Role of Hydrophobic Interactions in the Adsorption of Poly(ethylene

Feb 19, 2009 - When the end group of PEG chain is not hydrophobic enough, PEG chains do not interact with the vesicles so that they have slight effect...
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J. Phys. Chem. B 2009, 113, 3365–3369

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Role of Hydrophobic Interactions in the Adsorption of Poly(ethylene glycol) Chains on Phospholipid Membranes Investigated with a Quartz Crystal Microbalance Guangming Liu, Li Fu, and Guangzhao Zhang* Hefei National Laboratory for Physical Sciences at Microscale, Department of Chemical Physics, UniVersity of Science and Technology of China, Hefei, P. R. China ReceiVed: NoVember 24, 2008; ReVised Manuscript ReceiVed: January 22, 2009

We have investigated the adsorption of poly(ethylene glycol) (PEG) with different end groups onto phospholipid membranes at the liquid/solid interface by use of a quartz crystal microbalance with dissipation in real time. On a SiO2-coated surface, the adsorption of lipid vesicles results in a solid-supported lipid bilayer. Our experiments demonstrate that PEG chains with enough hydrophobic end groups (PEG-C18H37) can insert in the bilayer and form aggregates on the membrane surface. On the other hand, the adsorbed vesicles are intact on a gold surface. When the end group of PEG chain is not hydrophobic enough, PEG chains do not interact with the vesicles so that they have slight effect on the vesicle stability. However, PEG-C18H37 chains with enough hydrophobic end groups lead to a vesicle-to-bilayer transition because of the insertion of the chains and their aggregation on the membrane surface. In addition, the studies on effect of polymer concentration show that PEG-C18H37 can readily induce the rupture of vesicles at a concentration above the critical micelle concentration. Introduction Cell membranes are complex dynamical structures primarily consisting of a bilayer in which two layers of phospholipid molecules are arranged in a way that the hydrophilic heads shield the hydrophobic lipid tails from the water.1-5 As a permeability barrier, the membrane can protect the cell from the environment and maintain membrane protein stability and function.1-5 Many cellular processes such as endocytosis, exocytosis, fertilization, signal transduction, viral infection, intracellular transport, and cell aggregation are mediated by such a biomembrane.6 Note that membrane proteins inserted within and across the bilayer play an essential role in such processes. For example, membrane fusion, a ubiquitous life process, is generally controlled by the fusion proteins inserted in the core of cell membrane.7-9 Probably because of the complex nature of and their interactions with other cellular systems, direct investigations on biological membranes are extremely difficult to address. Therefore, model membranes are essential for studying membrane related phenomena. The most commonly used model system is a phospholipid bilayer decorated with polymers.10-41 Firestone et al.27,28 have investigated the interactions between a series of poly(ethylene oxide)-b-poly(propylene oxide) (PEO-PPO) diblock copolymers and lipid bilayers by use of X-ray scattering and differential scanning calorimetry and revealed that the association of diblock copolymer-lipid bilayers is determined by the number of molecular repeat units in the hydrophobic PPO block. Using Langmuir isotherm and fluorescence microscopy measurements, Maskarinec et al.26 have investigated the insertion of PEO-PPO-PEO triblock copolymers into a lipid monolayer at the air-water interface. They demonstrated that the insertion alters the phase behavior and morphology of the monolayer. In principle, polymers that will be inserted in a lipid bilayer should first adsorb and bind on the bilayer. One fundamental problem is the effect of molecular interactions on the insertion. * To whom correspondence [email protected].

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Previous studies reveal that the hydrophobic anchor groups in an amphiphilic polymer profoundly influence its insertion in lipid membranes.11,12 However, the role of hydrophobic interactions remains poorly understood. In parallel, some studies demonstrate that adsorption can change the shape profile of the vesicle by altering the bending energy.30,31,33,36,37 Actually, even for the simple lipid membrane system, the mechanism of the interactions between polymer chains and membranes has not been elucidated yet. In the present study, we have lipid vesicles adsorbed on SiO2 or gold surfaces. Because of competition between the adhesion energy of the lipid-solid surface and the bending energy of the vesicle42-45 the vesicles fuse into a supported membrane in the former case but retain vesicles in the latter case. Afterward, we introduced poly(ethylene glycol) (PEG) with different end groups to the solid-supported lipid bilayer (s-SLB) or vesicles at the solid/liquid interface. By use of a quartz crystal microbalance with dissipation (QCM-D) which can provide information about a film at interfaces,46,47 we have investigated the adsorption of PEG chains in real time. The transformation of adsorbed lipid vesicles during the adsorption of PEG has also been studied. Note that no charges are involved in PEG chains, so that the role of hydrophobic interactions can be well extracted. The aim of the present study is to understand the role of hydrophobic interactions in the adsorption of polymer chains on membranes. Experimental Section Materials. Poly(ethylene glycol) (HO(CH2CH2O)113OH), poly(ethylene glycol) methyl ether (HO(CH2CH2O)104CH3), and poly(ethylene glycol) stearyl ether or Brij700 (HO(CH2CH2O)100C18H37) were purchased from Aldrich and used as received. The molecular weights (Mn) of the samples thereafter designated as PEG-OH, PEG-CH3 and PEG-C18H37 are about 5000, 4600, and 4700 g/mol, respectively. Namely, the main difference between the samples is in their end groups. L-R-phosphatidylcholine (Aldrich, 99%) was from egg yolk.

10.1021/jp810304f CCC: $40.75  2009 American Chemical Society Published on Web 02/19/2009

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The buffer used was 10 mM Tris (100 mM NaCl, pH 8.0). Milli-Q water was used for the preparation of buffer solution. Preparation of Phospholipid Vesicles. Unilamellar vesicles were prepared by use of a protocol reported elsewhere.42,48,49 After a certain mass of L-R-phosphatidylcholine was dissolved in chloroform, the lipid/chloroform mixture was dried onto the wall of a continuously rolled flask under flowing N2, and then left for 12 h to ensure the removal of all of the solvent. The dried lipids were resuspended in a Tris buffer under a N2 atmosphere and sonicated by use of a 1/8-in. microtip of a Scientz sonicator (Ningbo Scientz Biotechnology Co., Ltd.) until the suspension turned clear. To protect the sample from heating, the sample was sonicated in an ice/water mixture and the sonication and cooling were alternatively performed, that is, the sample was cooled for 5 s after 5 s of sonication. The total sonication time was ∼30 min. Eventually, the sample was spun for removing Ti particles from the sonicator tip and large aggregates by a centrifuge. The hydrodynamic radius (Rh) of the monodisperse vesicles measured by dynamic laser light scattering (ALV-5022F) was ∼70 nm. QCM-D Measurements. QCM-D with an AT-cut crystal was from Q-sense AB. The fundamental resonant frequency of the crystal was 5 MHz. The crystal was mounted in a fluid cell with one side exposed to the solution.50 The mass sensitivity constant of the crystal was 17.7 ng/cm2 Hz. The measurable frequency shift was within (1 Hz in aqueous medium. A quartz crystal is excited to oscillate in the thickness shear mode at its fundamental resonant frequency (f0) when a RF voltage near the resonant frequency is applied across the electrodes. A small layer added to the electrodes would induce a decrease in resonant frequency (∆f) which is proportional to the mass change (∆m) of the layer. In vacuum or air, if the added layer is rigid, evenly distributed, and much thinner than the crystal, ∆f is related to ∆m and the overtone number (n ) 1, 3, 5,...) by the Sauerbrey equation,51

∆m ) -

Fqlq ∆f f0 n

(1)

where f0 is the fundamental frequency and Fq and lq are the specific density and thickness of the quartz crystal, respectively. The dissipation factor (D) is defined by50

D)

Ed 2πEs

(2)

where Ed is the energy dissipated during one oscillation and Es is the energy stored in the oscillating system. D is measured based on the fact that the voltage over the crystal decays exponentially as a damped sinusoidal when the driving power of a piezoelectric oscillator is switched off.50 By switching the driving voltage on and off periodically, we can simultaneously obtain a series of changes of the resonant frequency and the dissipation factor. When QCM-D is applied to soft matter systems, the changes in mass and structure of the soft materials on the resonator surface as well as the viscoelastic properties of the film can be obtained by using the Voigt model.52 ∆f and ∆D values from the fundamental overtone were usually noisy because of insufficient energy trapping and thus discarded.53 The SiO2 coated resonator was cleaned by exposing to UVproduced ozone in air for 30 min followed by immersing into a 2% SDS solution for 10 min of sonication in an ultrasonic

Figure 1. The changes in frequency (∆f) and dissipation (∆D) as a function of time for the adsorption of lipid vesicles onto a SiO2-coated resonator surface, where the overtone number (n) is 3.

Figure 2. The changes in frequency (∆f) and dissipation (∆D) as a function of time for the adsorption of PEG-OH and PEG-CH3 onto the solid-supported lipid bilayer at a concentration of 0.05 mg/mL, where the overtone number (n) is 3.

bath. The resonator was rinsed with Milli-Q water and dried under N2. The gold coated resonator was cleaned by using Piranha solution composed of one part H2O2 and three parts H2SO4 for 3 h at room temperature, rinsed with Milli-Q water, and blown dry with N2 before use. All measurements were conducted at 25 ( 0.02 °C. Results and Discussion On Lipid Bilayer. First, we have examined the adsorption of lipid vesicles onto a SiO2-coated resonator surface. Figure 1 shows the changes in frequency (∆f) and dissipation (∆D) as a function of time. It can be seen that ∆f rapidly decreases to a minimum. Then, it gradually increases and levels off. Meanwhile, ∆D has an initial increase followed by a gradual decrease. As reported before about the vesicle-to-bilayer transition of eggyolk phosphatidylcholine on a SiO2 surface,42-44 the time dependences of ∆f and ∆D indicate the formation of s-SLB. Specifically, the initial decrease in ∆f and increase in ∆D indicate that the intact vesicles are adsorbed onto the clean substrate. The following increase in ∆f and decrease in ∆D reflect that the vesicles rupture and fuse into a bilayer because the more densely packed bilayer has a smaller ∆D and the release of trapped water molecules after a dense packing leads ∆f to increase. ∆f rests at ca. -23 Hz after the rinsing, corresponding to a fully covering bilayer.42-44 Figure 2 shows the changes in frequency (∆f) and dissipation (∆D) as a function of time for the adsorption of PEG-OH and PEG-CH3 onto the s-SLB surface at a concentration (C) of 0.05 mg/mL. The addition of PEG-OH solution into QCM cell does not lead to any significant change in either ∆f or ∆D, suggesting a slight adsorption of PEG-OH chains on the bilayer surface. In other words, the interactions between them are weak. Both ∆f and ∆D return to the original points after rinsing, indicating that no PEG-OH chains are adsorbed on the s-SLB surface. Note

Hydrophobic Interactions in the Adsorption of PEG

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Figure 4. The changes of elastic shear modulus (µ) of the solidsupported lipid bilayer during the rearrangement of PEG-C18H37 chains at the concentrations of 0.05 and 1.0 mg/mL.

Figure 3. The changes in frequency (∆f) and dissipation (∆D) as a function of time for the adsorption of PEG-C18H37 onto the solidsupported lipid bilayer at the concentrations of 0.05 and 1.0 mg/mL, where the overtone number (n) is 3.

that PEG-OH does not have a hydrophobic end group. Therefore, the results indicate that no PEG chains are directly adsorbed on the surface. The adsorption of PEG-CH3 is analogous to that of PEG-OH, although the methyl in the former is more hydrophobic than hydroxyl in the latter. Note that the transient spikes in ∆f and ∆D are attributed to the fluctuation of temperature during the solution exchange. Accordingly, either PEG-OH or PEG-CH3 chains slightly interact with the membranes. To understand the role of the hydrophobic interactions, we have also investigated the adsorption of PEG chains with a much more hydrophobic end group (-C18H37). Figure 3 shows the changes in frequency (∆f) and dissipation (∆D) as a function of time for the adsorption of PEG-C18H37 onto the s-SLB surface at the concentrations of 0.05 and 1.0 mg/mL. The critical micelle concentration (CMC) of PEG-C18H37 is ∼0.1 mg/mL,54 so PEGC18H37 chains exist as individuals at the concentration of 0.05 mg/mL. After PEG-C18H37 solution is introduced at C ) 0.05 mg/mL, ∆f sharply decreases. This can be attributed to the insertion of PEG-C18H37 chains in the lipid bilayer and the aggregation between the inserted chains and the incoming chains on the membrane surface. Note that the chains can form polymer aggregates on the membrane surface even at the concentration below CMC due to the attraction between the chains. Subsequently, ∆f slowly increases, indicating that the trapped water molecules in the polymer aggregates are slowly released out due to the rearrangement of polymer chains on the membrane surface. On the other hand, it is known that a dense and rigid structure exhibits a lower dissipation of energy, whereas a looser and more random structure leads to a higher dissipation.52 The sharp increase of ∆D in the initial stage indicates that PEGC18H37 chains insert into the bilayer and they further form random aggregates with the incoming chains. The subsequent decrease in ∆D suggests that the adsorbed chains gradually pack more densely upon the rearrangement of the chains. After rinsing, ∆f almost returns to the original point, indicating that most of the polymer aggregates are removed from the outer surface of membrane and only a small number of polymer chains are incorporated in the bilayer. Thus, the difference in adsorption between PEG-C18H37 and PEG-OH or PEG-CH3 is largely due to the insertion and aggregation of the chains induced by the hydrophobic end groups. However, ∆D does not return to the original point. This is because the hydrophilic tails of the incorporated PEG-C18H37 chains protruding from the s-SLB

Figure 5. The changes in frequency (∆f) and dissipation (∆D) as a function of time for the adsorption of lipid vesicles onto a gold-coated resonator surface, where the overtone number (n) is 3.

surface have a significant influence on ∆D.47 At C ) 1.0 mg/ mL, PEG-C18H37 chains form micelles in equilibrium with free chains. The initial sharp decrease in ∆f and increase in ∆D indicate the rapid adsorption of PEG-C18H37 micelles and the insertion of the free chains. Subsequently, ∆f and ∆D slightly change, implying no obvious rearrangement of polymer chains on the membrane surface. The rinsing with buffer only leads to small changes in ∆f and ∆D, indicating that the adsorbed PEGC18H37 micelles and the incorporated chains are stably attached on the s-SLB surface. Note that the aggregates formed on the membrane surface in this case are different from those at the lower concentration (0.05 mg/mL). They might form wormlike micelles and bind strongly to the membrane surface so that they are not easily removed upon rinsing. Figure 4 shows the changes of elastic shear modulus (µ) of s-SLB during the adsorption of PEG-C18H37 chains at their concentrations of 0.05 and 1.0 mg/mL, where µ is obtained from the fitting of ∆f and ∆D at n of 3, 5, 7 based on the Voigt model.52 It can be seen that µ gradually decreases with time at C ) 0.05 mg/mL, indicating that the rearrangement of the adsorbed chains renders the s-SLB more fluid so that the bending rigidity decreases.55-57 This is consistent with the theoretical prediction.58-60 At C ) 1.0 mg/mL, µ almost keeps constant with time further indicating no obvious rearrangement of the chains on the membrane surface. On Lipid Vesicles. Figure 5 shows the changes in frequency (∆f) and dissipation (∆D) as a function of time for the adsorption of lipid vesicles onto a gold-coated resonator surface. ∆f rapidly decreases and leaves off, indicating the saturation of lipid vesicles on the gold surface. The rinsing has a slight effect on the saturation, indicating that the vesicles are strongly adsorbed.

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Figure 6. The changes in frequency (∆f) and dissipation (∆D) as a function of time for the adsorption of PEG-OH and PEG-CH3 onto the layer formed by lipid vesicles at a concentration of 0.05 mg/mL, where the overtone number (n) is 3.

Figure 7. The changes in frequency (∆f) and dissipation (∆D) as a function of time for the adsorption of PEG-C18H37 onto the layer formed by lipid vesicles at the concentrations of 0.05 and 1.0 mg/mL, where the overtone number (n) is 3.

The large ∆f (ca. -90 Hz) is due to the trapped water molecules in the adsorbed vesicles.42 On the other hand, since the adsorbed vesicles are soft and easily deformed, the internal friction increases, leading to the significant increase in energy dissipation.42 As reported before,42,44 the facts indicate that intact lipid vesicles are absorbed on the gold surface. Figure 6 shows the changes in frequency (∆f) and dissipation (∆D) as a function of time for the adsorption of PEG-OH and PEG-CH3 onto the layer formed by lipid vesicles at a concentration of 0.05 mg/mL. The introduction of PEG-OH only leads to a very small change in ∆f, indicating a slight adsorption of PEG-OH chains. In other words, PEG chains do not exhibit any significant adsorption on the lipid vesicle surface. The larger shift of ∆D can be attributed to the formation of loops or tails of a few PEG-OH chains on the vesicle surface, which has marked effect on the energy dissipation.47 The adsorption of PEG-CH3 chains onto the vesicle surface is similar. Therefore, either PEG-OH or PEG-CH3 chains do not exhibit significant interactions with the vesicle membrane so that no vesicle-tobilayer transformation occurs. Figure 7 shows the changes in frequency (∆f) and dissipation (∆D) as a function of time for the adsorption of PEG-C18H37 onto the layer formed by lipid vesicles at the concentrations of 0.05 and 1.0 mg/mL. After adding PEG-C18H37, ∆f decreases

Liu et al.

Figure 8. The frequency changes of vesicle rupture fit by using exponential decay function at the concentrations of 0.05 and 1.0 mg/ mL, where the overtone number (n) is 3.

and ∆D increases sharply in the initial stage, indicating a rapid binding of PEG-C18H37 chains or micelles onto the surface of lipid vesicles. The most important event is that ∆f and ∆D exhibit a minimum and a maximum, respectively. The increase in ∆f and decrease in ∆D after their extrema indicate that the vesicles rupture and fuse into a bilayer because a lot of water molecules are released out during the vesicle-to-bilayer transition. Theoretical studies indicate that the adsorption of vesicles onto a surface is governed by the competition between adhesion energy (Fa) and bending energy (Fb), where Fa ) -WA* and Fb )(k/2)IdA(C1 + C2 - C0)2.42-45,55-57 W and A* are the effective contact potential and the contact area, respectively, k is the bending rigidity of the membrane, C1 and C2 are the two principal curvatures, and C0 is the spontaneous curvature.55-57 The integration is performed over the surface area A of the vesicle. The former is the energy to deform the shape, which is gained by the vesicle adsorption. The latter is the energy to hold the vesicle shape. When the latter is dominated by the former, a planar bilayer results via fusion of the vesicles.45 During the adsorption of PEG-C18H37 chains on the membrane surface, W and A* should hold constant, thereby resulting in a constant Fa. The adsorption of polymer chains only leads to a fluctuation of local membrane curvature,30,31,36 so Fb mainly depends on the bending rigidity. Clearly, the rupture of vesicles is likely dominated by the change of bending rigidity. This can elucidate the rupture of vesicles at C ) 0.05 mg/mL because of the decrease of bending rigidity during the rearrangement of the chains, but it can not explain the results at C ) 1.0 mg/mL since the elastic shear modulus slightly changes at the concentration (Figure 4). Indeed, our results demonstrate that the insertion of the chains and their aggregation on the membrane surface might profoundly influence the stability of vesicles, that is, they lead to the rupture of vesicles due to the strong interactions between the chains and membrane. This conclusion can be further validated by comparing the relative rates of vesicle rupture induced by the adsorption of PEG-C18H37 at different concentrations. The frequency changes of vesicle rupture were fit by using an exponential decay function (∆f ) ∆f0 + A exp(-t/τ)) as shown in Figure 8, where A is a constant and t is the time. The characteristic decay constants (τ) are 3.7 and 2.5 min for C ) 0.05 mg/mL and 1.0 mg/mL, respectively, indicating that the vesicle rupture in the latter is faster than that in the former. This is understandable because the free PEGC18H37 chains at C ) 1.0 mg/mL are more than those at C ) 0.05 mg/mL; a much more rapid insertion of free polymer chains

Hydrophobic Interactions in the Adsorption of PEG is expected at a higher concentration. On the other hand, the polymer aggregates may be directly adsorbed onto and interact with the surface of vesicles at the concentration of 1.0 mg/mL, leading to a fast rupture of vesicles. However, the chains should first insert in the vesicle membrane and then form aggregates with the incoming chains on the membrane surface at C ) 0.05 mg/mL, which results in a relatively slow rate of vesicle rupture. Conclusion The adsorption of PEG chains onto the phospholipid membranes has been investigated by use of QCM-D in real time. Lipid vesicles adsorbed on a SiO2 surface form a solid-supported lipid bilayer (s-SLB) but retain intact vesicles on a gold surface. PEG chains with weakly hydrophobic end groups do not interact with either s-SLB or the vesicle membrane. However, PEG chains with enough hydrophobic end groups (PEG-C18H37) can insert in the bilayer and form aggregates on the membrane surface. For the intact vesicles on a gold surface, the adsorption of PEG-C18H37 leads to a vesicle-to-bilayer transition due to the insertion of polymer chains and the formation of polymer aggregates on the membrane surface. The present study reveals that the hydrophobic end group plays an important role in the rupture of vesicles. To further investigate the interactions between polymer chains and vesicle membranes, isothermal titration calorimetry and laser light scattering measurements are desirable. Acknowledgment. The financial support of the National Distinguished Young Investigator Fund (20725414) and Ministry of Science and Technology of China (2007CB936401) is acknowledged. References and Notes (1) Cabiaux, V.; Wolff, C.; Ruysschaert, J. M Int. J. Biol. Macromol. 1997, 21, 285. (2) Cevc, G.; Richardsen, H. AdV. Drug DeliVery ReV. 1999, 38, 207. (3) Im, W.; Brooks, C. L., III. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 6771. (4) Sun, J. J.; Vernier, G.; Wigelsworth, D. J.; Collier, R. J. J. Biol. Chem. 2007, 282, 1059. (5) Dorairaj, S.; Allen, T. W. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 4943. (6) Alberts, B.; Bray, D.; Lewis, J.; Raff, M.; Roberts, K.; Watson, J. D. Molecular Biology of the Cell; Garland: New York, 1989. (7) Engelman, D. M. Science 1996, 274, 1850. (8) Lipowsky, R. Nat. Mater. 2004, 3, 589. (9) Shillcock, J. C.; Lipowsky, R. Nat. Mater. 2005, 4, 225. (10) Lasic, D. D.; Needham, D. Chem. ReV. 1995, 95, 2601. (11) Ringsdorf, H.; Schlarb, B.; Venzmer, J. Angew. Chem., Int. Ed. 1988, 27, 113. (12) Polozova, A.; Winnik, F. M. Biochim. Biophys. Acta 1997, 1326, 213. (13) Kono, K. Adv. Drug DeliVery ReV. 2001, 53, 307. (14) Hayashi, H.; Kono, K.; Takagishi, T. Bioconjugate Chem. 1999, 10, 412. (15) Kono, K.; Yoshino, K.; Takagishi, T. J. Controlled Release 2002, 80, 321. (16) Sheth, S. R.; Leckband, D. E. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 8399. (17) Zhu, X.; Yan, C.; Winnik, F. M.; Leckband, D. E. Langmuir 2007, 23, 162.

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