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Migration of Deformable Vesicles Induced by Ionic Stimuli Atsuji Kodama, Mattia Morandi, Ryuta Ebihara, Takehiro Jimbo, Masayuki Toyoda, Yuka Sakuma, Masayuki Imai, Nicolas Puff, and Miglena I. Angelova Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b02105 • Publication Date (Web): 29 Aug 2018 Downloaded from http://pubs.acs.org on August 30, 2018

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Migration of Deformable Vesicles Induced by Ionic Stimuli

Atsuji Kodama,† Mattia Morandi,† Ryuta Ebihara,† Takehiro Jimbo,† Masayuki Toyoda,† Yuka Sakuma,† Masayuki Imai,† Nicolas Puff,‡,§ and Miglena I. Angelova‡,§ †

Department of Physics, Graduate School of Science, Tohoku University, Aoba, Aramaki, Aoba, Sendai 980-8578, Japan ‡

§

Laboratoire Matière et Systèmes Complexes, Universitè Paris Diderot, Paris 7, F-75205 Paris Cedex 13, France

Physics Department, Universitè Pierre et Marie Curie, Paris 6, F-75005 Paris, France

KEYWORDS: Giant Unilamellar Vesicle, Phospholipid, Electrolyte, Deformation, Membrane Elasticity Theory, Migration, Diffusiophoresis

ACS Paragon Plus Environment

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Abstract We have investigated the dynamics of phospholipid vesicles composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) triggered by ionic stimuli using electrolytes such as CaCl2, NaCl and NaOH. The ionic stimuli induce two characteristic vesicle dynamics, the deformation due to the ion-biding to the lipids in the outer leaflet of the vesicle and the migration due to the concentration gradient of ions, i.e., diffusiophoresis or the interfacial energy gradient mechanisms. We examined the deformation pathway for each electrolyte as a function of time and analyzed it based on the surface dissociation model and the area difference elasticity model, which reveals the change of the cross section area of the phospholipid by the ion-binding. The metal ions such as Ca2+ and Na+ encourage inward budding deformation through decreasing the cross section area of a lipid, whereas the hydroxide ion (OH-) encourages outward budding deformation through increasing that of a lipid. When we micro-injected these electrolytes toward the vesicles, the strong coupling between the deformation and the migration of the vesicle was observed for CaCl2 and NaOH, while for NaCl, the coupling was very weak. This difference probably originates from the binding constants of the ions.

Introduction Phospholipid vesicles exhibit unique dynamics in response to ionic stimuli through the interactions between ions and lipids. The association of the ion with the head group of the phospholipid induces deformations of vesicles. A typical example is the effect of pH on the vesicle shape [1-6]. By increasing pH of the external solution of phospholipid vesicles, the vesicles deform toward outward budding shape, i.e., increasing the area of outer leaflet, whereas by decreasing pH, the vesicles deform toward stomatocyte shape, i.e., decreasing the area of the outer leaflet [1,2]. These deformations were well explained by an electrostatic spontaneous curvature induced by the adsorption of hydroxide ions on the trimethylammonium group of PC lipid [3]. In addition, when a local pH gradient is applied to anionic phospholipid vesicles, the vesicle membrane forms tubes, where the growth direction of the tube depends on the value of pH [4-6]. The observed membrane deformations are well described by local modifications of the equilibrium lipid density and the spontaneous membrane curvature in the outer leaflet.


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Another well-known deformation induced by the electrolyte is the effect of calcium ion on membrane curvature [7-10]. When giant unilamellar vesicles (GUVs) composed of anionic lipids, such as 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) and phosphatidylinositol-4,5-bisphosphate (PI(4,6)P2), are exposed to the Ca2+ solution, the formation of tubule invaginations is observed, indicating generation of negative spontaneous curvature. On the other hand, by injecting a Ca2+ solution toward a membrane

tether

pulled

from

the

GUV

composed

of

1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) and DOPS, the tether-retraction force and the equilibrium radius of the tether decrease, indicating that Ca2+ generates a positive membrane curvature. Thus, the effect of Ca2+ on the membrane curvature is still controversial. To address the membrane deformation induced by the association of ions, atomistic molecular dynamics (MD) simulations provide a detailed microscopic picture of the phospholipid-ion interactions [11-14]. When metal ions are introduced to the phosphatidylcholine bilayer, the cations are bound to the carbonyl or to the phosphate oxygens of the polar head group. An important prediction of MD simulations is the change of the average area per lipid caused by the ion-binding. The binding cations modify the tilt of the lipid dipole, induce rearrangements around lipid head-groups, and affect the order of acyl chains, resulting in the decrease of the area per lipid. This area modification of the lipid in the outer leaflet induces the spontaneous curvature of the bilayer. In addition to the vesicle deformations induced by the ionic stimuli, the concentration gradient of electrolytes drives phospholipid vesicles due to the diffusiophoresis [15-17] or the interfacial energy gradient mechanisms [18-20]. In the former case, the surface potential of lipid vesicle (usually ~ −10 mV for neutral phosphatidylcholine (PC) lipids [17,21]) enhances the concentration gradient of the electrolyte in the electric double layer, which causes a tangential flow on the vesicle surface (chemiphoresis). Moreover, the asymmetry of the diffusion coefficients between the cation and the anion coupled with the concentration gradient causes the internal electric field (electrophoresis) [22,23]. These two contributions drive vesicles (diffusiophoresis). In the latter case, the electrolytes react with the lipids directly, such as hydrolysis [18], ion exchange [19] or solubilization [20], which causes the interfacial


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energy gradient. The vesicles move toward regions where its interfacial energy would be reduced. A coupling between the deformation and the migration of vesicles induced by the electrolyte stimuli gives a new technique to control the vesicle transportation. If we can control the shape of the migrating vesicle by the ionic stimuli, the vesicles acquire an ability to mitigate the geometrical constraints. In this study we examine the vesicle migration coupled with the deformation. First, to describe the vesicle deformation induced by the ion-binding, we examine deformations of quiescent vesicles by varying the concentration of electrolytes in the external solution (electrolyte penetration experiments). We follow the morphological change of the vesicle in the penetration experiments using a fluorescence microscope. From the 2D microscope images we reconstruct 3D images using the Surface Evolver package (hereinafter called SE) [24], where we assume the axis symmetry of the GUV. The observed morphological change is described by the area difference elasticity (ADE) model [25-27]. With the aid of the ADE model and the ion adsorption model based on the mass action law and the Poisson-Boltzmann equation [28,29], we estimate the area change of lipid induced by the ion-binding. Then we examine the coupling between the deformation and the migration of vesicles by micro-injecting electrolytes toward non-spherical neutral vesicles (electrolyte micro-injection experiment). The concentration gradient generated by the micro-injection drives the deformable vesicles, which is followed by the microscope observation. These investigations will give a physical basis for the vesicle migration coupled with the deformation. Theoretical background for vesicle deformation induced by ion-binding Membrane elasticity theory Here we present a geometrical model to describe the shape deformation of GUV caused by the ion-binding based on the ADE model [25,26], since ADE model well describes vesicle shapes and shape transitions [27,30]. The elastic energy of ADE model normalized by the bending energy of a sphere is expressed by 2 2 W 1 κ c κr = ( 2H ) dA + 2Ad 2 ( ∆A − ∆A0 )  8πκ c 8πκ c  2 ∫ , 2 2 1 κr = ( 2H ) dA + κ ( ∆a − ∆a0 ) 16π ∫ c


(1)

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where κc and κr are the local and the non-local bending moduli, respectively, and H is the mean curvature. The first and the second terms express the binding energy and the area difference elastic energy, respectively. The preferred area difference between the outer and inner leaflet, ∆A0, is given by ∆A0 = (N o − N i )a , where No and Ni are number of lipids in the outer and inner leaflet, respectively, and a is the cross section area of a lipid molecule. The geometrical area difference between the inner and outer leaflet, ∆A, is given by ,

(2)

where d is the distance between the neutral surfaces of the inner leaflet and outer leaflet. The ADE energy arises from the deviation in the geometrical area difference ∆A from ∆A0. The two area differences, ∆A and ∆A0 are normalized by the area difference of a sphere as ∆a =

∆A 8π dR0

∆a0 =

(3)

∆A0 , 8π dR0

(4)

where R0 = A / 4π , V and A are the volume and the area of the vesicle, respectively. According to the ADE model, the vesicle shape is determined by two geometrical parameters, the reduced volume given by v = V /(4π R03 / 3) , and the normalized preferred area difference, ∆a0. A morphology diagram obtained by the ADE model (κr/ κc=3) is shown in Fig. 1. The ratio, κr/κc=3 is usually used to describe phospholipid vesicle morphologies [30]. Solid lines indicate the first-order discontinuous transitions between stomatocyte and oblate (Dsto/obl), oblate and prolate (Dobl/pro), and prolate and pear (Dpro/pea). Two limiting shape lines, stomatocyte limiting shape (inner spherical vesicle is connected to the outer spherical vesicle through a very narrow neck) and budded limiting shape (two spherical vesicles are connected by a very narrow neck), are shown by Lsto and Lbud, respectively. When we add electrolytes into GUV suspension, the ion binding of lipids in the outer leaflet induces the change of the preferred area difference, which causes the deformation of GUVs. The shape transition (e.g. from prolate to oblate) takes place when the deformation trajectory crosses the shape boundary line (e.g. Dobl/pro).


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Ion-binding model We derive the expressions for the preferred area difference caused by the ion binding [28,29]. For simplicity, here we focus on the ion-binding between the neutral phospholipid PC and ion Iw (w is the valence of the ion, e.g., Ca2+: w = +2, Na+: w = +1 and OH−: w = −1.) expressed by

PC+ISw ↔PC-Iw ,

(5)

where ISw is the ion at the vesicle surface, and PC and PC-Iw are the free and the ion-binding PC lipids in the vesicle membrane, respectively. Here we introduce the degree of association, θ, given by (6)

,

where No and NPCI are numbers of lipids and ion-binding lipids in the outer leaflet, respectively. Since the permeability of ions through the phospholipid membranes is order of 10-16 m/sec [31,32], we assumed that the ions can not pass through the lipid bilayer. Thus, we focus on the ion binding to the outer monolayer. By the ion-binding, the cross section area of a lipid molecule increases by a factor of (1+ε). Then the preferred area of the ion-binding outer leaflet, A0o , is given by (7)

.

Since the preferred area of the inner leaflet does not change by the external electrolyte solution, the normalized preferred area difference of the ion-binding vesicle is expressed by ,

(8)

where ∆a0ini is the normalized preferred area difference of the vesicle before ion-binding. The important feature of this ion-binding model [eq. (8)] is that even for the very small degree of association (θ ~10-4), the vesicle shows remarkable deformation owing to the numerical coefficient R0/2d ~ 104 [33]. The ion-binding equilibrium, eq. (5), is governed by the binding constant, w K = [PC-I w] . [PC][Is ]

(9)

According to the Boltzmann law, the surface concentration is expressed by


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.

(10)

where [Iw± ] is the concentration of the ion in the bulk solution, e is the elementary charge, kB is Boltzmann constant, T is the temperature, and ψ(0) is the electrostatic potential at the vesicle surface. Using eqs. (8), (9) and (10), the degree of association is expressed by

θ=

K[I w ]exp{−weψ (0)/ kBT } . 1+ K[I w ]exp{−weψ (0)/ kBT }

(11)

When we add electrolytes in the external solution of the vesicles, the ions bind to the lipids of the vesicle following eq. (11). Since the surface potential is estimated by ζ-potential measurements and the binding constants are obtained from literatures, we can calculate the degree of association θ through eq. (11). The binding of ions to the outer leaflet of the vesicle modifies ∆a0 [eq. (8)], which induces the vesicle deformation according to the phase diagram (Fig. 1). A unique point of the vesicle deformation is that the shape transition takes place at the phase boundary. Thus if we estimate the reduced volume of the vesicle at the shape transition, we can obtain the hidden parameter ∆a0 from the phase diagram, which makes it possible to estimate the change of the cross section area of a lipid, ε, using eq. (8) as shown in the following. Experimental Chemicals. In this study, we used a phospholipid, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC: purity > 99%), purchased from Avanti Polar Lipids Inc. (Alabaster, USA) without further purification. The lipid was dissolved in chloroform at 10 mM and stored at –20°C as stock solution. The vesicles were labeled with a fluorescent phospholipid, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (Rh-DOPE), purchased from Avanti Polar Lipids Inc. (Alabaster, USA). The stock solution of Rh-DOPE dissolved in chloroform at 0.08 mM was stored at –20°C. For electrolyte solutions, we purchased sodium chloride (NaCl), calcium chloride dihydrate (CaCl2·2H2O), and sodium hydroxide (NaOH) in special grade from Wako Pure Chemicals Industries (Osaka, Japan). These chemicals were dissolved in ultrapure water purified with Direct-Q 3 UV (Millipore, USA) at desired concentrations. Preparation of giant unilamellar vesicles.


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GUVs composed of DOPC were prepared using the gentle hydration technique [34,35]. First, the desired amounts of DOPC and Rh-DOPE (0.125 mol% to DOPC) taken from each stock solution were mixed in a glass vial. Then we prepared the thin lipid film in the vial using a nitrogen gas stream with rotating the vial by hand. To remove the organic solvent in the lipid film completely, we put the lipid film under vacuum for 1 day, where we wrapped the sample vial in an aluminium foil. The pre-warmed lipid film was hydrated with 3 ml of ultrapure water at 60 °C for 12 hours, which resulted in the formation of GUVs with radii of 5 − 30 µm. According to the ADE model, the vesicle shape is determined by two geometrical parameters, the normalized preferred area difference, ∆a0, and the reduced volume, v. Since these two parameters of the vesicle are determined at the hydration process in the sample preparation, each vesicle has individual ∆a0 and v. Then, various shapes of vesicles are seen in the vesicle suspension. We selected non-spherical (prolate, oblate, stomatocyte, and tube) vesicles to examine the vesicle migration coupled with the deformation. Here it should be noted that the dye-labeled lipid, Rh-DOPE, does not affect the vesicle migration significantly as demonstrated in the previous paper [16]. For the electrolyte penetration experiments, we used 4°C and 24 hours as the hydration temperature and time, respectively. This hydration condition improved the efficiency for preparing non-spherical vesicles. Electrolyte penetration experiments. To examine the vesicle deformation caused by the ion-binding, we prepared an electrolyte penetration cell as shown in Fig. S1 (Supporting Information, S1). The sample cell was composed of a reservoir chamber and a sample chamber separated by a membrane filter. Both chambers had a thickness of h = 1 mm and a diameter of 6 mm. The membrane filter had a pore size of 0.2 µm (OmniporeTM Membrane Filter JGWP02500, Merck Milipore Ltd, Massachusetts, USA). The sample and reservoir chambers were filled with a suspension of vesicles and a 1 mM electrolyte solution, respectively. Here, since no convection flow in the sample chamber was confirmed by monitoring the motion of small vesicles, we assumed the simple diffusion of the electrolytes in the sample chamber. The osmotic flow of electrolytes through the filter is described by

j = − P∆c ,

(12)


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where j is the molar flux density, ∆c is the difference of molar concentrations between the sample and reservoir chambers, and P is the permeability of the membrane filter [36]. The time dependences of the concentrations in the reservoir, cr(t), and the sample, cs(t), chambers are expressed by

∂cr (t) = − P [c (t)− c (t)] s h r ∂t

(13)

∂cs (t) = − P [c (t)− c (t)] . r h s ∂t

(14)

Then we obtained the concentration of electrolyte in each chamber by

cr (t) =

c0  P  1+ exp(− t) h  2 

(15)

cs (t) =

 c0  1− exp(− P t) , h  2 

(16)

where c0 is the initial concentration of the electrolyte in the reservoir chamber. To estimate the permeability of the electrolytes through the membrane filter, we measured the mean osmolality of the solution in the sample chamber as a function time using osmometer OM815 (Vogel, Germany), where the initial electrolyte solutions in the reservoir chamber were CaCl2 (266 mOsm/l), NaCl (186 mOsm/l), and NaOH (184 mOsm/l). The osmolality in the sample chamber increased with time and reached an equilibrium value at ~1000 sec (Fig. S2 in supporting information S2). From the concentration profile, we obtained the permeability of each ion as PNaCl= 3.63 µm/s, PCaCl2= 2.54 µm/s and PNaOH= 6.56 µm/s. Here we should note that the concentration of the electrolyte in the sample chamber is not homogeneous since the thickness of the sample chamber is 1 mm and the diffusion coefficients of the ions are

DNa+ = 1.334 × 10−9 m 2 /s , DCa2+ = 0.792 × 10 −9 m 2 /s , DOH− = 5.27 × 10−9 m 2 /s , and DCl− = 2.032 × 10 −9 m 2 /s [37]. Then we calculated concentration profiles at position z (the distance from the membrane filter in the thickness direction) and time t in the sample chamber by

Pc0 exp(− P t) 2 h exp  − z  G(z,t) =  4Dt  4π Dt

(17)

t

c(z,t) = ∫ G(z,t − s)ds . 0


(18)

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The calculated concentration profiles for 1 mM CaCl2 at various elapsed times are shown in Fig. S3 (Supporting information, S3). The vesicle deformations induced by various electrolytes in the sample chamber were followed by using an Axio Observer.Z1 inverted fluorescent microscope (Carl Zeiss, Germany) with a 20× objective (LD Plan-Neofluar 20× N.A. = 0.40) and recorded using a CMOS camera, ORCA-Flash 4.0 (Hamamatsu Photonics, Japan) at a time interval of 50 ms. Electrolyte micro-injection experiments. To examine the vesicle migration coupled with the deformation, we performed the micro-injection experiments. The sample chamber for the micro-injection experiments was a hole in a silicone rubber sheet, which was placed onto a glass slide. The hole had a diameter of 12 mm and a thickness of 1 mm. The micro-pipette used for micro-injection was a Femtotips II with an inner diameter of 0.5 µm ± 0.2 µm (Eppendorf, Germany). The position of the micro-pipette was controlled using a hydraulic micro-manipulator MMO-202ND (Narishige, Japan), and the micro-injection was performed using a Femtojet system (Eppendorf, Germany). The vesicle suspension was carefully transferred into the sample chamber from the glass vial at room temperature. The micro-pipette filled with the injection solution was then set into the chamber and kept still for 10 min to equilibrate the sample before each micro-injection experiment. The geometry of the micro-injection experiment is shown in Fig. S4 (Supporting Information, S4). The observed vesicle dynamics in response to the micro-injection was followed by the microscope system described in the penetration experiment section. ζ-potential measurements. The electrostatic potential at the vesicle surface plays a crucial role to estimate the degree of association. As a measure of the surface potential, we measured ζ-potential that is the electrostatic potential at the slipping plane in the electric double layer. For ζ-potential measurements, we used homogeneous large unilamellar vesicle (LUV) suspension, since the GUV suspension contains various small aggregates of lipids. We prepared large unilamellar vesicles (LUVs) in the following manner. The thin lipid film composed of DOPC and Rh-DOPE (0.125 mol% to DOPC) was prepared on the wall of a test tube using a nitrogen gas stream at 50°C and put under a vacuum overnight to


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ensure the complete removal of the organic solvent. The lipid film was hydrated with 3 ml of ultrapure water at 50°C (concentration of DOPC: 1 mM) and then the suspension was vortexed for 1 min and sonicated for 15 min while maintaining the temperature at 50°C, resulting in the formation of multi-lamellar vesicles (MLVs). The MLVs were extruded 21 times through a polycarbonate filter with 100 nm pores using an extruder, Avanti Mini-Extruder purchased from Avanti Polar Lipids Inc. (Alabaster, USA), to prepare LUVs. Pencer et al., reported that using this extrusion technique, the obtained LUVs have spherical shape with the diameter of 60-70 nm and the polydispersity of 0.3-0.4 [38]. The LUV suspensions were mixed with electrolyte solutions at the desired concentrations for the ζ-potential measurements. The ζ-potential measurements were performed using a ζ-potential analyzer, ELSZ-2000 (Otsuka electronics, Japan) at 25°C. The 0.25 mM LUV suspension in predetermined electrolyte solution (0.01 − 10 mM) was loaded in the sample cell for the ζ-potential measurement. We carried out the measurements 6 times for CaCl2 and 3 times for NaCl and NaOH, and the obtained electrophoretic

mobility,

µ,

was

converted

to

ζ-potential,

ζ,

using

Helmholtz-Smoluchowski equation [39] given by

µ=

ε 0ε r ζ. η

(19)

Results and Discussions Vesicle deformation induced by the ion-binding First, to characterize the vesicle deformation induced by the ion-binding, we followed deformations of quiescent vesicles by the penetration experiment. The electrolytes in the reservoir chamber penetrate into the sample chamber and the concentration of the electrolytes in the sample chamber at position z and time t is expressed by eqs. (17) and (18). Here we examined vesicle deformation pathways induced by CaCl2, NaCl, and NaOH solutions. i) Deformation by CaCl2 When 1mM CaCl2 solution in the reservoir chamber penetrated into the sample chamber, a prolate GUV located at z = 0.9 mm showed deformations as shown in Fig. 2(a) and Movie S1 (Supporting Information, S5). The initial prolate vesicle transformed to an oblate shape at ~45 sec, where the origin of time is the onset time of the CaCl2


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solution in the reservoir chamber. Subsequently the oblate vesicle transformed to a stomatocyte shape at ~95 sec and eventually we obtained a stomatocyte-limiting shape vesicle at ~105 sec. Thus, the area of the outer leaflet of DOPC GUV decreases by the ion-binding of CaCl2. We reconstructed the stable 3D vesicle images [Fig. 2(a)] from the observed 2D images by using the SE package, where we assumed that vesicles have stable shapes obtained by minimizing the ADE energy. The reliability of this 3D reconstruction technique was confirmed by comparing the experimental 3D vesicle images obtained by fast confocal microscope imaging with the prediction of ADE model [30,37]. Within the SE package, we evaluated vesicle volume V and area A, as well as the normalized geometrical area difference of the two monolayers ∆a using eqs. (2) and (3). This 3D analysis shows that the reduced volume maintained a constant value of v = 0.86 during our observation (~ 100s), i.e., V = 1550 µm3 and A = 716 µm2 at t = 0 s, and V = 1520 µm3 and A = 705 µm2 at t = 105 s. Due to the osmotic pressure difference between inside and outside of the GUV, the volume of the vesicle decreases with time, which is expressed by dV = −AP v c(z,t) , lip m dt

(20)

where vm (= 18 ×10 −6 m3) is the water molar volume, and Plip (= 4 ×10−5 m/s) is the permeability of the lipid membrane [34]. When the prolate vesicle having V = 1550 µm3 and A = 716 µm2 is subjected to the maximum concentration difference cz(t) = 1 mM, the time needed to change the reduced volume by 1% is ca. 2 ×10 4 sec. Thus, the volume change in this penetration experiment is negligible, which is consistent with our 3D analysis. Figure 3 shows obtained the time dependences of ∆a for the deformation of the prolate vesicle induced by CaCl2 [Fig. 2(a)]. The normalized geometrical area differences, ∆a, showed a stepwise profile. The initial prolate vesicle (t = 0) had values of (v, ∆a) ~ (0.86, 1.07). The vesicle kept the prolate shape for a while and then transformed to an oblate shape with (v, ∆a) ~ (0.86, 1.03) at t ~ 45 s. Subsequently, the oblate vesicle transformed to a stomatocyte shape with (v, ∆a) ~ (0.86, 1.01) at t ~ 95 s. Finally, this stomatocyte vesicle reached a stomatocyte-limiting shape with (v, ∆a) ~ (0.86, 0.69) at ~105 sec. According to our ion-binding model, when the electrolyte concentration in the bulk solution increases, the degree of association, θ, changes continuously depending on the ion concentration and the surface potential [eq. (11)].


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Then, the normalized preferred area difference of the ion-binding vesicle, ∆a0I , changes continuously with maintaining the reduced volume constant [eq. (8)]. When the ∆a0I reaches to the phase boundary (Fig.1), the vesicle shows the discrete change of the normalized geometrical area difference, ∆a. Therefore, the observed discrete shape deformations strongly support that the deformation is governed by the ADE model, where the shape transition takes place when the deformation trajectory in (∆a0, v) plane crosses the shape boundary line [30,40]. Then the observed shape deformation trajectory induced by CaCl2 (v = 0.86) is shown by a red broken line in Fig. 1, from which we can fix the transition points on the discontinuous transition lines in (v, ∆a0) phase diagram, (v, ∆a0pro/obl ) = (0.86, 0.93) for the prolate/oblate transition, (v,

∆a0obl/sto ) = (0.86, 0.48) for the oblate/stomatocyte transition, and (v, ∆a0sto−lim ) =(0.86, 0.30) for the stomatocyte-limiting shape transition. Substituting these values into eq. (8), the effect of the ion-binding of CaCl2 on the cross-section area of a lipid, εCaCl2 , is expressed by ,

(21)

where θsto-lim and θpro/obl are the degrees of association at the stomatocyte-limiting shape

transition, and the prolate/oblate transition, respectively. We estimated the degree of association, θ, at each transition point using eq. (11). The binding constants of the phosphatidylcholine lipid for Ca2+ and Cl− are 40 ± 5 M-1 and 0.20 ± 0.1 M-1, respectively [41], indicating that Ca2+ ions preferentially bind to the head group. Here we assume that the effect of Ca2+-lipid binding on the cross-section area, εCa, is dominant for the observed shape deformation, i.e. εCaCl2 ≈ εCa. We calculated the time dependence of Ca2+ concentration around the GUV located at z = 0.9 mm using eqs. (17) and (18) as shown in Fig. 4, where we adopted DCa2+ = 0.792 × 10 −9 m 2 /s and

PCaCl = 2.54 µm/s. The Ca2+ concentration increases exponentially and the GUV showed 2

the prolate-oblate transition at c = 5 ×10−5 mM (t = 45 s) and the stomatocyte-limiting shape transition at c = 0.005 mM (t = 105 s). The ζ-potential of DOPC vesicle was measured as a function of CaCl2 concentration (Fig. 5). Taking into account that

ζ-potential of DOPC vesicle labeled with Rh-DOPE in pure water is -25 mV, the ζ-potential was not affected by CaCl2 solution for c < 0.01 mM and increased logarithmically in the range of 0.01 – 10 mM. In this penetration experiment, the

ζ-potentials at the prolate-oblate and the stomatocyte-limiting shape transitions had a


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same value of −25 mV. Substituting these values into eq. (11), we obtained the degrees of association as θpro/obl = 1.4 ×10−5 and θsto-lim = 1.45 ×10 −3 . These very small values of

θ justify our simple assumption on the law of mass action for the ideal solution [eq. (9)], where the inter ion-binding-lipid distance is comparable with the Debye screening length. Since the initial prolate vesicle had R0= 5.09 ×10 −6 m and d = 2.26 ×10 −9 m, we calculated ε for Ca2+-lipid ion binding as εCa = −0.40 ( ±0.1 ). The error originates from the ambiguity of the vesicle position in z direction. The analysis of another deformation pathway gave εCa = −0.39 ( ±0.1 ), which shows good reproducibility of our analysis. This experimental lipid area modification parameter by Ca2+ ion binding showed good agreement with the result of the atomistic molecular dynamics simulation, εCa_sim = −0.7 at θ = 0.18 [12]. ii) Deformation by NaCl We set 1 mM NaCl solution in the reservoir chamber and followed the deformation of a prolate GUV located at z = 0.7 mm in the sample chamber. As shown in Fig. 2(b) and Movie S2 (Supporting Information, S6), the initial prolate vesicle transformed to an oblate shape at ~82 sec. Subsequently the oblate vesicle transformed to a stomatocyte shape at ~135 sec and eventually we obtained a stomatocyte-limiting shape vesicle at ~160 sec. This deformation pathway was similar to that induced by CaCl2 as shown in Fig. 2(a), indicating that the area of outer leaflet decreases with the ion binding of NaCl. The reconstructed stable 3D vesicle shapes are displayed in Fig. 2(b). Figure 3 shows the time dependence of ∆a for the vesicle deformation induced by NaCl [Fig. 2(b)], where the reduced volume maintained a constant value of 0.81, i.e., V = 1540 µm3 and at t = 0 s, and V = 1500 µm3 at t = 161 s. The initial prolate vesicle (t = 0) had the values of (v, ∆a) ~ (0.81, 1.11). The prolate vesicle transformed to an oblate shape with (v, ∆a) ~ (0.81, 1.02) at t ~ 82 s. Subsequently, the oblate vesicle transformed to a stomatocyte shape with (v, ∆a) ~ (0.81, 0.81) at t ~ 135 s. Finally, this stomatocyte vesicle reached a stomatocyte-limiting shape with (v, ∆a) ~ (0.81, 0.63) at ~160 sec. The observed shape deformation trajectory induced by NaCl is shown by a green broken line in Fig. 1. Using the reduced volume v = 0.81, we fix the transition points on the discontinuous transition lines in (v, ∆a0) phase diagram, (v, ∆a0pro/obl ) = (0.81, 0.97) for the prolate/oblate transition, (v, ∆a0obl/sto ) = (0.81, 0.60) for the oblate/stomatocyte


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transition, and (v, ∆a0sto−lim ) =(0.81, 0.34) for the invaginated-limiting shape transition. Substituting these values into eq. (8), the effect of the ion-binding of NaCl on the cross-section area of a lipid, εNaCl, is expressed by .

(22)

The binding constants of the phosphatidylcholine lipid for Na+ and Cl are 1.25 ± 0.05 M-1 [42] and 0.20 ± 0.1 M-1 [41], respectively. Taking into account the negative

ζ-potential of DOPC vesicle (−25 mV), we assume that the effect of Na+ - lipid binding on the cross-section area, εNa, is dominant for the observed shape deformation, i.e. εNaCl + ≈ εNa. Then, we calculated Na concentration around the GUV located at z = 0.7 mm

using DNa+ = 1.33 × 10−9 m 2 /s , and PNaCl= 3.63 µm/s, and plotted against time as shown in Fig. 4. The Na+ concentration increases exponentially and then levels off a little bit. The GUV showed the prolate-oblation transition at c = 0.03 mM (t = 82 s), the oblate-stomatocyte transition at c = 0.07 mM (t = 135 s) and the invaginated-limiting shape transition at c = 0.09 mM (t = 160 s). The ζ-potential of DOPC vesicle decreased from −25 mV (0.01 mM) to −30 mV (0.1 mM) and then increased logarithmically to −10mV (10 mM) as shown in Fig. 5. Then the ζ-potentials at the prolate-oblate, oblate-stomatocyte and invaginated-limiting shape transition had −28, −30, and −30 mV, respectively. Substituting these values into eq. (11), we obtained the degrees of association as θpro/obl = 1.1×10 −4 , θobl/sto = 2.8 ×10 −4 and θsto-lim = 3.6 ×10 −4 . Since the initial prolate vesicle had R0 = 4.38 ×10 −6 m, we estimated ε for Na+-lipid ion-binding as

εNa = −2.6, which shows large deviation from the result of the atomistic molecular dynamics simulation, εNa = −0.39 [12]. Then, we performed the NaCl penetration experiments several times and observed similar deformation pathway (prolate → oblate → stomatocyte), which means that the change of the preferred area difference induced by Na+ binding was almost the same as that by the binding of Ca2+ (Fig. 1). On the other hand, the binding constants of Ca2+ and Na+ are 40 M-1 and 1.24 M-1. The large difference in the binding constant is responsible for the value of εNa = −2.6. This

εNa = −2.6 (< −1) indicates that Na+ - lipid binding affects the cross section area of PC lipids surrounding the ion-binding lipid. The effect of the ion-binding on the surrounding lipids becomes more prominent for the low electrolyte concentration (long Debye screening length, λ), which might be responsible for the difference between εNa =


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−2.6 in the experiment (c ~ 0.1 mM: λ ~ 30 nm) and εNa = −0.39 in the simulation (c = 200 mM: λ ~ 0.7 nm). iii) Deformation by NaOH We set 1 mM NaOH solution in the reservoir chamber and followed the deformation of a prolate GUV located at z = 0.7 mm in the sample chamber. As shown in Fig. 2(c) and Movie S3 (Supporting Information, S7), the initial prolate vesicle transformed to a pear shape at ~33 sec and then the pear vesicle transformed to a budded-limiting shape at ~38 sec. Thus, the area of outer leaflet of the GUV increases by the ion-binding of NaOH. The reconstructed stable 3D vesicle shapes are displayed in Fig. 2(c). During the deformation, the reduced volume maintained a constant value of 0.86, i.e., V = 470 µm3 at t = 0 s, and V = 460 µm3 at t = 38 s. Figure 3 shows time dependences of ∆a for the vesicle deformation induced by NaOH [Fig. 2(c)]. The initial prolate vesicle (t = 0) had the values of (v, ∆a) ~ (0.86, 1.07). The prolate vesicle maintained the prolate shape for a while and then transformed to a pear shape with (v, ∆a) ~ (0.86, 1.08) at t ~ 33 s. Subsequently, the pear vesicle transformed to a budded-limiting shape with (v, ∆a) ~ (0.86, 1.28) at t ~ 38 s. The observed shape deformation trajectory agrees with the previous report [1,2] and is indicated by a blue broken line in Fig. 1. Using the reduced volume v = 0.86, we fix the transition points on the discontinuous transition lines in (v, ∆a0) phase diagram, (v, ∆a0pro/pear ) = (0.86, 1.75) for the prolate/oblate transition, and (v,

∆a0bud−lim ) =(0.86, 1.82) for the budded-limiting shape transition. Then the effect of the ion-binding of NaOH on the cross-section area of a lipid, εNaOH, is expressed by .

(23)

We estimated εNaOH in the same ways as the case of Ca2+ ion. The binding constants of the phosphatidylcholine lipid for Na+ is 1.25 ± 0.05 M-1 [42]. To our knowledge, the binding constant for OH

is not reported so far [43], since the surface potential of PC

lipids in monolayers remained unchanged in the region from pH 4 to pH 12 [44,45]. Here we assumed that the binding constant of PC lipids for OH is 10 M 1, since pKa of

choline

is

13.9

[46]

and

the

micelle

of

1,l-phenylhydroxypropyl-2-

dimethylalkylammonium, amphiphilic molecule having choline-like residue, has pKa ≈ 12.7 [47]. Then OH

ions preferentially bind to the head group. In addition, we

assumed that the effect of OH−-lipid binding on the cross-section area, εOH, is dominant


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for the observed shape deformation, i.e. εNaOH ≈ εOH. We calculated time dependence of OH concentration around the GUV located at z = 0.7 mm using

DOH− = 5.27 × 10−9 m 2 /s , and PNaOH= 6.56 µm/s as shown in Fig. 4. The OH concentration increases exponentially and then levels off a little bit. The GUV showed the prolate-pear transition at c = 0.037 mM (t = 33 s) and the budded-limiting shape transition at c = 0.045 mM (t = 38 s). We measured the ζ-potential of DOPC vesicle as a function of NaOH concentration (Fig. 5). The ζ-potential decreased a little bit in the range of 0.01 – 0.1 mM and then increased logarithmically with respect to the concentration. Then both ζ-potentials at the prolate-pear and the budded-limiting shape transitions were estimated to be −30 mV. Substituting these values into eq. (11), we obtained the degrees of association as θpro/obl =1.1 × 10

4

and θbud-lim =1.4 × 10

4

. Since

the initial prolate vesicle had R0= 5.05 ×10 −6 m, we estimated ε for the OH -lipid ion binding as εOH = 2.4. The ion-binding of OH− significantly increases the cross section area of the PC lipid. We also performed the 1 mM NaOH penetration experiment for a long tubular GUV located at z = 0.9mm. The deformation of the long tubular GUV having v = 0.28 and R0= 15.3×10 −6 m was shown in Fig. 6(a) and Movie S4 (Supporting Information, S8). The long tube started to modulate at ~37 sec and then transformed to a chain of spindles at ~39 sec. Eventually the chain of spindles were deformed to a chain of spheres at ~43 sec, where the large spheres were connected by chains of very small spheres. After the formation of the chain of spheres, the vesicle maintained its shape during our observation (until 90 sec). Thus this observation indicates that the tube – chain of spheres (pearling) transition is first-order discontinuous transition, which is caused by the increase of ∆a0 due to the OH - lipid binding. In this case, the initial tubular vesicle had θ ≈ 0 (c ≈ 0) and at t = 43 sec the chain vesicle felt OH

concentration of 0.029

mM, resulting in θ ~ 1.0 ×10−4 . Then, using εOH = 2.4 and R0= 15.3×10−6 m, the change of the preferred area difference by the OH binding is estimated as .

(24)

Here, we calculated stable shapes of the tube GUV by changing ∆a using the SE package. The initial stable tubular GUV with v = 0.28 was obtained by minimizing the bending energy as shown in Fig. 6(b), where the vesicle has ∆atube = 2.67. Then, we increased ∆a from 2.67 to 3.51 (=2.67+0.84) gradually and examined the equilibrium


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vesicle shape. With increasing ∆a, the tubular GUV forms spheres from both ends. The first sphere is formed at the lower end (∆a = 2.77) and the vesicle is composed of a spherical part and a tubular part connected by a narrow neck. The second sphere is formed at the upper end (∆a = 2.84) and the third sphere is formed at the lower end (∆a = 2.91). In this way, spheres are alternately formed at the lower and the upper end sides as shown in Fig. 6(b) and Movie S5 (Supporting Information, S9). Finally the vesicle transforms to the chain of spheres at ∆achain = 3.49, which agrees well with the observed chain of spheres as shown in Fig. 6(a) (t = 44.0 sec). Here we should note that the intermediate vesicle shapes obtained in Fig. 6(b) do not appear in the experiment [Fig. 6(a)], which indicates that the intermediate shapes are metastable due to the area difference elastic energy term. The good agreement between ∆a0tube − ∆a0chain = −0.84 and ∆a tube − ∆achain = −0.82 supports our ion-binding model.

Here we mention about the effect of hydrolysis of PC lipids by OH deformation. It is well known that in the high pH region OH

on the

ions hydrolyze the

glycerol group in PC lipids [48], which might be responsible for the observed vesicle deformation. If the observed deformation is dominated by the hydrolysis, this deformation should be irreversible. To examine this hydrolysis effect, we micro-injected 50 mM NaOH solution to a prolate vesicle as shown in Movie S6 (Supporting Information, S10). The prolate vesicle showed the pearling deformation (deformation to three spheres connected by tethers) immediately after the micro-injection, which is consistent with the above observations. When we stopped the injection, the pearling vesicle recovered the initial prolate shape immediately. Again, this reversible deformation supports our ion-binding model. Migration of deformable vesicles induced by micro-injection of electrolytes In the previous study, we demonstrated that spherical DOPC GUVs migrate toward the tip of micropipette while maintaining the spherical shape when we micro-inject CaCl2, NaCl, and NaOH solutions [17,18]. These migrations are well described by the diffusiophoresis mechanism for CaCl2 and NaCl [17] and the interfacial energy gradient mechanism for NaOH [18]. When the vesicle has some excess area (non-spherical shape), the migration of vesicle might be coupled with shape deformation as described above. It should be noted that in the penetration experiments we observed no vesicle migration. This discrepancy originates from the difference in the concentration gradient,


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since according to the diffusiophoresis and the interfacial energy gradient mechanisms, the migration velocity is proportional to the concentration gradient. In the penetration experiments, 1 mM electrolyte diffused to the sample chamber through the membrane filter and the distance between the membrane filter and the target vesicle was 0.7 – 0.9 mm. On the other hand, in the micro-injection experiments, we injected 50 mM electrolyte to the target vesicle and the distance between the tip of the pipette and the vesicle was approximately 50 µm. Thus, the concentration gradient of ions in the penetration experiment was much smaller than that in the penetration experiment. Here we show the dynamics of the non-spherical GUVs in response to the micro-injection of 50 mM CaCl2, NaCl, and NaOH. When we micro-injected 50 mM CaCl2 to a prolate GUV (Fig. 7(a) and Movie S7 in Supporting Information, S11), the vesicle started to migrate toward the tip of pipette with maintaining the prolate shape until 4.0 sec and then deformed to an oblate vesicle at 7.0 sec. The oblate vesicle continued the migration with slightly elongating to the tip. For the micro-injection to an oblate vesicle, the vesicle showed a transformation to a stomatocyte shape during the migration toward the tip (Fig. 7(b) and Movie S8 in Supporting Information, S12). Thus the preferred area difference of the vesicle, ∆a0, decreases by the micro-injection of CaCl2 solution to the DOPC GUV, which is consistent with the results of the penetration experiments for CaCl2. Thus for the micro-injection of CaCl2, we can couple the diffusiophoresis of vesicle with the vesicle deformation by the ion-binding. On the other hand, when we micro-injected NaCl to a prolate vesicle (Fig. 7 (c) and Movies S9 in Supporting Information, S13) and a stomatocyte vesicles (Fig. 7 (d) and Movies S10 in Supporting Information, S14), both vesicles migrated toward the tip without shape deformation. Thus NaCl did not induce the vesicle deformation during the migration. Recalling that in the penetration experiments, NaCl induces decrease of ∆a0 (prolate to stomatocyte-limiting shape transition), this result indicates no coupling between the diffusiophoresis and the deformation for NaCl. Although more experimental work is necessary, at present we consider that the difference in the binding constant, i.e. K = 40 for Ca2+ and K = 1.25 for Na+, is responsible for the discrepancy. Thus to deform the vesicle, Na+ ion requires a higher concentration than the case of Ca2+. In fact, in the penetration experiments, the transition to the stomatocyte-limiting shape took place at 0.09 mM for Na+, while 0.005 mM for Ca2+. Then in the


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micro-injection experiment, it takes longer time to deform the vesicle by Na+ ions than by Ca2+. In addition, the time constant for the vesicle deformation is order of seconds [30]. Combination of the binding constant and the time constant makes it difficult to deform the vesicle during the vesicle migration to the tip in the micro-injection of NaCl experiment. Finally, we examined the coupling between the vesicle migration caused by the interfacial energy gradient and the vesicle deformation by the ion-binding. When we micro-injected NaOH to a prolate vesicle, the vesicle showed a pearling transition during the migration toward the tip (Fig. 7(e) and Movies S11 in Supporting Information, S15). For the micro-injection to a stomatocyte vesicle, the vesicle transformed to a budded-limiting shape during the migration (Fig. 7(f) and Movie S12 in Supporting Information, S16). Thus ∆a0 of DOPC vesicles increases by the micro-injection of NaOH, which is consistent with the results of the penetration experiments for NaOH. The coupling between the migration and the deformation was observed for NaOH having the binding constant of K~10. Here we should noted that the velocity of the migrating vesicle by the micro-injection of NaOH (Movies S11 and S12) was much faster than those observed for the micro-injections of CaCl2 (Movies S7 and S8) and NaCl (Movies S9 and S10). For example, velocities of migrating non-spherical vesicles just before reaching the tip of the micro-pipette were 32 – 35 µm/s for NaOH, 7 – 10 µm/s for CaCl2, and 8 – 9 µm/s for NaCl. The observed migration velocity for NaOH was consistent with that of spherical vesicles driven by the OH stimulus [18], while the observed migration velocities for CaCl2 and NaCl were roughly one-third of the migration velocity of spherical vesicles driven by the CaCl2 and NaCl stimuli [17]. This difference might originate from the difference in the migration mechanisms, i.e., the interfacial energy gradient for NaOH and the diffusiophoresis for CaCl2 and NaCl, although further quantitative experiments are necessary. Conclusion Ionic stimuli toward phospholipid GUVs induce unique dynamics. The electrolytes bind to the head group of the lipids and modify the cross section area of the lipids in the outer leaflet, which causes the deformation of the vesicle through the change of the preferred area difference, ∆a0. We quantified the effect of the ion-binding on the vesicle deformation based on the area difference elasticity (ADE) model and the surface


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dissociation model using the mass action law and the Poisson-Boltzmann equation. An important theoretical prediction is that even for the very small degree of association (θ ~10-4), the vesicle shows remarkable deformation. In fact our experiments showed that electrolytes at very low concentrations (c ~ 10-2 mM) induces vesicle deformations. In addition, the vesicle deformation depends on the ions. The metal ions such as Ca2+ and Na+ encourage inward budding deformation through decreasing the cross section area of a lipid, whereas the hydroxide ion encourages outward budding deformation through increasing that of a lipid.

Another unique dynamics induced by the ionic

stimuli is the vesicle migration driven by the concentration gradient through the diffusiophoresis or the interfacial energy gradient mechanism. The target of this study is to examine the control of the migrating vesicle shape, i.e. coupling between the migration and the deformation of vesicles. For CaCl2 and NaOH we succeeded to couple the vesicle migration with the deformation, whereas for NaCl vesicles migrated toward the tip of the micro-pipette without deformation. This difference probably originates from the binding constants of ions. Although we need more quantitative studies, this observation opens the door to develop new vesicle based micro-meter size transportation system in which vesicles can change their shapes to fit the geometrical obstacles in the microfluidic circuits.


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ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS publications website at DOI: XXX. Schematic representation of the cell used in the electrolyte penetration experiments, Time dependence of the mean osmolarity in the sample chamber of the penetration experiment for CaCl2, NaCl and NaOH solutions, Time dependence of the concentration profile in the sample chamber for CaCl2, Schematic representation of the micro-injection experiments. (PDF) Movie S1 (AVI) Movie S2 (AVI) Movie S3 (AVI) Movie S4 (AVI) Movie S5 (AVI) Movie S6 (AVI) Movie S7 (AVI) Movie S8 (AVI) Movie S9 (AVI) Movie S10 (AVI) Movie S11 (AVI) Movie S12 (AVI) AUTHOR INFORMATION Corresponding Authors E-mail: [email protected] ORCID Masayuki Imai: orcid.org/0000-0002-1400-7794 Notes The authors declare no competing financial interest.


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Acknowledgement Authors, A. Kodama, Y. Sakuma, and M. Imai, received funding from JSPS KAKENHI Grand Numbers, JP16H02216, K325800233, and JSPS KAKENHI ‘‘Fluctuation & Structure’’ Grand Number JP25103009, and the Core-to-Core Program ‘‘Non-equilibrium dynamics of soft matter and information’’ from JSPS.


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References [1] Farge, E.; Devaux, P. Shape changes of giant liposomes induced by an asymmetric transmembrane distribution of phospholipids. Biophys. J. 1992, 61, 347. [2] Mui, B. L.-S.; Do¨bereiner, H.-G.; Madden, T. D.; Cullis, P. R. Influence of transbilayer area asymmetry on the morphology of large unilamellar vesicles. Biophys. J. 1995, 69, 930. [3] Lee, J. B.; Petrov, P. G.; Döbereiner, H.-G. Curvature of Zwitterionic Membranes in Transverse pH Gradients. Langmuir 1999, 15, 8543-8546. [4] Khalifat, N.; Puff, N.; Bonneau, B.; Fournier, J.-B.; Angelova, M. I. Membrane Deformation under Local pH Gradient: Mimicking Mitochondrial Cristae Dynamics. Biophys. J., 2008, 95, 4924-4933. [5] Fournier, J.-B.; Khalifat, N.; Puff, N.; Angelova, M. I. Chemically Triggered Ejection of Membrane Tubules Controlled by Intermonolayer Friction. Phys. Rev. Lett., 2009, 102, 018102. [6] Bitbol, A. F.; Puff, N.; Sakuma, Y.; Imai, M.; Fournier J.-B.; Angelova, M. I. Lipid membrane deformation in response to a local pH modification: theory and experiments. Soft Matter, 2012, 8, 6073-6082. [7] Chen, Y.-F.; Tsang, K.-Y.; Chang, W.-F.; Fan Z.-A. Differential dependencies on [Ca2+] and temperature of the monolayer spontaneous curvatures of DOPE, DOPA and cardiolipin: effects of modulating the strength of the inter-headgroup repulsion. Soft Matter, 2015, 11, 4041-4053. [8] Simunovic, M.; Lee, K. Y. C.; Bassereau, P. Screening of the calcium-induced spontaneous curvature of lipid membranes. Soft Matter, 2015, 11, 5030-5036. [9] Graber, Z. T.; Shi, Z.; Baumgart, T. Cations induce shape remodeling of negatively charged phospholipid membranes. Phys. Chem. Chem. Phys., 2017, 19, 15285-15295. [10] Doosti, B. A.; Pezeshkian, W.; Bruhn, D. S.; Ipsen, J. H.; Khandelia, H.; Jeffries, G. D. M.; Lobovkina, T. Membrane tubulation in lipid vesicles triggered by the local application of calcium ions. Langmuir, 2017, 33, 11010-11017. [11] Böckmann, R. A.; Hac, A.; Heimburg, T.; Grubmüller, H. Effect of Sodium Chloride on a Lipid Bilayer. Biophys. J. 2003, 85, 1647–1655. [12] Cordomí, A.; Edholm, O.; Perez, J. J. Effect of Ions on a Dipalmitoyl Phosphatidylcholine Bilayer. A Molecular Dynamics Simulation Study. J. Phys. Chem. B 2008, 112, 1397-1408.


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[13] Vácha, R.; Siu, S. W. I.; Petrov, M.; Böckmann, R. A.; Barucha-Kraszewska, J.; Jurkiewicz, P.; Hof, M.; Berkowitz, M. I.; Jungwirth, P. Effects of Alkali Cations and Halide Anions on the DOPC Lipid Membrane. J. Phys. Chem. A 2009, 113, 7235–7243. [14] Kruczek, J.; Chiu, S.-W.; Jakobsson, E.; Pandit, S. A. Effects of Lithium and Other Monovalent Ions on Palmitoyl Oleoyl Phosphatidylcholine Bilayer. Langmuir 2017, 33, 1105−1115. [15] Anderson, J. L. Transport Mechanisms of Biological Colloids. Ann. NY Acad. Sci. 1986, 469, 166–177. [16] Prieve, D. C.; Anderson, J. L.; Ebel, J. P.; Lowell, M. E. Motion of a Particle Generated by Chemical Gradients. Part 2. Electrolytes. J. Fluid Mech. 1984, 148, 247– 269. [17] Kodama, A; Sakuma, Y.; Imai, M.; Kawakatsu,T.; Puff, N.; Angelova, A. I. Migration of Phospholipid Vesicles Can Be Selectively Driven by Concentration Gradients of Metal Chloride Solutions, Langmuir 2017, 22, 19698-10706. [18] Kodama, A.; Sakuma, Y.; Imai, M.; Oya, Y.; Kawakatsu, T.; Puff, N.; Angelova, M. I. Migration of Phospholipid Vesicles in Response to OH− Stimuli. Soft Matter 2016, 12, 2877–2886. [19] Miura, T.; Oosawa, H.; Sakai, M.; Syundou, Y.; Ban, T.; Shioi, A. Autonomous Motion of Vesicle via Ion Exchange. Langmuir 2010, 26, 1610–1618. [20] Igarashi, T.; Shoji, Y.; Katayama, K. Anomalous Solubilization Behavior of Dimyristoylphosphatidyl- Choline Liposomes Induced by Sodium Dodecyl Sulfate Micelles. Anal. Sci. 2012, 28, 345–350. [21] Morini, M.A.; Sierra, M.B.; Pedroni, V.I.; Alarcon, L.M.; Appignanesi, G.A.; Disalvo, E.A. Influence of temperature, anions and size distribution on the zeta potential of DMPC, DPPC and DMPE lipid vesicles. Colloids Surf. B: Biointerfaces 2015, 131, 54–58. [22] Ebel, J. P.; Anderson, J. L.; Prieve, D. C. Diffusiophoresis of Latex-Particles in Electrolyte Gradients. Langmuir 1988, 4, 396–406. [23] Velegol, D.; Garg, A.; Guha, R.; Kar, A.; Kumar, M. Origins of Concentration Gradients for Diffusiophoresis. Soft Matter 2016, 12, 4686–4703. [24] Brakke, K. A., 1992. The Surface Evolver. Exp. Math. 1:141-165; the software package is available at http://www.susqu.edu/brakke/evolver/evolver.html.


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[25] Miao, L.; Seifert, U.; Wortis, M.; Döbereiner, H.-G. Budding transitions of fluid-bilayer vesicles: The effect of area-difference elasticity. Phys. Rev. E, 1994, 49, 5389-5407. [26] Heinrich, V.; Svetina S.; Žekš, B. Nonaxisymmetric vesicle shapes in a generalized bilayer-couple model and the transition between oblate and prolate axisymmetric shapes. Phys. Rev. E, 1993, 48, 3112-3123. [27] Döbereiner, H.-G.; Evans, E.; Kraus, M.; Seifert U.; Wortis, M. Mapping vesicle shapes into the phase diagram: A comparison of experiment and theory. Phys. Rev. E, 1997, 55, 4458-4474. [28] Ninham, B. W.; Parsegian, V. A. Electrostatic potential between surfaces bearing ionizable groups in ionic equilibrium with physiologic saline solution. J. Theor. Biol., 1971, 31, 405-428. [29] Chan, D. Y. C.; Mitchell, D. J.; Ninham, B. W. A self-consistent study of ion adsorption and discrete charge effects in the electrical double layer. J. Chem. Phys. 1980, 72, 5159-5162. [30] Sakashita, A; Urakami, N.; Ziherl, P.; and Imai, M. Three-Dimensional Analysis of Lipid Vesicle Transformations. Soft Matter, 2012, 8, 8569-8581. [31] Deamer, D. W.; Bramhall, J. Permeability of Lipid Bilayers to Water and Ionic Solutes. Chem. Phys. Lipids. 1986, 40, 167-188. [32] Paula, S.; Volkov, A. G.; Van Hoek, A. N.; Haines, T. H.; Deamer, D. W. Permeation of Protons, Potassium Ions, and Small Polar Molecules Through Phospholipid Bilayers as a Function of Membrane Thickness. Biophys. J. 1996, 70, 339-348. [33] Sakuma, Y; Imai, M. Model system of self-reproducing vesicles. Phys. Rev. Lett. 2011, 107, 198101. [34] Reeves, J. P.; Dowben, R. M. Formation and Properties of Thin-Walled Phospholipid Vesicles. J. Cell. Physiol. 1969, 73, 49–60. [35] Akashi, K.; Miyata, H.; Itoh, H.; Kinosita, K. Preparation of Giant Liposomes in Physiological Conditions and Their Characterization under an Optical Microscope. Biophys. J. 1996, 71, 3242–3250. [36] Boroske, E.; Elwenspoek, M.; Helfrich W. Osmotic Shrinkage of Giant Egg-Lecithin Vesicles. Biophys. J. 1981, 34, 95-109.


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[37] Cussler, E. L. Diffusion - Mass Transfer in Fluid Systems; Cambridge University Press: Cambridge, 2009. [38] Pencer, J.; Jackson, A,; Kučerka, N.; Nieh, M.-P.; Katsaras J. The Influence of Curvature on Membrane Domains. Eur. Biophys. J. 2008, 37, 665-671. [39] Aveyard, R.; Haydon, D. A. An introduction of the principles of surface chemistry; Cambridge University Press: Cambridge, 1973. [40] Jimbo, T.; Sakuma, Y.; Urakami, N.; Ziherl, P.; Imai, M. Role of Inverse-Cone-Shape Lipids in Temperature-Controlled Self-Reproduction of Binary Vesicles. Biophys. J, 2016, 110, 1551-1562. [41] Tatulian, S. A. Binding of alkaline-earth metal cations and some anions to phosphatidylcholine liposomes. Eur. J. Biochem. 1987, 170, 413-420. [42] Klasczyk, B.; Knecht, V.; Lipowsky, R.; Dimova, R. Interactions of Alkali Metal Chlorides with Phosphatidylcholine Vesicles. Langmuir 2010, 26, 18951–18958. [43] Petelska reported pK=8.3 for ionized choline group in PC lipid using a titration technique [Petelska, A. D.; Figaszewski, Z. A. Effect of pH on the Interfacial Tension of Lipid Bilayer Membrane. Biophys. J. 2000, 78, 812–817]. Here we did not adopt this value, since pK=8.3 is inconsistent with the other observations [44-46] and our ζ-potential data (Fig. 5). [44] Papahadjopoulos, D. Surface properties of acidic phospholipids: interaction of monolayers and hydrated liquid crystals with uni- and bi-valent metal ions. Biochim. Biophys. Acta 1968, 163, 240-254. [45] Tocanne, J. F.; Teissié, J. Ionization of phospholipids and phospholipid-supported interfacial lateral diffusion of protons in membrane model systems. Biochim. Biophys. Acta 1990, 1031, 111-142. [46] Dawson, R. M. C.; Elliott, D. C.; Elliott, W. H.; Jones, K. M. Data for Biochemical Research Clarendon Press: Oxford, 1959. [47] Bunton, C. A.; Robinson, L. B.; Stam, M. F. Hydrolysis of p-nitrophenyl diphenyl phosphate catalyzed by a nucleophilic detergent. J. Am. Chem. Soc. 1970, 92, 7393-7400. [48] Kensil, C. R.; Dennis, E. A. Alkaline Hydrolysis of phospholipids in model membranes and the dependence on their state of aggregation. Biochemistry 1981, 20, 6079-6085.


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Fig. 1.

Morphology diagram obtained by the ADE model (κr/κc=3). Solid lines

indicate the first-order discontinuous transitions between stomatocyte and oblate (Dsto/obl), oblate and prolate (Dobl/pro), and prolate and pear (Dpro/pea). Two limiting shape lines, stomatocyte limiting shape and budded limiting shape, are shown by Lsto and Lbud, respectively. The schematic image for each morphology is shown in the phase diagram. The broken lines are deformation trajectories for CaCl2 (red), NaCl (green) and NaOH (blue) observed in the penetration experiments.


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Fig. 2. Deformations of DOPC GUVs with prolate shape in the penetration experiments (a): CaCl2, (b): NaCl, and (c): NaOH. The electrolyte was added at 0.0 sec at each observation and the elapsed time from the addition of the electrolyte is shown at bottom-left in each image. The lower column shows 3D vesicle images reproduced by SE program.


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1.3

CaCl2 NaCl NaOH

1.2 1.1

∆a

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 0.9 0.8 0.7 0.6 0

40

80 120 Times (s)

160

Fig. 3 Time dependences of ∆a for CaCl2, NaCl and NaOH in the penetration experiments.


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0.14 0.12 Concentration (mM)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0.1 0.08 0.06 0.04 CaCl2 NaCl NaOH

0.02 0

0

50

100 150 Time (s)

200

250

Fig. 4 Time dependence of ion concentration around the GUV in the penetration experiments calculated using eqs. (17) and (18), CaCl2: Ca2+ at z = 0.9 mm, NaCl: Na+ at z = 0.7 mm, and NaOH: OH at z = 0.7 mm.


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40 CaCl2 NaCl NaOH

30 20 ζ-potential (mV)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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10 0 -10 -20 -30 -40

0.01

0.1 1 Concentration (mM)

10

Fig. 5 Measured ζ-potentials of LUVs composed of DOPC labeled with 0.125 mol% of Rh-DOPE as a function of the concentration of CaCl2 (red), NaCl (green), and NaOH (blue). Each line is obtained by the least-squares approximation.


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(a)

(b)

Fig 6 (a) Deformation of a long tubular GUV having v = 0.28 observed in NaOH penetration experiments. The electrolyte was added at 0.0 sec at each observation and the elapsed time from the addition of the electrolyte is shown at bottom-left in each image. (b) 3D tubular vesicle images calculated by minimization of the bending energy using SE program, where we fix v = 0.28 and varied ∆a=2.67 to 3.51.


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Fig. 7. Dynamics of DOPC GUVs in response to the micro-injections of 50 mM CaCl2: (a) prolate vesicle and (b) oblate vesicle, 50 mM NaCl: (c) prolate vesicle and (d) stomatocyte vesicle, and 50 mM NaOH: (e) prolate vesicle and (f) stomatocyte vesicle, using the injection pressures of 5 hPa for (a), (b), (d) and 15 hPa for (c), (e), (f). The micro-injection started at 0.0 sec in each experiment and the elapsed time from the start of the micro-injection is shown at top-left in each image. The yellow arrow heads show the tips of the micro-pipettes.


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