Migration of Phospholipid Vesicles Can Be Selectively Driven by

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Migration of phospholipid vesicles can be selectively driven by concentration gradients of metal chloride solutions Atsuji Kodama, Yuka Sakuma, Masayuki Imai, Toshihiro Kawakatsu, Nicolas Puff, and Miglena I. Angelova Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b02617 • Publication Date (Web): 12 Sep 2017 Downloaded from http://pubs.acs.org on September 17, 2017

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Migration of phospholipid vesicles can be selectively driven by concentration gradients of metal chloride solutions Atsuji Kodama,† Yuka Sakuma,† Masayuki Imai,*,† Toshihiro Kawakatsu,† 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 vesicle, Diffusiophoresis, Phospholipid, Electrolyte, Concentration gradient, Micro-injection

ACS Paragon Plus Environment

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Abstract

We have investigated the migrations of phospholipid vesicles under the concentration gradients of metal ions. We micro-injected metal chloride solutions, monovalent (NaCl and KCl), divalent (CaCl2 and MgCl2), and trivalent (LaCl3) salts, toward phospholipid giant vesicles (GVs) composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). For NaCl, CaCl2, and MgCl2 solutions, the GVs migrated straight toward the tip of the micro-pipette in response to the concentration gradients, whereas for KCl and LaCl3, GVs moved to the opposite direction. Our motion tracking of lipid domains in a vesicle membrane showed no unidirectional flow in the membrane during the vesicle migration, indicating that the Marangoni mechanism is not responsible for the observed vesicle migration. We calculated the diffusiophoretic velocities for symmetric and asymmetrical electrolytes by solving the Stokes’ equation numerically. The theoretical diffusiophoretic velocities well described the observed migration velocities. Thus we can control the migration of vesicle in response to the concentration gradient by adapting the electrolytes and the lipids.

INTRODUCTION The vesicle is an important carrier in the transportation of biological and artificial wet systems.1,2 For example, in secretory pathway ingredients are loaded into the phospholipid vesicles by the membrane budding-fission process and transported to the destination organism.3 The vesicle based transportation has high potential to establish the drag delivery system, since the phospholipid vesicle has good affinity to the biological systems.4 To deliver the cargos,


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however, the vesicles must be guided to the destination by external signals, such as chemical concentration gradient. So far, the migrations of vesicles triggered by chemical stimuli have been reported for several systems.5–8 When ionic surfactant vesicles composed of didodecyldimethylammonium bromide (DDAB) are mixed with a KI aqueous solution, the iodide anions decompose the membrane to small aggregates, and generate a directional convection on the vesicle membrane. Then the DDAB vesicles start to move using the convection.5 Similarly, heterogeneous solubilization of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) vesicles by addition of sodium dodecyl sulfate (SDS) solution causes motion of the vesicles due to the instability of the surface tension.6 Recently we reported the migration of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) vesicles induced by the micro-injection of NaOH,7 where the hydrolysis of the phospholipids by NaOH solution9 decreases the surface tension of the vesicle. Under the pH gradient induced by the micro-injection of NaOH the vesicles move toward a direction where the surface energy decreases, i.e. high pH region. In these cases, the chemicals react with the lipids and generate the surface tension gradients on the vesicle membrane, which drives the vesicles. Thus the original membranes are irreversibly consumed by the chemical reactions. In general, the motion of particles in response to the concentration gradient of electrolytes that have no specific interactions with the particles has been investigated in detail and well described by the flow in the particle/medium interfacial region.10 When the particles have fluid nature such as a liquid droplet, the surface tension gradient caused by the concentration gradient at the particle surface generates a unidirectional flow of the particle content, which is called the “Marangoni effect”.11 This directional flow drives the particles. For charged solid particles, the concentration gradient of electrolytes causes spontaneous electric field due to difference in the


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diffusion coefficients of the anion and the cation, and the gradient of hydrostatic pressure in the electric double layer, which governs the particle motion, i.e., “diffusiophoresis”.12–16 In the case of vesicles, the internal fluid is enclosed by an incompressible 2D fluid membrane, which suppresses the unidirectional flow at the vesicle surface.17 Thus, vesicles should be driven by the diffusiophoresis mechanism. However, it is well known that electrolyte solutions strongly influence physicochemical properties of biomolecules.18,19 Hofmeister reported the effect of various salts on the aqueous solubility of proteins. On the basis of the magnitude of their effects, the ions have been ordered into sequences, which are called the Hofmeister series. For cations, the strength of salting out of proteins follows the series, K+ > Na+ > Cs+ > Li+ > Mg2+ > Ca2+ > Ba2+. The interactions of metal ions with phospholipid bilayers have been also investigated20,21 and binding constants of ions to phospholipid bilayers were found to follow the Hofmeister series.22,23 The Hofmeister series plays a significant role in a broad range of phenomena in biological systems, although the precise origin of action of the ions in the series has not been clarified yet. Thus such specific interactions between metal ions and lipid membranes might affect the classical diffusiophoresis mechanism in biological systems, i.e. vesicle motions in the concentration gradients of electrolytes. In the present study we examine vesicle migrations induced by the concentration gradients of electrolytes, where the interactions between the membrane and the electrolytes is purely electrostatic.24 We generate the concentration gradients of the metal chlorides by micro-injecting the solutions including monovalent (Na+ and K+), divalent (Ca2+ and Mg2+) and trivalent (La3+) cation toward free standing DOPC giant vesicles (GVs)24 labeled with a fluorescent phospholipid,

1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine

rhodamine

B

sulfonyl) (Rh-DOPE). After the correction of the injection flow contribution, the obtained


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migration velocities are compared with the theoretical prediction based on the diffusiophoresis mechanism.14,15 Here the theoretical migration velocities under the concentration gradients for symmetric and asymmetric electrolytes by solving Stokes’ equation numerically. The systematic and quantitative investigations reveal the mechanism of vesicle migrations induced by the concentration gradients of electrolytes.

THEORY In the diffusiophoresis mechanism, the concentration gradients of electrolytes on a charged solid particle gives rise to an electrostatic stress caused by a difference in the mobilities between the cation and the anion (electrophoresis), and a hydrostatic pressure modulated by the electrostatic potential (chemiphoresis).10,16 The imbalance between the electrostatic stress and the hydrostatic pressure drives the vesicle. Here we assume that the electrolyte is expressed by M X (e.g., CaCl2 has Z+ = 2 and Z− = 1), where Z+ and Z– denote the valences of the cation

and the anion, respectively. The Stokes’ equation that describes the fluid velocity, V is expressed by −∇ + ∇ + − ∇! = 0, (1) where η is the viscosity of the exterior fluid, C+ and C– denote the local number densities of the cation and the anion, respectively, e is the elementary charge, ψ is the electrostatic potential. The coordinates x (parallel to the particle surface) and y (normal to the particle surface) are described in the Supporting Information, S1 and the concentration gradient of the electrolyte is applied to the x direction. The second term and the third term in eq 1 express the hydrostatic pressure and the electrostatic body force, respectively. At equilibrium, the ion distribution is expressed by ± , = ∓ exp∓± , ,

(2)


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! , − ! , (3) #$ % where kB is the Boltzmann constant, and T is the absolute temperature. Φ denotes the normalized , =

electrostatic potential expressed by eq 3, where ψ∞ denotes the electrostatic potential at y = ∞. As y → ∞, the number densities of the cation and the anion approach to Z–C∞(x) and Z+C∞(x), respectively, where C∞(x) denotes the unperturbed number density of the electrolyte. Since we focus on x-component of the velocity, the hydrostatic-pressure term is estimated by integrating eq (1) with respect to y-coordinate using eq (2) and we obtain the x-component of the Stokes’ equation by substitute the hydrostatic-pressure term.12 d '( d! = − d d

d + #$ %) exp − − 1 + exp − 1+ . d The fluxes of the cation and the anion are given by the Nernst-Plank equation

(4)

. . ∇!2, (5) #1 % where i = + and – denote the cation and anion, respectively, and D+ and D– are the diffusion -. = −/. 0∇. +

coefficients of i species. In an electrically neutral solution, Z+N+ = Z−N−, the gradient of the electrostatic potential is expressed by d! #$ % d =3 d d / − / 3= , / + / where β is the diffusivity difference factor. Using eq 6, eq 4 yields −

(6) (7)

d '( − d = −3#$ % d d

(8) d + #$ %) exp − − 1 + exp − 1+ d In addition based on the Poisson-Boltzmann equation, the relationship between the normalized electrostatic potential, Φ, and the distance from the particle surface, y, is expressed by


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99%), and cholesterol (ovine wool, purity > 98%) purchased from Avanti Polar Lipids Inc. (Alabaster, USA) were used to prepare vesicles without further purification. These chemicals were dissolved in chloroform at 10 mM and stored at –20°C as stock solutions. The vesicles were labeled with a fluorescent phospholipid, 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 metal chloride solutions used in this study we purchased sodium chloride (NaCl), potassium chloride (KCl), calcium chloride dihydrate (CaCl2·2H2O), magnesium chloride hexahydrate (MgCl2·6H2O), and lanthanum chloride heptahydrate (LaCl3·7H2O) 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. To visualize the injection flow, we used spherical polystyrene latex particles with a radius of Rc = 1.5 µm purchased from Magsphere Inc. (Pasadena, USA). To remove preservative (0.1% sodium azide)


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we dialyzed the particle suspension using cellulose tubes with a pore size of ∼ 50 Å (Viskase Companies Inc., Darien, USA). The polystyrene latex particles were also used to examine the diffusiophoresis of the solid colloid particles. Preparation of giant vesicles. GVs composed of DOPC were prepared using gentle hydration technique.26,27 First, the desired amounts of DOPC (1 µmol) 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 GVs with radii of 5 − 30 µm. To examine the membrane flow in a vesicle, we prepared the ternary GVs composed of DOPC/DPPC/cholesterol (3/1/1; molar ratio) using the same protocol as described above, which shows the phase separation at 25°C. The motion of liquid-ordered (Lo) domains was visualized by the fluorescent phospholipid, Rh-DOPE at a concentration of 0.375 mol% to the lipids, which is partitioned into liquid-disordered (Ld) phase. ζ-potential measurements. The electrostatic potential at the vesicle surface plays a crucial role in the diffusiophoresis mechanism. 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 prepared large unilamellar vesicles (LUVs). 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 ensure the complete removal of


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the organic solvent. The lipid film was vortexed for 1 min and sonicated for 15 min both at 50°C in an ultrapure water at a concentration of 1 mM of DOPC, resulting in the formation of multilamellar 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. The LUV suspensions were mixed with metal chloride 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 LUV suspension at 0.25 mM concentration in metal chloride solution (0 − 10 mM) was loaded in the sample cell for the measurement. We carried out the measurement 3 times for each sample and the obtained electrophoretic mobility, µ, was converted to ζ-potential,

ζ, using Helmholtz-Smoluchowski equation28 given by

89 8: E. (15) The LUVs of DOPC labeled with Rh-DOPE in pure water had the ζ-potential of –25 mV. It M=

should be noted that the ζ-potential of DOPC vesicles without Rh-DOPE in pure water was –15 mV. We examined the effect of the charged dye on the vesicle migration. By the micro-injection of the metal chloride solutions, the pure and the labeled GVs showed the similar migration behavior but the labeled GVs had faster migration velocities than those of the pure GVs. In this study, we used labeled GVs, since we expected two advantages; i) The labeled vesicles have larger migration velocities than the unlabeled vesicles due to their surface potentials, which decreases the contribution from the injection flow, and ii) the fluorescence images make it easy to measure the velocities of the migrating vesicles. The comparison of the migration velocities between the GVs with and without the dye is described in the Supporting Information, S2. Micro-injection experiments.


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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 9 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 viral at room temperature. The micro-pipette filled with the injection solution was then set into the chamber and waited for 10 min to equilibrate the sample before each micro-injection experiment. The geometry of the microinjection experiment is shown in Figure S4 in the Supporting Information, S3. In the microinjection experiments, we have to minimize the drift of vesicles caused by the injection flow. For this purpose we controlled the injection using the compensation pressure pc = 5 hPa of the Femtojet system. However, at the minimized injection condition the compensation pressure was unstable. Thus the injection flow generated by the micro-injection varied even if we used the same pc = 5 hPa and the observed vesicle migration velocity was affected by the injection flow. Then we controlled the injection flow by monitoring the compensation pressure and checked the injection flow by eye. We visualized the stable injection flow using colloids dispersed in the medium and estimated the velocity of the injection flow around the tip of the micro-pipette. To obtain velocities of migrating vesicles, we corrected a contribution of the stable injection flow. Details of the correction procedure were described in the Supporting Information, S4.7 The observed vesicle dynamics in response to the micro-injection was followed by using an Axio Observer.Z1 inverted fluorescent microscope (Carl Zeiss, Germany) with a 20× objective (LD


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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. Concentration profile of electrolytes generated by micro-injection. After the correction of the injection flow, the unperturbed electrolyte concentration, C∞, around the tip of the micro-pipette is approximately governed by diffusion process. Here we calculated a time evolution of the concentration profile around the tip of the pipette by solving the diffusion equation with convoluting the inner radius of the pipette, ε, as ′, ′, O′, P ^ ] \

= 444

Q9 exp I−

R − ′9 +

′ − S cos W + O′ − S sin W K 4/ P − Y [ Y

SdSdWdY,

(16)

4Z/ P − 9 9 9 where D = (Z++Z–)/(Z–/D++Z+/D–) is the diffusion coefficient of the solute, S0 is the flux of the injected solution from the pipette, and ξ and χ (polar coordinates) designate the position in the pipette mouth.7 Then we obtained a relationship, S0 = 2DC0/ε. The concentration profile reaches its steady state within ∼ 1.0 sec. Since it took ∼ 1.0 sec to start the migration of vesicles after the micro-injection, we expressed the solute concentration profile around the tip of the pipette by the steady state solution, \ ]

Q9 SdSdW ′, ′, O′ = 44 . (17) +

′ − S cos W + O′ − S sin W 4Z/ 5 ′ − ′ 9 9 9 We calculated the steady state profiles in the case of micro-injections of NaCl, KCl, CaCl2, and LaCl3 at the concentration of 10 mM. The flux S0 is related to the concentration of the injecting solution by S0 = 2DC0/ε, since eq (17) should give C0 at the tip of the micro-pipette, i.e., C∞ (0, 0, z’→0) = S0ε/(2D) = C0. Then the concentration profile is independent of the diffusion coefficients, i.e. injecting electrolytes. The steady concentration profiles with C0 = 10 mM and ε = 0.25 × 10−6 m is shown in Figure 2.


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RESULTS AND DISCUSSION Migration of DOPC vesicles induced by micro-injection of metal chloride solutions. According to the diffusiophoresis mechanism, vesicles in the concentration gradients of electrolytes show migrations depending on the surface potential, ζ0 and the diffusivity difference factor, β.10,13 Here we examined the migration of vesicles induced by micro-injections of various types of electrolytes, symmetric monovalent salts NaCl, and KCl, asymmetric divalent salts CaCl2, and MgCl2, and asymmetric trivalent salt LaCl3. The cationic metal ions modify the surface potential through the binding to the head group of the phospholipid, since in pure water the DOPC vesicle has the ζ-potential of −25 mV. The diffusivity difference factors for the examined salts are given by β = −0.207 (NaCl), −0.0188 (KCl), −0.343 (CaCl2), −0.390 (MgCl2), and −0.363 (LaCl3) using the diffusion coefficient of each ion, /_` = 1.334 × 10–9 m2/s, /a = 1.957 × 10–9 m2/s, /b`c = 0.792 × 10–9 m2/s, /dec = 1.334 × 10–9 m2/s, /f`g = 0.619 × 10–9

m2/s and /bh = 2.032 × 10–9 m2/s.29

The observed typical dynamics of DOPC GVs in response to the micro-injections of various metal chlorides at 10 mM are shown in Figure 3a–e, where the injection flux was kept constant for each salt. For the micro-injections of NaCl [Figure 3a: Movie S1 in the Supporting Information, S5], CaCl2 [Figure 3b: Movie S2 in the Supporting Information, S6], and MgCl2 [Figure 3c: Movie S3 in the Supporting Information, S7] the GVs and small lipid aggregates started to migrate straight toward the tip of the micro-pipette (high concentration side) with gradually increasing their velocities in ∼ 1.0 sec after we started the micro-injections. We reported similar vesicle migration induced by the micro-injection of NaOH.7 The most significant difference of the vesicle migration between NaOH and NaCl is the strength of the


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pulling force. In the case of NaOH, vesicles having excess areas protrude the tubes toward the tip of the micro-pipette during the migrations, whereas for NaCl, non-spherical vesicles migrate toward the tip maintaining the initial shapes. This difference originates from the hydrolysis of lipids by hydroxide ions. On the other hand, for KCl [Figure 3d: Movie S4 in the Supporting Information, S8] and LaCl3 [Figure 3e: Movie S5 in the Supporting Information, S9], GVs moved to the opposite side of the micro-pipette, i.e., low concentration side. Thus the vesicle motions induced by the micro-injections strongly depend on the electrolytes. It should be noted that in the previous paper7 the migrations of vesicles induced by monovalent ions were hidden by the strong injection flows. In this study we paid much attention to reduce the injection flow (Supporting Information, S10). Before we explain the observed vesicle migrations based on the diffusiophoresis mechanism, we examine the effect of the micro-injection on the membrane flow, i.e., the possibility of the Marangoni effect. Membrane flow during vesicle migration. If the micro-injection of the electrolyte induces the symmetry breaking membrane flow in the vesicle, the directional flow drives the vesicle. To examine the flow in the vesicle membrane induced by the micro-injection, we prepared phase separated vesicles composed of DOPC/DPPC/Cholesterol = 3/1/1 (molar ratio) labeled with Rh-DOPE, where the dye is expelled from the Lo phase. At the room temperature, the ternary vesicles have small circular Lo domains as shown in Figure 4.30 We can visualize the membrane flow through the motion of the domains.30 Before the micro-injection the small domains show Brownian motion on the vesicle surface [traces of domain motions are shown by solid lines in Figure 4a and Figure 4b]. By micro-injecting 10 mM MgCl2, the GVs migrated toward the tip of a micro-pipette, whereas Lo domains on the vesicle continued the random motion as shown in Figure 4c and Figure 4d


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(Movie S6 in the Supporting Information, S11). Thus no asymmetric directional flow was induced by the micro-injection, which indicates that the Marangoni effect is not responsible for the observed vesicle migration. Migration velocity analyzed by diffusiophoresis mechanism. To examine the observed vesicle migration based on the diffusiophoresis mechanism, we estimated the migration velocities of vesicles. In the measurements we focused on the isolated vesicles with the radii of approximately 5 µm located on the pipette axis (e.g., x’ axis in Figure S4) to avoid interference from neighbor vesicles. When the vesicles were subjected to the microinjection, they moved straight on the x’ axis (e.g., Movie S1 in the Supporting Information, S5). We measured the velocities of migrating vesicles in the distance range between 5 µm (marked as A in Figure 2) and 35 µm (marked as B in Figure 2) from the tip of the micro-pipette, where the vesicles feel the same concentration gradient. In the experiments the vesicles migrated from A to B for KCl and LaCl3 (moving away from the tip) and from B to A for NaCl and CaCl2 (approaching the tip). By subtracting the contribution of the injection flow from the measured migration velocity, we obtained the corrected migration velocity of the migrating vesicle. Figure 5a shows the corrected migration velocities of GVs induced by the micro-injections of NaCl, KCl, CaCl2, and LaCl3 as a function of the distance between the tip of the micro-pipette and the center of the vesicles, x’0. For the micro-injections of NaCl and CaCl2 the velocities of the migrating DOPC GVs exponentially increased as the GVs approached the tip and reached the maximum velocities of 40 ∼ 50 µm/s before they touched the pipettes. In the case of KCl, the GVs moved to a low concentration side but after the correction of the injection flow, the migration velocities had small positive values of several µm/s (move to high concentration side). On the other hand, for LaCl3 the corrected migration velocities of GVs had negative values of ∼


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−10 µm/s (move to low concentration side). To examine the corrected migration velocities based on the diffusiophoresis mechanism, we plotted them against dlnC∞/dx’0 in Figure 5b. We expressed the unperturbed concentration gradient of the vesicle, dlnC∞(x’0)/ dx’0, by averaging the concentration gradient all over the surface of the vesicle, since dlnC∞/dx’0 depends on the position of the vesicle surface. Here the convection due to the vesicle migration did not affect the unperturbed electrolyte concentration profile, C∞(x’) (Supporting Information, S12). The plots show good linear relationships between the migration velocities and dlnC∞/dx’0 for all examined metal chloride solutions, which agrees well with the predictions by the diffusiophoresis mechanism [eq 13]. The slopes of the plots are determined by the diffusivity difference factor, β, and the surface potential, ζ0. Since in our experiments the values of β are given by −0.207 (NaCl), −0.0188 (KCl), −0.343 (CaCl2), and −0.363 (LaCl3), the fitting parameter to describe the profile is only ζ0. We described the migration velocity profile [Figure 5b] using eqs 8, 9, and 14 by tuning the value of ζ0. It should be noted that ζ0 depends on the concentration of the metal chloride ion around the vesicle, i.e., distance from the tip of the pipette. For simplicity we assumed that the vesicle at distance x’0 has a ζ-potential ζ0(x’0) obtained by averaging the ζpotential all over the surface of the vesicle. The theoretical diffusiophoretic velocity profile for each metal chloride solution is shown by solid lines in Figure 5b. The theoretical velocity profiles well describe the corrected migration velocities. The estimated surface potentials, ζ0, used for the calculations of the diffusiophoretic velocities [solid lines in Figure 5b] are plotted as a function of the metal chloride concentration in Figure 6. As a measure of the surface potential, we measured ζ-potentials of DOPC vesicles in NaCl, KCl, CaCl2, and LaCl3 solutions as a function of their concentrations, which are also plotted in Figure 6. The measured ζ-potentials substantially agree with the surface potential profiles predicted


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from the diffusiophoresis mechanism, although we observed small deviations, i.e., −20 mV for both NaCl and CaCl2. To examine the validity of these analysis we performed control experiments on migrations of solid particles induced by micro-injections of electrolytes (Supporting Information, S13). For solid particles, the migrations under the concentration gradients of electrolytes are governed by the diffusiophoresis mechanism.14,15 In our experiments, the migration velocities of polystyrene latex particles (diameter = 3 µm) induced by the injections of NaCl and CaCl2 were well described by the diffusiophoresis mechanism assuming the concentration dependence of the surface potential, ζ0 (Figure S9 in the Supporting Information, S13). Then, we measured ζ-potentials of polystyrene latex particles in NaCl, and CaCl2 solutions as a function of their concentrations. We observed similar small deviations between the measured ζ-potential and the estimated surface potential, ζ0, i.e., −10 mV for NaCl and −15 mV for CaCl2. The agreement in the deviations between the vesicle migration and the solid particle migration indicates that the deviation originates from the technical issues, such as errors in the estimations of the injection flow, the concentration gradient of electrolytes, and the ζ-potential. Although we cannot fix contributions of these factors quantitatively, the agreements between the measured ζ-potential profiles and the surface potential profiles estimated from the diffusiophoresis mechanism support that the diffusiophoresis mechanism is responsible for the observed vesicle migrations. The diffusiophoretic velocity is composed of two contributions, “electrophoresis” and “chemiphoresis”. The electrophoretic mechanism is based on the electric field that is generated spontaneously when a concentration gradient of an electrolyte is established. This field is caused by unequal diffusion coefficients of the ions and expressed by


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/ − / dln #$ % dln = 3 (18) / + / d d Then the vesicle with the surface potential, ζ0, is driven by this electric field. The chemiphoretic i =

#$ %

mechanism is caused by the hydrostatic pressure gradient in the electrical double layer. In the electrical double layer on a charged surface, the pressure is higher than in the bulk due to the attraction of ions. Then a concentration gradient tangential to the surface results in a gradient of pressure, which causes a tangential flow in the electric double layer. In general, chemiphoretic effect always directs the vesicle toward the direction of higher electrolyte concentration, whereas electrophoretic contribution can move the vesicle in both directions depending on the sign of

βζ0. Since the calculated chemiphoretic velocities for our systems are order of several µm/s, the observed migration velocities with several tens of µm/s are governed by the electrophoresis mechanism. All electrolytes used in this study have negative β [−0.207 (NaCl), −0.0188 (KCl), −0.343 (CaCl2), −0.390 (MgCl2), and −0.363 (LaCl3)], which indicates that the migration direction is determined by ζ0. The positive surface potential for LaCl3 is responsible for the observed negative migration velocity and the small diffusivity difference factor for KCl accounts for the observed small migration velocity.

CONCLUSIONS To drive vesicles in response to the concentration gradient of the electrolyte is an important technique to establish vesicle based transport systems. We have two mechanisms to migrate vesicles under the concentration gradient of electrolytes having no specific interactions, the Marangoni effect and the diffusiophoresis. The diffusiophoresis is a plausible candidate to drive the vesicle, since the internal fluid is enclosed by an incompressible 2D fluid membrane. Phospholipids, however, frequently show specific interactions with electrolytes, so-called


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Hoffmeister series. Then it is not trivial that the phospholipid vesicles follow the diffusiophoresis mechanism. In this study we demonstrated that the migration of the phospholipid vesicle is induced by the micro-injection of various metal chloride solutions. For NaCl, KCl, CaCl2, and MgCl2 DOPC vesicles move to the higher concentration direction, whereas for LaCl3 DOPC vesicles move to the lower concentration direction. These characteristic migration behaviors are well described by the diffusiophoresis mechanism, where the surface electrostatic potential and the asymmetry in diffusion coefficients of the cation and the anion in the electrolyte determine the migration velocity. Thus the specific interactions between the lipid membrane and the electrolytes (Hofmeister series) do not affect the vesicle motion significantly. Although more systematic experimental work including negatively and positively charged lipids is necessary, we can control vesicle migration by tuning the surface potential and the diffusion coefficients of ions, i.e., by adapting electrolytes and lipids. Our observation gives a fundamental understanding to develop more sophisticate vesicle motion, such as self-diffusiophoresis vesicles.32


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ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS publications website at DOI: XXX. Coordinates for theoretical description of diffusiophoresis, Comparison of migration velocities between labeled and non-labeled GVs, Coordinates for micro-injection experiments, Estimation of injection flow induced by micro-injection, Explanations of movies of migrating vesicles under micro-injections of NaCl, MgCl2, CaCl2, KCl, and LaCl3, Diffusiophoretic motions of vesicles under micro-injections of monovalent salt, Explanations of movie on motion of Lo domains on DOPC/DPPC/Cholesterol vesicle in response to micro-injection of MgCl2 solution, Perturbation of concentration profle caused by migrating vesicle, Migration of solid particles induced by micro-injection of electrolytes. (PDF) Movie S1 (AVI) Movie S2 (AVI) Movie S3 (AVI) Movie S4 (AVI) Movie S5 (AVI) Movie S6 (AVI) AUTHOR INFORMATION


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Corresponding Authors E-mail: [email protected] ORCID Masayuki Imai: orcid.org/0000-0002-1400-7794 Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by 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) Anderson, J. L. Transport Mechanisms of Biological Colloids. Ann. NY Acad. Sci. 1986, 469, 166–177. (2) Allen, R. D. The Microtubule As an Intracellular Engine. Sci. Am. 1987, 256, 42–49. (3) Alberts, B., Johnson, A., Lewis, J., Morgan, D., Raff, M., Roberts, K., Walter, P., Eds.; Molecular Biology of the Cell 6th ed.; Garland Science: New York, 2014. (4) Allen, T. M.; Cullis, P. R. Liposomal Drug Ddelivery Systems: From Concept to Clinical Applications. Adv. Drug Deliv. Rev. 2013, 65, 36-48. (5) Miura, T.; Oosawa, H.; Sakai, M.; Syundou, Y.; Ban, T.; Shioi, A. Autonomous Motion of Vesicle via Ion Exchange. Langmuir 2010, 26, 1610–1618. (6) 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. (7) 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. (8) Nawa, E.; Yamamoto, D.; Shioi, A. Chemotactic Amoeboid-Like Shape Change of a Vesicle under a pH Gradient. Bull. Chem. Soc. Jpn. 2015, 88, 1536–1544.


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(9) 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. (10) Anderson, J. L. Colloid Transport by Interfacial Forces. Annu. Rev. Fluid Mech. 1989, 21, 61–99. (11) Young, N. O.; Goldstein, J. S.; Block, M. J. The Motion of Bubbles in a Vertical Temperature Gradient. J. Fluid Mech. 1959, 6 (3), 350–356. (12) Anderson, J. L.; Lowell, M. E.; Prieve, D. C. Motion of a Particle Generated by Chemical Gradients Part 1. Non-Electrolytes. J. Fluid Mech. 1982, 117, 107–121. (13) 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. (14) Lechnick, W. J.; Shaeiwitz, J. A. Measurement of Diffusiophoresis in Liquids. J. Colloid Interface Sci. 1984, 102 (1), 71–87. (15) Ebel, J. P.; Anderson, J. L.; Prieve, D. C. Diffusiophoresis of Latex-Particles in Electrolyte Gradients. Langmuir 1988, 4, 396–406. (16) Velegol, D.; Garg, A.; Guha, R.; Kar, A.; Kumar, M. Origins of Concentration Gradients for Diffusiophoresis. Soft Matter 2016, 12, 4686–4703. (17) Landau, L. D.; Lifshitz, E. M. Fluid Mechanics 2nd ed. Course of Theoretical Physics Vol. 6; Butterworth-Heinemann: Oxford, 1987. (18) Cacace, M. G.; Landau, E. M.; Ramsden, J. J. The Hofmeister Series: Salt and Solvent Effects on Interfacial Phenomena. Q. Rev. Biophys. 1997, 30, 241–277.


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(19) Kunz, W.; Henle, J.; Ninham, B. W. “Zur Lehre von Der Wirkung Der Salze” (about the Science of the Effect of Salts): Franz Hofmeister’s Historical Papers. Curr. Opin. Colloid Interface Sci. 2004, 9, 19–37. (20) Leontidis, E.; Aroti, A.; Belloni, L.; Dubois, M.; Zemb, T. Effects of Monovalent Anions of the Hofmeister Series on DPPC Lipid Bilayers Part II: Modeling the Perpendicular and Lateral Equation-of-State. Biophys. J. 2007, 93, 1591–1607. (21) Vácha, R.; Siu, S. W. I.; Petrov, M.; Böckmann, R. A.; Barucha-Kraszewska, J.; Jurkiewicz, P.; Hof, M.; Berkowitz, M. L.; Jungwirth, P. Effects of Alkali Cations and Halide Anions on the DOPC Lipid Membrane. J. Phys. Chem. A 2009, 113, 7235–7243. (22) Eisenberg, M.; Gresalfi, T.; Riccio, T.; McLaughlin, S. Adsorption of Monovalent Cations to Bilayer Membranes Containing Negative Phospholipidst. Biochemistry 1979, 18, 5213–5223. (23) Klasczyk, B.; Knecht, V.; Lipowsky, R.; Dimova, R. Interactions of Alkali Metal Chlorides with Phosphatidylcholine Vesicles. Langmuir 2010, 26, 18951–18958. (24) 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. (25) Most of examined GVs were giant unilamellar vesicles (GUVs) and no significant difference between GVs and GUVs was observed in the migration behavior. (26) Reeves, J. P.; Dowben, R. M. Formation and Properties of Thin-Walled Phospholipid Vesicles. J. Cell. Physiol. 1969, 73, 49–60.


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(27) 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. (28) Aveyard, R.; Haydon, D. A. An introduction of the principles of surface chemistry; Cambridge University Press: Cambridge, 1973. (29) Cussler, E. L. Diffusion - Mass Transfer in Fluid Systems; Cambridge University Press: Cambridge, 2009. (30) Veatch, S. L.; Keller, S. L. Separation of Liquid Phases in Giant Vesicles of Ternary Mixtures of Phospholipids and Cholesterol. Biophys. J. 2003, 85, 3074–3083. (31) Dimova, R.; Bezlyepkina, N.; Jordö, M. D.; Knorr, R. L.; Riske, K. A.; Staykova, M.; Vlahovska, P. M.; Yamamoto, T.; Yang, P.; Lipowsky, R. Vesicles in Electric Fields: Some Novel Aspects of Membrane Behavior. Soft Matter 2009, 5, 3201–3212. (32) Gupta, S.; Sreeja, K. K.; Thakur, S. Autonomous Movement of a Chemically Powered Vesicle. Phys. Rev. E - Stat. Nonlinear, Soft Matter Phys. 2015, 92, 1–8.


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Figure 1. Normalized electrostatic potential and relative velocity for asymmetric electrolyte. (a) Normalized electrostatic potential, Φ, as a function of the distance from the particle surface, y, for various normalized surface potentials Φ0 = 1.97, 0.97, 0.00, –0.97, and –1.97. (b) Relative velocity, Vx(∞) – Vx(0), as a function of the distance from the particle surface for various normalized surface potentials Φ0 = 1.97, 0.97, 0.00, –0.97, and –1.97 at β = –0.50, and (c) for various β = 0.25, 0.00, –0.25, and –0.50 at Φ0 = –0.97. In these calculations we adopted the electrolyte (Z+, Z–) = (2, 1) with C∞ = 1 mM and dC∞/dx = –0.01 µm–1.


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Figure 2. Concentration profile of the micro-injected solution at the steady state as a function of the distance from the tip of micro-pipette calculated from eq 17. The concentration of injected solutions at the tip of the micro-pipette, C0 is 10 mM. In the micro-injection experiments (Figure 5), we measured the migration velocity in the distance range between around 5 µm (marked as A) and 35 µm (marked as B), where the vesicles migrated from A to B (moving away from the tip) for KCl and LaCl3 and B to A (approaching the tip) for NaCl and CaCl2.


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Figure 3. Migration of vesicles induced by micro-injection of (a): NaCl, (b): CaCl2, (c): MgCl2, (d): KCl, and (e): LaCl3 at the concentration of 10 mM using the injection pressure of 5 hPa. The yellow arrow heads indicate the tips of the micro-pipettes. The elapsed times since the start of the micro-injections are shown at the upper-left of each image. The scale bars are 30 µm.


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Figure 4. Traces of the Lo domain (black region) motions on a GV composed of DOPC/DPPC/Cholesterol (3/1/1) labeled with Rh-DOPE (a) before and (c) during the microinjection of 10 mM MgCl2. The GV migrated toward the tip of a micro-pipette during the microinjection. The observation was carried out at 25°C where the vesicle showed Lo / Ld phase separation. Each trajectory on the both images was the trace for 1.1 sec at the interval of 50 ms. The scale bar is 30 µm. The enlarged images around a domain marked as 02 in (a) and 02* in (c) are shown as (b) and (d), respectively.


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Figure 5. (a) Migration velocities of GVs as a function of the distance from the tip of the micropipette to the center of the vesicle, x’0, in response to micro-injections of NaCl (red circle), KCl (orange triangle), CaCl2 (yellow-green square), and LaCl3 (blue open diamond) at the concentrations of 10 mM using injection pressures of 5 hPa. (b) Migration velocities of GVs as a function of dlnC∞/dx’0 [same data in (a)]. Solid lines are theoretical calculation results based on eqn (8), (9), and (14) (NaCl: red line, KCl: orange line, CaCl2: yellow-green line, LaCl3: blue line). The positive velocity means that vesicle migrates toward the tip of micro-pipette. Error in the velocities originates from the pixel size in the fluorescence images and we indicated that with an error bar at the leftmost data point in each profile to make the plot visible.


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Figure 6. Measured ζ-potentials (Z.P.) of LUVs composed of DOPC labeled with 0.125 mol% of Rh-DOPE as a function of the concentration of NaCl (red circle), KCl (yellow triangle), CaCl2 (yellow-green square), and LaCl3 (blue diamond). Lines (NaCl: red line, KCl: yellow line, CaCl2: yellow-green line, LaCl3: blue line) show the surface potential, ζ0 (S.P.) used in the calculations of theoretical velocities in Figure 5b.


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Migration of Phospholipid Vesicles Can Be Selectively Driven by

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