Effect of Osmotic Stress on Membrane Fusion on Solid Substrate

Apr 30, 2013 - National Laboratory of Solid State Microstructures and Department of Physics, ... allowed us to separate the osmotic effects from the s...
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Effect of Osmotic Stress on Membrane Fusion on Solid Substrate Tao Zhu,† Zhongying Jiang,*,†,§ El Mi Ra Nurlybaeva,∥ Jie Sheng,† and Yuqiang Ma*,†,‡ †

National Laboratory of Solid State Microstructures and Department of Physics, Nanjing University, Nanjing 210093, China Laboratory of Soft Condensed Matter Physics and Interdisciplinary Research, Soochow University, Suzhou 215006, China § School of Electronics and Information and College of Chemistry and Biological Science, Yi Li Normal University, Yining 835000, China ∥ Kazakh National Technical University named after K. I. Satpaev, Almaty, 050013, Kazakhstan ‡

S Supporting Information *

ABSTRACT: There is currently a lack of comprehensive understanding of osmotic effect on lipid vesicle fusion on solid oxide surface. The question has both biological and biomedical implications. We studied the effect by quartz crystal microbalance with dissipation monitoring using NaCl, sucrose as osmolytes, and two different osmotic stress imposition methods, which allowed us to separate the osmotic effects from the solute impacts. Osmotic stress was found to have limited influence on the fusion kinetics, independently of the direction of the gradient. Further atomic force microscopy experiments and energy consideration implied that osmotic stress spends the majority of chemical potential energy associated in directed transport of water across membrane. Its contribution to vesicle deformation and fusion on substrate is therefore small compared to that of adhesion.

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INTRODUCTION Osmotic stress or osmotic gradient is generated by the unequal ionic or molecular distribution across a membrane. Hyperosmotic and hypoosmotic stresses reflect that osmolarity, a measure of solute concentration, of the external compartment is higher and lower than that of the membrane-trapped compartment, respectively. Osmotic stress participates in influencing plenty of membrane activities. It may induce dramatic membrane morphology transformation, such as pearling of neurons under hypoosmotic environment.1 It may also inspire budding and fission in phase-separated lipid vesicles.2 However, the relations between some membrane activities, i.e. membrane fusion and osmotic stress, are still not clear. Membrane fusion, an important biophysical process essential for life function,3 can be initiated either by contact between floating vesicles in bulk solution or adsorption of vesicles on solid substrate. The former is commonly observed for the vesicles with diameter less than 100 nm, where the vesicles fuse to release high bending energy.4,5 The latter is mostly determined by the vesicle−substrate interaction, where the fusion is driven by adsorption-induced stress.6−9 Different results have been obtained concerning osmotic effect on both fusion processes. On the one hand, Seitz and Burridge et al. reported that hyperosmotic stress enhances vesicle fusion on hydrophilic surface in atomic force microscopy (AFM) and fluorescence microscopy studies.10,11 Reimhult et al. furthermore showed that hypoosmotic stress accelerates fusion on © XXXX American Chemical Society

substrate using quartz crystal microbalance with dissipation monitoring (QCM-D).8 But its promotion effect is weaker than that of the hyperosmotic. On the other hand, Schonherr et al. observed that a significant number of osmotic-stress-imposed vesicles remain stable on substrate for hours by AFM measurement.9 Another AFM study by Wang et al. exhibited that hyperosmotic stress alone does not promote surfaceattached vesicle rupture or fusion.12 As well as vesicle fusion on substrate, vesicle fusion in solution was reported to be promoted by hyperosmotic and hypoosmotic stresses in some research,5,13,14 while being inhibited or not influenced by the two in the others.15−17 The previous studies generally changed the medium outside the vesicle to impose the osmotic stress for fusion.8−16 This external method changes the osmotic gradient and the specific medium involved in vesicle−vesicle and vesicle−substrate interactions simultaneously, which may complicate the fusion kinetics observed. Besides that, most previous studies adopted only one solute (osmolyte) to produce osmotic stress.8−13,15−17 Specific membrane−solute interaction18,19 may affect the universality of the conclusion obtained. In this study, an internal osmotic stress imposition method was developed in addition to the external method to study the effect of osmotic stress on vesicle fusion on substrate. The Received: August 29, 2012 Revised: April 20, 2013

A

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a nitrogen flow and under vacuum in sequence. The lipid film was then hydrated by adding 1 mL of Tris buffer (the hydration buffer) with occasional shaking for 3 h. Following that, the suspension was extruded using a mini-extruder (Avanti) with a 100-nm pore polycarbonate membrane. The resulting vesicle solution was stored at 4 °C, diluted with another Tris buffer (the redistribution buffer) before experiment, and used within a day. The lipid molar concentration, vesicle size, and zeta potential were determined by total phosphorus, nanoparticle tracking, and zeta potential analyses, respectively (Supporting Information (SI)). QCM-D Experiments. QCM-D characterizes vesicle fusion on substrate by the shifts of the resonant frequency (Δf) and the energy dissipation (ΔD). In ideal condition, Δf is in proportion to the mass change (Δm) on sensor crystal by the Sauerbrey relation21

internal method keeps the external vesicular medium identical within each parallel comparison. Using NaCl and sucrose as the representative membrane-impermeable ionic and organic osmolytes, and fully considering their specific impacts, the experimental observations were found to arrive unanimously at the conclusion that osmotic stress alone does not have strong influence on the fusion kinetics. The physical origin was explored. This study provides new insight into the role that osmotic stress plays in vesicle adsorption and membrane fusion on substrate.

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MATERIALS AND METHODS

Materials and Buffer Preparation. 1,2-Dioleoyl-sn-glycero-3phosphocholine (DOPC), sucrose, NaCl, and tris(hydroxymethyl)aminomethane (Tris) were purchased from Sigma Adrich. Tris buffers (10 mM Tris, pH 7.8) were prepared using purified water (minimum resistivity 18.2 MΩ) from a Milli-Q Water System. The osmolarities (Π) of the buffers were adjusted by the NaCl concentration (CNaCl) or sucrose concentration (Csucrose) (Table 1).

Δm = CQCMΔf

where CQCM is the mass sensitivity constant of the crystal (CQCM = −17.7 ng/cm2/Hz). ΔD is related to the viscoelasticity of the adsorbed layer. The experiments were performed using SiO2 coated sensor crystals in a QCM-D flow chamber E1 (Qsense) at 25 °C. First, a steady QCM-D baseline was achieved in the Tris buffer (the redistribution buffer), then the formerly prepared vesicle solution was diluted with the redistribution buffer by 30 times. After 100 s of mixing, the resulting vesicle solution (0.13 mg/mL) was pumped into the QCM-D chamber to start vesicle fusion on SiO2. When Δf and ΔD curves approached equilibrium, the residual vesicles were washed away by the redistribution buffer. All Δf and ΔD listed in this work were measured at the seventh overtone. Error bars are the standard deviations of 3 measurements. AFM Experiments. AFM experiments were conducted on a NanoWizard AFM (JPK) in contact mode. V-Shaped silicon nitride cantilevers with a spring constant of 0.06 N/m and a tip diameter of 20 nm (DNP-S10, Bruker) were used. First, borosilicate cover glass (Fisher) was prepared by soaking in sodium dodecyl sulfate solution, rinsing with purified water, and baking at 400 °C for 5 h. The glass substrate was then glued to the bottom of a liquid cell. Vesicle adsorption was carried out in either heteroosmotic (200 mOsm and −200 mOsm produced by sucrose) or isoosmotic (the buffer B1)

Table 1. Tris Buffers Used in the Experiment no.

CNaCl (mM)

Csucrose (mM)

approximately Π (mOsm)

B1 B2 B3 B4 B5 B6 B7 B8 B9

150 175 200 225 250 150 150 150 150

0 0 0 0 0 50 100 150 200

300 350 400 450 500 350 400 450 500

(1)

Preparation of Vesicles. The unilamellar vesicles were prepared by the extrusion method.20 A complete dry lipid film was formed by evaporating 200 μL of DOPC chloroform solution (20 mg/mL) under

Figure 1. Schematic representation of the external (left) and internal methods (right). The upper part shows how osmolarity imbalance is generated in these two methods. And the lower part presents the detailed lists of the buffers (Table 1) used in the corresponding experiments (Hydration buffer, orange; redistribution buffer, green). The corresponding experimental results are displayed in Figures 2, 3, and 4, respectively. B

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conditions at 0.3 μg/mL for 2 h. AFM images were acquired at a scan rate of 1.7 Hz with a resolution of 256 pixels/line using the minimized force (by minimizing the set point). A first-order polynomial was subtracted from each line to compensate for sample tilt. The widths of individually adsorbed vesicles were estimated following reference 9. Briefly, the mean value of the vesicle widths from cross-sectional plots at four different relative orientations was calculated and corrected by subtraction of the tip diameter.

the osmotic stress, tc and teq decrease with the increase of the hyperosmotic gradient, and increase with the increase of the hypoosmotic gradient. And for the experiments that used sucrose to produce the osmotic stress, tc and teq remain almost constant with the increase of the hyperosmotic and hypoosmotic gradients. However, the external method adopted above may not be optimal for studying osmotic effect on membrane fusion. The method changes the osmotic gradient and the medium for vesicle−substrate and vesicle−vesicle interactions at the same time. The latter may also greatly influence the fusion kinetics.22−25 Thus a new osmotic stress imposition method was proposed to avoid the defect of the external method. In the internal method, the external medium is kept constant, while the internal medium is varied. Specifically, the osmolarity of the hydration buffer was changed in a series of 300, 350, 400, 450, and 500 mOsm, while the osmolarity of the redistribution buffer was fixed at 500 and 300 mOsm for the hyperosmotic and hypoosmotic stress experiments, respectively (Figure 1). Figure 4 presents tc and teq versus ΔΠ profiles in the internal method measurements. Neither hyperosmotic nor hypoosmotic gradient produced by NaCl or sucrose changes tc or teq. The experimental observations in both external and internal method measurements above are independent of the osmotic stress exposure time before fusion. The exposure time was set at 10, 1000, and 3500 s besides 100 s, and no fusion kinetics change was found (SI). Besides that, no equilibrium Δf or ΔD difference was found among the SLBs formed in the different osmotic conditions. Further fluorescence recovery after photobleaching experiments demonstrate that all the SLBs are continuous and fluid (SI). Comparison of the Observations from the Two Methods. In both the external and internal methods, the solute concentration is partly changed at the same time as the osmotic gradient. The difference is that the external method changes the solute concentration outside the vesicles, which may greatly influence vesicle−substrate and vesicle−vesicle contact and hence vesicle fusion; while the internal method changes the solute concentration inside the vesicles, whose impact on fusion is expected to be small. In the following, the specific impacts that arise from the solute concentration changes are studied in detail to get a general picture of the role that osmotic stress plays in vesicle fusion on substrate. Characterization of the fusion kinetics under the different solute concentrations in isoosmotic condition provides information about the impact of the solute on fusion. Because the osmotic gradient is kept constant at zero, the solute concentration becomes the only variable responsible for fusion kinetics change. As illustrated in Figure 5, tc and teq decrease with the increase of the NaCl concentration (CNaCl), while they remain almost unchanged with the increase of the sucrose concentration (Csucrose). These are consistent with the results obtained in the previous studies. It is reported that the rise of the NaCl concentration promotes phosphocholine (PC) vesicle fusion on SiO2 substrate by raising the ionic strength in the bulk solution, which screens the repulsive electrostatic interaction between the negatively charged surface and vesicle (DOPC vesicles have a zeta potential of −5.9 ± 1.2 mV in 10 mM NaCl, pH 7.8 Tris buffer).23−25 Though there is no direct report on the sugar impact on vesicle fusion on substrate, the influence of sugar on floating vesicle fusion in solution has been studied intensively. Its inhibition effect on the process has shown to decrease with the decreasing sugar polymerization

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RESULTS External and Internal Osmotic Stress Imposition Methods. The unilamellar lipid vesicles were formed in the hydration buffer, and were exposed to the redistribution buffer before fusion on substrate. The hydration and redistribution buffers can be taken approximately as the internal and external (vesicular) mediums, respectively. Osmotic stress can therefore be produced by varying the osmolarity between the two buffers, with the osmotic gradient (ΔΠ) equal to the osmolarity difference between the external and internal buffers. Previous studies on vesicle fusion generally kept the internal medium constant, while varying the external medium to adjust the imposed osmotic stress.8−16 This external method is convenient as all the samples used in a parallel comparison can be extruded in one batch. We also adopted this method first to study the osmotic effect on vesicle fusion on substrate. As exhibited in Figure 1, the osmolarity of the redistribution buffer was changed in a series of 300, 350, 400, 450, and 500 mOsm, while the osmolarity of the hydration buffer was fixed at 300 and 500 mOsm for the hyperosmotic and hypoosmotic stress experiments, respectively. Figure 2 illustrates Δf and ΔD versus time (t) curves obtained in typical osmotic-stress-imposed vesicle fusion

Figure 2. Δf−t and ΔD−t curves in typical fusion experiments. Data are from the external method measurements that used NaCl to produce the hyperosmotic stress.

experiments. −Δf and ΔD initially increase with t, and decrease after the critical time point tc, and finally equilibrate at teq (∼ 25 Hz and 0.4 × 10−6), indicating that vesicles fuse into a complete supported lipid bilayer (SLB) through the critical vesicular surface coverage pathway.7,8 In this pathway, vesicles adsorb on substrate intact until a critical surface coverage, then rupture and fuse into a SLB. The pathway is followed generally by the zwitterionic lipid vesicles on SiO2 substrate in high ionic strength condition.22,23 tc reflects the time for vesicle adsorption to reach the critical surface coverage. teq reflects the time for vesicles to fuse into a complete SLB. tc and teq are the temporal characteristic parameters of the fusion process.23 Figure 3 presents tc and teq versus ΔΠ profiles in the external method measurements. For the experiments that used NaCl to produce C

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Figure 3. Plots of tc (○) and teq (●) versus ΔΠ in the external method measurements. The hyperosmotic positive ΔΠ and hypoosmotic negative ΔΠ were produced either by (a) and (b) NaCl, or (c) and (d) sucrose. The dashed lines are guides to the eyes in Figures 3−6.

Figure 4. Plots of tc (○) and teq (●) versus ΔΠ in the internal method measurements. The osmotic stress was produced either by (a) and (b) NaCl, or (c) and (d) sucrose.

degree.26 And single and double sugars have no impact on vesicle fusion in solution.27 First, because sucrose (concentration) does not influence vesicle fusion on substrate, the experimental observations in its corresponding external and internal method measurements (Figure 3c and d and Figure 4c and d) indicate directly the

osmotic effect on the process. That is, the osmotic stress does not affect fusion kinetics on substrate. Second, Figure 6a displays tc and teq versus the external NaCl concentration (Cex) profiles in the external method measurements (Figure 3a and b) and the isoosmotic experiments (Figure 5a). tc, as well as teq, in the hyperosmotic, hypoosmotic, and isoosmotic conditions D

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kinetics disparity. Change of tc and teq observed in the external measurements is due to the change of the external NaCl concentration rather than the osmotic stress. Lastly, Figure 6b shows the results of a series of solute-asymmetric isoosmotic fusion experiments, which changed the internal NaCl concentration (Cin) and the external sucrose concentration simultaneously to maintain a zero osmotic gradient. Since sucrose does not influence fusion, the approximately constant tc andteq observed with the increasing Cin demonstrates that the impact of the concentration on fusion is negligible. This conforms to the expectation, as the internal concentration is irrelevant to the ionic strength in the vesicle−substrate and vesicle−vesicle interaction medium. Thus the corresponding internal method measurements (Figure 4a and b) present directly that the osmotic stress does not influence the fusion kinetics. To sum up, the experimental observations obtained through the internal method all indicate directly the osmotic effect on fusion, while those obtained through the external method may be affected by the specific solute impacts. After all, both hyperosmotic and hypoosmotic stresses were found to be ineffective in influencing vesicle fusion kinetics on substrate.

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DISCUSSION Water flows across membrane in response to osmotic stress, inducing vesicle volume change.28,29 Hyperosmotic stress expels water from the inside, shrinking the vesicles; while hypoosmotic stress draws water inside, swelling the vesicles. We also studied the volume change of adsorbed vesicles in presence of osmotic stress based on the QCM-D measurements. Because water is highly dissipative, its content change would alter the dissipation per unit mass (ΔD/Δf) of the surface-attached vesicles.22,30 Figure 7a and c illustrate ΔD versus Δf curves in the hyperosmotic and hypoosmotic experiments. ΔD/Δf slope of the intact vesicle adsorption (the solid lines, vesicles adsorb with little rupture before tc) decreases with the increase of the hyperosmotic gradient, and increases with the increase of the hypoosmotic gradient, indicating the loss and gain of water volume in vesicles under the two osmotic conditions, respectively. Accompanying the volume change, the height of the adsorbed vesicles also changes as exhibited in Figure 7b and d. It was shown that the intercept with the X axis, extrapolated from −ΔD/Δf versus −Δf curve of intact vesicle adsorption, yields a Δf value (corresponding to zero ΔD) that can be plugged into the Sauerbrey relation to give an equivalent height (he) of the adsorbed vesicles.31 This intercept decreases with the increase of the hyperosmotic gradient, and increases with the increase of the hypoosmotic gradient, demonstrating the fall and rise of the adsorbed vesicle height in the hyperosmotic and hypoosmotic conditions, respectively. Though ΔD/Δf slope and intercept may be influenced specifically by the solute concentration change as shown in the isoosmotic experiments (Figure 7e and f), the changes are much smaller than those observed in the heteroosmotic condition. Thus the soluteinduced volume and height changes were neglected in the former discussions. Vesicle deformation is known to be critical to vesicle fusion on solid substrate. In isoosmotic condition, vesicle deformation is generated by the initial vesicle adsorption and the subsequent squeezes of newly adsorbed neighboring vesicles, which are both driven by the gain in energy of vesicle adhesion.32 When the deformation reaches a critical value, vesicles rupture and fuse with each other to release the membrane curvature stress

Figure 5. Influence of CNaCl and Csucrose on tc (□) and teq (■). The experiments were conducted in isoosmotic condition, where the hydration and redistribution buffers were identical. The two buffers were the buffer B1, B2, B3, B4, B5 in (a) and B1, B6, B7, B8, B9 in (b).

Figure 6. Discrimination between the fusion kinetics changes that arise from the osmotic stress and NaCl concentration. (a) Average tc, teq as a function of Cex in the hyperosmotic (□, ■), hypoosmotic (○, ●) external method measurements, and isoosmotic experiments (Δ, ▲). (b) Influence of Cin on tc and teq (Δ, ▲). The experiments were conducted in solute-asymmetric isoosmotic condition, where the osmotic gradient was maintained at zero by equilibrating the osmolarity produced by NaCl in the internal medium with that of sucrose in the external medium. Specifically, the hydration and redistribution buffers were changed in pairs of the buffer B1, B1; B2, B6; B3, B7; B4, B8; B5, B9.

coincide with each other under the same Cex. This reveals that the osmotic gradient disparity does not produce actual fusion E

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Figure 7. Extraction of the adsorbed vesicle volume and height information from QCM-D measurements in (a) and (b) hyperosmotic; (c) and (d), hypoosmotic; and (e) and (f) isoosmotic conditions. (a)−(d) are from the internal method measurements that used sucrose to produce the osmotic stress. (e) and (f) are from the isoosmotic experiments under the different sucrose concentrations. The solid and dot lines in ΔD−Δf plots denote ΔD vs the temporally corresponding Δf profiles obtained before and after the critical time point tc in each experiment. The symbols and dashed lines in −ΔD/Δf versus −Δf plots represent the data from the solid lines in the corresponding ΔD versus Δf curves and their linear least-squares fits. The inserts present the extracted he.

generated by the deformation.6,32 The width-to-height ratio of individual vesicle on substrate, which reflects the magnitude of deformation and curvature stress in the curved lipid bilayer, has been used in many theoretical and experimental researches to explore the vesicle rupture and fusion tendency.32−34 We also characterized the deformation of individual vesicles on glass substrate under heteroosmotic and isoosmotic conditions by AFM in liquid. The experiments were conducted in low vesicle concentration (0.3 μg/mL) where vesicles simply adsorb without rupture or fusion. As shown in Figure 8, the widthto-height ratio of the substrate-attached vesicles remains constant irrespective of the osmotic conditions. This is consistent with the result obtained by Boxer et al., who also observed that osmotic stress does not change the deformation of individually adsorbed vesicles.9 The independence of vesicle deformation on osmotic stress can be understood from an energy perspective. On the one hand, it is known that the osmotic-associated chemical potential

energy (μ), which is stored in the form of osmolarity imbalance across membrane, is equal to the work done in the volume change (directed water transport) of a vesicle acting as an ideal osmometer28,29 μ=

∫V

Ve

pdV

0

(2)

where p is osmotic pressure, V is the vesicle volume, and the subscripts 0 and e represent the initial and equilibrium time, respectively. p is given by29 p = −ΔΠRT

(3)

where R is the gas constant and T is the temperature. ΔΠ is changed with the vesicle volume following the Boyle−Van’t Hoff equation28 ΠV = Π 0V0 F

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Figure 8. AFM height images of the substrate-attached vesicles under (a) hyperosmotic (200 mOsm produced by sucrose), (b) hypoosmotic (−200 mOsm produced by sucrose), and (c) isoosmotic (the buffer B1) conditions. The section analyses show the profiles along the corresponding lines indicated in the height images. (d) Width (w) versus height (h) plot of the individually adsorbed vesicles under the hyperosmotic (□), hypoosmotic (○), and isoosmotic (Δ) conditions. The linear least-squares fit of the data is shown with a dashed line.

Because the total vesicle volume fraction is low (10−5), transmembrane water flow would not change the osmolarity outside the vesicle. Ve is therefore obtained by substituting Π with the osmolarity of the external medium in eq 4 (osmotic gradient turns to be zero when an ideal osmometer arrives at equilibrium).28 Accordingly, μ is estimated to be around 10−17 J for the 200 and −200 mOsm osmotic gradient. In reality, vesicle does not act as an ideal osmometer. Due to the possible vesicle deformation and accompanying bending energy change (Eo) during the volume change, the osmotic-associated chemical potential energy can not be released completely in doing work of transporting water molecules across membrane (W).28,35 W is equal to the work done in the actual vesicle volume change36

W=

∫V

Vr

0

pdV

osmotic stress to deformation makes it ineffective in influencing the fusion process, as observed in our experiments. Nevertheless, it must be pointed out that the energy consideration above is only a rough estimation. Further computer simulation may be needed to perfectly understand the energy relations in the process.

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CONCLUSION The generally adopted external and newly developed internal methods were used to explore osmotic effect on vesicle fusion on substrate. It is shown that the experimental observations obtained through the external method are complicated by the impacts arising from solute concentration changes. However, these specific impacts are minimized in the internal method, whose observations directly present the osmotic effect on fusion. Osmotic stress alone was found to have almost no influence on the fusion kinetics. Further QCM-D and AFM analyses provided statistic information that reflects the volume and height, and individual information that presents the deformation of vesicles on substrate under the various osmotic conditions. Combining with a simple energy consideration, osmotic stress was proved to be insufficient to generate strong deformation change, as the majority of chemical potential energy associated is released in directed transport of water across membrane. It is therefore concluded that the independence of fusion kinetics on osmotic stress is a result of the incapability of osmotic stress to change vesicle deformation upon adsorption. This study provides new insight into osmotic effect on vesicle fusion on substrate. The osmotic-associated chemical potential energy may release in multiple forms, including changing vesicle water volume, bending membrane, and others. The distribution of the released potential energy is crucial in deciding the role that osmotic stress plays in the corresponding process, which has not been clarified in the previous fusion research.9,38 Besides that, a new osmotic stress imposition strategy was developed for the future studies. The internal method should be better suited than the external to studying

(5)

where the subscript r represents reality. An estimation of W can be done by supposing that the vesicle volume is proportional to the third power of the equivalent adsorbed vesicle height in Figure 7. W is then calculated to be more than 94% of μ (between 200 and −200 mOsm ΔΠ), which reveals that only a very small portion of osmotic-associated chemical potential energy can be used in changing vesicle deformation (Eo is around 10−19 J). On the other hand, the vesicle deformation (bending) energy attributed to vesicle adhesion to substrate (Ea) is given by9

Ea = wA

(6)

where w is the effective contact potential and A is the contact area between a vesicle and substrate. For PC vesicle adsorption on SiO2, w is reported to be 10−3 J/m2.37,38 A is in the order of the vesicle radius square. Thus Ea is estimated to be about 10−17 J, which is 2 orders of magnitude larger than Eo. This exhibits that the vesicle deformation arising from the vesicle adhesion is much larger than that from the osmotic stress. Because vesicle fusion on substrate is mainly controlled by the deformationinduced membrane curvature stress, the small contribution of G

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osmotic effect in membrane interaction with another object,39 as the interaction medium is kept unchanged. Last, the conclusions obtained may have implications for biology and biomedicine studies. The former cell research suggested that osmotic stress may play a vital role on the substrate-induced cell fusion.40 At the same time, many other biosensor and drug carrier studies attempted to promote vesicle fusion on biomimetic surface and nanoparticle by osmotic stress.41−43 However, our study implies that osmotic stress alone may not play the desired role in the processes above. Other factors such as ionic strength should be considered in biology research and biomedical engineering.

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(9) Schonherr, H.; Johnson, J. M.; Lenz, P.; Frank, C. W.; Boxer, S. G. Vesicle Adsorption and Lipid Bilayer Formation on Glass Studied by Atomic Force Microscopy. Langmuir 2004, 20, 11600−11606. (10) Seitz, M.; Ter-Ovanesyan, E.; Hausch, M.; Park, C. K.; Zasadzinski, J. A.; Zentel, R.; Israelachvili, J. N. Formation of Tethered Supported Bilayers by Vesicle Fusion onto Lipopolymer Monolayers Promoted by Osmotic Stress. Langmuir 2000, 16, 6067−6070. (11) Burridge, K. A.; Figa, M. A.; Wong, J. Y. Patterning Adjacent Supported Lipid Bilayers of Desired Composition to Investigate Receptor-Ligand Binding under Shear Flow. Langmuir 2004, 20, 10252−10259. (12) Wang, X.; Shindel, M. M.; Wang, S. W.; Ragan, R. Elucidating Driving Forces for Liposome Rupture: External Perturbations and Chemical Affinity. Langmuir 2012, 28, 7417−7427. (13) Cohen, F. S.; Akabas, M. H.; Finkelstein, A. Osmotic Swelling of Phospholipid Vesicles Causes Them to Fuse with a Planar Phospholipid Bilayer Membrane. Science 1982, 217, 458−460. (14) Lerebours, B.; Wehrli, E.; Hauser, H. Thermodynamic Stability and Osmotic Sensitivity of Small Unilamellar Phosphatidylcholine Vesicles. Biochim. Biophys. Acta 1993, 1152, 49−60. (15) Malinin, V. S.; Frederik, P.; Lentz, B. R. Osmotic and Curvature Stress Affect PEG-Induced Fusion of Lipid Vesicles but Not Mixing of Their Lipids. Biophys. J. 2002, 82, 2090−2100. (16) Mui, B. L. S.; Cullis, P. R.; Evans, E. A.; Madden, T. D. Osmotic Properties of Large Unilamellar Vesicles Prepared by Extrusion. Biophys. J. 1993, 64, 443−453. (17) Markosyan, R. M.; Melikyan, G. B.; Cohen, F. S. Tension of Membranes Expressing the Hemagglutinin of Influenza Virus Inhibits Fusion. Biophys. J. 1999, 77, 943−952. (18) Petrache, H. I.; Zemb, T.; Belloni, L.; Parsegian, V. A. Salt Screening and Specific Ion Adsorption Determine Neutral-Lipid Membrane Interactions. Proc. Natl. Acad. Sci., U. S. A. 2006, 103, 7982−7987. (19) Andersen, H. D.; Wang, C.; Arleth, L.; Peters, G. H.; Westh, P. Reconciliation of Opposing Views on Membrane-Sugar Interactions. Proc. Natl. Acad. Sci., U. S. A. 2011, 108, 1874−1878. (20) Zhu, T.; Xu, F.; Yuan, B.; Ren, C.; Jiang, Z.; Ma, Y. Effect of Calcium Cation on Lipid Vesicle Deposition on Silicon Dioxide Surface under Various Thermal Conditions. Colloids Surf., B 2012, 89, 228−233. (21) Sauerbrey, G. Verwendung von Schwingquarzen zur Wagung dunner Schichten und zur Mikrowagung. Z. Angew. Phys. 1959, 155, 206−222. (22) Seantier, B.; Breffa, C.; Felix, O.; Decher, G. DissipationEnhanced Quartz Crystal Microbalance Studies on the Experimental Parameters Controlling the Formation of Supported Lipid Bilayers. J. Phys. Chem. B 2005, 109, 21755−21765. (23) Seantier, B.; Kasemo, B. Influence of Mono- And Divalent Ions on the Formation of Supported Phospholipid Bilayers via Vesicle Adsorption. Langmuir 2009, 25, 5767−5772. (24) Garcia-Manyes, S.; Oncins, G.; Sanz, F. Effect of pH and Ionic Strength on Phospholipid Nanomechanics and on Deposition Process onto Hydrophilic Surfaces Measured by AFM. Electrochim. Acta 2006, 51, 5029−5036. (25) Boudard, S.; Seantier, B.; Breffa, C.; Decher, G.; Felix, O. Controlling the Pathway of Formation of Supported Lipid Bilayers of DMPC by Varying the Sodium Chloride Concentration. Thin Solid Films 2006, 495, 246−251. (26) Oliver, A. E.; Hincha, D. K.; Crowe, J. H. Looking beyond Sugars: The Role of Amphiphilic Solutes in Preventing Adventitious Reactions in Anhydrobiotes at Low Water Contents. Comp. Biochem. Physiol., Part A: Mol. Integr. Physiol. 2002, 131, 515−525. (27) Kiselev, M. A.; Wartewig, S.; Janich, M.; Lesieur, P.; Kiselev, A. M.; Ollivon, M.; Neubert, R. Does Sucrose Influence the Properties of DMPC Vesicles? Chem. Phys. Lipids 2003, 123, 31−44. (28) Pencer, J.; White, G. F.; Hallett, F. R. Osmotically Induced Shape Changes of Large Unilamellar Vesicles Measured by Dynamic Light Scattering. Biophys. J. 2001, 81, 2716−2728. (29) Nelson, P. Biological Physics; Clancy Marshall: New York, 2008.

ASSOCIATED CONTENT

S Supporting Information *

Details of total phosphorus, nanoparticle tracking, zeta potential analyses, and fluorescence recovery after photobleaching experiments; raw data of QCM-D experiments and additional ΔD versus Δf, −ΔD/Δf versus −Δf plots; AFM characterization of the concentration-dependent vesicle deposition behaviors. This information is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: (086) 13813955964; e-mail: [email protected] (Z.J.). Tel: (086) 02583592900; e-mail: [email protected] (Y.M.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Chunlai Ren for valuable comments concerning the manuscript. This work was supported by the National Basic Research Program of China (2012CB821500) and the National Natural Science Foundation of China (91027040, 31061160496, 11104192, and 21264016).

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