable related to the fact that the effect of ILs on phospholipid vesicles is dependent on the alkyl chain length, resulting in a deeper permeation of ILs with a longer alkyl chain into phospholipid membranes (Figure 1). Nevertheless, the 21 ACS Paragon Plus Environment

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obtained results suggest that [P4441][OAc] at high concentration induces interactions with intact vesicles immobilized on silicon nitride surface.

On the other hand, as shown in Figure 7B, there was only a slight effect of [P4441][OAc] on the SLB. In fact, only a small amount of phospholipids was removed from the surface. In addition, there was no observable change in the maximum-extinction wavelength after the water rinse. This seems to be related to the fact that phospholipids were already in the form of SLBs and they would not be as much influenced as the intact adsorbed vesicles. Clearly, our studies demonstrate that [P4441][OAc] at a concentration of 50 mM does not have a strong effect on the POPC liposomes.

The present study allowed us to compare the two studied models, i.e. intact adsorbed vesicles and SLBs. From our results, we can conclude that when the effect of IL on phospholipid vesicles is strong or, conversely, negligible both lipid models can be utilized, and similar results will be achieved. However, when the effect is unclear or less obvious the intact vesicles are much more suitable for such interaction studies, because the change in the maximum-extinction wavelength is easier to quantitatively understand and more complete and detailed information on possible analyte-membrane interactions will be obtained.

CONCLUSION In the present study, a NPS technique was used to investigate the effect of one amidinium- and two phosphonium-based ILs on POPC liposomes, acting as a biomimicking cell model. First, a simple and relatively fast method for phospholipid vesicle adsorption on titanium dioxide and silicon nitride sensor surfaces was developed. Our results showed that the buffer composition and the pretreatment 22 ACS Paragon Plus Environment

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conditions played an important role in the vesicle attachment process. Successful immobilization was only obtained with HEPES buffer in the presence of sodium hydroxide in the preconditioning step or with HEPES buffer containing calcium chloride. The addition of calcium chloride was found to improve the stability of the adsorption and the sensor-to-sensor reproducibility. Moreover, by changing the pretreatment conditions we were able to immobilize phospholipids as intact, adsorbed vesicles as well as disrupted into SLBs on the same sensor surface, concluding that silicon nitride sensors seem to be an excellent choice for such analyte-liposome interaction studies. The studies between IL and phospholipids showed that depending on the IL, real-time lipid removal or binding processes could be observed. [DBNH][OAc] did not have any significant effect on the phospholipid vesicles, which remained as intact vesicles or formed SLBs. The strongest and the most significant effect was observed with [P14444][OAc], which caused profound changes in the phospholipid layer architecture leading to vesicle rearrangement and consequent phospholipid removal. Interestingly, when the intact vesicles were exposed to [P4441][OAc] at a very high concentration, only a mild effect was observed. The effect of ILs on phospholipid vesicles is influenced by the alkyl chain length, i.e. IL with longer alkyl chain induces deeper permeation into phospholipids. Taken together, the findings in this work suggest that intact adsorbed vesicles are a suitable model for interaction studies between ILs and biomembranes in order to achieve more detailed information of possible analyte-membrane interactions. Furthermore, the NPS results presented here confirm our previous observations using SAXS and zeta potential determinations showing that [P14444][OAc] has strong interactions with model phospholipid membranes, but only weak interactions was observed between [P4441][OAc] and MLVs of POPC. The NPS-based methodology provided an excellent and powerful tool for elucidation of the nuanced changes in the interactions between industrially relevant ILs and biomembranes, shedding new light into membrane binding, permeation, and IL-induced phospholipid removal.


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AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] ORCID iD Joanna Witos: 0000-0003-0021-1599 Giacomo Russo: 0000-0002-2964-389X Suvi-Katriina Ruokonen: 0000-0002-1905-3554 Susanne K. Wiedmer: 0000-0002-3097-6165

Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS Financial support from Magnus Ehrnrooth Foundation and Academy of Finland (project number 266342) are gratefully acknowledged.


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FIGURE CAPTIONS

Figure 1. Structures of amidinium- and phosphonium-based ILs.


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Figure 2. Change in the maximum-extinction wavelength (∆λ) as a function of time for 0.15 mM extruded POPC lipid vesicles after four different pretreatment procedures on titanium dioxide sensor. The sensor was pretreated with: A) 0.1 M NaOH, water, HEPES buffer pH 7.4 (I=10 mM), HEPES buffer pH 7.4 (I=10 mM) containing CaCl2 (5.26 mM); B) HEPES buffer pH 7.4 (I=10 mM) containing CaCl2 (5.26 mM); C) 0.1 M NaOH, water, HEPES buffer pH 7.4 (I=10 mM); and D) HEPES buffer pH 7.4 (I=10 mM). All measurements were carried out under continuous flow.


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Figure 3. Change in the maximum-extinction wavelength (∆λ) as a function of time for 0.15 mM extruded POPC lipid vesicles after four different pretreatment procedures on silicon nitride sensor. The sensor was pretreated with: A) 0.1 M NaOH, water, HEPES buffer pH 7.4 (I=10 mM), HEPES buffer pH 7.4 (I=10 mM) containing CaCl2 (5.26 mM); B) HEPES buffer pH 7.4 (I=10 mM) containing CaCl2 (5.26 mM); C) 0.1 M NaOH, water, HEPES buffer pH 7.4 (I=10 mM); and D) HEPES buffer pH 7.4 (I=10 mM). All measurements were carried out under continuous flow.


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Figure 4. Change in the maximum-extinction wavelength (∆λ) as a function of time for 0.15 mM POPC MLVs after different pretreatment procedures. The silicon nitride sensor was pretreated with: A) HEPES buffer pH 7.4 (I=10 mM) containing CaCl2 (5.26 mM) and B) 0.1 M NaOH, water, HEPES buffer pH 7.4 (I=10 mM). All measurements were carried out under continuous flow.


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Figure 5. Influence of [DBNH][OAc] at a concentration of 250 mM on: A) intact, adsorbed vesicles and on B) the formation of SLBs immobilized on silicon nitride sensor. All measurements were carried out under continuous flow.


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Figure 6. Influence of [P14444][OAc] at a concentration of 1 mM on: A) intact, adsorbed vesicles and on B) the formation of SLBs immobilized on silicon nitride sensor. All measurements were carried out under continuous flow.


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Figure 7. Influence of [P4441][OAc] at a concentration of 50 mM on A) intact, adsorbed vesicles and on B) the formation of SLBs immobilized on silicon nitride sensor. All measurements were carried out under continuous flow.


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TABLE OF CONTENTS


Unraveling Interactions between Ionic Liquids and Phospholipid

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