Compatible solutes: Ectoine and hydroxyectoine improve functional nanostructures in artificial lung surfactants

Biochimica et Biophysica Acta 1808 (2011) 2830–2840

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Compatible solutes: Ectoine and hydroxyectoine improve functional nanostructures in artificial lung surfactants Rakesh Kumar Harishchandra a, Amit Kumar Sachan a, b, Andreas Kerth c, Georg Lentzen d, Thorsten Neuhaus d, Hans-Joachim Galla a,⁎ a

Institute of Biochemistry, Westfälische Wilhelms Universität, Wilhelm Klemm Str. 2, 48149 Münster, Germany Institute of Medical Physics and Biophysics, Westfälische Wilhelms Universität, Robert-Koch Str. 31, 48149 Münster, Germany Institute of Chemistry, Martin-Luther-University Halle–Wittenberg, von-Danckelmann-Platz 4, 06120 Halle (Saale), Germany d Bitop AG, Stockumer Strasse, 58453 Witten, Germany b c

a r t i c l e

i n f o

Article history: Received 17 February 2011 Received in revised form 5 August 2011 Accepted 16 August 2011 Available online 22 August 2011 Keywords: Langmuir film balance Domain structures Surfactant protein C Height profile Vesicle insertion Infrared Reflection Absorption Spectroscopy

a b s t r a c t Ectoine and hydroxyectoine belong to the family of compatible solutes and are among the most abundant osmolytes in nature. These compatible solutes protect biomolecules from extreme conditions and maintain their native function. In the present study, we have investigated the effect of ectoine and hydroxyectoine on the domain structures of artificial lung surfactant films consisting of dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG) and the lung surfactant specific surfactant protein C (SP-C) in a molar ratio of 80:20:0.4. The pressure–area isotherms are found to be almost unchanged by both compatible solutes. The topology of the fluid domains shown by scanning force microscopy, which is thought to be responsible for the biophysical behavior under compression, however, is modified giving rise to the assumption that ectoine and hydroxyectoine are favorable for a proper lung surfactant function. This is further evidenced by the analysis of the insertion kinetics of lipid vesicles into the lipid–peptide monolayer, which is clearly enhanced in the presence of both compatible solutes. Thus, we could show that ectoine and hydroxyectoine enhance the function of lung surfactant in a simple model system, which might provide an additional rationale to inhalative therapy. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Ectoine and hydroxyectoine are small, low molecular weight organic molecules, which are zwitterionic and have strong water binding properties. They are synthesized by various aerobic chemoheterotrophic and halophilic/halotolerant bacteria [1–4]. Within the bacterial cells they are enriched either by synthesis or by accumulation from the surrounding during adverse environmental conditions such as extreme temperature, excess dryness and high salinity [5]. These solutes are biologically inert and do not interfere with the overall cellular functions even if they are present at high concentrations within the cytoplasm, hence these compounds are called ‘compatible solutes’ [3]. In addition to the function as osmoprotectants, ectoine and hydroxyectoine are distinguished as effective stabilizers of biomolecules such as proteins [5,6], nucleic acids [7] and biomembranes [8,9]. There are several theories proposed to explain the protective function of the compatible solutes which

Abbreviations: DPPC, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; DPPG, 1,2dipalmitoyl-sn-glycero-3-phosphoglycerol; AFM, atomic force microscopy; LB, Langmuir–Blodgett; LC, liquid condensed; LE, liquid expanded; LUV, large unilamellar vesicle; IRRAS, Infrared Reflection Absorption Spectroscopy ⁎ Corresponding author. Tel.: + 49 251 8333200; fax: + 49 251 8333206. E-mail address: [email protected] (H.-J. Galla). 0005-2736/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bbamem.2011.08.022

includes water replacement hypothesis [10], transfer free energy, excluded volume, contact interaction [11–13] as well as preferential exclusion [14,15]. Among these the “preferential exclusion model” has gained considerable interest, which states that the solutes are excluded from the immediate hydration shell for example of a protein. Consequently, a preferential hydration of the protein surface occurs, thereby enhancing the stability of the native conformation and thus making denaturation thermodynamically less favorable. Although, in recent times studies have shown the possible direct interactions of osmolytes with the protein backbone as a stabilizing effect [15–17]. Studies have been carried out exploring the protective ability of ectoine on whole cells as well as on biomolecules. However, among the biomolecules protein–solute interaction has been extensively studied and lately compatible solutes are finding interest in the field of biomembranes. Graf et al. [8] using red blood cells as the model cell system showed that the cells were protected against lysis by the SDS detergent in the presence of ectoine. Further, we have recently shown the fluidizing effect of ectoine and hydroxyectoine on lipid monolayers as well as bilayer systems [9]. Our results showed that ectoines essentially act on the line tension occurring at the phase boundaries and thus fluidize the monolayer. Hence ectoines were regarded as line tension active species. Compatible solutes have been shown to prevent protein misfolding and aggregate formation,

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which are key pathological features for neurodegenerative diseases [18–20]. Recently, ectoine was reported to inhibit nanoparticleinduced signaling responsible for pro-inflammatory reaction in lung epithelial cells in vitro and in vivo [21]. Ectoine-based inhalation solutions are under clinical trials and are tested for the treatment and prophylaxis of asthma in humans. Since the ectoines will be used to treat asthma it would be worthwhile to understand their effects on the properties of lung surfactant materials, which is the first layer that ectoines would encounter in the alveoli. The lung surfactant is a lipid–peptide monolayer responsible for lowering the surface tension at the interface thus resulting in effortless breathing [22]. The lung surfactants are considered as highly dynamic in nature since they are under repeated compression and expansion cycles. During this process a continuous exchange of materials between the monolayer and the pulmonary hypophase takes place [23–25]. Hence to study the effects of compatible solute, we used a model lung surfactant system consisting of zwitterionic dipalmitoylphosphatidylcholine (DPPC) and negatively charged dipalmitoylphosphatidylglycerol (DPPG) along with SP-C in a molar ratio of 80:20:0.4, which is very well characterized and found to mimic the aspects of the natural lung surfactant system [26–28]. Here, we present new results on the influence of compatible solutes on both static and dynamic processes of lung surfactant system using a biophysical approach. 2. Materials and methods 2.1. Chemicals The lipids, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG) were purchased from Avanti Polar Lipids Inc. (Alabaster, AL). 2-(4,4-Difluoro-5methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (β-BODIPY® 500/510 C12-HPC, BODIPY-PC) was obtained from Molecular Probes (Eugene, OR). Ectoine ((S)-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid) and hydroxyectoine ((4S,5S)-2-methyl-5-hydroxy-1,4,5,6tetrahydropyrimidine-4-carboxylic acid) were obtained from bitop AG (Witten, Germany). All lipids were used without further purification. Chloroform and methanol were high pressure liquid chromatography grade and purchased from Sigma-Aldrich (Steinheim, Germany) and Merck (Darmstadt, Germany), respectively. Water was purified and deionized by a multicartridge system (MilliPore, Billerica, MA) and had a resistivity of N18 MΩm. The buffer solution used to hydrate lipid films consisted of N-2-(hydroxyethyl)piperazine-N′-2ethansulfonic acid (HEPES) from Merck (Darmstadt, Germany) supplemented with sodium salt of ethylenediaminetetraacetic acid (Na-EDTA) from Sigma-Aldrich. Lipids were dissolved in chloroform/ methanol solution (1:1, v/v). The preparation of vesicles was carried out with a LiposoFast-miniextruder from Avestine Europe GmbH, (Mannheim, Germany). The porcine surfactant proteins SP-C was purified from bronchoalveolar lavage fluid by butanol extraction [29]. The protein was free of contaminants as was evidenced by electrospray ionization mass spectrometry.

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2.3. Fluorescence light microscopy Domain structures of lipid samples doped with 0.5 mol% BODIPY-PC were visualized by means of video-enhanced epi-fluorescence microscope (Olympus STM5-MJS, Olympus, Hamburg, Germany) equipped with a xyz-stage and connected to a CCD camera (Hamamatsu, Herrsching, Germany). The images were captured at desired surface pressures by stopping the barrier. All the measurements were performed on a subphase containing pure water or water containing ectoine or hydroxyectoine at 20 °C. 2.4. Langmuir–Blodgett (LB) transfer For atomic force microscopy investigations, Langmuir–Blodgett (LB) films were prepared by spreading the lipid–protein mixture on a film balance (Riegler & Kirstein, Mainz, Germany) with an operational area of 39 cm 2. All transfers were performed on pure water or water containing ectoine or hydroxyectoine at 20 °C by controlling the surface pressure. Prior to spreading, a freshly cleaved mica sheet (Electron Microscopy Science, Munich, Germany) was dipped into the subphase. After an equilibration period of 10–15 min the monolayer was compressed with a velocity of 1.5 cm 2/min until the surface pressure of 52.5 mNm − 1, just below the plateau region, was reached to avoid large shift in the area per molecule prior to film transfer. The monolayer was equilibrated at the specified surface pressure for another 20 min, during which there will be a slight shift of area per lipid molecule (corresponding to an area of 38–40 Å 2), and then transferred onto mica with a velocity of 0.7 mm/min. 2.5. Atomic force microscopy (AFM) AFM images of the LB films transferred onto mica sheets were obtained at ambient conditions (20 °C) using a NanoWizard II AFM (JPK Instruments, Berlin, Germany) operating in intermittent contact mode in air. PointProbe Plus Non-Contact/Tapping Mode-High Resonance Frequency-Reflex Coating (PPP-NCHR) tips (Nanosensors™, Neuchatel, Switzerland) with a spring constant of 42 Nm− 1 and a resonance frequency of 330 kHz was used. The images were obtained at 512 × 512 pixel resolution and the scan rate was 0.5–0.8 Hz. The image processing and a detailed section height analysis of the images were performed using JPK image processing software. 2.6. Vesicle preparation DPPC/DPPG (4:1, molar ratio) was dissolved in chloroform/methanol (1:1, v/v) and dried under a stream of nitrogen at 50 °C. Traces of solvent were removed by keeping under vacuum at 50 °C overnight. The lipid films were hydrated by adding a pre-warmed buffer containing 25 mM HEPES and 0.1 mM EDTA, pH 7.0. The vesicle suspension (5 mM) was kept for 30 min at 50 °C in a water bath and was vortexed for 30 s at regular intervals to get multilamellar vesicles (MLVs). The resulting MLVs were converted into large unilamellar vesicles (LUVs) at 50 °C by membrane extrusion using the LiposoFast extruder containing a polycarbonate membrane with a pore diameter of 100 nm.

2.2. Surface pressure–area isotherms

2.7. Vesicle insertion studies

Film balance experiments were performed on an analytical Wilhelmy film balance (Riegler and Kirstein, Mainz, Germany) with an operational area of 144 cm 2. Surface pressure–area measurements were performed on pure water or water containing ectoine or hydroxyectoine at 20 °C. The monolayer spread onto the subphase was composed of DPPC/DPPG/SP-C (0.4 mol%) prepared in a chloroform/ methanol solution (1:1, v/v). After an equilibration time of 10– 15 min for the solvent to evaporate, the monolayer was compressed at a rate of 3 cm 2/min.

The vesicle insertion experiments were performed using a lipid– peptide monolayer composed of DPPC/DPPG/SP-C (0.4 mol%). The lipid–peptide monolayer was formed at the air/water interface of a Wilhelmy-film balance (Riegler and Kirstein, Mainz, Germany), with an operational area of 39 cm 2, by spreading the respective samples from chloroform/methanol (1:1 v/v) onto the aqueous subphase. The subphase consisted of 25 mM HEPES and 3 mM CaCl2 (pH 7.0) and was stirred continuously by a magnetic bar. The monolayer was compressed with a computer-controlled barrier to a defined surface

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pressure and maintained for 10 min. The vesicle suspensions were then injected through an injection port into the subphase with syringe. The final lipid concentration in the subphase was 20 μmol/L. The lipid vesicle insertion was studied at 20 °C, by monitoring the change in surface pressure with respect to time, at constant surface area. The studies were carried out over a period of 3600 s. The insertion velocity was quantified by determining the slope of the pressure–area isotherm. 2.8. Infrared Reflection Absorption Spectroscopy (IRRAS) The experimental setup has been described before in detail [30]. Briefly, all experiments were performed with a Wilhelmy film balance (Riegler and Kirstein, Berlin, Germany) consisting of two Teflon troughs of different size, which are positioned on a movable platform to be able to shuttle between sample and reference trough. Infrared spectra were recorded with an Equinox 55 FT-IR spectrometer (Bruker, Karlsruhe, Germany) connected to an XA 511 reflection attachment (Bruker) with an external narrow band MCT detector. The angle of the incident infrared beam (s-polarized) was 40° with respect to the normal of the water surface. The spectral resolution was 4 cm − 1 using a zero filling factor of 2. For each spectrum 1000 scans were co-added over a total acquisition time of 4.5 min. The reflection absorption spectrum (−log(R / R0)) was calculated by ratioing the reflectance spectra of the monolayer in the sample trough to the single-beam reflectance spectrum of the reference trough surface as background. Five individual Infrared Reflection Absorption (IRRA) spectra were usually averaged in order to increase the signal to noise ratio. The temperature of the subphase was maintained at 20 ± 0.5 °C. 3. Results We have investigated the effect of ectoine as well as of hydroxyectoine on the phase behavior of artificial lung surfactant monolayers composed of two lipid components, dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) as well as the lung surfactant specific surfactant protein C (SP-C). Lipid–peptide monolayers have been investigated by using the Langmuir film balance technique to monitor the surface pressure–area isotherms as well as by the epi-fluorescence microscopy to study the distribution of fluid and rigid domains. Further, atomic force microscopy was used to inspect the topology of films transferred onto the solid support. The insertion of lipid vesicles made of DPPC and DPPG into the preformed lipid–peptide monolayer was performed in order to investigate the influence of compatible solutes on this dynamic process of the lung surfactant system. Finally, IRRAS experiments were

3.1. Phase behavior monitored by the film balance technique and epi-fluorescence microscopy Typical isotherms of DPPC/DPPG/SP-C films on an aqueous subphase containing ectoine or hydroxyectoine are shown in Fig. 1. The isotherm of this artificial surfactant layer on pure water has a typical shape as was reported earlier [27,31]. Briefly, in the absence of the protein, SP-C, the surface pressure increases to values up to 70 mNm − 1 where an irreversible break down of the monolayer occurs. For an SP-C containing monolayer the surface pressure increases under compression up to a value of around 55 mNm − 1 and then remains constant upon further compression which has been explained by the formation of three dimensional bilayer stacks underneath the surface monolayer. The formation of these protrusions allows the compression of the monolayer without increasing the surface pressure. Interestingly, ectoine (Fig. 1a) or hydroxyectoine (Fig. 1b) up to a concentration of 100 mM in the aqueous phase does not change the overall behavior, although, a slight influence is observed at higher surface pressures. This is in contrast to the effect of ectoine and hydroxyectoine on pure DPPC or a mixture of DPPC and DPPG monolayers where a strong fluidization and expansion of the monolayers has been reported [9]. Furthermore, when closely analyzing the surfactant monolayer compressed to high surface pressure the plateau region remains unaffected, except at 100 mM hydroxyectoine, where the plateau is not stable and becomes shortened. The epi-fluorescence microscopy was performed using BODIPY-PC, a fluorescent dye that is preferentially soluble in the fluid phase of a lipid monolayer and the images are presented in Fig. 2. Dark domains represent a liquid condensed (LC, rigid) phase; bright regions represent the liquid expanded (LE, fluid) phase of a monolayer. It is clearly visible that the distribution and size of the domains are strongly influenced by ectoine in contrast to hydroxyectoine. The influence of ectoine and hydroxyectoine on the domains can be best visualized at low surface pressure (5 mNm − 1). The analysis showed that the number of dark rigid domains increases with increasing concentration of compatible solutes especially in the presence of ectoine. In parallel, the relative area fraction covered by fluid domains also increases in presence of both compatible solutes (for data see Supporting information). These fluid domains have been identified earlier as a region for the formation of staggered bilayer structures during compression to the plateau pressure at around 55 mNm − 1. Interestingly, the monolayer on hydroxyectoine shows only a slight decrease in domain size and the size remains almost constant over the different concentrations. 70

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Fig. 2. Video-enhanced epi-fluorescence microscopic images of DPPC/DPPG/SP-C (0.4 mol%) monolayers on subphases containing 1 mM, 10 mM and 100 mM of ectoine and hydroxyectoine. The measurements were performed at 20 °C. Images taken at different surface pressures are shown. All samples were doped with 0.5 mol% of fluorescent dye, BODIPY-PC. The scale bar is 50 μm.

3.2. Atomic force microscopic investigation of the film topology

(Fig. 4b and e) and 100 mM ectoine (Fig. 4c and f) are 31.8 (six bilayer steps) and 30 nm (five bilayer steps), respectively, suggesting that the protrusion formation efficiency on an average is conserved throughout the different ectoine concentrations. In the presence of 1 mM ectoine, fluidized regions exhibit a spot like dispersed pattern (Fig. 4a), whereas in the presence of 10 mM and especially 100 mM ectoine the network structure is more clearly pronounced and the fluid regions are well organized around the condensed rigid domains. Histogram analysis of images taken on 100 mM ectoine shows a roughly 2 fold (n = 3) increase in number of events for bilayered (6 nm) and tetralayered heights (12 nm) as compared to images taken on 1 mM and 10 mM ectoine containing subphases. The topography image and corresponding histogram analysis of surfactant monolayers formed on different concentrations of hydoxyectoine are presented in Fig. 5. The statistical data of the surfactant film transferred from 1 mM, 10 mM and 100 mM hydroxyectoine (Fig. 5a–f) reveals maximum visible step heights of 40.6 nm (7 bilayers), 40.2 nm (7 bilayers) and 17.3 nm (3 bilayers), respectively.

Atomic force microscopy (AFM) is a powerful tool to study the topography of the biomaterials at high lateral resolution and the technique was used in this study to investigate the influence of compatible solutes on the SP-C facilitated protrusion formation efficiency. DPPC/DPPG/SP-C films compressed on subphases containing water or compatible solutes were transferred on to mica at a surface pressure near the plateau region and scanned under intermittent contact mode. The AFM height channel image and the corresponding histogram of DPPC/DPPG/SP-C film on pure water are presented in Fig. 3a and b, respectively. Bright structures are the protrusions formed in areas of fluidized lipid as demonstrated in the fluorescence microscopic pictures. The protrusion regions of SP-C containing lipid film on pure water attain to a maximum visible stack height of 22 nm corresponding to four bilayers. The surfactant monolayer on 1 mM ectoine (Fig. 4a and d) shows similar step heights of four reaching up to 21.7 nm. Similarly, the protrusion heights formed at 10 mM 20

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Fig. 4. AFM topography images of DPPC/DPPG/SP-C (0.4 mol%) monolayer on subphases containing a) 1 mM, b) 10 mM, and c) 100 mM of ectoine. d), e) and f) show the respective statistical histogram analyses indicating the number of bilayer stacks. LB films were transferred on to mica at a surface pressure close to the plateau region at 20 °C. The AFM images were scanned in intermittent contact mode.

The reduction in the frequency of more highly stacked layers in 100 mM hydroxyectoine is expected since the plateau region was not clearly formed as observed in the film balance studies. Furthermore, examining the surface pattern of the transferred film from the subphases containing 1 mM and 10 mM hydroxyectoine shows that the squeeze out of surfactant material and the formation of network

structures are enhanced with respect to the transferred film from water subphase. The network structures are clearly formed at all the hydroxyectoine concentrations. Interestingly, the AFM image of surfactant film on100 mM hydroxyectoine shows the uniformly distributed laterally stretched patches of the protrusion domains with a height close to a single bilayer.

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Fig. 5. AFM topography images of DPPC/DPPG/SP-C (0.4 mol%) monolayer on subphases containing a) 1 mM, b) 10 mM, and c) 100 mM of hydroxyectoine. d), e) and f) show the respective statistical histogram analyses indicating the number of bilayer stacks. LB films were transferred on to mica at a surface pressure close to the plateau region at 20 °C. The AFM images were scanned in intermittent contact mode.

3.3. Vesicle insertion in SP-C containing monolayers Surfactant specific proteins SP-B and SP-C have been found to play an important role in the dynamics of lung surfactant [32]. They promote absorption of lipids from membrane suspensions present in the hypophase to pure or monolayer covered air/water interfaces [33–35]. Moreover, they induce controlled squeeze-out of surface

material during monolayer compression [36,37]. To determine the influence of compatible solutes on surfactant material insertion, we first studied the pressure–time isotherms of vesicle (DPPC/DPPG, 4:1) insertion into DPPC/DPPG/SP-C monolayers at 20 °C. Fig. 6 shows the insertion process in the absence of compatible solutes. On vesicle insertion, the surface pressure increases with time and reaches a final equilibrium pressure of ~ 50–52 mNm − 1. The insertion

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Fig. 6. Pressure–time isotherms of DPPC/DPPG/SP-C (0.4 mol%) monolayers compressed to different initial start pressures and injecting DPPC/DPPG (4:1), vesicles into the subphase containing 25 mM HEPES, 3 mM CaCl2 (pH 7) at 20 °C. a) Pressure–time isotherms and b) insertion velocities obtained at each pressure. The standard deviation was calculated from three measurements at each start pressure (n = 3).

velocity depends strongly on the start pressure fixed before vesicle insertion with a maximum incorporation seen around 20–30 mNm − 1. The insertion process observed for DPPC/DPPG/SP-C monolayers on 10 mM ectoine and hydroxyectoine is presented in Fig. 7. The vesicle insertion process was found to be significantly enhanced on both ectoine (Fig. 7a and c) and hydroxyectoine (Fig. 7b and d) containing subphases at a concentration of 10 mM. The pressure–time isotherms at 10 mNm − 1 and 15 mNm − 1 show a sigmoidal curve (biphasic) with an initial lag phase for attaining the critical surface pressure (~25 mNm − 1) followed by a second phase leading in a 2-fold increase up to an equilibrium pressure of 50–52 mNm − 1. The influence of higher concentration (100 mM) of compatible solutes on the vesicle insertion process and the corresponding pressure–time isotherms are presented in Fig. 8. The isotherms on 100 mM ectoine (Fig. 8a and c) show similar insertion phenomena to that on 10 mM ectoine. At the start pressures of 10 mNm − 1 and 15 mNm − 1, a biphasic mode of vesicle insertion is observed. However, at initial start pressures above 25 mNm − 1, only monophasic insertion processes were seen. Interestingly, the insertion process on 100 mM hydroxyectoine (Fig. 8b and d) was considerably slowed down. The insertion velocities of vesicle insertion on 100 mM hydroxyectoine were comparable to those obtained on subphases without compatible solutes. 3.4. IRRAS to investigate the interaction of compatible solutes with lipid head groups IRRAS provides unique information about the structural conformation and orientation of lipid chains in situ in monolayers at the air/ water interface [38]. Here, IRRAS was used to study the interaction of compatible solutes with the DPPC monolayer at the air/water interface mainly concentrating on the head group regions, that is the carbonyl group ν(C_O) and the antisymmetric phosphate νas(PO2−) stretching vibrational bands of DPPC. Both groups are sensitive to hydration and interaction with solute molecules [39,40]. IRRA spectra of DPPC films compressed on subphases containing water or compatible solutes were collected at two different surface pressures (5 mNm − 1 and 40 mNm − 1). The IRRA spectra in the region of ν(C_O) and νas(PO2−) vibrations are presented in Fig. 9. The C_O stretching vibrational band is in the region between 1750 cm − 1 and 1700 cm − 1, while the antisymmetric PO2− stretching vibrational band is in the region between 1245 cm − 1 and 1222 cm − 1 [39]. At 5 mNm − 1, the ν(C_O) band in the DPPC

monolayer on water is centered at 1733 cm − 1. The presence of 100 mM ectoine and 100 mM hydroxyectoine slightly shifts the center of the ν(C_O) band to a higher frequency around 1735 cm − 1 and 1737 cm − 1, respectively (Fig. 9a). A shift of the band center towards a higher wavenumber indicates dehydration, while a shift towards lower wavenumbers indicates hydration. In contrast, no shift of the ν(C_O) band was observed at higher surface pressure of 40 mNm − 1 in the presence of compatible solutes (Fig. 9b). The νas(PO2−) stretching vibrational band of DPPC monolayer on water is centered around 1233 cm− 1 at 5 mNm − 1. In the presence of ectoine the band positions remained unchanged while hydroxyectoine shifts the center of the νas(PO2−) band towards a lower wavenumber of 1228 cm− 1 (Fig. 9c). Interestingly, at 40 mNm − 1 a decrease of the wavenumber was observed for both ectoine (1239 cm− 1) and hydroxyectoine (1231 cm− 1) compared to lipid spread on water (1242 cm− 1). Although, the shift of the wavenumber was smaller for ectoine (3 cm− 1), but in the presence of hydroxyectoine the shift was larger, 11 cm− 1 (Fig. 9d). 4. Discussion The compatible solutes, ectoine and hydroxyectoine, are produced and stored by various microorganisms to protect themselves against the stressful conditions such as salinity, extreme temperatures, and desiccation [1,41]. These solutes protect the cells mainly by stabilizing the biomolecules. In the past, the protective action of compatible solutes has been extensively studied on protein molecules and the underlying mechanism is explained by ‘preferential exclusion model’ [14]. Thus, the protective abilities of ectoine and hydroxyectoine have been explored in the fields from protein chemistry to dermatology and respiratory diseases. In a recent study, Sydlik et al. [21] reported the protection of pulmonary epithelial cells against the nanoparticle-induced neutrophilic inflammation and suggested the potential use of ectoine in treatments of lung inflammation. In the present study, we aimed to investigate the influence of ectoine and hydroxyectoine on the model lung surfactant system. Therefore, a simplified model system of lung surfactant consisting of DPPC/DPPG/SP-C monolayers, which is found to closely mimic the natural lung surfactant, is used [26,28]. First, the influence of ectoine and hydroxyectoine on the phase behavior of surfactant monolayers was investigated at the air–water interface using Langmuir film balance and video-enhanced epi-fluorescence microscopy. Both ectoine and hydroxyectoine showed no pronounced effect on the isotherms

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of DPPC/DPPG/SP-C monolayer. Moreover, the typical plateau region formed at ~ 55 mNm − 1 that corresponds to the formation of multilamellar structures [31] is maintained, except at 100 mM hydroxyectoine where the plateau is short and not stable suggesting the squeeze out process to be affected. To further elucidate the influence of compatible solutes, we examined their effect on the formation of domain structures using epi-fluorescence microscopy. It has been reported that the line tension, which is the interfacial energy present at the domain edges, is an important parameter in determining the membrane lateral organization as well as the distribution of domain sizes [42]. The fluidizing effect of ectoine and hydroxyectoine on lipid monolayers has been shown previously and they were reported to be a line tension active species [9]. It is clearly visible from the fluorescence images that ectoine shows prominent effects on the distribution as well as on the size of domains with increasing concentration as compared to hydroxyectoine. Intriguingly, the effect of 100 mM hydroxyectoine on DPPC/DPPG/SP-C monolayers is not much influenced and the domain size remains almost same over the different surface pressures. The increase in the fluidity of the surfactant monolayer results in the higher lateral mobility of the lipid molecules, thereby the effect is observed as an increase in number of LC domains and decrease in their domain size. Further, we elucidated the influence of compatible solutes on the formation of surface-confined reservoirs induced by SP-C during

compression to higher surface pressures. Atomic force microscopy (AFM) has been extensively used to study the topography of lung surfactant system and contributed substantial information [26,28]. The AFM studies showed that ectoine and hydroxyectoine significantly enhance the number of bilayer step formation in protrusion regions. In contrast to ectoine, hydroxyectoine is more effective at low concentrations of 1 mM and 10 mM. The enhanced squeeze out of the surfactant materials can be related to the increase in fluidity of the lipid–peptide monolayer induced by compatible solutes. Similar observations were reported for cholesterol and unsaturated phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) containing surfactant monolayers where increase in the fluidity, essentially due to POPE, was shown to promote the protrusion formation [43]. Diemel et al. also showed the fluidity of surfactant monolayer containing the unsaturated phospholipids resulted in enhanced protrusion formation [44]. Therefore, the ability of compatible solutes to increase the fluidity, which in turn results in enhanced multilayer structure formation, is appropriate for the stabilization of lung surfactant during breathing cycle. Interestingly, at 100 mM concentration of hydroxyectoine comparatively large amount of mainly stretched single-bilayer structures were observed, which is expected as the plateau region was not stable as revealed by the film balance studies. This can be speculated due to the possible direct interaction of hydroxyectoine with lipid head group at high concentrations,

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which might result in extended bilayer structures and thereby prevent the curvature necessary to form protrusions. Further the influence of compatible solutes on the vesicle adsorption and insertion processes of lung surfactant monolayers was investigated. The process of vesicle insertion into the surfactant monolayer has been extensively studied and the underlying mechanism is shown to occur in two steps: transport of vesicles to the lipid head groups and insertion into the interfacial monolayer at the air–water interface in the presence of calcium [45–47]. The calcium mainly bridges the vesicles to the lipid head group of the monolayer. This is followed by the insertion process at the disordered (fluid) regions [47]. When the vesicle insertion kinetics were performed on the subphase containing low concentration (10 mM) of compatible solutes the insertion rate was significantly enhanced compared to the solute free subphase. This enhanced insertion can be attributed to the fluidity of the monolayers resulting in the increased disordered regions, which is essential for insertion process. The initial vesicle insertion is regarded to be protein-independent and after reaching a critical surface pressure the insertion is facilitated by the surfactant proteins [48]. Hence, a biphasic insertion process has been observed at lower initial surface pressures. Increasing the concentration of ectoine to 100 mM did not show any further rise in insertion rate and the insertion process was comparable to that of 10 mM. Interestingly, at 100 mM hydroxyectoine the vesicle insertion velocity decreased and was similar to the compatible solute free subphase. The

reduction in the vesicle insertion at higher concentrations can be associated to the possibility of strong direct interaction of compatible solutes with the lipid head groups. This aspect is supported by the IRRAS of compatible solutes–DPPC interaction at the air/water interface. It is known that both, carbonyl and antisymmetric PO2− stretching vibrational bands, are extremely sensitive to environmental factors [39]. The IRRA spectra of the C_O stretching vibrational band did not show much influence of compatible solutes on DPPC, especially at higher surface pressures. This might be due to the inaccessibility of the carbonyl group at higher pressure for direct interaction. The νas(PO2−) vibrational spectrum is very interesting and provides valuable information about ectoines–lipid headgroup interaction. At higher surface pressure there was a large decrease in the wavenumber of the νas(PO2−) vibrational band in the presence of hydroxyectoine. The decrease in the wavenumber is related to the formation of hydrogen bonds with the oxygen atom of PO2− which would lead to the weakening of the vibrational force constants. Thus the difference in the effect between the ectoine and hydroxyectoine can be attributed to the presence of an additional OH group in hydroxyectoine resulting in an additional capability to strongly interact and form hydrogen bonds with the phosphate group of DPPC molecules. A similar observation has been reported by Ciesik et al. [49,50] who studied the interaction of 3-pentadecylphenol (PDP) with both dry and hydrated DPPC systems using ATR-IR spectroscopy. They found that the OH group of PDP strongly interacts with the phosphate group

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in dry DPPC bilayers by forming the hydrogen bonds with PO2−. Interestingly, under the same conditions PDP did not form hydrogen bonds with the ester carbonyl group and the ν(C_O) band was shifted to higher wavenumbers [49,50]. These studies further support our results and the assumption of a direct interaction of the OH group in hydroxyectoine with the phosphate groups of lipids. Consequently, the possible direct interaction of hydroxyectoine with lipid head groups at higher concentration would prevent the efficient adsorption and insertion of vesicle into the monolayer. Same explanation holds good for the protrusion formation at higher concentrations of compatible solutes. These observations can be compared to the AFM studies where 100 mM ectoine did not show any difference in the number of step heights in comparison to 10 mM, while at higher concentration of hydroxyectoine a significant decrease in the number of bilayer step heights was seen. Therefore, the above observations give an indication that the exclusion model holds good when low concentrations of compatible solutes are used for lipid monolayers while at higher concentrations, in addition to the exclusion process the compatible solutes might have a direct interaction with lipid head groups, thereby leading to a decrease in the protrusion formation as well as vesicle insertion kinetics.

In conclusion, ectoine and hydroxyectoine show that they certainly favor the biophysical aspects of lung surfactant in a simple model system. Also, the results provide a motivation for further studies involving natural lung surfactant system. This study gives us a prospect that both these compatible solutes can be a promising candidate for use in the treatment of lung disorders and could increase the efficiency of lung surfactant functioning. Importantly, the concentration of compatible solutes must be considered before the application on respiratory systems. In addition to the present study, we have previously reported that even the concentration as low as 1 mM has a considerable influence on lipid monolayers [9] and similar observation using 1 mM ectoine has been shown to prevent the nanoparticle-induced lung inflammations [21]. Supplementary materials related to this article can be found online at 10.1016/j.bbamem.2011.08.022. Acknowledgements Dr. R. K. Harishchandra was a PhD student and has been supported with a stipend by our International Graduate School in Chemistry at the University of Münster, Germany. We would like to thank Prof.

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