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Aescin Incorporation and Nanodomain Formation in DMPC Model Membranes Ramsia Sreij, Carina Dargel, Lara Hernandez Moleiro, Francisco Monroy, and Thomas Hellweg Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b02933 • Publication Date (Web): 06 Oct 2017 Downloaded from http://pubs.acs.org on October 11, 2017

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Aescin Incorporation and Nanodomain Formation in DMPC Model Membranes Ramsia Sreij,†,‡ Carina Dargel,†,‡ Lara H. Moleiro,† Francisco Monroy,¶,§ and Thomas Hellweg∗,† †Physical and Biophysical Chemistry, Department of Chemistry, Bielefeld University, Universitässtraße 25, Bielefeld, Germany ‡Contributed equally to this work ¶Department of Physical Chemistry I, Complutense University, Avda. Complutense s/n, 28040, Madrid, Spain §Unit of Translational Biophysics, Institute of Biomedical Research Hospital Doce de Octubre (imas12), Av. Andaluc´ıa s/n, 28041 Madrid, Spain E-mail: [email protected] Phone: +49(0)521 1062055. Fax: +49(0)521 1062981

Abstract The saponin aescin from the horse chestnut tree is a natural surfactant well known to self-assemble as oriented-aggregates at fluid interfaces. Using model membranes in the form of lipid vesicles and Langmuir monolayers, we study the mixing properties of aescin with the phase-segregating phospholipid 1,2-dimyristoyl-sn-glycerophosphocholine (DMPC). The binary membranes are experimentally studied on different length scales ranging from the lipid headgroup area to the macroscopic scale using small-angle X-ray scattering (SAXS), photon correlation spectroscopy (PCS) and differential scanning calorimetry (DSC) with binary bilayer vesicles and Langmuir

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tensiometry (LT) with lipid monolayers spread on the surface of aescin solutions. The binary interaction was found to strongly depend on aescin concentration in two well differentiated concentration regimes. Below 7 mol%, the results reveal phase segregation of nanometer-sized aescin-rich domains in an aescin-poor continuous bilayer. Above this concentration, aescin-aescin interactions dominate which inhibit vesicle formation but lead to the formation of new membrane aggregates of smaller sizes. From LT studies in monolayers, the interaction of aescin with DMPC was shown to be stronger in the condensed phase than in liquid expanded phase. Furthermore, a destructuring role was revealed for aescin on phospholipid membranes, similarly to the fluidizing effect of cholesterol and non-steroidal anti-inflammatory drugs (NSAIDs) on lipid bilayers.

Introduction The anti-oedemigenous and vasoprotective acitivities of the extract of the horse chestnut tree aesculus hippocastanum are completely ascribed to the saponin aescin. 1–3 Saponins are plant derived amphiphilic glycosides with an either steroidic or triterpenic aglyconic part. To this hydrophobic part a different number, mostly one or two, sugar chains are attached. 4–6 The amphiphilic saponin structure entails different chemical functionalities like a high surface activity, and in many cases cytotoxic and haemolytic properties. 7 In plants saponins function primarily as defense molecules against fungals or insects. 5 Since saponins are present in foods like legumes, peanuts, pepper or garlic, they are introduced naturally by ingestion into the human body. The many effects of saponins are well summarized in many review articles as well as in a large number of pharmacology works. 8–11 Amongst others the cholesterol-protecting effect of saponins in human health was proven recently by Vinarova et al. 12 Structural investigations of the interaction of different other saponins like α-hederin or Quil A with lipid bilayers have been recently addressed by other groups, for instance, saponin penetration into the bilayer, 13 phase separation into saponin-poor and saponin-rich regions 5 and strong interaction with the phospholipid headgroups. 14 Most of 2 ACS Paragon Plus Environment

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the studies were done on giant unilamellar vesicles (GUVs), and some of them confirmed with thermodynamical data from Langmuir monolayers.

Aescin is one of few existing saponins that can be isolated as a mixture of structurally similar substances from the naturally occuring saponin mixtures. It exists in a water soluble α-form and can be easily crystallized in the β-form. 1 Only the latter one is commercially available. β-aescin itself is a mixture of mainly six compounds, differing in the residues at C-22 (R2 , acetic or protonic) and C-21 (R1 , angelic or tiglic acid) of the scaffold as well as in a residue of one glucose molecule (R3 , see Figure 1). 1,3,15–17 Golemanov et al. assigned the aescin purchased from Sigma (CAS 6805-41-0), which will be the object of our study, even as a single pure chemical compound. 4 Similar to other saponins aescin is used against different deseases such as chronic venous insuffency (CVI), haemorrhoids and peripheral oedema formation due to its diverse biological effects. 1,15,18 Nevertheless, negative side effects such as a haemolytic activity leading to membrane perturbations with the potential to induce membrane permeabilization are also observed. 19 These effects were investigated earlier in vivo by different groups using cell cultures. 20,21 Hence, the existence of the biological effects exhibited by aescin is well established. Nevertheless, the understanding of the physical chemistry of the interaction between saponin and cell membrane components is still poor on the molecular level. Small unilamellar phospholipid vesicles (SUVs) with a diameter of 80 nm consisting of the phospholipid 1,2-dimyristoyl-sn-glycerophosphocholine (DMPC) can be used to mimic cell membranes. DMPC is one of the most abundant lipids in mammalian membranes 22 and vesicles are easy to prepare. Hence, DMPC vesicles are optimal to investigate the effects of different agents on biomimetic membranes. Molecules interacting with a lipid membrane have the potential to stongly influence an important feature of lipid bilayers, namely to impact the temperature driven main phase transition from a well-ordered gel phase Lβ 0 to a flexible liquid crystalline phase Lα with


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Figure 1: Structure of substances belonging to the class of β-aescin. Modified from Wilkinson. 16 increasing temperature. Such an influence was ascertained for example for cholesterol or ibuprofen, which insert into the membrane and distort the arrangement of the lipid molecules in the bilayer. 2,23,24 Moreover, a pretransition from Lβ 0 into a partially distorted ripple phase Pβ 0 occurs for DMPC around Tp ≈ 14 − 15 ◦ C whereas the main phase transition takes place at Tm ≈ 23 − 24 ◦ C. 24–27 Thus, all the three phases are experimentally accessible in aqueous vesicle solution. In this study, a combination of various methods is used to resolve the influence of the saponin aescin on small unilamellar DMPC-vesicles (SUVs). Since aescin is assumed to be a pure compound, thus free from other saponins, observed effects can be directly assigned to the interaction of the aescin molecule with the lipid model membrane. We have proven that the interaction between aescin and the lipid model membrane is strongly dependent on aescin content. Furthermore, we revealed the existence of two characteristic concentrations in the studied concentration range of added aescin (0-10 mol%) to the lipid-saponin mixture, which determine three consecutive regions showing a different structural behavior within these mixtures. In addition, penetration kinetics, membrane incorporation into and further interaction of aescin with a DMPC monolayer is investigated by Langmuir tensiometry (LT) and evaluated 4 ACS Paragon Plus Environment

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in the rigid liquid condensed (LC) and flexible liquid expanded (LE) phase of the monolayer. We have shown that aescin impacts the LC phase more strongly than the LE phase and can comparatively incorporate more aescin molecules. Furthermore, the presence of two-step kinetics reveals the existence of two first-order phase transitions in the surface pressure-time (Π − t) isotherms, which indicates probable formation of domains in the monolayer. 28–30 Investigations of lipid bilayers in the form of vesicles by differential scanning calorimetry (DSC) have proven a concentration dependent phase separation leading to domain formation into aescin-rich and aescin-poor regions within the lipid bilayer. The analysis of the endotherms confirms furthermore the presence of two competing effects involved in this process. The first one gives rise to a peak broadening and only a slight shifting of Tm to lower temperatures, similar to the effect arising from cholesterol incorporation. 23 The second, and more prominent, effect is the strong fluidization of the lipid membrane due to destabilization of the phospholipid by intermolecular cooperativity with aescin, similar to the incorporation of non-steroidal anti-inflammatory drugs (NSAIDs). 31 In our particular case, the strong interaction of the sugar groups with the phospholipid headgroups alters their hydration shells, eventually causing a disorganization of the lipid structure. Additionally, we have used photon correlation spectroscopy (PCS) and wide-angle X-ray scattering (WAXS) to reveal the incorporation of aescin molecules into DMPC SUVs in the concentration range below 1 mol%. These findings are supported by the visual appearance of the samples studied in a systematics of the phase behavior of the mixtures of aescin with DMPC in vesicle suspension. While vesicles with an aescin content below 1 mol% are found to be stable in solution, samples containing aescin between 1 and 6-7 mol% are found to form preferably large aggregates which precipitate over time. This may result from enhanced attractive correlations of the growing aescin domains with increasing saponin content present in solution. By these structural analysis not only domain formation in the nm-range and enhanced fluidization was confirmed but also their relative amount and progression with temperature could be determined. These observations hold until the second concentration limit found at 6-7 mol%. An aescin content


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above 6-7 mol% induces the formation of smaller structures at low temperature. While the presence of domains was shown by WAXS, a remaining bilayer contribution could be verified by DSC. The size of the formed structures was estimated by small-angle X-ray scattering (SAXS).

Materials and Methods Chemicals. 1,2-dimyristoyl-sn-glycerophosphocholine (DMPC) was purchased from Lipoid GmbH (> 99 %, Ludwigshafen, Germany). The pure saponin aescin (≥ 95 %, CAS number 6805-41-0) and chloroform were obtained from Sigma Aldrich (Munich, Germany). The purity of the aescin was confirmed by mass spectrometry. All samples were prepared with purified water (Sartorius arium VF pro, Göttingen, Germany). Since aescin dissolves poorly in water and decreases the pH significantly, a phosphate buffer with pH 7.4 was used to guarantee a constant pH. The buffer concentration depends on the used DMPC mass concentration (see Table 1). Vesicle preparation. For vesicle preparation DMPC was dissolved in chloroform, subsequently evaporated with a rotary evaporator and stored over night at 60 ◦ C for complete chloroform evaporation. The generated thin lipid film was then hydrated with an aescin solution in phosphate buffer at the desired concentration. The obtained multilamellar vesicles (MLVs) were enlarged with five freeze-thaw cycles in liquid nitrogen and warm water. Afterwards the MLVs were transformed into small unilamellar vesicles (SUVs) by extrusion (at least 15 x) using an extruder (Avanti Polar Lipids Inc., Alabama, USA) with a polycarbonate membrane with a diameter of 500 ˚ A (Whatman, Avanti Polar Lipids, Alabama, USA). The method dependent used mass concentrations are listed in Table 1. In the following, the used aescin content χ(aescin) in mol% is defined in respect to the used amount of DMPC (χ(aescin)=naescin /(naescin +nDM P C )). Differential scanning calorimetry (DSC). DSC experiments were carried out to in-


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Table 1: Method dependent DMPC mass concentration w and related phosphate buffer concentration cbuffer . method DSC PCS WAXS/SAXS

w / g·L−1 15 0.5 15

cbuffer / mM 50 10 50

vestigate the thermotropic phase behavior of the lipid vesicles on a differential scanning heat-flow calorimeter (Q100, TA Instruments, New Castle, USA, for calibration see 32 ). The samples (20 mg) were hermetically sealed into aluminum pans (TA Instruments). The thermograms were recorded between 14 and 40 ◦ C with a scan rate δ of 0.25 or 0.50 ◦ C·min−1 , referenced to air. The difference in heat flow ∆P = ∆PS − ∆PR is measured between sample S and reference R as a function of temperature. The heat capacity ∆Cp = Cp,S − Cp,R is then given by ∆Cp =

∆P . δ

(1)

Photon correlation spectroscopy (PCS). PCS experiments were carried out to determine the hydrodynamic radius RH of the vesicles. All samples were extruded through the same membrane. The samples were measured after extrusion within a few days. At the used DMPC mass concentration the samples were found to be stable for several month. For the PCS experiments the samples were filtered through hydrophilic syringe filters ( 400 nm, Whatman, FP30/0.45, Sigma-Aldrich, Munich, Germany) and filled into cuvettes. The samples were placed in a temperature-controlled matching bath filled with decaline. The experiments were carried out on a goniometer setup (ALV-Laser Vertriebsgesellschaft mbH, Langen, Germany) equipped with an argon ion laser (λ=514.5 nm, Spectra Physics, Santa Clara, USA). The signal was read by an ALV 5000 E-Multiple-τ -correlator also from ALV run in pseudo-cross mode. The autocorrelation function was recorded for scattering angles between 50 and 90 ◦ in steps of 5 ◦ at 18 and 32 ◦ C. Temperature-dependent measurements were carried out between 10 and 40 ◦ C at an angle of 60 ◦ using the same instrument. The obtained data were analyzed using the CONTIN algorithm. 33 7 ACS Paragon Plus Environment

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In PCS measurements the intensity of light scattered by an ensemble of diffusing particles is detected at a specific angle of 2θ, whereby the latter can be normalized to the wavelength independent scattering vector q =

4πn λ0

sin(θ). n is the refractive index and λ0

the incoming wavelength. The field-time autocorrelation function g1 (τ ) of the scattered light equals a monoexponential decay (Equation 2) if diluted monodisperse particles are present in solution. 34 g1 (q, τ ) = exp(−ΓT τ )

(2)

Here, ΓT is the relaxation rate. In polydisperse samples Equation 2 has to be replaced by the following expression to consider all present particle sizes: Z

∞

G(Γ) exp(−ΓT τ ) dΓ.

g1 (q, τ ) =

(3)

0

Hence, g1 (q, τ ) is the Laplace transform of the relaxation rate distribution functions G(Γ). G(Γ) can be used to compute the mean relaxation rate, from which the apparent translational diffusion coefficient DTapp can be obtained. Purely diffusional motion follows the q dispersion given in Equation 4.

Γ = DTapp · q 2

(4)

Hence, plotting Γ vs. q 2 allows to obtain DTapp from the slope. The Stokes-Einstein equation (Equation 5) inversely relates DTapp with the vesicles hydrodynamic radius RH .

DTapp =

kB T 6πηRH

(5)

Here, kB is the Boltzmann constant, T the temperature, and η the solvent viscosity. Langmuir tensiometry (LT) experiments. All LT experiments were carried out at the air/water interface in a Langmuir trough (KSV/NIMA Small, 77.5 cm2 , Biolin Scientific


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Holding AB, Stockholm, Sweden). The Langmuir trough is equipped with a Wilhelmy-plate system for measuring the surface tension and two moving hydrophilic Delrin barriers that allow unilateral compression of the monolayer. For a given state, the surface pressure is defined as the difference in surface tension γ with respect to the initial value of the bare surface γ0 , this is Π = γ0 − γ. DMPC was dissolved in chloroform, and the solution spread at the air/buffer interface. Monolayer equilibration is reached when the surface pressure is observed to maintain a constant value, after complete chloroform evaporation occurred. The surface pressure-area (Π − A) isotherms were obtained at a compression rate of 5 mm/min. For subphase-exchange experiments, the obtained monolayer was compressed from the initial diluted state (near to zero pressure) up to a nominal pressure of Πi . The subphase (50 mM phosphate buffer) was then exchanged against aescin solution (10−4 M in 50 mM phosphate buffer) using a peristaltic pump for one hour. The surface pressure vs. time measurements were recorded continuously from the beginning of the subphase exchange until a constant pressure Πeq was obtained. The experiments were carried out at 4 and 38 ◦ C for different Πi . Small- and wide-angle X-ray scattering (SAXS/WAXS). SAXS and WAXS experiments were performed on an inhouse X-ray scattering system (XEUSS, Xenocs, Sassenage, France) with a CuKα X-ray source (λ=0.154 nm, GeniX Ultra low divergence, Xenocs, Sassenage, France) and a Pilatus 300K hybrid-pixel detector (Dectris Ltd., Baden Deattwil, Switzerland). The samples were measured in a flow-through Kapton capillary ( 1 mm, GoodFellow GmbH, Bad Nauheim, Germany) positioned in a Linkam stage (Linkam Scientific, Tadworth, UK). The sample-to-detector distance was calibrated using silver behenate. The covered range of magnitude of the scattering vector is for SAXS qSAXS =0.008-0.6 ˚ A−1 , and for WAXS q=0.2-2 ˚ A−1 . The experiments were performed for WAXS between 10 and 50 ◦ C, and for SAXS only at 10 ◦ C. The scattered intensity was normalized to the incident intensity, measuring time, transmission, background and sample thickness. It was calibrated to absolute scale using glassy carbon (type 2, sample P11 35 ). The scattered intensity of a particle solution I(q) = N · I0 · (∆ρ)2 · (V )2 · P (q) · S(q) is


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dependent on the intensity of the incident beam I0 , the scattering volume of the particle V , the scattering length density difference ∆ρ = ρparticle − ρsolvent , i.e. the contrast between solvent and particle, and on the particle form factor P (q) thus on the shape of the particle. 36,37 For dilute systems, the structure factor S(q) amounts to 1. A correlation distance d can be computed from the definition of the scattering vector (q =

4π λ

sin(θ) at a scattering angle of

2θ) and the Bragg equation using: d∼ =

2π qmax

(6)

This equation is applied to calculate the head-to-head correlation distance dx from the WAXS data. qmax is derived from the maximum of a Lorentzian fit to the scattering peaks. From the scattering data at low angles (SAXS) information about the overall shape of the scattering particles is obtained by analyzing the pair distance distribution function (p(r)) in real space. p(r) is the Fourier transform of the angle dependent scattered intensity I(q). It was obtained using the inverse Fourier transform method as implemented in the GIFT software package provided by O. Glatter. 38 It is the convolution square (spatial correlation function) of the excess scattering length density distribution function ∆p(r). 39 Furthermore, the p(r) function can be used to determine RG by integration: R p(r)r2 dr 2 . RG = R 2 p(r)r2 dr


(7)

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Results and discussion Phase behavior The binary mixtures of aescin and DMPC in aqueous solution show a peculiar concentrationand temperature-dependent phase behavior determined by the presence of lipid vesicles and aescin aggregates. We studied this phase behavior at a DMPC mass concentration of 15 g L−1 and varying aescin contents between 0 and 10 mol% in 50 mM phosphate buffer at pH 7.4. Under these conditions a first structural transition was observed at 1 mol% aescin (see supporting information; Figure S1). Below this concentration the presence of vesicles was confirmed by PCS. These vesicles were found to be stable for several hours at this DMPC mass concentration and during heating and cooling cycles. Between 1 and 6-7 mol% the formation of vesicles was also observed which, differently to the ones observed at lower aescin content, tend to form larger aggregates that precipitate over time. The supernatant of the precipitate becomes more transparent with increasing aescin content. Also, a temperature-dependent behavior was observed. Specifically, this supernatant becomes opaque with increasing temperature, which indicates further entropic aggregation within this lipid-rich phase. At aescin contents above 6-7 mol% no phase separation is observed and all samples are completely transparent at low temperature and turn opaque at high temperature. In this concentration regime, at low temperatures, the clear solutions indicate formation of much smaller structures. A similar behavior was already shown for different detergents. 40,41 Photographs covering a broad concentration regime are shown in the supporting information in Figure S1.


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Differential scanning calorimetry (DSC) In order to get insight into the thermal transition of the hybrid DMPC-aescin vesicles, we choose DSC as the adequate tool to monitor the thermotropic behavior of the vesicle systems. 2,42 The resulting thermograms for vesicles containing up to 7 mol% aescin are shown in Figure 2(a). The obtained data was background corrected by subtraction of a linear baseline. The positions of the peak maxima were analyzed and are plotted in Figure 2(b).

(a)

endo

0.2

DMPC 0.2 mol%

Cp / J K-1 g-1

0.4 mol% 0.8 mol% 1.0 mol% 2.0 mol% 4.7 mol% 7.0 mol% 21

22

23

24

25

26

27

T / °C 24.8

(b)

24.4

Tmax / °C

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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24.0 23.6 23.2

narrow signal broad signal

22.8 0

1

2

3

4

5

6

7

(aescin) / mol%

Figure 2: (a) DSC thermograms (with δ = 0.25 ◦ C min−1 ) of DMPC vesicles (15 g L−1 ) with different amounts of aescin between 0 and 7 mol%. The whole signal is a superposition of a sharp (aescin-poor) and a broad (aescin-rich) component, as indicated for vesicles containing 1 mol% aescin. (b) Main phase transition temperatures Tm resulting from the thermograms in dependence of the aescin amount χ(aescin). The Tm values were obtained from the maximum values of the single peaks. The endothermic transition signal becomes progressively asymmetric under increasing concentration of aescin and consists of a superimposed sharp and a broad component, as indicated for the signal with 1 mol% of aescin (see Figure 2(a)). While the narrow signal 12 ACS Paragon Plus Environment

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decreases, the broad signal increases in intensity with increasing aescin concentration and both shift to lower temperatures. The existence of such a second broad contribution was also reported for cholesterol incorporation to the same lipid system. 2,23 In the study of McMullen et al. with DMPC/cholesterol mixtures the superimposed endothermic signal consists of a prominent sharp and flat broad signal. 23 The former signal is assigned to cholesterol-poor phase and the latter one to cholesterol-rich regions. In that previous work, the temperature obtained from the narrow signal, being assigned to the phospholipids Tm , was shifted slightly but not significantly to lower temperatures. The incorporation of high amounts of cholesterol (20 mol%) even extended the signal slightly to lower and but more pronounced to higher temperatures beyond the main sharp signal. This effect was explained by a disrupting impact on the cooperative arrangement of the phospholipid molecules in the hydrophobic part of the bilayer. On the other hand, the incorporation of more polar membrane additives, e.g. nonsteroidal anti-inflammatory drugs (NSAIDs), especially Diclofenac, seems to impact the main phase transition of the lipid by shifting it to lower temperatures. 31,43 Also, a second endothermic contribution appearing at much lower temperatures was reported for that system, which was assigned to a phase segregation process leading to Diclofenac-rich and Diclofenac-poor regions. Let’s notice that other than the effect of cholesterol, which interacts mainly with the hydrophobic part of the membrane, it has been shown for the NSAID ibuprofen by Boggara et al. that such polar additives impact mainly the outer hydrophilic phospholipid headgroups instead of the hydrophobic core of the membrane. 44 The strong interaction with the phospholipid headgroups leads to strong fluidization of the membrane that changes deeply the thermodynamic behavior of the lipid bilayer, which results in a strong destabilization of the gel phase and thus in a lowered Tm . 45 The comparison of those cases 23,31,44 with the results obtained in our present study for the aescin molecule allows to find similarities between the different cases. The narrow contribution observed for binary DMPC bilayers mixed with aescin can be assigned to an aescin-poor lipid phase and the broad contribution to aescin-rich regions in the bilayer. Similarly to the


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fluidizing impact of cholesterol in ordered bilayers of saturated phospholipids, 46 the incorporation of aescin to DMPC ordered bilayers elicits a minor but visible displacement of the maximum of the narrow contribution to lower temperatures. The steric similarity of the backbone of the saponin and sterols leads to the assumption that there is a disordering interaction of the saponins backbone with the hydrophobic moiety of the lipid membrane. This disordering might lead to a decrease in Tm and a broadening of the endothermal melting transition. Unfortunately, the used DSC instrument does not allow a more precise analysis of the peak-broadening of this signal. The decreasing signal intensity indicates that the amount of aescin-poor domains diminishes already significantly at an incorporation of 2 mol% aescin. A further analysis of the endothermic signal indicates the appearance of phase separation with increasing saponin content between 0.4 and 4.7 mol%. With increasing aescin content the phase separation becomes more pronounced, which suggests that also the aescin-rich regions grow. The effect of phase separation becomes more prominent above a concentration of 1 mol%. Similar to the NSAIDs interactions with the bilayer, we assume that in this concentration regime the saponin molecules mainly interact with the hydrophilic phosphocholine groups of the bilayer. Possibly, these cohesive interactions occuring between the sugar groups and the phospholipid headgroups are driven by hydrogen bonds. Demana et al. confirmed in a previous study with the saponin Quil A using diffuse reflecting infrared spectroscopy (DRIFT) that the hydroxy groups of the saponins sugar moieties may form hydrogen bonds with the carbonyl and negatively-charged phosphate groups of the phospholipid. 14 Furthermore, they show that the free carboxylic-group of the saponin may electrostatically bind with the phosphocholines positively charged quaternary-ammonium group, contrarily to NSAIDs where the molecules are incorporated entirely into the hydrophobic core of the membrane. 44 Above an aescin amount of 6-7 mol% the DSC signal is so broad that the main transition peak of the phospholipid practically disappears. Nevertheless, a broad endothermic band remains in the temperature range between 22 to 24 ◦ C, which can be confirmed for the concentrations between 6 and 10 mol% of aescin in the supporting information (see Figure


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S2(a)). Even though direct structural information cannot be extracted from DSC data, the formation of completely new structures above 6 mol% is proven. However, also at such a high concentration a small signal resulting from a cooperative bilayer phase transition seems still to remain (see Figure S2(b)).

Langmuir tensiometry (LT) experiments The interaction of aescin with a DMPC monolayer was studied by subphase exchange experiments performed in a Langmuir trough. The Langmuir monolayer of DMPC adsorbed at the air/buffer interface was studied in both, the liquid expanded phase (LE) and in the liquid condensed phase (LC). 47 The two surface pressure-area isotherms for 4 ◦ C and 38 ◦ C are compared in Figure S3. The high temperature monolayer shows a continuous compressible behavior compatible with the LE phase. However, at low temperature a first-order transition from LE to LC phase is observed upon monolayer compression at Πcoex ≈15 mN m−1 . The LE/LC-transition is actually pseudo first-order as the surface pressure increases along the coexistence plateau due to kinetically impeded nucleation and growth of the domains of the LC phase at the expense of the continuous LE phase, as observed in monolayers of similar phospholipids. 47 The penetration capacity of aescin in these DMPC monolayers was tested in subphase-exchange experiments in which aescin is gently incorporated to the adsorption layer from the bulk solution. Briefly, the DMPC monolayers were first compressed to an initial pressure (Πi ) and then equilibrated. Afterwards the aqueous subphase was exchanged against an aescin solution and the surface pressure recorded over time. The surface pressure-time (Π(t))-adsorption kinetics were recorded for both temperatures at similar initial pressures Πi until final equilibration at an equilibrium plateau pressure (Πeq ) (see Figures 3(a) and (b) for 4 ◦ C and 38 ◦ C, respectively). The comparison of Figures 3(a) and (b) indicates faster adsorption at higher temperature, which is due to faster diffusional adsorption of aescin from the solution. The plots show a rapid incorporation of the soluble aescin into the DMPC monolayer, as the surface


Langmuir

(a)

40

/ mN m-1

40

/ mN m-1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

30

Πeq 20

10

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(b)

30

20

10

Πi

38°C

4°C

0

0 0

1

2

3

4

0.0

t/h

0.2

0.4

0.6

t/h

Figure 3: Surface pressure-time adsorption kinetics at (a) 4 ◦ C and (b) 38 ◦ C for different initial pressures Πi ≈5, 15 and 25 mN m−1 after start of buffer exchange against aescin solution until reaching equilibrium pressure Πeq . The arrows mark the intermediate cusp. pressure starts to increase from the very begining, without displaying an induction period. Further aescin adsorption leads to an intermediate cusp at a discrete pressure before reaching Πeq (cusps marked by arrows in Figure 3). These intermediate cusps in the Π(t) adsorption kinetics may result either from an adsorption barrier for aescin before eventual monolayer penetration, 7 or alternatively, the induction of a phase transition in the DMPC upon aescin adsorption. 28 Since the kinetic plots are recorded in well-defined homogeneous phases (LC phase if Πi >15 mN m−1 at 4 ◦ C, otherwise LE phase), effects related to LE/LC phasetransition of the lipid monolayer can be reasonably discarded. Therefore, an adsorption barrier leading to a transient metastable state is assumed before reaching the final penetration/reorganization of aescin inside the DMPC monolayer, or transient domain-formation within the monolayer, or both with time. 13,28 Then, after a given characteristic delay time during these cusps, which is observed longer at lower temperature as expected for diffusive transport, the surface pressure raises again up to the final equilibrium value. From an equilibrium standpoint, using the data in Figure 3 the pressure difference ∆Π = Πeq − Πi can be calculated as a free energy difference between the initial pure lipid state (Πi ) 16 ACS Paragon Plus Environment

Page 17 of 35

and the final equilibrium plateau (Πeq ), 29 corresponding to the mixed aescin-lipid monolayer after aescin has penetrated the DMPC monolayer from the solution. Figure 4 shows the dependence of such a penetration pressure ∆Π as a function of the initial pressure of the monolayer Πi . Similar values of ∆Π are measured in the LE phase for both temperatures below ≈15 mN m−1 . This indicates a similar facilitated penetration of aescin in the disordered LE phase, independently of temperature. In the LC phase (Πi >15 mN m−1 at 4 ◦ C) higher values of ∆Π are measured indicating a larger interaction effect of aescin in the LC phase of DMPC, relative to the characteristic LE-phase at 38 ◦ C. From the extrapolate of these curves to zero ∆Π one obtains the maximum interaction pressure (MIP). 29,30 The MIP corresponds to the maximum surface pressure and thus the minimum surface area of the monolayer at which the insertion of the aescin molecules into lipid monolayer is energetically favorable. Insertion above the MIP value is not possible. 29 In the present case, the MIP of the saponin into the lipid monolayer is dependent on the physical state of the phospholipid: the LC-phase of DMPC (MIPLC ≈62±2 mN m−1 ) is quite more penetrable by aescin than the LE-phase (MIPLE ≈38±3 mN m−1 ). This confirms the depicted structural scenario by DSC whereby

4°C 38°C

30

!" / mN m-1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

20

10 empty symbols : LC filled symbols : LE

MIP

0 0

20

40 i/

mN m

60

-1

Figure 4: Insertion pressure ∆Π = Πeq − Πi upon aescin incorporation into a DMPC monolayer at 4 ◦ C and 38 ◦ C as a function of initial pressure Πi obtained from Π(t) adsorption kinetics (see Figure 3). Filled symbols correspond to the LE phase, open symbols to the LC phase. The maximum insertion pressure (MIP) at ∆Π = 0 is marked in grey.


Langmuir

the saponin was assumed to tightly interact with the headgroups of the phospholipids in the dense assembly of the bilayer, rather compatible with the density of the LC-phase in every monolayer leaflet. Despite the equivalence between monolayers and bilayers is strictly qualitative, 48 the higher MIP value may be the result of a more pronounced structural impact of aescin molecules on the bilayer-like LC phase than in the LE phase.

Photon correlation spectroscopy (PCS) The vesicles hydrodynamic radius RH as well as the presence of aggregates were determined by PCS. 49,50 Figure 5(a) shows experimental data that indicate the temperature dependent swelling of pure DMPC vesicles, which is due to the area dilation occuring upon bilayer melting. Tm was calculated from the inflection point of a sigmoidal Boltzman fit

51

RH / nm

50 49 48 Tm = 23.6 °C

47

(a)

46 12

16

20

24

28

32

36

40

T / °C 4

18 °C

32 °C

3

RH / nm

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 35

2 1 0

(b)

-1 0.0

0.5

1.0

1.5

2.0

2.5

3.0

(aescin) / mol%

Figure 5: (a) Swelling curve of DMPC vesicles (w = 0.5 g L−1 ) measured with PCS. A value for Tm can be estimated from the inflection point of a Boltzmann fit (solid line). (b)Relative change of the vesicles hydrodynamic radius (∆RH ) as function of aescin content χ(aescin) below, at 18 ◦ C (◦), and above Tm , at 32 ◦ C (•). The lines are spline functions to facilitate data interpretation.


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Langmuir

and determined at 23.6±0.16 ◦ C, which is in agreement with values from literature. 2 The data evidence constant values below and above Tm , indicating that the vesicles are in osmotic equilibrium and no vesicle aggregation occurs in these regimes. Indeed, temperature driven aggregation effects upon specific thermal treatment were reported for dipalmitoylsn-glycerophosphocholine (DPPC) 51 and DMPC 52 vesicles approximately 10 ◦ C below the phospholipid Tm . To exclude temperature driven aggregation effects, the influence of aescin on DMPC vesicles was studied at 18 ◦ C and 32 ◦ C (see Figure 5(b)). Vesicles with an aescincontent up to 3 mol% show a narrow size distribution with a polydispersity index (PDI) of around 0.1 and no aggregation effects detected over time. Here, the PDI is defined as the standard deviation devided by the statistical average over the vesicle population. Let’s notice that these experiments were performed at a DMPC mass concentration of 0.5 g L−1 , where aggregation occurs at higher aescin concentrations. By increasing the aescin content further aggregation becomes visible and the PDI increases, so that samples with aescin contents higher than 3 mol% are not considered anymore (PDI > 0.3). 53 Figure 5(b) shows data for the bilayers in Lβ 0 at 18 ◦ C and in the Lα phase at 38 ◦ C. ∆RH increases with increasing aescin mole fraction for about 2 nm and reaches a constant level above the before mentioned aescin amount of 1 mol%. From the data we deduce direct incorporation of the aescin molecules into the vesicles bilayer in both, the Lβ 0 and the Lα phase until an aescin amount of 1 mol% causing RH to increase. Also, Wojciechowski et al. indicate easy penetration of certain saponins into a phospholipid membrane. 13 From previously shown DSC and LT results we note that at higher contents aescin starts to assemble locally. We observe here that these formed domains do not influence RH significantly anymore. Furthermore, in the Lβ 0 phase (at 18 ◦ C) an elevated RH -value was found close to the previously discussed concentration limit at 0.8 mol%. This is indicated by an arrow in Figure 5(b). This elevation may result from probable hydration effects of the bilayers surface. At 0.8 mol% of aescin the vesicle surface with homogeneously spread, protruding aescin head groups are maximally hydrated by water molecules. By local assembly of aescin


Langmuir

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molecules within the lipid membrane the impact of hydration in RH decreases. Thereby RH at 18 ◦ C seems to decrease again due to probable reduction of the hydration shell before remaining at a constant level. Due to higher mobility of the solvent molecules as well as the membrane itself this effect might not be clearly visible at 38 ◦ C.

Wide-angle X-ray scattering (WAXS) Structural information about the head-to-head correlation distance in the lateral plane of the membrane can be obtained by the analysis of WAXS spectra. 54 WAXS offers a q-range suitable to resolve the mean lipid head-to-head correlation length within monolayers of the bilayer membrane. 36 The method is sensitive to the electron density difference (∆ρ) between colloid and solvent, thus it is very qualified to investigate the interfacial region of aggregates composed of e.g. lipid and saponin molecules. 39 By monitoring the correlation signal as a function of temperature the conversion of the rigid Lβ 0 via the Pβ into the liquid crystalline Lα phase is directly observed for phospholipid vesicles. To deduce this information from the WAXS spectra and to distinguish between simultaneously present phases in the lipid bilayer, the diffraction bands are fitted with two Lorentzian functions below and close to Tm and with one Lorentzian function above Tm , as shown in Figure 6 for pure DMPC vesicles. The broad distribution is assigned to defects arising from chain melting into gauche-conformations, whereas the narrower distribution is assigned to lipid molecules in the Lβ 0 phase within the bilayer membrane. 55 In Figure 6(a)-(c) (below and close to Tm ) we show the result of both Lorentzian function whose parameters, namely position, width and ratio change with temperature. While the narrow distribution progressively becomes smaller, the broader one becomes more prominent with increasing temperature. From the peak maxima of the individual Lorentzian functions for both, the narrow and the broad distribution, the head-to-head correlation distance dx is calculated using Equation 6. The results are presented in Figure 7 (see upper panel) for pure DMPCvesicles and vesicles containing different contents of aescin, covering all studied concentration


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Page 21 of 35

(a )

1 3 ° C

1 0

d a ta s u m b ro a d n a rro w

8 6 4 2 0 8

2 4 ° C

(b )

2 5 ° C

(c )

3 5 ° C

(d )

6

-1

4

-3

c m

2 0

I(q ) / 1 0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

6 4 2 0 4

2

0 1 .0

1 .5

q / Å

2 .0 -1

Figure 6: Lorentzian fits of the WAXS signals of pure DMPC vesicles (extruded through a membrane with a pore diameter of 500 ˚ A) in the Lβ 0 phase at (a) 13 ◦ C, close to Tm at (b) ◦ ◦ 24 C and (c) 25 C and in the Lα phase (d) at 35 ◦ C. The data is fitted with two Lorentzian functions below and around Tm and with one above Tm . The fit (black) is the sum of the narrow (dark grey) and broad (light grey) contribution. The dotted line represents the peak position of the fit at 13 ◦ C revealing the peak shift to lower q with increasing temperature.


Langmuir

regions. The integral fraction of the diffraction signal which describes the relative amount of each phase present in the membrane is shown in the lower panel of the image. The integral fraction is the integrated area of each signal divided by the sum of the area of both signals.

4 .6

/ Å

4 .3

d

4 .4

x

4 .5

(a ) p u re D M P C

(b ) 0 .6 m o l%

(c ) 4 .0 m o l%

(d ) 6 .5 m o l%

(e ) 1 0 m o l%

5 .0

b ro a d n a rro w s in g le

4 .8 4 .6 4 .4

d x / Å

4 .7

4 .1

4 .2

1 0 0

1 0 0

8 0

8 0

6 0

6 0

4 0

4 0

2 0

2 0

0

In t. fra c . / %

4 .2

In t. fra c . / %

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0

1 0 1 5 2 0 2 5 3 0 3 5 4 0 1 0 1 5 2 0 2 5 3 0 3 5 4 0

1 0 1 5 2 0 2 5 3 0 3 5 4 0 1 0 1 5 2 0 2 5 3 0 3 5 4 0

1 0

2 0

3 0

4 0

5 0

T / °C Figure 7: Fit results of WAXS signals of (a) pure DMPC vesicles and vesicles containing (b) 0.6 mol%, (c) 4.0 mol%, (d) 6.5 mol%, and (e) 10.0 mol% aescin as function of temperature. The upper image shows the lipids head-to-head distance dx , the lower image the corresponding integral fraction calculated from the area of the fits. The dots (•), are calculated from the narrow contribution, the circles (◦) from the broad contribution, and the triangles (N) from the single contribution in Figure 6. For pure DMPC vesicles, the diffraction pattern was fitted according to Figures 6(a)-(c) below Tm with two Lorentzian functions. The integral fractions show that the contribution from the broad peak increases with temperature up to a threshold temperature compatible with Tm , while the one of the narrow peak and thus representing the gel phase decreases. Above the threshold temperature, the bilayer has already undergone a phase transition into the full fluid phase, thus the WAXS signal can be fitted only by a single Lorentzian function. Hence, the integral fraction of the fluid phase amounts to 100 %. We argue, that the dx -values arising from the broad distribution nicely correspond to the ripple phase Pβ , into which the DMPC-molecules, which are in the Lβ 0 , pass continuously with increasing temperature. 56 At 20 ◦ C this transition seems already to be completed since dx of the broad distribution does 22 ACS Paragon Plus Environment

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Langmuir

not differ significantly from dx of the narrow distribution, thus the DMPC molecules are not in the Lβ 0 phase anymore, which implies that the two mentioned phases are not strictly separated in the membrane. By increasing the temperature further, monophasic re-entrance into the Lα phase occurs, as indicated by the integral fractions in the lower panel of Figure 7. The interaction of aescin with the membrane lipid is depicted in Figures 7(b)-(e). However, panel 7(e) will be discussed separately since at 10 mol% no vesicles exist anymore. The comparison of the data of different contents of aescin in Figures 7(b)-(d) with pure DMPC (Figure 7(a)) illustrates similarities and differences regarding the narrow and broad contribution and the trend of the integral fraction. While the phase transition described by the narrow contribution broadens over a wider temperature range compared to DMPC, the broad contribution becomes significantly elevated and less distinct. The progress of the integral fraction of 0.6 and 4.0 mol% is similar to the one of pure DMPC while the one of 6.5 mol% differs around DMPC’s Tm . It is worthy to mention that this concentration corresponds to the maximal stability limit of monomeric aescin, as previously determined at 6-7 mol%, and shows nicely the transition between vesicular structures, e.g. 4 mol% in Figure 7(c) and smaller structures, micelle-like, above this limit (e.g. 10 mol% in Figure 7(e)). To further discuss the smaller formed structures we note that not only the evolution of the integral fraction of 10 mol% in Figure 7 is significantly different from the vesicular structures but also the dx -values of the broad contribution are much more elevated and distinct and decrease continuously with temperature until the Lα phase-level of DMPC is reached. To further compare this concentration to the vesicular structures we show the progress of dx in the main lipid phases, namely L0β at low into Lα at high temperature, for DMPC and DMPC-containing (10 mol% and 4 mol%) aescin dispersions in Figure 8. The comparison of the dx -values below and above Tm shows a prominent impact and elevated dx -values at low temperatures. At high temperatures all dx -values conclude at the same level as pure DMPC. The elevation of the dx -values in the Lβ 0 phase indicates the incorporation of aescin molecules of significant amount with major structural impact in this


Langmuir

1 0 m o l% 4 m o l% D M P C

4 .5

4 .3

x

/ Å

4 .4

d

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 35

4 .2 4 .1 1 0

1 5

2 0

2 5

3 0

3 5

4 0

T / °C Figure 8: Progress of dx in the main lipid phases, summarized for pure DMPC, DMPC + 4 mol% and 10 mol% of aescin. phase. Here, former considerations of enhanced fluidization of the membrane upon aescin incorporation are assured since the dx -values of the Lβ 0 are brought closer to the ones of the Lα phase. The more prominent impact of incorporated aescin molecules into the gel phase was already discussed for bilayers via PCS and for monolayers via LT experiments. This argumentation holds for both concentrations considered. The main difference between the dx trends of 4 mol% and 10 mol% is the disappearance of the sharp phase transition around 25 ◦ C for 10 mol%. While the data confirm the remaining vesicular structures at 4 mol%, they point out structural differences upon incorporation of 10 mol%. Nevertheless, a very broadened phase transition of the lipid molecules remains with temperature, which was also confirmed by DSC experiments in Figure S2.


Page 25 of 35

Small-angle X-ray scattering (SAXS) SAXS experiments were performed in order to resolve the structure of the aescin-containing aggregates. From the scattering probed at low angles information about the global dimensions and shape of the scattering object is obtained. Figure 9(a) shows the SAXS scattering profiles of DMPC vesicles and of a DMPC/aescin mixture containing 10 mol% aescin. Figure 9(b) shows the calculated corresponding pair distance distribution functions (p(r)) obtained by using the IFT method. 38

-3

x 1 0

(a )

D M P C

x 1 0

7 6 5

6

-1

D M P C 5

1 0 m o l%

p (r)

0

1 0

a e s c in

4 4 3

1 0

3

-1

2 2 1

1 0

-4

8

(b ) 7

1

1 0

I(q ) / c m

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

1

-2

1 0 m o l% 0

0 .0 0 1

q / Å

0

0 .0 1 -1

1 0 0

2 0 0

3 0 0

4 0 0

5 0 0

a e s c in 6 0 0

7 0 0

0 8 0 0

r / Å

Figure 9: (a) SAXS scattering curves of DMPC vesicles and a DMPC/aescin mixture containing 10 mol% aescin at 10 ◦ C. (b) Pair-distance distribution functions p(r) of these samples. The line represents a convolution between the fitted data and calculated approximation by GIFT. 38 The vesicles prepared by extrusion through a 500 ˚ A membrane are found with a largest size of 800 ˚ A, as happens usually with the extrusion method. 57 Furthermore, it is obvious from the data that a high aescin concentration (10 mol%) leads to the formation of much smaller structures with a maximum size of 300 ˚ A at 10 ◦ C. The calculated radii of gyration RG by Equation 7 are for the DMPC-vesicles 308 ± 8 ˚ A and for the DMPC/aescin mixture


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98 ± 4 ˚ A, a value compatible with the existence of micelles. Errors correspond to the mean deviation of RG calculated by GIFT.

Conclusion In our current work we have investigated DMPC vesicles in the presence of different amounts of aescin at a lipid mass concentration of 15 g L−1 . At a first sight, the mere visual inspection of the samples reveals changes induced by the aescin. Based on the different experiments with DSC, PCS, WAXS and SAXS with bilayer DMPC vesicles, we conclude the existence of three different concentration regimes in the studied concentration range between 0 and 10 mol%: 0 to 1 mol% aescin, 1 to 6-7 mol% aescin and beyond 6-7 mol%. In the first regime we obtain stable vesicles incorporating aescin and the onset of a phase separation. In this regime large parts of the bilayer are probably saponin-free. The incorporation of aescin to DMPC bilayers was proven by DSC, PCS and WAXS experiments. In the second regime the phase behavior is dominated by a rather prominent phase separation process. Here, the strong interaction between the sugar groups of the saponin and the lipids phosphocholine group leads to prominent domain formation and probable fluidization of the bilayer. This explanation is compatible with the thermodynamic behavior observed by DSC and confirmed on the molecular level by WAXS. The incorporation of aescin seems to strongly impact on the gel phase structure, whereas the fluid phase was not significantly affected on the molecular scale. In the third regime the formation of smaller structures, micelle-sized, occurs with a RG of approx. 98 ˚ A at 10 ◦ C. From the WAXS and DSC experiments with bilayered vesicles, we show that the formed structures are still temperature-sensitive and undergo a thermal phase transition. By investigating the properties of aescin incorporation into a monolayer in a Langmuir trough, we have shown that the saponin-lipid interaction strongly depends on the initial state of the lipid monolayer indicating differential binary interactions depending on the density status of the phospholipid. The saponin incorporates into both the LC and LE phase


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Langmuir

but with different kinetics. The incorporation into the LE phase is slower than into the LC phase, but the interaction of the aescin molecules with the DMPC monolayer is much stronger in the LC phase due to higher structural impact on the lipids LC phase conformation. The experimental results in this work reveal altogether the existence of net cohesive interactions with the phospholipid at discrete aescin concentration, resulting in the appearence of binary aescin-lipid aggregates based on the bilayered structure of the phospholipid. At high aescin concentration this binary interaction is overcome by the aescin-aescin attraction resulting in a different self-assembly scenario dominated by aescin-based micelle-like aggregates.


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Acknowledgement We gratefully acknowledge financial support by the Deutsche Forschungsgemeinschaft (DFG) (HE2995/7-1, INST 215/432-1 FUGG). Further, the authors thank Prof. Dr. Thomas Koop to place the DSC at our disposal.

Supporting Information Available Photographs on the phase behavior (Figure S1), and additional information on DSC (Figure S2) and Langmuir trough experiments (Figure S3) are available.

References (1) Sirtori, C. R. Aescin:

pharmacology, pharmacokinetics and therapeutic profile.

Pharmacological Research 2001, 44(3), 183–193. (2) Malcolmson, R. J.; Higinbotham, J.; Beswick, P. H.; Privat, P. O.; Saunier, L. DSC of DMPC liposomes containing low concentrations of cholesteryl esters or cholesterol. Journal of Membrane Science 1997, 123, 243–253. (3) Sipos, W.; Reutterer, B.; Frank, M.; Unger, H.; Grassauer, A.; Prieschl-Grassauer, E.; Doerfler, P. Escin inhibits type I allergic dermatitis in a novel porcine model. International Archives of Allergy and Immunology 2013, 161, 44–52. (4) Golemanov, K.; Tcholakova, S.; Denkov, N.; Pelan, E.; Stoyanov, S. D. The role of the hydrophobic phase in the unique rheological properties of saponin adsorption layers. Soft Matter 2014, 10, 7034–7044. (5) Lorent, J. H.; Quetin-Leclercq, J.; Mingeot-Leclercq, M.-P. The amphiphilic nature of saponins and their effects on artificial and biological membranes and potential conse-


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quences for red blood and cancer cells. Organic & Biomolecular Chemistry 2014, 12, 8803–8822. (6) Böttger, S.; Hofmann, K.; Melzig, M. F. Saponins can perturb biologic membranes and reduce the surface tension of aqueous solutions: A correlation? Bioorganic & Medicinal Chemistry 2012, 20, 2822–2828. (7) Wojciechowski, K. Surface activity of saponin from Quillaja bark at the air/water and oil/water interfaces. Colloids and Surfaces B: Biointerfaces 2013, 108, 95–102. (8) Mackie, A. M.; Singh, H. T.; Owen, J. M. Studies on the distribution, biosynthesis and function of steroidal saponins in echinoderms. Comparative Biochemistry and Physiology Part B: Comparative Biochemistry 1977, 56B(1), 9–14. (9) Francis, G.; Kerem, Z.; Makkar, H. P. S.; Becker, K. The biological action of saponins in animal systems: a review. British Journal of Nutrition 2002, 88, 587–605. (10) Price, K. R.; Johnson, I. T.; Fenwick, G. R.; Malinow, M. R. The chemistry and biological significance of saponins in foods and feedingstuffs. Critical Reviews in Food Science and Nutrition 1987, 26(1), 27–135. (11) Zhou, D.; Jin, H.; Lin, H.-B.; Yang, X.-M.; Cheng, Y.-F.; Deng, F.-J.; Xu, J.-P. Antidepressant effect of the extracts from Fructus Akebiae. Pharmacology, Biochemistry and Behavior 2010, 94, 488–495. (12) Vinarova, L.; Vinarov, Z.; Atanasov, V.; Pantcheva, I.; Tcholakova, S.; Denkov, N.; Stoyanov, S. Lowering of cholesterol bioaccessibility and serum concentrations by saponins: in vitro and in vivo studies. Food & Function 2015, 6, 501–512. (13) Wojciechowski, K.; Orczyk, M.; Gutberlet, T.; Geue, T. Complexation of phospholipids and cholesterol by triterpenic saponins in bulk and in monolayers. Biochimica et Biophysica Acta 2016, 1858, 363–373. 29 ACS Paragon Plus Environment

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(14) Demana, P. H.; Davies, N. M.; Hook, S.; Rades, T. Analysis of Quil A-phospholipid mixtures using drift spectroscopy. International Journal of Pharmaceutics 2007, 342, 49–61. (15) Wang, T.; Fu, F.; Zhang, L.; Han, B.; Zhu, M.; Zhang, X. Effects of escin on acute inflammation and the immune system in mice. Pharmacological Reports 2009, 61, 697–704. (16) Wilkinson, J. A. .; Brown, A. M. G. Horse chestnut aesculus hippocastanum: potential applications in cosmetic skin-care products. International Journal of Cosmetic Science 1999, 21, 437–447. (17) Zhou, X.-y.; Fu, F.-y.; Li, Z.; Dong, Q.-j.; He, J.; Wang, C.-h. Escin, a natural mixture of triterpene saponins, exhibits antitumor activity against hepatocellular carcinoma. Planta Medica 2009, 75, 1580–1585. (18) Pittler, M. H.; Ernst, E. Horse-chestnut seed extract for chronic venous insufficiency. A criteria-based systematic review. Archives of Dermatology 1998, 134(11), 1356–1360. (19) Augustin, J. M.; Kuzina, V.; Andersen, S. B.; Bak, S. Molecular activities, biosynthesis and evolution of triterpenoid saponins. Phytochemistry 2011, 72, 435–457. (20) Dourmashkin, R. R.; Dougherty, R. M.; Harris, R. J. C. Electron microscope observations on rous sarcoma virus and cell membranes. Nature 1962, 194, 1116–1119. (21) Bangham, A. D.; Horne, R. W.; Glauert, A. M.; Dingle, J. T.; Lucy, J. A. Action of saponin on biological cell membranes. Nature 1962, 196, 952–955. (22) Koynova, R.; Caffrey, M. Phases and phase transitions of the phosphatidylcholines. Biochimica et Biophysica Acta 1998, 1376, 91–145. (23) McMullen, T. P.; McElhaney, R. N. New aspects of the interaction of cholesterol with


Page 30 of 35

Page 31 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

dipalmitoylphosphatidylcholine bilayers as revealed by high-sensitivity differential scanning calorimetry. Biochimica et Biophysica Acta 1995, 1234, 90–98. (24) Du, L.; Liu, X.; Huang, W.; Wang, E. A study on the interaction between ibuprofen and bilayer lipid membrane. Electrochimica Acta 2006, 51, 5754–5760. (25) Kiselev, M. A.; Wartewig, S.; Janich, M.; Lesieur, P.; Kiselev, A. M.; Ollivon, M.; Neubert, R. Does sucrose influence the properties of DMPC vesicles? Chemistry and Physics of Lipids 2003, 123, 31–44. (26) Heimburg, T. A model for the lipid pretransition: coupling of ripple formation with the chain-melting transition. Biophysical Journal 2000, 78, 1154–1165. (27) Mortensen, K.; Pfeiffer, W.; Sackmann, E.; Knoll, W. Structural properties of a phosphatidylcholine-cholesterol system as studied by small-angle neutron scattering: ripple structure and phase diagram. Biochimica et Biophysica Acta 1988, 945, 221– 245. (28) Hossain, M. M.; Suzuki, T.; Kato, T. Phases and phase transitions in Gibbs monolayers of an alkyl phosphate surfactant. Journal of Colloid and Interface Science 2005, 288, 342 – 349. (29) Calvez, P.; Bussières, S.; Demers, E.; Salesse, C. Parameters modulating the maximum insertion pressure of proteins and peptides in lipid monolayers. Biochimie 2009, 91, 718–733. (30) Calvez, P.; Demers, E.; Boisselier, E.; Salesse, C. Analysis of the contribution of saturated and polyunsaturated phospholipid monolayers to the binding of proteins. Langmuir 2011, 27, 1373–1379. (31) Moreno, M. M.; Garidel, P.; Suwalsky, M.; Howe, J.; Brandenburg, K. The membrane-


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

activity of Ibuprofen, Diclofenac, and Naproxen: A physico-chemical study with lecithin phospholipids. Biochimica et Biophysica Acta 2009, 1788, 1296–1303. (32) Riechers, B.; Wittbracht, F.; H¨ utten, A.; Koop, T. The homogeneous ice nucleation rate of water droplets produced in a microfluidic device and the role of temperature uncertainty. Physical Chemistry Chemical Physics 2013, 15, 5873–5887. (33) Provencher, S. W. CONTIN: A general purpose constrained regularization program for inverting noisy linear algebraic and intergal equations. Computer Physics Communications 1982, 27, 229–242. (34) Berne, B. J.; Pecora, R. Dynamic Light Scattering; Wiley, New York, 2000. (35) Zhang, F.; Ilavsky, J.; Long, G. G.; Quintana, J. P.; Allen, A. J.; Jemian, P. R. Glassy carbon as an absolute intensity calibration standard for small-angle scattering. Metallurgical and Materials Transactions A 2010, 41A, 1151–1158. (36) Guinier, A.; Fournet, G. Small-angle scattering of X-rays; Wiley, New York, 1955. (37) Kratky, O.; Porod, G. Diffuse small-angle scattering of X-rays in colloid systems. Journal of Colloid Science 1948, 4, 35–70. (38) Bergmann, A.; Fritz, G.; Glatter, O. Solving the generalized indirect Fourier transformation (GIFT) by Boltzmann simplex simulated annealing (BSSA). Journal of Applied Crystallography 2000, 33, 1212–1216. (39) Orthaber, D.; Glatter, O. Synthetic phospholipid analogs: a structural investigation with scattering methods. Chemistry of Physics of Lipids 2000, 107, 179–189. (40) Helenius, A.; Simons, K. Solubilization of membranes by detergents. Biochimica et Biophysica Acta 1975, 415, 29–79.


Page 32 of 35

Page 33 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

(41) Urbaneja, M. A.; Alonso, A.; González-Ma˜ nas, J. M.; Go˜ ni, F. M.; Partearroyo, M. A.; Tribout, M.; Paredes, S. Detergent solubilization of phospholipid vesicle-Effect of electric charge. Biochemical Journal 1990, 270, 305–308. (42) Genz, A.; Holzwarth, J. F.; Tsong, T. Y. The influence of cholesterol on the main phase transition of unilamellar dipalmytoylphosphatidylcholine vesicles: A differential scanning calorimetry and iodine laser T-jump study. Biophysical Journal 1986, 50, 1043–1051. (43) Lygre, H.; Moe, G.; Holmsen, H. Interaction of ibuprofen with eukaryotic membrane lipids. Acta Odontologica Scandinavica 2003, 61, 303–309. (44) Boggara, M. B.; Krishnamoorti, R. Small-angle neutron scattering studies of phospholipid NSAID adducts. Langmuir 2010, 26, 5734–5745. (45) Boggara, M. B.; Faraone, A.; Krishnamoorti, R. Effect of pH and ibuprofen on the phospholipid bilayer bending modulus. Journal of Physical Chemistry B 2010, 114, 8061–8066. (46) Finegold, L. X. Cholesterol in Membrane Models; CRC Press, Boca Raton, 1992. (47) Arriaga, L. R.; López-Montero, I.; Ignés-Mullol, J.; Monroy, F. Domain-growth kinetic origin of nonhorizontal phase coexistence plateaux in Langmuir monolayers: compression rigidity of a raft-like lipid distribution. The Journal of Physical Chemistry B 2010, 114, 4509–4520. (48) Marsh, D. Genral features of phospholipid phase transitions. Chemistry of Physics of Lipids 1991, 57, 109–120. (49) Cho, N.-J.; Hwang, L. Y.; Solandt, J. J. R.; Frank, C. W. Comparison of extruded and sonicated vesicles for planar bilayer self-assembly. Materials 2013, 6, 3294–3308.


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(50) Hunter, D. G.; Frisken, B. J. Effect of extrusion pressure and lipid properties on the size and polydispersity of lipid vesicles. Biophysical Journal 1998, 74, 2996–3002. (51) Howard, F. B.; Levin, I. W. Lipid vesicle aggregation induced by cooling. International journal of molecular sciences 2010, 11, 754–761. (52) Br¨ uning, B.; Stehle, R.; Falus, P.; Farago, B. Influence of charge density on bilayer bending rigidity in lipid vesicles: a combined dynamic light scattering and neutron spin-echo study. European Physical Journal E 2013, 36, 77. (53) Ahlin, P.; Kristl, J.; Kristl, A.; Vreˇcer, F. Investigation of polymeric nanoparticles as carriers of enalaprilat for oral administration. International Journal of Pharmaceutics 2002, 239, 113–120. (54) Karlovská, J.; Williams, A. A.; Macri, R. V.; Gandour, R. D.; Funari, S. S.; Uhr´ıková, D.; Balgav` y, P. Synchrotron SAX and WAX diffraction study of a hydrated very long-chain, dendritic amphiphile + DPPC mixture. Colloids and Surfaces B: Biointerfaces 2007, 54, 160–164. (55) Cunningham, B. A.; Brown, A.-D.; Wolfe, D. H.; Williams, W. P.; Brain, A. Ripple phase formation in phosphatidylcholine: Effect of acyl chain relative length, position, and unsaturation. Phys. Rev. E 1998, 58, 3662–3672. (56) Akabori, K.; Nagle, J. F. Structure of the DMPC lipid bilayer ripple phase. Soft Matter 2015, 11, 918–926. (57) Arriaga, L. R.; López-Montero, I.; Monroy, F.; Orts-Gil, G.; Farago, B.; Hellweg, T. Stiffening effect of cholesterol on disordered lipid phases: A combined neutron spin echo + dynamic light scattering analysis of the bending elasticity of large unilamellar vesicles. Biophysical Journal 2009, 96, 3629–3637.


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Graphical TOC Entry Aescin Incorporation & Fluidization

Lo

h Hig

wC

Mid

c

on

Phase separation

Con

c

c Con

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Langmuir

Aggregation & Phase Separation

Micelles


Aescin Incorporation and Nanodomain Formation ... - ACS Publications

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