RESEARCH ARTICLE
Dimensions, s abili y, and de o mabili y o DOPC-
choles e ol gian unilamella esicles o med by d ople
ans e
[ e sion 3; pee e iew: 2 app o ed, 2 app o ed wi h ese a ions]
Elisa Robe i 1, Elisa Linda Pe ocelli2, Da io Cecchi 1, S e ano Palagi 1
1The BioRobo ics Ins i u e, San 'Anna School o Ad anced S udies, Pisa, Tuscany, 56127, I aly
2Uni e si à degli S udi di Roma La Sapienza, Rome, Lazio, 00185, I aly
Fi s published: 24 Ma 2025, 5:77
h ps://doi.o g/10.12688/open eseu ope.19149.1
Second e sion: 04 Aug 2025, 5:77
h ps://doi.o g/10.12688/open eseu ope.19149.2
La es published: 07 Oc 2025, 5:77
h ps://doi.o g/10.12688/open eseu ope.19149.3
3
Abs ac
Backg ound
Unde s anding cell memb ane-like lipid bilaye s is c ucial o s udying
undamen al biological mechanisms. Gian Unilamella Vesicles (GUVs)
a e key ools o his in es iga ion and ha e applica ions in bo h
syn he ic biology and, mo e ecen ly, in mic o obo ics. The e ec s o
choles e ol, a key componen o cellula memb anes, on syn he ic
phospholipid memb ane models like GUVs a e howe e no ully
unde s ood, as hey may a y wi h lipid composi ion and p oduc ion
me hod.
Me hods
We examined he size dis ibu ion, empo al s abili y and
de o mabili y o GUVs p epa ed wi h he d ople ans e me hod
using di e en Dioleoylphospha idylcholine (DOPC) o choles e ol
a ios in he oil phase (namely 100:0, 85:15, 71:29, 60:40). Phase-
con as mic oscopy assessed size and s abili y, while de o mabili y
was es ed by loading he GUVs wi h an aqueous e o luid and
applying a uni o m magne ic ield o induce hei elonga ion. Image
analysis was conduc ed using Fiji and a cus om Julia sc ip .
Resul s
The median diame e s inc eased wi h he con en o choles e ol,
Open Pee Re iew
App o al S a us
1234
e sion 3
( e ision)
07 Oc 2025
e sion 2
( e ision)
04 Aug 2025
iew iew
e sion 1
24 Ma 2025 iew iew iew iew
Nikole a I ano a , Uni e si y o Chemical
Technology and Me allu gy, So ia, Bulga ia
1.
Ma ín E. Villanue a , Uni e si é lib e de
B uxelles, B uxelles, Belgium
2.
Josep Jul e , Ins i u de Rece ca San Pau
(IR SANT PAU), Ba celona, Spain
3.
Da id W. E e e , Massey Uni e si y,
Palme s on No h, New Zealand
4.
Any epo s and esponses o commen s on he
a icle can be ound a he end o he a icle.
Open Resea ch Eu ope
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Open Resea ch Eu ope 2025, 5:77 Las upda ed: 07 OCT 2025
oge he wi h he dimensional dis ibu ion. In e ms o s abili y,
choles e ol gene ally educed GUV median diame e o e ime, while
i a yingly in luenced he numbe o esicles. As o de o mabili y,
beyond he expec ed elonga ion dependen on he in ensi y o he
applied magne ic ield, he e we e no s a is ically signi ican
di e ences in GUV de o mabili y in he p esence o absence o
choles e ol.
Conclusions
Ou indings sugges ha choles e ol can lead o inc eased a e age
diame e o GUVs made wi h DOPC h ough d ople ans e , while
a yingly a ec ing hei ime-s abili y and no a ec ing hei
de o mabili y. This s udy shows how small adjus men s on a
s aigh o wa d p o ocol like he d ople ans e me hod, p o ide a
simple and e ec i e way o ailo ing GUV p ope ies. Edi s in he oil
phase enable p ecise uning o GUV memb anes p o iding a ool o
bo h undamen al s udies and applica ions such as a i icial cells o
mic o obo s.
Plain language summa y
Cell memb anes play a key ole in he o e all unc ioning o cells and
hei en i onmen al in e ac ions. Fo a be e unde s anding o hei
mechanisms, a common ool is ep esen ed by Gian Unilamella
Vesicles (GUVs). These mic ome ic esicles a e p oduced h ough he
sup amolecula assembly o phospholipids, he main molecule o cell
memb anes, and used o explo e basic biological p ocesses, build
syn he ic cells, and e en design mic o obo s. He e we s udy he
e ec s ha choles e ol, a key componen o na u al cell memb anes,
has on GUVs’ memb ane p ope ies. While hese can a y wi h he
p oduc ion me hod and he ypes o lipids, in his s udy we ocus on
one o he mos widely adop ed me hods (known as d ople ans e )
and one o he mos common phospholipids
(dioleoylphospha idylcholine o DOPC). Ou esul s sugges ha he
e ec o choles e ol on he size and s abili y o GUVs depends on i s
concen a ion, while hei lexibili y seems no o be a ec ed. By
explo ing hese p ope ies, we aim o deepen ou unde s anding o
cell memb anes and imp o e he design o syn he ic sys ems o
u u e applica ions.
Keywo ds
GUVs, d ople ans e , choles e ol, a i icial cells, mic o obo s,
de o mabili y
This a icle is included in he Ho izon 2020
ga eway.
Open Resea ch Eu ope
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Open Resea ch Eu ope 2025, 5:77 Las upda ed: 07 OCT 2025
Co esponding au ho s: Elisa Robe i ([email p o ec ed]), S e ano Palagi ([email p o ec ed])
Au ho oles: Robe i E: Da a Cu a ion, Fo mal Analysis, P ojec Adminis a ion, Supe ision, Valida ion, W i ing – O iginal D a
P epa a ion, W i ing – Re iew & Edi ing; Pe ocelli EL: Concep ualiza ion, Da a Cu a ion, Fo mal Analysis, In es iga ion, Me hodology,
P ojec Adminis a ion, So wa e, Valida ion, W i ing – O iginal D a P epa a ion; Cecchi D: Fo mal Analysis, Valida ion, W i ing – Re iew
& Edi ing; Palagi S: Concep ualiza ion, Da a Cu a ion, Fo mal Analysis, Funding Acquisi ion, Me hodology, P ojec Adminis a ion,
So wa e, Supe ision, Valida ion, Visualiza ion, W i ing – Re iew & Edi ing
Compe ing in e es s: No compe ing in e es s we e disclosed.
G an in o ma ion: This p ojec has ecei ed unding om he Eu opean Resea ch Council (ERC) unde he [Eu opean Union’s Ho izon
2020 esea ch and inno a ion p og amme][Eu opean Union’s Ho izon Eu ope esea ch and inno a ion p og amme] (G an ag eemen
No. [948590]).
The unde s had no ole in s udy design, da a collec ion and analysis, decision o publish, o p epa a ion o he manusc ip .
Copy igh : © 2025 Robe i E e al. This is an open access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License
, which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed.
How o ci e his a icle: Robe i E, Pe ocelli EL, Cecchi D and Palagi S. Dimensions, s abili y, and de o mabili y o DOPC-choles e ol
gian unilamella esicles o med by d ople ans e [ e sion 3; pee e iew: 2 app o ed, 2 app o ed wi h ese a ions] Open
Resea ch Eu ope 2025, 5:77 h ps://doi.o g/10.12688/open eseu ope.19149.3
Fi s published: 24 Ma 2025, 5:77 h ps://doi.o g/10.12688/open eseu ope.19149.1
This a icle is included in he Eu opean
Resea ch Council (ERC) ga eway.
Open Resea ch Eu ope
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Open Resea ch Eu ope 2025, 5:77 Las upda ed: 07 OCT 2025
In oduc ion
Unde s anding cell lipid bilaye s is c ucial o deciphe ing un-
damen al biological mechanisms, conside ing hei cen al ole
in a ious cellula p ocesses. Insigh s in o lipid bilaye beha -
iou enhance ou unde s anding o gene al cellula mechanisms
and he unc ional di e si ies among di e en cell ypes. To
his end, syn he ic lipid bilaye s ha e long se ed as a unda-
men al ool o model biological memb anes (Dimo a, 2019;
Kahya & Schwille, 2006; Lau ence e al., 2022; Menge &
Keipe , 1998; Topa lak & Mansy, 2019; Wubshe & Liu, 2023)
and hese sup amolecula assemblies a e also o g ea in e -
es om an applica ion pe spec i e. Due o hei selec i e pe -
meabili y and ease o ab ica ion, lipid bilaye s ha e been
u ilised in syn he ic biology o he pas wo decades (Robinson
e al., 2021; Van de Cau e e al., 2023) and, mo e ecen ly, in
he ield o mic o obo ics (Sa o e al., 2017; Vu uku i e al.,
2020). Gian Unilamella Vesicles (GUVs), liposomes wi h
diame e s anging om 1 o 100 mic ome es, ha e a single
phospholipid bilaye ha is commonly used as a lipid mem-
b ane model o cha ac e iza ion s udies and echnological
applica ions.
In cells, he composi ion o he plasma memb ane a ies
acco ding o he cell unc ion (Casa es e al., 2019), bu also in
esponse o pa hological (Woodman & Kim, n.d.), nu i ional
(Agos oni e al., 2013), and pha macological ea men con-
di ions (Esc ibá e al., 2015). Cha ac e izing he e ec s o
a ia ions in he composi ion o lipid bilaye s can yield alu-
able insigh s in o cell memb ane beha iou , while also acili a ing
applica ions such as esicle-based a i icial cells and mic o o-
bo s. Whe he he goal is o unde s and cell memb ane beha -
iou o o de elop esicle-based mic o obo s and a i icial cells,
i is c ucial o examine how he memb ane composi ion in lu-
ences he dimensions, s abili y, and de o mabili y o GUVs.
One impo an p ope y in luencing he mechanical beha iou
o cells and esicles is he bending igidi y o he memb ane,
which quan i ies i s esis ance o changes in cu a u e (Wubshe
& Liu, 2023). I is well known ha bending igidi y is in lu-
enced by a mul i ude o ac o s such as he hyd ophobic ails’
chain leng h and sa u a ion (Rawicz e al., 2000), he empe a-
u e (Pan e al., 2008b), he p esence o cha ged lipids (Dimo a,
2014; Faizi e al., 2019) he concen a ion o suga s o sal s
(Dimo a, 2014), and he p esence o s e ols like choles e ol
(Chen & Rand, 1997; Dimo a, 2014; G acià e al., 2010a; Ka al
e al., 2022).
Choles e ol is a c ucial elemen o biological sys ems, cons i-
u ing up o 50 mol% o he o al lipid con en o he memb ane
(Ka al e al., 2020a) al hough i s p opo ion a ies be ween
cell ypes. Fo example, lymphocy e memb anes and ce ain
me as a ic cance cells end o exhibi lowe choles e ol con-
en . High choles e ol con en gene ally inc eases memb ane
igidi y and dec eases de o mabili y, whe eas memb anes wi h
lowe le els a e mo e lexible, enhancing hei abili y o de o m
and pass h ough he na ow in e cellula spaces in issues
(Szlasa e al., 2020; Zhao e al., 2019). Fo syn he ic memb anes,
such as GUVs, he e ec o choles e ol has been ex ensi ely
deba ed in he li e a u e. I is widely accep ed ha choles e ol
has di e en e ec s on memb anes depending on hei cons i-
u ing phospholipids (Pan e al., 2008a). The bending igidi y o
sa u a ed lipids memb anes inc eases wi h choles e ol con en ,
while in memb anes composed o double unsa u a ed lipids, like
dioleoylphospha idylcholine (DOPC), he igidi y seems o be
independen o choles e ol ac ion (Dimo a, 2014; G acià e al.,
2010a; Pan e al., 2008a; So e e al., 2009). None heless, some
e idence sugges s he bending igidi y o DOPC memb anes
inc easing alongside he choles e ol con en as o sa u a ed
lipids memb anes (Ashka e al., 2019; Chak abo y e al., 2020).
These disc epancies likely e lec di e ences in measu emen
echniques: equilib ium app oaches (e.g., licke spec oscopy)
cap u e he s a ic bending modulus, while non-equilib ium
me hods (e.g., neu on spin echo, NSE) a e mo e sensi i e o
memb ane iscosi y on nanosecond–mic osecond imescales.
Thus, some o he epo ed igidi y changes in DOPC–choles e ol
memb anes may in ac o igina e om al e ed memb ane
iscosi y a he han changes in long- imescale elas ic s i ness
(Hein ich & Nagle, 2025).
To assess he ole o choles e ol in lipid memb ane models,
he e ec s we e p e iously es ed on LUVs (la ge unilamel-
la esicles ab ica ed wi h he swelling me hod (Chak abo y
e al., 2020; G acià e al., 2010b), e ealing i s in luence on
bo h he a e age size o esicles and he memb ane’s bending
modulus (Ka al e al., 2022). While his echnique o e s a sim-
ple and eliable me hod o ob ain GUVs, i s use is p e e ably
a oided when aiming a encapsula ing solu ions and ca -
gos wi hin he esicles. Fo he ab ica ion o a i icial cells o
mic o obo s, a mo e ad an ageous al e na i e is p o ided by he
d ople ans e me hod (Pau o e al., 2003), which allows o
highe e iciency o encapsula ion o aluable in e nal GUV
solu ions, such as cell- ee p o ein syn hesis eac ions (Ga enne
e al., 2021; Shimane & Ku uma, 2022) o mic opa icles
(Vu uku i e al., 2020). Because he me hod is based on he dis-
solu ion o phospholipids in o an oil phase and he o ma ion o
bilaye s om hei spon aneous adso p ion a oil-wa e in e -
aces, i is impo an o conside ha o he o ganic molecules
p esen in o added o he oil phase, like choles e ol, may o may
no con ibu e o he memb ane. Indeed, memb anes o GUVs
ob ained ia he d ople ans e me hod may ha e a choles-
e ol con en ha does no ma ch ha in he lipid solu ion. The
ac ual choles e ol concen a ion can be signi ican ly lowe han
he nominal one (Weakly e al., 2024). Consequen ly, choles-
e ol canno be assumed o a ec d ople ans e -made GUVs in
he same manne i a ec s GUVs p oduced by o he me hods.
Amendmen s om Ve sion 2
Majo e isions ha e been implemen ed in he In oduc ion
and Conclusion sec ions. In addi ion, ou new e e ences ha e
been inco po a ed in o bo h sec ions o s eng hen he con ex
and suppo o he discussion. Fu he mo e, a new pa ag aph
en i led S a is ical Analysis in Me hods sec ion has been added,
desc ibing in de ail he me hods used o he s a is ical
e alua ion o he da a.
Any u he esponses om he e iewe s can be ound a
he end o he a icle
REVISED
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In his wo k, we s udy how he addi ion o choles e ol a ec s
GUVs p epa ed wi h he d ople ans e me hod in e ms o size
dis ibu ion, empo al s abili y and de o mabili y o he esicles.
We use he commonly adop ed double unsa u a ed DOPC as
he memb ane’s main cons i uen and mine al oil as an o ganic
sol en . Choles e ol is added in di e en p opo ions o he
DOPC-oil lipid solu ion and esicles a e p oduced keep-
ing all o he p ocess pa ame e s un a ied. To assess he equi-
lib ium de o ma ion o he esicles, we used a cus om se up
equipped wi h wo pe manen magne s, gene a ing a uni o m
magne ic ield, and an inne solu ion con aining e o luid.
Gi en he well-known s abilizing e ec s o choles e ol (Zhang
e al., 2019), we also examined he impac o his lipid on he
empo al s abili y o ou memb anes.
Me hods
The ollowing ma e ials we e ob ained om Sigma-Ald ich® (S .
Louis, MO): DOPC (1,2-dioleoyl-sn-glyce o-3-phosphocholine, in
chlo o o m, C44H84NO8P, ca . 850375C-1G, CAS: 4235-95-4),
suc ose ≥99.5% (GC) (α-D-Glucopy anosyl β-D- uc o u anoside,
C12H22O11, ca . S9378-500G, CAS: 57-50-1), D-(+)-Glucose
≥99.5% (GC) (C6H12O6, ca . G8270-100G, CAS: 50-99-7),
and choles e ol powde (3β-Hyd oxy-5-choles ene, C27H46O,
ca . C3045-5G, CAS: 57-88-5). The aqueous e o luid, con-
aining app oxima ely 20% by weigh o 10 nm magne -
i e nanopa icles s abilized wi h ci a e, was pu chased om
Q luidics (F ance). Addi ionally, he neodymium N42 nickel-
pla ed pe manen magne s we e sou ced om Supe magne e
(ca . Q-15-15-08-N). The expe imen al se up u ilized o de o m-
abili y es s was designed using Compu e -Aided Design (CAD)
so wa e (e.g. Au odesk Fusion 360) and subsequen ly 3D p in ed
in VisiJe M3 C ys al ma e ial (3D Sys ems P oje MJP 3600
HD Max). Gene F ames (250 µm hickness and 1 cm2 a ea)
o mic oscope glass slides we e ob ained om The mo Fishe
Scien i ic (ca . AB0576).
P epa a ion o s ock solu ions
Two lipid s ock solu ions we e p epa ed in mine al oil, one
a 2.5 mg/mL (3.18 mM) DOPC and one a 0.79 mg/mL
(2 mM) choles e ol. Lipids dissol ed in chlo o o m we e ans-
e ed on o a glass ial, hen he chlo o o m was e apo a ed
h ough a ni ogen low, allowing he o ma ion o a hin ilm o
d ied phospholipids. Finally, he mine al oil was added, and he
lipids we e dissol ed by hea ing a 80°C o one hou . The
s ock was hen s o ed a 4°C.
Two inne solu ions and one ou e solu ion we e p epa ed using
milliQ wa e . One inne solu ion was p epa ed wi h 240 mM
suc ose, and he o he wi h 1:2 ( / ) e o luid:suc ose 240 mM,
while he ou e solu ion was p epa ed wi h 240 mM glucose.
The solu ions wi hou e o luid we e il e ed wi h a
0.22 µm mic o il e and hen s o ed a +4°C.
P epa a ion o GUVs h ough d ople ans e
GUVs we e p epa ed ollowing a modi ied e sion o he
d ople ans e me hod (Pau o e al., 2003) desc ibed in ou
p e ious wo k (Cecchi e al., 2025). 200 µL o lipid s ock
solu ion we e ans e ed in a 2 mL ube, sonica ed o 10 min-
u es in a ba h sonica o (Ul asonic cleane B own Sonic
2510E-MTH), and cooled on ice o 15 minu es. An emul-
sion was hen p epa ed by adding 10 µL o inne solu ion o
he ube, o exing he mix u e o 25 seconds a 3000 pm
(Vo ex WIZARD IR, VWR ca . # 444-0746) and chilling i
o 10 minu es on ice. 300 µL o ou e solu ion we e ans-
e ed in a 1.5 mL ube and placed on ice. Then 100 µL o
lipid s ock solu ion we e laye ed on op o he ou e solu ion o
allow o he spon aneous o ma ion o a lipid monolaye a
he wa e /oil in e ace. The emulsion was hen laye ed on op
o he in e ace and cen i uged o 20 minu es a 3300g a
4°C. Finally, he oil phase was emo ed wi h a pipe e and he
liposomes we e esuspended om he pelle .
Fo he p epa a ion o GUVs wi h choles e ol, oil mixes com-
posed o DOPC and choles e ol a di e en mola a ios
we e p epa ed ollowing he p e iously epo ed analysis on
GUVs made wi h he swelling me hod (Ka al e al., 2022):
85:15, 71:29, and 60:40. DOPC concen a ion was kep cons an
a 1.25 mg/mL (1.59 mM) and he choles e ol concen a ion
a ied acco ding o he mola a io.
Obse a ion and da a analysis o GUVs
A e emo ing he oil phase wi h a mic opipe e and esus-
pending he liposomes, a 25 µL aliquo o he sample was
placed on o a glass slide wi hin a Gene F ame and co e ed
wi h a co e slip. Samples we e hen obse ed in phase con-
as h ough he in e ed mic oscope Nikon Eclipse TE2000-U,
equipped wi h a 20x/0.40 Ph1 ADL objec i e a a wo king
dis ance (WD) o 3.1. Images o he en i e ame a ea we e
cap u ed. To in es iga e he e ec s o choles e ol on he size
dis ibu ion o GUVs and o ensu e consis en obse a ion and
s a is ical analysis o how choles e ol in luences esicle size
dis ibu ion, iplica e samples o he ou composi ions (DOPC:
Chol 100:0, 85:15, 71:29, and 60:40) we e p oduced and
analysed. To ensu e compa abili y du ing analysis, app oxi-
ma ely 120 images we e acqui ed o each sample. Fo he
analysis o esicle s abili y, iplica e samples o DOPC:Chol
100:0, 85:15, 71:29, and 60:40 we e s o ed o e nigh a 4°C and
obse ed again on he ollowing day. In his case, 100 images
we e cap u ed o each sample. The acqui ed images we e ana-
lysed wi h Fiji ImageJ (h ps://imagej.ne /so wa e/ iji/). Fo
each image, GUVs we e iden i ied, and hei a eas we e meas-
u ed using he ools p o ided by ImageJ. The collec ed da a
we e analysed wi h a cus om sc ip implemen ed in he Julia
language (h ps://julialang.o g/) ha calcula es he diame e
o he GUVs om hei a ea (assuming hem as ound), e alu-
a es whe he he di e ences be ween samples a e s a is ically
signi ican , i s he da a o a dis ibu ion unc ion, and plo s
he da a and analysis esul s (Robe i e al., 2025). Finally,
we ha e calcula ed he o al su ace a ea o all obse ed
esicles as he sum o he indi idual su ace a eas (assum-
ing hem as sphe ical), and compa ed he esul s ob ained o
as-p epa ed samples wi h hose o o e nigh -s o ed samples.
This comp ehensi e app oach aims o assess he impac o
choles e ol on he esicle size dis ibu ion and s abili y.
Obse a ion and da a analysis o magne o-GUVs
The cha ac e iza ion o magne ic GUVs and he obse a-
ion o hei de o mabili y unde he in luence o an ex e nal
Page 5 o 22
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Figu e 1. Schema ics o he GUVs’ dimensions and he ela ed
es adius.
Figu e 2. Rep esen a i e phase-con as images o A) DOPC:chol 100:0, B) DOPC:chol 85:15, C) DOPC:chol 79:21, D) DOPC:chol 60.40. Scale
ba s = 50 μm.
magne ic ield we e ca ied ou using a digi al 3D op ical mic o-
scope (Hi ox RH-2000). To obse e de o ma ion, he sample
was subjec ed o an ex e nal magne ic ield gene a ed by a pai
o pe manen magne s kep a speci ic dis ances by a cus om
3D p in ed se up (Robe i e al., 2024). The magne s a e o i-
en ed wi h he poles in he same di ec ion and placed a equal
dis ances o he obse a ion a ea a he cen e o he de ice. The
supe posi ion o he magne ic ields esul s in a homogeneous
magne ic ield in he obse a ion a ea. 25 µL o he sample we e
placed on a mic oscope glass slide inside a Gene ame and
co e ed wi h a co e slip. All samples we e ini ially obse ed
wi hou he applica ion o a magne ic ield. Subsequen ly, he
glass slide was placed a he cen e o he magne is’ se up and
he magne ic ield in ensi y was a ied by changing he dis-
ance be ween he magne s. Th ee di e en dis ances be ween
he magne s we e conside ed o ou analysis: 13, 10 and
4 cm, esul ing in magne ic ields o 2.0, 4.7, and 48.0 mT,
espec i ely, which we named H1, H2 and H3 (Robe i
e al., 2025). The images we e subsequen ly analysed using
ImageJ and each GUV was ea ed as an ellipse, collec ing da a
on a ea, majo axis, and mino axis. Da a we e hen analysed
wi h a sc ip implemen ed in Julia (Robe i e al., 2025).
The esicles a e assumed o ha e a sphe ical shape a es
and o achie e an ellipsoidal shape (namely a p ola e sphe oi-
dal shape) unde he ac ion o a magne ic ield. The long axis
o he ellipsoid is aligned wi h he o ien a ion o he magne ic
ield (see Figu e 1).
Gi en he majo and mino axis a and b o he sphe oid, i s
olume can be calcula ed as
πV = ab
42
3
. Assuming ha he
olume o he esicle does no change wi h he magne ic
elonga ion, we can es ima e he es adius as
3 2
0
R ab=
(and
hus he es diame e ). This means, howe e , ha he appa en
su ace o he esicles inc eases, as he memb ane sp eads
ou . We hus calcula e he su ace a ea o a sphe e ha ing a
adius equal o he es adius,
2
0
4
s
S Rπ=
, and assumed i o
be he es appa en su ace a ea o he esicle. We also cal-
cula ed he su ace a ea o he esicles in he obse ed p o-
la e sphe oidal shape as o Sps = 2π b2 (1 + a/be a csin e), whe e
e2 = 1 – b
2
/a2. As we a e in e es ed in he e ec o he lipidic
composi ion o he memb ane on esicles de o mabili y a
equilib ium, we hen e alua ed he su ace a ea de o ma ion as
σ = Sps/Ss – 1. S a is ical signi icance es s on σ a e pe o med
assuming a logno mal dis ibu ion.
S a is ical analysis
S a is ical analyses we e pe o med o e alua e he in luence o
choles e ol con en on he size dis ibu ion and empo al s abil-
i y o esicles as well as on hei magne ically induced de o -
ma ion. Fi ing he diame e da a o logno mal dis ibu ions,
we also calcula e mode (i.e. he peak o he dis ibu ion), median,
and pe cen iles o he dis ibu ion o each sample. Assum-
ing logno mal dis ibu ions o he alues o diame e s and o
σ, he s a is ical signi icance es s (F- es and - es ) a e no pe -
o med on he ac ual da a bu on hei na u al loga i hm (which
ha e no mal dis ibu ions). Size dis ibu ion analyses we e
ca ied ou ac oss samples wi h di e en choles e ol concen a-
ions a bo h 0 and a e o e nigh incuba ion using wo-sample
- es s (see GUVs_concen a ion_s abili y.cs , Unde lying Da a).
S abili y was u he assessed, epo ing he peak, median
and numbe o GUVs a 0 and a e o e nigh incuba ion o
each concen a ion. Two-sample - es s we e pe o med be ween
samples a p is ine and o e nigh s o ed samples (co espond-
ing p- alues a e epo ed in GUVs_s abli y_T0 sON.cs , Unde -
lying Da a). Mo eo e , he minimum de ec able a io be ween
g oups (σ₁/σ₂), as de e mined by he log- ans o med - es a
80% powe , was inco po a ed in o he analysis o de ine he
smalles e ec size ha could be eliably iden i ied unde ou
expe imen al condi ions (see GUVs_de o mabili y_s a , Unde ly-
ing Da a).
Resul s
Size dis ibu ion
We p epa ed GUVs by he d ople ans e me hod a di e en
DOPC:choles e ol a ios, namely 100:0 (no choles e ol),
85:15, 71:29, and 60:40, in he lipid solu ion (Figu e 2). Righ
a e p epa a ion ( 0), we obse ed 247, 190, 279, and 394
Page 6 o 22
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Figu e 3. Compa ison o esicles p epa ed wi h lipid solu ions (LSs) a di e en concen a ions o choles e ol, as p epa ed ( 0)
and a e o e nigh s o age (o.n.). A) measu ed diame e s o he esicles; B) numbe o obse ed esicles; C) median (solid lines), mode
(peak – dashed lines) and 5 h o 95 h pe cen ile (colou ed a eas) o he logno mal dis ibu ions i ed o he diame e s da a.
esicles, espec i ely, in he sample aliquo (see Figu e 3B
le side / b igh colou ). Assuming a logno mal dis ibu ion
o he diame e s wi hin each sample (see Me hods), we ound
ha he dimensional di e ences be ween all combina ions o
samples a e s a is ically signi ican (p < 0.005; p alues a e
epo ed in Unde lying Da a). Mo eo e , as he concen a-
ion o choles e ol in he lipid solu ion inc eased, he maximum
obse ed diame e o esicles inc eased.
Fi ing logno mal dis ibu ions o he diame e s da a, we ound
ha he median diame e (geome ic mean o he dis ibu ion)
inc eased wi h he con en o choles e ol in he lipid solu ion
consis en wi h wha was desc ibed in p e ious s udies (Ka al
e al., 2020b; Ka al e al., 2022). The es ima ed median diam-
e e s a e 12.1, 14.5, 17.3 and 21.5 µm, espec i ely o 100:0,
85:15, 71:29, and 60:40 dis ibu ions (see Figu e 3C, solid
lines – le side / b igh colou ). The colou ed ba s in Figu e 3C
ep esen he dimensions o esicles wi hin he 5 h and 95 h
pe cen ile o he dis ibu ions: i can be no iced ha he main
di e ence among samples wi h inc easing concen a ions o
choles e ol in he lipid solu ion is he inc ease in he 95 h
pe cen ile alue and, hus, in he wid h o he dis ibu ion.
Tempo al s abili y
To in es iga e he impac o choles e ol on he s abili y o he
GUV memb ane, samples we e s o ed o e nigh a 4°C and
obse ed a he phase-con as mic oscope he ollowing day. Fo
each sample, we eco ded he numbe o esicles obse ed in
a new aliquo and hei dimensional dis ibu ion (see Figu e 3 –
igh sides / da k colou s). Among he o e nigh -s o ed aliquo s,
he sample 100:0 has a signi ican ly di e en size dis ibu ion
om all choles e ol-con aining samples (p < 0.005), whe eas all
choles e ol-con aining samples p esen no s a is ically signi i-
can di e ences among hem (p alues a e epo ed in Unde -
lying Da a). When choles e ol was absen (sample 100:0), he
o al numbe o obse ed GUVs almos hal ed and he median
diame e om he i ed logno mal dis ibu ion inc eased wi h
a gene al shi owa ds la ge diame e s. Con e sely, o he
choles e ol-con aining samples, a gene al shi owa ds smalle
diame e s was obse ed wi h a dec ease in he uppe limi o he
dis ibu ion (95 h pe cen ile) compa ed o he no-choles e ol sam-
ples. Wi h he highes choles e ol concen a ion (60:40), bo h
he numbe o esicles and he median diame e dec eased o e -
nigh . Fo he in e media e cases (85:15 and 79:21), ins ead,
he numbe o GUVs inc eased while he median diame e
dec eased. In pa icula , o he 85:15 samples, he numbe o
esicles mo e han doubled and he median diame e dec eased.
The di e ences in size dis ibu ions be ween he p is ine and
o e nigh aliquo a e s a is ically signi ican o all samples
(p<0.005; p alues a e epo ed in Unde lying Da a).
Finally, we es ima ed he o al su ace a ea As o esicles and
obse ed a dec ease in all he samples (especially in hose
wi h he highes choles e ol concen a ion) excep o he
85:15 sample whe e, su p isingly, he o al su ace a ea almos
doubled o e nigh (Table 1).
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De o ma ion unde magne ic ields
As e o luid-loaded esicles unde go magne ic ield-dependen
elonga ion (Nuñez-Magos e al., 2021), we exposed GUVs
o uni o m magne ic ields in o de o in es iga e a po en ial
co ela ion be ween hei de o mabili y and he p esence o
choles e ol in hei memb ane (see Obse a ion and da a
analysis o magne o-GUVs). Following he applica ion o he
magne ic ield, he esicles exhibi a p ola e shape, elonga ing
in he di ec ion o he ield. Addi ionally, hey end o agg ega e
due o magne ic dipole-dipole in e ac ions hus assembling
in chains, as shown in Figu e 4. Fo each measu ed esicle,
we calcula ed he su ace a ea de o ma ion σ wi h espec o
an ideal sphe ical es con igu a ion. The esul s, epo ed in
Figu e 5, show ha , as expec ed, he de o ma ion inc eases
wi h he in ensi y o he applied magne ic ield, wi h some
esicles unde going a su ace a ea de o ma ion o abou 40% a
he highes -in ensi y magne ic ield. Al hough i can be obse ed
in Figu e 5 ha 100:0 samples ha e one o ew ou lie s wi h
la ge de o ma ions, whe eas his is no obse ed in he 60:40
samples, he di e ences in su ace a ea de o ma ion be ween
he wo samples a e no s a is ically signi ican (p<0.005;
p alues a e epo ed in Unde lying Da a). This sugges s ha
choles e ol does no a ec he mechanical p ope ies o lipid
bilaye s. None heless, gi en he obse ed a iance in alues
o σ and aiming a a s a is ical powe o 80%, we would ha e
been able o de ec a ios be ween median alues o σ o abou
1.4 o mo e (see Unde lying Da a).
Discussions and conclusions
Choles e ol is a c ucial componen o cellula memb anes
ha in luences hei physical p ope ies by modi ying he
spa ial o ganisa ion o phospholipid acyl chains. Due o his
signi icance, choles e ol has been employed in he p oduc ion
o GUVs, bo h as models in biophysics s udies and as semi-
pe meable shells o a i icial cells. The e ec o choles e ol
on esicles a e age size and hei memb ane’s bending modu-
lus has been p e iously analysed o esicles p epa ed h ough
he swelling me hod (Chak abo y e al., 2020; G acià e al.,
2010b; Ka al e al., 2022). In ou s udy, we e alua ed he e ec
o choles e ol on GUVs p epa ed wi h he d ople ans e
me hod, whe e phospholipids and choles e ol a e dissol ed
in an oil phase and he bilaye ’s assembly is media ed by he
wa e /oil in e ace. GUVs p oduced using he d ople ans e
me hod may con ain signi ican ly less choles e ol han he lipid
solu ions om which hey a e p epa ed, especially when com-
pa ed o o he p oduc ion echniques such as elec o o ma ion
o gen le hyd a ion. Fo his eason, we did no expec he mem-
b ane composi ion o ma ch ha o he o iginal lipid solu ion
as p e iously epo ed (Weakly e al., 2024). None heless, we
expec he choles e ol con en in he memb ane o depend on
ha in he lipid solu ion. Gi en he unce ain pa i ioning o
choles e ol be ween he oil and he lipid bilaye , we aimed o
es ima e choles e ol’s in luence on d ople ans e -based GUVs,
i any.
To his end, we examined he dimensional dis ibu ion, empo al
s abili y, and de o mabili y o GUVs wi h a ious lipid
composi ions p oduced h ough he d ople ans e me hod.
Speci ically, we ab ica ed esicles wi h DOPC and di e en
choles e ol a ios (100:0, 85:15, 71:29, 60:40). Ou obse a ions
e ealed ha an inc ease in choles e ol a io co ela es wi h a
highe a e age diame e o GUVs. This aligns wi h he p e i-
ously obse ed ole o choles e ol in lipid bilaye s. Indeed, due
o i s hyd ophobici y, choles e ol is likely embedded wi hin
he bilaye , educing he ans-gauche isome iza ion o he
neighbou ing lipid acyl chains. This dec eases hei dynamics
and luidi y, he eby s abilizing he memb anes and leading
o la ge -diame e esicles (Yang e al., 2016). We also ana-
lysed how choles e ol a ec s he empo al s abili y o GUV
memb anes. All ou samples we e s o ed o e nigh and
obse ed he ollowing day using a phase con as mic oscope.
GUVs wi hou choles e ol (DOPC:Chol 100:0) we e ewe
in numbe bu la ge in size compa ed o he eshly p epa ed
samples ( 0), possibly indica ing a endency o usion. On he
o he hand, choles e ol-con aining GUVs unde go a educ ion
o he mean diame e o e nigh , mo e e iden in he sam-
ple wi h he highes choles e ol concen a ion. The 85:15
and 71:29 samples showed an inc ease in esicle numbe s
compa ed o 0, while he 60:40 sample aced a educ ion in
numbe s a e an o e nigh incuba ion. Al hough we could
no e i y his, i is possible ha in lowe choles e ol samples
la ge GUVs spli in o smalle ones, while GUVs wi h he
highes choles e ol con en bu s in o ei he lipid agg ega es o
small liposomes unde ec able a he mic oscope.
Table 1. O e nigh a ia ions in GUV popula ions. Median diame e s we e calcula ed assuming a
logno mal dis ibu ion (see Me hods). All alues in he able ep esen cumula i e esul s om iplica e
expe imen s.
DOPC:
choles e ol
Median
diame e a = 0
Median diame e a e
o e nigh s o age
Coun a
= 0
Coun a e
o e nigh s o age
A
A
o.n.
s
s
= 0
100:0 12.1 µm 16.1 µm 247 129 82 %
85:15 14.5 µm 11.9 µm 190 491 198 %
71:29 17.3 µm 12.3 µm 279 389 66 %
60:40 21.5 µm 13.4 µm 394 289 20 %
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To in es iga e whe he choles e ol addi ion enhances o hinde s
GUV de o mabili y, we applied a magne ic ield o p o ide
mechanical s ess o he lipid memb anes. Wi h he use o wo
pe manen magne s and by encapsula ing aqueous e o luid,
we ab ica ed magne ic GUVs and exposed hem o h ee
in ensi ies o a uni o m magne ic ield. The GUVs, ini ially
sphe ical, became ellipsoidal unde he magne ic ield, wi h
comp ession inc easing alongside he magne ic ield in ensi y
(Figu e 5). In e es ingly, GUVs emained s able and did no
b eak unde he magne ic ield. Since i was no possible o
obse e any signi ican di e ence in GUVs de o mabili y, we
concluded ha choles e ol does no ha e a clea in luence on
memb ane igidi y, a leas no o an ex en obse able wi h ou
echnique. This inding is consis en wi h a ecen s udy
(Hein ich & Nagle, 2025), which highligh ed ha equilib ium-
based me hods, such as he one applied he e, ypically
e eal no changes in he bending modulus (Kc) o DOPC
memb anes upon choles e ol inco po a ion. This is likely due
Figu e 4. B igh - ield op ical mic oscope images depic DOPC:chol 60:40 (A–C) and DOPC:chol 100:0 (D–F) unde going de o ma ion a
di e en magne ic ield in ensi ies: A and D: H1; B and E: H2; C and F: H3. Scale ba s: 100 μm.
Figu e 5. Compa ison o σ alues o DOPC:chol 100:0 and 60:40 unde inc easing magne ic ields.
Page 9 o 22
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Pa ly
A e all he sou ce da a unde lying he esul s a ailable o ensu e ull ep oducibili y?
Yes
A e he conclusions d awn adequa ely suppo ed by he esul s?
Pa ly
Compe ing In e es s: No compe ing in e es s we e disclosed.
Re iewe Expe ise: Lipid esicle s uc u es
I con i m ha I ha e ead his submission and belie e ha I ha e an app op ia e le el o
expe ise o con i m ha i is o an accep able scien i ic s anda d, howe e I ha e
signi ican ese a ions, as ou lined abo e.
Au ho Response 06 Aug 2025
Elisa Robe i
1. We hank he e iewe o poin ing ou he e o in he ex ; we ha e e ised i
acco dingly ( e sion 3). Rega ding he use o DLS, ou GUVs a e oo la ge o be measu ed
wi h his echnique. The e o e, sui able echniques a e mic oscopy and low cy ome y.
2. In e sion 2 o he manusc ip , we e ised he s a ical analysis and added a CSV ile in he
Unde lying Da a con aining a pai wise compa ison o he concen a ions a 0 and o.n..
3.I is likely ha he obse ed dec ease in bo h numbe and size o e nigh is due o he
spli ing o esicles in o nanoliposomes, which canno be de ec ed by mic oscopy and
canno he e o e be analyzed.
4.The o al su ace a ea is calcula ed based on liposomes wi hou e o luid, which a e no
subjec ed o de o ma ion induced by he magne ic ield. These a e isola ed GUVs ha do
no come in con ac wi h each o he . Rega ding he size o GUVs con aining e o luid and
hus subjec ed o he de o mabili y es , he Ex ended Da a con ain g aphs showing he
equi alen diame e s o he magne ic GUVs, calcula ed om i ed ellipses unde he
assump ion o p ola e sphe oidal shape. These g aphs indica e ha , in gene al, GUVs
con aining e o luid end o be la ge han emp y ones.
5.We expec ha usion e en s be ween GUVs could occu o e long imescales, pa icula ly
du ing ex ended s o age o incuba ion, e en hough we did no obse e usion wi hin he
sho imescale o he de o mabili y expe imen s. The e o e, usion does no in luence
de o mabili y measu emen s, and he obse ed mechanical esponses can be a ibu ed o
he in insic p ope ies o he indi idual esicles. None heless, we belie e ha usion could
explain he change in numbe and size o esicles du ing o e nigh s o age.
6.The sugges ed a icle speci ically discusses mul ilamella esicles con aining
sphingomyelin, which is no p esen in ou sys em. Fo his eason, we do no conside i
Open Resea ch Eu ope
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ele an o ou s udy.
7. In e sion 3 o he manusc ip we ha e included a oo no e o explain he las column
heading o Table 1.
Compe ing In e es s: No compe ing in e es s we e disclosed.
Re iewe Repo 21 July 2025
h ps://doi.o g/10.21956/open eseu ope.20721. 55563
© 2025 Jul e J. This is an open access pee e iew epo dis ibu ed unde he e ms o he C ea i e Commons
A ibu ion License, which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he
o iginal wo k is p ope ly ci ed.
Josep Jul e
1 Ins i u de Rece ca San Pau (IR SANT PAU), Ba celona, Spain
2 Ins i u de Rece ca San Pau (IR SANT PAU), Ba celona, Spain
3 Ins i u de Rece ca San Pau (IR SANT PAU), Ba celona, Spain
Technical issues:
1- Me hodology: no s a is ical analysis subsec ion has been conside ed in he me hodology. why?
How many independen eplica es we e done?
2- How many independen eplica es we e done in each pack o expe imen s (g ouped in
igu es/ ables); o ins ance, in igu es 2 and 3, he au ho s showed ep esen a i e images and
collec ed size da a bu did no men ion how many independen expe imen s we e done. The
au ho s did no men ione how was he ep oducibili y in his case.
2-Why he assesmen o physical cha ac e is ics o GUVs was no ca ied ou unde oom o
physiological empe a u es o/n? May he empe a u e in luence he indica ed physical
cha ac e is ics o GUVs? Possibly, his could be a leas conside ed as one o he main limi a ions
o his s udy. Please discuss
3- May he au ho s conside he possibili y o use al e na i e ools o de emine sucha as low
cy ome y o assess numbe and size ds ibu ion o newly-gene a ed GUVs?
4- We e he a ios be ween choles e ol and POPC moie is wi hin he ange o physiological?
5- A schema ic lux diag am (new igu e) could be sui able o isualize he echnical p o ocol o
ob ain GUVs.
6- Me hodology, sugges ion: ypan blue dye is commonly used in cell cul u es o assess cell
iabili y; howe e , his dye is also a sui able luo och ome o s aining and isualize GUs unde
con ocal mic oscopy allows high esolu ion and con as , and enables he c ea ion o sha p, high-
esolu ion images and 3D econs uc ions, ha would en ich he analysis o he geome y o
gene a ed GUVs.
7- In ligh o p e ious s udies, he au ho s used he double unsa u a ed DOPC as a ep esen a i e
phospholipid o s udy he impac o inc easingly highe concen a ion choles e ol on memb ane
cha ac e is ics; i is no discussed he whe he o he phospholipids may beha e simila ly in he
o ma ion o GUVs and o s udy he in luence on GUVs ac oss choles e ol concen a ion.
Open Resea ch Eu ope
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Is he wo k clea ly and accu a ely p esen ed and does i ci e he cu en li e a u e?
Yes
Is he s udy design app op ia e and does he wo k ha e academic me i ?
Pa ly
A e su icien de ails o me hods and analysis p o ided o allow eplica ion by o he s?
Yes
I applicable, is he s a is ical analysis and i s in e p e a ion app op ia e?
No
A e all he sou ce da a unde lying he esul s a ailable o ensu e ull ep oducibili y?
No
A e he conclusions d awn adequa ely suppo ed by he esul s?
Yes
Compe ing In e es s: No compe ing in e es s we e disclosed.
Re iewe Expe ise: lipid me abolism
I con i m ha I ha e ead his submission and belie e ha I ha e an app op ia e le el o
expe ise o con i m ha i is o an accep able scien i ic s anda d, howe e I ha e
signi ican ese a ions, as ou lined abo e.
Re iewe Repo 21 July 2025
h ps://doi.o g/10.21956/open eseu ope.20721. 56207
© 2025 Villanue a M. This is an open access pee e iew epo dis ibu ed unde he e ms o he C ea i e
Commons A ibu ion License, which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium,
p o ided he o iginal wo k is p ope ly ci ed.
Ma ín E. Villanue a
1 Uni e si é lib e de B uxelles, B uxelles, Belgium
2 Uni e si é lib e de B uxelles, B uxelles, Belgium
3 Uni e si é lib e de B uxelles, B uxelles, Belgium
In he p esen pape , he au ho s ha e in es iga ed he in luence o choles e ol on he p ope ies
o Gian Unilamella Vesicles (GUVs) p epa ed ia he d ople ans e me hod using a ying
DOPC:choles e ol a ios. They sys ema ically examined how choles e ol con en a ec s GUV size
dis ibu ion, empo al s abili y, and de o mabili y. The s udy combines phase-con as mic oscopy,
magne ic de o ma ion assays using e o luid-loaded esicles, and quan i a i e image analysis o
assess hese physical cha ac e is ics. The esul s show ha inc easing choles e ol con en leads o
Open Resea ch Eu ope
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la ge median diame e s and b oade size dis ibu ions, while s abili y o e ime seems a ec ed in
a composi ion-dependen manne . The au ho s conclude ha choles e ol does no signi ican ly
a ec esicle de o mabili y unde magne ic s ess and p opose ha adjus ing he lipid
composi ion in he oil phase o e s a simple ye e ec i e s a egy o une GUV p ope ies.
While he p oposed app oach has ce ain po en ial, I ha e a ew conce ns on how he esul s a e
displayed and in e p e ed. I will go h ough hem poin by poin :
The ba plo indica ing numbe o esicles a 0 and o e nigh in Figu e 3 indica es ha he
lowes numbe o esicles is p esen ed a 0 by he 85:15 DOPC:Cho mix u e. Howe e , i
one ollows he co esponding pa o he ex in he Resul s sec ion, i is unde s ood ha
he 71:29 mix u e displays he lowes amoun o esicles. One o hem should be co ec ed.
1.
Concluding ha choles e ol has no clea in luence on memb ane igidi y is somewha
p oblema ic, e en i his appea s o be he case o he speci ic sys em s udied. As he
au ho s a e awa e, choles e ol is well known o igidi y lipid bilaye s, a phenomenon
ex ensi ely documen ed in he li e a u e. In ac , one o he ci ed e e ences (Ka al e al.
2022) clea ly highligh s how he bending modulus a ies wi h di e en PC/choles e ol
mix u es, bo h in bilaye s and in GUVs, using a ious expe imen al echniques. Gi en his,
he au ho s should discuss in g ea e de ail he possible sou ces o he obse ed lack o
di e ence in mechanical esponse. Could i be ha he me hod used p ima ily p obes a ea
s ain o su ace de o ma ion, which ela es mo e closely o he a ea comp essibili y
modulus (Ka)? In DOPC/choles e ol sys ems, choles e ol has a s onge e ec on bending
igidi y (κ) han on Ka. I he magne ic ield p ima ily induces shape de o ma ion a he han
su ace comp ession, his may explain he absence o a signi ican choles e ol e ec .
Addi ionally, i he GUV popula ion a ies in size, memb ane p e- ension, o exac
composi ion, such he e ogenei y could obscu e sub le mechanical di e ences due o
choles e ol.
2.
On he o he hand, assuming ha he me hod eliably suppo s he conclusion ha
inco po a ing up o 40 mol% choles e ol does no al e he mechanical p ope ies o he
GUVs, he au ho s p o ide li le cla i ica ion on how he p epa a ion p o ocol i sel migh
in luence hese mechanical ou comes. This aspec is pa icula ly ele an , as he me hod o
GUV o ma ion can signi ican ly a ec memb ane s uc u e and p ope ies. Unlike
elec o o ma ion, he d ople ans e me hod elies on monolaye - o-bilaye assembly, an
in e acially d i en p ocess ha may lead o di e en molecula a angemen s. I is
he e o e plausible ha his echnique esul s in asymme ic memb anes, e en o ela i ely
simple DOPC/choles e ol mix u es. As epo ed by Feigenson e al. (2022, BBA Ac a), such
asymme y can p omo e he o ma ion o induced o de ed domains as a means o minimize
midplane ee ene gy. These s uc u al di e ences could, in u n, impac measu emen s o
bending igidi y and may help explain he absence o signi ican mechanical changes
obse ed in he s udy.
3.
Is he wo k clea ly and accu a ely p esen ed and does i ci e he cu en li e a u e?
Yes
Is he s udy design app op ia e and does he wo k ha e academic me i ?
Yes
A e su icien de ails o me hods and analysis p o ided o allow eplica ion by o he s?
Yes
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I applicable, is he s a is ical analysis and i s in e p e a ion app op ia e?
Pa ly
A e all he sou ce da a unde lying he esul s a ailable o ensu e ull ep oducibili y?
Yes
A e he conclusions d awn adequa ely suppo ed by he esul s?
Pa ly
Compe ing In e es s: No compe ing in e es s we e disclosed.
Re iewe Expe ise: Memb ane biophysics, Biomemb ane models, Physical Chemis y,
Nano echnology.
I con i m ha I ha e ead his submission and belie e ha I ha e an app op ia e le el o
expe ise o con i m ha i is o an accep able scien i ic s anda d, howe e I ha e
signi ican ese a ions, as ou lined abo e.
Au ho Response 06 Aug 2025
Elisa Robe i
1. We hank he e iewe o poin ing ou his e o . The ex has been upda ed in e sion 3.
2.We would like o cla i y ha , as also s a ed in he in oduc ion, he li e a u e does no
p esen a unanimous iew on he e ec o choles e ol on memb ane igidi y in syn he ic
sys ems. While many s udies epo a igidi ying e ec o choles e ol, se e al o he s show
no signi ican in luence, o e en con adic o y esul s. These di e ences appea o depend
no only on he me hod used o measu e mechanical p ope ies (e.g., mic opipe e
aspi a ion, luc ua ion analysis, elec ode o ma ion) bu also on he echnique used o
ab ica e he esicles (e.g., elec o o ma ion s. gen le hyd a ion). Fo his eason, we do no
claim ha choles e ol has no e ec in gene al, bu a he ha in ou speci ic sys em and
unde ou expe imen al condi ions, he e ec , i p esen , is oo small o be de ec ed. Fo
wha conce n he ela ionship be ween su ace de o ma ion and Ka o κ, since no all
memb anes necessa ily exhibi he same ini ial ension, σ can be co ela ed wi h bo h
pa ame e s, Ka and κ. 3.Fi s , as cla i ied in e sion 2 o he manusc ip , he ac ual
choles e ol con en in he memb ane is likely lowe han ha in he ini ial lipid mix u e.
The e o e, we expec no o each 40% choles e ol in he memb ane o GUVs. Mo eo e , we
did no explici ly accoun o he o ma ion o asymme ic esicles. Indeed, since he lipid
solu ions used o bo h he inne and ou e lea le s we e iden ical and composed o a single
phospholipid (DOPC), we a e easonably con iden ha he inal lea le composi ions a e
app oxima ely symme ic.
Compe ing In e es s: No compe ing in e es s we e disclosed.
Re iewe Repo 12 Ap il 2025
h ps://doi.o g/10.21956/open eseu ope.20721. 52406
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© 2025 I ano a N. This is an open access pee e iew epo dis ibu ed unde he e ms o he C ea i e
Commons A ibu ion License, which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium,
p o ided he o iginal wo k is p ope ly ci ed.
Nikole a I ano a
1 Uni e si y o Chemical Technology and Me allu gy, So ia, Bulga ia
2 Uni e si y o Chemical Technology and Me allu gy, So ia, Bulga ia
3 Uni e si y o Chemical Technology and Me allu gy, So ia, Bulga ia
The main goal o he p esen ed s udy was o esea ch he s abilizing e ec o choles e ol a
di e en concen a ions in bilaye s composed o unsa u a ed lipid.
The in oduc ion includes in o ma ion abou lipid bilaye s and hei in ol emen in cell
memb anes. The unc ions o choles e ol and he e ec o he p esence o unsa u a ed lipids such
as DOPC in lipid bilaye s a e discussed. The size and s abili y o Gian Unilamella Vesicles (GUVs)
we e moni o ed wi h Phase-con as mic oscopy, while he de o ma ion was examined unde
magne ic ield condi ions.
The main ocus o my e iew is on he esul s and conclusions ob ained in my scien i ic ield o
wo k ela ed o MD simula ions. The p oposed wo k o e iew is well s uc u ed wi h clea ly
s a ed conclusions, bu he s udied e ec is no ully con i med by he applied me hod. MD
simula ions ha e epo ed he sa u a ion (lack o changes) o some bilaye pa ame e s a
choles e ol con en abo e 30%. I is no ewo hy ha he au ho s did no commen on he phase
s a e o lipids a he ela i ely low empe a u e 4°C. This may also be he eason o he obse ed
weak dependence o s abili y on choles e ol concen a ion.
Sugges ed commen s could be added o he Discussion and conclusions sec ion.
Is he wo k clea ly and accu a ely p esen ed and does i ci e he cu en li e a u e?
Yes
Is he s udy design app op ia e and does he wo k ha e academic me i ?
Pa ly
A e su icien de ails o me hods and analysis p o ided o allow eplica ion by o he s?
Yes
I applicable, is he s a is ical analysis and i s in e p e a ion app op ia e?
Yes
A e all he sou ce da a unde lying he esul s a ailable o ensu e ull ep oducibili y?
Yes
A e he conclusions d awn adequa ely suppo ed by he esul s?
Yes
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Compe ing In e es s: No compe ing in e es s we e disclosed.
Re iewe Expe ise: Molecula Dynamics Simula ion, Physical Chemis y, Compu a ional Chemis y
I con i m ha I ha e ead his submission and belie e ha I ha e an app op ia e le el o
expe ise o con i m ha i is o an accep able scien i ic s anda d, howe e I ha e
signi ican ese a ions, as ou lined abo e.
Au ho Response 17 Jul 2025
Elisa Robe i
We hank he e iewe o he commen and o gi ing us he oppo uni y o cla i y his
concep . DOPC has a low ansi ion empe a u e (Tm = -17°C). A 4°C, DOPC is abo e i s Tm
and hus emains in he liquid-diso de ed (Ld) phase. The ansi ion empe a u e o
phospholipids is in luenced by he p esence o choles e ol bo h in cell memb anes and
syn he ic ones. A high empe a u es, choles e ol ends o educe memb ane luidi y by
in e e ing wi h he mo emen o phospholipids chains, making he memb ane less
pe meable o small molecules. Ins ead, a low empe a u es choles e ol p e en s he
memb ane om becoming oo igid and s i [1]. To da e, no s udies ha e been epo ed in
he li e a u e ega ding he e ec o choles e ol a 4 °C. Ne e heless, i can be
hypo hesized ha choles e ol inc eases memb ane luidi y by p e en ing he igh packing
and c ys alliza ion o phospholipid a y acid chains. Indeed, a low empe a u es, he
p esence o choles e ol p omo es g ea e la e al mobili y o lipids, he eby inhibi ing he
o ma ion o a igid, o de ed s uc u e [2].
In any case, his easoning does no apply o ou liposomes, which a e composed o DOPC
wi h a phase ansi ion empe a u e (Tm) well below 4 °C. As a esul , no phase ansi ion
occu s a his empe a u e, and choles e ol likely does no ac o modula e he memb ane
om an o de ed o a diso de ed s a e in his con ex . Bibliog aphy:
[1] G. M. Coope , “S uc u e o he Plasma Memb ane,” in The Cell: A Molecula App oach.
2nd edi ion, Sinaue Associa es, 2000. Accessed: Jun. 05, 2025. [Online]. A ailable:
h ps://www.ncbi.nlm.nih.go /books/NBK9898/
[2] E. L. CROCKETT, “Choles e ol Func ion in Plasma Memb anes om Ec o he ms:
Memb ane-Speci ic Roles in Adap a ion o Tempe a u e1,” Ame ican Zoologis , ol. 38, no.
2, pp. 291–304, Ap . 1998, doi: 10.1093/icb/38.2.291.
Compe ing In e es s: No compe ing in e es s we e disclosed.
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