Solubilization of Phosphatidylcholine Vesicles by Hydrophobically

Aug 9, 2007 - Key Laboratory of Colloid and Interface Science, Institute of ... by the polymers increases to a greater degree at higher AA fraction, o...
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J. Phys. Chem. B 2007, 111, 10123-10129

10123

Solubilization of Phosphatidylcholine Vesicles by Hydrophobically Modified Poly(acrylamide)-co-(Acrylic Acid): Effects of Acrylic Acid Fraction and Polymer Concentration Yanru Fan, Yuchun Han, and Yilin Wang* Key Laboratory of Colloid and Interface Science, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100080, People’s Republic of China ReceiVed: May 9, 2007; In Final Form: June 27, 2007

The interaction of hydrophobically modified copolymers of acrylamide and acrylic acid, designated as PAMC12-AA (X%) (X% indicates the percentage of acrylic acid unit and X ) 5, 10, 20), with dimyristoylphosphatidylcholine (DMPC) vesicles has been studied. Complementary techniques including isothermal titration microcalorimetry (ITC), differential scanning calorimetry (DSC), turbidity measurement, calcein leakage measurement, dynamic light scattering (DLS), and transmission electron microscopy (TEM) were used to get comprehensive information. The results show that PAM-C12-AA leads to solubilization of DMPC vesicles. There is a critical concentration (Cs) for PAM-C12-AA to induce obvious vesicle disruption. This concentration is very close to the critical aggregation concentration (CAC) for the polymer self-aggregation. The Cs values are found to be similar for the three polymers. However, the disruption of DMPC vesicles induced by the polymers increases to a greater degree at higher AA fraction, owing to the increasing strength of interaction between the polymer and the lipid bilayer.

Introduction Mixed systems of lipids with water-soluble polymers have provided major improvements in the lowering of protein adsorption, the increase of circulation time in vivo, and the responsiveness to external stimuli for targeted release.1-6 Similar to surfactants, polymers can change the permeability of the membrane, modify the curvature energy, and even solubilize lipids into mixed micelles. However, differing from surfactants, the structure of the polymer can be designed to change in a continuous manner, by gradually changing the charge density or the hydrophobicity of long chains, making it more favorable to modulate the stability of vesicles.7 In recent years, amphiphilic polymers composed of both hydrophobic and hydrophilic parts have received increasing attention because they can easily adsorb onto the surface of lipid vesicles. Winnik’s group8-10 has studied the effect of polymer architecture on the interaction between temperature-sensitive hydrophobically modified poly(N-isopropylacylamide) and liposomes and found that the anchoring process of the polymers occurs on zwitterionic lecithin liposomes and on negatively charged phosphatidic acid liposomes. Tribet’s group1,7 has investigated the kinetics of vesicle disruption induced by hydrophobically modified poly(acrylic acid) and proposed tentative mechanisms. Meanwhile, it was found that stabilization of vesicles is usually obtained by using slightly hydrophobic polymers that do not significantly perturb the bilayer. In contrast, polymers with high hydrophobicity destabilize the lipid bilayers. These previous works concerning amphiphilic polymers and phospholipids have considered various structure factors influencing their interaction, including the isopropylamine graft and the content and length of the hydrophobes and the labeled aromatic groups. However, the effect of acrylic acid groups * To whom correspondence [email protected].

should

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addressed.

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incorporated in hydrophobically modified poly(acrylamide) on their interaction with phospholipids1,7 has not been paid much attention, although they play an important role. In the present study, a series of dodecyl-modified copolymers containing acrylamide (AM) and acrylic acid (AA) designated as PAM-C12-AA (X%, X ) 5, 10, 20) were used. The rheological properties of these polymers on addition of surfactants have been studied by Li and Kwak.11,12 The different AA incorporation in the polymers has been found to play an important role in viscosity enhancement. It is noted that these polymers have both hydrophobic anchors and ionic groups, and the charge density of polymers can be varied in a continuous manner by gradually changing the AA fraction. Therefore, the interaction between these polymers and the phospholipid vesicles is intriguing to us. Would the addition of PAM-C12-AA to dimyristoylphosphatidylcholine (DMPC) vesicles disrupt vesicles or stabilize vesicles? How would the different AA fractions influence the interaction of the copolymers with DMPC vesicles? In order to obtain comprehensive information, we used complementary techniques including isothermal titration microcalorimetry (ITC), differential scanning calorimetry (DSC), turbidity measurement, calcein leakage measurement, dynamic light scattering (DLS), and transmission electron microscopy (TEM). Use of the ITC method to study polymer-phospholipid interaction has been rarely seen in the literature.13 Moreover, the results from different methods support each other, and the possible interaction mechanism was discussed. Experimental Section Materials. The hydrophobically modified polymers PAMC12-AA (X%) (X ) 5, 10, 20) are random terpolymers of acrylamide, N-dodecylacrylamide, and acrylic acid. X% indicates the respective percentage of acrylic acid. The polymers were prepared by radical polymerization. The detailed preparation

10.1021/jp0735637 CCC: $37.00 © 2007 American Chemical Society Published on Web 08/09/2007

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Figure 1. Molecular structures of DMPC and PAM-C12-AA (X%).

method was described in the literature.11 The polymer molecular weights were estimated from the capillary viscometry of dilute solution and are between 1.6 × 105 and 2.5 × 105 Da in all cases. These polymers can be assumed to have similar molecular masses and different charge densities. PAM-C12-AA (5%), PAM-C12-AA (10%), and PAM-C12-AA (20%) are abbreviated to 5% AA, 10% AA, and 20% AA, respectively. The phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphotidylcholine (DMPC) was purchased from Sigma and used without further purification. Calcein and Sephadex G-50 were obtained from Sigma and Pharmacia, respectively. The structures of polymers and DMPC are shown in Figure 1. All the experiments were carried out in sodium phosphate buffer with ionic strength I ) 10 mM and pH ) 7.0. Preparation of Vesicles. An appropriate amount of DMPC was dissolved in chloroform and then was dried under vacuum on a rotary evaporator to obtain a thin film on the bottom of the flask. The dried film was subsequently suspended in a certain volume of buffer to a final desired concentration and votexed at the required temperature above Tc (gel-to-liquid crystal transition temperature) for several minutes. After that, the crude phospholipid suspension was sonicated for about 10 min using a cell sonifier at power 500 W alternating 5 s bursts and 2 s rest periods. These freshly prepared vesicle dispersions were then directly used. Isothermal Titration Calorimetry (ITC). The ITC method was used to measure the heat flow relating to the adsorption of polymers onto liposome surface and further interaction, which was taken in a TAM 2277-201 microcalorimetric system (Thermometric AB, Ja¨rfa¨lla, Sweden) with a stainless steel sample cell of 1 mL at 298.15 ( 0.01 K. The reference cell was filled with buffer. The sample cell was initially loaded with 0.7 mL of buffer or 1 mM of DMPC vesicle dispersion. Aliquots of concentrated polymer solutions of 10 g/L were injected into the stirred sample cell. The measurement of the heat was done as programmed. Differential Scanning Calorimetry (DSC). Calorimetric scans were recorded on a TA Instruments Q100 interfaced to a refrigerated cooling system at a heating rate of 2 °C/min. Weighted amounts of the various samples were sealed in aluminum pans and equilibrated at 5 °C for 10 min. The scans were recorded over a range from 5 to 50 °C. The lipid phase transition temperature (Tm) was taken as the temperature at the peak of endothermic transition. The lipid phase transition enthalpies (∆Hm) were determined by integration of the peak using the software provided by TA Instruments. Calcein Leakage. Calcein-loaded vesicles were prepared by hydrating DMPC with buffer containing 25 mM calcein. The unentrapped dye was removed by running through a Sephadex G-50 column eluted with calcein-free buffer. The liposome fractions were collected and diluted to the required lipid concentration. The liposome and polymer solutions were rapidly mixed, and the fluorescence intensity was measured as a function of time with excitation at 495 nm and emission at 520 nm on

Fan et al. a Hitachi model F-4500 spectrophotometer. Calcein leakage was calculated according to % release ) (Fs - Fb)/(FTX - Fb) × 100, where Fs is the fluorescence intensity of the prepared sample in the presence of polymer, Fb is the background fluorescence intensity of the calcein-containing vesicles in the absence of polymer, and FTX is the maximum fluorescence intensity obtained by adding TX-100 to disrupt all the vesicles. Turbidity Measurements. Concentrated polymer solution was added in a stepwise manner to 1 mL of vesicle dispersion of 1 mM concentration. After each addition, the sample was equilibrated for 10 min, and thereafter, the absorbance was measured using a Hitachi UV-vis spectrophotometer (model U-4500). All the measurements were conducted at 298.2 ( 0.5 K. Dynamic Light Scattering (DLS). Measurements were carried out at 298.2 ( 0.5 K with a LLS spectrometer (ALV/ SP-125) that employs a multi-τ digital time correlator (ALV5000). A solid-state He-Ne laser (output power ) 22 mW at λ ) 632.8 nm) was used as a light source. The samples were introduced into the 7 mL glass bottle through a 0.45 µm Millipore filter prior to measurements. Thereafter, DLS measurements were performed at a scattering angle of 90°. The autocorrelation function of scattering data was analyzed via the CONTIN method. Transmission Electron Microscopy (TEM). TEM samples were prepared using a negative-staining method. A drop of the solution was placed onto a carbon-coated copper grid (300 mesh), blotted with filter paper, and then negatively stained with 1% uranyl acetate ethanol solution. After drying, the samples were imaged under a JEM-200CX electron microscope. Results and Discussion Turbidity. Turbidity measurement is a simple and direct method for detecting the changes of aggregate size. Figure 2a shows the changes of turbidity of the polymer-DMPC mixtures with an increase in the polymer concentration at fixed DMPC vesicle concentration of 1 mM. The blank curve of polymerfree titration is also provided, which was performed by adding the same volume of buffer instead of polymer solution into 1 mM DMPC vesicle dispersion. As can be seen, for the polymerfree system, initially the turbidity decreases obviously, and then it changes very slowly as more buffer is added. The initial decrease may be due to the disintegration of some vesicle clusters and the dilution of the vesicles. In the presence of polymer, the decrease in turbidity of the first several points may be due to the same reason for the buffer titration. Then, after a critical concentration, the turbidity drops more steeply until reaching a rather low constant value. As shown in Figure 2a, the critical point corresponds to the change in the slope of turbidity decline, which can be clearly observed in 10% AA and 20% AA curves, although it is not obvious for 5% AA. It is proposed that the more significant decrease afterward in turbidity may result from vesicle disruption into bilayer fragments or mixed micelles, as supported by the later DLS and TEM results. Therefore, the critical concentration is defined as Cs here. The Cs values for the three polymers are quite close to each other and are all ∼0.35 g/L. Besides, Figure 2a shows that, at the same polymer concentration beyond Cs, the turbidity values decrease from 5% AA to 20% AA. This may suggest that the polymer with higher AA fraction interacts more strongly with DMPC bilayers and leads to a greater extent of vesicle disruption. It is noted that the turbidity of the 5% AA/DMPC mixture decreases rather gradually compared to the other two polymers. This phenomenon reveals that the polymer with lower

Solubilization of Phosphatidylcholine Vesicles

Figure 2. (a) Turbidity curves for these three polymers titrated into 1 mM DMPC and the blank curve of buffer titrated into 1 mM DMPC vesicles. (b), (c), and (d) Calorimetric curves for 20% AA, 10% AA, and 5% AA titrated into 1 mM DMPC vesicles and buffer solution, respectively.

AA fraction interacts with the DMPC vesicles rather gently and the DMPC vesicles are disrupted to a lower extent at the same polymer concentration. ITC. Microcalorimetry is a very powerful tool to investigate weak interactions between molecules because of its high sensitivity.14,15 Parts b, c, and d of Figure 2 show the variation of the observed enthalpy ∆Hobs versus the polymer concentration for the polymers titrated into buffer and 1 mM DMPC vesicles. The polymer-DMPC interaction induces significant differences in the calorimetric curves compared to those of the polymerbuffer system, especially at lower polymer concentrations. For the titration of the polymer into buffer, the observed enthalpies ∆Hobs increase to more negative values at the initial several

J. Phys. Chem. B, Vol. 111, No. 34, 2007 10125 points and then decrease gradually to less negative, which generate a critical concentration corresponding to the maximum ∆Hobs. For the curves of polymer-DMPC interaction, a critical concentration is also observed in each curve, which is similar for all the polymers and very close to the critical concentration in the buffer system within experimental error. On the other hand, the ∆Hobs values exhibit obvious differences between 20% AA and the other two polymer concentrations. For 5% AA and 10% AA, initially ∆Hobs abruptly decreases from significantly exothermic to less exothermic values, and then it continues to decrease to much less exothermic values successively in a similar trend to that of the polymer-buffer system. Compared to those of the polymer-buffer system, the ∆Hobs values are much more exothermic before the transition point and relatively less exothermic after that. For 20% AA, a similarly changing trend to that of the buffer curve is detected in the whole concentration range, except that the ∆Hobs value is always more exothermic than the corresponding buffer curve. For the titration of polymer into buffer, the break point may correspond to the critical concentration for the polymer selfassociation, which is defined as CAC here, as shown in Figure 2. It is well-known that hydrophobically modified polymers comprise both hydrophilic and hydrophobic parts, so they often associate into micelle-like aggregates through intra- or intermolecular hydrophobic interactions.5 Here, despite the different AA fractions, all the polymers have similar CACs. This result may suggest that the hydrophobic branches along the backbone play a dominant role in the polymer association. On the other hand, the appearance of a maximum ∆Hobs at CAC for all the polymers can be explained as follows. PAM-C12-AA polymers have both hydrophobes and carboxyl groups. At pH 7.0, based on our previous pH titration results,16 the ionization degrees for the acrylic acid groups of these three polymers are all ∼73% at pH 7.0. Before CAC, when the concentrated polymer solution is titrated into buffer, the polymer aggregates are disrupted and the polymer molecules are further hydrated. The further hydration of the polymer molecules during the aggregate disruption should be the main factor leading to the exothermic enthalpy. This exothermic enthalpy decreases with the decrease of the AA content in the polymers. Beyond CAC, since the polymer aggregates can be partially kept after titration, the disruption extent of the polymer aggregates tends to decrease. Then, the extent of the molecule hydration becomes weak gradually, so the observed exothermic values are getting smaller. For the titration of the polymers into DMPC, the break points of Cs, which are in agreement with the Cs defined from the turbidity curves, correspond to the critical concentrations for the polymer-induced vesicle solubilization. The Cs values are very close to CAC in each plot. The solubilization of DMPC vesicles may start when the number of polymer molecules is sufficient enough to form mixed micelles with DMPC molecules. This condition may be similar to the requirement for the polymer self-aggregation because hydrophobic interaction is the primary driving force for both processes. The difference in enthalpy changes between the polymer-buffer curve and the polymer-DMPC curve can be attributed to the DMPC-polymer interaction. As aforementioned, the carboxylic acid groups in PAM-C12-AA are largely deprotonated for all three polymers. According to Seki and Tirrell,17 attractive forces between a partially ionized PAM-C12-AA and DMPC bilayer may arise from (i) charge-dipole interactions involving polymer-bound carboxylate anions and the phosphocholine dipole, (ii) hydrophobic interaction between the hydrophobic anchors of the polymer and the hydrocarbon chains of lipid, and (iii) hydrogen

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Fan et al. TABLE 1: Tm and ∆Hm Values Obtained from DSC for DMPC Vesicles in the Absence and in the Presence of Polymers

Figure 3. Main phase transition from DSC scans for 1 mM DMPC vesicles without polymer and with the polymers 5% AA, 10% AA, and 20% AA at a concentration of 0.5 g/L.

bonding involving un-ionized carboxyl groups of the polymer and the phosphodiester head group of the lipid. These kinds of interactions may all result in exothermic effect. Meanwhile, there are also several factors leading to endothermic effect. One is the electrostatic repulsion between partly ionized polymers, which will hinder more free polymers absorbing onto the polymer-bound vesicle surface with the same charge. The other is relating to the disordering effect of the lipid bilayers and the dehydration of DMPC headgroups induced by polymer binding.18 The enthalpy of the interaction between the polymer and DMPC is the sum of all the above heat effects. In the present study, these three copolymers have the same length and amount of the side hydrocarbon chains. Thus, their difference in ∆Hobs is mainly related to the AA incorporation in the polymers. For both 5% AA and 10% AA, before Cs, the three factors inducing exothermic effect are predominant, while both the disturbance of bilayer and the electrostatic repulsion are not obvious. Therefore, the resultant enthalpy of interaction between the polymers and DMPC is exothermic. After Cs, the solubilization of vesicles occurs, and the disturbance of the ordered DMPC bilayers and the structure reorganization of DMPC vesicles will produce more endothermic effect, which results in less negative ∆Hobs. In the case of 20% AA, the charge-dipole interaction and the hydrogen bonding with DMPC molecules are stronger than the other two polymers, owing to its highest AA fraction. Therefore, the exothermic effect still plays a dominant role after Cs. DSC. In order to probe the effect of these polymers on the ordered state within the hydrocarbon core of DMPC bilayers, we performed DSC measurement. Figure 3 presents a series of DSC scans of pure DMPC and the DMPC-polymer mixtures. Clearly, the incorporation of the polymers in DMPC vesicles broadens the peak of endothermic transition, and the peak becomes broader with the increase of AA fraction. For clarity, the results of phase transition enthalpy (∆Hm) and phase

polymer

Tm/°C

∆Hm/J‚g-1

none 5% AA 10% AA 20% AA

22.6 23.0 23.7 -

0.649 0.138 0.091 -

transition temperature (Tm) for gel to liquid-crystalline transition are given in Table 1. The Tm value of 20% AA cannot be obtained because its peak is not obvious. It can be seen that ∆Hm is reduced significantly with the addition of polymer and the values become much lower at higher AA fraction. In addition, Tm becomes a little larger with the addition of polymer and with the increase of AA fraction. In general, the thermotropic phase behavior of lipid membranes is strongly influenced by the presence of guest molecules. The characteristics of the gel-liquid phase transition are determined by the nature of interaction between the membrane constitutes and by the structure of the guests.2 The present guest molecules are PAM-C12-AA polymers. Firestone and coworkers2,19 have suggested that the width of a transition is an important indicator of the size of the cooperative unit that undergoes the transition and is inversely proportional to the cooperativity of the phase transition. Thus, the broader transition peak in the present DMPC-polymer mixture compared to pure DMPC may reflect a highly noncooperative event in the bilayer. The much broader peak induced by the polymer with higher AA fraction signals the increasing noncooperativity, which results in vesicle disruption. According to the literature,17 there are three steps in the polymer-lipid interactions: (i) adsorption of the polymer on the lipid bilayer surface, (ii) insertion of the polymer into the bilayer, and (iii) complete disruption of the bilayer, accompanied with the formation of mixed polymer-lipid micelles or other aggregates. The first kind of interaction shows no effect on ∆Hm, while the other two factors should reduce ∆Hm; the complete disruption of the bilayer should reduce ∆Hm to zero. On the basis of the above discussion, the DSC results may indicate that all the three polymers insert into the DMPC bilayer and further induce the disruption of the DMPC bilayer. Moreover, the degree of vesicle disruption becomes greater in the order of 5% AA to 20% AA. Because the polymer concentration used is just above Cs, it is speculated that the vesicles may still exist in the polymer-DMPC mixtures, and their contents become lower with the increase of AA fraction. Normally, the incorporation of a guest species into a lipid bilayer acts as a “defect”, increasing the disorder (increasing local membrane fluidity) of the membrane and, thus, lowering the Tm.2,20 Nevertheless, if the guest molecules fit well with the membrane, the local order and the rigidity of the bilayer may be raised due to synergistical hydrophobic interaction, leading to a higher Tm and a reduction in the width of transition.2,20 In the present study, however, we got a little higher Tm and an obvious broader peak in the polymer-DMPC system. On the basis of the above discussion, the increase of Tm in the present system may be due to other reasons. Previously, there were some proposals that dehydration of phosphatidylcholine could increase Tm.21,22 Therefore, here, a hypothesis could be made that the DMPC headgroups are dehydrated by the bound polymer through hydrogen bonding and charge-dipole interaction between carboxyl groups of the polymer and the phosphodiester head group of the lipid, which may replace some of the hydrogen bonding of DMPC with water.

Solubilization of Phosphatidylcholine Vesicles

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Figure 5. Distributions of hydrodynamic radius (Rh) for DMPCpolymer mixed system at 1 mM DMPC and different polymer concentrations indicated in the figure.

Figure 4. Time-course of calcein release from 1 mM DMPC vesicles in the presence of polymers with various concentrations. (d) is derived from (a), (b), and (c) at the polymer concentration of 0.4 g/L.

Calcein Release. Calcein release measurements have been performed in order to correlate the polymer-lipid interaction with the DMPC membrane permeability. The influences of the polymer concentration and the AA fraction are both studied. The addition of polymers into vesicles induces a dye release, which can be reflected in a fluorescence increase.23 The plots in Figure 4 show a similar manner for all the mixed systems. It can be seen that abrupt and rapid increases in leakage are found after addition of the polymers. The increases level off within 10 min, with a following slight increase in release. Parts a-c of Figure 4 reveal that the release of calcein is strongly dependent on the concentration of the polymer. With the increase of the polymer concentration from 0.1 to 1 g/L, the time required to attaining plateau decreases and the maximum release increases. In order to illustrate the effect of AA fraction more

clearly, the curves derived from the data in parts a-c of Figure 4 are summarized in Figure 4d. Here, only the results at 0.4 g/L polymer with different AA fractions are present, because an analogous trend is observed at other polymer concentrations. Figure 4d shows that both the rate of leakage and the percent of release increase with the increase of AA fraction. According to Tribet and co-workers,1 the observed release of calcein has two origins. First, vesicles could disappear gradually while keeping a low permeability until their breakdown. Second, permeability with no membrane disruption could be modulated by the degree of perturbations of the membrane, which must vary with the density of bound polymer. In the present case, at the lower polymer concentration, there is no obvious transformation and solubilization of vesicles. Therefore, the dramatic increase in leakage within a short time may be mainly caused by a fast disturbance of ordered bilayer from the polymer insertion into the vesicle surface, which belongs to the second origin. At the higher polymer concentration, some vesicles are obviously disrupted, as indicated in the turbidity and DSC results. Hence, the higher degree of dye leakage is a result of the vesicle-micelle transition, which belongs to the first origin. Furthermore, the dissimilarity in release rate and extent with the different polymers may be generated from the strength of interaction between the polymer and DMPC. The polymer with higher AA proportion has a higher charge density and a larger amount of protonated AA groups, which definitely strengthen their interaction with DMPC vesicles to a higher degree. This could further lead to more significant disruption of DMPC vesicles and certainly more dye release. Aggregate Size and Morphology. All three polymer-DMPC mixtures have been studied by DLS and TEM. Figure 5 presents the size distribution of 1 mM DMPC with the polymers at different polymer concentrations. Here, only the concentration

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Fan et al.

Figure 6. TEM images of the DMPC-polymer mixtures with 1 mM DMPC and different polymer concentrations: (a) 0.2 g/L, (b) 0.5 g/L, and (c) 1 g/L.

dependence in the 20% AA-DMPC mixture is provided because the situation in other polymer-DMPC systems is similar. As can be seen, in the absence of polymer, the distribution is almost unimodal with an average particle radius of ca. 90 nm. In the presence of polymer, the distribution is bimodal with one peak at 10 nm and a second peak at ∼100 nm. It is suggested that the large particles with average radius of 100 nm are composed of vesicle fragments, mixed vesicles, or other large aggregates induced by preferential association of the polymers with the DMPC bilayers. The small particles of ca. 10 nm should be mixed micelles constituted of solubilized DMPC molecules and the polymers. Because the amplitudes in Figure 5 are based on scattered intensity but a larger particle scatters much more light than a small one,24 the population of particles at 10 nm is actually much greater and that of particles at 100 nm is actually much less, which is also supported by the turbidity results. Furthermore, as can be seen in parts a and b of Figure 5, with the increase of AA fraction and polymer concentration, the amount of small particles becomes larger as indicated by the enlarging of the left peak, which may mean that the disruption of vesicles is enhanced significantly. Further proof on the aggregate morphology is shown in TEM results. Figure 6 only presents some representative TEM micrographs from the 10% AA/DMPC systems at different polymer concentrations, because the aggregates with similar morphology were found in these three kinds of polymer-DMPC mixtures, except that the amount of each kind of aggregate may be different. As can be seen, the morphology of the vesicles depends on the ratio of the polymer to DMPC. Figure 6a shows that, at 0.2 g/L polymer concentration, most of the vesicles are intact in coexistence with some faceted vesicles. The average diameter of vesicles is in agreement with DLS results. When the polymer concentration is just above the CS, some vesicles are broken up and bilayer fragments are formed, as indicated by the black arrows in Figure 6b. When the polymer concentration increases to 1.0 g/L, the broken bilayer fragments become dominant (Figure 6c). It is noted that some of these aggregates are rod-like with their size from about 30 to 100 nm, as illustrated in Figure 6c with arrows. At this concentration, a small fraction of vesicles still coexists. Johnsson et al.25 have studied the DMPC-pluronic samples by Cryo-TEM, and both bilayer disks and spherical micelles have been clearly observed besides vesicles. However, because of the limit of negatively staining TEM, small micelles could not be detected in the present study although they actually exist. On the basis of the above results of DLS and TEM, it may be suggested that the differences in AA fractions in the polymer do not induce significant variation in the kinds and morphologies of aggregates while interacting with vesicles, but they indeed lead to the difference in the amount of each kind of aggregate. It is noteworthy that the vesicles and lamellae are still found at

1 g/L of polymer concentration, indicating that PAM-C12-AA polymers could not induce complete solubilization of DMPC vesicles. Conclusions The effects of PAM-C12-AA copolymers on the structure and permeability of DMPC vesicles have been investigated. The interaction of the copolymers with DMPC is complicated, mainly including charge-dipole interaction, hydrogen bonding, and hydrophobic interaction. The complementary experiments reveal that all three polymers lead to the solubilization of DMPC vesicles despite the different AA fractions. The possible critical polymer concentrations for vesicle solubilization are obtained, and they are similar for the three polymers and also close to the critical aggregation concentration for the polymer selfaggregation. The ITC results show that the stronger interaction between 20% AA and DMPC produces a more exothermic effect than the other two polymers with lower AA fractions. The DSC results prove that the polymers significantly perturb the ordered state within the DMPC bilayer, which further leads to the vesicle disruption. All the results show that the disruption of DMPC vesicles induced by the polymers increases to a greater extent at higher AA fraction, owing to the increasing strength of the interaction between the polymer carboxyl groups and the headgroups of DMPC. However, the solubilization ability of these polymers to DMPC is not strong enough to reach a complete solubilization of DMPC vesicles at the studied polymer concentration range. Acknowledgment. We are grateful for financial support from the National Natural Science Foundation of China (Grants 20633010 and 20573123). We greatly appreciate Professor Jan C. T. Kwak for providing the polymer samples. References and Notes (1) Vial, F.; Rabhi, S.; Tribet, C. Langmuir 2005, 21, 853. (2) Firestone, M. A.; Seifert, S. Biomacromolecules 2005, 6, 2678. (3) Kono, K.; Zenitani, K.; Takagishi, T. Biochim. Biophys. Acta 1994, 1193, 1. (4) Linhardt, J. G.; Tirrell, D. A. Langmuir 2000, 16, 122. (5) Tribet, C. Biochimie 1998, 80, 461. (6) Ringsdorf, H.; Venzmer, J.; Winnik, F. M. Angew. Chem., Int. Ed. Engl. 1991, 30, 315. (7) Ladavie`re, C.; Toustou, M.; Gulik-Krzywicki, T.; Tribet, C. J. Colloid Interface Sci. 2001, 241, 178. (8) Polozova, A.; Winnik, F. M. Biochim. Biophys. Acta 1997, 1326, 213. (9) Ringsdorf, H.; Sackmann, E.; Simon, J.; Winnik, F. M. Biochim. Biophys. Acta 1993, 1153, 335. (10) Polozova, A.; Yamazaki, A.; Brash, J. L.; Winnik, F. M. Colloids Surf., A 1999, 147, 17. (11) Li, Y.; Kwak, J. C. T. Colloids Surf., A 2003, 225, 169. (12) Li, Y.; Kwak, J. C. T. Langmuir 2004, 20, 4859. (13) Kawakami, K.; Nishihara, Y.; Hirano, K. J. Phys. Chem. B 2001, 105, 2374.

Solubilization of Phosphatidylcholine Vesicles (14) Wieprecht, T.; Apostolov, O.; Beyermann, M.; Seelig, J. Biochemistry 2000, 39, 442. (15) Suurkuusk, M.; Singh, S. K. Chem. Phys. Lipids 1998, 94, 119. (16) Song, X. Y.; Cao, M. W.; Han, Y. C.; Wang, Y. L.; Kwak, J. C. T. Langmuir 2007, 23, 4279. (17) Seki, K.; Tirrell, D. A. Macromolecules 1984, 17, 1692. (18) Heerklotz, H.; Epand, R. M. Biophys. J. 2001, 80, 271. (19) Firestone, M. A.; Thiyagarajan, P.; Tiede, D. M. Langmuir 1998, 14, 4688. (20) Grasso, D.; Milardi, D.; La Rosa, C.; Rizzarelli, E. New J. Chem. 2001, 25, 1543.

J. Phys. Chem. B, Vol. 111, No. 34, 2007 10129 (21) Schroeder, U. K. O.; Tirrell, D. A. Macromolecules 1989, 22, 765. (22) Chapman, D.; Williams, R. M.; Ladbrooke, B. D. Chem. Phys. Lipids 1967, 1, 445. (23) Johnsson, M.; Bergstrand, N.; Edwards, K.; Stålgren, J. J. R. Langmuir 2001, 17, 3902. (24) Eum, K. M.; Langley, K. H.; Tirrell, D. A. Macromolecules 1989, 22, 2755. (25) Johnsson, M.; Silvander, M.; Karlsson, G.; Edwards, K. Langmuir 1999, 15, 6314.