# Incorporation of short-chain alcohols into fluid bilayers and its effect on membrane dynamic properties as seen by neutron scattering

Fernanda Alvarado Galindo, Joachim Venzmer, Sylvain Prévost, Ingo Hoffmann, Michael Gradzielski

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Fernanda Alvarado Galindo, Joachim Venzmer, Sylvain Prévost, Ingo Hoffmann, Michael Gradzielski. Incorporation of short-chain alcohols into fluid bilayers and its effect on membrane dynamic properties as seen by neutron scattering. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 2024, 702 (1), pp.135014. 10.1016/j.col SURFA.2024.135014. hal-05520004

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# Incorporation of short-chain alcohols into fluid bilayers and its effect on membrane dynamic properties as seen by neutron scattering

Fernanda Alvarado Galindo<sup>a,c,*</sup>, Joachim Venzmer<sup>b</sup>, Sylvain Prévost<sup>a</sup>, Ingo Hoffmann<sup>a</sup>, Michael Gradzielski<sup>c</sup>

## HIGHLIGHTS

* Incorporation of solvent molecules into the lipid bilayer.

* Liposomes extruded with alcohols exhibit size reduction.

* Reduction of bilayer bending rigidity induced by alcohols.

## GRAPHICAL ABSTRACT

<figure><img src="image_4.png" /></figure>

## ARTICLE INFO

Dataset link: https://doi.ill.fr/10.5291/ILL-DA-TA.9-10-1722, https://doi.ill.fr/10.5291/ILL-D-ATA.9-10-1707

MSC:

0000

1111

Keywords:

Liposomes

Short-chain alcohols

Scattering invariant

Bending rigidity

SANS

NSE

## ABSTRACT

In this work, we investigate the effect of concentrated alcoholic solutions (up to 40%w) on extruded 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes using scattering techniques. Extrusion in alcoholic aqueous solutions (methanol, ethanol, or butanol) reduces liposome size, evidenced from the decrease in hydrodynamic radius ($R_H$) obtained from dynamic light scattering. Short-chain alcohols such as ethanol and butanol soften the lipid membranes, as observed by the decrease of the unrelaxed bending modulus ($\bar{k}$) obtained by neutron spin-echo spectroscopy. Thus, softer membranes are easily ruptured during extrusion, leading to a reduction in vesicle radius corroborated by a reduction in both $R_H$ and radius of gyration as seen by dynamic and static light scattering. Moreover, model-free analysis of small-angle neutron scattering (SANS) curves suggests that solvent molecules become incorporated into the lipid membrane. Analysing deviations in the scattering invariant from values predicted by mass balance, we find that the volume fraction of liposomes increases, modifying the scattering length density of the assemblies. By quantifying these changes, we estimate that 12% to 18% of the liposome membrane is composed of solvent molecules (alcohol + water), depending on the type of alcohol and its concentration present in solution, with the exception of glycerol where next to no incorporation was observed. Finally, structural parameters obtained through light scattering and changes in contrast profiles calculated from analysis of the invariant were corroborated through modelling of the SANS

<footnote><sup>a</sup> Institut Max von Laue-Paul Langevin (ILL), 71 Av. des Martyrs - CS 20156, Grenoble cedex 9, 38042, France</footnote>

<footnote><sup>b</sup> Evonik Operations GmbH, Essen, Germany</footnote>

<footnote><sup>c</sup> Stranski-Laboratorium für Physikalische und Theoretische Chemie, Institut für Chemie, Technische Universität Berlin, Straße des 17. Juni 124, Sekr. TC 7, Berlin, D-10623, Germany</footnote>

<footnote>* Corresponding author at: Institut Max von Laue-Paul Langevin (ILL), 71 Av. des Martyrs - CS 20156, Grenoble cedex 9, 38042, France. E-mail addresses: f.alvaradogalindo@campus.tu-berlin.de (F. Alvarado Galindo), hoffmann@ill.fr (I. Hoffmann).</footnote>
curves. Modelling suggests that a reduction of bilayer thickness takes place for liposomes dispersed in ethanolic and butanolic solutions, but not in the presence of methanol or glycerol.

# 1. Introduction

When amphiphilic molecules, namely phospholipids (PLs), are dispersed in aqueous solution, they give rise to bilayers that self-assemble into concentric closed structures known as liposomes, where the precise structure of the aggregates formed depends strongly on the preparation path. Nowadays, PL liposomes are commonly used as delivery systems in pharmacology and cosmetics as they are able to incorporate hydrophobic or amphiphilic active ingredients within their bilayer [1–5]. At an industrial scale, they are often prepared by the ethanol injection method, where the lipids are molecularly dissolved in ethanol (or another organic solvent) and slowly injected into water. The change in solvent quality by admixing water and alcohol promotes lipid self-assembly into liposomes [6]. At this stage, the alcohol content in the dispersion is >10 %w. Thereafter, shear is applied to form unilamellar vesicles (ULVs), reducing polydispersity and increasing colloidal stability of the assemblies. In general it is very important to have the ability to exercise size control, as it is a key parameter to control the properties of liposomes. The effect of shear on bilayer systems has been studied in good detail since the 1990s and corresponding shear phase diagrams then show how one can switch from unilamellar to multilamellar vesicles of more or less well-ordered planar lamellar phases [7]. It might be noted here that for these transitions of bilayer structures typically the total deformation applied [8] and the shear rate play a major role for the finally observed structures, with smaller vesicles formed at higher shear rates [9]. Finally, in a typical commercial formulation the dispersions where ULVs and alcohol molecules co-exist are stored for several weeks prior to removal of the organic solvent and incorporation to the final product.

Despite the numerous studies dedicated to understanding the influence of alcohols on phospholipid bilayers, alcohol-lipid interactions are not completely clarified, yet, especially when being present during the preparation process. The current consensus is that alcohols of the form C_nH_(n×2+1)OH (1 < n < 4) affect the structure of liposomes by increasing the area per lipid molecule and thereby decreasing the bilayer thickness [10–13]. Lipid exchange and flip-flop processes are also enhanced by the presence of short-chain alcohols [14,15]. In parallel, the mechanical properties of the bilayer are affected. A thinner membrane, induced by the alcohol, should be softer and this effect should become more prominent with increasing alcohol concentration and alcohol chain-length, as suggested by micropipette aspiration and maxwell displacement current experiments [16,17], where a decrease in the bending modulus was reported. The bending modulus (κ) is related to the fluidity of the bilayer and while a less rigid membrane leads to stronger repulsion between bilayers due to undulation forces [18–20], it also leads to increased bilayer permeability [21,22]. Although there are a various of techniques available to assess the bending rigidity [23], in this work we focus on using neutron spin-echo spectroscopy to do so.

Moreover, it is generally accepted that short-chain alcohols gather within the lipid head-group region [15,24–26], effectively dehydrating the interface due to preferential membrane-alcohol hydrogen bond interactions. Ethanol for example, tends to bond at the phosphate acceptor sites whereas butanol tends to bond at the carbonyl oxygen [27,28]. However, X-ray scattering studies hint at the possibility of alcohol molecules being present in the liposome’s hydrophobic bilayer core [29].

In this regard, we study the extent of alcohol incorporation into the lipid bilayer of DOPC liposomes dispersed in solutions containing primary alcohols (methanol, ethanol and butanol) or glycerol, a potential alternative to primary alcohols for the formation of liposomes. Butanol is employed since it is a well-known co-surfactant that readily

incorporates in the lipid membrane [13]. We take advantage of the non-perturbing nature of light and neutron scattering techniques, in particular, light scattering to determine particle size, small-angle neutron scattering (SANS) to give in addition information on the bilayer thickness and a qualitative estimate of alcohol presence in the fluid bilayers, and NSE spectroscopy to study the influence of alcohols on the bending modulus of the membrane.

# 2. Materials and methods

## 2.1. Materials

1,2-dioleoyl-sn-glycero-3-phosphocholine (18:1-18:1 PC, DOPC, Avanti) at PC > 99% was purchased from Sigma Aldrich, France. Lipids were used as received. Deuterium oxide (D₂O, 99.9%) and high purity alcohols, namely methanol, ethanol, butanol and glycerol, were also purchased from Sigma Aldrich, France.

## 2.2. Methods

### Preparation of phospholipid liposomes

Phospholipid liposomes at a lipid mass ratio of 0.2 %w were obtained by dispersing DOPC in water or alcoholic solution with a magnetic stirrer, followed by extruding the lipid suspension through a 200 nm pore diameter polycarbonate membrane at least 21 times with an Avanti mini extruder (Avanti Polar Lipids, United States) at room temperature (above the transition temperature of DOPC Tₘ,₉₉₄ = -16.5 °C) [30]. The solutions consisted of water or water-alcohol mixtures using alcohols at different mass ratios, namely 10, 20 and 40 %w for methanol and glycerol, 10 and 20 %w for ethanol and 0.1, 1 and 5 %w for butanol. The lower mass ratios for butanol are due to its lower solubility. Samples for small-angle neutron scattering experiments were prepared with the same procedure using (partially) deuterated solvents: D₂O, D₂O-deuterated alcohol or D₂O-hydrogenated alcohol. For neutron spin-echo studies, liposomes were also prepared by extrusion at a lipid mass ratio of 1 %w in fully deuterated solvent.

### Light Scattering

Dynamic (DLS) and static (SLS) light scattering experiments were performed at temperature T = 25 °C with an ALV/CGS-3 Compact Go-niometer system with a HeNe Laser (wavelength λ = 632.8 nm). The samples were diluted 1:50 in the corresponding solvents for accurate DLS and SLS analysis. Fluctuations in scattered light during DLS experiments were measured with a ALV7004/FAST real time multiple tau digital correlator at a 90° angle. The measurements were conducted 5 times with a duration of 30 s each.

The intensity autocorrelation functions g⁽²⁾ were fitted with the cumulant analysis of second order [31] from which diffusion coefficients (D) were calculated. Thereafter, the hydrodynamic radius (R_H) was calculated from the Stokes-Einstein relation R_H = k_B T/6πη D where k_B is the Boltzmann constant and η the solvent viscosity. η was corrected for the different alcohol ratios (Table A.2).

SLS was measured at different scattering angles θ in the range of 41°–140° corresponding to 0.0009 1/Å < q < 0.0024 1/Å, where q is the magnitude of the scattering vector q = (4πn_s/λ)sin(θ/2) and n_s the refractive index of the solvent. Radius of gyration (R_g) and forward scattering (I₀ : I(q = 0)) were obtained using the Guinier approximation (Eq. (1)), valid for qR_g ≪ 1.

$$I(q) = I_0 P(q) \approx I_0 \exp \left( - \frac{q^2 (R_g)^2}{3} \right) \quad (1)$$
From $I_0$ the mass-average molecular weight of the liposomes is calculated from the relation

$$MW_{liposome} = \frac{I_0}{Kc}. \qquad (2)$$

where K is the optical constant $K = (4\pi^2 n_s^2 (dn/dc)^2)/(\lambda^4 N_a)$, c the mass concentration in the sample, $N_a$ Avogadro's constant and $dn/dc$ the refractive index increment.

## Small-Angle Neutron Scattering

SANS experiments were performed on the D11 instrument at the Institut Laue-Langevin (ILL) in Grenoble, France. Samples were loaded in 2 mm quartz cuvettes and positioned in the sample holder under temperature control of 25 °C. Three settings with three different sample-to-detector distances (38 m, 16.5 m and 1.7 m) were used with a wavelength of 4.6 Å to access the data collection in a q-range of 0.0006 1/Å–0.6744 1/Å. Empty cuvette, backgrounds and transmissions were measured for all samples. For absolute scale and normalization, a 1 mm sample with distilled water was used as an isotropic scatterer.

In a system with 2 micro-phases and a sharp interface the scattering invariant ($Q_{inv}$) [32,33]

$$Q_{inv} = \int_{0}^{\infty} q^2 I(q) dq \qquad (3)$$

is directly linked to the volume fractions of the components

$$Q_{inv} = 2\pi^2 \phi_1 \phi_2 \Delta S L D^2, \qquad (4)$$

where $\phi$ is the volume fraction and $\Delta SLD$ is the difference in scattering length between the two components. Eq. (4) holds true independent of the shape and structure of the two components.

The invariant is partitioned in three as described by Eq. (5), with the second term representing the experimentally available information.

$$Q_{inv} = \int_{0}^{q_{min}} q^2 I(q)_{Guinier} dq + \int_{q_{min}}^{q_{max}} q^2 I(q) dq + \int_{q_{max}}^{\infty} q^2 I(q)_{Porod} dq \quad (5)$$

The Guinier region, first contribution to Eq. (5), is used to extrapolate the scattered intensity to $q = 0$. For dilute solutions, $I(q)_{Guinier}$ should vary according to Eq. (1) [34] and accounts for only ~0.4% of the $Q_{inv}$ of our systems.

The third component of Eq. (5) refers to the Porod regime. At very high q-values (when $qR \gg 1$) it is assumed that there is no shape information left of the system and all scattering contributions come from reflections of the sharp interfaces. In this region, the scattering follows the Porod law, where the power law exponent is fixed to -4 and the scattered intensity is described by Eq. (6) [32]

$$I(q) = \frac{1}{q^4} 2\pi(\Delta SLD)^2 S_v, \qquad (6)$$

where $S_v$ is the specific surface area. For our systems, the Porod region accounts for ~2% of our calculated $Q_{inv}$. Overall, less than 3% of the invariant from our samples come from Guinier and Porod extrapolations, which ascertains the reliability in the calculation of $Q_{inv}$. See Figure A.12 for examples of the extrapolations.

Here, we treat our 3 component system consisting of water, alcohol and lipids as a pseudo-2 component system, made of liposomes and solvent. All of the lipid is assumed to reside in the liposomes and a fraction of both alcohol and water can penetrate into the vesicles while the remainder forms the solvent, so that the volume fraction of the liposomes is given by

$$\phi_{lipo} = \phi_{PL} + \alpha\phi_{A} + \beta\phi_{wat} \qquad (7)$$

and the volume fraction of the solvent phase is given by

$$\phi_{solvent} = (1 - \alpha)\phi_{A} + (1 - \beta)\phi_{wat}, \qquad (8)$$

where $\alpha$ and $\beta$ are the fractions of alcohol and water that penetrate into the vesicle membrane. As a consequence, the SLDs of the two components read

$$SLD_{lipo} = \left[ \frac{1}{\phi_{lipo}} \right] \left[ \phi_{PL} SLD_{PL} + \alpha \phi_{A} SLD_{A} + \beta \phi_{wat} SLD_{wat} \right] \qquad (9)$$

and

$$SLD_{solvent} = \left[ \frac{1}{\phi_{solvent}} \right] \left[ (1 - \beta) \phi_{wat} SLD_{wat} + (1 - \alpha) \phi_{A} SLD_{A} \right]. \qquad (10)$$

If no solvent were to penetrate the liposomes, $\alpha$ and $\beta$ through Eqs. (7)–(10) would equal zero and the theoretical invariant ($Q_{theo}^{theo}$) was calculated as such.

## Neutron Spin-Echo Spectroscopy

Neutron Spin-Echo (NSE) experiments were performed on the IN15 instrument at the ILL. Wavelengths of 12 Å, 10 Å, 8 Å and 6 Å and detector angles of 3.5°, 5.5°, 7° and 7.5° were used to span a q-range from 0.023 1/Å–0.154 1/Å. NSE is useful to study membrane dynamics such as membrane undulations related to the bending rigidity of the bilayer $\kappa$. Experimentally, the intermediate scattering function $S(q,t)$ is obtained, which is related to the probability for scattering a neutron with a given momentum and energy transfer. In the framework of the classical Zilman–Granek theory for thin membranes $S(q,t)$ should follow a stretched exponential decay as described by the relation $S(q,t) = \exp(-(\Gamma_{ZG}q^3t)^{2/3})$. When accessing sufficiently long Fourier times, diffusion also contributes to $S(q,t)$, thus it should be taken into account to compare vesicles of different sizes. Recently, an extended version of the theory taking into account effects from finite size, spherical geometry and translational diffusion has been published [35].

In short, $S(q,t)$ is given by

$$S(q,t) = \langle P(q) \exp \left( -\frac{q^2 R^2 \langle \Delta u^2(t) \rangle}{2} \right) \exp \left( -D(R)q^2t \right) \rangle, \qquad (11)$$

where the angular brackets indicate an average over the vesicle radius $R$, with a Gaussian size distribution with 0.3 relative standard deviation was used and its mean value was taken from the hydrodynamic radius obtained from DLS. $D(R)$ is the translational diffusion coefficient which depends on the vesicle radius through the Stokes-Einstein equation. For the form factor of the vesicle $P(q)$ we use the approximation for a thin spherical shell. The mean square displacement $\langle u^2(t) \rangle$ is given by

$$\langle u^2(t) \rangle = \frac{1}{2\pi} \sum_{l=2}^{l_{max}} (2l + 1) \langle |u_l|^2 \rangle (1 - \exp(-\omega_l t)), \qquad (12)$$

with the relaxation rate of mode l

$$\omega_l = \frac{\kappa}{\eta R^3} \frac{(l-1)(l+1)(l+2)(l(l+1))}{4l^3 + 6l^2 - 1} \qquad (13)$$

and its mean square amplitude

$$\langle |u_l|^2 \rangle = \frac{k_B T}{\kappa (l+2)(l-1)(l(l+1))}. \qquad (14)$$

The time and length scales observed in NSE are such that in its high q limit the observed bending rigidity is the unrelaxed bending rigidity $\bar{\kappa} = \kappa + 2K_m d^2$ [36] rather than the relaxed bending rigidity $\kappa$ [37], where $K_m$ is the monolayer compression modulus and $d$ is the height of the neutral surface. While the transition becomes visible at lower $q$, it has proven challenging to extract reliable values of $\kappa$ and we limit ourselves to reporting values of $\bar{\kappa}$.

# 3. Results and discussion

## 3.1. Size reduction of DOPC liposomes when extruded in alcoholic aqueous solution

First, we analyse the effect on liposome size caused by alcohols of different chain-length and with a different number of -OH groups. We prepared 0.2% w DOPC liposomes by extrusion in water-alcohol solutions containing either methanol, ethanol, butanol or glycerol at varying alcohol mass ratios. The hydrodynamic radii ($R_H$) of the assemblies obtained by dynamic light scattering (DLS) are presented in Fig. 1.

In water, DOPC vesicles have an $R_H$ of 95 nm that tends to decrease upon addition of the organic solvents. Methanol at a mass ratio of
<figure><img src="image_5.png" /><figcaption>**Fig. 1.** Hydrodynamic radii ($R_H$) vs. alcohol content of 0.2 %w DOPC liposomes extruded in different D₂O/alcoholic solutions determined by DLS measured at $\theta = 90^\circ$ at 25 °C. $R_H$ decreases in the presence of methanol, ethanol and butanol in a fashion that is somewhat independent of alcohol concentration or alkyl chain-length. Particle size remains fairly unchanged for liposomes in glycerol-containing solutions across all glycerol:water ratios. Liposomes were extruded using a 200 nm membrane pore size and analysed within 3 days after preparation.</figcaption></figure>

20 %w or 40 %w reduces liposome radius up to a half. In the case of ethanol, the $R_H$ is cut down to ~67 nm across all ethanol concentrations. Moreover, we observe a marked change when 5 %w water is replaced by butanol, as the rather low butanol concentration suffices to decrease $R_H$ to ~60 nm, i.e., to a much lower size.

The changes in hydrodynamic radius observed by DLS are supported by static light scattering (SLS) experiments carried out on the same samples, where we observe a reduction in the radius of gyration $R_g$ obtained from Guinier fits, as well as the reduction of apparent molecular weight calculated with Eq. (2). We note that the strong reduction of $R_H$ for the samples at the highest methanol and butanol concentrations is not as pronounced in $R_g$. This is evident from the deviation of the shape factor $R_g/R_H$ from 1, as reported in Table A.2 summarizing light scattering results. For reference, a value of 1 is characteristic of hollow spheres as for hollow spheres both $R_H$ and $R_g$ correspond to the radius of the sphere [38]. In our case the values are close or even bigger than 1. However, these slightly higher than expected values may simply be attributed to measurement precision and shape fluctuations of the vesicles.

For glycerol, we expected a different behaviour compared to primary alcohols due to its higher polarity (larger dipole moment). As shown in Fig. 1, $R_H$,glycerol remain fairly unchanged at ~100 nm across all glycerol concentrations. It is possible that glycerol has no effect on particle size as it is similarly known to have little influence on membranes. For example, studies suggest that the interaction between glycerol and the lipid head-groups is similar to that of water with the head-groups [39].

## 3.2. Liposome size differs with alcohol addition before or after extrusion

Before moving further, it is important to note that the impact of alcohol on liposome size differs depending on the stage of the preparation at which it is introduced. We made two lipid dispersions using two different solutions: one with pure water and the other with a water-alcohol mixture. Both dispersions underwent extrusion and ethanol was added to the initially alcohol-free dispersion to equalize their final ethanol content. Consequently, one set of liposomes was extruded with ethanol molecules, while the other was subjected to shearing solely in the presence of water.

After performing DLS analysis on both dispersions, it is observed that $R_H$ is not affected when ethanol is added after extrusion since the radius stays constant at around 100 nm across all solvent compositions

<figure><img src="image_6.png" /><figcaption>**Fig. 2.** Hydrodynamic radii ($R_H$) vs. ethanol content of 0.2 %w DOPC liposomes in ethanolic solution determined by DLS measured at $\theta = 90^\circ$ at 25 °C. On the one hand, liposomes extruded in the water-alcohol solution (▼) exhibit size reduction with increasing ethanol content. On the other hand, liposomes initially sheared in pure water, followed by ethanol addition after extrusion (▶), show little to no effect on $R_H$. Both samples were extruded with a 200 nm membrane pore size and measured on the day of preparation.</figcaption></figure>

(▶ in Fig. 2). The lack of apparent size change upon ethanol addition to a vesicular dispersion has also been observed by other groups [40]. On the contrary, vesicles extruded in the water-ethanol solution show a reduction in hydrodynamic radius at increasing ethanol content, which evolves to 79 nm and 66 nm in 10 %w and 20 %w ethanol, respectively, from the $R_H$ of 95 nm when dispersed in pure water. Such an observation is also in agreement with other work found in the literature [41–43]. Lastly, we corroborate the particle size trend with SLS experiments, where we observe reduced $R_g$ values in one case (lipids extruded in water-ethanol solution), but not the other (ethanol added to liposomes extruded in water). A summary of both DLS and SLS results is found in Table A.3.

Apparently, the presence of the alcohol during the extrusion process has a large effect on vesicle size and might be a consequence of membrane softening when the alcohol ratio in the solution increases [16]. Such an effect would allow for stronger bending of the softer membranes formed in the presence of alcohol, which then may go hand in hand with easier rupturing and deformation during the extrusion process and thus leading to a smaller size.

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

To complement the information acquired by light scattering, we carried out small-angle neutron scattering (SANS) experiments to elucidate the impact of short-chain alcohols on particle morphology and to define the alcohol location in the dispersion. For that purpose, extruded DOPC 0.2 %w vesicles in either water or alcoholic solutions were characterized with this technique. The alcohols employed were (deuterated) methanol, ethanol, butanol and glycerol at the same concentrations used for the light scattering experiments (Fig. 1).

### 3.3.1. The invariant analysis for alcohol localization

To determine the alcohol location we perform a model-free analysis of the SANS curves. This involves obtaining the scattering invariant ($Q_{inv}$), a parameter independent of shapes, interactions, or the overall arrangement of the domains that is a constant directly proportional to the average fluctuation in contrast, as denoted by Eq. (4).

In the event that alcohol or solvent molecules incorporate into the membrane, the volume fraction of the lipid phase ($\phi_{lip}$) would increase, altering the contrast between the phases ($\Delta SLD$). For fluid bilayers in deuterated solvent (D₂O-deuterated alcohol), incorporation
<figure><img src="image_7.png" /><figcaption>Fig. 3. Difference between experimental and theoretical values of the scattering invariant vs. alcohol content (%w) in solution. $\Delta Q_{inv} = Q_{inv}^{theo} - Q_{inv}$. (a) Difference for samples in h-alcohol/D₂O solutions are below 10% except at 5% butanol and 40% methanol, where $Q_{inv} > Q_{inv}^{theo}$. (b) shows a magnification of (a). (c) Difference for samples in D₂O/deuterated alcohol solutions show $Q_{inv}^{theo} > Q_{inv}$ for all samples. Experimental $Q_{inv}$ contracts further than predicted by theory, probably due to alcohol incorporation to the bilayer. Dashed lines are added for visual guideline. Error bars are the same size or smaller than the symbols.</figcaption></figure>

of solvent molecules of $SLD_{solvent} \gg SLD_{lipids}$ to the membrane would increase the scattering length density of the liposomes ($SLD_{lipo}$) (Eq. (9)) and reduce $\Delta SLD$, as well as the invariant given that $Q_{inv} \propto \Delta SLD^2$. In partially deuterated solvent (D₂O-hydrogenated alcohol), $Q_{inv}$ would increase primarily through the gain in $\phi_{lipo}$.

Such changes would cause a deviation of the invariant from values predicted by mass balance ($Q_{inv}^{theo}$), calculated with Eqs. (7)–(10) assuming no solvent incorporation into the lipid bilayer (i.e. $\alpha$ and $\beta = 0$). To follow such deviations, one can calculate the difference between experimental and theoretical values as $(Q_{inv}^{theo} - Q_{inv})/Q_{inv}^{theo} = (\Delta Q_{inv})/Q_{inv}^{theo}$. Depending on the solvent composition, the deviations should be either positive, as for vesicles in partially deuterated solvent, or negative for those dispersed in fully deuterated solvent. An exemplification of the expected trend is presented in Figure A.7 in the Appendix.

For our investigations, we used partially and (where available) fully deuterated solvents of the alcoholic solutions presented in Section 3.1 to differentiate the contributions from water and alcohol in $\phi_{lipo}$ upon solvent incorporation. To facilitate the analysis, the scattering length density of D₂O was determined from the $Q_{inv}$ value obtained for liposomes ($1.34 \times 10^{20} \text{ nm}^{-4}$), by solving for it in Eq. (15). The objective was to account for potential H/D exchange and to remove the contributions of hydrating water on the invariant, isolating only those arising from solvent incorporation into the bilayer. Therefore, the resulting SLD value of $6.14 \times 10^{10} \text{ cm}^{-2}$ was adopted for calculation of the $Q_{inv}^{theo}$ values.

$$Q_{inv,D_2O} = 2\pi^2\phi_{lipo}\phi_{D_2O}(SLD_{lipo} - SLD_{D_2O})^2 \quad (15)$$

We compare the experimental $Q_{inv}$ obtained from SANS curves to $Q_{inv}^{theo}$ by calculating $\Delta Q_{inv}$. For vesicles in partially deuterated solvents, $\Delta Q_{inv}$ is mostly negative as presented in Fig. 3(a). The exceptions are at 10% ethanol and at the lowest butanol concentrations, where $\Delta Q_{inv}$ is

~0% and ~3%, respectively. Most samples have a $\Delta Q_{inv}$ between ~2% and ~7%, being ethanol the one with the smallest difference ($\Delta Q_{inv} = -2\%$ at 20% ethanol). The highest discrepancies are observed at the highest methanol and butanol concentrations, where the difference is ~37% and ~20%, respectively.

The small differences in $\Delta Q_{inv}$ could be derived from changes in the solvent composition, as the contrast changes with replacement of heavy water by hydrogenated alcohol. However, this does not clarify the elevated discrepancy at the highest methanol (40 %w) and butanol (5 %w) mass ratios. Therefore, we propose that solvent incorporation is at the root of all discrepancies, with ethanol having the weakest tendency to become part of the liposome (smallest $\Delta Q_{inv}$). Additionally, butanol at similar concentrations is reported to incorporate to the bilayer [13].

$\Delta Q_{inv}$ is positive when $Q_{inv} < Q_{inv}^{theo}$ and this is observed for samples in fully deuterated solvent. For these samples, $Q_{inv}$ deviates 7% or more from $Q_{inv}^{theo}$ for all alcohol species and concentrations as depicted in Fig. 3(c). The exception comes at the lowest butanol concentrations where $\Delta Q_{inv}$ is ~0.8% and ~0.9% at 0.1 %w and 1 %w butanol, respectively.

On the one hand such a disparity between $Q_{inv}$ and $Q_{inv}^{theo}$ can be a consequence of either deviations of the volume fraction of the liposomes ($\phi_{lipo}$) or of the solvent SLDs. If there was material lost during extrusion, this would lower $\phi_{lipo}$ and thus $Q_{inv}$. However, the loss in the invariant would be observed in both solvent contrasts, which is not the case. On the other hand, one could argue that in fully deuterated solvent a reduction of $Q_{inv}$ is due to a loss in contrast derived from a lower $SLD_{solvent}$ or a gain in $SLD_{lipo}$. Yet, corrections for $SLD_{D_2O}$ were made and samples were measured within 3 days of preparation, so we assume that there is no loss in $SLD_{solvent}$.

Therefore, it is likely that an increase in $SLD_{lipo}$ is the core of the deviations observed in $\Delta Q_{inv}$. Most importantly, the change in contrast
is attributed to swelling of the lipid bilayer by solvent molecules becoming incorporated into it, causing an increment in $\phi_{lipo}$. Consequently, this leads to an elevated $Q_{inv}$ (and negative $\Delta Q_{inv}$) for samples in partially deuterated solvent and a reduction in $Q_{inv}$ (and positive $\Delta Q_{inv}$) in fully deuterated solvent due to the changes in the contrast discussed at the beginning of the section.

To ascertain the composition of the solvent incorporated to the membrane, we employed Eqs. (7)–(10) to determine the volume fraction of alcohol ($\alpha$) and $D_2O$ ($\beta$) responsible for the increase in $\phi_{lipo}$ and subsequent deviations of $\Delta Q_{inv}$. This analysis was conducted for samples with two contrasts, where two equations exist for determining the two unknown $\alpha + \beta$. In the case of glycerol where only one solvent contrast was available, we calculate the overall solvent fraction that penetrates the bilayer ($\alpha + \beta$). The volume fraction of incorporated solvent was then normalized with respect to the lipid fraction using the equation:

$$ \phi_{l,liipo} = x_i \phi_i / (\phi_{lipid} + \sum x_i \phi_i), \quad (16) $$

where $i$ is either $D_2O$ or alcohol and $x$ is the corresponding fraction calculated with the system of equations ($\alpha$ or $\beta$).

The results are graphed and presented in Fig. 4, however the numerical results are also reported in Table A.4 in the Appendix. With the correction made to $SLD_{D_2O}$, water hydration is not considered in Fig. 4. This explains why the fraction of $D_2O$ is zero in liposomes dispersed in pure water. This does not mean that the liposomes are impermeable, but rather emphasizes the magnitude of solvent incorporation into the membrane.

Our results reveal the simultaneous incorporation of water and alcohol molecules into the lipid membrane when there is a primary alcohol present in the solution. For example, in 10%w methanol, the initial fraction of $D_2O$ in the liposomes ($\phi_{D_2O,liipo}$) is 9% and that of alcohol ($\phi_{alc,liipo}$) is 8%. As the amount of methanol in the dispersion increases, $\phi_{D_2O,liipo}$ is progressively supplanted by the alcohol as shown in Fig. 4(a). At 40% methanol, the lipid bilayer is depleted of water and replaced by methanol as $\phi_{D_2O,liipo} = 0.2\%$ whereas $\phi_{alc,liipo} = 20\%$.

Moreover, at our lowest ethanol concentration (10%w), estimated $\phi_{D_2O,liipo}$ is 12.5% and drops to 6.3% at the highest concentration (20%) (see Fig. 4(b)). Meanwhile, $\phi_{alc,liipo}$ remains fairly unchanged as we calculate fractions of 4.9% and 5.4%, respectively, for the aforementioned solvent compositions. Butanol (Fig. 4(c)) is more peculiar as even minute amounts of the alcohol (0.1%w) suffice to increase $\phi_{D_2O,liipo}$ from 0 to ~10% whereas $\phi_{alc,liipo}$ shows a dramatic increase from nearly 0 to 12.2% at 5%w butanol.

For DOPC liposomes in glycerol (Figure A.8) the solvent fraction (water + glycerol) lodged in the lipid bilayer is less than 5% across all glycerol concentrations. This suggests little to no effect on the membrane by glycerol, consistent with what has been reported in the literature [44] and with MD simulation results where glycerol is not seen to diffuse through DOPC bilayers [45]. Although the interaction of glycerol with PLs could provoke bilayer thinning and lateral area expansion (i.e. head-group (HG) area expansion) as single -OH solvents do, studies demonstrate that glycerol substitutes water molecules in a fashion that properties of the lipid system remain unaffected [39,46].

Overall, $\phi_{D_2O,liipo} > \phi_{alc,liipo}$ at the lowest alcohol concentrations and interestingly, $\phi_{alc,liipo}$ increases at the expense of $\phi_{D_2O,liipo}$ as more water is replaced by alcohol in the solution. In other words, as the alcohol content in solution increases, organic molecules gradually replace water in the bilayer, consistent with their dehydration properties on membranes [47,48].

One point of debate is that alcohol tends to replace water within the head-group region, preferentially at the phosphate and carbonyl oxygens [27] and not within the hydrophobic bilayer core [24]. Still, more studies at higher alcohol concentrations are needed to rule out this possibility. As an example, we find X-ray scattering investigations that indicate the possibility of the presence of alcohol and/or water

molecules in the hydrophobic membrane core [29] for liposomal dispersions at 5%w ethanol. For our case we provide clear evidence that water and alcohol are associated with the lipid bilayer without clearly locating their position within the bilayer, but assuming mainly a presence in the head group region.

### 3.3.2. Modelled SANS curves

Thanks to light scattering results and model-free analysis of the SANS curves we obtain structural parameters of the liposomes as well as composition and contrast for the components present in the dispersion. On the one hand, we estimated the size of the vesicles from light scattering experiments. On the other hand, we obtained the volume fractions and scattering length densities through analysis of the scattering invariant.

To define the appropriate form factor contributions, we estimated the lamellarity $N_{bi}$ of the assemblies. To do so, we calculated the molecular weight (MW) of a unilamellar vesicle (ULV) and compared it to the experimental MW calculated from SLS results. The bilayer thickness $\delta$ was necessary to calculate $MW_{ULV}$ and so we used the position of the form factor minima at high-q values (between 0.12 1/Å–0.22 1/Å) as a first approximation to calculate it, given that $\delta \sim 2\pi/q$. The values were thereafter refined during the data modelling.

Given that $N_{bi}$ indicates the presence of multilamellar vesicles, we used a combination of form factors for uni- and multilamellar vesicles to model SANS curves using the SasView programme [49]. The contributions from the ULV and MLV models were set so that the average number of bilayers would match the molecular weight of the MLVs defined by SLS. Details of the contributions and parameters used for modelling can be found in Table A.6 in the Appendix.

As an example, we present in Fig. 5 the experimental and modelled (black dashed-lines) SANS curves for the liposomes dispersed at the lowest alcohol mass ratios. The free parameters for the models were bilayer thickness and thickness of the solvent layer between the lipid bilayers, which was adjusted to best match the correlation peak near $q = 0.1 1/Å$. The size distribution was set to 0.30 using a LogNorm distribution. The complete set of modelled curves are presented in Figures A.10 through A.11.

Firstly, we note that the thickness of the bilayers becomes thinner in the presence of alcohol, except in the presence of methanol, where there is an initial increase of 10% followed by a progressive decrease of $\delta$ down to 33 Å. It is possible that methanol, at its lowest concentration, promotes higher lipid packing if it is unable to link lipid head groups effectively with its single -OH and methyl group [50]. This could explain the initial increase in $\delta$, which decreases at higher methanol concentrations due to increasing membrane dehydration. A similar phenomenon occurs with glycerol, where $\delta$ increases at the lowest concentration and progressively decreases at increasing mass ratios. Such behaviour could be related to a mostly unaffected membrane rigidity of the liposomes by these two solvents, as will be presented in Section 3.4.

For the rest of the alcohols, $\delta$ reduces at increasing alcohol concentrations as reported in Table A.5. A reduction in $\delta$ must be coupled to an increase in the head-group area observed by MD simulations and NMR studies [10,26,28,51–54] that can be attributed to alcohol molecules residing near the glycerol backbone of the phospholipids and also agrees with other studies where interdigation of the lipid alkyl chains, and reduction of $\delta$, was reported [55].

Moreover, it can be seen that the parameters derived from the analysis appropriately describe the experimental scattering intensity of the DOPC liposomes. It corroborates the changes in the SLD of the components derived from the invariant analysis and the degree of lamellarity estimated by SLS. The latter is confirmed when the curves modelled only with the ULV form factor (Figure A.9a) fail to describe the experimental data. Another criteria that supports the multilamellarity of the assemblies is the power law dependence of the curves. At the mid-q region (between 0.007 1/Å and 0.03 1/Å for
<figure><img src="image_8.png" /><figcaption>**Fig. 4.** Calculated volume fractions of D₂O (x) and alcohol (◄) in the lipid phase vs. alcohol content (%w) in solution. Using the invariant as a starting point, a system of equations was conceived to examine in which proportion would solvent molecules need to be within the bilayer (increasing the volume fraction of the liposome phase) as to modify Qᵢₘ, obtained from SANS experiments. The results are for DOPC liposomes (0.2 %w) in (a) methanolic, (b) ethanolic and (c) butanolic alcoholic solutions.</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>**Fig. 5.** Kratky plots of the SANS curves for 0.2 %w extruded DOPC liposomes in D₂O or hydrogenated alcohol-D₂O solutions. The dashed-lined are curves modelled with the parameters obtained from light scattering experiments (R<sub>g</sub>, lamellarity) and analysis of the scattering invariant (volume fractions, SLDs) without performing data fitting. Modelled and experimental data have a good match, validating the multilamellarity estimated by SLS. See Table A.6 for a summary of the parameters used for modelling.</figcaption></figure>

our systems), a q⁻² decay is characteristic of either flat surfaces or unilamellar liposomes where surface properties dominate. In our case the dependence lies between -2.1 and -2.9, which is indicative of compact volume properties such as those of MLVs [56]. A summary of the structural parameters obtained from the SANS curves, including the power law exponents and R<sub>g</sub> are reported in Table A.5.

### 3.4. Membrane softening with short-chain alcohols

Light scattering and SANS corroborate increased curvature and bilayer thinning of the fluid bilayers at the studied alcohol mass ratios.

Since the membrane thickness is reduced, we also expect changes in its dynamic properties, in particular, reduced bending rigidity, which can be studied using neutron spin-echo spectroscopy (NSE) applying Eqs. (11) to (14).

The bending rigidity quantifies the energy needed to bend a lipid bilayer, thus describing its resistance to changes in curvature. Most of the bending undulations that can be observed on the NSE time and length scale are fast enough so that their timescale is determined by the unrelaxed bending rigidity ķ. However, at q < 0.05 1/Å, the observed bending rigidity decreases, which most likely means that at those low q values we observe a transition from ķ to κ. In addition, the vesicles studied here are oligolamellar with the exception of the sample with 5% butanol and show a correlation peak at about 0.09 1/Å in their static scattering pattern, which results in de Gennes narrowing [57] and higher apparent bending rigidities around the peak position [58] which leaves a rather narrow window in q, either at about 0.5 1/Å or above 0.12 1/Å to observe ķ without the influence of de Gennes narrowing.

The viscosities that were used in the fits are reported in table A.7. The radii were taken from DLS and the relative standard deviation was fixed at 0.3 as for SANS. The fits are shown in fig. A.13. The obtained bending rigidities are summarized in Fig. 6.

For the sample with 5% added butanol which has been shown by SANS to give unilamellar vesicles a plateau of ķ is observed above a q value of about 0.06 1/Å as expected. However, the plateau value of about 20 k<sub>B</sub>T is extremely low compared to about 150 k<sub>B</sub>T which have been previously observed for POPC in D₂O [35]. The plateau values of the other samples are obscured by de Gennes narrowing around the interlamellar correlation peak at about 0.9 1/Å. Nevertheless, it can be seen that the membrane rigidity is reduced by the addition of 20% ethanol relative to the sample with pure D₂O as solvent while methanol and glycerol have rather little effect. Assuming that the effect of de Gennes narrowing is rather small at 0.13 1/Å, the highest q where a reliable value could be determined and at 0.06 1/Å, which
<figure><img src="image_10.png" /><figcaption>**Fig. 6.** Unrelaxed bending modulus $\bar{\kappa}$ obtained from NSE for DOPC liposomes (1 %w) in fully deuterated alcoholic solutions as a function of $q$. For the unilamellar vesicles in butanol a plateau is observed for $q > 0.05 \text{ 1/Å}$ at a very small value of $\bar{\kappa}$ of about 20 $k_B T$. For the other samples de Gennes narrowing [57] obscures the plateau value around the correlation peak at ~0.09 1/Å. Nevertheless it can be seen that $\bar{\kappa}$ is reduced for ethanol and remains mostly unaffected for methanol and glycerol relative to pure D₂O solvent.</figcaption></figure>

is the onset of the plateau region, it can be seen that $\bar{\kappa}$ is between 100 and 200 $k_B T$ for D₂O, methanol and glycerol, which is close to the value previously reported for unilamellar POPC vesicles. At first sight it seems somewhat surprising that multilamellarity has relatively little effect on $\bar{\kappa}$. However calculating $\sqrt{\langle |u_2|^2 \rangle} R^2$ for a 100 nm vesicle with 150 $k_B T$ gives about 1.7 nm. Comparing this value to $2\pi/q_{peak}$ with $q_{peak} = 0.09 \text{ 1/Å}$, which gives about 7 nm, it seems reasonable that multilamellarity has relatively little effect on the bending rigidity aside from de Gennes narrowing.

## 4. Conclusions

The primary alcohols employed for this study influence the morphology of extruded phospholipid liposomes as observed from the reduction of particle size and bilayer thickness ($\delta$) studied by light scattering and SANS, respectively. In particular, the presence of ethanol and butanol in the bulk solution results in softening of the lipid membrane as indicated by the reduction in the unrelaxed bending modulus $\bar{\kappa}$ observed from neutron spin-echo experiments.

More importantly, we explored the location of the alcohols and water within the DOPC liposomes through model-free analysis of SANS curves. We provide qualitative information regarding solvent (alcohol + water) incorporation into the bilayers of the liposomes. Deviations in the scattering invariant ($Q_{in}$) from values calculated from mass balance ($Q_{in}^{theo}$) suggest that solvent molecules reside within the lipid bilayer, causing an increase in the volume fraction of the liposomes. As a result, we estimate that 12%v to 18%v of the swollen bilayers consists of solvent. At the lowest analysed alcohol concentrations, we observe that heavy water is incorporated into the membrane in a higher proportion with regards to alcohol. As water in the bulk solution is progressively replaced by alcohol, the volume fraction of alcohol within the lipid membrane ($\phi_{alc,lipo}$) increases at the expense of water ($\phi_{D_2O,lipo}$). The volume fractions and scattering length densities estimated by the invariant analysis, as well as the structural information obtained from light scattering, allowed us to model SANS curves that appropriately describe the experimental data, showing that despite being extruded 20 times, the vesicles formed are still of multi-lamellar nature.

These findings open the door to debate whether higher than previously thought alcohol volumes can penetrate the membrane and be partially confined there when dispersions have ≥10 %w of alcoholic solvent. Interestingly, glycerol affects liposomes in a very different

fashion, with minimal impact neither on the size nor the membrane rigidity, and with little to no glycerol incorporation into the bilayer. This corroborates the known fact that glycerol can effectively replace water without altering solvent properties. Very interestingly, the bending modulus of the lipid bilayers, as determined by NSE, becomes increasingly reduced with rising alkyl length of the alcohols, while glycerol shows no such effect.

To our knowledge, this is the first report that systematically studies the influence of short-chain alcohols at elevated concentrations on PL liposomes by this combination, SANS and NSE, of neutron scattering techniques.

### CRediT authorship contribution statement

**Fernanda Alvarado Galindo:** Writing – review & editing, Writing – original draft, Investigation, Formal analysis. **Joachim Venzmer:** Conceptualization. **Sylvain Prévost:** Investigation. **Ingo Hoffmann:** Writing – review & editing, Investigation, Formal analysis. **Michael Gradzielski:** Writing – review & editing, Supervision, 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 can be downloaded using the DOIs for the two experiments: https://doi.ill.fr/10.5291/ILL-DATA.9-10-1722 and https://doi.ill.fr/10.5291/ILL-DATA.9-10-1707 after an embargo period of four years. Requests for data earlier than this should be emailed to the corresponding author.

### Acknowledgements

The authors gratefully acknowledge the Institut Laue-Langevin (ILL), Grenoble, France, for the allocation of SANS and NSE beamtime (experiments 9-10-1732 and 9-10-1707). We gratefully acknowledge the Partnership for Soft Condensed Matter (PSCM) for access to support laboratories. This project has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 847439.

## Appendix A. Supplementary data

Supplementary material related to this article can be found online at https://doi.org/10.1016/j.colSURFA.2024.135014.

## References

[1] G. Gregoriadis, Targeting of drugs, Nature 265 (1977) 407–411, http://dx.doi.org/10.1038/265407a0.

[2] M. Malmsten, Soft drug delivery systems, Soft Matter 2 (2006) 760–769, http://dx.doi.org/10.1039/B608348J.

[3] P. Liu, G. Chen, J. Zhang, A review of liposomes as a drug delivery system: Current status of approved products, regulatory environments, and future perspectives, Molecules 27 (4) (2022) 1372, http://dx.doi.org/10.3390/molecules27041372.

[4] R. Sreij, S. Prévost, C. Dargel, R. Dattani, Y. Hertle, O. Wrede, T. Hellweg, Interaction of the saponin aescin with ibuprofen in DMPC model membranes, Mol. Pharm. 15 (10) (2018) 4446–4461, http://dx.doi.org/10.1021/acs.molpharmaceut.8b00421, PMID: 30102549.

[5] E.B. Souto, A.S. Macedo, J. Dias-Ferreira, A. Cano, A. Zielińska, C.M. Matos, Elastic and ultradeformable liposomes for transdermal delivery of active pharmaceutical ingredients (APIs), Int. J. Mol. Sci. 22 (18) (2021) 9743, http://dx.doi.org/10.3390/ijms22189743.
[6] D.D. Lasic, The mechanism of vesicle formation, Biochem. J. 256 (1) (1988) 1–11, http://dx.doi.org/10.1042/bj2560001.

[7] D.R. Olivier Diat, F. Nallet, Effect of shear on a lyotropic lamellar phase, J. Phys. II 3 (9) (1993) 1427–1452, http://dx.doi.org/10.1051/jp2:1993211.

[8] J.I. Escalante, M. Gradzielski, H. Hoffmann, K. Mortensen, Shear-induced transition of originally undisturbed lamellar phase to vesicle phase, Langmuir 16 (23) (2000) 8653–8663, http://dx.doi.org/10.1021/la000242c.

[9] M. Bergmeier, M. Gradzielski, H. Hoffmann, K. Mortensen, Behavior of ionically charged lamellar systems under the influence of a shear field, J. Phys. Chem. B 103 (1999) 1605–1617.

[10] A.A. Gurtovenko, J. Anwar, Interaction of ethanol with biological membranes: The formation of non-bilayer structures within the membrane interior and their significance, J. Phys. Chem. B 113 (7) (2009) 1983–1992, http://dx.doi.org/10.1021/jp808041z, PMID: 19199697.

[11] J. Mou, J. Yang, C. Huang, Z. Shao, Alcohol induces interdigitated domains in unilamellar phosphatidylcholine bilayers, Biochemistry 33 (33) (1994) 9981–9985, http://dx.doi.org/10.1021/bi00199a022, PMID: 8061008.

[12] N.J. Wittenberg, L. Zheng, N. Winograd, A.G. Ewing, Short-chain alcohols promote accelerated membrane distention in a dynamic liposome model of exocytosis, Langmuir 24 (6) (2008) 2637–2642, http://dx.doi.org/10.1021/la703171u, PMID: 18278956.

[13] M.D. Smith, S.V. Pingali, J.G. Elkins, D. Bolmatov, R.F. Standaert, J.D. Nickels, V.S. Urban, J. Katsaras, B.H. Davison, J.C. Smith, L. Petridis, Solvent-induced membrane stress in biofuel production: molecular insights from small-angle scattering and all-atom molecular dynamics simulations, Green Chem. 22 (2020) 8278–8288, http://dx.doi.org/10.1039/DG0C01865A.

[14] M.H. Nguyen, M. DiPasquale, B.W. Rickard, C.B. Stanley, E.G. Kelley, D. Marquardt, Methanol accelerates DMPC flip-flop and transfer: A SANS study on lipid dynamics, Biophys. J. 116 (5) (2019) 755–759, http://dx.doi.org/10.1016/j.bpj.2019.01.021.

[15] M.H. Nguyen, D. Dziura, M. DiPasquale, S.R. Castillo, E.G. Kelley, D. Marquardt, Investigating the cut-off effect of n-alcohols on lipid movement: a biophysical study, Soft Matter 19 (2023) 5001–5015, http://dx.doi.org/10.1039/D2SM01583H.

[16] H.V. Ly, M.L. Longo, The influence of short-chain alcohols on interfacial tension, mechanical properties, area/molecule, and permeability of fluid lipid bilayers, Biophys. J. 87 (2) (2004) 1013–1033, http://dx.doi.org/10.1529/biophysj.103.034280.

[17] M. Weis, M. Kopáni, J. Jakubovský, L. Danihel, Ethanol and methanol induced changes in phospholipid monolayer, Appl. Surf. Sci. 253 (5) (2006) 2425–2431, http://dx.doi.org/10.1016/j.apsusc.2006.04.053.

[18] V.I. Gordeliy, V. Cherezov, J. Teixeira, Strength of thermal undulations of phospholipid membranes, Phys. Rev. E 72 (2005) 061913, http://dx.doi.org/10.1103/PhysRevE.72.061913.

[19] W. Helfrich, Steric interaction of fluid membranes in multilayer systems, Z. Nat.forsch. A 33 (3) (1978) 305–315.

[20] W. Helfrich, R. Servuss, Undulations, steric interaction and cohesion of fluid membranes, Nuovo Cimento D 3 (1) (1984) 137–151, http://dx.doi.org/10.1007/BF02452208.

[21] D. Lombardo, M.A. Kiselev, Methods of liposomes preparation: Formation and control factors of versatile nanocarriers for biomedical and nanomedicine application, Pharmaceutics 14 (3) (2022) 543, http://dx.doi.org/10.3390/pharmaceutics14030543.

[22] H. Komatsu, S. Okada, Effects of ethanol on permeability of phosphatidylcholine/cholesterol mixed liposomal membranes, Chem. Phys. Lipids 85 (1) (1997) 67–74, http://dx.doi.org/10.1016/S0009-3084(96)02634-5.

[23] R. Dimova, Recent developments in the field of bending rigidity measurements on membranes, Adv. Colloid Interface Sci. 208 (2014) 225–234, http://dx.doi.org/10.1016/j.cis.2014.03.003, Special issue in honour of Wolfgang Helfrich.

[24] S.E. Feller, C.A. Brown, D.T. Nizza, K. Gawrisch, Nuclear overhauser enhancement spectroscopy cross-relaxation rates and ethanol distribution across membranes, Biophys. J. 82 (3) (2002) 1396–1404, http://dx.doi.org/10.1016/S0006-3495(02)75494-5.

[25] M. Patra, E. Salonen, E. Terama, I. Vattulainen, R. Faller, B.W. Lee, J. Holopainen, M. Karttunen, Under the influence of alcohol: The effect of ethanol and methanol on lipid bilayers, Biophys. J. 90 (4) (2006) 1121–1135, http://dx.doi.org/10.1529/biophysj.105.062364.

[26] J. Chanda, S. Bandyopadhyay, Perturbation of phospholipid bilayer properties by ethanol at a high concentration, Langmuir 22 (8) (2006) 3775–3781, http://dx.doi.org/10.1021/la053398r, PMID: 16584255.

[27] A.N. Dickey, R. Faller, How alcohol chain-length and concentration modulate hydrogen bond formation in a lipid bilayer, Biophys. J. 92 (7) (2007) 2366–2376, http://dx.doi.org/10.1529/biophysj.106.097022.

[28] R.M. Konas, J.L. Daristotle, N.B. Harbor, J.B. Klauda, Biophysical changes of lipid membranes in the presence of ethanol at varying concentrations, J. Phys. Chem. B 119 (41) (2015) 13134–13141, http://dx.doi.org/10.1021/acs.jpcb.5b06066, PMID: 26390264.

[29] L. Toppozini, C.L. Armstrong, M.A. Barrett, S. Zheng, L. Luo, H. Nanda, V.G. Sakai, M.C. Rheinstädter, Partitioning of ethanol into lipid membranes and its effect on fluidity and permeability as seen by X-ray and neutron scattering, Soft Matter 8 (2012) 11839–11849, http://dx.doi.org/10.1039/C2SM26546J.

[30] D.V. Lynch, P.L. Steponkus, Lyotropic phase behavior of unsaturated phosphatidylcholine species: relevance to the mechanism of plasma membrane destabilization and freezing injury, BBA - Biomembranes 984 (3) (1989) 267–272, http://dx.doi.org/10.1016/0005-2736(89)90292-7.

[31] B.J. Frisken, Revisiting the method of cumulants for the analysis of dynamic light-scattering data, Appl. Opt. 40 (24) (2001) 4087–4091, http://dx.doi.org/10.1364/AO.40.004087.

[32] G. Porod, Die röntgenkleinwinkelstreuung von dichtgepackten kolloiden systemen, Kolloz Z. 124 (2) (1951) 83–113, http://dx.doi.org/10.1007/BF01512792.

[33] P. Hickl, M. Ballauff, Interpretation of the structural parameters derived from the analysis of polymer colloids by small-angle X-ray and neutron scattering, Phys. A 235 (1) (1997) 238–247, http://dx.doi.org/10.1016/S0378-4371(96)00344-5, Proceedings of the Workshop on Colloid Physics.

[34] A. Guinier, G. Fournet, Small-Angle Scattering of X-rays, John Wiley & Sons, 1955.

[35] R. Granek, I. Hoffmann, E.G. Kelley, M. Nagao, P.M. Vlahovska, A. Zilman, Dynamic structure factor of undulating vesicles: Finite-size and spherical geometry effects with application to neutron spin echo experiments, Eur. Phys. J. E 47 (2) (2004) http://dx.doi.org/10.1140/epje/s10189-023-00400-9.

[36] U. Seifert, S.A. Langer, Viscous modes of fluid bilayer membranes, Europhys. Lett. 23 (1) (1993) 71–76, http://dx.doi.org/10.1209/0295-5075/23/1/0121.

[37] M.C. Watson, Y. Peng, Y. Zheng, F.L.H. Brown, The intermediate scattering function for lipid bilayer membranes: From nanometers to microns, J. Chem. Phys. 135 (19) (2011) http://dx.doi.org/10.1063/1.3657857.

[38] W. Schärtl, Light Scattering from Polymer Solutions and Nanoparticle Dispersions, Springer Laboratory, 2007.

[39] A. Nowacka, S. Douezan, L. Wadsö, D. Topgaard, E. Sparr, Small polar molecules like glycerol and urea can preserve the fluidity of lipid bilayers under dry conditions, Soft Matter 8 (2012) 1482–1491, http://dx.doi.org/10.1039/C1SM06273E.

[40] H. Komatsu, S. Okada, Ethanol-induced aggregation and fusion of small phosphatidylcholine liposome: participation of interdigitated membrane formation in their processes, Biochim. Biophys. Acta (BBA) - Biomembr. 1235 (2) (1995) 270–280, http://dx.doi.org/10.1016/0005-2736(95)80014-7.

[41] E. Toutou, N. Dayan, L. Bergelson, B. Godin, M. Eliaz, Ethosomes — novel vesicular carriers for enhanced delivery: characterization and skin penetration properties, J. Control Release 65 (3) (2000) 403–418, http://dx.doi.org/10.1016/S0168-3659(99)00222-9.

[42] J. López-Pinto, M. González-Rodríguez, A. Rabasco, Effect of cholesterol and ethanol on dermal delivery from dppc liposomes, Int. J. Pharm. 298 (1) (2005) 1–12, http://dx.doi.org/10.1016/j.jipharm.2005.02.021.

[43] R.R. López, P.G. Font de Rubinat, L.-M. Sánchez, T. Tsering, A. Alazzam, K.-F. Bergeron, C. Mounier, J.V. Burnier, I. Stiharu, V. Nerguizian, The effect of different organic solvents in liposome properties produced in a periodic disturbance mixer: Transcutol®, a potential organic solvent replacement, Colloid Surf. B 198 (2021) 111447, http://dx.doi.org/10.1016/j.colsurf.2020.111447.

[44] C. Malardier-Jugroot, D.T. Bowron, A.K. Soper, M.E. Johnson, T. Head-Gordon, Structure and water dynamics of aqueous peptide solutions in the presence of co-solvents, Phys. Chem. Chem. Phys. 12 (2010) 382–392, http://dx.doi.org/10.1039/B915346B.

[45] Z.E. Hughes, C.J. Malajczuk, R.L. Mancera, The effects of cryosolvents on DOPC-β-sitosterol bilayers determined from molecular dynamics simulations, J. Phys. Chem. B 117 (12) (2013) 3362–3375, http://dx.doi.org/10.1021/jp400975y, PMID: 23445456.

[46] A.V. Egorov, A.P. Lyubartsev, A. Laaksonen, Molecular dynamics simulation study of glycerol-water liquid mixtures, J. Phys. Chem. B 115 (49) (2011) 14572–14581, http://dx.doi.org/10.1021/jp208758r, PMID: 22004353.

[47] J. Paxman, B. Hunt, D. Hallan, S.R. Zarbock, D.J. Woodbury, Drunken membranes: Short-chain alcohols alter fusion of liposomes to planar lipid bilayers, Biophys. J. 112 (1) (2017) 121–132, http://dx.doi.org/10.1016/j.bpj.2016.11.3205.

[48] F.W. Stetter, T. Hugel, The nanomechanical properties of lipid membranes are significantly influenced by the presence of ethanol, Biophys. J. 104 (5) (2013) 1049–1055, http://dx.doi.org/10.1016/j.bpj.2013.01.021.

[49] M. Doucet, et al., SasView, 2021, URL https://www.sasview.org/. Version 5.0.4.

[50] C.J. Malajczuk, B.I. Armstrong, S.S. Stachura, R.L. Mancera, Mechanisms of interaction of small hydroxylated cryosolvents with dehydrated model cell membranes: Stabilization vs destruction, J. Phys. Chem. B 126 (1) (2022) 197–216, http://dx.doi.org/10.1021/acs.jpcb.1c07769, arXiv:https://doi.org/10.1021/acs.jpcb.1c07769, PMID: 34967634.

[51] K.J. Tierney, D.E. Block, M.L. Longo, Elasticity and phase behavior of DPPC membrane modulated by cholesterol, ergosterol, and ethanol, Biophys. J. 89 (4) (2005) 2481–2493, http://dx.doi.org/10.1529/biophysj.104.057943.

[52] J.A. Barry, K. Gawrisch, Direct NMR evidence for ethanol binding to the lipid-water interface of phospholipid bilayers, Biochemistry 33 (26) (1994) 8082–8088, http://dx.doi.org/10.1021/bi00192a013, PMID: 8025114.

[53] D. Pinisetty, D. Moldovan, R. Devireddy, The effect of methanol on lipid bilayers: An atomistic investigation, Ann. Biomed. Eng. 34 (9) (2006) 1442–1451, http://dx.doi.org/10.1007/s10439-006-9148-y.
[54] I. Söderberg, Phase behavior and structure in the soybean phosphatidylcholine-ethanol-water system, in: B. Lindman, J.B. Rosenholm, P. Stenius (Eds.), Surfactants and Macromolecules: Self-Assembly at Interfaces and in Bulk, Steinkopff, Darmstadt, 1990, pp. 285–289.

[55] T. Adachi, H. Takahashi, K. Ohki, I. Hatta, Interdigitated structure of phospholipid-alcohol systems studied by x-ray diffraction, Biophys. J. 68 (5) (1995) 1850–1855, http://dx.doi.org/10.1016/S0006-3495(95)80361-9.

[56] H. Frielinghaus, Small-angle scattering model for multilamellar vesicles, Phys. Rev. E 76 (2007) 051603, http://dx.doi.org/10.1103/PhysRevE.76.051603.

[57] P.G. de Gennes, Liquid dynamics and inelastic scattering of neutrons, Physica 25 (1959) 825–839, http://dx.doi.org/10.1016/0031-8914(59)90006-0.

[58] L. Chiappisi, I. Hoffmann, M. Gradzielski, Membrane stiffening in chitosan mediated multilamellar vesicles of alkyl ether carboxylates, J. Colloid Interface Sci. 627 (2022) 160–167, http://dx.doi.org/10.1016/j.jcis.2022.07.006.