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Dynamics of Liposomes in the Fluid Phase

Sudipta Gupta,1 Judith U. De Mel,1 Gerald J. Schneider1,2
1 Department of Chemistry, Louisiana State University, Baton Rouge, LA 70803, USA
2 Department of Physics & Astronomy, Louisiana State University, Baton Rouge, LA 70803,
USA

1. Abbreviations
1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC); 1,2-distearoyl-sn-glycero-3-
phosphocholine (DSPC); 1,2-dipalmitoyl-sn-glycero-phosphocholine (DPPC); 1,2-dioleoyl-sn-
glycero-phosphocholine (DOPC); 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC); L-α-
Phosphatidylcholine (SoyPC); egg lecithin (EYPC); dimyristoylphosphatidyl-ethanolamine
(DMPE); galactosyl-diacylglycerol (G-DG); digalactosyl-diacylglycerol (DGDG); 1-stearoyl-2-
oleoyl-sn-glycero-3-phosphocholine (SOPC); diarachidonyl phosphatidyl-choline (DAPC);
1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC); 1,2-dilinoleoyl-sn-glycero-3-
phosphocholine (DLPC); 1-palmitoyl-2-oleoylphosphatidic acid (POPA); poly(ethylene glycol)
(PEG); nanoparticles (NPs); phosphatidylserine (PS); phosphatidylglycerol (PG);
phosphatidylethanolamine (PE); phosphatidylcholine (PC); 1,2 –dimyristoyl –sn-glycero-3-
phosphoglycerol (DMPG); Electron Spin Resonance (ESR); sodium dodecylsulphate (SDS); 4-
(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); 2-(N-morpholino) ethanesulfonic
acid (MES); 3-(N morpholino) propanesulfonic acid (MOPS); piperazine-N , N′ -Bis (2-
ethanesulfonic acid) (PIPES); tris(hydroxymethyl)-aminomethane (TRIS), ethylene-diamine-
tetraacetic acid (EDTA); specific gravity from density meter (SG); Differential Scanning
Calorimetry (DSC)

2. Abstract

We review the currently available material on the morphology and dynamics of phospholipids
assembled into liposomes. Key information obtained from neutron scattering, NMR, and other
techniques plays a crucial role to understand the vital role of lipids in sustaining life in living
organisms. We concentrate on the dynamics in the biologically important fluid phase in the time

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range from picoseconds to seconds, which includes a discussion of the center of mass diffusion of
liposomes, membrane fluctuations, lateral, rotational and flip-flop motions of the lipids. We
emphasize on the sensitivity of the dynamics on interactions with a variety of biologically
relevant molecules such as cholesterol. By a comparison of data from literature, we witness a
good agreement of the results from different techniques and studies.

3. Introduction

Liposomes are spherical vesicles that encapsulate an aqueous core by a bilayer formed by
amphiphilic phospholipids. Phospholipids constitute the major part of the cell membrane. The
physical properties of cell membrane are optimized for both cellular barrier as well as selective
gateways. It protects the interior of the cell membranes from a potential hostile environment.
Lipid membranes are model systems of a two-dimensional surfaces in three-dimensional space
and allow us to study them at molecular and atomic level. They exhibit unique viscoelastic
properties essential for the biological functioning of the living cell. It’s a grand challenge to
understand the fundamentals of self-organization of such a multi-component system that leads to
complex interactions, structures, and dynamics. For example the role of a lipid bilayer
undergoing spontaneous curving, bending as observed in large unilamellar vesicles, that leads to
the formation of multilamellar vesicles, vesicle budding, vesicle fusion and synaptic vesicle
exocytosis, is still not well understood [1]. Lipids contribute to cell division [2] and reproduction
[3] but the corresponding physical principles and methodologies are still not solved. Much
overlooked dynamics such as lipid rotation have shown to be affected by hydrostatic pressure,
lipid phase and additives such as cholesterol content [4, 5]. Lipid flip-flop motion is another
dynamic process which occurs in relatively larger time scales. It is crucial for maintaining lipid
composition in the inner and outer monolayers of the membrane [6]. All of these processes form
the basis to understand the evolution of life. On the other hand, the importance of vesicles and
membranes in biotechnical application is unchallenged, like, vesicles used for drug delivery
cargo [7], or integration of membranes with electronic/optoelectronic devices to build biosensors
[8].

2
This review concentrates on the dynamics in the biologically very important fluid phase, often
more accurately referred to as liquid crystalline phase, . It is separated from the gel phase,
( ) by a certain melting transition temperature, . In the fluid phase the hydrocarbon chains
of the lipid tails are liquid like, disordered and randomly oriented [9]. The lipid membrane has a
unique molecular structure that resembles both liquid-like viscous component and elastic liquid-
crystalline part [10]. The conformation of the lipid chain is related to the thermodynamics and to
the mechanical properties of the membrane, like, bending rigidity and elasticity that defines the
spontaneous curvature of the bilayer [9, 11]. Hereafter we concentrate on spherical liposomes on
a length-scale of the order of 50 to 200 nm. Often the literature distinguishes large unilamellar
vesicles (LUVs, 50 to 200 nm) and giant unilamellar vesicles (GUVs, diameters on the order 1 to
10 μm). As illustrated in figure 1, the variability of these so-called liposomes results from the
lipid flexibility, including head group chemistry, hydrocarbon chain lengths of the fatty acids and
their level of unsaturation. Its combination determines many properties, like [11-13].

Figure 1: Schematic drawing of phospholipids and phospholipid head groups.

3
4. Morphology of vesicles

The morphology of liposomes is defined by the bending elasticity of the lipid bilayer [9]. Based
on the spontaneous curvature model introduced by Helfrich [12] the curvature energy, , of a
closed 2D surface is given by

= 2 − +
2
(1)

The mean curvature, = 1⁄ ! + 1⁄ ⁄2, and the Gaussian curvature, = 1⁄ ! , can

be deduced from the two radii, ! and . The parameters, and , are the bending rigidity and
Gaussian bending rigidity, respectively. The spontaneous curvature, , defines the asymmetry
of the shape fluctuations, and is assumed to depend on the environment chemistry and the bilayer
architecture [9].

Seifert et al. proposed the formation of bowl (tomatocytes), oblate, prolate, and pear shaped
vesicles by changing different parameters like the reduced volume, # = $ ⁄$ , , or
temperature, [13]. Domain induced budding was proposed by Jülicher et al. [14], whereas
lateral phase separation was introduced by Seifert [15]. These objects have been discovered
experimentally by phase contrast microscopy [16-18]. Hereafter, we focus on the spherical
structures.

Detailed information on the structure of liposomes and membranes has been obtained by
scattering techniques. Nagele et al. utilized X-ray diffraction to deduce information on the
number of bilayers [19, 20]. Different theoretical approaches exist to describe the structure factor
of multilamellar liposomes, e.g., by Frielinghaus [21]. More detailed information on the
distribution of molecules in the bilayer itself has been obtained by a joint refinement of X-ray
and neutron diffraction data [22]. This method determines a quasimolecular structure consisting
of fragments representing the methylene regions by Gaussian distributions.

Our discussion implies that the lipids are aggregated to liposomes, i.e., the system itself is at
(lipid) concentrations, Φ, greater than the critical micellar concentration (CMC) of the lipids,
often also referred to as critical vesicular concentration (CVC) or critical aggregation
concentration (CAC). Many different experimental approaches permit to determine the CMC

4
[23]. Mole fractions seem to be lesser than mM usually [24]. The textbook by Marsh provides a
comprehensive overview and reports CMC values in the range of 10-9 to 10-3 M [24]. The
experiments reviewed below used conditions Φ >> CMC. Hence, we neglect the contribution of
the free lipids to the experimental results in our discussion.

5. Dynamics of Liposomes at Different Length- and Time-Scales

As figure 2 illustrates, many different processes are anticipated in liposomes, even if a

spherically symmetric morphology is assumed. Individual molecular motions and their collective
dynamics capture a broad range of length- and time scales and may even be viewed in terms of
hierarchical structures and dynamics. Lipid membrane motions can vary from picoseconds to
several hours spanning many decades of the time scale as shown in figure 2 [25]. Therefore,
using multiple techniques becomes a vital part in unveiling membrane dynamic processes. In the
subsequent sections we will discuss these motions in detail.

For the sake of simplicity, we have omitted vesicle fusion from our discussion. As will be
detailed later and illustrated in figure 2, translational diffusion of liposomes refers to the
Brownian diffusion of the center of mass of liposomes in the respective solvent (section 6.2).
Liposomes can be considered as flexible colloids. Section 5.2 reports experimental findings and
discusses the relevance to the biological function. In view of the motions of the bilayer itself,
several processes can be identified such as shape fluctuations [26], thickness fluctuations and
membrane undulations. Membrane fluctuations represent collective motion of the bilayer that
may lead to breathing mode (symmetric deformation), deformations modes (asymmetric
deformation), undulations, etc. [11]. Membrane undulation includes fluctuation of the membrane
in the 2D plane due to thermal noise. It is related to the membrane rigidity as discussed in section
5.2. On the other hand, the membrane thickness fluctuations represent individual motion of the
leaflets that lead to thickness fluctuation perpendicular to the 2D plane [27, 28]. We exlore
theoretical and experimental details in section 5.3. At faster times additional lipid lateral [29]
[30] and lipid rotational motion along its symmetry axis become visible (sections 6.5 and 6.6
respectively )[5]. In case of the so called “flip-flop” motions, the exchange of lipids across the
bilayer occurs at much slower times as explained in section 6.7. Time scale of individual lipid
dynamics does not have a direct proportionality to their size but rather depends on the process

5
and the length scale of focus. For instance, individual lipids also show vibrational motions which
can be accessed by FT-IR methods at a time scale approx. 10-10 s while being able to flip-flop
which may take up to several hours [31].

Length-scales of dynamic processes in liposomes are important in understanding the scope of

dynamics and some must be expressed with respect to time. Membrane fluctuations such as
undulations, thickness fluctuations occur typically at a mesoscopic length scale (10 to 1000 Å)
[32] and lateral diffusion at micrometer scale [33]. Wanderling et.al have shown a slow diffusion
of individual lipids in a confined cylindrical space in addition to the well-known conformational
motions of the fatty acid chains and fast uniaxial rotations of H atoms around the C bonds. All of
these dynamics occur in few Angstrom to a few nanometers length scale.[34] [35].

Figure 2: Different dynamics at different time scales. Acyl chain motions[35]Lateral

diffusion[36]wobbling[37]

6
5.1. Liposome translational diffusion

Starting at the largest length-scale and the simplest structural case, liposomes can be considered
as spherical colloidal particles that undergo Brownian motion in aqueous solution [11]. Dynamic
light scattering is a technique commonly used to measure the translational diffusion coefficient,
&' . The Stokes-Einstein relation, &' = () ⁄ 6+, - , connects &' and the length-scale,
represented by the hydrodynamic radius, -. The thermal fluctuations that cause the Brownian
motion of the liposomes enter via the thermal energy, () , with the Boltzmann constant, () , and
the temperature, T (in K). The parameter, , refers to the viscosity of the solvent.

Depending on their preparation, the hydrodynamic radii of liposomes can vary from 20 nm to ~
50 µm [38, 39]. From the Stokes-Einstein relation, we can estimate the associated range of
translational diffusion coefficients from 1.2 × 100!! to 5 × 100!2 m /4. ( - ≈ 50 nm then &' ≈
10-12 m2s-1.) Beyond the case of an infinitely diluted system, colloidal interactions may become
important. For example, Batchelor [40] and later Cichocki and Felderhof [41] calculated the
long-time self-diffusion coefficient, &56 , of hard sphere colloids, &56 = & 1 − 2.09729 , with,
& , the diffusion coefficient at infinite dilution and, 9, the volume fraction of the colloids, not to
be mixed up with the previously introduced concentration of lipids, Φ. The simple linear
relationship shows that at a hypothetical liposome concentration of around 0.47 the self-diffusion
would tend to be zero.

At the first glance, it seems to be simple to determine the diffusion coefficient, including the
availability of a selection of tools to permit the correct numerical values. Often, certain
limitations seem to require more effort than expected. For example, pulsed field gradient (PFG)
NMR is a common technique to measure the self-diffusion coefficient of macromolecules. In
case of micelles the self-diffusion measured by DLS and PFG-NMR usually agree [42].
However, in case of liposomes, Odeh et al. report a larger diffusion coefficient for the pure
liposomes than one would expect from other techniques like DLS [43]. The authors explain the
not-well defined signals of the PFG NMR experiment by the inherent rigidity of the bilayer and
demonstrated the importance of adding a hydrotropic agent, like poly(ethylene glycol) (PEG), to
obtain the true diffusion coefficient. In case of PEGylated liposomes Leal et.al. report &' =

7
5.2 × 100! m /4, which corresponds to a size of 84 nm and to the values expected from DLS
[44]. However, usually, colloidal science introduces a structure factor contribution to accomplish
agreement, this these observations deserve further investigation.

5.2. Membrane Fluctuations (Undulations)

As illustrated by figure 2, membranes undergo out-of-plane fluctuations, often referred to as

undulations, due to thermal excitations. The spectrum of fluctuation frequencies determines
essentially the bending elasticity, :, of the membrane. The value is characteristic for certain
chemical compositions and morphologies and is seen as one of the most important physical
parameters. It is related to the stability and shape of liposomes, the inter-liposome interactions,
as well as interactions of the liposomes with foreign objects, like, drugs, nanoparticles, polymers,
planar substrates, etc. [9]. It is also responsible for the phenomenon of polymorphism of the
lipids that allow them to aggregate into different structures and phases, such as liquid crystalline
phases under external perturbations like, changes in pH, temperature, pressure, volume, etc. [11].

The lipid membrane has the unique molecular structure that resembles both a liquid-like viscous
component and the elastic liquid-crystalline part. The conformation of the lipid chain is related to
the thermodynamic and mechanical properties of the membrane, like, bending rigidity and
stretching elasticity that defines the spontaneous curvature of the bilayer, whereas, the shear
modulus inside a fluid membrane is zero [9, 12, 45, 46]. The stretching elasticity accounts for the
increase in the average distance between the molecules perpendicular to the bilayer neutral plane.
The corresponding area compressibility modulus, ;, is proportional the relative change in
surface area, Δ ⁄ = = ⁄ ;, for a given lateral stress, = . Micromechanical analysis and X-ray
diffraction reveal that the stretching of the lipid bilayer is limited to rather small deformations
and rupture occurs even at a small change in area (Δ ⁄ ~ 1%) [47]. Therefore, lipid bilayers
behave like a planar surface and can undergo a smooth shape deformation along the bilayer
surface normal that depends on the bending rigidity, :, of the lipid bilayer.

Neutron Spin Echo (NSE) spectroscopy has been frequently utilized to determine the bending
rigidity, :, of the lipid bilayer [10, 27, 28, 48-54]. NSE measures the time dependent structure

8
factor, > ?, @ , as a function of the momentum transfer, ? . Assuming quasielastic scattering =
4+/B sin F , with the wave-length, B, of the incident neutrons and the scattering angle, F. The
reciprocal relationship ? = 2+/ℓ connects the momentum transfer with the size or length-scale,
ℓ, of the scattering objects in the sample. Measuring > ?, @ allows to distinguish different
processes by their length- and time scale, which is essential for liposomes, e.g., to distinguish the
slow translational diffusion of the liposomes from the faster motion of lipids. Individual NSE
instruments can capture a time-range from 0.01 ns to 500 ns and a length-scale range of around
1-30 nm [55].

NSE has often been used to measure the dynamics of membranes. The common approach to
extract information on the undulations utilizes a simplified theory for an ensemble of membrane
patches following the Zilman-Granek (ZG) model [56]. It assumes the intermediate scattering
function
> ?, @ /S
H I = exp N−OΓQ @R T
> ? J
(2)

is connected to a Q-dependent decay rate, ΓQ , which depends on the intrinsic bending modulus,

: [10, 48, 57, 58]

ΓU () ()
= 0.0069γ V
? S ,
(3)
:

The term () represents the thermal energy with the Boltzmann constant, () , and the
temperature, T. The temperature dependent solvent viscosity, , , can be determined
independently using a viscometer [59]. While there seems to be no rigorous theoretical proof,
recent experiments seem to justify the established procedure of using , instead of the solution
S \] ^
viscosity. Usually, : /() ≫ 1 is assumed that leads to X ≈ 1 − Z[ _`
ln ?b ≈ 1, with the

length-scale, b , of the fluctuations of the membrane [10, 27, 48, 56, 60].

Zilman and Granek first introduced a prefactor 0.025 (instead of 0.0069). Then 0.0058 was
calculated assuming a ratio, c ⁄ 2c^ ≈ 0.6, between the distance of the neutral surface from the

9
bilayer midplane, c , and the thickness of the monolayer, c^ . However, this moves the neutral
surface into the headgroup region of the bilayer, which turned out to be impossible. It finally led
to the assumption that c = c^ and which resulted in a numerical prefactor 0.0069 [10, 48]. Since
different publications use different prefactors, the comparison of the bending moduli requires a
recalculation. Therefore, table 1 summarizes the original values and calculates : for the
prefactor 0.0069, the most recent value. While the ZG model seems to be more appropriate for
vesicles with a radius R ≥ 20 nm [61], in some cases the Milner-Safran seem to be more suitable
for microemulsion droplets [62]. The relaxation spectrum according to ZG theory relaxes at a
slower rate for rigid membranes as observed in phospholipids, in comparison to that observed in
most microemulsions droplets and their sponge phases [63-65]. The faster relaxation of bending
modes in microemulsions results in super flexible systems, with : /() ≲ 1. In the fluid phase,

: was found to decrease linearly with temperature [10], increases dramatically with decrease in
size [66], decreases with decrease in pH [67], and varies abruptly in presence of different
additives, cf. table 1.

Equation 2 assumes ZG motion but neglects translational diffusion of liposomes. As defined by

the diameter and the related diffusion coefficient, &' , a substantial contribution to the
intermediate structure factor within the typical time-window of NSE experiments can be
expected [48]. We can incorporate the influence of diffusion in the dynamic structure factor by
assuming that ZG and center of mass motion are statistically independent, which leads to a
product ansatz [48, 68]
> ?, @
= exp −&' ? @ exp e−OΓU @RS f.
> ?
(4)

Though there is no rigorous theoretical proof that diffusion and ZG are statistically independent,
this assumption permits to determine &' independently from ΓQ , either by analyzing the time and
? -dependence of > ?, @ or by complementary experiments, such as DLS [58].

For the fluid phases, , of h-DOPC, h-DMPC, h-DPPC, h-DSPC, and h-POPC liposomes,
values of : ≈ 20 () are reported [27, 52, 53] [69]. (The exact values are listed in table 1).
A much smaller value : = 8.4 ± 1 (g was observed for h-SoyPC [58]. Linear increases in
10
 : with decreasing temperature and approaching the transition temperatures are reported for dt-
DMPC, dt-DPPC and dt-DSPC, all in phase, by Nagao et al. [10].

Several experimental techniques can measure membrane fluctuations. Typical values seem to
be on the order of : ≈ 10-20 () [70, 71]. Examples are flickering spectroscopy that used
image processing methods and Fourier analysis to study thermally induced shape fluctuations on
DMPC, EYPC, DMPE, G-DG and DGDG membranes in the fluid state [70, 71]. Another
approach, like entropic tension micropipette includes aspiration of vesicles in a micropipette that
changes the area available for mechanical fluctuations [47, 72, 73]. From the relation between
the fluctuation area and suction pressure, thereby the effective entropic tension one can
determine :. Lipids like DGDG, DMPC, SOPC and DAPC were studied in the phase. It
should be noted that apart from, :, the other two elastic parameters, ;, and the corresponding
thermal expansion coefficient, h; , can be determined in general by aspiration pipet method [47,
73]. Mechanical fluctuations can also be implemented using electric deformation [74, 75].

Several studies indicate interactions of nanoobjects with liposomes impact the membrane
rigidity. Examples include a reduction of : by 3 (g due to a decoration of 40% of the
surface of liposomes by silica nanoparticles [48]. In stark contrast a stiffening due to
nanoparticles on oil-water interfaces decreases membrane fluctuations [48]. Unlike
cholesterol, nanoparticles seem to disturb the arrangement of lipids in the bilayer. Arriaga et
al. observed a systematic increase of : from 19 ± 2 (g for pure h-POPC in the phase to
37 ± 2 (g at 50 mol% cholesterol, the maximum cholesterol content in a stable bilayer, by
NSE and DLS [76]. The observed stiffening effect of cholesterol on h-POPC bilayers was
explained assuming structural condensation caused by hydrogen-bonding complexes between
the phospholipids and cholesterol in the disordered fluid phase forming an assembly of
dynamical network with a net increase in mechanical properties [77, 78].

Changes induced by antimicrobial peptides like melittin on the rigidity on lipid bilayer were
investigated using NSE spectroscopy [53]. The effect of pore formation of h-DOPC in
HEPES buffer was studied using melittin where three distinct dynamical regimes were

11
observed. First, at low concentrations of melittin, as defined by peptide to lipid molar ratio,
P/L = 0.2%, it is absorbed only at the surface of the membrane causing a slight decrease in
the bending rigidity, :. The decrease of : is explained by the perturbation of chain packing
by peptide adsorption [53]. Second, with increasing the concentration, pores start to form up
to P/L > 1%. At a critical concentration P/L* = 0.4% about half of the LUVs are perforated.
These NSE results are supported by DLS and 31 P NMR [53]. At the critical concentration
P/L*, : started to increase slightly due to high pore rigidity. Third, at higher P/L
concentration, there is a rapid increase in :, where the repulsive interpore interactions
become significant. At P/L = 2%, the membrane fluctuations appeared to be damped by the
melittin-induced pores and at P/L ≥ 6%, fusion of vesicles was observed [53].

A gradual increase of disordering or fluidizing of h-DMPC with increasing the concentration of

nonsteroidal anti-inflammatory drugs like aspirin was observed by NSE spectroscopy [52]. A
33% decrease in bending rigidity from, : = 17.4 ± 0.9 (g for pure h-DMPC to : = 11.7 ±
0.4 (g for 13.5 wt% of aspirin at 20°C in the phase was found. Following the X-ray
diffraction studies by the Alsop et al. [79], it was concluded that the residence of aspirin near the
head group along with increase in disorder due to the formation of gauche defects in the
hydrocarbon chains is responsible for the softening or plasticizing effect of the membrane [52].

The effect of model nonsteroidal anti-inflammatory drug like ibuprofen was studied using NSE,
SANS and MD simulation as a function of pH and temperature [67]. It was observed that for
pure h-DMPC in the phase at 30°C, the : of the lipid bilayer remains relatively unchanged
at high pH and decreases significantly by approximately 32%, when the pH decreases from 8 to
2. It was predicted that owing to the decrease in hydration of the lipid head group, the
simultaneous decrease in : has its origin in the electrostatic and structural perturbations of the
lipid head [67]. It was also observed that at pH ≈ 2, : decreases by ≈ 18%, for an increase in
temperature from 30 to 37°C, due to the lipid chain transforming from more gel-like to more
fluid-like state causing an decrease in bilayer thickness by ≈ 0.4 nm [67]. On the other hand,
the introduction of Ibuprofen was found to decrease the : of the lipid bilayer at all pH values,
although : remains unchanged with change in pH for a fixed Ibuprofen concentration [67].

12
Such a decrease in : was explained as a combination of bilayer thinning, decrease in head
group hydration and lowering of area compressibility modulus [67].

The effect of another drug like aescin on the membrane rigidity of h-DMPC liposomes in the
fluid phase and rigid gel phase was studied by SAXS, SANS, DLS and NSE
spectroscopy [54]. Saponins aescin are a type of glycosidic biosurfactants produced from plants
and are used to treat diseases, like, chronic venus insufficiency (CVI), hemorrhoids and
peripheral oedemic formation. From SANS a slight increase in the bilayer thickness along with
increase in the radius of gyration, j, of the liposome was observed. SAXS reveals more
prominent effects over the entire lipid thickness, where polar region of the aescin interacts with
the lipid head group in the phase at 10°C. In the phase at 40°C insertion of the aescin
deep into the bilayer thickness was observed. A decrease in : in the phase, whereas an
increase in : in the phase, with increasing aescin content was reported [54]. It was
concluded that the H-bond formation between the hydroxy group of aescins sugar counterpart
with the carbonyl and negatively-charged phosphate groups of h-DMPC causes a reduction of :

in the phase. In the phase the incorporation of large triterpenic backbone of aescin in the
bilayer causes an increase in rigidity [54].

Recently NSE spectroscopy has provided valuable information on the mechanism of lipid raft
formation in liposomes. The lipid raft constitutes of a liquid ordered phase (LO) formed from a
high melting lipid like DSPC rich phase and the liquid disordered phase (LD) formed from
cholesterol forming the so called “raft” domains [49]. Using contrast variation, either the LO or
the LD phase, can be contrast matched with the solvent [49]. Such an approach enables the
extraction of bending rigidities for each the LO and LD phases independently. A mixture of
cholesterol, DSPC and POPC in 22:39:39 ratio in a D2O/H2O mixture led to a diameter of the
rafts of approximately 13 nm in unilamellar vesicles with a diameter of around 60 nm, as
determined by separate SANS experiments. The bending modulus of the raft domains, : =
126.5 ± 29.9 (g , is substantially different from that of the surrounding medium, : = 18.4 ± 9.8
(g , at 20°C [49]. These values were supported by MD simulations [49]. It was concluded that
this mismatch in bending moduli also accounts for the evolution of lateral heterogeneities in

13
different classes of lipids and in biological membranes [49]. In contrast, mixtures of h-
DMPC/DSPC show a similar bending rigidity, : ≈ 20 () , as in case of the single lipid
liposomes [28]. If the temperature is lowered, a substantial increase of : can be observed once
one of the lipids reached the gel phase (DSPC), while DMPC is still in its fluid phase [28]. It was
assumed that the effective : depends on the average bilayer density [28]. The variations in :

for different systems with and without additives are presented in table 1.

Table 1: Bending rigidity, : or, k: , from different papers in the fluid phase. The NSE
prefactor denotes the value in equation 3, used to calculate, : . The last column represents the
normalized : from NSE to match the prefactor 0.0069.
Phospholipids in Lipid l °m ln ° m` Phase Delta Diamete pq (rs l) Methods Prefactor pq (rs l)
water (D2O for NSE, chains (metho T= T- r [nm] in
H2O for other ds) Tm equation Recalculated
techniques) 3 for prefactor
0.0069 in
equation 3
10 wt % dt-DPPC 16:0 / 50 41.3 NA 46.4 ± 0.2 NSE 0.0069 46.4 ± 0.2
16:0 (SG) [10]
[80] 8.7

14
10 wt % dt-DSPC 18:0 / 60 54.4 NA 49.6 ± 1.8 NSE 0.0069 49.6 ± 1.8
18:0 (DSC) 5.6 [10]
[81]
65 NA 42.0 ± 1.2 NSE 0.0069 42.0 ± 1.2
10.6 [10]

70 NA 46.4 ± 0.2 NSE 0.0069 46.4 ± 0.2

15.6 [10]

10 wt % dt-DMPC 14:0 / 30 23.6 NA 35.6 ± 1.9 NSE 0.0069 35.6 ± 1.9

14:0 (DSC) 6.4 [10]
[81]
35 NA 34.0 ± 1.8 NSE 0.0069 34.0 ± 1.8
11.4 [10]

40 NA 30.0 ± 1.3 NSE 0.0069 30.0 ± 1.3

16.4 [10]

50 NA 25.4 ± 1.0 NSE 0.0069 25.4 ± 1.0

26.4 [10]

65 NA 20.6 ± 1.0 NSE 0.0069 20.6 ± 1.0

41.4 [10]

1 wt % h-DMPC 14:0 / 37 23.6 110 [52] 17.4 ± 0.9 NSE 0.0058 24.6 ± 1.3
14:0 (DSC) 13.4 [52]
[81]
1 wt % h-DMPC + 37 110 [52] 11.7 ± 0.9 NSE 0.0058 16.6 ± 1.3
13.5 wt% asprin 6.4 [52]

2 wt% h- at pH 14:0 / 30 23.6 Lu 100 nm 26.2 ± 0.8 NSE 0.025 18.0 ± 0.5
DMPC 7.4 14:0 (DSC) pores [67]
(different [81] 6.4 [67]
pH) at pH Lu 100 nm 26.8 ± 0.9 NSE 0.025 18.4 ± 0.6
4.9 pores [67]
6.4 [67]
at pH Lu 100 nm 24.6 ± 0.8 NSE 0.025 18.2 ± 0.5
3.3 pores [67]
6.4 [67]
at pH Lu 100 nm 18.0 ± 0.5 NSE 0.025 12.3 ± 0.3
1.6 pores [67]
6.4 [67]
2 wt% h- at pH 30 Lu 100 nm 12.3 ± 0.3 NSE 0.025 8.4 ± 0.2
DMPC, 31 7.4 pores [67]
mol% 6.4 [67]
ibuprofen at pH Lu 100 nm 12.8 ± 0.3 NSE 0.025 8.8 ± 0.2
(different 4.9 pores [67]
pH) 6.4 [67]
at pH Lu 100 nm 12.2 ± 0.3 NSE 0.025 8.4 ± 0.2
1.6 pores [67]
6.4 [67]
1.5 wt% h-DMPC 14:0 / 40 23.6 16.4 100 ± 5 17 ± 0.3 NSE 0.025 11.7 ± 0.2
14:0 (DSC) [54] [54]
[81]

1.5 wt% h- at 0.4 40 23.6 Lu 16.4 100 ± 5 26 ± 1 NSE 0.025 17.8 ± 0.7
DMPC, mol% (DSC) [54] [54]
aescin [81]
at 0.8 14:0 / Lu 16.4 100 ± 5 31 ± 2 NSE 0.025 21.3 ± 1.4
mol% 14:0 [54] [54]

5 wt% h-SoyPC 16:0 / 30 -18.5 48.5 120 ± 8.4 ± 1 NSE 0.0069 8.4 ± 1
18:2 (DSC) 15 [58] [58]
[82]

15
5 wt% h-DOPC 18:1 / 20 -16.5 36.5 107 ± 4 20 ± 3 NSE 0.0069 20 ± 3
18:1 (DSC) [58] [58]
[83]

10 wt% dt- 14:0 / 15 20.5/5 / -5.5/- 100 nm 124 ± 12 NSE 0.0058 175 ± 17
DMPC/DSPC 14:0 0.5 35.5 pores [28]
and [28]
18:0 /
18:0 25 20.5/5 / 4.5/- 100 nm 119 ± 8 NSE 0.0058 168 ± 11
0.5 25.5 pores [28]
[28]

30 / 9.5/- 100 nm 120 ± 8 NSE 0.0058 169 ± 11

20.5 pores [28]
[28]

35 / 14.5/- 100 nm 61 ± 2 NSE 0.0058 86 ± 3

15.5 pores
/
[28]
40 19.5/- 100 nm 36 ± 2 NSE 0.0058 51 ± 3
10.5 pores [28]
[28]

45 / 24.5/- 100 nm 24 ± 2 NSE 0.0058 34 ± 3

5.5 pores [28]
[28]

55 / 34.5/4. 100 nm 21 ± 1 NSE 0.0058 30 ± 2

5 pores [28]
[28]

65 / 44.5/1 100 nm 20 ± 1 NSE 0.0058 28 ± 1

4.5 pores [28]
[28]

0.11 wt% h-DOPC 18:1 / 25 -16.5 87 [48] 23 ± 1 NSE 0.0069 23 ± 1

18:1 (DSC) 41.5 [48]
[83]
0.11 wt% h-DOPC, 25 87 [48] 20 ± 1 NSE 0.0069 20 ± 1
0.17 wt% Silica NPs 41.5 [48]

6 mM h-DOPC 30 46.5 92 ± 1 26.8 ± NSE 0.0058 38 ± 2

(HEPES buffer) [53] 1.6 [53]

6 mM h- P/L = 30 Lu 46.5 92 ± 1 24.9 ± NSE 0.0058 35 ± 2

DOPC 0.19 [53] 1.5 [53]
(HEPES
buffer) at P/L = Lu 46.5 92 ± 1 19.1 ± NSE 0.0058 27 ± 1
peptide 0.40 [53] 1.0 [53]
(melittin) :
lipid (P/L) P/L = Lu 46.5 93 ± 1 21.4 ± NSE 0.0058 30 ± 2
molar ratio 0.60 [53] 1.3 [53]
(%)
P/L = Lu 46.5 93 ± 1 19.8 ± NSE 0.0058 28 ± 2
0.80 [53] 1.3 [53]

P/L = Lu 46.5 91 ± 1 20.6 ± NSE 0.0058 29 ± 2

1.7 [53] 1.2 [53]

P/L = Lu 46.5 90.5 ± 1 28.0 ± NSE 0.0058 40 ± 3

2.4 [53] 2.2 [53]

P/L = 18:1 / -16.5 Lu 46.5 90 ± 1 42.0 ± NSE 0.0058 59 ± 7

4.0 18:1 (DSC) [53] 4.7 [53]
[83]
2 mg/ml h-POPC 16:0 / 22 -2.6 81 ± 2 19 ± 2 NSE 0.025 13.0 ± 1.4
18:1 (DSC) 24.6 [76] [69] [76]

16
2 mg/ml h-POPC, 50 22 [84] 102 ± 5 37 ± 2 NSE 0.025 25.4 ± 1.4
mol% cholesterol 24.6 [76] [76]

14:0 / 30 23.6 6.4 NA 27.5 ± 3 Flickering - -

14:0 (DSC) [70]
1 mM DMPC [81]
30 6.4 NA 50.1 ± 6 Flickering - -
1 mM DMPC, 20 [70]
mol% cholesterol
30 6.4 NA 95.5 ± 19 Flickering - -
1 mM DMPC, 30 [70]
mol% cholesterol
18:1 / 30 -15 to - Lu 37 to NA 27.4 ± Flickering - -
1 mM EYPC 16:0 71 [85] 45 3.5 [70]
16:3 / 30 -50 Lu 80 NA 3.5 to 9.5 Flickering - -
18:3 (DSC/ [70]
XRD)
1 mM G-DG [86]
18:3 / -30 Lu - NA 2.9 ± 0.9 Flickering - -
18:3 (DSC/ [71]
XRD)
10 mg/ml DGDG room [86]
18:1 / -15 to - Lu 37 to NA 19.4 ± 5 Flickering - -
10 mg/ml EYPC 16:0 room 7 [85] 45 [71]
14:0 / 50 [87] Lu 10 NA 15.2 ± 2 Flickering - -
14:0 [71]
10 mg/ml DMPE PE 60
18:0 / 9 Lu 9 NA 22.5 ± Micropipette - -
18:1 (DSC) 1.5 [72]
10 mg/ml SOPC 18 [87]
14:0 / 23.6 Lu 5.4 NA 13.4 ± Micropipette - -
14:0 (DSC) 1.4 [72]
10 mg/ml DMPC 29 [81]
20:4/ -69 Lu 87 NA 10.9 ± Micropipette - -
20:4 (DSC) 1.2 [72]
10 mg/ml DAPC 18 [87]
18:3 / -30 Lu 53 NA 10.8 ± Micropipette - -
18:3 (DSC/ 0.7 [72]
XRD)
10 mg/ml DGDG 23 [86]
18:0 / 9 Lu 12 NA 8.4 ± 0.8 Flickering - -
18:1 (DSC) [72]
1 mg/ml SOPC 21 [87]
18:1 / -16.5 Lu 37.5 NA 3.9 ± 1.3 Flickering - -
18:1 (DSC) [74]
1 mg/ml DOPC 21 [83]
16:0 / -2.6 Lu 26.6 NA 9.5 ± 2.1 Flickering - -
18:1 (DSC) [74]
1 mg/ml POPC 24 [84]
18:0 / 9 Lu 12 NA 7.9 ± 1.3 Electric - -
18:1 (DSC) [74] Deformation
1 mg/ml SOPC 21 [87]
18:1 / -16.5 Lu 37.5 NA 4.2 ± 0.8 Electric - -
18:1 (DSC) [74] Deformation
1 mg/ml DOPC 21 [83]
16:0 / -2.6 Lu 26.6 NA 14.1 ± 2.8 Electric - -
18:1 (DSC) [74] Deformation
1 mg/ml POPC 24 [84]
18:1 / -15 to - Lu - NA 6 ± 1.2 Electric - -
16:0 7 [75] Deformation
(DSC)
10 mg/ml EYPC room [85]
12:0 / -2 Lu - NA 8.2 ± 1.6 Electric - -
12:0 (DSC) [75] Deformation
10 mg/ml DLPC room [87]

1
EYPC is a complex mixture of phosphatidylcholines, exhibits Tm = -15 to -17 °C

17
 18:3 / -30 Lu - NA 2.5 ± 0.5 Electric - -
18:3 (DSC/ [75] Deformation
XRD)
10 mg/ml DGDG room [86]
16:0 / -2.6 Lu 22.6 20-50 39.52 ± Flickering - -
0.2 mg/ml POPC 18:1 20 (DSC) µm [38] 0.7 [38]
0.2 mg/ml POPC in [84] Lu 22.6 20-50 39.09 ± Flickering - -
HEPES buffer 20 µm [38] 0.7 [38]
0.2 mg/ml POPC in Lu 22.6 20-50 40.61 ± Flickering -
Histidine buffer 20 µm [38] 0.4 [38]
0.2 mg/ml POPC in Lu 22.6 20-50 33.07 ± Flickering -
MES buffer 20 µm [38] 0.4 [38]
0.2 mg/ml POPC in Lu 22.6 20-50 34.70 ± Flickering -
MOPS buffer 20 µm [38] 0.7 [38]
0.2 mg/ml POPC in Lu 22.6 20-50 37.98 ± Flickering -
PIPES buffer 20 µm [38] 1.0 [38]
0.2 mg/ml POPC in Lu 22.6 20-50 35.79 ± Flickering -
TRIS buffer 20 µm [38] 0.4 [38]
0.2 mg/ml POPC in Lu 22.6 20-50 31.01 ± Flickering -
100 mM NaCl 20 µm [38] 0.8 [38]
0.2 mg/ml POPC in Lu 22.6 20-50 24.00 ± Flickering -
100 mM NaCl, 2 mM µm [38] 0.3 [38]
EDTA, 10 mM TRIS 20
0.2 mg/ml POPC in Lu 22.6 20-50 24.62 ± Flickering -
100 mM NaCl, 10 µm [38] 1.2 [38]
mM TRIS 20

5.3. Thickness Fluctuations of the Bilayer

In addition to undulations, (spontaneous) fluctuations of the thickness of the bilayer were

proposed theoretically in the early 1980s [88-91] and are considered to be the primary
mechanism underlying pore formation in membranes [92, 93], which leads to passive
permeability of the membrane for drugs [94]. The amplitude of the fluctuations is around 3.7 Å,
and seems to be less dependent on the length of the alkyl chains, at least for a variation from 14
to 18 carbon atoms in the fatty acid tails [27]. The relaxation occurs well in the time window of
NSE and seems to have characteristic fluctuation times on the order of 100 ns [27], which is the
same order of magnitude at which conformational transitions in proteins occur [95].

Since all protons contribute to the signal and overshadow the information on thickness
fluctuations of the bilayer, Woodka et al. employed partially deuterated and fully hydrogenated
DMPC, DPPC, and DSPC [27]. In this pioneering study the time-dependent structural
correlations between the head groups were studied by contrast matching of the tail by the solvent
[27]. Thickness fluctuations seem to be very common in soft matter and biological systems. For
example, Farago et al. reported the occurrence of similar processes in the lamellae of SDS-water-
penthanol-dodecane microemulsions in the lyotropic phase [96].

18
In order to describe the observations by the ZG model, a semi-empirical approach was
introduced that accounts for the additional Q-dependence of the decay rate ΓU ⁄? S due to the
structural correlations [10, 27, 97]
ΓU ΓJ 1 1
= S+ y z
? S ? w^x ? 1 + ? − ?
S b
(5)

The second term introduces the relaxation time of the thickness fluctuations, w ^x . The thickness
of the bilayer can be obtained from the position, ? , of the structural correlation of the head
groups. The amplitude of the fluctuations in the reciprocal space is described by the parameter
b 0! .

As illustrated in figure 3 a, SAXS and SANS studies on DMPC, DPPC and DSPC show the
minimum at ? = ? is determined by the bilayer thickness only, whereas the peak at ? > ?
reflects both the bilayer thickness and thickness fluctuation amplitude of the membrane [98]. As
summarized by figure 2b, from the modeling of NSE data with equation 2 the Q-dependence of
the decay rate ΓU ⁄? S shows a distinct peak close to ? in case of the tail contrast matched
samples. The solid lines represent the calculations by equation 4. Differences between the full
contrast experiments – tail and heads visible in case of hydrogenated (h) lipids in D2O – and tail
deuterated (dt) samples is obvious. This peak is a direct evidence of the substantial contribution
of a process in addition to the ZG behavior and is ascribed to the thickness fluctuations [28].

(a) (b)

19
Figure 3. (a) SANS scattering diagrams on dt-DMPC/dt-DSPC at three different
temperatures. Reproduced from the literature [28]. (b) The decay rate, }U ⁄? S , from NSE
relaxation spectrum for tail-deuterated (dt) and fully hydrogenated (h) phospholipids in the
fluid phase. The solid lines represent calculated results by equation 4. The data for h-DMPC
[52], h-DOPC [58], dt-DPPC [27] and the lipid mixtures dt-DMPC (41.5wt%)/dt-DSPC
(48.3wt%) in h-DMPC (4.49wt%)-h-DSPC (1.1wt%), and, h-DMPC (46.1wt%)/h-DSPC
(53.8wt%) [28] are adapted from the literature.

Recently, Bingham et al. [99] introduced a theoretical relation between thickness fluctuations
and viscoelastic properties of membranes. Following that prediction Nagao et al. [10] have
included both the elastic and viscous components in the Lorentzian expression as

ΓU ΓJ ; ()
= S+
? S ? |? () + 4|? ;
S
?−?
(6)

Here ; is the area compressibility modulus, is the area per lipid molecule and µ is the local
viscosity of the membrane. The obtained membrane viscosity was found to decrease with
increase in temperature for DMPC, DPPC and DSPC, ranging from 10 to 100 nPa·s [10]. The
most important consequence of equation 6 is the connection of the viscoelastic properties of the
membrane with the bending and thickness fluctuations. Generically speaking, the viscosity of
lipid membranes seem to vary from 1 to 700 nPa·s·m. Nagao report a linear increase with
temperature and the results suggest that values of around 100 nPa·s·m are reached at for
DMPC, DPPC, DSPC [10].

5.4. Lipid Lateral Motions

Lipid lateral motion embraces all time-dependent processes perpendicular to the symmetry axis
of the lipids. These may include independent motion of single lipids or groups of lipids, or
collective dynamics. Hereafter, we consider only motions of those lipids confined to one leaflet
of the bilayer and omit flip-flop processes, which will be discussed in section 6.7. Mode coupling
theory and MD simulations indicate that the lateral motion of the lipid entity (head and tail) is
restricted by cages formed by neighboring lipids [100]. In addition, MD simulations predict a
20
sub-diffusive motion of the lipids due to the crowded environment that leads to a non-Gaussian
dynamics [101].

Several experimental techniques permit the measurement of lateral motions at different length-
and time-scales. Busch et al. used quasielastic neutron scattering (QENS) to access the fast-
localized motion of h-DMPC lipid molecules at time-scale from few ps to few ns [29]. The
authors conclude that the entire lipid molecules undergo localized diffusive motions along with
its neighbors [29, 102]. In agreement with MD simulations they found the lipid molecules form a
dynamically assembling patch [29, 102]. A more detailed study of the lateral diffusion was
conducted by Sharma et al. on h-DMPC [52, 103, 104] using QENS. They authors used a
separation ansatz to distinguish the lateral from the faster internal motion. It was concluded that
&~•' is essentially determined by three contributions, the change in free volume, €•‚ , direct
obstructions, €ƒ„ ' , and interactions, €…†' , between the additives and lipids [103]. Recently, more
details have been obtained extracting the mean square displacement by a procedure established
earlier. It shows a certain motion of the tails, which can be considered as a trapped motion of the
tails [105-107].

A very successful tool to measure lateral diffusion is fluorescence recovery after photo bleaching
(FRAP). Fluorescent molecules are embedded in the bilayer and bleached in one region. By
recovery of the fluorescence intensity, Brownian motion of the fluorescent molecules (lateral
diffusion) is quantified [108]. Under such circumstances, microscopes can be used to track the
motion of organic fluorophores, typically on a time- and length-scale of ms to s and mm-µm,
respectively. Typical diffusion coefficients of around 0.67 – 1.62 Å2ns-1 are obtained [109],
values which are well compatible with those obtained by other techniques, such as QENS, PFG
NMR and RET, cf. table 2. PFG-NMR has been extensively used to explore lateral diffusion
[110]. Most of the studies concentrate on oriented bilayers, powders or hydrated multilamellar
structure, which are omitted here. Astonishingly only a few attempts target the lateral diffusion
of lipids in spherical liposomes ins the liquid, Lu , phase.

The effect of additives like cholesterol, myristic acid, farnesol, and sodium glycocholate on the
lateral dynamics of h-DMPC was studied using QENS [111] [104]. While a decrease of the lipid

21
lateral mobility of h-DMPC with increasing cholesterol content was observed, the other additives
did not significantly impact the mobility of lipids [104] [111]. It was concluded that cholesterol
affects the packing density of the tail regime causing a decrease of free volume, and thereby
lowering the overall in-plane lipid mobility [111]. Observations of enhanced membrane rigidities
(table 1) may also be associated with a closer packing of lipids.

A systematic study of the impact of the addition of antimicrobial peptides alamethicin and
melittin on the dynamics of h-DMPC by QENS in the fluidic phase discovered a reduced
lateral diffusion and a stiffening of the membrane, with melittin being a stronger stiffening agent
than alamethicin [103]. For comparison, at T = 6.85 °C and 19.85 °C, both are in the gel
phase, there was no change in the lateral diffusion on addition of alamethicin, however there is
an increase in &~•' on addition of melittin (table 2). The addition of cholesterol decreases the
mobility of h-DMPC [111] and lowers the binding affinity of melittin to the bilayer, but also
saturates the liposomes that prohibits further incorporation of melittin into the bilayer [104]. In
this case, &~•' is not affected by the addition of melittin as observed in table 2. On the other
hand, addition of drugs like, aspirin causes a slight increase in &~•' both in and phases in
h-DMPC due to the plasticizing effect of aspirin [52], causing a decrease in the membrane
rigidity as observed before (table 1).

Table 2: Comparison between values of lateral diffusion coefficient, &~•' , from different
techniques, for the fluid and gel phases.
Phospholipids in water (D2O for QENS) T (° C) Lipid Phase ‡ˆ‰Š (Å2ns-1) Methods

22
 6.85 0.7 ± 0.1 [103] QENS

100 mM h-DMPC 19.85 1.4 ± 0.1 [103] QENS

36.85 5.0 ± 0.2 [103] QENS

6.85 0.7 ± 0.1 [103] QENS

100 mM h-DMPC, 0.5 mol% alamethicin 19.85 1.3 ± 0.1 [103] QENS

36.85 4.9 ± 0.1 [103] QENS

6.85 1.2 ± 0.1 [103] QENS

100 mM h-DMPC, 0.5 mol% melittin 19.85 1.9 ± 0.1 [103] QENS

36.85 3.3 ± 0.1 [103] QENS

19.85 1.5 ± 0.1 [52] QENS

7wt% h-DMPC
36.85 6.0 ± 0.1 [52] QENS

19.85 2.5 ± 0.1 [52] QENS

7wt% h-DMPC, 6.5 wt% asprin
36.85 6.3 ± 0.1 [52] QENS

19.85 0.7 ± 0.1 [104] QENS

74 mM h-DMPC
36.85 7.7 ± 0.3 [104] QENS

19.85 1.1 ± 0.1 [104] QENS

74 mM h-DMPC, 0.2 mol% melittin
36.85 2.4 ± 0.1 [104] QENS

19.85 0.7 ± 0.1 [104] QENS

74 mM h-DMPC, 20 mol% cholesterol
36.85 2.8 ± 0.1 [104] QENS

19.85 0.7 ± 0.1 [104] QENS

74 mM h-DMPC, 0.2 mol% melittin, 20
mol% cholesterol 36.85 2.8 ± 0.1 [104] QENS

12wt% h-POPC in H2O (MLV) 48.85 1.9 ± 0.1 [110] PFG-MAS

BOD diffusion in h-DMPC (MLV) 27 0.67 – 1.62 [109] FRAP

h-DMPG (ULV) in H2O 10 0.04 [112] RET

23
 23 0.11 [112] RET

35 0.23 [112] RET

10 0.31 [112] RET

h-DOPC (ULV) in H2O 23 0.69 [112] RET

35 1.6 [112] RET

3wt% h-DPPC 25 0.68 [113] PFG - NMR

5.5. Lipid rotational motion

The notion lipid rotational motion embraces several possible processes [114]. Considering lipids
as circular cylindrical entities, (i) rotations around their symmetry axes, which is assumed to be
perpendicular to the bilayer plane (axial rotation), can occur. While free lipids could also rotate
around their longitudinal axes, the bilayer restricts this attempt to a confined space (wobbling).
The term anisotropic diffusion embraces axial rotation [5] and wobbling [115]. Lipids are
flexible molecules, in which the (iii) lipid head group and fatty acid tails may show a different
rotational motion from the entity. Furthermore, (iv) tails may rotate (flexing) (lipid hydro-carbon
chain as an entity), but also the rotational isomerization around C-C bonds.

Axial rotation of phospholipids can be measured using several techniques (Dielectric

spectroscopy, ESR and NMR) [116] [5]. Roberts and Redfield identified molecular rotation and
wobbling by 31P Field Cycling NMR experiments [115]. A more recent study of Klauda et al.
shows how the 31P field cycling NMR data agrees with molecular dynamic simulations while the
dynamics are described by a rigid-body model [117]. In fluid phase, cumulative effect of the
rotational isomerism of C-C bonds in the fatty acid chains gives rise to a shorter length of the
fatty acid chain and as well as more disordered structure. These individual rotations around C-C
bonds are in fact crucial in obtaining the fluid phase of bio membranes.

5.6. Lipid flip-flop motion

The notions flip-flop or transverse diffusion of lipids refer to the change of lipids from one to the
other bilayer. Many biological membranes maintain distinct asymmetry between the inner and

24
outer monolayers of the bilayer in terms of lipid composition. This could only be explained by a
transverse movement of lipids although it seems energetically unfavorable [118]. In cells, this
movement is controlled enzymatically by energy driven or independent flippases (inward
movement) and floppases (outward movement) [119].

A direct method of measuring the flip-flop motion in fluid phase vesicles is by using Electron
Paramagnetic Resonance (EPR) [120]. Fluorescence spectroscopy has shown detergents can
accelerate flip dynamics in fluorescence labelled erythrocytes [121]. However, it has been
observed that using fluorescent or spin-labelled lipids do not accurately represent the native lipid
flip-flop dynamics [122]. Therefore, it is important to explore techniques that obtain flip-flop
dynamics without or minimum perturbation to the lipid bilayer. Recently, Marquardt et.al. have
studied translocation rates in DPPC vesicles in both gel and fluid phases under various
temperatures using 1H NMR coupled with a paramagnetic shift reagent where they found that in
the gel phase translocation is much slower compared to fluid phase [123]. Molecular dynamics
simulations have provided important insights on flip-flop dynamics in relationship to water
permeation, acyl chain length and unsaturation, integral membrane proteins and the presence of
cholesterol [124] [125-127]. Time–resolved SANS on DMPC, POPC and POPA vesicles have
shown that acyl chain length and saturation change the flip-flop motion rates. Moreover, it has
also shown cholesterol entirely inhibits flip-flop in DMPC vesicles [128]. SANS have also been
employed in showing that methanol can increase the rate of flip-flop in DMPC LUVs in fluid
phase [129].

6. Future perspectives

This review summarizes the dynamics of liposomes. A variety of processes can be envisioned. In
many cases, differences in time- and/or length-scale can help to distinguish them from each
other. In order to understand how the molecular mechanisms may contribute to the membrane
properties at least 12 orders of magnitude need to be captured to track the dynamics from the
motion of single elements (~ 0.9 nm) to the translational diffusion of liposomes (~ 20 nm to
several μm for LUVs). Numerous experimental and simulation techniques are necessary to
capture such a broad time- and length scale range.

25
We have discussed in detail the rich morphology of the liposomes and the different dynamics of
the liposomes over a broad length and time scale. In order to understand the dynamics at
different time scales one needs to consider specific techniques. We have shown that scattering
experiments like SANS and SAXS are vital to understand the structure and morphology of the
liposomes, whereas, neutron spectroscopic techniques like NSE and QENS are vital to
understand the membrane fluctuations (undulation), thickness fluctuations and lipid lateral
motions in nano to picosecond time scale. The length scale dependent dynamics measured from
different scattering techniques are noteworthy to validate different scaling behavior from theory
and simulations. Other techniques like, DLS and PFG-NMR provides us valuable information
about the center of mass diffusion of the liposome in millisecond time scale. The lipid rotational
motion was successfully perturbed using Dielectric spectroscopy, ESR and NMR. The slower
flip-flop motion of the lipid can be best measured using EPR techniques. Understanding the
dynamics of lipid bilayer is gaining more importance in order to tune the viscoelastic properties
of the lipid bilayer. In this context a joint study of NSE and QENS is very important.

7. Acknowledgments

The neutron scattering work is supported by the U.S. Department of Energy (DOE) under
EPSCoR Grant No. DE-SC0012432 with additional support from the Louisiana Board of
Regents. This paper was prepared as an account of work sponsored by an agency of the United
States Government. Neither the United States Government nor any agency thereof, nor any of
their employees, makes any warranty, express or implied, or assumes any legal liability or
responsibility for the accuracy, completeness, or usefulness of any information, apparatus,
product, or process disclosed, or represents that its use would not infringe privately owned
rights. Reference herein to any specific commercial product, process, or service by trade name,
trademark, manufacturer, or otherwise does not necessarily constitute or imply its endorsement,
recommendation, or favoring by the United States Government or any agency thereof. The
views and opinions of authors expressed herein do not necessarily state or reflect those of the
United States Government or any agency thereof.

26
References

• of special interest.
•• of outstanding interest.

[1] Bonifacino JS, Glick BS. The Mechanisms of Vesicle Budding and Fusion. Cell. 2004;116:153-
66.
[2] Storck EM, Ozbalci C, Eggert US. Lipid Cell Biology: A Focus on Lipids in Cell Division. Annu
Rev Biochem. 2018;87:839-69.
[3] McCusker D, Kellogg DR. Plasma membrane growth during the cell cycle: unsolved mysteries
and recent progress. Curr Opin Cell Biol. 2012;24:845-51.
[4] Bernsdorff C, Wolf A, Winter R, Gratton E. Effect of hydrostatic pressure on water
penetration and rotational dynamics in phospholipid-cholesterol bilayers. Biophysical Journal.
1997;72:1264-77.
[5] Aguilar LF, Pino JA, Soto-Arriaza MA, Cuevas FJ, Sánchez S, Sotomayor CP. Differential
Dynamic and Structural Behavior of Lipid-Cholesterol Domains in Model Membranes. PLOS
ONE. 2012;7:e40254.
[6] Menon AK, Herrmann A. Lipid Flip-Flop. In: Roberts GCK, editor. Encyclopedia of Biophysics.
Berlin, Heidelberg: Springer Berlin Heidelberg; 2013. p. 1261-4.
[7] Andaloussi SE, Mager I, Breakefield XO, Wood MJ. Extracellular vesicles: biology and
emerging therapeutic opportunities. Nat Rev Drug Discov. 2013;12:347-57.
[8] Nikoleli GP, Nikolelis DP, Siontorou CG, Karapetis S, Nikolelis MT. Application of Biosensors
Based on Lipid Membranes for the Rapid Detection of Toxins. Biosensors (Basel). 2018;8.
[9] Lipowsky R, Sackmann E. Structure and Dynamics of Membranes, From Cells to Vesicles. 1st
ed: North Holland; 1995.
•• [10] Nagao M, Kelley EG, Ashkar R, Bradbury R, Butler PD. Probing Elastic and Viscous
Properties of Phospholipid Bilayers Using Neutron Spin Echo Spectroscopy. J Phys Chem Lett.
2017:4679-84. The authors for the first time calculated membrane viscosity from NSE.
[11] Katsaras J, Gutberlet T. Lipid Bilayers: Structure and Interactions. Berlin, Heidelberg,:
Springer-Verlag; 2001.
•• [12] Helfrich W. Elastic Properties of Lipid Bilayers: Theory and Possible Experiments. Z
Naturforsch. 1973;28c:693-703. The authors for the first time calculated the curvature energy
for membranes.
•• [13] Seifert U, Berndl K, Lipowsky R. Shape transformations of vesicles: Phase diagram for
spontaneous- curvature and bilayer-coupling models. Physical Review A. 1991;44:1182-202.
The authors for the first time theoretically proposed different morphologies of vesicles.
• [14] Julicher F, Lipowsky R. Domain-induced budding of vesicles. Phys Rev Lett. 1993;70:2964-
7. The authors identified phase separation within the fluid membrane can lead to domain
induced budding.
[15] Seifert U. Curvature-induced lateral phase segregation in two-component vesicles. Phys
Rev Lett. 1993;70:1335-8.
[16] Käs J, Sackmann E. Shape transitions and shape stability of giant phospholipid vesicles in
pure water induced by area-to-volume changes. Biophysical Journal. 1991;60:825-44.

27
[17] Käs J, Sackmann E, Podgornik R, Svetina S, Žekš B. Thermally induced budding of
phospholipid vesicles — a discontinuous process. Journal de Physique II. 1993;3:631-45.
[18] Döbereiner HG, Käs J, Noppl D, Sprenger I, Sackmann E. Budding and fission of vesicles.
Biophysical Journal. 1993;65:1396-403.
[19] Nagle JF. Introductory Lecture: Basic quantities in model biomembranes. Faraday Discuss.
2013;161:11-29.
[20] Nagle JF, Tristram-Nagle S. Structure of lipid bilayers. Biochimica et Biophysica Acta (BBA) -
Reviews on Biomembranes. 2000;1469:159-95.
[21] Frielinghaus H. Small-angle scattering model for multilamellar vesicles. Phys Rev E Stat
Nonlin Soft Matter Phys. 2007;76:051603.
•• [22] Wiener MC, White SH. Structure of a fluid dioleoylphosphatidylcholine bilayer
determined by joint refinement of x-ray and neutron diffraction data. III. Complete structure.
Biophysical Journal. 1992;61:434-47. First joint refinement of X-ray and neutron diffraction data
to understand the distribution of molecules in the lipid bilayer.
[23] Ananthapadmanabhan KP, Goddard ED, Turro NJ, Kuo PL. Fluorescence Probes for Critical
Micelle Concentration. Langmuir. 1985;1:352-5.
[24] Marsh D. Handbook of Lipid Bilayers 2nd ed: CRC Press; 2013.
[25] Dufourc EJ. Sterols and membrane dynamics. Journal of Chemical Biology. 2008;1:63-77.
[26] Berndl K, Käs J, Lipowsky R, Sackmann E, Seifert U. Shape Transformations of Giant
Vesicles: Extreme Sensitivity to Bilayer Asymmetry. Europhysics Letters (EPL). 1990;13:659-64.
•• [27] Woodka AC, Butler PD, Porcar L, Farago B, Nagao M. Lipid bilayers and membrane
dynamics: insight into thickness fluctuations. Phys Rev Lett. 2012;109:058102. The authors for
the first time discovered thickness fluctuations in lipid bilayers.
[28] Ashkar R, Nagao M, Butler PD, Woodka AC, Sen MK, Koga T. Tuning membrane thickness
fluctuations in model lipid bilayers. Biophys J. 2015;109:106-12.
•• [29] Busch S, Smuda C, Pardo LC, Unruh T. Molecular mechanism of long-range diffusion in
phospholipid membranes studied by quasielastic neutron scattering. J Am Chem Soc.
2010;132:3232-3. The authors for the first time identified lateral motions from QENS data.
[30] Singer SJ, Nicolson GL. The Fluid Mosaic Model of the Structure of Cell Membranes.
Science. 1972;175:720.
[31] Blume A. Properties of lipid vesicles: FT-IR spectroscopy and fluorescence probe studies.
Current Opinion in Colloid & Interface Science. 1996;1:64-77.
[32] Bloom M, Evans E. Observation of Surface Undulations on the Mesoscopic Length Scale by
NMR. In: Peliti L, editor. Biologically Inspired Physics. Boston, MA: Springer US; 1991. p. 137-47.
[33] Bloom M, Evans E, Mouritsen OG. Physical properties of the fluid lipid-bilayer component
of cell membranes: a perspective. Quarterly Reviews of Biophysics. 2009;24:293-397.
[34] Wanderlingh U, D’Angelo G, Branca C, Conti Nibali V, Trimarchi A, Rifici S, et al. Multi-
component modeling of quasielastic neutron scattering from phospholipid membranes. The
Journal of Chemical Physics. 2014;140:174901.
[35] Frey L, Hiller S, Riek R, Bibow S. Lipid- and Cholesterol-Mediated Time-Scale-Specific
Modulation of the Outer Membrane Protein X Dynamics in Lipid Bilayers. Journal of the
American Chemical Society. 2018;140:15402-11.
[36] Lenaz G. Lipid fluidity and membrane protein dynamics. Bioscience Reports. 1987;7:823.

28
[37] Klauda JB, Roberts MF, Redfield AG, Brooks BR, Pastor RW. Rotation of Lipids in
Membranes: Molecular Dynamics Simulation, ³¹P Spin-Lattice Relaxation, and
Rigid-Body Dynamics. Biophysical Journal. 2008;94:3074-83.
[38] Bouvrais H, Duelund L, Ipsen JH. Buffers Affect the Bending Rigidity of Model Lipid
Membranes. Langmuir. 2014;30:13-6.
[39] Andar AU, Hood RR, Vreeland WN, Devoe DL, Swaan PW. Microfluidic preparation of
liposomes to determine particle size influence on cellular uptake mechanisms. Pharm Res.
2014;31:401-13.
[40] Batchelor GK. Diffusion in a dilute polydisperse system of interacting spheres. Journal of
Fluid Mechanics. 2006;131.
[41] Cichocki B, Felderhof BU. Diffusion of Brownian particles with hydrodynamic interaction
and hard core repulsion. The Journal of Chemical Physics. 1991;94:556-62.
[42] Gupta S, Stellbrink J, Zaccarelli E, Likos CN, Camargo M, Holmqvist P, et al. Validity of the
Stokes-Einstein Relation in Soft Colloids up to the Glass Transition. Phys Rev Lett.
2015;115:128302.
[43] Odeh F, Heldt N, Gauger M, Slack G, Li Y. PFG-NMR Investigation of Liposome Systems
Containing Hydrotrope. Journal of Dispersion Science and Technology. 2006;27:665-9.
[44] Leal C, Rögnvaldsson S, Fossheim S, Nilssen EA, Topgaard D. Dynamic and structural aspects
of PEGylated liposomes monitored by NMR. Journal of Colloid and Interface Science.
2008;325:485-93.
[45] Lipowsky R. The conformation of membranes. Nature. 1991;349:475.
[46] Safinya CR, Sirota EB, Roux D, Smith GS. Universality in interacting membranes: The effect
of cosurfactants on the interfacial rigidity. Phys Rev Lett. 1989;62:1134-7.
[47] Evan Evans, David Needham. Physical properties of surfactant bilayer membranes: thermal
transitions, elasticity, rigidity, cohesion and colloidal interactions. Journal of Physical Chemistry.
1987;91:4219-28.
• [48] Hoffmann I, Michel R, Sharp M, Holderer O, Appavou MS, Polzer F, et al. Softening of
phospholipid membranes by the adhesion of silica nanoparticles--as seen by neutron spin-echo
(NSE). Nanoscale. 2014;6:6945-52. The authors observed reduction in membrane rigidity on
adhesion of silica nanoparticle by NSE.
•• [49] Nickels JD, Cheng X, Mostofian B, Stanley C, Lindner B, Heberle FA, et al. Mechanical
Properties of Nanoscopic Lipid Domains. J Am Chem Soc. 2015;137:15772-80. The authors for
the first time identified that the rigidity of the lipid rafts are different than the sarrounding
lipids using contrast variantion NSE.
[50] Bradbury R, Nagao M. Effect of charge on the mechanical properties of surfactant bilayers.
Soft Matter. 2016;12:9383-90.
• [51] Nagao M, Chawang S, Hawa T. Interlayer distance dependence of thickness fluctuations in
a swollen lamellar phase. Soft Matter. 2011;7. The authors for the first time related thickness
fluctuation with bilayer thickness.
[52] Sharma VK, Mamontov E, Ohl M, Tyagi M. Incorporation of aspirin modulates the
dynamical and phase behavior of the phospholipid membrane. Phys Chem Chem Phys.
2017;19:2514-24.

29
•• [53] Lee JH, Choi SM, Doe C, Faraone A, Pincus PA, Kline SR. Thermal fluctuation and elasticity
of lipid vesicles interacting with pore-forming peptides. Phys Rev Lett. 2010;105:038101. First
systematic observation of pore formations and the corresponding rigidity in lipid bilayer.
• [54] Sreij R, Dargel C, Geisler P, Hertle Y, Radulescu A, Pasini S, et al. DMPC vesicle structure
and dynamics in the presence of low amounts of the saponin aescin. Phys Chem Chem Phys.
2018;20:9070-83. For the first time identifies a decrease in κŒ in the L• phase, whereas an
increase in κŒ in the Lu phase, with increasing aescin content was reported.
[55] Holler S, Moreno AJ, Zamponi M, Bačová P, Willner L, Iatrou H, et al. The Role of the
Functionality in the Branch Point Motion in Symmetric Star Polymers: A Combined Study by
Simulations and Neutron Spin Echo Spectroscopy. Macromolecules. 2017;51:242-53.
•• [56] Zilman AG, Granek R. Undulations and Dynamic Structure Factor of Membranes. Phys
Rev Lett. 1996;77:4788-91. First theoretical prediction of membrane undulation.
[57] Watson MC, Brown FL. Interpreting membrane scattering experiments at the mesoscale:
the contribution of dissipation within the bilayer. Biophys J. 2010;98:L9-L11.
• [58] Gupta S, De Mel JU, Perera RM, Zolnierczuk P, Bleuel M, Faraone A, et al. Dynamics of
Phospholipid Membranes beyond Thermal Undulations. J Phys Chem Lett. 2018;9:2956-60. First
time observation of localized dynamics within NSE time window.
[59] Kandil ME, Harris KR, Goodwin ARH, Hsu K, Marsh KN. Measurement of the Viscosity and
Density of a Reference Fluid, with Nominal Viscosity atT= 298 K andp= 0.1 MPa of 29 mPa·s, at
Temperatures between (273 and 423) K and Pressures below 275 MPa. Journal of Chemical &
Engineering Data. 2006;51:2185-96.
[60] Takeda T, Kawabata Y, Seto H, Komura S, Ghosh SK, Nagao M, et al. Neutron spin–echo
investigations of membrane undulations in complex fluids involving amphiphiles. Journal of
Physics and Chemistry of Solids. 1999;60:1375-7.
[61] Hoffmann I, Hoffmann C, Farago B, Prevost S, Gradzielski M. Dynamics of small unilamellar
vesicles. J Chem Phys. 2018;148:104901.
[62] Milner ST, Safran SA. Dynamical fluctuations of droplet microemulsions and vesicles.
Physical Review A. 1987;36:4371.
[63] Brodeck M, Maccarrone S, Saha D, Willner L, Allgaier J, Mangiapia G, et al. Asymmetric
polymers in bicontinuous microemulsions and their accretion to the bending of the membrane.
Colloid and Polymer Science. 2014;293:1253-65.
[64] Lipfert F, Holderer O, Frielinghaus H, Appavou MS, Do C, Ohl M, et al. Long wavelength
undulations dominate dynamics in large surfactant membrane patches. Nanoscale.
2015;7:2578-86.
[65] Monkenbusch M, Holderer O, Frielinghaus H, Byelov D, Allgaier J, Richter D. Bending
moduli of microemulsions; comparison of results from small angle neutron scattering and
neutron spin-echo spectroscopy. Journal of Physics: Condensed Matter. 2005;17:S2903-S9.
[66] Ahmadpoor F, Sharma P. Thermal fluctuations of vesicles and nonlinear curvature
elasticity--implications for size-dependent renormalized bending rigidity and vesicle size
distribution. Soft Matter. 2016;12:2523-36.
• [67] Boggara MB, Faraone A, Krishnamoorti R. Effect of pH and Ibuprofen on the Phospholipid
Bilayer Bending Modulus. The Journal of Physical Chemistry B. 2010;114:8061-6. First
observation of the effect of pH and ibuprofen on the bending rigidity.

30
[68] Mell M, Moleiro LH, Hertle Y, Fouquet P, Schweins R, Lopez-Montero I, et al. Bending
stiffness of biological membranes: what can be measured by neutron spin echo? Eur Phys J E
Soft Matter. 2013;36:75.
[69] Arriaga LR, Lopez-Montero I, Orts-Gil G, Farago B, Hellweg T, Monroy F. Fluctuation
dynamics of spherical vesicles: frustration of regular bulk dissipation into subdiffusive
relaxation. Phys Rev E Stat Nonlin Soft Matter Phys. 2009;80:031908.
[70] H.P. Duwe, J. Kaes, E. Sackmann. Bending elastic moduli of lipid bilayers : modulation by
solutes. Journal de Physique France. 1990;51:945-61.
[71] M. Mutz, W. Helfrich. Bending rigidities of some biological model membranes as obtained
from the Fourier analysis of contour sections. Journal de Physique France. 1990;51:991-1001.
[72] Evans E, Rawicz W. Entropy-driven tension and bending elasticity in condensed-fluid
membranes. Phys Rev Lett. 1990;64:2094-7.
[73] Rawicz W, Olbrich KC, McIntosh T, Needham D, Evans E. Effect of Chain Length and
Unsaturation on Elasticity of Lipid Bilayers. Biophysical Journal. 2000;79:328-39.
[74] Niggemann G, Kummrow M, Helfrich W. The Bending Rigidity of Phosphatidylcholine
Bilayers: Dependences on Experimental Method, Sample Cell Sealing and Temperature. Journal
de Physique II France. 1995;5:413-25.
[75] Kummrow M, Helfrich W. Deformation of giant lipid vesicles by electric fields. Physical
Review A. 1991;44:8356-60.
• [76] Arriaga LR, Lopez-Montero I, Monroy F, Orts-Gil G, Farago B, Hellweg T. Stiffening effect
of cholesterol on disordered lipid phases: a combined neutron spin echo + dynamic light
scattering analysis of the bending elasticity of large unilamellar vesicles. Biophys J.
2009;96:3629-37. First observation of stiffening effect of cholesterol in the Lu phase of lipid
bilayer.
[77] Ole G. Mouritsen, Kent Jørgensen. Dynamical order and disorder in lipid bilayers. Chemistry
and Physics of Lipids. 1994;73:3-25.
[78] Presti FT. The role of cholesterol in membrane fluidity. In Membrane Fluidity in Biology.
New York: Academic Press; 1985.
[79] Alsop RJ, Maria Schober R, Rheinstadter MC. Swelling of phospholipid membranes by
divalent metal ions depends on the location of the ions in the bilayers. Soft Matter.
2016;12:6737-48.
[80] Biltonena RL, Lichtenberg D. The use of differential scanning calorimetry as a tool to
characterize liposome preparations. Chemistry and Physics of Lipids. 1993;64:129.
[81] Lewis RNAH, Mak N, McElhaney RN. A differential scanning calorimetric study of the
thermotropic phase behavior of model membranes composed of phosphatidylcholines
containing linear saturated fatty acyl chains. Biochemistry. 1987;26:6118–26.
[82] Hernandez-Borrell J, Keough KMW. Heteroacid phosphatidylcholines with different
amounts of unsaturation respond differently to cholesterol. Biochimica et Biophysica Acta
(BBA) - Biomembranes. 1993;1153:277-82.
[83] Ulrich AS, Sami M, Watts A. Hydration of DOPC bilayers by differential scanning
calorimetry. Biochimica et Biophysica Acta (BBA) - Biomembranes. 1994;1191:225-30.
[84] Keough KMW. Modifications of lipid structure and their influence on mesomorphism in
model membranes: the influence of hydrocarbon chains. Biochemistry and Cell Biology.
1986;64:44-9.

31
[85] New RRC. Liposomes : a practical approach. Oxford ; New York: Oxford University Press;
1990.
[86] Graham Shipley G, Green JP, Nichols BW. The phase behavior of monogalactosyl,
digalactosyl, and sulphoquinovosyl diglycerides. Biochimica et Biophysica Acta (BBA) -
Biomembranes. 1973;311:531-44.
[87] Silvius JR. Thermotropic Phase Transitions of Pure Lipids in Model Membranes and Their
Modifications by Membrane Proteins. New York: John Wiley & Sons; 1982.
[88] Bach D, Miller IR. Glyceryl monooleate black lipid membranes obtained from squalene
solutions. Biophysical Journal. 1980;29:183-7.
[89] Hladky SB, Gruen DW. Thickness fluctuations in black lipid membranes. Biophysical Journal.
1982;38:251-8.
[90] Miller IR. Energetics of fluctuation in lipid bilayer thickness. Biophysical Journal.
1984;45:643-4.
[91] Jacob N. Israelachvili, Wennerstroem H. Entropic forces between amphiphilic surfaces in
liquids. The Journal of Physical Chemistry 1992;96:520–31.
[92] Movileanu L, Popescu D, Ion S, Popescu AI. Transbilayer Pores Induced by Thickness
Fluctuations. Bulletin of Mathematical Biology. 2006;68:1231-55.
[93] Bennett WF, Sapay N, Tieleman DP. Atomistic simulations of pore formation and closure in
lipid bilayers. Biophys J. 2014;106:210-9.
[94] Orsia M, Essex JW. Permeability of drugs and hormones through a lipid bilayer: insights
from dual-resolution molecular dynamics. Soft Matter. 2010;6:3797–808.
[95] Henzler-Wildman K, Kern D. Dynamic personalities of proteins. Nature. 2007;450:964-72.
[96] Farago B, Monkenbusch M, Goecking KD, Richter D, Huang JS. Dynamics of Microemulsions
as Seen by Neutron Spin-Echo. Physica B-Condensed Matter. 1995;213:712-7.
[97] Nagao M. Observation of local thickness fluctuations in surfactant membranes using
neutron spin echo. Phys Rev E Stat Nonlin Soft Matter Phys. 2009;80:031606.
[98] Lee V, Hawa T. Investigation of the effect of bilayer membrane structures and fluctuation
amplitudes on SANS/SAXS profile for short membrane wavelength. J Chem Phys.
2013;139:124905.
[99] Bingham RJ, Smye SW, Olmsted PD. Dynamics of an asymmetric bilayer lipid membrane in
a viscous solvent. Europhysics Letters (EPL). 2015;111:18004.
[100] Flenner E, Das J, Rheinstadter MC, Kosztin I. Subdiffusion and lateral diffusion coefficient
of lipid atoms and molecules in phospholipid bilayers. Phys Rev E Stat Nonlin Soft Matter Phys.
2009;79:011907.
[101] Akimoto T, Yamamoto E, Yasuoka K, Hirano Y, Yasui M. Non-Gaussian fluctuations
resulting from power-law trapping in a lipid bilayer. Phys Rev Lett. 2011;107:178103.
[102] Falck E, Rog T, Karttunen M, Vattulainen I. Lateral diffusion in lipid membranes through
collective flows. J Am Chem Soc. 2008;130:44-5.
• [103] Sharma VK, Mamontov E, Tyagi M, Qian S, Rai DK, Urban VS. Dynamical and Phase
Behavior of a Phospholipid Membrane Altered by an Antimicrobial Peptide at Low
Concentration. J Phys Chem Lett. 2016;7:2394-401. The authors proved that the disruption of
lateral motions in the lipid bilayer by QENS, but not the pore formation due to peptides is the
principal interaction.

32
[104] Sharma VK, Mamontov E, Anunciado DB, O'Neill H, Urban VS. Effect of antimicrobial
peptide on the dynamics of phosphocholine membrane: role of cholesterol and physical state
of bilayer. Soft Matter. 2015;11:6755-67.
[105] Gupta S, De Mel JU, Perera RM, Zolnierczuk P, Bleuel M, Faraone A, et al. Dynamics of
Phospholipid Membranes beyond Thermal Undulations. The Journal of Physical Chemistry
Letters. 2018;9:2956-60.
[106] Gerstl C, Schneider GJ, Fuxman A, Zamponi M, Frick B, Seydel T, et al. Quasielastic
Neutron Scattering Study on the Dynamics of Poly(alkylene oxide)s. Macromolecules.
2012;45:4394-405.
[107] Schneider GJ, Nusser K, Neueder S, Brodeck M, Willner L, Farago B, et al. Anomalous chain
diffusion in unentangled model polymer nanocomposites. Soft Matter. 2013;9:4336.
[108] Pincet F, Adrien V, Yang R, Delacotte J, Rothman JE, Urbach W, et al. FRAP to Characterize
Molecular Diffusion and Interaction in Various Membrane Environments. PloS one.
2016;11:e0158457-e.
[109] Johnson ME, Berk DA, Blankschtein D, Golan DE, Jain RK, Langer RS. Lateral diffusion of
small compounds in human stratum corneum and model lipid bilayer systems. Biophysical
Journal. 1996;71:2656-68.
[110] Gaede HC, Gawrisch K. Lateral Diffusion Rates of Lipid, Water, and a Hydrophobic Drug in
a Multilamellar Liposome. Biophysical Journal. 2003;85:1734-40.
[111] Busch S, Unruh T. The influence of additives on the nanoscopic dynamics of the
phospholipid dimyristoylphosphatidylcholine. Biochim Biophys Acta. 2011;1808:199-208.
• [112] Kuśba J, Li L, Gryczynski I, Piszczek G, Johnson M, Lakowicz JR. Lateral Diffusion
Coefficients in Membranes Measured by Resonance Energy Transfer and a New Algorithm for
Diffusion in Two Dimensions. Biophysical Journal. 2002;82:1358-72. Lateral diffusion in
membranes was measured by resonance energy transfer.
[113] Kawaguchi T, Kita R, Shinyashiki N, Yagihara S, Fukuzaki M. The Bi-modality Diffusion of
Water Molecules in Liposome/Water Dispersion Systems Analyzed by Pulsed Field Gradient
Spin Echo NMR Method. Transactions of the Materials Research Society of Japan. 2016;41:359-
62.
[114] Moore PB, Lopez CF, Klein ML. Dynamical Properties of a Hydrated Lipid Bilayer from a
Multinanosecond Molecular Dynamics Simulation. Biophysical Journal. 2001;81:2484-94.
[115] Roberts MF, Redfield AG. High-Resolution 31P Field Cycling NMR as a Probe of
Phospholipid Dynamics. Journal of the American Chemical Society. 2004;126:13765-77.
[116] Essmann U, Berkowitz ML. Dynamical Properties of Phospholipid Bilayers from Computer
Simulation. Biophysical Journal. 1999;76:2081-9.
[117] Klauda JB, Roberts MF, Redfield AG, Brooks BR, Pastor RW. Rotation of Lipids in
Membranes: Molecular Dynamics Simulation, 31P Spin-Lattice Relaxation, and Rigid-Body
Dynamics. Biophysical Journal. 2008;94:3074-83.
[118] Contreras FX, Sánchez-Magraner L, Alonso A, Goñi FM. Transbilayer (flip-flop) lipid motion
and lipid scrambling in membranes. FEBS Letters. 2010;584:1779-86.
[119] Devaux PF, Herrmann A, Ohlwein N, Kozlov MM. How lipid flippases can modulate
membrane structure. Biochimica et Biophysica Acta (BBA) - Biomembranes. 2008;1778:1591-
600.

33
•• [120] McConnell HM, Kornberg RD. Inside-outside transitions of phospholipids in vesicle
membranes. Biochemistry. 1971;10:1111-20. First direct measurement of flip-flop motions in
the Lu phase by EPR.
[121] Pantaler E, Kamp D, Haest CWM. Acceleration of phospholipid flip-flop in the erythrocyte
membrane by detergents differing in polar head group and alkyl chain length. Biochimica et
Biophysica Acta (BBA) - Biomembranes. 2000;1509:397-408.
[122] Allhusen JS, Conboy JC. The Ins and Outs of Lipid Flip-Flop. Accounts of Chemical
Research. 2017;50:58-65.
• [123] Marquardt D, Heberle FA, Miti T, Eicher B, London E, Katsaras J, et al. (1)H NMR Shows
Slow Phospholipid Flip-Flop in Gel and Fluid Bilayers. Langmuir : the ACS journal of surfaces and
colloids. 2017;33:3731-41. Detailed description of the flip-flop motions in the gel and fluid
phase by 1H-NMR.
[124] Inokuchi T, Arai N. Relationship between water permeation and flip-flop motion in a
bilayer membrane. Physical Chemistry Chemical Physics. 2018;20:28155-61.
[125] Ogushi F, Ishitsuka R, Kobayashi T, Sugita Y. Flip-Flop Motions of Lipid Molecules in Mixed
Bilayer Systems. Biophysical Journal. 2010;98:489a.
[126] Sapay N, Bennett WFD, Tieleman DP. Molecular Simulations of Lipid Flip-Flop in the
Presence of Model Transmembrane Helices. Biochemistry. 2010;49:7665-73.
[127] Choubey A, Kalia RK, Malmstadt N, Nakano A, Vashishta P. Cholesterol translocation in a
phospholipid membrane. Biophysical journal. 2013;104:2429-36.
• [128] Nakano M, Fukuda M, Kudo T, Matsuzaki N, Azuma T, Sekine K, et al. Flip-Flop of
Phospholipids in Vesicles: Kinetic Analysis with Time-Resolved Small-Angle Neutron Scattering.
The Journal of Physical Chemistry B. 2009;113:6745-8. First time showed that the acyl chain
length and headgroup size determines the flip-flop motions by time-resolved SANS.
[129] Nguyen MHL, DiPasquale M, Rickeard BW, Stanley CB, Kelley EG, Marquardt D. Methanol
Accelerates DMPC Flip-Flop and Transfer: A SANS Study on Lipid Dynamics. Biophysical Journal.
2019.

34