Estimation of Mechanical Strength of Unilamellar and Multilamellar

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Estimation of Mechanical Strength of Unilamellar and Multilamellar AOT/Water Vesicles and Their Rupture Using Micropipet Aspiration† G. Hema Sagar‡ and Jayesh R. Bellare*,‡,§ Department of Chemical Engineering, School of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Powai, Mumbai - 400076 India ReceiVed: March 31, 2009; ReVised Manuscript ReceiVed: July 9, 2009

Vesicles prepared from surfactant sodium dioctyl sulfosuccinate (AOT) were characterized by micropipet aspiration for determining membrane bending rigidity and area expansion modulus and mechanism of rupture. Unilamellar vesicles (ULV) and multilamellar vesicles (MLV) were studied. The mechanical properties calculated using micropipet aspiration for ULV were found to be 5-10 kBT, Ka ) 100 ( 20 mN/m and for MLV were 8-15 kBT, Ka ) 120 ( 30 mN/m. These properties fall with the range of lipid (PC) membrane measurements (values). However, the membrane rigidity of multilamellar vesicles was found to be ∼3 times larger than that of unilamellar vesicles. The apparent area expansion moduli of multilamellar vesicles are of the order 1.4 times, sustained far greater areal strain before rupture compared to that of unilamellar vesicles. A dynamic structural change in MLV is demonstrated upon stress by micropipet aspiration. MLV at stress undergoes various stages of deformation. The fluctuation in size and shape of MLV led to separation of bilayers from the stack and decrease in vesicle diameter facilitating in formation of new equilibrium MLV, for it to sustain the specified membrane tension, a new mechanism that is demonstrated experimentally. Introduction Amphiphilic molecules, owing to their solubility, aggregate to form various ordered structures1-3 such as vesicles. Vesicles show potential as carriers for drug molecules and as templates for material applications. In addition, vesicular structures are used as rheology modifiers4 and in chemical reactions.5 However, the major concern in the application of vesicles is the metastability of their aqueous dispersions. Vesicular bilayer stability can be enhanced by polymerization or polymer insertion,6,7 addition of cosurfactant, or incorporation of cholesterol.8,9 Vesicles are classified based on size and lamellarity, small unilamellar vesicles (SUVs) with a diameter in the 20-100 nm range, large unilamellar vesicles (LUVs) with a diameter larger than 100 nm, and multilamellar vesicles (MLVs) which span from 0.1 to several dozens of micrometer. In case of double tailed surfactant solutions, vesicles form spontaneously; the transition from the unilamellar to multilamellar phase is observed with increasing surfactant concentration.10 Because of its concentric bilayers arrangement, low curvature, and rheological behavior, a multilamellar vesicle is suitable for numerous applications. Recently reported studies establish the use of MLVs as drug delivery vehicles,11 chemical micro reactors,12 and as carriers of submicrometer particles.13 Various preparation techniques have been reported for MLVs over the years. Lamellar solutions, when subjected to shear, exhibit a structural transition above a threshold shear rate, and form quasimonodispersed multilamellar droplets.14,15 The formation of MLV from the lamellar phase of AOT in brine can be controlled by steady, oscillatory shearing. Increasing the stress amplitude or decreasing the frequency leads to formation of onionlike †

Part of the “H. Ted Davis Special Section”. * To whom correspondence should be addressed. E-mail: [email protected]. ‡ Department of Chemical Engineering. § School of Biosciences and Bioengineering.

structures;16 the average MLV size decreases in stress-controlled experiments with increasing applied shear stress. Spherulites are multilamellar vesicles formed by shearing of lamellar phases which have similar properties as that of the lamellar phase as regards the solubilization of water or oil, interlamellar spacing and bending rigidity. Several interesting aspects related to mechanical properties, area compressibility modulus, bending rigidity and mechanical stiffness of ULVs and MLVs can be controlled by changes in membrane fusion, size, shape, and stability at various physiological conditions. Since membranes are extremely flexible, well-characterized local force and submicrometer detection of the membrane deformation of individual vesicles is required to measure the bending modulus. A number of experimental methods have been designed to measure the bending rigidity of the membrane, such as shape fluctuation recorded by microscopy17,18 micropipet suction,19 micropipet aspiration, and others.20 However, most reports are related to various microstructures formed in the sodium dioctyl sulfosuccinate (AOT)-water system. It is known that AOT/ water can form vesicles (unilamellar, multilamellar) when dissolved in water, but their mechanical properties have not yet been reported. In this work, we have used micropipet aspiration technique for the determination of mechanical properties of ULVs and MLVs, based on the anionic surfactant AOT. We have also conducted a microscopic observation of time-dependent changes in the morphology of multilamellar vesicle when subjected to micropipet aspiration. Materials and Methods Materials. AOT purchased from Sigma-Aldrich Cheme was used without further purification. The deionized water used for experiments had a resistivity of 18.1 MΩ/cm from a Milli-Q System (Dubuque, IA).

10.1021/jp902909z CCC: $40.75  2009 American Chemical Society Published on Web 09/18/2009

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Vesicle Preparation. To prepare SUVs and MLVs, AOT was dissolved in water with a weight fraction of 2% and was gently stirred by magnetic stirrer for 4 days. Stirring gently causes the formation of unilamellar and multilamellar vesicles. These samples were visualized under a optical microscope before proceeding for measurement of mechanical properties. Micropipet Aspiration and Data Analysis. All imaging was performed using an Olympus IX81 motorized inverted microscope, equipped with differential interference contrast (DIC) optics. The aspiration of vesicles was observed with a 60× water immersion objective (N.A. 1.30) under bright-field illumination. Microaspiration of both ULV and MLV experiment was recorded with a Nikon digital camera, image processing was done offline using FORTRAN code written to extract images at required time intervals from the recorded video. The micropipets were made from 1 mm (o.d) borosilicate glass tube capillaries (World precision instruments TW100-4) by pulling capillary glass tubing into thin tapered shafts with a glass puller (Narishige Model PN-30), and fracturing the tips flat with inner diameters of 6-8 µm with a microforge (Narishige MF 900). The micropipet holder was connected to a water column filled with distilled water. The level of the water column was referenced to the microscope stage (i.e., the zero pressure point). Negative pressure was applied by lowering the water column. The micropipet was advanced to the center of the field of view by a joystick-controlled electronic micromanipulator (Narishige, Tokyo). The position of the micropipet is controlled with a micromanipulator (model MHW-3, Narishige, Japan). Once a vesicle was located, the micropipet was brought into contact with the vesicle. Negative pressure was applied, and the pressure was then held constant to maintain an equilibrium state. After a vesicle aspirated into the micropipet, aspiration pressure was increased in a stepwise manner. For every step, the following observations were recorded: Dv, diameter of the spherical portion of the vesicle outside the pipet, length L portion inside the pipet, and aspiration pressure ∆P. The aspiration pressure, ∆P, applied to a vesicle was calculated for the change in height of a water manometer connected to the micropipet. When a giant vesicle is aspirated into a micropipet21 with i.d., Rp, and an applied pressure ∆P, that leads to a projection length of membrane ∆L. The membrane tension,σ, calculated using eq 1, depends on the suction pressure and the geometry of both the vesicle and micropipet. The change in projected area of a vesicle ∆A aspirated in a micropipet was determined using the approximate relationship shown in eq 2

σ)

∆PRp Rp 21Rv

(

(

)

∆A ≈ 2πRp∆L 1 -

Figure 1. Micrograph for micropipet aspiration of unilamellar vesicles. (a) ULV at time t ) 0 s. (b) ULV at t ) 15 s; arrow represents projection length of vesicles in the micropipet at a negative pressure, ∆P. The scale bar corresponds to 50 µm.

(1)

Rp Rv

)

(2)

Results and Discussion The aspiration of a vesicle held in a micropipet induces a well-defined tension and area expansion of the vesicle; measurement of change in length of the membrane projection in the pipet in response to change in aspiration (suction) pressure is performed. A plot of tension versus area change gives directly two parameters that characterize the mechanical properties of the ULV and MLV, namely, the apparent area compressibility modulus and bending rigidity.

Figure 2. A typical micrograph for aspiration of multilamellar vesicles. (a) Aspiration time t ) 0; stacking of bilayers in multilamellar vesicles is shown by arrow. (b) The arrow mark represents projection length of vesicle in the micropipet, at a negative pressure, ∆P. The scale bar corresponds to 50 µm.

Unilamellar and Multilamellar AOT/Water Vesicles

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Figure 3. A typical plot of membrane tension (σ) versus areal strain R ) ∆A/A0 for calculation of bending rigidity (k) and apparent area compressibility modulus (Ka) for (a) ULV (c) MLV; histogram represents apparent area compressibility modulus from micropipet aspiration experiments for ∼20 vesicles normal distribution function has been superimposed (b) ULV (d) MLV.

Bending Rigidity. In the low-tension regime, the logarithmic term dominates and shows that almost all increase in R come from smoothing out thermal undulations. The bending rigidity is determined by plotting ln σ versus R, calculated as obtained in eq 3

∆A kBT σ ≈ ln A0 8πk σ0

(3)

Here ∆A is the vesicle area difference between the area at tension σ and initial tension σ0, the initial area is A0, and bending rigidity is k, where kBT is the thermal energy (kB is Boltzmann’s constant). When a ULV is sucked in a glass micropipet, fluctuations of the membrane are strongly reduced, and the shape of the vesicle becomes spherical. Since in low tension regime the aspiration pressures are weak, undulation are flattened out. The initial pressure required to pull the vesicle into the micropipet is termed as threshold pressure. The threshold pressure for ULV is lower in comparison with MLV. When aspiration is larger than a certain threshold pressure, the vesicle continuously flows into the micropipet. Micrograph of ULV at the start of aspiration and an elapsed times of 15 s is shown (Figure 1). Figure 3a

shows plots of the membrane tension as function areal strain R for ULV. The bending rigidity of ULV calculated for 20 vesicles of diameters 30-70 µm were in the range of 5-10 kBT. However for a few unilamellar vesicles the bending rigidity falls in the range of multilamellar vesicle calculated using micropipet aspiration. The initial aspiration pressure, that is, threshold pressure, required to aspirate an MLV (Figure 2) is much higher owing to its robust structural arrangement of bilayer. In Figure 3c, there is a jump in the slope of membrane tension versus areal strain, which could be due to the lateral adjustments of bilayers close to high tension regime. The bending rigidity calculated based on aspiration data in low tension regime for MLV lies in the range 8-18 kBT for vesicles of diameters 60-100 µm, 3-fold higher that of unilamellar vesicles. The bending deformation of multilamellar vesicle takes several seconds to reach equilibrium. This slow motion leads to a lateral rearrangement of AOT molecules, and so the deformation may contain both single-layer bending and bilayer coupling bending. This seems to be a reason of the larger k values in multilamellar vesicles compared to unilamellar vesicles.21 In addition, there is a possibility of an interlamellar interaction effect in the multilayers plays a significant role. Increased repulsion between bilayers with excess water could

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Figure 4. Differential interference contrast micrographs for peeling of multilamellar vesicle at different times of aspiration pressure and membrane tension where Dp ) 25 µm, Dv ) 100 µm. (a) t ) 5 s σ ) 0.97 mN/m. (b) t ) 265 s, σ ) 3.6 mN/m: arrow mark indicates bilayer separation of MLV (c) t ) 307 s; arrow indicated separation of aggregates and bilayer separation. The scale bar corresponds to 50 µm.

Figure 5. Differential interference contrast micrographs of the morphological changes of a MLV at different aspiration time and aspiration pressure where pipette diameter Dp ) 25 µm, vesicle diameter Dv ) 100 µm. (a) t ) 20 s, giving membrane tension σ ) 0.97 mN/m; (b) t ) 190 s, σ ) 2.6 mN/m; (c) t ) 242 s. Arrow marks show the rupture of the outer layer close to the critical pressure. The scale bar corresponds to 20 µm.

also come from stronger undulation fluctuations22,23 resulting from a smaller bending rigidity modulus, k, of the thinner bilayer.8 Interbilayer interactions in a multilamellar vesicle are too weak to alter their structure when compared to unilamellar vesicle. This increases its undulation fluctuation, which increases the water spacing of the interbilayer interactions, which are weaker when the bilayers are further apart. Interlamellar interaction effect in the multilamellar vesicles, which are essentially unoriented stacks of bilayers. When multilamellar vesicles are stressed because undulations and line tension in the outer layer of MLV not only push the bilayers closer to one another by decreasing water spacing, DW, but also remove water by decreasing A, the local stress generated in the outer layer of MLV due to aspiration pressure is transformed perpendicular to the axis, which results in an increase of the diameter of the vesicles that are perpendicular in direction to the axis and a marginal decrease in initial volume. The average thickness of each bilayer along the z direction is larger than the bilayer, which was perfectly flat. The average interbilayer water spacing, D′W, occurs by balancing the sum of these pressures with whatever osmotic pressure is exerted on the system. Area Compressibility Modulus. As Figure 3a,c demonstrates, the membrane tension exhibits a linear dependence on area dilation as the vesicle area is expanded in the high tension

regime. The slope of the linear correlation is the apparent area compressibility modulus Ka, Ka ) σ /R. Figure 3b shows conglomerated results for 20 ULV aspiration data. The population of area compressibility modulus for ULV has a range of 80-100 mN/m with a few ULV with area compressibility ranging in 110-120 mN/m, which suggests that occasionally rigid vesicles are observed; however the average apparent area compressibility modulus for ULV was 100 ( 20 mN/m. Figure 3d shows cumulated results of the apparent area compressibility modulus for MLV; the average apparent area compressibility modulus was 120 ( 30 mN/m. On the contrary to ULV results, MLV tests suggest that the area compressibility modulus separate into two groups, MLV, which are nonrigid with apparent area compressibility modulus in the range 90-100 mN/m comparable to ULV, and MLV that are rigid as result of its complex structure. When membrane tension is of higher order, close to rupture (critical tension), unilamellar vesicle is sucked into the micropipet. However experimental observation suggest that ULV have a critical tension from 3.5-5 mN/m; MLV have a higher critical tension of the order 5-7.5 mN/m due its compact packing of bilayer. The crossover tension, τx ) (Ka/k)(kBT/8) for multilamellar vesicles, was 0.38 mN/m and for unilamellar vesicle it was 0.49 mN/m. The cross-over tension for MLV was less than that of unilamellar vesicles but falls within the range of

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Figure 6. Schematic drawing of various development stages of peeling phenomenon for MLV during micropipet aspiration. (a) MLV (N bilayers stacked) projected length into the micropipet increases and reaches a critical point and the shape of MLV vesicles transform from spherical to elliptical with increase in pressure; beyond a critical pressure increase in aspiration pressures results in rupture of outer layer, exposing the inner layers. After the bilayers peel out of the MLV, a new equilibrium MLV evolves. (b) MLV (bilayers stacked around large aqueous core) shapes change in MLV with the rupture of the outer layer followed by the complete disappearance of MLV.

appropriate high-tension (membrane stretching) and low-tension (membrane smoothing) regime. The area compressibility modulus depends on the elastic parameters of the constituent’s layers and equilibrium area. The elastic deformation involves bending energy, which is small compared to stretching energy in the high tension regime. Our experimental observations suggest that MLV has higher area compressibility modulus to that of ULV. For MLV (N bilayers large stack separated by thin layers of solvent), the area compressibility modulus should be the sum of the area compressibility modulus of the constituent bilayers. It is well established that the intermembrane repulsive force in the multilamellar membrane increases with an increase in the undulation motion of the membrane.24 When MLV is under tension, stress on the outer layer,N, transfers to the adjacent layer and it follows to the core. Since the interbilayer distances are much smaller than the dimension of the vesicles the curvature varies slowly, deformation of vesicle is lesser due relative expansion and compression of layers in MLV. MLV under stress does not result automatically in lateral compression of the surrounding bilayers, as each pair of neighboring bilayers behaves asymptotically as if it were not affected by the presence of the other membranes within the stack. Because of this, structural transition may result in transient compression of the surrounding bilayer with slow relaxation of the tension by lower lateral expansion25 and bulging. Instead, the entire membrane could expand laterally or bulge to accommodate the area increase. Bulging may be suppressed by

interactions between neighboring bilayers, thereby increasing the equatorial radius. This suggests that interbilayer forces have only negligible influence on lateral compressibility and based on the known compressibility modulus, changes in the bilayer thickness26 can be calculated. Peeling of MLV. A series of snapshots of MLV subjected to micropipet aspiration at various time intervals are shown in Figure 4a-c. When a vesicle is in contact with the micropipet, zero time is defined and the MLV starts to experience shape fluctuations. When a step increment in negative pressure is given, MLV size decreases continuously until it reaches an equilibrium diameter Deq; formation of small aggregates in the neighborhood of MLV are also observed. These aggregates either are aspirated into the micropipet or separated from MLV by the time the size of MLV reaches Deq. The shape of MLV fluctuates strongly and can evolve initially from a spherical to a distorted and then back to spherical shape. We have measured the structural transition of the MLV, and the average size of the MLV is calculated as the average of the minor, Dmin, and major, Dmax, dimensions of the MLV as measured from micrographs. Average size MLV is calculated from the micrographs obtained at different time intervals of aspiration. We have observed a continuous decrease of average MLV size which reduced to 50% of the initial size in ∼220 s, which is a rapid process. In addition to size, shape distortion was quantified with aspect ratio a ) Dmax/Dmin, which was estimated to be 1 (spherical) initially at the start of aspiration. At time interval of t ) 0, aspect ratio was 1 and 0.8 (where MLV is distorted) at

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t ) 637 and 811 s, shown in Figure 4. There is rapid change in the aspect ratio with time, reflecting a change in the shape of MLV. However the projection length increased in spite of changes in average size and aspect ratio. The projection length was zero when the peeling of bilayers had started. Detachment of a set of bilayers from MLV is shown in Figure 4b. MLV normally constitutes of N bilayers stacked, while we observed MLV with a large aqueous core volume enclosed by bilayers were subjected to micropipet aspiration. The behavior of these MLV were unusual as that of N bilayer stacked MLV. The average size of MLV decreased marginally, but the aspect ratio of the MLV and its aqueous core decreased continuously until its disappearance. In Figure 5a-c, the aspect ratio reduced from 1 to 0.6 just before the MLV rupture. In a typical observation, initially the thickness of the stack of bilayers around the aqueous core is concentric at time t ) 0. Increment in suction pressure results in aqueous core distortion from spherical to elliptical with the aqueous core moving towards the mouth of the micropipet; rearrangement of the bilayer took place in the vicinity. When MLV bilayer stack thicknesses reached a minimum ∼5 µm near the mouth of the micropipet, membrane rupture was observed. The rupture of MLV structure happens in the outer layer when membrane tension ∼3 mN/m, followed by complete suction of aqueous core and bilayers into the micropipet. It is reported that there are two distinct mechanisms of peeling, simple and buckled, when MLV is contact with solid surface.27 We believe that the micropipet aspiration of MLV stimulates peeling, which is discrete by the removal of a set of bilayers of the MLV. Peeling of MLV may take any of the two routes, layer by layer separation or few outer layers of the MLV, with the MLV as a result of peeling of lower size compared to mother MLV as shown in Figure 6. We have discussed two different peeling phenomena observed during aspiration of MLV, explained in detailed in Figure 6 a,b. Peeling process happens as there is rupture of outer layer upon stress due to micropipet aspiration.28 The detachment of layers is due to spontaneous opening of pores in the outer layer, exposing the inner layers to external fluid. The outermost bilayer is unbound due to thermal fluctuation and peels off from the stack. These scenarios observed are for partially dehydrating fluid samples, where interbilayer forces that distort the structure; behavior is unpredictable for higher aspiration pressures, since bilayers are stacked as oriented arrays. The destabilization in the outer membrane induces the interlamellar reorientation of the tightly packed bilayer membranes, followed by division of MLV, suggesting that MLV consist of membrane folded or onion-like structure29 from which a giant vesicle and tubule are evolved. Various stages observed during peeling of MLV (with bilayers enclosed aqueous core) are shown Figure 6b; peeling behavior was quite unusual in that of MLV with N bilayers stacked. Complete disappearance of MLV happens just before the rupture of outer layer. The stress on the outer layer transfers to the aqueous core, pushing bilayers in the direction of the micropipet. We suspect that the multilayer system stops water uptake upon reaching the so-called equilibrium spacing, with the outermost

Sagar and Bellare membrane tending to peel off. Vesicle rupture could be due opening of pores or due to MLV outer layer in contact with a bilayer edge. Conclusion The present study reports mechanical properties of unilamellar and multilamellar AOT vesicles, estimated using micropipet aspiration technique. These mechanical properties of vesicle membranes are important for various applications because of their flexibility. We have demonstrated a peeling process of MLV; a shape transition in MLV due to stress eventually leads to a MLV of lower size that is experimentally supported. These results provide evidence for the existence of peeling process of MLV during dynamic structural change. We conclude that the dynamic structural change of MLV surface is useful for the investigation of the process of formation of other microstructures from MLV. The observation described here should provide important insights into behavior of vesicles in stressed state, fusion and releasing kinetics in MLV for various studies. References and Notes (1) Hajduk, D. A.; Kossuth, M. B.; Hillmyer, M. A J. Phys. Chem. B 1998, 102, 4269. (2) Yu, K.; Eisenberg, A Macromolecules 1998, 31, 3509. (3) Won, Y.-Y.; Davis, H. T.; Bates, F. S. Science 1999, 283, 960. (4) Fernandez, P.; Norbert, W.; Frechen, T.; Angelika, K. Colloid Surf., A 2005, 262, 204. (5) Bota, A.; Varga, Z.; Goerigk, G. J. Appl. Crystallogr. 2007, 40, 259. (6) Koltover, I.; Salditt, T.; Radler, J. O.; Safinya, C. Science 1998, 281, 78. (7) Cornelissen, J. J. L. M.; Fischer, M.; Sommerdijk, N. A. J. M.; Nolte, R. J. M. Science 1998, 280, 1427. (8) Evans, E.; Rawicz, W. Phys. ReV. Lett. 1990, 64, 2094. (9) Petrov, A. G.; Bivas, J. Prog. Surf. Sci. 1984, 18, 359. (10) Segota, S.; Tezak, D. AdV. Colloid Interface Sci. 2006, 121, 51. (11) Honeywell-Nguyen, P. L.; Bouwstra, J. A. Drug DiscoVery Today: Technol. 2005, 2, 67. (12) Gauffre, F.; Roux, D. Langmuir 1999, 15, 3738. (13) Arrault, J.; Grand, C.; Poon, W. C. K.; Cates, M. E. Europhys. Lett. 1997, 38, 625. (14) Akiko, I.; Ramon, P.; Ushiki, H.; Rouch, H.; Louis, L. J. Phys. Soc. Jpn. 2004, 73, 2449. (15) Diat, O.; Roux, D.; Nallett, F. Phys. ReV. E. 1995, 51, 3296. (16) Gerhard, F.; Norman, J. W.; Kaler, Eric W Langmuir 2003, 19, 8709. (17) Meleard, P.; Gerbeaud, C.; Pott, T.; Fernandez-Puente, L.; Bivas, I.; Mitov, M. D.; Dufourcq, J. Biophys. J. 1997, 72, 2616. (18) Duwe, H. P.; Sackman, E. Physica A 1990, 63, 410. (19) Needham, D.; Nunn, R. S. Biophys. J. 1990, 58, 997. (20) Evans, E.; Rawicz, W. Phys. ReV. Lett. 1990, 64, 2094. (21) Evans, E. A.; Parsegian, V. A. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 7132. (22) Evans, E.; Skalak, R. Mechanics and Thermodynamics of Biomembranes; CRC Press Inc.: Boca Raton, FL, 1980; pp 1-254. (23) Mishima, K.; Nakamae, S.; Ohshima, H.; Kondo, T. Chem. Phys. Lipids 2001, 110. (24) Shillcock, Julian C.; Lipowsky, R. Nat. Mater. 2005, 4, 225. (25) Straume, M.; Litman, B. J. Biochemistry. 1978b, 26, 5121. (26) Rand, R. P.; Fuller, N.; Parsegian, V. A.; Rau, D. C. Biochemistry. 1988, 27, 7711. (27) Hamada, T.; Yoshikawa, K. Chem. Phys. Lett. 2004, 396, 303. (28) Evans, E.; Heinrich, V. C. R. Phys. 2003, 4, 265. (29) Douliez, J. P.; Lavenant, L.; Renard, D. J. Colloid Interface Sci. 2003, 266, 477.

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