# Effect of cardiolipin on the lamellarity and elongation of liposomes hydrated in PBS

Alice Piccinini, Joachim Kohlbrecher, Dihia Moussaoui, Anja Winter, Sylvain

Prévost

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Alice Piccinini, Joachim Kohlbrecher, Dihia Moussaoui, Anja Winter, Sylvain Prévost. Effect of cardiolipin on the lamellarity and elongation of liposomes hydrated in PBS. Journal of Colloid and Interface Science, 2024, 669, pp.844-855. 10.1016/j.jcis.2024.04.211. hal-05105608

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Regular Article

# Effect of cardiolipin on the lamellarity and elongation of liposomes hydrated in PBS

Alice Piccinini<sup>a,b,c</sup>, Joachim Kohlbrecher<sup>d</sup>, Dihia Moussaoui<sup>e</sup>, Anja Winter<sup>b,*</sup>, Sylvain Prévost<sup>a,*</sup>

<figure><img src="image_3.png" /></figure>

GRAPHICAL ABSTRACT

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ARTICLE INFO

Keywords:

Cardiolipin

Small-angle neutron scattering (SANS)

Small-angle X-ray scattering (SAXS)

Unilamellar vesicles (ULV)

Bilamellar vesicles (BLV)

Multilamellar vesicles (MLV)

Phosphatidylcholine (PC)

Elongation

Lamellarity

Osmotic stress

PBS

ABSTRACT

Lamellarity and shape are important factors in the formation of vesicles and determine their role in biological systems and pharmaceutical applications. Cardiolipin (CL) is a major lipid in many biological membranes and exerts a great influence on their structural organization due to its particular structure and physico-chemical properties. Here, we used small-angle X-ray and neutron scattering to study the effects of CL with different acyl chain lengths and saturations ($CL_{14:0}$, $CL_{18:1}$, $CL_{18:2}$) on vesicle morphology and lamellarity in membrane models containing mixtures of phosphatidylcholine and phosphatidylethanolamine with different acyl chain lengths and saturations ($C_{14:0}$ and $C_{18:1}$). Measurements were performed in the presence of Phosphate Buffer Saline (PBS), at 37°C, to better reflect physiological conditions, which resulted in strong effects on vesicle morphology, depending on the type and amount of CL used. The presence of small quantities of CL (from 2.5%) reduced inter-membrane correlations and increased perturbation of the membrane, an effect which is enhanced in the presence of matched shorter saturated acyl chains, and mainly unilamellar vesicles (ULV) are formed. In extruded vesicles, employed for SANS experiments, flattened vesicles are observed partly due to the hypertonic effect of PBS, but also influenced by the type of CL added.

Our experimental data from SAXS and SANS revealed a strong dependence on CL content in shaping the membrane microstructure, with an apparent optimum in the PC:CL mixture in terms of promoting reduced correlations, preferred curvature and elongation. However, the use of PBS caused distinct differences from previously published studies in water in terms of vesicle shape, and highlights the need to investigate vesicle formation under physiological conditions in order to be able to draw conclusions about membrane formation in biological systems.

<footnote><sup>a</sup> Institut Laue-Langevin – The European Neutron Source, 38042 Grenoble, France</footnote>

<footnote><sup>b</sup> School of Life Sciences, Keele University, ST55BG Staffordshire, United Kingdom</footnote>

<footnote><sup>c</sup> Dept. of Medical Biotechnology and Translational Medicine Università degli Studi di Milano, 20054 Milan, Italy</footnote>

<footnote><sup>d</sup> Paul Scherrer Institut, 5232 Villigen, PSI, Switzerland</footnote>

<footnote><sup>e</sup> ESRF – The European Synchrotron, 38043 Grenoble, France</footnote>

<footnote>* Corresponding authors.</footnote>

<footnote>E-mail address: a.winter@keele.ac.uk (A. Winter).</footnote>

<footnote>https://doi.org/10.1016/j.jcis.2024.04.211</footnote>

<footnote>Received 20 December 2023; Received in revised form 25 March 2024; Accepted 29 April 2024</footnote>

<footnote>Available online 7 May 2024</footnote>

<footnote>0021-9797/© 2024 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).</footnote>
# 1. Introduction

Cardiolipin (CL) is a major component of biological membranes, significantly influencing their microstructure and properties [50]. Alongside cardiolipin, biological membranes contain different phospholipids (PL), such as phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidic acid (PA), and phosphatidylinositol (PI), in addition to proteins and other molecules. Together, cardiolipin, phospholipids, and proteins form a selective barrier around the cell or cellular organelle to control the exchange of nutrients, ions, and gasses, and provide a platform where different cellular tasks can be performed. This includes maintaining a proton gradient as needed for ATP synthesis via the respiratory complex [6,12,43], allowing selected ion flows across the membrane via ligand-gated ion channels [24], or initiating cell death via the mitochondrial apoptosis pathway [42]. Therefore, the lipid composition of biological membranes is varied and depends on their location and purpose in the cell, leading to a high variability along different species and organelles [15,8,34,4].

In mammalian cells, CL was found most abundant in the inner mitochondrial membrane (IMM), where it is present at ca. 14% by weight, alongside PC and PE at 44% and 34% by weight, respectively [8]. CL is also one of the main constituents of *E. coli* membranes, which represent ≈10% by weight of the total lipids content. On the other hand, in organelles such as the endoplasmic reticulum (ER) and plasma membrane of mammalian cells, CL represents less than 1% by weight [35]. Additionally, CL is known to be asymmetrically distributed in a bilayer and is predominantly found in the matrix-side leaflet (inner leaflet) of the mitochondria [8,13,7]. The over-abundance of CL in the IMM is related to its role of maintaining mitochondrial morphology, which is characterized by the presence of cristae [2,17], which warrant a curvature of the IMM.

Many of CL's biological functions can be attributed to its physicochemical properties and interactions with other lipids in biological membranes. CL is a diphosphatidylglycerol containing four acyl chains that cause the peculiar molecular shape of an inverted truncated cone [50]. The packing parameter of CL and its acyl chain order parameter are maximized at close-to-flat regions and minimized in the most negative curvature region for CL, which may explain CL's tendency to induce a negative membrane curvature by spreading its acyl chains. This is exacerbated by the presence of unsaturated bonds along the acyl chains, providing them with a “kink” and leading to a dramatic change in packing parameter [10,5,18]. The most abundant CL types which are present in biological systems contain acyl chains $C_{16:1}$, $C_{18:1}$, and $C_{18:2}$, with $C_{18:1}$ and $C_{16:1}$ most common in *S. cerevisae*, and $C_{18:2}$ found to be most common in rat liver and in the bovine heart [39].

Another important function of CL is to increase membrane fluidity by decreasing the order of lipid packing [47], which may be accompanied by a slight increase in membrane thickness [29]. CL can act on membrane fluidity by modifying the membrane at a structural level, modulating the viscoelasticity of the membrane, with transverse or lateral diffusion [28]. This may relate to its role as a protein scaffold, often found linked with proteins through non-covalent interactions [38], resulting in increased efficiency or activity of the bound peptides or proteins [45,22]. Additionally, CL was found to selectively interact with antimicrobial ionones [23], with lytic activities of hydrophilic ionones found to be strongly correlated with the presence of $C_{18:1}$, which tends to increase the membrane curvature making it more sensitive to membrane rupture [22].

To study the membrane structure and the interaction of lipids with other biomolecules, the formation of lipid vesicles or liposomes in an artificial environment is required. Liposomes can be classified based on the number of lipid layers: ULV (unilamellar vesicles), BLV (bilamellar vesicles) and MLV (multilamellar vesicles). ULVs can be further classified by size as small (SUV, (20 nm to 100 nm), large (LUV, (>100 nm)), and giant unilamellar vesicles (GUV (>1000 nm)) [44]. The type and

size of vesicle obtained from a lipid mixture depends on several factors, such as preparation method, lipid composition and buffer system used. One of the most common methods of lipid vesicle preparation is the thin film method, which consists of dissolving lipids in an organic solvent that will be evaporated to form a thin film, which is then hydrated by employing an appropriate solvent ($H_2O$ or buffer). The vesicles obtained by this method are mostly MLVs [25], and it is possible to reduce the vesicle's lamellability through sonication or extrusion via small pore size membranes [41].

Multilamellarity and morphology of vesicles are often studied using Cryo-electron microscopy (Cryo-EM) (e.g. [41,46,26,36]) and Small-Angle Scattering (SAS) (e.g. [41,26,36]), with Cryo-EM utilised to explore the integrity and shape of the vesicle in a vitrified sample, and SAS used to obtain information about overall shape, morphology and lamellability of the vesicles, as well as the characteristics of the bilayer, in an *in-solution* sample. While both techniques are capable of determining the shape and eventual multilamellarity of the membranes, SAS presents the additional advantage of allowing the overall assignment of vesicles as ULV, BLV, and MLV as a fraction of the whole sample, overall shape of the vesicles, as well as investigating specific features of the membrane such as thickness and asymmetric lipid distribution of the bilayer.

In our study, we employed SAS to investigate CL's role in membrane structure, namely whether different concentrations of CL in the vesicles cause different changes to the membrane's microstructure and morphology, or how the presence of other phospholipids modulates the impact that CL has on a membrane. In this work, we studied simplified PC vesicles and more complex model membranes which were hydrated in low-salt Phosphate Buffer Saline (PBS), then exposed to osmotic stress by reaching the typical PBS's salt concentration and measured at 37 °C, to simulate biologically relevant conditions. We employed SASX and SANS to study the influence of CL on simplified membrane models containing DMPC and DOPC (acyl chain length $C_{14:0}$ and $C_{18:1}$, respectively). For SANS, an isotopically labelled version of DMPC (d54DMPC) was employed, containing perdeuterated acyl chains which in $D_2O$ are almost invisible in comparison to the contrast of hydrogenated chains, so that the scattering signal would mainly be driven by CL and the headgroup of d54DMPC. This experimental setup allows the identification of effects on the hydrophobic part of the bilayer caused by CL. Our SASX results on non-extruded vesicles demonstrate that up to 20% by weight CL content strongly reduces the stacking of layers. We also demonstrate the importance of acyl length, saturation state, and matching of acyl chains on vesicle formation, with an increase in disorder of lamellar packing attributed to unsaturated chains and mismatched acyl lengths. Using SANS, we show that vesicles containing CL which had undergone osmotic stress assume a flattened shape (elongated and thin vesicles), and the addition of CL in these vesicles at a concentration ≥20% or higher induces variations on the vesicle morphology and reduction in lamellability.

We observed a trend in our fitted data, whereby d54DMPC vesicles with $C_{14:0}$ and $C_{18:1}$ are best fitted using more flattened triaxial elongated vesicles. In contrast, d54DMPC vesicles containing or $C_{18:2}$ displayed very different SANS profiles and could only be fitted satisfactorily over the whole Q-range using a triaxial elongated vesicle with 4 layers model, likely assuming peanut-shaped vesicles. An increase in CL content beyond 20% was not able to significantly change the axis ratios in these fitted geometric bodies, suggesting that biologically relevant amounts of CL are sufficient to exert their function on a membrane. Lastly, we investigated the effect of CL on more complex membranes, *E. coli* polar lipid extract and IMM models, and show the importance of the overall lipid membrane composition on the impact CL has on the membrane structure. Most importantly, our results highlight throughout that vesicles prepared in PBS show different behaviour to vesicles traditionally prepared in water, highlighting the importance of carefully choosing an artificial system as close as possible to the biologically relevant environment, in order to better relate findings obtained using
biophysical methods to results gained from biochemical or cellular approaches.

## 2. Materials and methods

### 2.1. Materials

Lipids (d54-DMPC, CL<sub>14:0</sub>, CL<sub>18:1</sub>, CL from bovine heart (CL<sub>18:2</sub>), DMPE, DOPC, DOPE, *E. coli* Polar Lipid Extract), were purchased from Avanti Polar Lipids (Table S1) and were stored at -20 °C until used. The “full” PBS buffer pH 7.4, was composed of 137 mmol NaCl, 2.7 mmol KCl, 10 mmol Na<sub>2</sub>HPO<sub>4</sub>, 1.8 mmol KH<sub>2</sub>PO<sub>4</sub>; whereas a “light” PBS buffer, denoted here after “l-PBS”, had the same composition except for NaCl at 13.7 mmol to reduce the ionic strength during extrusion. Ultra-pure H<sub>2</sub>O (18.2 MΩ cm) or heavy water (D<sub>2</sub>O), (Eurisotop, 99.9% D) were used to prepare solutions; D<sub>2</sub>O was employed for SANS experiments to increase the contrast with hydrogenated compounds and reduce incoherent scattering that causes a loss of neutrons and creates an additional background. All chemicals including organic solvents CHCl<sub>3</sub>, salts and CH<sub>3</sub>OH were bought from Sigma-Aldrich and used as received.

### 2.2. Vesicle preparation

Lipids were dissolved in CHCl<sub>3</sub>:MeOH at a volume ratio 2:1. Solvents were evaporated under nitrogen flow and dried under vacuum overnight. Lipids were hydrated with l-PBS to give a concentration from 1 mg ml<sup>-1</sup> to 5 mg ml<sup>-1</sup> depending on the experiment. These solutions were vortexed, then several freeze-thaw cycles using liquid nitrogen were performed and water at 50 °C. For extruded vesicles, the solutions were extruded an odd number of times (depending on the experiment) on polycarbonate membranes (Whatman Cat no. WHA10417304) with support membranes PE drain discs (Whatman, Cat no. 230300) using an Avanti Polar extruder (Cat. no 610000) on a hot plate at around 50 °C.

Extrusion was done no more than 24 h before the experiment. After the extrusion of lipids solutions, NaCl was added to reach a final concentration of 137 mmol (“full” PBS). For sonicated lipids employed for SAXS, an ultrasonic bath at medium intensity for 15 minutes was employed. The lipids were hydrated in light PBS and completed to reach the concentration of the full PBS after sonication, just before the SAXS experiments were performed, for consistency with the SANS data collected and to ensure measurement in biologically relevant PBS buffer.

### 2.3. Small-angle X-ray scattering (SAXS)

SAXS was performed at ESRF – The European Synchrotron, Grenoble, France on the Bio-SAXS beamline BM29, equipped with a Pilatus3 X 2M 2D detector, and using energy of 12.5 keV corresponding to a wavelength of 1 Å to cover a Q-range of 5.7 × 10<sup>-3</sup> to 5.7 × 10<sup>-1</sup> Å<sup>-1</sup>, where Q is the magnitude of the wavevector: $Q = \frac{4\pi}{\lambda} \sin(\frac{\theta}{2})$, with λ the wavelength and θ the scattering angle. The detector distance was set at 2.867 m. To reduce radiation damage, solutions were pushed in a flow-through quartz capillary while measuring. Data were reduced by the beamline workflow with PyFAI. 10 frames were systematically acquired for all samples and buffers, and buffers were always measured in between each sample measurement. After visual inspection, the frames were averaged, creating uncertainties from the standard-deviation between intensity values, and subtracting the average of frames from buffers measured before and after the sample. Data can be obtained on demand, and raw data remain available at DOI:10.15151/ESRF-ES873732891.

### 2.4. Small-angle neutron scattering (SANS)

Monochromatic SANS was performed on D11 at the ILL (Institut Laue-Langevin – the European Neutron Source, Grenoble, France), and

on SANS-1 at the Paul-Scherrer-Institut (PSI, Villigen, Switzerland) using quartz cuvettes (Hellma GmbH, Müllheim, Germany) of 1 mm (# 120-QS) or 2 mm (# 110-QS) path length for D11 and SANS-1, respectively. Samples were measured using a thermalized sample-changer at 37 °C, at a wavelength of 6.0 Å (with relative full width at half maximum of 10%), and at three different sample-to-detector distances.

On D11, a 1.5 × 1.5 m<sup>2</sup> multi-tube detector was used at distances of 1.7, 10.5 and 38 m with collimations of 2.5, 10.5 and 40.5 m to obtain a Q-range of 1.24 × 10<sup>-3</sup> to 5.3 × 10<sup>-1</sup> Å<sup>-1</sup>. Data from the three distances were combined and reduced using Grasp [9]. ILL data can be found on demand at DOI:10.5291/ILL-DATA.8-02-922 and DOI:10.5291/ILL-DATA.INTER-543. On SANS-1, the 0.96 × 0.96 m<sup>2</sup> detector (pixels of 7.5 × 7.5 mm<sup>2</sup> was placed at distances of 1.4, 6 and 18 m with collimations of 4.5, 6 and 18 m, allowing to reach a Q-range of 3 × 10<sup>-3</sup> to 4 × 10<sup>-1</sup> Å<sup>-1</sup>. Data were reduced with BerSANS [20]. However, data for d54DMPC vesicles containing 80% CL<sub>18:2</sub> could not be collected because sample preparation was extremely challenging, with vesicles being near impossible to extrude.

## 3. Results and discussion

### 3.1. CL content up to 20% reduces the stacking of layers

Lipid membrane stacking is strongly influenced by the packing parameter and membrane fluidity. Investigating the joint effect of cardiolipin and PBS on membrane stacking will reveal crucial information as to how membranes are formed and resulting vesicle morphology. Using X-rays, the main contrast from which the scattering pattern originates is between the acyl chains (with a lower electron density than buffer) and the headgroup (with a higher electron density than buffer), making SAXS an ideal technique to obtain detailed information on the organisation of lipids across the bilayer and on correlations between bilayers. The extrusion process is often used to purposely reduce inter-membrane correlations in vesicles, therefore, in order to study the spontaneous formation of vesicles while preserving inter-membrane correlations, we employed non-extruded but gently ultrasonicated vesicles for SAXS studies. Our goal was to mimic physiological conditions, which is important when conclusions about CL's role in the biological context are being made, therefore, the experiment was performed at 37 °C and utilizing PBS. No aggregations and opalescence was observed between sample preparation and sample measurement (around 4 hours), therefore indicating stability of the vesicles.

We first examined the effect of low CL content (≤20% by weight) on a PC bilayer with regard to vesicle lamellarity and size using SAXS. Vesicles were composed of DMPC (≡C<sub>14:0</sub>-PC) and DOPC (≡C<sub>18:1</sub>-PC) at 1 mg ml<sup>-1</sup> as well as different types of CL (CL<sub>14:0</sub>, CL<sub>18:1</sub>, or CL<sub>18:2</sub>) in percentages of 0%, 2.5%, 10% and 20% by weight. DMPC and DOPC vesicles were measured alone and as a mixture of lipids, with matching chains (DMPC:CL<sub>14:0</sub>, DOPC:CL<sub>18:1</sub>) and mismatching chains (DMPC:CL<sub>18:1</sub>, DOPC:CL<sub>14:0</sub>).

Our data show that PC vesicles hydrated in PBS buffer displayed stacked membranes, as can be seen from the sharp Bragg peaks at around 1 × 10<sup>-1</sup> Å<sup>-1</sup> and 2 × 10<sup>-1</sup> Å<sup>-1</sup> for first and second-order peaks, respectively, for both DMPC (Fig. 1A and B in purple) and DOPC (Fig. 1C and D in purple). These correspond to almost identical centre-to-centre distances of 63 Å despite their different acyl chain lengths, with similar distances previously reported [36,30], alongside an increase in lamellarity upon addition of salt or PBS buffer [41,36,37].

The introduction of CL in all vesicles causes the scattering curves to become smoother with Bragg peaks broadening or entirely disappearing, indicating a reduction in stacking. Remarkably, the addition of CL<sub>14:0</sub> to DMPC vesicles at 2.5% already leads to non-correlated membranes (disappearance of the Bragg peak), and at 5% and above the spectra display a well-defined form factor of vesicles at around 3 × 10<sup>-2</sup> Å<sup>-1</sup>, i.e. a radius of ≈100 Å. 5% of CL<sub>14:0</sub> in DMPC vesicles induces the formation of elongated vesicles, as seen by the change of slope in
<figure><img src="image_5.png" /><figcaption>**Fig. 1.** Effect of CL on PC vesicles with matching and mismatching acyl chains as determined by SAXS (BM29, ESRF). A) DMPC:CL<sub>14:0</sub>, B) DMPC:CL<sub>18:1</sub>, C) DOPC:CL<sub>14:0</sub>, D) DOPC:CL<sub>18:1</sub>. DMPC and DOPC-only vesicles are shown in purple, with CL content of 2.5% (light gray), 5% (cyan), 10% (blue) and 20% (dark gray). The high *Q*-range of the data was fitted according to the model as described in section 3.1.1, while fitting parameters are described in SI Table (S2). Low-*Q* data were fitted only for DMPC:CL<sub>14:0</sub> vesicles, for samples containing from 5 to 20% of CL. Fits are shown with a solid line. An incremental multiplication factor of 10 along *I* was applied for clarity. (For interpretation of the colours in the figure(s), the reader is referred to the web version of this article.)</figcaption></figure>

the low-*Q* region towards *Q*⁻¹, which was confirmed by fitting the data with a cylindrical shell model (SI Table S2), and for 10 to 20% the form factor is that of a spherical vesicle. In the high-*Q* region, data were modelled using SASfit [21], employing a form factor of discs with 3 homogeneous layers (a single inner layer for the acyl chains and two outer layers for the solvated headgroups exposed to solvent) and a structure factor to account for multi-lamellarity (paracrystalline model with polydispersity implemented on the discrete number of layers) ([31] (SI Table S2)). Fit parameters revealed a rather constant lipid layer thickness of 35 Å for all examined vesicles but with a rapidly decreasing number of fitted stacked layers when a small amount of CL is present, e.g. decreasing from 38 for DMPC only vesicles to 11 for DMPC:CL<sub>18:1</sub> vesicles with CL at 2.5% then to 3 with CL at 5% (Fig. 1 B) (SI Table S2). The fitted number of stacked layers is then kept constant which indicates that CL even at low concentrations promotes the formation of vesicles with less layers and eventually drives the assembly towards bilamellar vesicles (BLV) or even ULV. Low-*Q* data of DMPC vesicles containing 2.5% of CL<sub>14:0</sub> and of other combinations of PC and CL (Fig. 1 B-D) do not present interesting features, such as form factor oscillations or a plateau. Instead, the scattering intensity presents as a single power-law around Q-2.7 (range -2.5 to -3.2), and thus were not included in the fit model. The justification for the model employed for fitting high-*Q* data is described below.

Aside from the amount of CL present in the vesicles, the type of the acyl chain and whether they are matching also seem to be deter-

mining factors regarding vesicle morphology. In the case of matching saturated acyl chain length (C<sub>14:0</sub>) (Fig. 1 A), a stronger tendency to form small unilamellar vesicles (ULVs) was demonstrated probably due to an easier packing of lipids. The presence of mono-unsaturated C<sub>18:1</sub> chains, either as DOPC or CL<sub>18:1</sub>, causes a transition from large stacks of bilayers to less correlated layers, with the broadening of the peak in vesicles made up of 5%, 10% and 20% CL (Fig. 1 B-D). A visual inspection of the humps at around 1 × 10⁻¹ Å⁻¹ and 2 × 10⁻¹ Å⁻¹ for DOPC:CL<sub>14:0</sub> and at around 7.5 × 10⁻² Å⁻¹ and 1.5 × 10⁻¹ Å⁻¹ for both DMPC and DOPC with CL<sub>18:1</sub> reveals that the bilayer correlation decreases from DMPC:CL<sub>18:1</sub> (still strongly correlated) to DOPC:CL<sub>18:1</sub> to DOPC:CL<sub>14:0</sub> (very weak bilayer correlation). A comparison of vesicles containing 20% of CL with matching and mismatching chains revealed that DMPC:CL<sub>14:0</sub> is the only one able to form ULV with a very well-defined form factor. On the other hand, in the case of DMPC with CL<sub>18:1</sub> or DOPC with CL<sub>14:0</sub> and CL<sub>18:1</sub>, correlated membranes are still evident. Our conclusions from a qualitative analysis were confirmed by fitting the data (SI Table S2), showing that the inclusion of CL induces a reduction of the number of layers (N) as well as an increase in the disorder of the stacking (Δ). This is especially evident when comparing vesicles formed only of saturated chains, such as DMPC:CL<sub>14:0</sub> (Fig. 1 A) with vesicles containing unsaturated chains (Fig. 1 B-D).

Our results show that the presence and amount of CL, as well as the type of acyl chains have a strong effect on vesicle membranes, reducing inter-membrane correlations via both an increased disorder and a re-
duced number of stacked layers. The ability of CL to reduce correlations between membranes is potentially further enhanced by its ability to increase the “waviness” of the membrane due to its inverted cone shape [10], which could either be defined by dynamic undulations or more static ripples. The membrane undulation of CL-containing GUV has previously been shown by EM, supporting the notion of a preference of CL for larger curvatures [46]. In previous reports the lamellarity of vesicles was shown to be dependent on the membrane curvature of the vesicles and its adhesion energy [41], which is thought to decrease upon addition of CL, therefore leading to repulsion between membranes and an increased bending rigidity which results in lamellar separation and thus ULV formation [41,26]. The increased bending rigidity ($K_c$) of CL with respect to PC was confirmed by other studies, where the $K_c$ of CL 14:0 was reported 50% larger than DMPC (X-ray diffuse scattering) [3], and the $K_c$ of CL 18:1 30% larger compared to DOPC (determined with Neutron Spin-Echo experiment (NSE)) [32].

Furthermore, a dependency on saturation state and chain length was also observed previously. In the case of PC lipids with short and saturated acyl chains, CL was thought to increase the bilayer order parameter, thus reducing the membrane fluidity [3]. However, in the case of PC lipids with longer and unsaturated acyl chains, CL decreases the acyl chain order parameter therefore increasing fluidity [11,48,29]. In addition, a recent study on bicelles identified further factors as crucial parameters responsible for affecting membrane structure, such as the $T_m$, the charge density and total lipid concentration. In the case of zwitterionic lipids, MLVs were found together with ribbons and ULV structures, while charged lipids formed perforated lamellae and ULVs, which became more uniform in the presence of unsaturations [25].

Our experimental data elaborate on many of the experimental and modelling studies which demonstrate a strong dependence on cardiolipin content in shaping the membrane microstructure. In particular, we highlight the ability of CL to reduce the correlation between membranes, reduced stacking, already at the lowest CL content explored (2.5% by weight), indicating a very profound effect of CL on the membrane structure and organisation. Our data also show that CL influences the overall shape of the vesicles by inducing elongation. Considering that we used gently sonicated vesicles in SAXS (bath sonication), where vesicles will have largely formed without applying a high energy method such as extrusion, vesicle formation most likely was largely influenced by the packing parameter, which lead to elongated vesicles in certain vesicles. This may be an important feature of the effects of CL on the vesicles, and therefore, we explored this further in SANS experiments, which are able to reveal additional information on the overall shape.

### 3.1.1. *Justification of the SAXS model employed*

Data analysis was performed with SASfit [21]. High-Q regions of SAXS profiles contain information on the scattering length density (SLD) profiles normal to the membranes. Therefore, the model must at minimum describe the electron-poor hydrophobic core and the two electron-rich polar shells. Thus, the high-Q of the SAXS data were fitted with a three-layer symmetrical model, two identical external ones for the headgroups and a central one for the tails. The low-Q region often contained no particular feature and therefore was not considered, except for DMPC:CL₁₄:₀ 5, 10 and 20%, which do contain interesting features and were fitted with a long cylindrical shell model (in the case of 5% of CL), a form factor of vesicles with a core-shell model containing 3 shells (in the case of 10 and 20% of CL)(SI Table S2). The core-shell models contain 3 shells: two for the headgroup and one for the tails, which were employed when the vesicle form factor was evident, confirming the presence of ULVs. On the other hand, fitting of vesicles containing 5% of CL with a cylindrical structure indicates the presence of elongated vesicles which also contain correlated membranes. SAS fitting of lipid membrane is often achieved with a multi-Gaussian scattering density profile model [31], however for our SAXS data, we found that the simple 3-slab model was sufficient to capture the main features of the

form factor, as necessary to determine the structure factor (stacking and disorder).

## 3.2. Different types of CL cause distinctive changes in vesicle morphology

To further explore the effect of CL on membrane organization, SANS experiments were carried out on mixed vesicles of CL and d54DMPC. Usually, SANS provides straightforward information on the overall shape and size of vesicles and on the bilayer thickness, as the contrast often originates from using hydrogenated lipids in a deuterated solvent. However, due to the presence of a perdeuterated tail and a perhydrogenated headgroup in d54DMPC, these vesicles, when measured by SANS with $D_2O$ as a solvent, lead to two peculiarities: (i) for vesicles containing only d54DMPC, the main contrast occurs between the D-rich tails and the H-rich headgroups, leading to a particular scattering pattern and (ii) for vesicles containing an additional hydrogenated lipid, the latter dominates the contrast, i.e. data can be interpreted mostly based on the CL content of the bilayer (Figure S4). Using defined vesicle mixtures of perdeuterated d54DMPC and hydrogenated CL, we aimed to study vesicle lamellarity, vesicle shape and CL distribution in the membrane.

For our study, we employed vesicles consisting of d54DMPC, which has its chains essentially matched-out in $D_2O$, in the presence of 20, 50 and 80% by weight of hydrogenated CL₁₄:₀, CL₁₈:₁, or CL₁₈:₂. Experiments were performed at 37 °C on vesicles extruded with a 50 nm membrane (at 50 °C in the presence of PBS), which allowed us to mimic physiological conditions. No aggregations and opalescence was observed between sample preparation and sample measurement (around 3 hours), therefore indicating stability of the vesicles. The use of PBS and presence of osmotic stress are crucial factors for our study, as they are known to induce lamellar stacking [40,41,36], which leads to formation of MLVs despite extrusion on a membrane with 50 nm pore size.

Due to lamellar stacking, extrusion was difficult, therefore, we utilized a “light-PBS” buffer during extrusion and adjusted the salt to reach the “full-PBS” salt contents after extrusion. However, seeing as NaCl is a membrane-impermeable solute, this resulted in a difference in salt content between the core of the vesicles (low-salt content) and the outside buffer (high-salt content), therefore increasing the osmotic stress. A calculation of the osmotic pressure attributed to “light-PBS” versus “full-PBS” revealed a difference of 3.4 atm (1.3 atm versus 4.7 atm for “light-PBS” versus “full-PBS”, respectively). Cells always experience osmotic stress, which is thought to be between 7 and 10 atm for the majority of cells [49], but this is usually regulated by several factors such as transporter proteins and channels which allow the regulated flux of ions in and out of the cell. However, the difference in osmotic pressure across model membranes without such regulators leads to membrane remodelling and a flattening of vesicles from a well-formed sphere [33] to a more oblate shape, often with invaginations, as was observed with fluorescence microscopy [27,51], light scattering [33,36] and EM [51,25,37]. GUVs containing DMPC and cholesterol already showed flattening and budding at an osmotic gradient of 0.37 atm, which was partially rescued by the addition of higher amounts of cholesterol [51]. Additionally, a degree of shrinkage was previously observed under hypertonic conditions, which leads to loss of volume of 20% or more [52]. We therefore expect the vesicles used in our SANS study to present features of stacking (ULV, BLV or MLV), but also characteristics of distortion of their morphology from a sphere to a more elongated vesicle.

Firstly a qualitative assessment of the data was performed to establish macroscopic differences between the studied cases. However, the peculiar contrast of d54DMPC makes a visual comparison between pure d54DMPC vesicles and vesicles enriched of CL difficult to perform, this is especially evident for vesicles with a low hydrogen content. On the other hand, as soon as the deuterated content decreases, and the scattering signal increases, a qualitative interpretation of the data can be made. All SANS curves obtained are classical for hollow vesicles, with
<figure><img src="image_6.png" /><figcaption>**Fig. 2.** Effect of an increased amount of CL in d54DMPC vesicles as observed by SANS (D11, ILL), fit with triaxial ellipsoidal model with 4 shells. A) with CL<sub>14:0</sub>, B) with CL<sub>18:1</sub>, C) with CL<sub>18:2</sub>. Shown are d54DMPC vesicles with 0% CL (purple), 20% (light gray), 50% CL (orange), 80% CL (red) and 100% CL (dark gray). An incremental multiplication factor of 3 along *I* was applied for clarity. D) Ratio of equivalent radii for d54DMPC vesicles with 20% (light grey), 50% CL (orange), 80% CL (red) and 100% CL (dark grey) CL<sub>14:0</sub>, CL<sub>18:1</sub> and CL<sub>18:2</sub>. (For interpretation of the colours in the figure(s), the reader is referred to the web version of this article.)</figcaption></figure>

most curves showing a bump at mid *Q* (4 × 10⁻² to 1 × 10⁻¹ Å⁻¹) that originates from the correlation between membranes (Fig. 2). Vesicles containing CL<sub>14:0</sub> and CL<sub>18:1</sub> show a similar shape of the scattering profile, while it's quite different for CL<sub>18:2</sub>. This suggests that different CL types have distinct effects on the morphology of the vesicles, which may mean that data fitting could involve several steps or that some datasets might require different fitting models.

An inspection of d54DMPC-only vesicles (Fig. 2 A-C purple curves) shows a bump at high-*Q* around 1 × 10⁻¹ Å⁻¹ to 2 × 10⁻¹ Å⁻¹ which is deformed into a square shape compared to the expected signal due to the peculiar contrast of d54DMPC. We interpret this deformation as evidence for the presence of strongly correlated membranes. Literature reports examples of d54DMPC vesicles which are ULVs, showing a classical form factor of unilamellar lipid vesicles with an absence of the stacking periodicity peak [14]. Therefore, several measurements of d54DMPC below and above its T<sub>m</sub> were tested, showing differences mainly in the low-*Q* region, while the high-*Q* region of the data remained largely unchanged, confirming that with the protocol employed in this paper, we produce multilamellar d54DMPC-only vesicles (SANS profile shown in SI S4). The fact that d54DMPC-only vesicles present stacking under our experimental setup may be due to the use of PBS. Additionally, we do not observe significant elongation or deformation of the vesicles due to osmotic pressure differences, as could be expected from previous studies using e.g. GUVs. In fact, d54DMPC-only vesicles are near-spherical in our study, possibly due to the high bending energy caused by stacking which would counteract osmotic pressure across the membranes. These findings present a particularly interesting avenue of enquiry considering the physiological environment of vesicles relevant

for biological studies, which we examined further utilising a systematic in-depth analysis to be published in due course.

Vesicles containing CL displayed scattering profiles different from d54DMPC-only vesicles, and we set out to fit our data using different models. We first fitted our datasets using a ULV+BLV model, however, we were unable to fully capture the low-*Q* and mid-*Q* ranges which are indicative of elongation. It is also possible that the observed membrane correlation results from flattened vesicles with a small radius resulting in two membranes very close to each other. Therefore, we next tried a biaxial ellipsoidal shell model, however, the slope at low-*Q* was still not fully captured, as well as features at mid-*Q* and a third dimension was added to result in a fitting model of a triaxial ellipsoidal shell. An ellipsoidal shell model indicates the presence of non-spherical, flattened vesicles, which may be due to the hypertonic effect after the addition of salt that causes a difference in osmotic pressure between the inside and outside of the vesicle. Additionally, it is known that extrusion also results in the formation of elongated vesicles [19], an effect which might have been enhanced by the difference in osmotic pressure on the vesicles. However, when considering that d54DMPC-only vesicles do not appear to be elongated, the effect of the osmotic gradient on vesicle shape may be dwarfed by the influence of CL. Additionally, it should be considered that d54DMPC-vesicles contain strongly correlated membranes with high bending energy, which would counteract elongation of the vesicles, as opposed to ULVs with a low bending energy (also see Supplementary Results for calculations). Therefore, it may be the case that because CL reduces stacking, the membrane overall becomes softer and more malleable, and therefore more susceptible to the influence of an osmotic pressure gradient across its membrane. We therefore
expect data fitting to require different models to capture all features of the different vesicles over the *Q*-range we measured.

### 3.2.1. Fitting of d54DMPC vesicles using a ULV+BLV model

From our SAXS experiments, we concluded that CL reduces the lamellarity of the vesicles leading to the presence of multiple populations of vesicles, ULVs and BLVs at various fractions. Therefore, we took in consideration the possibility of having different populations of vesicles in our SANS data: i) unilamellar vesicles (ULV), ii) bilamellar vesicles (BLV), and iii) model which includes two populations of vesicles BLV and ULV using SasView (https://www.sasview.org). BLVs, as opposed to ULVs, are characterised by a stacking periodicity peak at mid-*Q*. Although including BLV into our fitting model increases the complexity of the system while decreasing the heterogeneity of the system, clearly correlation between membranes is visible in our data, which may be best captured with BLV. Additionally, to fit these data, some simplifications were made, to avoid the risk of overfitting (for additional information on this model, please see Supplementary Results; fitting parameters are presented in (Supplementary Table S5)).

For the purpose of this study, given that the contrast of a bilayer simplifies when sufficient amounts of hydrogenated CL are added, we compared 3 models with less parameters, considering only ULV, or only BLV, or a mixture of both. For consistency, we applied the same model to all spectra including that of pure d54DMPC, and found a single population of BLV with an outer radius of 344 Å (SI Table S5 and SI Figure S2). Although the fit is less satisfactory, as an excess of the intensity at mid-*Q* at 0.03 Å⁻¹ to 0.04 Å⁻¹ is visible, this simpler model overall represents the content of this sample satisfactorily. However, as we can see from the high-*Q* region, we have correlated, stacked layers. Therefore, we next tried to improve upon this fit by including a strongly correlated membrane parameter and included this into a new model, a spherical vesicle with thermal disorder with 5 layers, which we describe in detail in Supplementary results. This fitting represents the best fitting for our data for d54DMPC-only vesicles, taking into account the whole *Q*-range of the SANS profile, confirming the presence of strongly correlated membranes, indicating the presence of MLVs. This is in line with our SAXS results, where DMPC sonicated in PBS, and exposed to osmotic stress show a strong correlation between membranes. We note that extrusion through a 500 Å membrane is not sufficient to destroy MLVs.

Analyses of mixed vesicles (both d54DMPC and CL present) examined in our SANS experiments using this model show a trend toward formation of ULV and BLV upon the addition of CL. Vesicles containing $CL_{14:0}$ at 20% by weight revealed the formation of ULV, whereas with $CL_{18:1}$ or $CL_{18:2}$ a mixture of BLV and ULV is observed. Vesicles containing ≥50% CL show the formation of BLV, independent from the CL type used (Figure S2 A - C, orange curves). This trend continues in vesicles containing 80% of $CL_{18:1}$. However, data from samples containing a high percentage of $CL_{18:1}$ in DMPC vesicles present features at mid-*Q* (starting from 1e-2 Å⁻¹) (SI Fig. 3 D) that are very difficult to capture with this model. Additionally, the low-*Q* range was not taken into account satisfactorily by this present model, suggesting additional features that may be able to be captured with a different, potentially more complex, model.

Turning to CL-only vesicles, we found that $CL_{14:0}$ vesicles could be fitted using ULVs, but $CL_{18:2}$ vesicles were mostly ULVs with a BLV content of 15%. On the other hand, $CL_{18:1}$ vesicles could be fitted as 100% BLV (Figure S2 A-C gray curves and SI Table S5). SANS spectra from extruded vesicles of pure CL show differences in the low and mid-*Q* regions which are dependent on the CL type (Figure S2 A-C dark gray curves). SANS profiles of $CL_{14:0}$ and $CL_{18:2}$ vesicles do not show any stacking periodicity peaks, however, a scattering profile atypical for a ULV is present. Our scattering curves present with a peculiar angular shape at mid-*Q* (0.1 to 0.2 Å⁻¹) which might suggest the presence of additional features. In contrast, $CL_{18:1}$ vesicles do show a peak at 6e-2 Å⁻¹, indicating a stronger correlation between membranes.

In summary, the overall good fitting that could be obtained with this ULV+BLV model captures the stacking periodicity peak, representative of the correlation between membranes, and overall shape of the data well. When CL is present in d54DMPC vesicles, BLV are formed irrespective of the CL chain length. However, the increase in intensity at low-*Q*, which suggest the presence of elongated and thin vesicles, is not well captured. Additionally, the fitted outer radius of the ULV was much smaller in many cases than the pore size of the membrane used for extrusion (e.g. 160 Å versus 500 Å for d54DMPC: $CL_{14:0}$ 50%). We would expect vesicles that are slightly smaller than the extruder pore size, as has been observed previously (120 Å versus 200 Å, [37] and references therein). Taking osmotic pressure and bending energy into account, a vesicle would shrink 1/10 in volume, leading to a reduction in vesicle diameter from 500 Å to around 230 Å (calculation can be found in Supplementary Materials). But the ULV size obtained from fitting is smaller still, and may be a parameter borne out of the fitting rather than a true physical dimension. Furthermore, several features present in the mid-*Q* region, where the stacking periodicity peak is present, could not be fitted satisfactorily, especially for $CL_{18:1}$. Therefore, we moved forward trying a model which includes a more elongated shape.

### 3.2.2. Fitting of d54DMPC vesicles using an elliptical biaxial ULV model

All vesicles containing d54DMPC and $CL_{14:0}$ or $CL_{18:1}$ presented with a smaller upturn of intensity at low-*Q* indicating elongation, where the vesicles lose their sphericity, leading to a lengthened structure, perhaps similar to a Rugby ball. In contrast, this upturn is completely absent with $CL_{18:2}$, which instead presents a plateau and a visible form factor oscillation at 1e-2 Å⁻¹, indicating a formation of less elongated vesicles, with probably a different overall shape. On the other hand, $CL_{14:0}$ has the steepest slope at low-*Q* suggesting very elongated vesicles (Figure S3 A dark gray).

As elongation was found to be a feature of our vesicles, we first conducted simulations utilizing an ellipsoidal-shell model considering different lengths of polar and equatorial radius to simulate different cases of elongations ($R_{pol}: R_{eq}$ with 40:20; 20:40; 20:100 (nm)), a thickness of 30 Å, and a polydispersity of 20% in the radius polar radius. This resulted in a shift of the form factor oscillation at low-*Q* (1e-2 Å⁻¹) preserving the formation of a plateau at low-*Q* (Supplementary Figure S5 A). Therefore, we believe that often in our mixed vesicles elongation occurs, especially for $CL_{14:0}$ and $CL_{18:1}$. The same was found applicable for CL-only vesicles with $CL_{14:0}$ and $CL_{18:1}$, but not $CL_{18:2}$.

We therefore, we carried out separate fitting of our data using an alternative model describing elongation. Elliptical shells (biaxial model) were tested, generally giving a good fit to our data (Figure S3), with axial ratios typically ranging from 4 to 10 for $CL_{14:0}$-containing vesicles, and 4 to 5 for $CL_{18:2}$-containing vesicles (Figure S3), making $CL_{14:0}$-containing vesicles slightly more elongated than $CL_{18:2}$-containing vesicles (SI Table S6). However, fits for low-*Q* regions for mixed vesicles with $CL_{18:1}$ were only slightly improved, and were considerably worse than in the ULV+BLV model for vesicles containing $CL_{18:2}$. Additionally, the fit for the high-*Q* region of $CL_{14:0}$ vesicles was worse than with the ULV+BLV model. This is due to the elliptical biaxial shell model only using one shell, therefore missing features of the bilayer that are captured by the ULV+BLV model. We did not observe a clear trend in axis ratio that was dependent on CL content, rather, the type of CL incorporated was the determining factor, hinting at a genuine property of the different CL species rather than a concentration effect.

The different degrees of elongation observed in vesicles made of the three different types of CL could be due to the different rigidity imposed by CL chain length and saturation state. A shorter saturated chain (in the case of $CL_{14:0}$) constitutes a rigid system that induces the elongation of vesicles, with extrusion carried out at ≈50 °C and SANS/SAXS measurements performed at 37 °C which is well above the melting temperature of DMPC at 24 °C. On the other hand, the unsaturation of CL would increase the flexibility of the system, arranging the vesicle in less (in the case of $CL_{18:1}$) or non-elongated form ($CL_{18:2}$).
<figure><img src="image_7.png" /><figcaption>**Fig. 3.** Effect of CL on IMM mimicking membrane models, as measured by SANS (SANS-1, PSI). Extruded vesicles of (DOPC:DOPE:CL₁₈:₁), where DOPE:DOPC ratio was kept constant at 1:1 while CL content increased from 0% CL (purple) to 20% (light gray), 50% CL (orange), and 80% CL (red). E) Extruded vesicles of (DOPE:POPG:CL₁₈:₁) mimicking *E. coli* membranes, where DOPE:POPG ratio was kept constant at 7:2 while CL content increased from 0% CL (purple) to 20% (light gray), 50% CL (orange), and 80% CL (red). An incremental multiplication factor of 3 along *I* was applied for clarity. Data presented in absolute scale. D) Ratio of equivalent radii for d54DMPC vesicles with 0% (purple), 20% CL (light grey), 50% CL (orange) and 80% CL (red) CL₁₄:₀, CL₁₈:₁ and CL₁₈:₂. (For interpretation of the colours in the figure(s), the reader is referred to the web version of this article.)</figcaption></figure>

An elongation of the vesicles could be caused by an increase in cur-
vature of the vesicles at certain points, which may be exacerbated by the
rigidity of the saturated acyl chains in CL₁₄:₀, and/or due to an asym-
metrical accumulation of CL and a promotion of negative curvature,
as previously suggested [29,50]. Therefore, studying the distribution of
CL between the outer and inner leaflets of a bilayer membrane could
help understand the effects of CL on the membrane microstructure.
Literature reports several studies which demonstrate an asymmetrical
distribution of CL in a membrane, with preferential distribution in the
inner leaflet [16]. Using isotopic labelling, SANS could resolve whether
a membrane composition is very asymmetrical or not, although it would
not inform on which leaflet is enriched in CL (see simulations in SI re-
sults and SI Figure S5 B). However, our simulations suggest that the
asymmetry would only cause minute changes in the resulting scattering
patterns in the high-Q region at low intensity, which would in turn re-
quire measurements at high concentrations of vesicles (≥20 mg ml⁻¹).
Based on our experiences, such a mixture would be difficult to achieve
via extrusion.

Seeing as our low-Q data could still not be fitted well, we resolved to
add another dimension and tried fitting our data with a triaxial elliptical
4 shells model.

3.2.3. Fitting of d54DMPC vesicles using an elliptical triaxial ULV model

The stacking periodicity peak present at mid-Q can be caused by
thin flattened vesicles, creating the same stacking effect as a BLV or
MLV would make. This could be captured by flattened ellipsoidal shapes
where one axis is rather small, much like a pill. Indeed, the low-Q part
of our experimental spectra are generally well-fitted using the triaxial
elliptical shell model (Fig. 2), suggesting an elongation of the mixed
d54DMPC:CL vesicles and the absence of spherical vesicles.

Due to the presence of strongly correlated membranes present in
the d54DMPC, the triaxial elliptical model does not fit the reference,
but for consistency fitting of the data with this model are shown. Fit-
ting of the d54DMPC-only vesicles again did not capture the features at
high-Q. However, fitting of scattering profiles containing mixed vesicles
with CL<sub>18:1</sub> and CL<sub>18:1</sub>-only vesicles were the best obtained by any fits
we attempted. Similarly, fits for mixed vesicles with CL<sub>14:0</sub> and CL<sub>14:0</sub>-
only vesicles best represented our data, but fits were not as good as
for CL<sub>18:2</sub>. There were some differences in the slope at low-Q which
was especially pronounced with CL<sub>14:0</sub>-only vesicles. The presence of
up to 20% of CL leads to a small change in the dimensions of the el-
lipsoid while after that amount increasing the amount of CL does not

increase its effect on the vesicles. The amount of CL in the vesicles does
not appear to influence the shape of the vesicles, nor can it rescue the
"collapsed" ellipsoidal vesicles into a more sphere-like shape.

Fits obtained for CL₁₈:₂-containing vesicles (both mixed and CL-
only) using this model were worse than with the ULV+BLV model, es-
pecially at mid-Q. Fits for d54DMPC:CL₁₈:₂ vesicles with 20% and 50%
were better using the biaxial elliptical shell model, but still not quite
right. The different behaviour of CL₁₈:₂ is visible even with qualitative
analysis, showing a plateau at low-Q as well as a different SANS pro-
file at mid-Q. Different models were tested for CL₁₈:₂ (ellipsoidal shell,
ellipsoidal cylinder shell, ULV, and ULV and BLV as well as random
multilamellar vesicles), and the results were the same, with intensity at
mid-Q missing. The fact that none of the models tested fully capture ex-
perimental data for CL₁₈:₂ well, with a lack of intensity at mid-Q at 0.01
Å⁻¹, suggests that this CL type behaves differently from the others. This
may be due to CL₁₈:₂ containing two unsaturations which may make
the membrane very flexible, soft and malleable, so that no discernible
shape can be reached.

To summarise, we fitted data of d54DMPC:CL vesicles using differ-
ent models which satisfactorily represent the main features of our data,
and we were able to discern a trend in our data sets. The model con-
taining two populations of vesicles (ULV+BLV model) did not take into
account the low-Q region of the data satisfactorily (keeping in mind
that we reach Q values as low as 0.003 Å⁻¹ with the instrumental setup
at D11 at ILL), and returned an outer radius of ULV much smaller than
the pore size of the membrane used for extrusion (in some instances
160 Å diameter vs 500 Å pore size). However, the fitted BLV size of
on average 500 and 800 Å would be expected from extrusion through
a membrane with 500 Å pore size. Therefore, the ULV+BLV model
was not considered to represent our samples adequately, although we
clearly observe a degree of stacking. The biaxial ellipsoid model lacks
three shells to represent headgroup-tail-headgroup, which may explain
the difference between the fitted curve and the experimental data at
high-Q. Fit values obtained with the triaxial elliptical shell model re-
turned equivalent dimensions of a sphere that correspond well with the
pore size of the membrane used for extrusion, and generally represented
the low-Q region well, and did capture the stacking in high-Q region.
d54DMPC vesicles with CL₁₄:₀ were overall best fitted using the triaxial
ellipsoidal model, based on qualitative assessment of the fits and data.
Similarly, d54DMPC vesicles containing CL₁₈:₂ were reasonably well
captured with the ULV+BLV model but the best fit was obtained with
the triaxial ellipsoid model with 4 layers. d54DMPC vesicles containing
<figure><img src="image_8.png" /><figcaption>**Fig. 4.** Schematic summarising the behaviour of CL in PC vesicles. DMPC vesicles without CL in PBS form MLVs, but addition of CL has several effects that lead to a reduction in the stacking of layers as well as increased vesicle elongation, with increase in membrane disorder and membrane fluidity and a reduction in membrane rigidity reported previously. Vesicles containing $CL_{14:0}$ and $CL_{18:1}$ form elongated ULVs, while vesicles containing $CL_{18:2}$ form more complex and less flattened shapes which are challenging to model.</figcaption></figure>

$CL_{18:1}$ were also best represented with a triaxial ellipsoidal model (Fig. 4). In all cases, CL content above 20% did not change the dimensions or shape of the vesicles significantly, indicating that CL is generally unable to return ellipsoidal vesicles to a spherical shape. The goodness of the fits of the different data series gives clues as to the likely shape of the vesicles and how different CL types might influence this. Therefore, $CL_{14:0}$ likely generates Rugby ball-shaped mixed vesicles with a biaxial ratio of around 10, whereas mixed vesicles containing $CL_{18:1}$ are triaxial ellipsoids reminiscent of pills with axial ratios of 8-10 for b/a and 4 for c/b. In contrast, vesicles containing $CL_{18:2}$ were only represented sufficiently using the triaxial ellipsoid model with 4 layers, which could indicate the formation of extended vesicles with a girth, similar to a peanut shell. Addition of CL generally makes the membrane softer, more flexible and reduces stacking, with $CL_{14:0}$ and $CL_{18:1}$ producing elongated vesicles due to the relative rigidity of the acyl chains. However, the acyl chains of $CL_{18:2}$ on the other hand, are flexible enough to allow a quite dramatic change in vesicle shape, from a spherical vesicle after extrusion to a quite complex extended shape. The effect of osmotic stress should not be underestimated in creating the different vesicle shapes, and a softer, more flexible membrane would allow water to leave the inside of the vesicle more easily, causing deformation and shrinkage. Interestingly, SANS profiles for d54DMPC-only vesicles, were qualitatively different from mixed vesicles or CL-only vesicles, and could not satisfactorily been fitted with most models. We developed a model with strongly correlated layers which fit the data best, suggesting that d54DMPC is a near-spherical MLV similar to an onion. This means that in all cases, CL has two effects on vesicle formation: it reduces stacking from MLV to ULV or BLV, and promotes formation of elongated vesicles.

### 3.3. Effect of CL in E. coli and IMM mimicking membrane models

So far we employed relatively simple membrane models to study the effects of CL on vesicle morphology and microstructure. We showed that CL has a profound effect on vesicle lamellarity and shape, alongside a clear dependence on the types of lipids used in vesicle formation. We propose that certain parameters are chiefly responsible for formation of the vesicles: saturation state of the lipids, chain lengths, and lipid composition which results in distinct packing parameters influencing liposome morphology. In an attempt to move towards more biologically relevant membrane models, we employed more complex vesicles containing DOPE:DOPC:$CL_{18:1}$ mimicking eukaryotic IMM membranes, and vesicles of DOPE:POPG:$CL_{18:1}$ mimicking E. coli membranes. To study the effects of increasing concentrations of CL, we utilised vesicles containing a constant ratio of PE:PC (1:1 ratio by weight) or PE:PG (7:2 ratio by weight) while increasing the amount of CL to 20, 50 and 80% by weight. In SANS experiments, the membrane model representing the IMM of a mammalian cell, PE:PC (1:1 ratio by weight), shows significant changes in the stacking periodicity peak upon increase of CL content, which is shifted towards lower Q by a factor of two (Fig. 3 A).

This indicates an increased distance between the two layers with the increase of the CL content up to 50%, which then remains unchanged for 80% CL content. Here, vesicles show a stacking periodicity peak which is sharper with respect to the $CL_{18:1}$ only vesicles (Fig. 3 B dark gray curve). On the other hand, PE-rich vesicles (the E. coli membrane model) without CL did not show significant changes in the vesicle scattering profiles as compared to vesicles containing a higher amount of CL, presenting a constant correlation between layers, and a constant thickness of ≈32 Å (Fig. 3 B). Therefore, in the presence of an excess of PE, which has a similar packing parameter as CL, CL has little effect on vesicle morphology.

Data were fitted with a triaxial ellipsoidal 4 shells model. The fitting of the data confirmed the presence of flattened vesicles, shaped similar to a pill, possibly caused by the osmotic stress the vesicles were subjected to. This characteristic shape of vesicles, long and thin, may justify the presence of the stacking periodicity peak in unilamellar vesicles, which is caused by the proximity of the bilayer instead of the presence of multilamellae. Fitting of the data revealed a trend to increase the smallest dimension, which is more pronounced in the IMM membrane mimicking model than for the E. coli, suggesting that after addition of CL the vesicles tend to be less flattened and have less correlated membranes. Therefore, fitting of the data confirmed the qualitative analyses that in the presence of an excess of PE, CL causes a change in the morphology of the vesicles.

Our results show that CL's effect on the membrane depends on the lipid composition of the bilayer: CL has little effect on layer distance in an E. coli membrane model which is PE-enriched (≈70% by weight), while having a bigger effect on the system representing the IMM of a mammalian system which is PE-poor (≈40% by weight), where it reduces stacking. CL was previously shown to induce a change in the packing and fluidity of a bilayer [47], which would have implications for membrane stacking and overall membrane flexibility. Neutron Spin-Echo and Molecular Dynamics found the bending modulus of pure $CL_{18:1}$ to be larger than for $C_{18:1}$-PC [32], suggesting that the stiffness of the lipid membrane appears to decrease when those lipids are mixed. In mixed vesicles of CL, PE, and PG, the difference in order parameter between lipids leads to an increase in its fluidity, that in the case of 17% of CL is concentrated in the IMM while with 35% of CL involves all the membrane, alongside an increase in the membrane curvature and thickness [29]. However, we show once more how the effect of CL on a lipid bilayer can vary according to the membrane lipid composition, leading in some cases to drastic changes or none at all.

### 4. Conclusions

The study of vesicle morphology under physiological conditions and osmotic stress is important for studying fundamental processes involved in both the formation and function of biological membranes, but also in the context of creating artificial vesicles that are utilized as e.g. drug delivery systems. Lipid composition, lamellarity, shape and size distribution
bution are all important characteristics of vesicles that can decide their suitability to fulfil certain tasks. Therefore, these characteristics need to be studied in detail utilising different available techniques.

We investigated the role of CL on the membrane micro-structure, specifically lamellarity and shape, by employing in-solution methods with different membrane models of different complexity. We determined an optimal range between 2.5% to 20% of CL content where the staking of layers is reduced or altogether prevented, leading to a significant reduction of the number of layers from MLVs, with the promotion of ULV. This range is also biologically relevant for many membranes, e.g. IMM and E. coli, containing 1 to 15% CL by weight, respectively, demonstrating the important role that CL plays in membrane organisation and enabling its biological function and structural capacity. The purposely reduction or prevention of membrane stacking is also important in many biotechnology applications that rely on ratio considerations of lipids and embedded agents, e.g. drug deliver systems, where the available lipid area may be important for cell interaction or presentation of a drug.

In our SANS experiments, lipid mixtures were first extruded using "light" PBS and then salt content adjusted to "full" PBS, which caused an osmotic gradient of around 3 atm across the vesicle membrane. This gradient alone was not found to be sufficient to cause a flattening of d54DMPC-only vesicles, possibly due to the counteracting effect of bending energy which is considerable in MLVs. However, a flattened elongated vesicle shape was evident with an initial amount of 20% CL which did not change significantly when more CL was used. Addition of more CL was not able to rescue the "collapsed" ellipsoidal vesicles into a more sphere-like shape. On the other hand, for SAXS, vesicles did not undergo significant osmotic stress in our experimental setup, because vesicle formation was promoted by very gentle ultrasonication in "full" PBS. It must be considered that SAXS data were collected at short distance, therefore not being in the Q-range useful for determining vesicle dimensions, making evaluation of the overall shape of the vesicles less reliable. Nonetheless, some SAXS profiles show evidence of elongation, confirming the ability of CL to induce morphological changes in vesicles.

Utilizing different membrane models, we also clearly demonstrate that the effect of CL on vesicle membranes is dependent on several factors, such as the overall lipid composition, chain length and saturation state, as well as the matching of acyl chains. This hints at a complex interplay between CL and other phospholipids regarding their physicochemical properties, such as shape, charge and packing parameters. Our results show that these properties lead to changes in lamellarity and curvature, further adding to the complexity of membrane formation. This is especially highlighted by the peculiar behaviour of CL<sub>18:2</sub>-containing vesicles, which did not form elongated vesicles and required different models to fit the SANS profiles. This led to the formulation of mainly two hypotheses: i) CL<sub>18:2</sub> assumes a molecular shape that we cannot understand fully, or ii) vesicles are formed that present a complex (and possibly malleable) shape due to the greater fluidity of this CL type, and its increased flexibility.

Our study enhances the understanding of how varying amounts and different types of CL affect the morphology of vesicles, both in its lamellarity and overall shape. This helps us understand the important role that CL plays in the formation and homeostasis of biological membranes and sheds light on the reason behind the differing CL contents found in different species and organelles. We highlighted the importance of several factors in this process, such as CL concentration, CL acyl chain length, saturation state of the lipids and overall lipid composition of the membrane. The use of PBS buffer was essential in our experiments to mimic physiological conditions, and the study of near-natural membrane systems containing mixtures of lipids and including CL is of utmost importance when attempting to draw conclusions regarding the biological function of membrane formation, vesicle morphology and vesicle shape.

Our observations from SANS and SAXS also led us to conclude that CL's particular physical-chemical properties and molecular shape may introduce additional microscopic features and changes in the microstructure of the bilayer which cannot be captured using conventional models, especially at low-Q range. Additionally, our experimental setup did not allow a firm conclusion regarding a possible asymmetric distribution of CL in the inner and outer leaflets.

However, our experimental setup did not allow conclusions regarding lateral membrane microstructure or membrane fluidity, which are important factors to consider in biological membranes that may contain large molecules and protein complexes. Further studies employing other biophysical and structural techniques will therefore be necessary, for instance an investigation of the morphology of CL-containing vesicles employing TEM. A possible increase in the roughness of the bilayer caused by CL could be further studied with interface techniques such as neutron reflectometry, whereas lipid raft formation could be investigated with atomic force microscopy (AFM). Additionally, membrane dynamics and fluidity could be observed using QENS, as has been done recently with cholesterol [1]. Prospective experiments would shed light on CL's ability to influence the membrane microstructure beyond the nanometer scale and may provide valuable insights into how membranes are formed and what role certain lipids, such as cardiolipin, may play in providing a local microenvironment for membrane proteins.

### CRediT authorship contribution statement

**Alice Piccinini:** Writing – review & editing, Writing – original draft, Methodology, Investigation, Formal analysis, Data curation. **Joachim Kohlbrecher:** Writing – review & editing, Resources, Investigation. **Dihia Moussaoui:** Resources, Investigation. **Anja Winter:** Writing – review & editing, Writing – original draft, Supervision, Project administration, Funding acquisition, Conceptualization. **Sylvain Prévost:** Writing – review & editing, Writing – original draft, Supervision, Investigation, Data curation, Conceptualization.

### Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

### Data availability

Data will be made available on request.

### Acknowledgements

This work benefited from the use of the PSCM (ILL) laboratories. This work benefited from the use of the SasView application, originally developed under NSF award DMR-0520547. SasView contains code developed with funding from the European Union's Horizon 2020 research and innovation programme under the SINE2020 project, grant agreement No 654000. We thank E. Kelley (NIST) for providing us with data used in Figure S4A. We thank Andrew Whitten for support on ANSTO instrument BILBY for data used in Figure S4B.

### Funding

AP received a PhD studentship from Institut Laue-Langevin and Keele University.

## Appendix A. Supplementary material

Supplementary material related to this article can be found online at https://doi.org/10.1016/j.jcis.2024.04.211.
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