BBA - Biomembranes 1866 (2024) 184253

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BBA - Biomembranes
journal homepage: www.elsevier.com/locate/bbamem

A closer look at calcium-induced interactions between
phosphatidylserine-(PS) doped liposomes and the structural effects caused
by inclusion of gangliosides or polyethylene glycol- (PEG) modified lipids
Philipp Grad a, Katarina Edwards a, Lars Gedda a, Víctor Agmo Hernández b, *ab

Department of Chemistry–Ångström, Uppsala University, Box 573, 75123 Uppsala, Sweden
Department of Medicinal Chemistry, Uppsala University, Box 574, 75123 Uppsala, Sweden

A R T I C L E I N F O

A B S T R A C T

Keywords:
Calcium ions
Liposome-liposome fusion
FRET
Cryo-EMPEGylated lipids
Gangliosides
Ion correlation

The effects of polyethylene glycol- (PEG) modified lipids and gangliosides on the Ca2+ induced interaction be­
tween liposomes composed of palmitoyl-oleoyl phosphatidylethanolamine (POPE) and palmitoyl-oleoyl phos­
phatidylserine (POPS) was investigated at physiological ionic strength. Förster resonance energy transfer (FRET)
studies complemented with dynamic light scattering (DLS) and cryo-transmission electron microscopy (Cryo-EM)
show that naked liposomes tend to adhere, rupture, and collapse on each other’s surfaces upon addition of Ca2+,
eventually resulting in the formation of large multilamellar aggregates and bilayer sheets. Noteworthy, thepresence of gangliosides or PEGylated lipids does not prevent the adhesion-rupture process, but leads to theformation of small, long-lived bilayer fragments/disks. PEGylated lipids seem to be more effective than gan­gliosides at stabilizing these structures. Attractive interactions arising from ion correlation are proposed to be a
driving force for the liposome-liposome adhesion and rupture processes. The results suggest that, in contrast withthe conclusions drawn from previous solely FRET-based studies, direct liposome-liposome fusion is not thedominating process triggered by Ca2+ in the systems studied.

1. Introduction
The aggregation and fusion of liposomes induced by Ca2+ has been asubject of study for several decades [1–6]. The phenomenon has beenused to mimic lipid-mediated cell and viral fusion events and explain therole that Ca2+ and other divalent ions play in these (e.g. [3,6]). In
several cases, addition of Ca2+ has also been used in order to facilitate
the formation of supported lipid bilayers (SLBs) via liposome adhesion
and spreading on a variety of surfaces, e.g., mica [7], silica [8],indium‑tin oxide [9], and titanium dioxide [10]. Although anionic(particularly phosphatidylserine- (PS) based) liposomes are considered
to be more sensitive to the presence of Ca2+, addition of the ion has beensuggested to improve and/or trigger fusion events and formation of SLBs
also for liposomes built from zwitterionic lipids alone [11,12].
The molecular mechanism by which Ca2+ facilitates the aggregationand fusion of liposomes has been discussed extensively. In the case of PScontaining liposomes, it is generally agreed that a high affinity of Ca2+
for the PS headgroup leads to liposome aggregation and fusion [3,13]. A
similar, although weaker, affinity has been observed between Ca2+ andother negatively charged (e.g. phosphatidylglycerol, PG) and zwitter­ionic (e.g. phosphatidylcholine, PC) lipid headgroups [14,15], ac­counting thus for the widespread effect of Ca2+. In the case of liposome
rupture and spreading on surfaces, it is generally believed that Ca2+
serves to screen the electrostatic repulsion between the surfaces and theliposomes, as well as to trigger liposome fusion events at the surface,which can facilitate the formation of SLBs [7,10].
In previous studies the most used approach to characterize liposomeliposome fusion (and the role of Ca2+) has been the monitoring of either
the lipid mixing or the intermixing of the aqueous contents [16]. For theformer, fluorescence assays based on Förster resonance energy transfer(FRET) between labeled lipids incorporated in part of the total liposome
population are often used [17], whereas for the latter liposomes filledwith an encapsulated fluorescent dye are mixed with liposomes filledwith an appropriate quencher [18]. Alternatively, the two populationsof liposomes can be filled with complementary reagents that give afluorescence response upon mixing [19]. In all cases, the changes influorescence intensity over time are then related to the degree ofliposome-liposome fusion. It is to be noted, however, that the results* Corresponding author.
E-mail addresses: katarina.edwards@kemi.uu.se (K. Edwards), lars.gedda@kemi.uu.se (L. Gedda), victor.agmo@ilk.uu.se (V. Agmo Hernández).
https://doi.org/10.1016/j.bbamem.2023.184253
Received 7 August 2023; Received in revised form 9 November 2023; Accepted 11 November 2023
Available online 16 November 2023
0005-2736/© 2023 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

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Fig. 1. A: Intensity weighted hydrodynamic radius (RH) distribution of POPE:POPS 75:25 liposomes before (solid line) and 30 min after (dashed line) the addition of
10 mM Ca2+. B: Determined degree of lipid mixing between POPE:POPS 75:25 liposomes as a function of time after Ca2+ addition.

from studies examining lipid mixing do not always agree with the con­clusions from studies based on monitoring the intermixing of theaqueous contents [20], implying that the results from these kind of ex­
periments need to be interpreted with caution.
In this report, the effect of Ca2+ on liposomes composed of palmitoyloleoyl phosphatidylserine (POPS) and phosphatidylethanolamine(POPE) is explored by FRET measurements complemented with dynamic
light scattering (DLS) and cryo- transmission electron microscopy (CryoEM) determinations. A mixture of PS and phosphoethanolamine (PE)lipids was chosen as this mixture is among the most commonly used inprevious FRET-based studies on Ca2+ induced liposome-liposome fusion(e.g. [2,4,5,21]). The results indicate that, in contrast with the conclu­
sions drawn from previous FRET-based studies, liposome-liposome
fusion is only one of two main processes triggered by the presence of
Ca2+, the other one being the adhesion, rupture, and collapse of lipo­
somes on each other.
In the present study we moreover explore how incorporation of lipids
modified with polyethylene glycol (PEGylated lipids) and gangliosides
affect the liposomes’ response to Ca2+. The presence of PEGylated lipids
has been reported to inhibit the fusion process, likely by preventing
close contact between the liposomes [21]. Our results indicate, however,that neither PEG nor gangliosides prevent liposome-liposome aggrega­tion, adhesion nor rupture. Instead, their presence result in the forma­tion of open bilayer structures with good colloidal stability.

All aqueous solutions were prepare using deionized water (18.2 MΩcm) from a Milli-Q plus system from Millipore (Bedford, USA).
2.2. Preparation of liposomes
Liposome components were weighed or pipetted from stock solutions
in chloroform or methanol. A lipid film was obtained by evaporation of
the organic solvent, first with a gentle stream of nitrogen followed byremoving the residual solvent in a vacuum oven (Lab instruments IL,
USA) overnight. The dried lipid film was suspended in HEPES buffer (10mM HEPES, 150 mM NaCl, pH 7.4). The lipid dispersion was thensubjected up to 10 freeze-thawing cycles (freezing with liquid Nitrogenand thawing with a water bath at 50 ◦ C) to ensure complete lipid mixing.
Unilamellar liposomes were then prepared by extruding the sample 31
times through a 100 nm pore size filter from Whatman plc (Kent, UK).
2.3. Dynamic light scattering (DLS)DLS experiments were performed using an ALV/CGS-3 compact
goniometer system and an ALV/LSE-5004 light scattering electronics
and multiple tau digital correlator from ALV-GmbH (Langen, Germany).
The laser beam had a wavelength of 532 nm with vertically polarized
light. All measurements were performed in triplicate for each sample atroom temperature with a run time of 2 min and at least two runs. Dataacquisition and fitting was performed with the ALV-correlator software(version 3.0). The regularized fit model with the (g2(t)) DLS-exponential
was used.

2. Materials and methods
2.1. Materials

2.4. Cryo-transmission electron microscopy (cryo-EM)

Total ganglioside extract (porcine brain, ammonium salt, >99 %),chloroform (pro analysis) and methanol (pro analysis) were purchasedfrom Merck KGaA (Darmstadt, Germany). 1,2-distearoyl-sn-glycero-3phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPEPEG2000, 98 %) ammonium salt was obtained as a kind gift from Lipoid
GmbH (Ludwigshafen, Germany). 1-palmitoyl-2-oleoyl-sn-glycero-3phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phos­
pho-L-serine (POPS), 1,2-distearoyl-sn-glycero-3-phosphoethanolamineN-[methoxy(polyethylene glycol)-750] (DSPE-PEG750), 1,2-distearoylsn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)1000] (DSPE-PEG1000) were bought from Avanti Polar Lipids(Alabaster, USA). Lissamine™ Rhodamine B 1,2-Dihexadecanoyl-snGlycero-3-Phosphoethanolamine (rhodamine-PE) and N-(7-Nitrobenz2-Oxa-1,3-Diazol-4-yl)-1,2-Dihexadecanoyl-sn-Glycero-3-Phosphoetha­
nolamine (NBD-PE) were purchased from Thermo Fischer Scientific.
4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, >99.5%), calcium chloride, and sodium chloride were purchased from Sigma
Aldrich (St. Louis, Missouri, US).

Specimens for cryo-EM investigations were prepared at controlledtemperature (25 ◦ C) and humidity (>90 %) within a custom-built
environmental chamber. A small (~1 μL) drop of sample was placed
onto a copper grid (300 mesh, Agar scientific) coated with a custommade holey polymer (cellulose acetate butyrate) film, and excess
liquid was blotted away with a filter paper. The sample was then vitri­fied by plunging the grid into liquid ethane held at a temperature justabove its freezing point. Thereafter the grid was mounted in a Gatan
CT3500 sample holder and transferred to a Zeiss TEM Libra 120 trans­mission electron microscope (Carl Zeiss AG, Oberkochen, Germany) forviewing. The sample was kept at a temperature below − 160 ◦ C during
the transfer and viewing processes. The microscope was operating at 80
kV and the observations were made in zero-loss bright-field mode.
Digital images were recorded under low dose conditions with a Bio­
Vision Pro-SM slow scan CCD camera from Proscan elektronische Sys­
teme GmbH (Scheurig, Germany). For the samples treated with Ca2+, 1
μL of 1 M CaCl2 was added to 100 μL of the liposome dispersion and2

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Fig. 3. Schematic representation of the structures formed after addition of Ca2+
to naked POPE:POPS (75:25) liposomes. Lipid mixing between ruptured (a) andcollapsed (b) vesicles is observed. The open structures on either side canaggregate with similar structures forming open bilayer sheets or large vesicles,in agreement with the Cryo-EM images.

mixed before applied on the grid and blotted. The total time between
mixing and plunging into ethane is the one used in results.
2.5. Lipid mixing assay
Lipid mixing was followed using the Förster resonance energy
transfer assay developed by Struck et al. [17] as described in Duzgunes
et al. [16]. Briefly, liposomes containing both acceptor, rhodamine-PE,
and donor, NBD-PE, fluorescent lipids with a proportion of 0.8 mol%each (labeled liposomes) were mixed with liposomes devoid of fluo­
rescent lipids (unlabeled liposomes) at a ratio of 1:9 (labeled:unlabeled)
to a total concentration of 50 μM lipid. Calcium chloride was then addedfrom a stock solution to a final concentration of 10 mM. Upon lipid
mixing, the average separation between donor and acceptor molecules
increases, resulting in a decrease in the energy transfer. The fluorescencesignal from the donor thus increases. The measurements were performed
at a temperature of 25◦ with constant stirring set to 500 rpm in a quartz
cuvette (Quartz SUPRASIL ®, Hellma Analytics, Mühlheim, Germany).
The fluorescence signal from the donor molecule (NBD-PE lipids) wasmonitored with a Fluorolog®-3 from Horiba (Kyōto, Japan) in the rightangle mode. The fluorescence emission intensity was measured at 530
nm with excitation at 460 nm [22,23].
The maximal fluorescence (I(max)) was determined by measuring
the fluorescence intensity of a dispersion containing 50 μM of calibra­tion liposomes, i.e. liposomes containing 0.08 mol% of NBD-PE andrhodamine-PE, mimicking thus the composition expected at 100 % lipid
mixing. The degree of lipid mixing over time (M(t)) was calculated by
the following equation [22,23]:

Fig. 2. Cryo-EM micrographs of liposomes composed of POPE:POPS with amolar composition of 75:25. A) Liposomes dispersed in buffer; B) In the pres­ence of 10 mM Ca2+ after 30 s (inset, same picture at lower magnification,
showing the formation of large bilayer sheets); C) In the presence of 10 mM
Ca2+ after 20 min. Scale bar = 100 nm. Note that the size of the scale bar is
unique for each image.

M(t) =

I(t) − I(0)I(max) − I(0)where I(t) is the fluorescence intensity at time t and I(0) is the3

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Fig. 4. A1-A3: Intensity weighted hydrodynamic radius (RH) distribution of POPE:POPS liposomes modified with 4 mol% (final molar composition POPE:POPS:
DSPE-PEG 71:25:4). A1) DSPE-PEG750, A2) DSPE-PEG1000 and A3) DSPE-PEG2000 liposomes before (solid line) and 30 min after (dashed line) the addition of
10 mM Ca2+. B1-B3: Calculated degree of lipid mixing for the corresponding PEGylated liposomes as a function of time after Ca2+ addition.

fluorescence intensity before the addition of CaCl2. Lipid mixing results
in an increased fluorescence intensity as the fluorescent lipids in thelabeled vesicles are diluted upon mixing with lipids in the unlabeledliposomes [16,17,22,23].

spherical and unilamellar (Fig. 2A). Upon addition of CaCl2 to a con­centration of 10 mM, the original population of liposomes disappears
and is replaced by two populations with radius ~ 200 nm and ~ 650 nm,
respectively, indicating liposome aggregation and/or fusion. FRET ex­
periments (Fig. 1B) confirm that full mixing of the lipid components isachieved within minutes after the addition of CaCl2. These results are inagreement with previous studies concerning the Ca2+-induced fusion
behavior of PE:PS liposomes [4,5,20]. Addition of extra 40 mM NaCl
instead of 10 mM CaCl2 did not induce any changes in the data retrieved
from DLS measurements (see Fig. S1 in the supplementary material),suggesting that no aggregation or fusion occur even though the ionicstrength was higher than in the case of CaCl2 addition. This observationis also in agreement with the results from previous publications [20],3. Results and discussion
3.1. Naked and PEG-stabilized POPE:POPS liposomes
Liposomes composed of POPE:POPS 75:25 were first characterizedusing DLS. The results shown in Fig. 1A indicate that the sample isdominated by liposomes with a hydrodynamic radius between 30 and
50 nm. Cryo-EM experiments suggest that the liposomes are mostly
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and suggest that the fusion-inducing effect of Ca2+ is not limited to adecrease in the repulsion between the charged lipid headgroups.
Cryo-EM images of the sample 30 s after addition of CaCl2 show thepresence of large aggregates in which closed large liposomes are“coated” and connected to each other by multiple bilayers (Fig. 2B). The
large spacing and general appearance of the latter suggest that they may
represent stacks of collapsed but still intact liposomes. Large open
bilayer sheets are also observed. The formation of these structures
suggests that liposomes do not only fuse with each other, but also
rupture, collapse and spread on each other’s surfaces. This phenomenon
has been observed previously in preparations of bovine brain PS lipo­
somes treated with Ca2+, leading to structures similar to the one
depicted in Fig. 2B [1,2]. The collapse and rupture of liposomes incontact with each other is believed to arise from a strong attractive interliposome interaction leading to a fast and extensive deformation of theliposomes [1,2]. It has been argued, however, that such attractive in­teractions become relevant only when the ionic strength in the medium
is very low (only 2 mM TES in the cited publications) leading to a strong
adsorption of Ca2+ on the liposome surface [3] and facilitating the for­mation of inter-liposomal PS-Ca2+-PS complexes [1]. It has indeed beenshown more recently that the binding of Ca2+ to PS-based membranes[13] is greatly diminished by increasing the ionic strength, with nosignificant adsorption/binding of Ca2+ being observed at NaCl concen­
trations above 100 mM. Since the NaCl concentration in the present caseis 150 mM, the strong attraction leading to liposome rupture cannot be
explained by Ca2+ adsorption and complex formation. An alternative
explanation is that the strong attraction between liposomes in our sys­
tem arises from ion correlation forces [24] caused by the accumulationof Ca2+ in the electric double layer. This potential explanation is dis­cussed in more detail in the following sections.
20 min after the addition of Ca2+ the dispersion consists mainly oflarge clusters formed by multilamellar structures (Fig. 2C), although insome cases large bilayer sheets were also observed (not shown). The factthat complete lipid mixing is observed in the FRET experiments eventhough individual small structures can still be identified in Cryo-EMsuggest that bilayer rearrangements take place in which lipids areexchanged. These rearrangements are likely to be similar to the multi­
layer stacking mechanism proposed by Rand et al. [1] to account for theformation of structures resembling those shown in Fig. 2B and C. The
proposed resulting structures are illustrated in Fig. 3.
Inclusion of 4 mol% PEGylated lipids did not prevent aggregation of
the liposomes, as illustrated by the DLS data obtained with lipids
modified with three different PEG molecular weights (Fig. 4 A1-A3). In
all cases, it is observed that addition of Ca2+ results in the formation of a
population with much larger hydrodynamic radius than the parent li­
posomes. This effect is most obvious for the low molecular weight PEG
chains, and is reduced as the PEG length increases. In spite of aggrega­tion taking place, the presence of PEG with molecular weights 1000 and2000 prevented the mixing of the lipid contents (Fig. 4 B2-B3), inagreement with previous reports [21]. For the shortest PEG-chain
studied (PEG750), mixing of the lipids was still observed (Fig. 4 B1),
although at a drastically reduced rate compared to what was observedwith the naked liposomes.
Very interestingly, cryo-EM analysis of samples containing PEG1000(Fig. 5) showed that the aggregates detected by DLS do not consist of
intact and separate liposomes, but of planar bilayer fragments often
attached to vesicular structures (Fig. 5B). 20 min after exposure to Ca2+,
also some isolated large bilayer fragments can be observed (Fig. 5C),
although they are found only in a few selected areas of the sample. Somestructures similar to hemifused liposomes are also identified in the
pictures (Fig. 5B and C). However, as indicated by the FRET experi­ments, it is likely that these structures consist of two (or more) liposomesthat have attached to each other without mixing of the lipids. The factthat the separation between bilayers at the contact area cannot be
resolved in the images, suggests that PEGylated lipids are excluded fromthis region leading to very close contact. This would result in

Fig. 5. Cryo-EM micrographs of liposomes composed of POPE:POPS:DSPEPEG1000 with a molar composition of 71:25:4. A) Liposomes dispersed inbuffer; B) In the presence of 10 mM Ca2+ after 30 s; C) In the presence of 10 mM
Ca2+ after 20 min. White thin arrows: Two or more liposomes in close contact
with each other. Thick white arrows: Two liposomes in close contact in theprocess of rupturing and opening. Black thin arrows: open bilayer structuresattached to partially open liposomes. Thick black arrows: Open bilayer struc­tures (lipodisks). Scale bar = 100 nm. Note that the size of the scale bar is
unique for each image.
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Fig. 6. Schematic representation of the structures formed after addition of Ca2+ to PEGylated POPE:POPS-based liposomes. PEG is excluded from the contact area
and accumulates at the points of vesicle rupture, stabilizing thus the formed open structures and resulting in the formation of disks.

Fig. 7. A: Intensity weighted hydrodynamic radius (RH) distribution of POPE:POPS:gangliosides 65:25:10 liposomes before (solid line) and 30 min after (dashed line)
the addition of Ca2+. B: Calculated degree of fusion between POPE:POPS:gangliosides 65:25:10 liposomes as a function of time after Ca2+ addition.

accumulation of PEGylated lipids in other parts of the membrane. As thelipid membrane is already close to the PEGylated lipid saturation limit(~5 mol% according to Edwards et al. [25]), this segregation of PEGy­
lated lipids would lead to the formation of open structures stabilized bya PEGylated rim (see Fig. 6), in a similar fashion to the formation of
PEGylated lipodisks described in previous publications [26]. This ex­
plains also why most of the observed adhered liposomes seem to haveruptured upon contact.
In order to remove the PEGylated lipids from the contact area, astrong attractive force between liposomes is required. It is also requiredthat this attractive force has longer range than the steric repulsion
provided by the PEG chains. Leckband et al. [27] have previously shownthat the presence of Ca2+ can lead to attractive long range hydrophobic
interactions between lipid bilayers due to induced membrane phasesegregation. However, in contrast with our observations, such attraction
was suggested to always lead to membrane fusion. Another plausible
explanation for the observed behavior would be that the addition of
Ca2+ results in strong attractive ion correlation forces [24]. Fig. S2 in the
supplementary material shows the estimated strength of Ca2+ induced
ion correlation forces between two spherical particles, with the same
surface charge density as the liposomes used in this study, interacting indeionized water. The calculations are based on the simplified approachpresented by Jönsson and Wennerström [28] complemented by thedescription of the repulsion between coaxial parallel uniformly charged
disks described by Ciftja [29], and lipid structural and interaction pa­
rameters found in the literature [30–32]. Details about the estimations
are found in the supplementary material. It is estimated that, at sepa­
rations down to ~1 nm between the surfaces, the strength of the ion
correlation attraction is enough to overcome both the electric double
layer and the steric repulsions between liposomes (calculated, respec­tively, according to Engström and Wennerström [33] and Li and Pincet[34]). In the high ionic strength media used in our experiments, it isexpected that the osmotic electric double layer repulsion will decreasemuch more than the attractive pressure from ion correlation forces,
given the preferential accumulation of multivalent ions near the lipo­
some surface. A roughly calculated total interaction (fig. S3 in the sup­plementary material) shows that under the experimental conditions a
net attraction (slightly weaker than in the case of deionized water) canbe expected at separations down to ~1 nm. In agreement with our dis­cussion, it has been previously shown experimentally that inclusion of
Ca2+ in the medium induces an attraction between phosphatidylglycerol
membranes at separations of about 3 nm, and this attraction could also
be attributed to ion correlation forces [35].
Even though the above estimations are based on rough6

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approximations, they show that it is indeed plausible that ion correlation
forces can lead to close contact between liposomes. It is likely that thestrong attraction will result in the segregation of PEGylated lipids fromthe area of contact. The cost in terms of mixing entropy when segrega­tion takes place can be well accounted for by the gain in configurational
entropy for the PEG chains if they leave the tight gap between theapproaching surfaces. As mentioned before, this PEG-lipid segregation
can lead to the formation of open structures. Thus, it is likely that theattractive ion correlation forces are the driving force behind the for­mation of the structures observed in Fig. 5B and C. Once open bilayer
structures are formed, the accumulation of PEG on the bilayer edges
would increase the range and strength of the repulsive steric forces,
avoiding thus further aggregation and/or fusion. Open structures haveindeed been shown to be stabilized by very low amounts of PEG lipids(down to 2 mol%) [26].
3.2. Ganglioside stabilized PE:PS liposomes
In order to compare the stabilizing effect of PEG and gangliosides,
the effect of Ca2+ on POPE:POPS liposomes modified with 10 mol%gangliosides was studied. Inclusion of gangliosides slowed, but did notcompletely prevent, the lipid mixing process as illustrated in Fig. 7. DLS
experiments (Fig. 7A) confirm the formation of larger structures after
Ca2+ addition, although structures smaller than the parent liposomes arealso identified. Cryo-EM investigations (Fig. 8) show that during the firstseconds after Ca2+ addition ganglioside modified liposomes behave very
similarly to their PEGylated counterparts, meaning that aggregation
occurs almost immediately but does not lead to fusion (Fig. 8B), and thatgangliosides appear to be excluded from the liposome-liposome contact
area. The adhered liposomes appear quite resilient to rupture, but after
20 min open bilayer patches or fragments are frequently observed(Fig. 8C). These have a relatively small size, and can thus be an expla­
nation for the small particles observed in DLS measurements. Similar
open bilayer structures have previously been shown to form whenmixing phosphocholine lipids with >20 mol% gangliosides [36] and to
consist of a planar lipid bilayer disks stabilized by gangliosides accu­
mulating at the edges. The fact that these structures form with only 10
mol% gangliosides after calcium-induced fusion suggest that ganglio­
sides are segregated from the region where adhesion occurs, leading to
the formation of high curvature patches that eventually become theedges of the open structures, similarly to what is believed to occur with
PEGylated lipids. However, in contrast with the results from liposomes
supplemented with DSPE-PEG1000 and DSPE-PEG2000, lipid mixing is
not completely avoided (Fig. 7B). This is likely a consequence of thestructural transformations resulting in open bilayer structures being
slower than in the case of PEGylated liposomes. Also, it has been shown
previously that gangliosides are not as effective as PEGylated lipids ingenerating open structures [36]. In agreement with the results from the
FRET measurements, some large, partially fused structures were indeed
found in the ganglioside-modified sample (see inset in Fig. 8C).
As in the case of PEGylated liposomes, it is likely that the driving
force causing ganglioside segregation and leading to liposome aggre­
gation, rupture and formation of open bilayer structures are ion corre­
lation forces acting between liposomes. The fact that the formed open
structures remain stable even in the presence of Ca2+ suggest that thestabilization provided by gangliosides has a strong non-electrostatic
component. Although our results are not conclusive in this respect, itis plausible to think that the stabilization arises either from steric or
hydration effects (or probably both) provided by the gangliosides.
The question remains, however, why the attractive forces leading toaggregation and rupture for both PEG- and ganglioside- decorated li­
posomes do not cause aggregation or stacking of the open bilayer
structures formed. The open structures can be considered as charged
planar surfaces and therefore it would be expected that the same
attractive forces as discussed for liposomes would act. The fact that thesestructures remain dispersed as individual particles suggests that a strong

Fig. 8. Cryo-EM micrographs of liposomes composed of POPE:POPS:Ganglio­
sides with a molar composition of 65:25:10. A) Liposomes dispersed in buffer;
B) In the presence of 10 mM Ca2+ after 30 s; C) In the presence of 10 mM Ca2+
after 20 min. Small open bilayer structures can be observed face-on (black ar­rows) or edge-on (white arrows). Scale bar = 100 nm. Note that the size of thescale bar is unique for each image.

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repulsive interaction arises upon their formation. Since the edge of thesestructures is rich in either PEGylated lipids or gangliosides, it isreasonable to assume that the ion correlation attraction between theplanar surfaces is counteracted by a strong steric repulsion between the
PEG or ganglioside coronas. The repulsion is expected to be stronger
than that between the PEGylated surfaces of liposomes, as the local
concentration of PEG or gangliosides is much higher in the case of thesurrounding corona. Besides this effect, the fact that there is no possi­
bility for further PEG or ganglioside segregation likely contributes toprevent the formation of stacks or other aggregated structures.

CRediT authorship contribution statement

4. Conclusions

Appendix A. Supplementary data

The results presented in this report suggest that Ca2+ can induce not
only fusion of PE:PS based liposomes, but also the rupture and/or
collapse of liposomes on each other’s surfaces even at high ionicstrengths. It is also shown that modifying the surface of the liposomeswith PEGylated lipids or gangliosides can slow down, but not completely
prevent, the adhesion and rupture of the liposomes. Indeed, once
adhesion occurs, the segregation of the gangliosides or PEGylated lipids
from the contact area serves to facilitate the formation of open bilayer
structures.
In the case of PEGylated or ganglioside-modified liposomes, theadhesion and rupture processes can remain undetected by FRET mea­
surements, as little or no lipid mixing occurs. In the case of “naked”
vesicles, the bilayer fragments formed eventually fuse leading to an in­crease in the measured fluorescence signal. The signal increase due tofragment fusion can potentially be misinterpreted as evidence of direct
liposome-liposome fusion. A scenario where a large proportion of theliposomes rupture before lipid mixing takes place would explain thediscrepancies observed between the results from mixing-of-lipids andmixing-of-contents approaches to follow liposome-liposome fusion.
Our findings also suggest that the molecular mechanisms ofCa2+–induced membrane fusion involve attractive forces strong enoughto overcome the long-range repulsive forces provided by PEGylated
lipids and gangliosides, and to induce segregation of the stabilizing
lipids. These long-range forces cannot be explained by the previously
proposed strong short-range attractive interactions between calcium
and PS. Instead, it seems likely that ion correlation forces are involved.
This would also explain the enhancement of the fusion behavior trig­
gered by calcium in systems where PS is not present.
Gangliosides seem to have a similar inhibitory effect as PEGylated
lipids concerning the Ca2+ induced fusion and/or adhesion and rupture.
The fact that the open structures formed in the presence of gangliosideremain dispersed as single entities (i.e., without aggregation) indicatesthat there is a steric component in the stabilization provided bygangliosides.
It is our view that the results of this study merit reevaluation of theconclusions drawn from earlier FRET-based studies.

Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bbamem.2023.184253.

Conceptualization: K.E. and V.A.H.; Methodology: P.G., K.E., L.G.,
and V.A.H.; Validation: P.G. and V.A.H.; Formal analysis: P.G., L.G., K.E.
and V.A.H.; Investigation: L.G., P.G. and V.A.H.; Resources: V.A.H. andK.E.; Writing—original draft preparation: P.G. and V.A.H.; Wri­ting—review and editing: P.G., L.G., V.A.H. and K.E.; Supervision: V.A.
H. and K.E.; Project administration: K.E.; Funding acquisition: K.E. All
authors have read and agreed to the published version of themanuscript.

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Declaration of competing interest
The authors declare no potential conflict of interest.
Data availability
Data will be made available on request.
Acknowledgements
We gratefully acknowledge Lipoid GmbH for providing DSPEPEG2000. Valuable assistance from David Mínguez Romero with theexperimental work is greatly appreciated.
The present work was financially supported by the Swedish ResearchCouncil (2016–03464) and the Swedish Cancer Society (20 0987).
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