Interaction of the Saponin Aescin with Ibuprofen in DMPC Model

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Interaction of the Saponin Aescin with Ibuprofen in DMPC Model Membranes Ramsia Sreij, Sylvain François Prévost, Rajeev Dattani, Carina Dargel, Yvonne Hertle, Oliver Wrede, and Thomas Hellweg Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00421 • Publication Date (Web): 13 Aug 2018 Downloaded from http://pubs.acs.org on August 16, 2018

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Molecular Pharmaceutics

Interaction of the Saponin Aescin with Ibuprofen in DMPC Model Membranes †

Ramsia Sreij,

‡,¶

Sylvain Prévost,

Hertle,

†

Carina Dargel,

Oliver Wrede,

†

†

Rajeev Dattani,

and Thomas Hellweg

‡

Yvonne

∗,†

†Physical and Biophysical Chemistry, Bielefeld University, Universitätsstr. 25, 33615

Bielefeld, Germany ‡ESRF-The European Synchrotron, 71, Avenue des Martyrs, 38043 Grenoble Cedex 9,

France ¶present address: Institut Laue-Langevin, 71 avenue des Martyrs, 38042 Grenoble Cedex 9,

France E-mail: [email protected]

Phone: +49 (0)521 1062055. Fax: +49 (0)521 1062981

Abstract In the present work we study the interaction of the saponin aescin with the nonsteroidal anti-inammatory drug (NSAID) ibuprofen at concentrations of 1.5-2.5 mM. These amounts are higher than usually used for medication (10-300 µM) to show possible structures and formulations for orally absorbed drug delivery systems. It is shown how the interaction of both substances, separately or together, alters the thermotropic phase behaviour of the DMPC bilayer in the presence of dierent amounts of aescin ranging from 20 µM to 1 mM. Methods of choice are dierential scanning calorimetry (DSC), and additionally wide-angle (WAXS) and small-angle X-ray scattering (SAXS). We found that these two additives, aescin and ibuprofen, alter the 1

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temperature-dependent structural appearance of the DMPC membrane depending on aescin and drug content. The presence of the saponin and the drug become visible on dierent length scales, i.e. ranging from a global structural change to inner-membrane interactions. DSC reveals that the drug and saponin alter the cooperativity of the DMPC phase transition in a concentration-dependent manner. Furthermore, there is a signicant dierence between the drug-containing compared to the drug-free systems. By WAXS we could resolve that aescin reverses the strong impact of ibuprofen on the WAXS diraction peak of DMPC. Both molecules interact strongly with the phospholipid head groups. This becomes visible in a changing area per lipid and shifting phase transition to higher temperatures. SAXS experiments reveal that the addition of ibuprofen leads to major morphological changes in the phospholipid bilayer. SAXS experiments performed on representative samples do not only cover the drug-saponin interaction within the bilayer from the structural perspective but also conrm the visually observed macroscopic concentration and temperature-dependent phase behaviour. Vesicular shape of extruded samples is conserved at low aescin contents. At intermediate aescin content aggregation between SUVs occurs whereby the strength of aggregation is reduced by ibuprofen. At high aescin contents DMPC bilayers are solubilized. The kind of formed structures depends on temperature and drug content. At low temperature separated bilayer sheets are formed. Their size increases with ibuprofen in a concentration-dependent manner. At high temperature, the drug-free system reorganizes into stacked sheet. Whereas sheets at 5 mol % ibuprofen seem to close to vesicles the ones with 10 mol % of the drug increase massively in size. Altogether, ibuprofen was found to rather enhance than inhibit structural and thermotropic membrane modications induced by the aescin on the DMPC model membrane.

Introduction Saponins are a diverse group of glycosidic compounds widely distributed in many plants. 1 Despite their large structural diversity saponins share some unique properties and biologi2


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cal activities including the ability to foam, as well as to lyse erythrocytes. 2 Their surface activity responsible for the biological properties is attributed to their characteristic structural features and amphiphilic nature. The molecular structure is constituted of a lipophilic sapogenin linked to one or more sugar chains. 2 Aescin, which is the key compound in this study, is a mixture of saponins extracted from the seed of the horse chestnut tree Aesculus

hippocastanum. 3 Its molecular structure is built by a triterpenic scaold to which a glucuronic acid and two glucose molecules are attached. The molecular structure of the β -form used in this work is shown in Figure 1(b), adapted from Wilkinson et al. and Sreij et al. 3,4 OH OH

+

N

OH

O

O

O

HO

OH O

O

HO

H O

O OH

OH

O O + P O O -

O

O

HO

OH OH

O

O

OH OH OH

O

O O

O

O

(a) DMPC (b) β -aescin (c) ibuprofen Figure 1: Molecular structures of (a) DMPC, (b) β -aescin, and (c) ibuprofen. It is well established for treatment for conditions such as e.g. varicose veins, hemorrhoids, inammation of veins, diarrhoea, fever, rheumatism and neuralgia. 3 The hemolytic activity of saponins is generally attributed to their interaction with cholesterol in the red blood cell membrane. 57 Cholesterol is a key compound in cell membranes in which its fraction can be as high as 50 mol% of the total lipid content. 8 In addition, there are some reports on the complex phase behavior in cholesterol-saponin mixtures. 9,10 Compounds isolated from 3



the aescin mixture have demonstrated potent anti-inammatory eects, especially on the early stages of induced inammation. 11,12 Furthermore, β -aescin has shown sucessfully to have the capability to reverse P-gp-dependent (P-glycoprotein) multidrug resistance through inhibition of the GSK3β /β -catenin pathway in cholangiocarcinoma. 13 Kimura et al. 14 have furthermore tested the eect of aescin on the blood glucose level of mice and have shown an inhibitory eect on the pancrease lipase. They determined IC50 values (i.e. the concentration required for 50 % inhibition) to lay for aescin between 14-64 µg/mL. Ibuprofen (2-(4-isobutylphenyl) propionic acid) belongs to the class of non-steroidal antiinammatory drugs (NSAIDs). 15 This family of compounds is classied into several subgroups, based on their chemical structure. These are for example salicylates (aspirin, diunisal), phenylacetic acids (diclofenac), propionic acids (ibuprofen, ketoprofen), indoles (indomethacin), oxicams (piroxicam, meloxicam), pyrazoles (phenylbutazone) and sulphonanilides (Nimesulide). 16,17 The therapeutic action of NSAIDs is based on their characteristic as non-specic inhibitors of the cyclooxygenase pathway of inammation. 16 They possess the anti-inammatory eect by inhibiting the cyclooxygenase (COX) enzymes catalyzing the biosynthesis of prostaglandines. However, the long-term use of these drugs causes not only resistance with time but also gastrointestinal toxicity due to non-selective inhibition of COX-1 and COX-2. 17,18 The gastrointestinal tract injury is due to ulcer formation and gastric bleeding by prolonged inhibition of COX-1. 19,20 While COX-1 posseses regulatory eects on platelet aggregation and gastric mucous biosynthesis, COX-2 is involved in pathological conditions such as inammation, pain, and fever. 19 The physiological activity results from the amphiphilic character of the molecules: they have a water-soluble head group and a hydrophobic portion. The surface activity of these compounds results in their interaction with the cell membrane, inducing changes of its structural and dynamical properties. 21,22 Amongst others, ibuprofen is a well-established NSAID, exhibiting anti-inammatory, antithrombotic, analgesic, and antipyretic properties. 23 Its molecular structure is presented in Figure 1(c). The eect of ibuprofen on human cells was investigated by Manrique-Monero 4


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et al. 24 by performing in vitro experiments in human promyelocytic leukemia and human carcinoma cells. Ibuprofen concentrations between 10 µM and 300 µM were studied and showed that already an ibuprofen concentration as low as 10 µM changes the shape of red blood cell membranes remarquably. A study by Gaikwad et al. on the anti-inammatory activity of the Madhuca longifolia seed saponin mixture indicated that the anti-inammatory eect of this saponin mixture may be due to eective inhibition of histamine/bradykininhe or prostaglandin synthesis, hence the mediators of inammation. 25 In consequence, studies dealing with both kinds of components and revealing the combined eect on biological membranes should indicate possible treatment conditions and health eects. Many NSAIDs such as ibuprofen are freely available for self-medication and are among others the most prescribed drugs for pain and fever-reduction. 24 Saponins such as aescin are available in daily foods and in dietary supplements and are consumed simultaneously to NSAIDbased medication. Investigation of the structural and thermotropic behavior of ibuprofen containing bilayers in the presence of the saponin aescin from the physico-chemical perspective should yield important insight into the interaction mechanism between these compounds in lipid bilayers. This may indicate on treatment conditions and give an overview on the structural picture of mixtures consisting of saponins, NSAIDs, and phospholipids. It is the scope of this study to investigate the concentration dependent structural properties of small-unilamellar vesicles (SUVs) containing ibuprofen in the presence of the saponin. Wheareas the ibuprofen content varies between 1.2 mM (5 mol %) and 2.5 mM (10 mol %), the aescin content was varied between 0-1.4 mM (0-6 mol %). These ibuprofen contents are higher than those usually used for treatment (10 µM-300 µM 24 ). They were used to show possible formulations for possible orally absorbed drug-delivery systems and also show the eect of ibuprofen on the lipid model membrane beyond error bars. The analysis methods of choice were dierential scanning calorimetry (DSC), wide-angle X-ray scattering (WAXS), and small-angle X-ray scattering (SAXS). The SUVs are based on the phospholipid 1,2dimyristoyl-sn -glycero-3-phosphocholine (DMPC). The use of this phospholipid enables easy 5



experimental access to its gel and uid phase state so that phase state specic interactions of aescin and ibuprofen can be monitored and translated to other lipids. In previous studies we have investigated the theormotropic and structural impact of incorporated aescin on SUVs based on DMPC. 4,26,27 We found that aescin incorporates easily into the membrane and that the interaction is strongly dependent on both, aescin content and lipid phase state. 4 The bilayer is softened below the phospholipids main phase transition temperature (Tm ;

Tm (DMPC) ≈ 23.6 ◦ C), and rigidied above it. 26 Bilayer softening was assumed to arise from hydrogen bonding and electrostatic interactions between the saponins sugar groups and the phospholipid head group, as already derived by other groups for dierent saponins. 28 Similarities were found to the interactions of NSAIDs with phospholipid bilayers. 16,29,30 Furthermore, we found that addition of aescin leads to phase segregation probably by formation of aescinrich and aescin-poor domains within the bilayer. In these former studies 4,26,27 an overall structural picture of DMPC-aescin mixtures was elaborated and a strong concentrationdependent phase behaviour was found. At lower aescin contents (up to 0.8 mol %) relatively stable unilamellar vesicles are formed. 4 At intermediate aescin contents (1 to 6-7 mol %) a broad region of coexisting structures was found. In this region vesicles begin to aggregate, deform and rst bilayer fragments become solubilized by the saponin. 4 At higher aescin contents (>6 mol %) we found by rst visual inspection 4 and up to know unpublished data that complete solubilization and a temperature-dependent structural reorganization occurs. Hence, in the present work we cover aescin contents at low and intermediate amounts before complete solubilization of the membrane. We present the evolution of structural parameters of the DMPC membrane as function of the temperature, and concentration of additives. The analysis techniques employed are DSC, SAXS, and WAXS. By DSC phase segregation becomes visible in an ibuprofen and aescin concentration dependent manner. WAXS shows that the presence of aescin alters the diraction spectrum of only ibuprofen and indicates therewith possible competing eects or interaction between these two compounds when they are in phospholipid environment. By SAXS a possible overall structural picture of the sam6


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ples is generated. Vesicles which tend to aggregate are present at low aescin contents. The size of these vesicles increases with increasing ibuprofen content. At the highest aescin content studied (5 mol %) SAXS indicates a structural reorganization into disks or large sheets; however well above the main phase transition temperature of the phospholipid these disks close and vesicles are recovered.

Materials and Methods Materials 1,2-dimyristoyl-sn -glycero-3-phosphocholine (DMPC) was purchased from Lipoid GmbH (> 99 %, Ludwigshafen, Germany). The pure saponin aescin (≥95 %, CAS number 6805-41-0), ibuprofen (≥98 %, 2-(4-Isobutylphenyl)propanoic acid) and chloroform were obtained from Sigma Aldrich (Munich, Germany). The purity of the aescin powder was conrmed by mass spectrometry. All samples were prepared with deionized, puried water (Sartorius arium VF pro, Göttingen, Germany). Since aescin dissolves poorly in water and decreases the pH signicantly, a 50 mM phosphate buer with pH 7.4 was used to guarantee a constant pH and solubility in aqueous solution. The DMPC mass concentration used was 15 g·L−1 .

Sample preparation DMPC was dissolved in chloroform, subsequently evaporated with a rotary evaporator and stored over night at 60 ◦ C for complete chloroform evaporation. In ibuprofen containing samples the drug was mixed together with DMPC and dissolved in chloroform. The thin lipid lm generated on the ask wall was subsequently hydrated with an aescin solution in phosphate buer at nal aescin concentration. The multilamellar vesicles obtained (MLVs) (at aescin contents below 6 mol%) were enlarged with ve freeze-thaw cycles in liquid nitrogen and warm water. Afterwards, the MLVs were transformed into small unilamellar vesicles (SUVs) by extrusion (21 ×) using an extruder (Avanti Polar Lipids Inc., Alabama, USA) 7



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with a polycarbonate membrane having a pore diameter of 500 Å (Whatman, Avanti Polar Lipids, Alabama, USA). In the following, the aescin content used (in mol %) is dened with respect to the total amount of DMPC, ibuprofen, and aescin (x(aescin) =naescin /(naescin +

nDM P C + nibuprof en )). The ibuprofen content is also given in respect to DMPC and ibuprofen (x(ibuprofen) =nibuprof en /(nDM P C + nibuprof en )).

Dierential scanning calorimetry (DSC) DSC experiments were carried out to investigate the thermotropic phase behavior of the lipid vesicles on a dierential scanning heat-ow calorimeter (Q100, TA Instruments, New Castle, USA, for calibration see 31 ). The samples (≈20 mg) were hermetically sealed into aluminum pans (TA Instruments). The sample was heated to 40 ◦ C, thermalized for 8 min, cooled to 7 ◦ C with a constant cooling rate of δ = 7 ◦ C/min and nally thermalized at 7 ◦ C for 8 min again prior to the experiment. The thermograms were then recorded between 7 ◦ C and 40 ◦ C with a scan rate of δ =0.50 ◦ C·min−1 , referenced to air (due to calibration conditions of the instrument). The dierence in heat ow ∆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,dif f =

∆P δ

(1)

Wide-angle X-ray scattering (WAXS) WAXS experiments were performed to obtain structural information on the sub-nm scale of the binary and ternary systems investigated. In scattering experiments, the scattering intensity is generally given by: I(q) = N ·(∆ρ)2 ·V 2 ·P (q)·S(q). Hence, the intensity detected depends on the scattering volume of the solute V , the scattering length density dierence

∆ρ = ρsolute − ρsolvent , the form factor P (q), and the structure factor S(q). The magnitude of the scattering vector is q =

4π λ

· sin(θ) with θ being half the scattering angle. Important 8


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parameters in structurally correlated systems such as the lipid bilayers investigated are e.g. the acyl chain correlation distance dW AXS . Both parameters can be derived from X-ray data when combining the scattering vector magnitude with the Bragg equation so that the following correlation is obtained:

d=

2πn . q0,n

(2)

Here, q0,n is the q−value of the nth-order Bragg diraction peak.

When the studied system is in the gel Lβ 0 phase, acyl chains of the lipid mixtures studied are packed in a two-dimensional hexagonal lattice. From this packing, the area per lipid (AL ) can be directly calculated from the position qW AXS of the chain-chain correlation peak by eq. 3, similar to the procedure by Eicher et al. 32,33

16π 2 AL = √ 2 3qW AXS

(3)

WAXS experiments of DMPC SUVs, samples containing 0.5 mol % and 1 mol % aescin and samples with 10 mol % ibuprofen with various amounts of aescin were performed in the temperature-range between 10 ◦ C and 50 ◦ C on the ID02 beamline at the ESRF synchrotron in Grenoble, France. 3436 The experiments were performed at a sample-to-detector distance of 0.12 m with a CCD Rayonix LX-170HS. The q−range covered was 0.6-4 Å−1 . Duration of the measurement was either 10x or 25×1 s for ibuprofen-free and ibuprofen containing samples, respectively. The wavelength used was 1 Å. Initial data treatment was done using the beamlines data reduction program package [DOI: 10.1107/S0021889807001100]. 36

WAXS experiments of the sample with 5 mol% aescin were performed on an inhouse SAXS/WAXS system (XEUSS, Xenocs, Sassenage, France) equipped with a CuKα source (λ =1.541 Å , GeniX ultra low divergence, Xenocs) and a Pilatus 300K hybrid pixel detec-

9



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tor (Dectris, Baden Deattwil, Switzerland). Data acquisition time was several hours per temperature. The 2D-data was analysed reduced the Foxtrot (V3.3.4.) software. The samples were measured in both cases in a ow-through Kapton capillary (1 mm, Good-Fellow GmbH, Bad Nauheim, Germany) positioned in a Linkam stage (Linkam Scientic, Tadworth, UK). The sample scattering was normalized with respect to incident intensity, sample thickness, acquisition time, transmission and background. The data was brought to absolute scale using either water or glassy carbon type 2 37 as standard.

Small-angle X-ray scattering (SAXS) Small angle scattering techniques yield information about the mesoscale structures in the sample. In the present case this is the vesicle shape and also length scales arising from vesicle vesicle interaction. The Porod regime is dened as q 1/Dmin with Dmin being the smallest dimension. 38 In this q -regime the scattered intensity I(q) is mainly the result of scattering from the interface between the aggregates and the solvent (eq. 4).

q 4 · I(q) = 2π∆ρ2 · ST

(4)

Here, ∆ρ2 is the contrast which describes the electron density dierence in the case of X-rays. ST is the total surface area of the aggregates per unit volume. As indicated by this equation, log(I(q)) decreases linearly with log(q ) with a slope of −4 in the Porod regime. When determining dierent slopes in the scattering curves in the log(I(q)) vs. log(q ) plot (e. g. I(q) ∼ q −m ), morphologies can be identied. The value of m reveals the systems overall structure. Values of m = 1, 2, 3, and 4 stand for cylinders, lamellae (disks), 3D fractal objects, and sharp interfaces (Porod's law), respectively. 39 Another approach to obtain model-independent information on the sample morphology is to calculate the pair distance distribution function p(r) from the scattering intensity. This function yields size and morphology in real space and was obtained by using the standard

10


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indirect Fourier transformation (IFT) method provided in the PCG software package from O. Glatter. 40 By integration over the p(r)-function, as shown in eq. 5, the radius of gyration is obtained.

R 2 RG

=

p(r)r2 dr R 2 p(r)dr

(5)

Small-angle scattering of a nite stack of correlated bilayers consist of sharp peaks at which the X-rays are scattered perpendicularly to the bilayers at values q0,h (eq. 2 with

n = h). The intensity is expressed by I(q) =∝ h|F (q)2 S(q)|i/q 2 . 41,42 q −2 is the Lorentz factor for unoriented powder samples. 41 S(q) (structure factor) takes the crystalline or quasicrystalline nature of the lattice of the bilayer stack in the liquid crystalline phase into account. R P (q) (form factor) along the z-axis is given by: F (q) = ρ(z) exp(iqz)z.. S(q) and P (q) are averaged with respect to bilayer uctuations. The simplest model which describes multilayered bilayers assumes a local spacing D with a mean value D and a mean square uctuation

δ 2 ≡ h(D − D)2 i. Zhang et al. 41 have shown that the electron density prole can be well represented by the modied Caillé theory (MCT). In this theory, the uctuations base on their energetics and on assumptions of arbitrary uctuations in the spacing between them. In this theory, the mean square uctuations (∆2n = n∆2 with bilayers separated by n − 1 intervening bilayers) diverge logarithmically with n. The tails of the scattering curves decay by S(q) ∼ (q −q0 )−1+η . The Caillé parameter η contains information about the bulk modulus of compression B and the bending modulus K and is given in eq. 6.

η=

2 qCaillé ·k ·T √ B 8π KB

(6)

This original Caillé theory was modied by Zhang et al. 41 in order to obtain better quantitative ts to the SAXS data. To extract Caillé parameters we analyzed some of the data by the generalized indirect Fourier transform (GIFT) method using the PCG software package. 40 S(q) was described by the modied Caillé theory with polydispersity. 43,44 This

11



model is based on four parameters: number of correlated bilayers (NoB), η , the interlamellar spacing dCaillé , and a diuse scattering term. SAXS curves were furthermore analysed by a model-dependent approach to conrm vesicular shape and to determine structural parameters of the samples. Accordingly, scattering curves of samples with low aescin contents were tted with the core multi shell (CMS) 4547 model with n = 3 shells, implemented in SASView. 48 Samples with higher aescin contents were analysed with the stacked disks 45,47,49,50 model, also implemented in SASView. 48 A schematic of structures and contrasts of both methods applied is shown in Figure S3 in the supporting information (SI). SAXS experiments for samples with 10 mol % ibuprofen and samples without ibuprofen but 0-1 mol % aescin were performed simultaneously to the WAXS experiments at the ID02 beamline at the ESRF synchrotron in Grenoble, France. 3436 The temperature was varied from 10 ◦ C to 50 ◦ C in steps of 5 ◦ C. The heating rate was 20 ◦ C/min. The q -range covered is 1.5·10−3 -0.6 Å−1 at detector distances of 1.4 m and 10 m. SAXS experiments of samples with 5 mol % ibuprofen and a sample without ibuprofen but 5 mol % aescin were measured at an inhouse SAXS/WAXS system (XEUSS, Xenocs, Sassenage, France) equipped with a CuKα source (λ =1.541 Å , GeniX ultra low divergence, Xenocs) and a Pilatus 300K hybrid pixel detector (Dectris, Baden Deattwil, Switzerland). The experiment was performed at two distances (0.8 m and 2.7 m). The q -range covered is 0.04-0.5 Å−1 . Data acquisition time was several hours per temperature and distance. The 2D-data was reduced using the Foxtrot (V3.3.4.) software. Inhouse SAXS/WAXS experiments were not performed simultaneously.

12


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Results and Discussion Phase behaviour Photographs of samples containing DMPC with ibuprofen amounts of 5 mol % and 10 mol % and varying aescin contents are shown in Figures S1 and S2 in the (SI), respectively. The aescin content covered ranges from 0-6 mol %. Photographs of samples without ibuprofen are reported elsewhere. 4 The samples were stored above the Tm of DMPC at 50 ◦ C and extruded above Tm through lters with 500 Å pores and subsequently lled into glass tubes. The samples were cooled to 6 ◦ C (shown in panels (a)), later heated to 37 ◦ C (panels (b)). The samples show a temperature and concentration dependent macroscopic phase behaviour which is observable by eye. At 5 mol % ibuprofen, stable SUVs are found for an aescin content 5 mol % samples are transparent at low temperature. This behavior is therewith similar to the ibuprofen-free system presented in the SI of a former study. 4 Transparent samples indicate that very small structures are formed at this saponin content. When increasing temperature, the sample becomes slightly bluish. This denotes reorganization to larger structures. At 10 mol % ibuprofen, stable SUVs are also found below 1 mol % for both temperatures. This is again indicated by the bluish color of the samples and no signicant color change upon temperature variation. At higher aescin contents, white coloring of the samples is observed which may indicate aggregation of vesicles, similar to samples with 5 mol % ibuprofen. When heating the samples, the white coloring reduces which denotes structural change in the samples to probably less correlated ones. Samples at aescin contents >5 mol % become transparent upon heating which may indicate formation of smaller structures such as e.g. sheets or micelles. The cooling-heating cycle was repeated several times for both samples to 13



conrm the reversibility of the phase behavior.

Dierential scanning calorimetry (DSC) The calorimetric analysis was performed in order to determine the heat ow associated with the lipid phase transitions. 51 It will be used to detect interactions between the incorporated molecules and DMPC. Here, especially the interactions inuencing the temperature-induced conformation changes from all-trans to partially-gauche conguration of the phospholipid hydrocarbon chains are visible. The analysis was performed for samples with aescin contents between 0 and 6 mol %. Figure 2 shows DSC endotherms for the binary system DMPC/aescin in panel (a) and the ternary systems with additional ibuprofen in panels (b) and (c) for 5 mol % and 10 mol % of the drug, respectively.

mol% aescin

mol% aescin

0.1

0.0 0.1

0.8 1.0 1.2 1.5 2.0

20

(a)

25

T / °C

0.1

0.8 1.0 1.2 1.5 2.0

-1

0.4

g

-1

g

0.6

0.2

0.6

-1

-1

0.6

0.5

-1

g

0.4

0.0

0.2

Cp, diff /J K

-1

0.2

mol% aescin

DMPC

Cp, diff /J K

0.3

DMPC

endo

DMPC

10 mol% ibuprofen

®

®

5 mol% ibuprofen

endo

endo

®

without ibuprofen

Cp, diff /J K

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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0.8 1.0 1.2 1.5 2.0

3.0

3.5

3.0

4.0

4.0

4.0

5.0

5.0

5.0

6.0

6.0

6.0

30

20

(b)

25

30

T / °C

20

(c)

25

30

T / °C

Figure 2: DSC endotherms for (a) the binary system DMPC/aescin and the ternary systems containing (b) 5 mol % and (c) 10 mol % of additional ibuprofen. The aescin content was varied for all samples between 0 mol % and 6 mol %, as indicated by the red numbers. The dotted blue line indicates the peak position of pure DMPC SUVs (blue endotherm). The scale bar applies for all panels. Please see the electronic version for color-encoding. The endotherms were recorded at a scan rate of 0.5 ◦ C/min from low to high temperature after fast and controlled cooling. The endothermic signal (blue curve) of the pure DMPC 14


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(a)

1.0 mol%

endo

®

without ibuprofen

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C

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(g)

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T / °C

Figure 3: Representative Lorentzian t to samples containing only aescin and 5 and 10 mol % ibuprofen in the aescin content range between 1 mol % and 1.5 mol %. Samples with (a)-(c) 1 mol % aescin, (d)-(f) 1.2 mol % aescin, and (g)-(i) 1.5 mol %. Aescin contents are given in red. Arrows point on characteristic features in the endotherms. The blue Lorentzian function corresponds to the aescin-poor phase transition of DMPC molecules, the green one to the one in an aescin-rich environment. Both types of phase transitions occur in the presence of ibuprofen in (b),(c), (e), (f), (h), and (g)). Please see electronic version for color-encoding. SUVs is composed of an intense sharp peak. This peak corresponds to the phosholipids main phase transition from the tightly packed Lβ 0 phase to the exible Lα phase 52 with

∆Hm =27 kJ mol−1 . 53 This transition shows in the DSC data as endothermic heat capacity. 16 The addition of aescin to the lipidic system induces the formation of an additional peak at lower temperatures. This peak increases in intensity with increasing aescin content, while the one corresponding to the main phase transition decreases, as can be seen for all samples in Figures 2(a)-(c). 4 Because the peaks are composed of multiple superimposed signals, onset temperatures were not determined but peak maxima were obtained by tting the data with Lorentzian functions. This analysis shows the evolution of the phase segregation and guarantees comparability of the peaks with multiple signals. The results are shown in Figures S3(a) and (b) in the SI for the right and left peaks, respectively. The analysis depicts signicant lowering of Tmax, right peak when (only) ibuprofen is added. Hence, the addition of only ibuprofen already has the ability to alter the cooperativity of the phase transition of the phospholipid: the gel phase is destabilized and the drug-lipid association 15



is conrmed. This is also in concordance with Moreno et al., 16 Du et al., 15 and ManriqueMoreno et al. 24 The addition of aescin seems to induce minor changes of Tmax, right peak which however remain within experimental error bars. This is valid for all ibuprofen concentrations studied. The main eect of aescin becomes visible at higher aescin contents in the second peak (Tmax, left peak ). Here, a lowering of around 1 ◦ C is observed for all ibuprofen contents studied and shows that this phase segregation is mainly induced by the aescin in the mixtures. Moreno et al. 16 and Manrique-Moreno et al. 24 showed similar eects, i.e. formation of two peaks, in the DSC thermograms when they investigated the phenomenon of phase separation for high contents of some NSAIDs (ibuprofen, naproxen and diclofenac) embedded in phospholipid membranes. They found in these studies that such DSC endotherms may result from phase segregation and that the key parameter leading to this phenomenon is the formation of strong hydrogen bonds between the hydrophilic portion of the drug and the phospholipids phosphocholine group, as also expected for the system studied in this work. 16,28 The presence of head-group interactions between phosholipid and saponin are expected for the system investigated here, as derived for the saponin Quil A by Demana et al. 28 Moreover, the tendency for phase segregation, and hence for aescin molecules to assemble into nanometer-sized domains can also be explained by the similarities of the structure of the hydrophobic domain to bile salt molecules. 5457 The presence of hydrophilic groups on the triterpenic backbone of aescin is assumed to lead to formation of aescin complexes in the form of dimers, trimers or tetramers. 27,57 In these assemblies, the polar parts and with that the polar side of aescin, is turned inside and is shielded from the hydrophobic environment formed by the hydrocarbon chains of DMPC. The apolar side is turned outward. Moreover, Tsibranska et al. demonstrated very recently by MD simulations that aescins tendency to self-assemble into compact clusters is promoted by long-range attractive interactions between hydrophobic aglycones, combined with intermediate dipole-dipole and short-range hydrogen bonds between sugar residues in aescin molecules. 58 Phase segregation phenomena become especially signicant and interesting between 116


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1.5 mol %. Endotherms in this concentration range are enlarged in Figure 3. The two peaks are presented by Lorentzian functions to help identifying them (right: blue and left: green ) shown in Figures 3(a)-(i) for aescin contents of 1.0 mol %, 1.2 mol %, and 1.5 mol %. Areas of these curves indicate small increasing values for the green and decreasing ones for the blue curve and are listed in Table S1 in the SI. Whereas the blue peak stems from DMPC molecules in an aescin-poor environment, the green one arises from aescin-rich ones. Therefore, we observe that the number of aescin-rich domains increases with increasing aescin content, as can be also seen in Figure 2 in a concentration-dependent manner. The lowering of the transition temperature of DMPC molecules in an aescin and ibuprofen-rich environment can be explained by the disturbed cooperative phase transition of the acyl chains by the inserted molecules: the well-ordered acyl chain packaging is disturbed and the transition takes place at lower temperature. The addition of higher amounts of aescin into the system induces broadening of the signal to higher temperature (yellow ) and the emergence of another (pink ) peak left to the green signal, as depicted for an aescin content of 3 mol % in Figures 4(a) and (b) with and without ibuprofen, respectively. The pink signal is especially pronounced at higher drug content and indicates destabilization of the phospholipid gel phase at already lower temperatures. Hence, higher amounts of aescin enhance the phospholipids ordered gel phase. This eect is independent of the ibuprofen content. The endothermic data show furthermore a broad signal which spreads mainly towards higher temperature (see yellow Lorentzian). The existence of such a broad signal and thus also the expansion of the signal to higher temperatures was reported for e.g. cholesterol containing systems 8 and may be caused by hydrophobic interactions between aescins backbone and the phospholipid bilayer. Observations of peak shifting of a main sharp signal and simultaneous appearance of a broad underlying signal were also made for cholesterol containing bilayers by Genz et al. 8 These authors explain the observation by insertion of the sti steroid scaold into the membrane which impacts the phospholipid conformation and 17



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Page 18 of 47

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Figure 4: DSC endotherms of samples containing 3 mol % aescin. (a) Sample without ibuprofen. (b) Sample with 10 mol % ibuprofen. Peaks were tted with three Lorentzian functions. The green function corresponds to the green one in Figure 3. Arrows point on characteristic features of the signals. Please see electronic version for color-encoding. induces earlier transition into the uid (Lα ) phase state. The broad underlying signal was assigned to the hydrophobic interactions between the lipophilic moieties of the components, which can also be expected for the interactions between aescin and DMPC. Altogether, the DSC data revealed appearing microphase separation which is related to ibuprofen and aescin in a concentration-dependent manner. The presence of ibuprofen seems not to inhibit but rather to enhance or modify the interaction of aescin and the DMPC model membrane. In respect to images in Figures S1 and S2 in the SI, we assume that segregation leads to stronger attractive interaction between vesicles. The presence of multiple peaks is especially enhanced at aescin contents between 1 mol % and 4 mol % aescin. These concentrations develop the most intense whitish color upon cooling.

Wide-angle X-ray scattering (WAXS) In the following we want to give insights into structural properties of these systems which can be derived from wide-angle X-ray scattering (WAXS) experiments. X-ray based tech-

18


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Figure 5: Wide-angle X-ray scattering (WAXS) spectra at temperatures around the main phase transition temperature Tm of DMPC and aescin containing samples. (a) DMPC and (b)-(f) in the presence of 10 mol% ibuprofen with 0.5, 1.0, 2.0, and, 5 mol% aescin, respectively. Samples without addition of the ibuprofen are shown in the panels (g)-(i) for 0.5, 1.0, and 5.0 mol% aescin, respectively. WAXS data in (a)-(h) were measured at the ID02 beamline. WAXS data in (i) were measured on the XEUSS inhouse system. Please see electronic version for color encoding. niques are sensitive to electron density dierences in the investigated structure, therefore, well suited to investigate the interfacial region of aggregates composed of e.g. amphiphilic molecules, with electron-rich polar head-groups and electron-poor apolar chains. 59

WAXS experiments were performed on the binary systems DMPC/aescin and on the ternary system DMPC/aescin and 10 mol% of ibuprofen. At this ibuprofen concentration the most prominent impact is expected. This method is very suitable to derive structural information about acyl chain correlations dW AXS in the lateral plane of the membrane on the Å-scale. 6062 In Figure 5, WAXS diraction patterns are shown in the q -range between 0.6

19



Figure 6: Scheme depicting in (a) the temperature dependent evolution of dW AXS below (left) and above(right) Tm and in (b) a possible impact on conguration of phospholipid molecules in the presence of ibuprofen molecules. Polar parts of ibuprofen (green) interact with DMPC's head groups (blue) from the inside of the bilayer, leading to multiple headto-head congurations. This becomes visible in the acyl-chain correlation peak analyzed by dW AXS . and 2.5 Å−1 for dierent temperatures, highlighting the diraction peak which corresponds to the distance dW AXS . As expected from literature 33,63 and shown in our our results, Figure 5 reveals a narrow peak at low temperatures, i.e. below Tm . Above Tm the peak suddenly broadens. This is observed for all samples with an aescin content below 1 mol % and holds for the data in panels (a)-(e) and (g) and (h). Here, the intensities of the signals have a nearly equal height and the transition from the Lβ 0 to the Lα phase is sharp. At higher aescin contents (>2 mol %), the signal intensity at temperatures 15 ◦ C and 20 ◦ C decreases signicantly with increasing temperature and the one at 25 ◦ C increases with respect to higher ones (see panels (e), (f), and (i)). This indicates a smoother transition from the gel into the uid phase. More quantitative information is discussed below (in Figure 7). The WAXS signals of the sample in panel (b), i.e. DMPC SUVs with 10 mol % ibuprofen, exhibit a shoulder on the right side of the peak in addition to the regular signal. It is located at larger q - values and therefore represents distances at smaller correlation length d. This

20


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may arise from the strong drug-phospholipid association as described by Moreno et al. 16 and Du et al. 15 Moreover, Boggara et al. 29 investigated ibuprofen incorporation into DMPC SUVs by means of contrast-variation small-angle neutron scattering (SANS). In their study they reveal that the small and rigid ibuprofen molecule is fully incorporated into the hydrophobic membrane part near the hydrocarbon chains. Furthermore, they state that there is strong interaction between the drugs polar groups with DMPC's phosphocholine head group from the inside of the bilayer. This eect leads to membrane thinning. When comparing these assumptions to our results, intercalation of DMPC molecules after ibuprofen incorporation is very likely. A possible conguration is depicted in Figure 6. This is in agreement with our DSC results. The incorporation of ibuprofen into the hydrophobic moieties disrupts the cooperative phase transition of the acyl chains and shifts the endothermic signal therewith to lower temperatures. When aescin is added, this shoulder disappears. Due to the fact that both molecules possess strong anity to interact with phospholipid head groups it is possible that competition for the interaction with the phosphocholine head groups takes place by changing hydration of head groups or by e.g. binding of ibuprofen molecules to aescin. This certainly aects the acyl chain arrangement and therewith the shape of the diraction peak. To get a closer look onto the ibuprofen-aescin interactions, the positions of the peak maxima were analyzed by eq. 2. Results are presented in Figure 7(a) for samples without and in Figure 7(b) for samples with ibuprofen. When comparing the results of the binary system DMPC/aescin in panel (a) to each other, we see that dW AXS of samples changes in a thermoresponsive manner. The most prominent impact occurs around Tm of DMPC, i.e. between 20 ◦ C and 30 ◦ C. To derive quantitative information on the transition temperature, each data set was tted with a sigmoidal t (solid lines). The temperature Tm(W AXS) was derived from the inection point of this t and the results are summarized in Figure 8(a).

Tm(W AXS) increases with increasing aescin content by a relatively strong increase upon an incorporation of 1 mol% aescin. Further incorporation shows continuous impact with minor 21



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T / °C Figure 7: dW AXS calculated from peak maxima in Figure 5 by eq. 2. The gure shows in (a) samples without and in (b) samples with 10 mol % ibuprofen for dierent aescin contents. Red numbers in (b) denote aescin conent in mol %. Solid lines are sigmoidal ts to the data. increase of this temperature as before. With respect to DSC thermograms and macroscopic images in Figure S2 in the SI, we would like to remind that an aescin content of around 1 mol % denotes the threshold concentration between stable SUVs and aggregates. Whereas below this content phase segregation is not or only weakly pronounced aescin domains are present in a pronounced form above 1 mol %. Hence, the evolution of Tm may indicate the evolution or increasing fraction of aescin domains inhibiting DMPC acyl chains to undergo conformational transition. Increasing values of Tm may be due to hydrophobic interactions, similar to the steroid-acyl chain interactions for cholesterol as shown by Genz et al. 8 These authors also showed by DSC that the main phase transition does not show a signicant temperature shift. This is similar to the evolution of the main peak shown in Figure S3(a) in the SI. When the phospholipid is in the gel phase, i.e. for DMPC at 10 ◦ C, 63,64 the area per 22


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Figure 8: (a) Tm(W AXS) obtained from the inection point of the sigmoidal t in Figure 7. (b) Area per lipid molecule AL calculated from the position of q0,n of the acyl chain correlation peak at 10 ◦ C by eq. 3 while assuming hexagonal packing. Dotted lines are guides to the eye. molecule AL can be calculated for the hexagonally packed molecules by eq. 3. 33 Results are presented for the samples measured at 10 ◦ C in Figure 8(b). Tristam-Nagle et al. reported a value of AL of 47±0.5 Å2 for gel phase DMPC, also from X-ray diraction experiments. 63 The addition of aescin leads to an increasing AL with increasing aescin content denoting successful incorporation of the saponin. Comparison of ibuprofen containing samples outlines dierences to the drug-free system. Figure 7(b) shows acyl chain correlations of ibuprofen containing samples and conrms thermoresponsiveness of the system studied on the Å-scale. The comparison of the dW AXS -values of the aescin-free sample with 10 mol % ibuprofen (blue) to DMPC (black) indicates that the main impact occurs at high temperature where the measured dW AXS correlations of the phospholipid molecules are signicantly reduced. Du et al. stated that the rigid drug can incorporate and intercalate into the lipid bilayer more easily when it is in its rather exible uid state. Here, we can conrm with our results that on the Å-scale the eect of ibuprofen incorporation is much more pronounced in the Lα phase. The addition of aescin is visible at all temperatures but especially around Tm , as indi23



cated by an arrow in Figure 7(b). Samples containing additional aescin exhibit values of

dW AXS in the Lα phase which are more similar to those of pure DMPC than to those of the sample with 10 mol % ibuprofen. As previously discussed, this eect may result from either competing interactions between saponin and aescin with the phosphocholine groups or probable attractive interaction between drug and saponin.

Results for Tm(W AXS) and AL are shown in blue color in Figures 8(a) and (b), respectively. The comparison of Tm(W AXS) in panel (a) to ibuprofen-free samples, shows that the increase of this transition temperature is more gentle for the ibuprofen containing samples at addition of initial aescin contents. In concordance with DSC results and what was derived from e.g. Figure 3 the presence of ibuprofen reduces the drastic eect of aescin on e.g. phase segregation. In respect to the evolution of AL compared to aescin-free samples similar values and trends are obtained for both systems. This denotes that the presence of aescin has a more pronounced impact on AL in the DMPC gel state than ibuprofen.

Small-angle X-ray scattering (SAXS)

Structural parameters of SUVs. SAXS experiments were performed at dierent temperatures to demonstrate the eect of added ibuprofen and aescin on structural parameters of unilamellar vesicles i.e. radius and bilayer thickness. This method will also be used to scrutinize sample morphology in a concentration-dependent manner with respect to drug and aescin content. At low aescin content, i.e. below 1 mol %, samples are expected to have vesicular shape after extrusion. Form factors of samples without the drug and with 10 mol % ibuprofen in gel state DMPC at 10 ◦ C and in uid phase DMPC at 40 ◦ C with 0 mol %, 0.5 mol %, and 1 mol % aescin are shown in Figures 9(a) and (b), respectively. They exhibit a typical shape of unilamellar vesicles with the overall spherical shape at low-q and the bilayer structure at high-q . Scattering curves for the same samples at additional temperatures ranging from 10 ◦ C to 50 ◦ C 24


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Figure 9: SAXS curves of DMPC SUVs and SUVs containing 10 mol % ibuprofen and 0.5 mol % and 1 mol % aescin with and without the drug at (a) 10 ◦ C and (b) 40 ◦ C. DMPC is on absolute scale (cm−1 ), all other data are scaled by the gray numbers. Solid lines are core multi shell (CMS) ts. SAXS curves of temperatures between 10 ◦ C and 50 ◦ C are presented in Figure S6 in the SI. Please see electronic version for color encoding. are shown in Figure S5 in the SI. SUVs with 5 mol % ibuprofen are shown in Figure S6 in the SI for temperatures between 10 ◦ C and 40 ◦ C. The SUV form factors were analyzed by the core multi shell (CMS) model from SASView. 48 The CMS model ts are represented by solid lines in the corresponding gures. A schematic of the model used is depicted in Figure S4 in the SI. The model is based on three shells whereby the inner and outer shell correspond to the DMPC head groups. The middle shell represents the hydrophobic portion of the bilayer which is composed of two units of the phospholipid hydrocarbon chains. The X-ray scattering length densities (XSLD) for DMPC were calculated based on molar volume and density measurements by Nagle et al. 64 The parameters necessary for the calculation are given in Table S2 in the SI. A detailed description of the calculations is given elsewhere. 27 Some of the parameters of the model are not independent and were therefore xed during the

25



Figure 10: Morphological parameters from CMS ts of SAXS scattering curves in Figure 9, S5 and S6. (a) Core radii Rcore and (b) hydrophobic thickness dtail . Both parameters were obtained through analysis of the SAXS curves by the CMS model. Dashed lines are guides to the eye. Rcore values above 40 ◦ C may have large error-bars because of the poor data statistics and limited q− range. t to obtain stable results. These are the XSLDs for the inner and outer shell (XSLD(head)) and the XSLD of the middle shell (XSLD(tail)). The specic values were calculated for each sample in dependence of the composition by taking into account the added compounds by their mole fraction. The XSLD of the middle shell considers the hydrophobic portions of DMPC and aescin and the ibuprofen molecule as entity. Ibuprofen has been found by other authors to be located in the hydrophobic portion of the bilayer. 15,29 The XSLDs of the inner and outer shells were calculated by taking the XSLD of the DMPC head group and the one of aescins head group into account. Here, XSLD(head) remains constant with temperature while XSLD(tail) is temperature-corrected due to the temperature-dependent volume change of DMPC. Specic values are summarized in Table S4 in the SI. The thickness of the head groups dhead was set constant for the inner and outer shells and all samples to

26


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a value of 7 Å. 65 The hydrophobic thickness dtail was determined through tting. Because the scattering intensity of SAXS curves is very sensitive to small deviations in XSLDs of the solvent, these values (XSLD(core) and XSLD(solvent)) were also tted. Initial values were calculated temperature-corrected ones for pure H2 O (see Table S3 in the SI). The elevation of these values may result either from from changed composition of the solvent by e.g. solute aescin molecules or salt. However, because the XSLD values are already slightly higher for the aescin-free samples we assume that the composition of the buer already has an impact on them. Moreover, a deviation of the calculated XSLD values for the membrane parts from the real ones may also cause such XSLD values for the solvent due to nally signicant dierence of all XSLDs determining the nal shape of the t. An additional scaling factor and a background were treated as adjustable parameters. The most crucial results of this analysis are the core radius Rcore of the vesicle and the hydrophobic thickness dtail . Both parameters were tted with polydispersity (PDI) by choosing a Schulz distribution for Rcore 66 and a Gaussian distribution for dtail . The values PDI(radius) for the distribution of the core radius are summarized in Table S4 in the SI and lay mostly between 0.18-0.35. Values of PDI(dtail ) did not change signicantly and remained around 0.1. The results of these two parameters (Rcore and dtail ) are summarized in Figures 10(a) and (b), respectively. Samples with only ibuprofen have smaller Rcore values than pure DMPC ones, as shown in panel (a). Similar observations were made by Boggara et al. 29 Because this eect is concentration-dependent it indicates that the amount of ibuprofen changes the phase state and the deformability of vesicles during the extrusion. Boggara et al. found that when ibuprofen is added at high pH value (pH > 7) the bilayer thickness decreases in the lipid uid phase due to reduction of the head group area through massive interactions with the head groups (and nearly no impact on hydrocarbon region) of the phospholipids. This is in agreement with our WAXS results and is also in concordance with results obtained for the hydrophobic thicknesses dtail shown in panel (b). Here, no signicant changes in dtail values are observable. Addition of aescin to all three systems studied leads to formation of larger 27



vesicles in all cases. This behavior is found to occur in dependence on concentration while swelling of vesicles with temperature remains for all samples. However, it must be noted that pure DMPC SUVs exhibit the most pronounced size increase around DMPC's Tm . A comparison of dtail values in panels of Figure 10(b) shows that aescin broadens the temperature-dependent transition from gel-like to uid-like DMPC molecules. The most prominent eect is visible around Tm between 20 ◦ C and 30 ◦ C and can be especially seen in panels (I) and (III) in the same gure. As already seen by WAXS, the transition temperature is shifted to higher ones with increasing aescin content. Tm values from sigmoidal ts to

dtail results are shown in Table S5 in the SI. Values and trends are in well agreement with WAXS results. Another typical structural feature are the inhomogeneities around the bilayer peak located at approx. 0.1 Å−1 . These inhomogeneities and peaks indicate the onset of aggregation of SUVs in the presence of aescin. This phenomenon becomes signicant with increasing aescin content. Therefore, we assume that the presence of aescin facilitates the formation of bridges between SUVs. The principle of aescin-induced bridge formation was discussed in a recent study for DMPC/aescin mixtures with intermediate amounts of the saponin. 27 The comparison of the samples in panels (b), (e), and especially in (c), and (f) in Figure S5 in the SI shows that this eect is further enhanced by the presence of ibuprofen. Additional information can be extracted from the low-q regime of the scattering curves by analyzing the scattering exponent m (I(q) ∼ q −m ). Furthermore, the model-independent analyses provides furthermore information on sample morphologies which are not directly visible by eye and do not show prominently in model-based tting. This was done for some representative samples, i.e. samples with and without 1 mol % ibuprofen at 10 ◦ C where the most well-dened shape is obtained. The corresponding analyses are shown in Figure 11(a) in the SI. In this case three dierent m values were found within the scattering curves. At high q , m =4 arises from the surface scattering of the bilayer, according to Porod's law. 39 In the intermediate q -regime m =3 results from the fractal scattering of the bilayer of the vesicle. At low q , m ≈1 is only identied for aescin containing samples and 28


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Figure 11: (a) SAXS curves of DMPC SUVs and SUVs containing 10 mol % ibuprofen with and without 1 mol % aescin at 10 ◦ C. DMPC is on absolute scale (cm−1 ) all other data are scaled by the gray numbers. Solid lines are ts obtained from the standard indirect Fourier transform (IFT) analysis. The dashed extensions at low q show the theoretical intensity. The slopes were determined for dierent parts of the scattering curves. The slopes -4 and -3 apply for all three scattering curves. (b) p(r) functions calculated from the standard indirect Fourier transform (IFT) analysis and are normalized from 0-1 for better comparability. For the aescin-free samples, the full form factor was analysed. For the other samples only the low-q part was analysed. The corresponding ts are shown in panels (a). cannot directly be assigned to the scattering contribution from a well-dened structure. However, the deviation from the dashed-line (extension of solid line at low q ) indicates the deviation from the scattering of a spherically shaped object. The observed increase in intensity results either from aggregation eects and with that from an underlying structure factor or from deformation hence elongation of the vesicle (m=1 stands for cylinders). This is in agreement with our previously made considerations for vesicle clustering. 27 Another interesting fact is that the aescin containing scattering curves exhibit more minima and posses with that well-dened form factor oscillations compared to the saponin-free samples. This indicates that the aescin containing samples seem to exhibit a lower polydispersity which is also visible in the PDI values shown in Table S4 in the SI for these samples. This can be induced by inhibited uctuations, i.e. reduced deformation of the vesicles due to damping of the undulations by aescin or due to attractive interactions to neighbouring 29



vesicles. The solid lines in this gure are the calculated ts from the IFT analysis. The dashed extension was not taken into account for the tting. The resulting p(r) functions are shown in Figure 11(b). The p(r) functions corresponding to pure DMPC SUVs and the ones with additional ibuprofen were analyzed for the full form factor. The tting procedure is described elsewhere. 26 The oscillation at low r below 5 Å results from the bilayer contribution. Hence, negative values of the p(r) function result from the XSLD dierence between solvent and inner part of the membrane. At larger r the vesicle shape is obtained and exhibits a rather uniform distribution. The radius of gyration (RG ) calculated by eq. 5 amounts to 335 Å for this sample. RG of ibuprofen containing SUVs exhibits a smaller value (RG =312 Å) and is in agreement with literature. 29,30 Also, both analyses methods (CMS and IFT) lead to similar conclusions in respect to size change. Ibuprofen incorporation induces bilayer thinning through strong interaction with the phospholipid so that the vesicles exhibit less deformation during the extrusion procedure compared to regular DMPC SUVs. 29,30 This is also visible in the p(r) function. The scattering curves of aescin containing samples were only analyzed in the low-q regime due to the inhomogeneous bilayer signal. The comparison of the

p(r) function of these samples to the one of pure DMPC SUVs shows that they exhibit larger values r and that their shape is leaned towards higher dimension; these structures have larger maximum sizes and have a slightly dierent shape compared to the DMPC SUVs. Here, the dierence between aesicn-containing samples is large whereas the one between both samples is marginal so that this deformation can be mainly attributed to result from incorporation of aescin. The calculated RG are 383 Å and 372 Å for the samples with and without ibuprofen, respectively, i.e. identical. Here, an increasing RG underlines again structural changes upon incorporation of the dierent components and that the sample containing ibuprofen is dierent from the one without the drug. This is in agreement with our previous results by DSC, WAXS and CMS analysis by SAXS. When the aescin content is increased above 1 mol %, aggregation of vesicles becomes signicant. The low-q scattering of a sample with 10 mol % ibuprofen and 2 mol % aescin is 30


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shown in Figure S7 in the SI. The typical vesicular shape is less distinct and other structural features become visible. This indicates the presence of multiple structures in the system at this concentration. 27

Structural parameters of samples with 5mol % aescin by stacked disk analysis.

Figure 12: SAXS scattering curves (a) without ibuprofen, (b) with 5 mol % ibuprofen and (c) with 10 mol % ibuprofen. All samples contain 5 mol % aescin. The covered temperature range ranges from 10 ◦ C to 40 ◦ C for (a) and (b), and to 50 ◦ C in (c). The data was measured at (a,b) the XEUSS instrument (c) the ID02 beamline. Solid lines in (a) and (b) at 10 ◦ C, 20 ◦ C and orange dashed line in (b) at 30 ◦ C and all lines in (c) correspond to the stacked disks model implemented in SASView. 48 The dashed extensions in (c) are extrapolations to I(0). Solid lines at 30 ◦ C and 40 ◦ C in (b) correspond to the core multi shell (CMS) model, also from SASView. 48 XSLDs used for tting are summarized in Table S4 (CMS) and Table S6 in the SI (stacked disks). The scattering curves at 40 ◦ C in (a) and (b) and 50 ◦ C in (c) are on absolute scale (cm−1 ). All other ones are scaled with respect to it, multiplied by multiples of 10. To analyse the structures around the threshold concentration located at higher aescin content, SAXS experiments were performed for samples with 5 mol % aescin. Form factors are shown for the binary system without ibuprofen and with 5 mol % and 10 mol % of the drug in Figures 12(a), (b), and (c), respectively. 31



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The binary ibuprofen-free system in panel (a) already indicates major structural reorganization with temperature change. Here, samples at 10 ◦ C and 20 ◦ C were analysed by the

stacked disk model from SASView. 48 Solid lines in Figure 12(a) are model ts. A schematic describing this model is shown in Figure S3(b) in the SI. The XSLDs were xed for the inner disk part (tail) and outer disk parts (head). They were calculated similar to the ones used for CMS model tting. The solvent XSLD was adjusted by tting due to same reasons as described before. All values and those for PDI(dtail ) are summarized in Table S6 in the SI. For all systems analysed by this model dhead was also xed to a value of 7 Å, 65 similar to CMS model tting. The results for Rdisk , PDI(Rdisk ), and dtail are summarized in Table 1. Table 1: Fitparameters Rdisk , PDI(dtail ), and dtail for the stacked disk model from SASView. 48 PDI(dtail ) was for all samples 0.

x(ibuprofen) mol % 0 0 5 5 5 10 10 10 10 10 10 10 10 10

x(aescin) mol % 5 5 5 5 5 5 5 5 5 5 5 5 5 5

T C 10 20 10 20 30 10 15 20 25 30 35 40 45 50

◦

Rdisk Å 390.0±0.3 496.4±0.5 545.2±8.6 424.3±7.8 173.3±1.2 550.1±2.9 510.0±12.0 186.2±0.9 204.4±1.5 353.9±3.6 456.4±3.3 539.3±3.0 602.8±3.4 897.5±4.5

PDI(dtail ) 0.1 0.2 0.2 0.1 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.1 0.1 0.1

dtail Å 27.7±0.03 28.3±0.02 28.6±0.03 29.6±0.03 25.8±0.03 29.9±0.02 28.7±0.02 27.0±0.02 26.0±0.02 24.4±0.01 23.8±0.05 23.1±0.01 23.4±0.04 23.3±0.04

From the good agreement of the t and the scattering data, it can be concluded that at this amount of aescin in the sample, the vesicle membrane is solubilized into bilayer fragments. The size of these sheets reaches for these conditions from 400-500 Å and is therewith dependent on temperature. The features around the bilayer form factor peak (∼1 Å) indicate the onset of aggregation and inter-particle interactions. To conrm the reversibility of these structures, the samples were heated and cooled several times. When 32


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increasing the temperature above Tm of DMPC, sharp lamellar correlation peaks appear. A distinct and well-dened global size information is not observable any more. Here, we assume formation of correlated lamellar bilayer stacks which is triggered by the main phase transition of the acyl chains in DMPC. Peaks were analysed by the modied Caillé theory (MCT) to obtain information on the number of correlated bilayers (NoB), the repeating distance dCaillé , and elasticity information through the Caillé parameter η (see eq. 6) The results are summarized in Table 2. Table 2: Fit results for the number of bilayers (NoB), the repeating distance dCaillé , and the Caillé parameter η .

x(ibuprofen) mol % 0 0 0

x(aescin) mol % 5 5 5

T C 25 30 40

◦

NoB Å 11.3 22.5 13.9

dCaillé 68.5 65.4 64.8

η Å 0.129 0.167 0.133

The MCT analysis reveals a systematic decrease of dCaillé with increasing temperature which can be explained by the temperature-induced volume change of DMPC. The NoB and

η is almost constant for 25 ◦ C and 40 ◦ C whereas the values for 30 ◦ C are strongly elevated. The analysis was repeated several times with dierent tting conditions and always similar values were obtained. SAXS curves at 10 ◦ C and 20 ◦ C (and 30 ◦ C) of the ternary system in panel (b) with 5 mol % ibuprofen were also analysed with the stacked disk model. Results are also shown in Table 1. Also here, multiple correlations can be found around 0.1 Å−1 which again indicate inter-particle interactions. However, the very pronounced one at 10 ◦ C in panel (a) is reduced by addition of ibuprofen. This indicates that the drug minimizes interparticle interactions. In a previous study we could show by cryo-transmission electron microscopy of a sample with 4 mol% aescin that in this intermediate concentration regime (1-6 mol % aescin) a large variety of structures coexist. Those are e.g. aggregated SUVs, deformed vesicles, elongated rod-shaped objects and small bilayer fragments. 27 When increasing temperature at this drug content above Tm SAXS curves could be represented 33



by CMS model. Fitting parameters are summarized in Table S4 in the SI. The parameters obtained at 30 ◦ C are Rcore =329.7 Å (PDI(Rcore )=0.3) and dtail =25.4 Å and at 40 ◦ C

Rcore =387.2 Å (PDI(Rcore )=0.3) and dtail =23.5 Å. The thermoresponsiveness of these samples is in well agreement with our results presented in Figure 10. The probable closure of sheets to SUVs or reorganization of micelles to vesicles was extensively discussed by other authors for dierent systems and varying conditions such as e.g. heating and cooling. 6773 The vesicle formation may be enhanced by ibuprofen and is not observed for aescin because of the reduced interactions to neighbouring sheets or vesicles. However, this hypothesis is based on a single experimental indication and this question will be addressed in detail in a future study when evaluating also the eect of higher amounts of aescin at similar drug content.

All of the scattering curves of samples with 10 mol % ibuprofen shown in Figure 12(c) were tted with the stacked disk model as described previously. The solid lines are model functions showing up to which points the data were tted. The dashed extensions indicate theoretical values of I(0). What can be seen from results in Table 1 is that the overall radii are larger than for the samples with less ibuprofen. This indicates that the presence of the drug tends to stabilizes formation of sheets and reduce formation of high-order bilayer stacks. Moreover, evident closure to vesicles is not seen. Nevertheless, from the model ts it can be concluded that the low−q scattering contribution and with that the global size information is not covered by the values given in Table 1. The continuously increasing intensity could be represented by (much) larger Rdisk values. This is simulated in Figure S8 in the SI for sheet sizes of 2000 Å. However, because neither a real form factor minimum can be seen nor a plateau is reached in the SAXS curves at low-q , a precise information of sheet radius is not accessible.

34


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Discussion and Conclusion In this work we have determined how the presence of the NSAID ibuprofen alters structural parameters of the model membrane built by the phospholipid DMPC at dierent amounts (0-6 mol %) of the saponin aescin. The analyses were performed by dierential scanning calorimetry (DSC), wide-angle (WAXS) and small-angle X-ray scattering (SAXS), and visual inspection of the samples. The complementary use of these techniques provides a broad set of information. While DSC shows acyl-chain correlation changes from an energetic perspective, WAXS yields complementary structural information between them. On the other hand, SAXS provides information about a global structural picture of the systems investigated. The visual inspection already revealed that the global structural picture of the systems studied is determined by the amount of aescin. The presence of ibuprofen altered thereby the temperature-dependent behaviour of the samples in a concentration dependent manner. Here, two threshold concentrations located at around 0.8-1 mol % and 6-7 mol % are observed. Below 1 mol % stable SUVs were identied by their bluish color which does not change upon temperature change. By SAXS we analysed structural parameters (Rcore and dtail ) of samples without and with low amounts of the saponin by model-dependent and modelindependent approaches. The model-based approach conrmed unilamellar structure and structural parameters were furthermore impacted by both, ibuprofen and aescin, contents in a concentration-dependent manner. Additionally, we found that samples with 1 mol % aescin show very low polydispersity. This structural appearance could also be found for the samples containing ibuprofen and this aescin content. This is an indication that the addition of ibuprofen does not completely inhibit or reduce the interaction of aescin with the biological membrane, as it is for example expected for cholesterol through strong complexation of the saponin to the steroid. 9,10 Moreover, DSC analysis revealed that at low aescin contents the DSC signal is mainly composed of one sharp peak similar to the lipid phase transition expected for unilamellar vesicles for samples below 1 mol % aescin for all systems studied. Only when approaching the threshold concentration of 1 mol % the additional peak denoting 35



strong phase segregation begins to appear at signicant intensity. The addition of ibuprofen and aescin has shown to impact the main phase transition temperature of DMPC which was analysed by DSC (acyl chain melting), WAXS (acyl-chain correlations) and SAXS (bilayer thickness reduction) which lead to similar results. WAXS has additionally shown that already low amounts of aescin, as 0.5 mol % aescin, alter the appearance of the diraction pattern for the ibuprofen containing system strongly suggesting that the eects from ibuprofen and aescin are synergistic. From this and the observed eects by the other methods it can be concluded that the overall impact experienced by the biological model membrane must be the result of the interaction of both molecules. This can be done by interaction of aescin with each other by e.g. complexation or, and which seems more likely from our point of view, interacting with the biological model membrane and altering membrane properties. However, by this methods the exact kind of interaction cannot be resolved and would require application of additional methods showing single-bond interactions such as e.g. FourierTransform Infrared (FT-IR) spectroscopy or solid state nuclear magnetic resonance (NMR) spectroscopy. Because these methods require extensive experimental resources and deserve an extended discussion they should be addressed in a separate work and are out of the scope of this study. The second concentration range studied in this work covers from 1 mol % to 6 mol % aescin. The macroscopic images shown in Figures S1 and S2 and ibuprofen-free samples shown elsewhere 4 already indicate that the samples posses not only a strong structure-related concentration dependence but also a temperature-dependent one. DSC reveals for these concentrations that phase segregation dominates the structural picture, i.e. that DMPC molecules undergoing the phase transition are located in dierent chemical environment. This was explained by very intense phase segregation or by formation of dierent structures such as e.g. a multilamellar environment. The coexistence of structures could be indicated by SAXS experiments on a sample with 2 mol % aescin and 10 mol % ibuprofen. This sample also showed a temperature dependence. However, because no clear form factor was identi36


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ed, a model-dependent analysis was not possible. When approaching the second threshold concentration of 5 mol % aescin, the samples without and with 5 mol % ibuprofen become transparent at low and turbid at high temperature. Here, dierent shades in turbidity indicate formation of dierent structures at dierent ibuprofen contents. Samples with 10 mol % ibuprofen even showed a slightly dierent temperature-dependent behaviour. Samples with 5 mol % aescin were investigated by SAXS, DSC, and WAXS. The broad acyl-chain melting signal seen in DSC can be explained by the rather broad diraction peak seen in WAXS. The broad WAXS signal indicates that the phospholipids are available in dierent states also already at low temperatures. Moreover, SAXS revealed that the samples undergo a macroscopic phase transition from sheets at dierent length scales to correlated membrane stack or very elongated or polydisperse sheets. This behaviour is dependent on ibuprofen (and aescin ) content and will be addressed more extensively in a future work.

Acknowledgement Financial support by the Deutsche Forschungsgemeinschaft (DFG) (HE2995/7-1, INST 215/4321 FUGG) is gratefully acknowledged. The authors thank the Synchrotron ESRF for allocation of beamtime at the ID02 beamline (proposal: SC-4393). Prof. T. Koop is gratefully acknowledged for placing the DSC at our disposal.

Conict of interest The authors have no conicts to declare.

Supplementary information Supplementary Information available. Photographs of ibuprofen containing samples. 37



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Fit parameters for CMS and SD model ts and model description SAXS scattering curves at dierent temperatures

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Graphical TOC Entry

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Interaction of the Saponin Aescin with Ibuprofen in DMPC Model

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