# Coexistence of DOPG model membranes and β-aescin micelles: a combined scattering and NMR study

Friederike Gräbitz-Bräuer, Carina Dargel, Ramsia Geisler, Pascal Fandrich, Viktor Sabadasch, Lionel Porcar, Andreas Mix, Thomas Hellweg

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## To cite this version:

Friederike Gräbitz-Bräuer, Carina Dargel, Ramsia Geisler, Pascal Fandrich, Viktor Sabadasch, et al. Coexistence of DOPG model membranes and β-aescin micelles: a combined scattering and NMR study. Colloid and Polymer Science, 2023, 301 (12), pp.1499-1512. 10.1007/s00396-023-05168-0. hal-05330179

HAL Id: hal-05330179

https://hal.science/hal-05330179v1

Submitted on 29 Oct 2025

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# Coexistence of DOPG model membranes and β-aescin micelles: a combined scattering and NMR study

Friederike Gräbitz-Bräuer<fnref n="1" /> · Carina Dargel<fnref n="1" />,<fnref n="2" /> · Ramsia Geisler<fnref n="1" />,<fnref n="3" /> · Pascal Fandrich<fnref n="1" /> · Viktor Sabadasch<fnref n="1" /> · Lionel Porcar<fnref n="4" /> · Andreas Mix<fnref n="5" /> · Thomas Hellweg<fnref n="1" />

Received: 14 March 2023 / Revised: 11 August 2023 / Accepted: 31 August 2023 / Published online: 25 September 2023
© The Author(s) 2023

## Abstract

The saponin β-aescin is well known for its self-aggregation above the critical micelle concentration (cmc) and its interaction with model membranes made of zwitterionic phospholipids including the formation of mixed bicelle systems. In this study, we investigate the interaction of β-aescin with small unilamellar vesicles (SUVs) made of the negatively charged lipid 1,2-dioleoyl-*sn*-glycero-3-phosphoglycerol (DOPG). The study is conducted at a pH value at which aescin is negatively charged as well, and mixtures up to an aescin content of 50 mol% (equivalent to a molecular ratio of 1:1) were investigated, so that the cmc of aescin is exceeded by far. Analysis of the system by scattering and NMR methods was performed with respect to two reference systems made of the bare components: DOPG SUVs and aescin micelles. Wide-angle X-ray scattering (WAXS) was used to determine molecular correlation distances for both kinds of molecules, and small-angle neutron and X-ray scattering (SANS and SAXS) revealed a structural picture of the system, which was further confirmed by diffusion-ordered nuclear magnetic resonance spectroscopy (DOSY-NMR). Contrary to the expected solubilization of the DOPG membrane, most probably none- or only weakly-interacting, separated DOPG SUVs and aescin micelles were found. The study additionally highlights the importance of using independent methods to characterize a rather complex colloidal system in order to obtain a complete picture of the structures formed.

**Keywords** Small angle X-ray scattering (SAXS) · Small angle neutron scattering (SANS) · Wide angle X-ray scattering (WAXS) · Small unilamellar vesicles (SUV) · Micelles · 1,2-dioleoyl-*sn*-glycero-3-phosphoglycerol (DOPG) · Aescin · Saponin · Bio-surfactant · GIFT · SasView · Diffusion ordered spectroscopy (DOSY)

Friederike Gräbitz-Bräuer and Carina Dargel have contributed equally to this work.

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Thomas Hellweg
thomas.hellweg@uni-bielefeld.de

Friederike Gräbitz-Bräuer
friederike.graebitz@uni-bielefeld.de

Carina Dargel
cdargel@uni-muenster.de

Ramsia Geisler
Geisler@med.uni-frankfurt.de

Pascal Fandrich
p.fandrich@uni-bielefeld.de

Viktor Sabadasch
viktor.sabadasch@uni-bielefeld.de

Lionel Porcar
porcar@ill.eu

Andreas Mix
a.mix@uni-bielefeld.de

<footnote n="1">¹ Physical and Biophysical Chemistry, Bielefeld University, Universitätstraße 25, Bielefeld 33615, Germany</footnote>

<footnote n="2">² Institute of Physical Chemistry, University of Münster, Corrensstraße 28/30, Münster 48149, Germany</footnote>

<footnote n="3">³ Department II of Internal Medicine, Hematology/Oncology, Goethe University, Theodor-Stern-Kai 7, Frankfurt am Main 60590, Germany</footnote>

<footnote n="4">⁴ Institute Laue-Langevin, 71 Avenue des Martyrs, Grenoble 38042, CEDEX 9, France</footnote>

<footnote n="5">⁵ Lehrstuhl für Anorganische Chemie und Strukturchemie (ACS) and Centre for Molecular Materials (CM2), Bielefeld University, Universitätstraße 25, Bielefeld 33615, Germany</footnote>
**Introduction**

Saponins are a vast group of plant-based biosurfactants, which usually have significant pharmacological activity [1–7]. Some of them are known to strongly interact with cell membranes and show haemolytic effects [1, 2, 8–10]. The saponin β-aescin from the horse chestnut tree (Aesculus hippocastanum L.; Hippocastanaceae), which is used in the present study, is known for its anti-edematous activity and shows also some anti-viral and anti-cancer activity [11–16]. Dargel et al. determined a critical micelle concentration (cmc) for β-aescin in phosphate buffer at pH=7.4 and 30 °C to be cmc<sub>aescin</sub> = (0.37 ± 0.11) mM [17].

In previous studies, the interaction of β-aescin with vesicles based on the synthetic, zwitterionic lipid DMPC (1,2-dimyristoyl-*sn*-glycero-3-phosphocholine) has been studied [18–22]. In these works, the major finding was that such vesicles made of a saturated synthetic lipid show extremely strong interaction with β-aescin [18–20] and are completely decomposed to lipid nanodisks (bicelles) [21, 22].

Moreover, the interaction was found to partly depend on the DMPC phase state. It is found that low amounts of β-aescin change the bilayer mechanics, and in the *L*<sub>α</sub> state, the bilayer shows an increased bending elastic constant. However, below the DMPC main phase transition temperature (*T*<sub>m</sub> = 24.5 °C [23, 24]), the membrane appears to be softened by the saponin. The aim of the present work is to study the interaction of β-aescin with the lipid DOPG (1,2-dioleoyl-*sn*-glycero-3-phosphatidyl-glycerol). Phosphatidylglycerols (PG) are major components in plant leaves and are also found in many algae and bacterial species [25]. Moreover, PGs are also present in mammalian cells [26, 27].

DOPG is unsaturated and always in the fluid state above
0 °C due to T_m = -18 °C [27, 28]. In addition, its head

group is negatively charged. Hence, a different interaction
with β-aescin compared to DMPC can be expected.

In the article at hand, we therefore address this question and study DOPG vesicles as model systems for bio-membranes in the presence of different amounts of β-aescin. The structures of DOPG and β-aescin are depicted in Fig. 1.

The interaction between the lipid and the saponin is scru-
tinized by combining NMR techniques and small/wide angle
X-ray scattering (SAXS/WAXS). Moreover, to generate a
different scattering contrast also small angle neutron scat-
tering (SANS) is exploited. The covered mole fraction of
β-aescin with respect to DOPG is X<sub>aescin</sub> = 0 mol% up to
X<sub>aescin</sub> = 50 mol%.

The major finding of this work is that DOPG vesicles
are not decomposed into bicelles even at a mole fraction of
X<sub>aescin</sub> = 50 mol%. This was unexpected and is in contrast to
previous results for saturated lipids and also in contrast to
the observed haemolytic effect of aescin. Even low amounts
of β-aescin are not interacting in a significant way with the
DOPG membrane. On the contrary, β-aescin forms micelles
for X<sub>aescin</sub> ≥ cmc<sub>aescin</sub>, which coexist with the vesicles.

**Materials and methods**

**Chemicals and sample preparation**

The phospholipid 1,2-dioleoyl-*sn*-glycero-3-phosphoglycerol (DOPG) was purchased from Lipoid GmbH (Ludwigshafen, Germany, ≥ 99% purity). The saponin β-aescin (≥ 95% purity, CAS-number 6805-41-0), chloroform, and deuteriumoxide (D₂O) were obtained from Sigma-Aldrich (Munich, Germany). All samples were prepared in a 50 mM phosphate buffer with pH/pD value of 7.4 in D₂O. All samples were produced with a lipid mass concentration of 15 mg/mL (equal to a molar concentration of 18.8 mM). The β-aescin content X<sub>aescin</sub> varied between 0 and 50 mol%, whereby X<sub>aescin</sub> relates

<figure><img src="image_4.png" /><figcaption>**Fig. 1** Molecular structure of (top) the charged phospholipid 1,2-dioleoyl-*sn*-glycero-3-phosphoglycerol (DOPG) and (bottom) the saponin β-aescin at pH value of 7.4. The oxygen atoms of the deprotonated acidic group of β-aescin are highlighted in red</figcaption></figure>
the amount of both substances, DOPG and aescin, and $X_{aescin}$ is the mole fraction of the mixture:

$$X_{aescin} = \frac{n_{aescin}}{n_{DOPG} + n_{aescin}} \quad (1)$$

For the preparation of DOPG vesicles with different $X_{aescin}$, DOPG was dissolved in chloroform at first. The chloroform was removed using a rotary evaporator leading to the deposition of a thin DOPG film on the inner surface of the flask used. These films were stored over night at 60 °C. After drying, the lipid film was rehydrated with an aescin-containing buffer solution at the desired aescin concentration. After rehydration, all samples were subjected to five consecutive freeze-thaw cycles (in liquid nitrogen and warm water). To generate SUVs of a defined size, all samples were subsequently extruded (at least 15 paths, with increasing $X_{aescin}$) through the same membrane with a pore size of 50 nm (Whatman, Avanti Polar Lipids Inc., Alabama, USA) using a conventional extruder (extruder from Avanti Polar Lipids Inc., Alabama, USA).

A table which relates the aescin mole fraction $X_{aescin}$ to the molar concentration of aescin can be found in the supporting information in Table S1. In the further text, the $X_{aescin}$ with aescin concentrations above the $cmc_{aescin}$ are written in italics and bold. In comparison to the DOPG-aescin-system, additionally pure aescin samples in the same phosphate buffer were prepared by dilution of a 18.8 mM stock solution. Only samples with aescin concentrations above the $cmc$ were considered.

## Small angle scattering (SAS)

The structure of colloidal particles like vesicular systems can be resolved by SAS experiments [29–32]. The magnitude of the scattering vector $q$ (Eq. 2) depends on the scattered radiation detected at a scattering angle of $2\theta$. By using different kinds of radiation, thus different wavelengths $\lambda$, a broad $q$-range becomes accessible (Eq. 2).

$$q = \frac{4\pi}{\lambda} \cdot \sin(\theta) \quad (2)$$

The total scattering intensity $I(q)$ is given by Eq. 3.

$$I(q) = N \cdot (\Delta SLD)^2 \cdot V^2 \cdot P(q) \cdot S(q) \quad (3)$$

$I(q)$ depends on the number of particles $N$ in a scattering volume $V$, the form factor $P(q)$, and structure factor $S(q)$. The scattering length density difference ($\Delta SLD$) differs for SAXS and SANS and also depends on the solvent used. For X-ray scattering, the lipid head groups are highlighted because of their rather high electron density. In contrast, neutrons are scattered by the nuclei, and especially the proton rich part of the membrane becomes visible in SANS. The scattering

length densities for DOPG and $\beta$-aescin are presented in Table S3. A combination of both methods provides a more detailed picture of the membrane.

The overall shape of the mixed vesicles is obtained from the pair distance distribution function $p(r)$ in real space, which is obtained from standard indirect Fourier transform (IFT) implemented in the GIFT program from SAS data [33]. This method is shape independent and works without assuming a shape of the aggregates [34, 35]. The limitation for this calculation with GIFT is $D_{max} \le \frac{\pi}{q}$.

Additional information on structural parameters of the SUVs are derived from model-dependent fitting with the core multi shell (CMS) model implemented in the program SasView [36–40]. SUV parameters such as the core radius $R_c$, its corresponding polydispersity $\sigma_{R_c}$, the membrane thickness $d_z$, and its corresponding polydispersity $\sigma_{d_z}$ are derived. By using three shells ($n = 3, n$: number of shells) in the model, the membrane thickness can be divided into the thickness of the hydrophilic parts, or heads (shells number 1 and 3, $d_{head}$) and the thickness of the hydrophobic part, or tail (shell number 2, $d_{tail}$). The membrane thickness $d_z$ can be calculated by Eq. 4.

$$d_z = 2 \cdot d_{head} + d_{tail} \quad (4)$$

Information about excess aescin micelles can be obtained from the ellipsoid model in SasView [36–38]. The polar radius ($R_p$) and the equatorial radius ($R_e$) of ellipsoids can be determined by fitting.

A customized model in SasView was implemented by Eq. 5 to determine the previously mentioned parameters for the SUV (model: CMS $n = 3$) and additionally parameters for an ellipsoid. The CMS + ellipsoid model is a sum of the CMS and ellipsoid models, which include the scale parameters $S$ and the fit functions $F$ themselves. $B$ is an additional background.

$$CMS + ellipsoid \equiv (S_{CMS} \cdot F_{CMS} + S_{ellipsoid} \cdot F_{ellipsoid}) + B \quad (5)$$

## Small angle neutron scattering (SANS)

SANS experiments were performed with samples prepared in D₂O buffer. Measurements were performed at the Institute Laue-Langevin (ILL) in Grenoble, France, using the D22 and D11 instruments. All samples in the range of $X_{aescin} = 0$ to 40 mol% were measured at the D22 instrument. The sample containing 50 mol% aescin was measured separately on the D11 instrument. For the D22 instrument, the samples were filled into 2 mm quartz cuvettes (Hellma Analytics, Müllheim, Germany) and measured at $T = 30$ °C in a 15-position sample holder. At a neutron wavelength of 6 Å with a sample to detector distance of 2.8 m and 17.6 m, and 12 Å at 17.6 m a $q$-range from $1.7 \cdot 10^{-3}$ Å⁻¹ to $0.4$ Å⁻¹ was
covered. The wavelength resolution was $\Delta \lambda/\lambda = 10\%$. For the D11 instrument, the sample was also filled into 2 mm quartz cuvettes (Hellma Analytics, Müllheim, Germany) and also measured at 30 °C in a similar 15-position sample holder. At a neutron wavelength of 6 Å with a sample to detector distance of 1.4 m, 8.0 m and 38.9 m a q-range from $1.6 \cdot 10^{-3}$ Å$^{-1}$ to 0.4 Å$^{-1}$ was covered. The wavelength resolution was $\Delta \lambda/\lambda = 10\%$. With the software GRASP [41], provided by the ILL, the initial treatment of the 2D data was carried out. The final data was normalized with respect to the empty cell, background, and transmission from direct beam measurements.

## Small angle X-ray scattering (SAXS)

SAXS experiments were performed on samples prepared in D₂O buffer on an inhouse SAXS/WAXS system (XEUSS, Xenocs, Sassenage, France) equipped with a CuKα source ($\lambda = 1.541$ Å, GeniX Ultra low divergence, Xenocs) and a Pilatus 300K hybrid pixel detector (Dectris, Baden Deatwil, Switzerland). To cover a q-range from $6 \cdot 10^{-3}$ to 0.4 Å$^{-1}$ sample-to-detector distances of 0.8 m and 2.7 m were used for vesicle samples. For aescin micelle samples, a q-range from 0.05 to 0.3 Å$^{-1}$ was covered by a sample-to-detector distance of 0.8 m. Because of the small radii of the aescin micelles, it is sufficient to examine the aescin micelle samples only with SAXS at the mentioned distance. All 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) at a temperature of 30 °C. The 2D data were analyzed using the Foxtrot software (V.3.3.4) [42] and normalized with respect to incident intensity, sample thickness, acquisition time, transmission, and background. By using glassy carbon type 2 as standard, the data were brought to absolute scale [43]. The normalized data were treated by the dynamic rebin formalism implemented in the program SAXSutilities to improve statistics at high q-values (min. steps: 1, $\Delta q: 0.005$ Å$^{-1}$) [44].

## Wide angle X-ray scattering (WAXS)

WAXS-measurements were performed for samples prepared in D₂O buffer on the inhouse SAXS/WAXS system described in the “Small angle X-ray scattering (SAXS)” section. Experimental details and the data reduction procedure can be found in the same section. All measurements were performed at a temperature of 30 °C and a sample-to-detector distance of 0.16 m to cover a q-range from 0.5 Å$^{-1}$ to 2 Å$^{-1}$. The peak observed in WAXS occurring at the position $q_{WAXS}$ is directly related to the lateral molecule correlation distance $d_{WAXS}$ via Eq. 6.

$$d_{WAXS} = \frac{2\pi}{q_{WAXS}} \qquad (6)$$

## Nuclear magnetic resonance spectroscopy (NMR)

Two dimensional NMR experiments like diffusion ordered NMR (DOSY-NMR) are excellently suited for determining the diffusion coefficient of molecules or structures in solution, like vesicles or micelles [45]. Thus, they can also provide insights into aggregation or incorporation processes in the liquid phase [46–48]. Species incorporated in the same aggregate exhibit the same diffusion coefficient. With the Stokes-Einstein equation (Eq. 7), the hydrodynamic radius $R_H$ can be determined from D with the dynamic viscosity of the solvent $\eta$, the temperature T, and the Boltzmann-constant $k_B$.

$$D = \frac{k_B T}{6\pi\eta R_H} \qquad (7)$$

The kinetic viscosity $\nu$ of the used phosphate buffer was determined at 30 °C using an Ubbelohde viscosimeter with a capillary constant of $k = 0.03088 \text{ m}^2\text{s}^{-2}$ (Schott Geräte, Mainz, Germany). The density of the phosphate buffer $\rho$ was measured with a DMA 4500 density meter from Anton Paar (Graz, Austria). $\eta$ was calculated from $\nu$ and $\rho$ ($\eta = \nu \cdot \rho$) and determined to $\eta_{buffer,30 \cdot C} = (1.04 \pm 0.01) \text{ mPa} \cdot \text{s}$.

Here, NMR experiments were performed on a BRUKER Avance NEO 600 FT NMR spectrometer (Bruker, Billerica, MA, USA), operating at a ¹H resonance frequency of 600.13 MHz. The instrument was equipped with a 5 mm CryoProbe Prodigy exhibiting a z-gradient coil delivering a maximum gradient strength of 6.57 G mm$^{-1}$ at 10 Å. ¹H-NMR spectra were referenced to the residual protons of the deuterated solvents. The temperature unit of the instrument was calibrated according to the manual of the manufacturer of the instrument.

Diffusion NMR experiments have been performed using the ledbpgp2s pulse sequence delivered by the manufacturer. Proton diffusion data have been collected with 16k data points and a spectral width of 6000 Hz. The relaxation delay was set to 5 s.

The diffusion delay time (big Delta, $\Delta$) was set to 100 ms. The gradient duration time (little delta, $\delta/2$) has been adjusted to values between 2300 and 3000 µs. The gradient strength within the diffusion experiments was incremented linearly using 16 steps. The diffusion data have been analyzed with the T1/T2 module of the BRUKER TopSpin® software package. The standard deviation of the experimentally determined gradient strength dependent signal intensities to the fitted decay function was $\le 3.9 \cdot 10^{-2}$.
¹H-NMR and ¹H-DOSY experiments were performed on samples containing pure DOPG vesicles, DOPG vesicles with X<sub>aescin</sub> = 1 and 50 mol%, and samples containing aescin micelles with c<sub>aescin</sub> = 18.8 mM, which is equivalent to X<sub>aescin</sub> = 50 mol%.

## Results

In this work, the influence of aescin on DOPG model membranes is studied with respect to aescin content X<sub>aescin</sub>. All DOPG-aescin mixtures with X<sub>aescin</sub> between 0 and 50 mol% were extruded and exhibit a bluish color typical for vesicle samples (see Fig. S1). No precipitation can be observed, even after several months, which means that formation of larger aggregates within the mixture can most probably be excluded independent on X<sub>aescin</sub>.

The visual inspection already indicates that the interaction of DOPG and aescin shows clear differences compared to the well-studied DMPC-aescin system, in which the DMPC membrane gets solubilized into small bicelle particles by aescin at concentrations above cmc of aescin [21, 22]. For the present system, increasing the aescin concentration above the cmc-border (which corresponds to 2 mol% aescin) seems to have none or only a very minor effect. This is unexpected on the basis of the results for DMPC model membranes. Thus, the aim of the experiments described in the following is to clarify the picture of interaction between the DOPG model membranes and the aescin as added surfactant. Several scattering techniques (WAXS, SANS, and SAXS) and DOSY-NMR are exploited to follow this aim.

## Lateral molecule correlation distance by WAXS ($d_{WAXS}$)

WAXS-measurements are used to explore molecule-molecule distances in the particles present in the DOPG-aescin mixtures. Because of the large scattering angles, correlation signals of molecules located in nearest proximity can be resolved. By that, e.g., the distance between lipid (and additive) molecules in a lipid membrane or surfactant molecules in micelles can be determined. From the position of the WAXS-signal $q_{WAXS}$, the correlation distance $d_{WAXS}$ (some authors call it headgroup distance when talking about lipid membranes) can be obtained by Eq. 6.

WAXS signals for mixtures composed of a fixed proportion of DOPG and varying amounts of aescin up to 50 mol% are presented in Fig. 2. The sample containing only the fixed amount of DOPG in the absence of aescin serves as a reference system (0 mol% aescin). For this system, a broad WAXS signal is obtained, which is in line with a lipid membrane adopting a liquid-crystalline L<sub>α</sub>-phase [49]. The

<figure><img src="image_5.png" /><figcaption>**Fig. 2** WAXS signals of DOPG and aescin mixtures (black dots) and aescin micelles with a concentration equivalent to X<sub>aescin</sub> = 50 mol% (blue dots). The aescin content X<sub>aescin</sub> is represented by black numbers on the right. X<sub>aescin</sub> ≥ cmc<sub>aescin</sub> are presented by bold, italic numbers. For better readability, the data is shifted by the grey numbers on the left. Lorentzian fits are presented with solid lines for $d_{WAXS,DOPG}$, dotted lines for $d_{WAXS,aescin}$, and dashed lines for the sum of the individual functions. The vertical dashed and dotted line describe the positions of $q_{WAXS,DOPG}$ and $q_{WAXS,aescin}$.</figcaption></figure>

position of the maximum of the signal $q_{WAXS}$, determined by a Lorentzian fit, is given in Table S2 in the supplementary information, and $d_{WAXS}$ calculated thereof with a value of ≈4.5 Å nicely corresponds to previous studies [49, 50].

Looking at the DMPC-aescin system, a contribution of aescin molecules to the WAXS signal of the mixture starting from any aescin content is also expected for the present system. Up to X<sub>aescin</sub> ≤ 10 mol%, only one Lorentzian fit is necessary to describe the scattering data. But, from an aescin content of 20 mol%, see Fig. 2), a second signal at lower *q*-value, corresponding to larger distances in real space and correlating with the larger volume of the aescin molecule compared to the DOPG (compare Fig. 1), arises. Hence, signals with X<sub>aescin</sub> ≥ 20 mol% are fitted by a sum of two Lorentzian functions representing the whole signals and providing two separate $q_{WAXS}$-values. These values are also
listed in Table S2, and the corresponding $d_{WAXS,DOPG}$ are presented in Table 1.

Compared to the DOPG-SUV-reference system, the position of the peak assigned to the DOPG molecules remains unchanged, so that $d_{WAXS, DOPG}$ does not change in dependence on $X_{aescin}$. This already indicates that the arrangement of most of the DOPG molecules stays unaffected by the addition of aescin molecules, even above the $cmc$, which is crossed at 2 mol% aescin. The correlation distance obtained from the position of the second signal does also not change as a function of $X_{aescin}$; it remains approximately constant at 6.1 Å. Since only the aescin content is increased and the integral share $I$ of the second Lorentzian function as well increases (see Table 1), the second contribution can most likely be assigned to structurally correlated aescin molecules. But it remains unknown whether the aescin molecules are integrated into the DOPG membrane of the SUV particles with a size of at most a few hundred nanometers or they are floating around as separate particles, e.g., micelles. From comparison with the DMPC-aescin system, the first option, incorporation into the DOPG membrane, is favored, as there, the appearance of the second WAXS signal can clearly be attributed to the incorporation of aescin into the lipid membrane [51]. But if this would also be the case for the present DOPG-aescin system, the incorporation into the DOPG membrane would most probably not lead to a solubilization of the lipid membrane, as indicated by the visual inspection of the samples.

To prove if the correlation distance of aescin molecules within an aescin micelle corresponds to the distance calculated from WAXS for the DOPG-aescin mixture, aescin micelles at a concentration equivalent to 50 mol% were prepared and investigated by WAXS. The signal obtained is also displayed in Fig. 2 and also yields a signal at $q_{WAXS} \approx 1$ Å⁻¹. Samples with lower aescin concentration starting from 20 mol% were additionally investigated (see Fig. S2), and all

obtained $d_{WAXS, aescin}$ values are listed in Table 1. Although the concentration of aescin corresponding to 20 mol% is already more than 5 times above the $cmc$ determined by Dargel et al. [17], meaningful data was only obtained from this content on, due to the small signal intensity compared to the WAXS signal intensity of the solvent itself (similar molecule-molecule-distances in both the particles and the solvent). In comparison to the mixed system, the $d_{WAXS, aescin}$ values for the micelles are, including the errors, slightly larger, but nevertheless the assignment of this signal to origin from packed aescin molecules becomes obvious.

What cannot be stated from WAXS measurements is the location and the (possible) interaction of the DOPG and aescin molecules. To determine the kind(s) of structure(s) present in the mixed solution, small-angle scattering with X-rays and neutrons was performed next.

Pair distance distribution function $p(r)$ from IFT on SANS data

Due to the larger length scale resolved by small-angle scattering methods compared to the WAXS experiments discussed in the “Lateral molecule correlation distance by WAXS ($d_{WAXS}$)” section, the shape of the particles present in the mixed DOPG-aescin solution can be determined thereof. First, only small-angle neutron scattering was used because of the good overall contrast between particles and solvent D₂O. For determination of the pair distance distribution function $p(r)$, which gives insights into the shape of the particles present, the indirect Fourier transformation (IFT) method was used applying the program GIFT [33].

The SANS data presented in Fig. 3 is very well suited for this analysis, because independent on $X_{aescin}$, a scattering plateau is reached at low $q$-values, indicating a well-defined particle size and allowing the whole particle dimension to be evaluated accurately. All curves measured show high similarity

<table><caption>**Table 1** Lateral molecule correlation distances $d_{WAXS}$ in DOPG-aescin mixtures with varying $X_{aescin}$ and aescin micelles with concentrations equivalent to $X_{aescin}$ obtained from Lorentzian fits to WAXS data (see Fig. 2). The relative integral $I$ of each fit is given in %</caption><thead><tr><th rowspan="2">X<sub>aescin</sub><br/>/ mol%</th><th colspan="4">Vesicle samples</th><th colspan="2">Micelle samples</th></tr><tr><th>d<sub>WAXS,DOPG</sub><br/>/ Å</th><th>I<br/>/ %</th><th>d<sub>WAXS,aescin</sub><br/>/ Å</th><th>I<br/>/ %</th><th>d<sub>WAXS,aescin</sub><br/>/ Å</th><th>I<br/>/ %</th></tr></thead><tbody><tr><td>0</td><td>4.54 ± 0.05</td><td>(100)</td><td></td><td></td><td></td><td></td></tr><tr><td>0.1</td><td>4.55 ± 0.05</td><td>(100)</td><td></td><td></td><td></td><td></td></tr><tr><td>1</td><td>4.55 ± 0.05</td><td>(100)</td><td></td><td></td><td></td><td></td></tr><tr><td>5</td><td>4.55 ± 0.05</td><td>(100)</td><td></td><td></td><td></td><td></td></tr><tr><td>10</td><td>4.54 ± 0.05</td><td>(100)</td><td></td><td></td><td></td><td></td></tr><tr><td>20</td><td>4.52 ± 0.05</td><td>(85)</td><td>6.07 ± 0.06</td><td>(15)</td><td>7.15 ± 0.18</td><td>(100)</td></tr><tr><td>30</td><td>4.52 ± 0.05</td><td>(73)</td><td>5.98 ± 0.06</td><td>(27)</td><td>6.73 ± 0.08</td><td>(100)</td></tr><tr><td>40</td><td>4.48 ± 0.05</td><td>(64)</td><td>6.21 ± 0.06</td><td>(35)</td><td>6.43 ± 0.07</td><td>(100)</td></tr><tr><td>50</td><td>4.49 ± 0.05</td><td>(61)</td><td>6.23 ± 0.06</td><td>(39)</td><td>6.31 ± 0.07</td><td>(100)</td></tr></tbody></table>
<figure><img src="image_6.png" /><figcaption>**Fig. 3** SANS data with different $X_{\text{aescin}}$ with IFT fits. $X_{\text{aescin}}$ is indicated by black numbers on the right. The grey, blue, and red solid lines are an approximation resulting from IFT calculation with the program GIFT. For better readability, the curves are scaled by different multiples of 10, marked in grey numbers on the left. Dotted lines are extrapolations of the IFTs beyond the fit range at low q-values</figcaption></figure>

to vesicle-like structures [19, 30, 50], and an influence of even an addition of 50 mol% aescin cannot be resolved by eye. To represent the formed structures in real space, the $p(r)$-function was calculated for each $X_{\text{aescin}}$ and displayed in Fig. 4a. The IFT fits to the scattering data are additionally shown in the same color coding as the $p(r)$-function in Fig. 3. The shape of the $p(r)$-functions nicely represents the presence of spherical particles, which might be polydisperse vesicles, for which normally such symmetric $p(r)$-functions are obtained [52–54].

At high r, the $p(r)$-functions all decay to zero, which on the one hand confirms the accurate determination of size and shape of the particles and on the other hand indicates that the size of the spherical particles seems to be unaffected by the addition of even 50 mol% aescin, which means a doubling of the total number of molecules present in the D₂O-solution. If all DOPG and aescin molecules are mixed to uniform particles, a size change in dependence on $X_{\text{aescin}}$ would have been expected, and moreover $I(0)$, the scattering intensity of the plateau would also increase with increasing $X_{\text{aescin}}$, since $I(0)$ is proportional to the particle concentration. Neither is the case, which means that complete mixing of DOPG and aescin molecules seems unlikely.

Having a closer look at the low r-values of the $p(r)$-function, a weak change in dependence on the increase of $X_{\text{aescin}}$ becomes visible. The $p(r)$-function of aescin micelles at a concentration equivalent to 50 mol% aescin obtained from SAXS measurements (see also Fig. 4 left for $p(r)$-function and Fig. S3 for the fit to the data) indicates that the changes in the $p(r)$-functions of the mixed system occur exactly in the size range of pure aescin micelles. To show this more clearly,

<figure><img src="image_7.png" /><figcaption>**Fig. 4** Left: Normalized $p(r)$ functions calculated by IFT for SANS data of DOPG vesicles with $X_{\text{aescin}}$ from 0 to 50 mol% (normalized to 1) and an aescin micelle with $c_{\text{aescin}}$ equivalent to $X_{\text{aescin}} = 50$ mol% (normalized to 0.1). The black box indicates the zoomed area displayed in the right panel. The underlying data and fits for the $p(r)$ functions are shown in Fig. 3 and for the micelle in Fig. S4(a). Right: Zoom and comparison $\Delta p(r)$ for $X_{\text{aescin}} \ge cmc_{\text{aescin}}$ (right axis). In addition, the $p(r)$ function for aescin micelles with a concentration equivalent to $X_{\text{aescin}} = 50$ mol% is plotted in green for comparison (left axis)</figcaption></figure>
again the pure DOPG sample is taken as reference, and the
difference between the *p(r)*-function at any *X*<sub>aescin</sub> > *cmc* to the pure DOPG as reference system is calculated. The
resulting functions are depicted in Fig. 4 right and clearly
show an increasing contribution to the *p(r)*-function of the
mixed system with increasing *X*<sub>aescin</sub>. These results now give
a clear indication for the coexistence of DOPG vesicles and
aescin micelles and an increase of the number of micelles
with increasing *X*<sub>aescin</sub>. To further clarify if both kinds of
particles are present without any interaction or are partially
mixed, the complementary scattering method SAXS was
used, which differs from SANS in a significantly changed
contrast over the lipid membrane, whereof the incorpora-
tion of aescin molecules into the DOPG membrane shall be
confirmed or excluded.

Model dependent fitting of SAXS and SANS data

In addition to the already presented SANS data, SAXS
measurements were performed on exactly the same mixed
DOPG-aescin samples. Both types of scattering data are
shown in Fig. 5. Whereas for SANS, the resolution of the
full particle size was possible, SAXS data does not show
a plateau at low *q* due to the limited *q*-range of our setup.
Moreover, the signal representing the particles in terms of
size and polydispersity is much better resolved in SANS
due to mainly two reasons: the better contrast of the liq-
uid-crystalline membrane compared to the solvent and the
much higher difference between scattering signal and back-
ground (flat for D₂O in SANS and strongly increased back-
ground with decreasing *q* in SAXS due to domination of

the background signal by the Kapton capillary). Hence, for
such particles, our inhouse SAXS setup is not perfectly well
suited to resolve the particle shape and size. But, in contrast
to SANS, the lipid membrane contrast is not homogeneous
in SAXS, and the chance of resolving an incorporation of
aescin into the lipid membrane is high. The scattering data in
Fig. 5b shows a strong change in dependence on the increase
of Xₐescin above the cmc of aescin. Below cmc, the homoge-
neous lipid membrane is clearly represented by the signal
around 0.1 Å⁻¹, and low polydispersity is indicated by the
deep minimum at 0.04 Å⁻¹.

An increase of $X_{\text{aescin}}$ above the cmc leads to a vanishing of the minimum, which might indicate an increase in membrane polydispersity either by adhesion or (partial) incorporation of aescin molecules. At highest $X_{\text{aescin}}$, a conventional (lipid) membrane structure cannot be guessed any more, and the strong changes in the SAXS signal undoubtedly are attributed to the addition of aescin molecules.

Still, two possible scenarios are taken into consideration:

I. Added aescin molecules are incorporated into the vesicular structures, and the change in the SAXS signal is attributed to this structural scenario.

II. Added aescin molecules at concentrations above cmc assemble into micelles and coexist with DOPG vesicle.

For option I, as already mentioned, an increase in I(0) is
expected, which was not confirmed. Moreover, we tried to fit
the SAXS data with a conventional three-shell sphere model
representing a vesicle with inner-membrane contrast [50],
but no good fits with reasonable parameters were obtained.

<figure><img src="image_8.png" /><figcaption>**Fig. 5** Comparison of a SANS- and b SAXS data of DOPG vesicle samples with different X<sub>aescin</sub>. The different X<sub>aescin</sub> are given by the black number on the right. The scattering data is scaled by the grey numbers indicating multiples of 10 on the left. Also shown are the CMS-fits by red lines, and the CMS + ellipsoid-fits by blue lines</figcaption></figure>
<table><caption>**Table 2** Core radii $R_C$, radii polydispersities $\sigma_{R_C}$, and membrane thicknesses $d_z$ obtained from CMS and CMS + ellipsoid-fitting to SANS and SAXS data. $d_z$ was calculated from the fitted parameters for the tails ($d_{\text{tail}}$, see Table S6) and the fixed head thickness of $d_{\text{head}} = 4.1$ Å [29]</caption><thead><tr><th>X<sub>aescin</sub><br>/ mol%</th><th>R<sub>C,SANS</sub><br>/ Å</th><th>σ<sub>R_C,SANS</sub><br>/ %</th><th>d<sub>z,SANS</sub><br>/ Å</th><th>d<sub>z,SAXS</sub><br>/ Å</th><th>Fitting model</th></tr></thead><tbody><tr><td>0</td><td>205 ± 4</td><td>38</td><td>34.3 ± 0.7</td><td>38.9 ± 0.8</td><td>CMS</td></tr><tr><td>0.1</td><td>224 ± 4</td><td>38</td><td>34.4 ± 0.7</td><td>39.0 ± 0.8</td><td>CMS</td></tr><tr><td>1</td><td>224 ± 4</td><td>32</td><td>34.2 ± 0.7</td><td>38.7 ± 0.8</td><td>CMS</td></tr><tr><td>5</td><td>205 ± 4</td><td>38</td><td>34.5 ± 0.7</td><td>38.9 ± 0.8</td><td>CMS + ellipsoid</td></tr><tr><td>10</td><td>207 ± 4</td><td>38</td><td>34.4 ± 0.7</td><td>38.9 ± 0.8</td><td>CMS + ellipsoid</td></tr><tr><td>20</td><td>207 ± 4</td><td>38</td><td>34.1 ± 0.7</td><td>38.8 ± 0.8</td><td>CMS + ellipsoid</td></tr><tr><td>30</td><td>208 ± 5</td><td>39</td><td>34.3 ± 0.7</td><td>38.8 ± 0.8</td><td>CMS + ellipsoid</td></tr><tr><td>40</td><td>209 ± 4</td><td>39</td><td>34.2 ± 0.7</td><td>38.9 ± 0.8</td><td>CMS + ellipsoid</td></tr><tr><td>50</td><td>207 ± 4</td><td>39</td><td>34.4 ± 0.7</td><td>39.0 ± 0.8</td><td>CMS + ellipsoid</td></tr></tbody></table>

Therefore, we tried to prove option II. If DOPG vesicles and aescin micelle coexist without strong interactions, the scattering signal of the mixed sample should be representable by the sum of the scattering signals of the reference systems. To not only add the scattering intensities, but to moreover extract precise information about the particle size and contrast parameters, model fits for the reference systems are performed, afterwards summed up, compared to the data, and finally optimized to it.

As named above, pure DOPG vesicles and aescin micelles with $X_{\text{aescin}}$ corresponding aescin concentrations were used as reference systems. To model the vesicular DOPG structures, the well-established core multi shell (CMS) model from the program SasView was used [36–40]. Using this model, the inner radius of the vesicles $R_C$ and their polydispersity $\sigma_{R_C}$ can be obtained. Furthermore, by using 3 shells, the thickness of the head groups $d_{\text{head}}$ (shell 1 and 3) and the thickness of the hydrophobic parts of the membrane $d_{\text{tail}}$ (shell 2) can be considered. Here, the thickness of the head group is fixed at $d_{\text{head}} = 4.1$ Å [29]. The scattering length densities (SLD) used are presented in Table S3. For fitting the data, the protocol as described by Dargel et al. was

used [50]. The fits to the DOPG reference sample are displayed in Fig. 5, and the fit parameters are given in Tables 2 and S4 to S6. All fit parameters are in good agreement with the study on DOPG and the saponin glycyrrhizin, in which the same fit routine was used [50].

Modeling the data of the micelle reference system was done according to a study by Dargel et al. [17], in which aescin micelles in H₂O-solution also measured by SAXS were fitted. An ellipsoidal micelle shape was found which is why also in this study an ellipsoidal model from SASView was used for fitting [36–38]. This model yields ellipsoidal parameters, the equatorial radius $R_e$ and the polar radius $R_p$. Furthermore, the scale and background can be adjusted. The SLD for aescin is presented in Table S3. The fits to the SAXS data are presented in Fig. S5, and the resulting parameters are shown in Tables 3 and S4. The ratio of $R_{p,SAXS}$ and $R_{e,SAXS}$ given in Table 3 shows that for smaller $X_{\text{aescin}}$, the micelles are elongated, and with increasing $X_{\text{aescin}}$, the micelles become more spherical. This trend was also described by Dargel et al. for aescin micelles in H₂O-buffer [17].

The scattering data of both model systems was now fitted, and the fit functions can be used to investigate hypothesis II. Both models are summed up to the customized CMS+ellipsoid-model, which combines the both individual models and adds an additional background (see Eq. 5). In simplified terms, each model consists of a fit function $F$, from which structure-relevant parameters can be extracted, and a scaling factor $S$. If both particle types are present independently of one another, it should not be necessary to change both $F$ and $S$ significantly after summation into the customized model in order to describe the scattering data of the mixed system adequately.

Figure 6 shows the fits to the reference systems as well as with the customized model for the mixed sample for two exemplary aescin contents of 20 mol% (the lowest concentration for which an effect of the aescin addition became visible by all methods used) and 50 mol% (the highest concentration investigated). Since aescin micelles were not measured by SANS, the fit function for the ellipsoidal micelle was determined from SAXS data and then converted

<table><caption>**Table 3** Ellipsoid parameters from fitting with the CMS + ellipsoid and ellipsoid models in SasView for SANS and SAXS data of DOPG vesicle samples and for SAXS data of aescin micelle samples</caption><thead><tr><th rowspan="3">X<sub>aescin</sub><br>/ mol%</th><th colspan="4">DOPG SUVs + aescin</th><th colspan="2">Aescin micelles</th></tr><tr><th>R<sub>p,SANS</sub></th><th>R<sub>e,SANS</sub></th><th>R<sub>p,SAXS</sub></th><th>R<sub>e,SAXS</sub></th><th>R<sub>p,SAXS</sub></th><th>R<sub>e,SAXS</sub></th></tr><tr><th>/ Å</th><th>/ Å</th><th>/ Å</th><th>/ Å</th><th>/ Å</th><th>/ Å</th></tr></thead><tbody><tr><td>5</td><td>12.2 ± 0.3</td><td>57.6 ± 1.2</td><td>13.0 ± 0.3</td><td>36.6 ± 0.8</td><td>13.7 ± 0.2</td><td>40.3 ± 0.4</td></tr><tr><td>10</td><td>13.1 ± 0.3</td><td>54.2 ± 1.1</td><td>13.3 ± 0.3</td><td>39.1 ± 0.8</td><td>15.9 ± 0.2</td><td>39.4 ± 0.4</td></tr><tr><td>20</td><td>14.0 ± 0.3</td><td>39.6 ± 0.8</td><td>19.4 ± 0.4</td><td>32.0 ± 0.6</td><td>19.7 ± 0.2</td><td>32.1 ± 0.3</td></tr><tr><td>30</td><td>14.3 ± 0.3</td><td>27.5 ± 0.6</td><td>21.3 ± 0.4</td><td>30.1 ± 0.6</td><td>21.8 ± 0.3</td><td>30.3 ± 0.3</td></tr><tr><td>40</td><td>14.6 ± 0.3</td><td>25.5 ± 0.5</td><td>23.4 ± 0.5</td><td>28.9 ± 0.6</td><td>23.9 ± 0.3</td><td>29.0 ± 0.3</td></tr><tr><td>50</td><td>23.9 ± 0.5</td><td>27.7 ± 0.6</td><td>26.1 ± 0.5</td><td>26.8 ± 0.5</td><td>26.9 ± 0.3</td><td>26.9 ± 0.3</td></tr></tbody></table>
**Fig. 6** Comparison of DOPG vesicles with $X_{\text{aescin}}$ = 0 mol% (blue) to vesicles with 20 and 50 mol% (green), and micelle samples with equivalent $X_{\text{aescin}}$ (grey). **a** SANS and **b** SAXS data; the different $X_{\text{aescin}}$ are given by the green numbers on the right. The scattering data is scaled by the grey numbers indicating multiples of 10 on the left. Also shown are the CMS-fits for pure DOPG vesicles by blue lines. For aescin containing samples are shown the CMS + ellipsoid-fits by red lines for pure addition and by green dotted lines after optimization. For aescin micelles, the SAXS data and ellipsoid-fits are shown in grey in **b**; furthermore, with SasView, calculated SANS patterns are shown in **a** [37]

<figure><img src="image_9.png" /></figure>

to a SANS curve using the known contrast parameters. Bare addition of the single model functions already led to a very good agreement between the CMS+ellipsoid-model and the experimental data of the mixed system. A final optimization of the fit parameters (scale and size variables) did not lead to a significant improvement of the fit quality (see Fig. 6).

The results obtained by this fitting strategy thus clearly prove that DOPG vesicles and aescin micelles coexist with each other in solution. From the comparison of the SANS and SAXS data of the mixed system, including the data for the reference systems, it becomes also clear why the influence of the increase in $X_{\text{aescin}}$ in the case of SANS has no significant impact. Due to the small size of the aescin micelles, their contribution to the total scattering intensity is small. This becomes obvious, for example, at small $q$ values in the case of SANS. The contribution of the scattering intensity of the aescin micelles is below 0.05 %. At high $q$ values, e.g., in the range of the membrane signal ($q \approx 0.1 \AA^{-1}$), the contribution for SANS is still at most 10%, here also because the lipid membrane itself shows a high scattering intensity. This is exactly not the case for SAXS, since due to multi-layered membrane contrast, interference effects lead to the formation of the previously described minimum-maximum structure of the scattering intensity. The scattering intensity of the aescin micelles is now added to this low-intensity pattern, which results in a high contribution of the aescin micelles concerning the total intensity. The scattering intensity in this $q$-range is increased significantly. This example impressively shows how important the complementary analysis of a system using different methods or different scattering contrasts can be and that the use of only one method can lead to a

completely wrong understanding of the system if the structural conception of the system cannot be substantiated by other, independent methods.

Two questions have not yet been answered by the methods used so far: are significant interactions between the DOPG and the aescin molecules observable, i.e., do the DOPG vesicles and the aescin micelles interact partially adhesively and diffuse through the solution as agglomerates at least temporarily? And is a part of the aescin molecules incorporated into the DOPG vesicles, so that interactions at closest spatial distance would have to be detected? Both questions should finally be clarified by using NMR methods.

## Spatial interaction and proximity of DOPG and aescin molecules by DOSY-NMR

The DOSY-NMR method was used to determine the diffusion coefficients $D$ of the DOPG and aescin molecules in the mixed solutions. Again, the two reference samples, pure DOPG vesicles and aescin micelles, were used. Via the NMR signals of unique protons in both reference systems and the comparison to the mixed system, the spatial interaction between the DOPG vesicles and the aescin micelles can be proven within the sensitivity limits of the method. Only the highest aescin content of 50 mol% was used due to the comparably low sensitivity of the method. The recorded DOSY spectra for all three systems are shown in Fig. 7. On top of the DOSY signals, additionally, the ¹H NMR signals of the mentioned samples are given. The peak allocation to the ¹H-signals is presented in Figs. S6 and S7.
<figure><img src="image_10.png" /><figcaption>**Fig. 7** ¹H DOSY plots of (red) pure DOPG vesicles, (blue) aescin micelles equivalent to $X_{aescin} = 50$ mol%, and (yellow) DOPG vesicles with $X_{aescin} = 50$ mol%. Highlighted are the signal at $\delta = 1.26$ ppm and the region between 3.1 ppm and 3.5 ppm</figcaption></figure>

The one dimensional ¹H-spectra show a severe overlap of resonances of both components. However, the signal at a chemical shift of $\delta = 1.26$ ppm is mainly caused by $-CH_2$ groups located in the DOPG tails, while the multiplets between $\delta = 3.1$ ppm and 3.5 ppm can be assigned to the glucose parts of the aescin molecule [17]. These substance specific signals are now used for the determination of the diffusion coefficients *D* of the DOPG SUVs and the aescin micelles and further for the subsequent comparison with the mixed system. For the pure DOPG vesicles, the diffusion coefficient is given by $D_{DOPG} = (8.5 \pm 1.1) \cdot 10^{-12} \text{ m}^2 \text{ s}^{-1}$, and for the aescin micelles, $D_{aescin}$ is found to be $(8.1 \pm 0.7) \cdot 10^{-11} \text{ m}^2 \text{ s}^{-1}$. The diffusion coefficients of both reference systems differ by one order of magnitude. Hence, DOPG vesicles and aescin micelles can be easily distinguished based on their *D* values. With the Stokes-Einstein equation (see Eq. 7), the hydrodynamic radii are determined to $R_{H,DOPG} = (241 \pm 26) \AA$ and $R_{H,aescin} = (26 \pm 3) \AA$. The values agree very well with the already determined values of $d_z$, $R_c$ for pure DOPG vesicles (see Table 2) and $R_c$ and $R_p$ for aescin micelles with $X_{aescin} = 50$ mol% (see Table 3).

Especially the signals focused above are now compared to the DOSY spectrum of the mixed system containing 50 mol% aescin (Fig. 7, yellow). If mixed particles are present, the

substance specific signals must show contributions of both particles, vesicles and micelles. A change in *D* is visible for both signals; for both cases, *D* is shifted in the direction of the “opposite reference system,” but there is no overlap with this “opposite” particle type (e.g., with the *D* for DOPG vesicles for the aescin signal at $\delta = 3.1$ ppm and 3.5 ppm).

These results indicate that in the mixed system, almost all aescin molecules are located in micelles and almost no aescin is incorporated into the DOPG vesicles. However, the low sensitivity of the NMR method must be taken into account here, so that an incorporated fraction may be below the detection limit. The shift of *D* in the direction of the opposite particle type nevertheless indicates that a slight association of aescin molecules and micelles to DOPG vesicles might be possible.

## Conclusion

Interactions of the lipid DOPG with the negatively charged head group and with the negatively charged saponin aescin were studied in this work in a $D_2O$-buffer solution at a pH value of 7.4. To correlate the species found in mixture with a ratio up to an aescin content $X_{aescin}$ of 50 mol%
(equivalent to a ratio of 1:1), DOPG SUVs and aescin micelles at concentrations appropriate to X<sub>aescin</sub> were investigated as well. In contrast to previous studies on DMPC vesicles, here no decomposition of the DOPG vesicles occurs at any aescin molar ratio.

An important concentration in this system is the *cmc* of
aescin which is reached at contents higher than 2 mol%.

Different scattering and the NMR method DOSY are
used to investigate the system. WAXS measurements
showed the contributions of correlated molecules from
both substances, DOPG and aescin. However, based on
this method, it was impossible to clarify whether the corre-
lation signal of the aescin molecules detectable well above
the *cmc* arises from aescin molecules incorporated into the
DOPG membrane or from independent aescin micelles.
Small angle scattering with neutrons and X-ray clearly
revealed the coexistence of both, DOPG SUVs and aescin
micelles. Especially the model-dependent fitting clearly
showed that the data of the mixed system can be repre-
sented by a sum of the signals of both reference systems.
Optimization of the fit function did not lead to a significant
improvement of the fit quality as well as a change in the fit
parameters. This observation leads to the assumption that
DOPG vesicles and aescin micelles do not, for instance,
stick to each other or that lipid molecules and aescin par-
tially mix. This is further proven by DOSY measurements
which presented only a slight change in the diffusion coef-
ficients of the single components in the mixed system com-
pared to the reference systems, indicating only a very weak
interaction and no incorporation of significant amount of
aescin in the lipid bilayer.

Compared to the well-investigated DMPC-aescin sys-
tem, a huge difference concerning the intermolecular
interactions between both substances is observed here.
We suggest that the avoidance of the mixing of both sub-
stances is mainly caused by the negative charges which
are located in the hydrophilic parts of both substances.
The lipid DMPC as well carries a negative charge in the
hydrophilic head group, but while interacting with the neg-
atively charged aescin, the charges might be compensated
within the membrane by the positive charge also present in
the DMPCs head group. Exactly this compensation within
the membrane is missing in this system, which might lead
to the observed repulsion of the molecules and formation
of the separate, mostly independent structures. Investiga-
tions from the theoretical point of view would be very
interesting here to clarify the reason for the very different
behavior of both systems.

We want to especially point out the importance of veri-
fication of results by independent methods in such multi-
component systems. Having a look at only the SANS data, a
change of the system upon addition of aescin is only hardly
detectable. One would assume that still, only DOPG SUVs

are present in the sample. But having a look at the SAXS
results too, strong changes became obvious.

A coexistence of vesicles and micelles was described previously [55–57], but to our knowledge, it has never been described for a phospholipid/saponin system, like the one used in the present work.

**Supplementary Information** The online version contains supplementary material available at https://doi.org/10.1007/s00396-023-05168-0.

**Acknowledgements** The authors thank the ILL for granting the beam-time at the D22 and D11 beamlines that was available as part of proposal 9-11-1824 and 9-13-898 (doi:10.5291/ILL-DATA.9-11-1824 and 10.5291/ILL-DATA.9-13-898). Furthermore, the authors thank Ralf Schweins for his support during the beamtime at the D11. The authors thank Otto Glatter for providing the PCG software package, the fruitful discussion, and help with the software.

**Author contributions** F.G.-B. has prepared the samples, conducted the SAXS/WAXS experiments, and analyzed all recorded data. Additionally, F.G.-B. has written important parts of the manuscript. C.D. supported F.G.-B. in the evaluation of the whole data set and also wrote important parts of the manuscript. R.G. has participated in the SANS experiments at the ILL, contributed to the initial data reduction, and helped with coordinating the project; she has also edited the manuscript. P.F. and V.S. participated in the experiments at the ILL and contributed to writing the manuscript. L.P. was the local contact during the SANS beamtime at the ILL and helped with setting up the sample environment, with the initial data reduction, and has revised the manuscript. A.M. performed the NMR experiments, discussed the data analysis, and has written parts of the manuscript. T.H. coordinated the project, discussed the data analysis with F.G.-B. and C.D., and has written important parts of the manuscript. All authors reviewed the manuscript.

**Funding** Open Access funding enabled and organized by Projekt DEAL.

**Data availability** Data can be obtained from the authors upon request.

Declarations

Ethics approval and consent to participate Does not apply.

**Consent for publication** Not applicable.

**Conflict of interest** The authors declare no competing interests.

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<figure><img src="image_11.png" /></figure>
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