Effect of Saline on Transitions in Poly(ethylene glycol)-Grafted

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Effect of Saline on Transitions in Poly(ethylene glycol)-Grafted Succinyl-Phosphoethanolamine Monolayers Bearing C16 Aliphatic Chains Muhammad Naeem Shahid and Valeria Tsoukanova* Department of Chemistry, York University, Toronto, ON, Canada M3J 1P3

bS Supporting Information ABSTRACT: To investigate the effect of saline on miscibility, phase, and conformational transitions in binary mixtures of a succinyl-phosphoethanolamine bearing C16 aliphatic chains, DPPE-succinyl, and a poly(ethylene glycol) (PEG)-phospholipid conjugate with a PEG molecular weight of 2000, DPPEPEG2000, we have compared the properties of monolayers spread on water and on phosphate buffered saline (PBS). A comparative analysis of monolayer surface pressure, surface potential, compressibility, and epifluorescence microscopy data has revealed that spreading on PBS induces unfavorable interactions between the two phospholipids, which stabilizes immiscible phases in mixed monolayers. Strikingly, the conformational transition in grafted PEG2000 chains on PBS could not be easily described by the existing interpretive schemes. Plausibly, this transition becomes partially impaired due to interactions with PBS. Thus, saline has a significant effect on miscibility, phase, and conformational transitions in these PEG-grafted monolayers bearing C16 aliphatic chains, which may have implications for understanding the behavior of PEG-grafted phospholipid surfaces in aqueous media of biological relevance.

1. INTRODUCTION Highly hydrated poly(ethylene glycol) (PEG) chains in a brush conformation grafted onto lipid surfaces is a key requirement in a variety of biomedical applications.1,2 The grafting of PEG chains onto lipid surfaces can be performed in a number of ways; in particular, through the incorporation of PEG-phospholipid conjugates into self-assembled lipid matrixes (monolayers, bilayers, and liposomes/vesicles). However, in aqueous media of biological relevance, grafted PEG chains might undergo dehydration due to interactions with saline.3 Studies performed in saline-containing aqueous solutions have reported that PEG hydration and conformational behavior can be affected by saline in a way not easily predictable by existing theories.4-6 Moreover, interactions with saline may influence the miscibility and phase of constituents of PEG-grafted lipid surfaces.7 Although much has been learned about the self-assembly of lipids and PEG-lipid conjugates,1,7-18 the effect of saline on their miscibility, phase, and conformational behavior at surfaces and interfaces has not yet been systematically studied. Among various model systems, PEG-lipid monolayers at the air/water interface have proven to provide ideal models of PEGgrafted lipid surfaces.1,8-18 The advantage of these monolayers as model systems is that they allow the precise control of parameters such as lateral (surface) pressure and area per lipid molecule, which is not possible with bilayers and liposomes/ vesicles. Wide range variations of PEG grafting density can be easily simulated in a well-controlled manner by the monolayer compression. Moreover, continuous observation of monolayer r 2011 American Chemical Society

morphology upon compression with imaging techniques offers a unique opportunity to monitor the phase behavior of constituents of PEG-grafted lipid surfaces and how it changes depending on various factors.8,11,15,16 Hence, the study of PEG-lipid monolayers provides a basis for understanding the phase and conformational behavior of any PEG-grafted lipid surface. To date, significant attention has been given to the conformational transitions in PEG chains grafted on monolayers at the air/water interface.1,8-16 By contrast, only a few studies have attempted to gain insight into the miscibility, phase, and conformational behavior of PEG-grafted lipid monolayers spread on aqueous subphases of biological relevance; in particular, phosphate buffered saline (PBS).17,18 In this study, we investigated the effect of saline on phase and conformational transitions in mixed monolayers of an N-succinyl-phosphoethanolamine bearing C16 aliphatic chains, DPPEsuccinyl, and a phosphoethanolamine N-derivatized in the headgroup with a PEG chain of molecular weight 2000, DPPEPEG2000. The latter is widely used in biomedical applications; in particular, in long-circulating vesicles for drug delivery.1,2 DPPE-succinyl belongs to the family of N-carboxyacylamidophosphoethanolamines (N-carboxyacylamido-PEs). These lipids can afford, via covalent coupling to the N-carboxyacylamido-PE group, the immobilization of various ligands on the lipid Received: October 14, 2010 Revised: February 17, 2011 Published: March 15, 2011 3303

dx.doi.org/10.1021/jp109877r | J. Phys. Chem. B 2011, 115, 3303–3314

The Journal of Physical Chemistry B

Figure 1. π-A isotherms for the pure DPPE-succinyl monolayer at the air/water interface (a) and on PBS subphase (b) at 20 ( 1 °C. Arrows indicate the LE-LC transition (see text for more detail) at πt,LE-LC of ∼5 and ∼25 mN/m for the monolayer on water and PBS, respectively. The inset shows the chemical structure for the DPPE-succinyl molecule.

surface.2,19 This makes them promising candidates for the design of patterned PEG-lipid surfaces and tunable lipid carriers for targeted delivery of therapeutics and diagnostic agents.2,7,19 The use of N-carboxyacylamido-PEs in these applications however requires understanding the miscibility, phase, and conformational transitions in PEG-grafted N-carboxyacylamido-PE lipid matrixes exposed to aqueous media of biological relevance, which can be obtained from monolayer studies. To the best of our knowledge, this is the first study of the effect of saline on PEGgrafted N-carboxyacylamido-PE monolayers. Binary mixtures of DPPE-succinyl and DPPE-PEG2000 spread on PBS provide ideal monolayer models to begin such a study. Among the family of N-carboxyacylamido-PEs, DPPEsuccinyl has the structure that closely resembles that of the phospholipid part of DPPE-PEG2000. The structural resemblance has been shown to enhance the miscibility of lipids and PEG-lipids.7,15 On the other hand, bearing a negative charge on the headgroup, DPPE-succinyl phospholipids attract cations from PBS. This might affect the phospholipid packing7,20 and the conformational behavior of grafted PEG chains,3,7 which remains largely unexplored, while such knowledge, apart from a fundamental interest, could hold predictive power for many biomedical applications. Hence, the present report will be aimed at elucidating the effect of saline on phase and conformational transitions in mixed DPPE-succinyl/DPPE-PEG2000 monolayers. The DPPE-PEG2000 content in mixed monolayers is varied within 1-9 mol %, the range typically used in drug delivery applications.1 For clarity of discussion, we will use a simple approach of comparing the properties of monolayers spread on water and on PBS. By analyzing together monolayer surface pressure, compressibility, surface potential, and epifluorescence microscopy data, we will demonstrate that in mixed monolayers, the phase transition in C16 aliphatic chains is coupled with the conformational transition in grafted PEG2000 chains. We will show that the latter becomes a dominant factor controlling the miscibility and phase behavior, in particular, the formation of the liquid-condensed phase, in mixed monolayers on PBS.

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Figure 2. π-A isotherms for the pure DPPE-PEG2000 monolayer at the air/water interface (a) and on PBS subphase (b) at 20 ( 1 °C. Arrows indicate the pseudoplateau corresponding to the conformational transition in grafted PEG2000 chains at πt,PEG of ∼10 and ∼12 mN/m, and the high-pressure transition at πt,high of ∼26 and ∼42 mN/m for the monolayer on water and PBS subphase, respectively (see text for more detail). The inset shows the chemical structure for the DPPE-PEG2000 molecule.

2. MATERIALS AND METHODS 2.1. Materials. The poly(ethylene glycol)-grafted phospholipid with PEG average molecular weight 2000, 1,2-dipalmithoylsn-glycero-3-phosphatidylethanolamine-N-[poly(ethylene glycol) 2000] (DPPE-PEG2000); 1,2-dipalmithoyl-sn-glycero-3phosphoethanolamine-N-(succinyl) (DPPE-succinyl); and the fluorescent probe, 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine-N-(lissamine rhodamine B sulfonyl) labeled at the headgroup (DOPE-Rh) were obtained from Avanti Polar Lipids and used as received. Chemical structures of DPPE-succinyl and DPPE-PEG2000 are presented in the insets to Figures 1 and 2, respectively. Phospholipid stock solutions were prepared at a concentration of 0.1-0.5 mg/mL by dissolving in chloroform. The stock solutions were mixed in various molar ratios to obtain spreading solutions containing 1, 3, 6, and 9 mol % PEG. All solutions were stored in the dark at 4 °C. Chloroform was of HPLC grade from Fisher Scientific. Phosphate-buffered saline (PBS) from Sigma containing 0.01 M phosphate salt, 0.12 M NaCl, and 0.0027 M KCl at pH = 7.4 and deionized water produced by a Milli-Q Synthesis water purification system were used as subphases for phospholipid monolayers. The specific resistivity of water was 18 106 Ω 3 cm (pH 6.2 in equilibrium with atmospheric carbon dioxide). 2.2. Methods. A KSV2000SP Langmuir trough (KSV Instruments Ltd., Finland) with an effective surface area of 75 760 mm was used to obtain the surface pressure (π-A) and surface potential (ΔV-A) isotherms. The trough was thermostatted to maintain the subphase temperature at 20 ( 1 °C. Monolayers were spread onto water or PBS subphase. After 15 min to allow for evaporation of the solvent, monolayers were compressed at a rate of 10 mm/min. A filter paper Wilhelmy plate was used to measure the surface pressure, π, to an accuracy of 0.1 mN/m. A vibrating plate surface potential meter, KSV-SPOT1 (KSV Instruments Ltd., Finland), was used to measure the surface 3304

dx.doi.org/10.1021/jp109877r |J. Phys. Chem. B 2011, 115, 3303–3314

The Journal of Physical Chemistry B potential, ΔV, to an accuracy of (3 mV. The area, A, in the isotherms is the mean molecular area.15,18 A series of seven measurements was performed to obtain each isotherm. Isotherm shift along the A axis did not exceed 5% within the series of measurements. Monolayer morphology was visualized by epifluorescence microscopy (EFM). Monolayer EFM images were acquired using a custom NIMA trough (NIMA Technology Ltd., UK) with an effective surface area of 70 460 mm interfaced with an upright Nikon Eclipse FN1 epifluorescence microscope (Nikon, Japan). The π-A isotherm measurement parameters were adjusted to precisely match those discussed above for the KSV2000SP trough. To observe the fluorescence from the rhodamine fluorophore labeled onto the headgroup of DOPERh, Nikon CFI infinity optics, a green excitation filter set (TRITC HYQ filter combination, 545CWL excitation filter, 570LP dichroic mirror, and 620CWL barrier filter), and a 10 objective (Nikon CFI Plan 10) were used. The images were captured by a CCD camera (ORCA ER(AG), Hamamatsu, Japan) directly onto a computer screen using Simple PCI 6 software (Compix Inc., PA). Image analysis in terms of the percentage of dark domains, % dark domains, was performed with the Quantify package of the Simple PCI 6.18

3. RESULTS 3.1. Surface Pressure (π-A) Isotherms. DPPE-succinyl used in our study as the matrix for the DPPE-PEG2000 forms expanded-type monolayers at 20 °C. On water, the lift-off of the DPPE-succinyl monolayer isotherm is observed at 1.25 nm2/ molecule as displayed by curve a in Figure 1. The isotherm exhibits a plateau with a midpoint at ∼5.5 mN/m. The monolayer collapses at A = 0.38 nm2/molecule and π = 56 mN/m. On PBS, the isotherm of the DPPE-succinyl monolayer appears significantly expanded compared with that on water (cf. curves a and b in Figure 1). The isotherm shows the same regions, yet they appear shifted to either larger molecular areas or higher surface pressures than those for the monolayer on water. Indeed, the lift-off of the isotherm b in Figure1 is observed at an area of ∼1.6 nm2/molecule. The isotherm plateau appears at ∼25 mN/m, which is much higher than the plateau pressure for the isotherm obtained on water. The monolayer collapses at A = 0.39 nm2/ molecule and π = 52 mN/m. Figure 2 shows the monolayer isotherms for the PEGphospholipid, DPPE-PEG2000, measured on water and PBS at 20 °C. The isotherm a obtained on water is similar to the previously reported data for DPPE-PEG2000 and has been discussed in detail elsewhere.12,14 Briefly, the isotherm shows a pseudoplateau with a midpoint at ∼10 mN/m. Above the pseudoplateau, another plateau-like region appears in its slope in the 1.6-1.3 nm2/molecule region at π of ∼26 mN/m and, thus, will be referred to as the high-pressure transition.12,14 The monolayer collapses at A = 0.8 nm2/molecule and π = 55 mN/m. Spreading the DPPE-PEG2000 monolayer on PBS results in a somewhat more expanded isotherm (cf. curves a and b in Figure 2). Indeed, the pseudoplateau in isotherm b (Figure 2) shifts to larger molecular areas. The high-pressure transition, although appearing in the same 1.6-1.3 nm2/molecule region, exhibits π of ∼42 mN/m, which is substantially higher than the plateau pressure for the same transition in the isotherm obtained on water. Above the high-pressure transition, the two isotherms converge.

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Figure 3 displays the isotherms of mixed DPPE-succinyl/ DPPE-PEG2000 monolayers containing 1-9 mol % PEG. The isotherms in Figure 3A obtained on water combine the characteristic features observed in the isotherms of the two pure components, DPPE-succinyl and DPPE-PEG2000. A plateau with a midpoint at ∼5.5 mN/m reminiscent of that of the DPPEsuccinyl isotherm a in Figure1 is observed for all the mixed monolayers. With increasing PEG content, the plateau noticeably shrinks while a curvature appears in the isotherm slope at ∼10 mN/m. As seen in Figure 3A, this curvature develops into a pseudoplateau at 9 mol % PEG that shows a close resemblance to the pseudoplateau observed in the DPPE-PEG2000 isotherm a in Figure 2. In the low-compressibility region, the effect of PEG content on the isotherms of mixed monolayers diminishes. Above 30 mN/m, the isotherms of mixed monolayers almost converge, being less than 0.02 nm2 apart from each other. The collapse point was detected for all the mixed monolayers on water at ∼56 mN/m. Figure 3B shows the isotherms of mixed DPPE-succinyl/ DPPE-PEG2000 monolayers obtained on PBS. Spreading on PBS results in isotherms with quite different shapes. At 1 and 3 mol % PEG, the isotherms of mixed monolayers are identical in shape to the DPPE-succinyl isotherm on PBS in Figure 1 (curve b), yet appear slightly shifted to larger molecular areas. Both isotherms exhibit the characteristic plateau with a midpoint at ∼25 mN/m. For monolayers containing 6 and 9 mol % PEG, the plateau in the isotherms in Figure 3B broadens remarkably while the surface pressure at the onset of the plateau decreases. Moreover, another change in the slope appears in these isotherms at ∼42 mN/m, which is reminiscent of the high-pressure transition in the DPPE-PEG2000 monolayer observed in curve b in Figure 2. The collapse pressure of mixed monolayers varied from 52 to 57 mN/m with increasing DPPE-PEG2000 content from 1 to 9 mol %. 3.2. Miscibility Analysis for DPPE-Succinyl/DPPE-PEG2000 Monolayers. According to the surface phase rule,21 continuous growth in collapse pressure with increasing mol % PEG is an indication of miscibility between the two components in the mixed monolayers on PBS. A somewhat similar trend is also observed for the mixed monolayers on water (Figure 3A). However, on water, the collapse pressures of monolayers of the pure components, DPPE-succinyl and DPPE-PEG2000, and their mixtures differ by merely ∼1 mN/m, thus making it difficult to draw an unequivocal conclusion on miscibility in these monolayers. More information on miscibility can be obtained from the analysis of their isotherms in Figure 3 in terms of the excess area of the mixture, Aexc,18 as given by Aexc ¼ A - χDPPE-succinyl ADPPE-succinyl þ χDPPE-PEG2000 ADPPE-PEG2000 Þ

ð1Þ

In eq 1, A is the mean molecular area in isotherms in Figure 3 at any given surface pressure, χDPPE-succinyl and χDPPE-PEG2000 are the mole fractions of the two components, and ADPPE-succinyl and ADPPE-PEG2000 are the molecular areas of DPPE-succinyl and DPPE-PEG2000 in their pure monolayers at the same surface pressure. By definition, Aexc is zero if the two components form an ideal mixture or behave as completely immiscible, whereas any deviation from zero, either positive or negative, evidences miscibility in mixed monolayers.18,21 The values of Aexc calculated for the mixed DPPE-succinyl/ DPPE-PEG2000 monolayers on water and PBS subphase are 3305


The Journal of Physical Chemistry B presented in Tables 1 and 2, respectively. As seen in Table 1 for monolayers on water, Aexc values are mostly negative. This evidently implies a closer packing of DPPE-succinyl and DPPE-PEG2000 molecules in the mixtures than in their pure monolayers, which is indicative of favorable interactions between the two components.18,21 For the mixed monolayers on PBS, Table 2 shows mostly positive Aexc values, with the excess area becoming more pronounced at higher PEG contents. This might point to repulsive interactions, poor miscibility between the two components in monolayers on PBS,21 or both. Thus, Aexc values listed in Tables 1 and 2 suggest that DPPE-succinyl and DPPEPEG2000 are miscible, yet they are likely to mix nonideally. The two components display different types of nonideal mixing behavior characterized by (i) contraction of mean molecular area (negative Aexc values) in the mixed DPPE-succinyl/DPPEPEG2000 monolayers on water and (ii) expansion of mean molecular area (positive Aexc values) in the mixtures on PBS. 3.3. Epifluorescence Microscopy of Monolayer Morphology. The monolayer morphology was visualized with EFM by detecting the fluorescence from a rhodamine-labeled phospholipid analog, DOPE-Rh, added to monolayer spreading solutions at a concentration of ∼0.5 mol %. DOPE-Rh has high affinity for the less ordered liquid-expanded (LE) phase and is largely excluded from the liquid-condensed (LC) phase.15 This enables elucidating the origin of the plateau(s) in π-A isotherms in Figures 1-3; in particular, whether it can be attributed to the LE-LC phase transition characterized by the coexistence of the LE and LC phases. 15,22,23 The latter will appear dark in the fluorescent LE background. Figure 4 shows EFM images captured from pure DPPEsuccinyl monolayers on water and PBS at the end of the plateau in their isotherms. Dark LC phase domains seen in image A in Figure 4 started to appear in the DPPE-succinyl monolayer on water at ∼4.5 mN/m. The domains progressively grew in diameter from ∼4 μm to ∼27 μm upon compression (images not shown). At ∼20 mN/m, the monolayer converted

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to a single dark phase. On PBS, the LC domains of DPPEsuccinyl appeared noticeably smaller than on water. Image B in Figure 4 displays domains 2-8 μm in diameter that remained almost unchanged throughout the compression across the Table 1. Excess Area, Aexc, Calculated Using Eq 1 for Mixed DPPE-Succinyl/DPPE-PEG2000 Monolayers on Water at Various Surface Pressuresa Aexc, nm2 π, mN/m 1 mol % PEG 3 mol % PEG 6 mol % PEG 9 mol % PEG 6

-0.02

-0.02

-0.02

12

-0.03

0

-0.04

-0.02 0

15

-0.04

-0.03

-0.06

-0.05

35 50

0 -0.01

-0.01 -0.01

-0.03 -0.02

-0.02 -0.01

Each value of Aexc is an average over a set of five isotherm measurements. For all mixed monolayers, standard deviations for Aexc were within (0.01 nm2. a

Table 2. Excess Area, Aexc, Calculated Using Eq 1 for Mixed DPPE-Succinyl/DPPE-PEG2000 Monolayers on PBS at Various Surface Pressuresa Aexc, nm2 π, mN/m 1 mol % PEG 3 mol % PEG 6 mol % PEG 9 mol % PEG 15

-0.01

25 30

0 0

40

-0.01

0.01

0

0.02

50

0

0.02

0.01

0.02

0.01

0.08

0.13

-0.01 0

0.05 0.03

0.10 0.07

a

Each value of Aexc is an average over a set of seven isotherm measurements. For all mixed monolayers, standard deviations for Aexc were within (0.01 nm2.

Figure 3. π-A isotherms for mixed DPPE-succinyl/DPPE-PEG2000 monolayers at the air/water interface (A) and on PBS subphase (B) with different mole percents of DPPE-PEG2000: 1, 3, 6, and 9 mol % as indicated in the figures. All isotherms were obtained at 20 ( 1 °C. Dotted lines and arrows indicate the surface pressure corresponding to the LE-LC transition, πt,LE-LC; the PEG2000 conformational transition, πt,PEG; and the highpressure transition, πt,high (see text for more detail). For the monolayers at the air/water interface (A), the transitions occur at πt,LE-LC ≈ 5 mN/m and πt,PEG ≈ 10 mN/m. For the monolayers on PBS (B), the transitions occur at πt,PEG ≈ 18-20 mN/m, πt,LE-LC ≈ 25 mN/m, and πt,high ≈ 42 mN/m. The transitions reminiscent of the pure DPPE-PEG2000 monolayer at πt,PEG and πt,high are seen only in the isotherms of mixed monolayers at 6 and 9 mol % PEG. 3306


The Journal of Physical Chemistry B plateau and low-compressibility region of the isotherm b in Figure 1. The pure DPPE-succinyl monolayer on PBS converted to a single dark phase at ∼45 mN/m (image not shown). EFM images captured from pure DPPE-PEG2000 monolayers on both water and PBS displayed continuous fluorescent fields from low surface pressures up to the point of monolayer collapse (images not shown). The images became progressively brighter upon compression, and no DOPE-Rh-excluded domains were observed. This indicates that DPPE-PEG2000 monolayers remain predominantly in the LE state at all surface pressures.14,16

Figure 4. Typical epifluorescence microscopy images of the pure DPPE-succinyl monolayer at the air/water interface (A) and on the PBS subphase (B). The images were captured at the end of the plateau in the DPPE-succinyl isotherm: (A) at π ≈ 6 mN/m and A ≈ 0.57 nm2/ molecule for the monolayer on water and (B) at π ≈ 26 mN/m and A ≈ 0.57 nm2/molecule for the monolayer on PBS. Dark areas correspond to the DOPE-Rh-excluded LC domains (see text for more detail). The stripes of different patterns (larger and smaller LC domains of essentially the same shape) seen in image B were sporadically observed in monolayers on PBS. They may be due to kinetic effects or an uneven DOPE-Rh distribution. The scale bar is 50 μm.

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Figure 5 shows EFM images of mixed DPPE-succinyl/ DPPE-PEG2000 monolayers captured on water (A-D) and PBS (E-H). The fluorescent LE and dark LC phases seen in images A-D in Figure 5 coexisted throughout the compression in the plateau region(s) of the mixed monolayer isotherms in Figure 3A. Then, similar to the pure DPPE-succinyl monolayer, a morphology change to a single dark LC phase occurred in the low-compressibility region for all the mixed monolayers on water (images not shown). On PBS, a somewhat different trend was observed. The LC phase domains on PBS appeared noticeably smaller than those on water (cf. images E-H and A-D in Figure 5). They ranged from 2 to 8 μm in diameter and did not change much upon compression. In sharp contrast to the monolayers on water, the mixed DPPE-succinyl/DPPE-PEG2000 monolayers on PBS never converted to a single LC phase (images not shown). This is demonstrated by the analysis of the EFM data in terms of % dark domains22,23 in Figure 6. Values for percent dark domains were calculated from EFM images taken at several points along the isotherms and are plotted as a function of surface pressure.18,22,23 As seen in Figure 6A, on water, all monolayers show steadily increasing percentage of dark LC phase domains from the point they start appearing at ∼4.5 mN/m until a value of 100% corresponding to the entire LC phase monolayer is attained. With increasing PEG content, the value of 100% is attained for percent dark domain at progressively increasing surface pressures (cf. curves b-e in Figure 6A). By contrast, on PBS, the nucleation of the LC phase domains began at ∼10 mN/m. At the collapse point, the mixed DPPE-succinyl/ DPPE-PEG2000 monolayers on PBS exhibited at the most ∼70% of the LC phase in their EFM images, as shown by percent dark domains values in curves b-e in Figure 6B.

Figure 5. Typical epifluorescence microscopy images of mixed DPPE-succinyl/DPPE-PEG2000 monolayers at the air/water interface (A-D) and on PBS subphase (E-H). The images display the morphology of mixed monolayers with different mole percents of DPPE-PEG2000: (A, E) 1, (B, F) 3, (C, G) 6, and (D, H) 9 mol %. The images were captured at the end of the LE-LC plateau in the mixed monolayer isotherms (see text for more detail); that is, at π ≈ 6 and 26 mN/m for monolayers on water and PBS subphase, respectively. The scale bar is 50 μm. 3307



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Figure 6. Percentage of dark DOPE-Rh-excluded domains per image as a function of surface pressure for the pure DPPE-succinyl and mixed DPPEsuccinyl/DPPE-PEG2000 monolayers at the air/water interface (A) and on PBS subphase (B). Data are presented in curves for monolayers with different mole percents of DPPE-PEG2000: (a) pure DPPE-succinyl; (b) 1, (c) 3, (d) 6, and (e) 9 mol % PEG. Dotted curves are guides to the eye. Error bars indicate (standard deviation.

3.4. Monolayer Surface Potential. Measurements of the surface potential, ΔV, were used in this study to assess, in a first approximation, the difference in the electric double-layer potential, ψ0, for monolayers on water and PBS. According to the Demchak-Fort model, the surface potential measured for an ionized monolayer contains contributions from (i) group dipole moments, μi; and (ii) the ψ0 potential, which can be written as

ΔV ¼

1 μi þ ψ0 ε0 A εi

∑

ð2Þ

where ε0 is the permittivity of vacuum, A is the area per molecule, and εi is the local effective dielectric constant.13,24,26 For the monolayers of DPPE-succinyl and DPPE-PEG2000, the dipole contributions to the measured surface potential are likely to come from the terminal CH3 groups, μCH3; CdO groups, μCd0; PE-succinyl headgroup of DPPE-succinyl, μPE-suc; phosphoethanolamine (PE) group of DPPE-PEG2000, μPE; the grafted PEG2000 chain, μPEG; and water molecules reorganized and polarized by the monolayer, μH2O.13,24,26,27 It is noteworthy that μi in eq 2 is a normal component of group dipole moment and is taken as a projection of dipole moment of a group onto the normal to the monolayer plane.13,26,27 At a given A, the orientation of group dipole moments and, consequently, their normal components, μi, will be the same in monolayers spread onto different subphases.25-27 This is especially true for the monolayers in the close-packed state where the orientational freedom of group dipoles is limited.25-27 In addition, in the close-packed state, both phospholipid headgroups and grafted PEG chains are largely dehydrated, whereas reorganization and polarization of water dipoles is limited to the first layer of molecules immediately adjacent to the monolayer.13,24-28 Hence, variations in μPEG and μH2O will likely be negligible for the close-packed monolayers on different subphases.13,24,27,28 By contrast, the ψ0 potential may be significantly affected by the subphase through the headgroup dissociation.25-27 Any difference in the monolayer surface potential measured on water and PBS subphase, ΔVH2O and

Figure 7. Surface potential, ΔV, as a function of mole percent of PEG for monolayers at the air/water interface (ΔVH2O) and on the PBS subphase (ΔVPBS) at 20 ( 1 °C. The values are for a mean molecular area of ∼0.42 ( 0.04 nm2. The dashed curves are guides to the eye. Error bars indicate (standard deviation.

ΔVPBS respectively, can therefore be related to the difference in ψ0 potentials as given by ΔVPBS - ΔVH2 O ψ0, PBS - ψ0, H2 O

ð3Þ

We would like to stress that eq 3 is used in the present study only to assess, in a first approximation, the difference in the electric double-layer potential on water (ψ0,H2O) and PBS (ψ0,PBS) for the monolayer at a mean molecular area of ∼0.42 ( 0.04 nm2. This area corresponds to the low-compressibility region of the π-A isotherms in Figure 3 and implies the closest possible packing of DPPE-succinyl and DPPE-PEG2000 in monolayers. At larger areas, changes in the contribution of μPEG and μH2O to 3308


The Journal of Physical Chemistry B the surface potential measured on different subphases undoubtedly should be considered.13,24,28 Figure 7 summarizes the ΔV data for the pure DPPE-succinyl and mixed DPPE-succinyl/DPPE-PEG2000 monolayers obtained on water and PBS. The data plotted in Figure 7 as a function of mol % PEG were taken from the ΔV-A isotherms recorded simultaneously with the π-A isotherms in Figures 1 and 3. For the clarity of discussion, the ΔV-A isotherms are not shown in the present report. The ΔV data presented in Figure 7 are the average of ΔV values recorded for each monolayer in a narrow range of molecular areas at ∼0.42 ( 0.04 nm2. As seen in the figure, for the pure DPPE-succinyl monolayer on water, ΔV attained a value of þ385 ( 7 mV in the close-packed state. Mixing with DPPE-PEG2000 results in a noticeable decrease in ΔV values, which is in good accord with the previously reported data.24,28 Interestingly, the monolayers on PBS displayed ΔV values ∼90 mV lower than those on water, which implies ΔVPBS - ΔVH2O ≈ ψ0,PBS - ψ0,H2O ≈ -90 mV. This enables us to conclude that, in the monolayers on PBS, the ψ0 potential is more negative than in monolayers on water.

4. DISCUSSION Previous studies have shown that the monolayer behavior of ionogenic amphiphiles, in particular, phospholipids with charged headgroups and C12-C16 aliphatic chains, may be significantly affected by the saline-containing subphase through the headgroup dissociation and electrostatic interactions with subphase counterions.29,30 For a series of phosphatidic acids, Helm et al.29 have demonstrated that, with increasing ionic strength of the subphase, the electrostatic contribution to the monolayer surface pressure can rise up to 10-15 mN/m due to the repulsion between dissociated headgroups and inclusion of counterions in the monolayer. A somewhat similar effect was observed for monolayers of DPPE-succinyl and DPPE-PEG2000 in our study. Like phosphatidic acids, both phospholipids (DPPE-succinyl and DPPE-PEG2000) bear a negative charge on the headgroup, as shown in the insets to Figures 1 and 2. In the pure monolayer, DPPE-succinyl appears to be much more affected by spreading on PBS than DPPE-PEG2000. As seen in Figure 1, the DPPEsuccinyl isotherm obtained on PBS is noticeably more expanded and exhibits a plateau pressure ∼16 mN/m higher than that on water. The isotherms of DPPE-PEG2000 in Figure 2 show a shift by increasing molecular area for the monolayer on PBS, yet the characteristic pseudoplateau12,14 appears at almost the same surface pressure. In the mixed monolayers, spreading on PBS has a much more pronounced effect on the DPPE-PEG2000 component of the mixture. Strikingly, the DPPE-PEG2000 pseudoplateau seen at ∼10 mN/m in mixed monolayers on water (Figure 3A) disappears upon spreading on PBS (Figure 3B). This makes it impossible to straightforwardly identify the transitions in mixed monolayers as reminiscent of those of the pure components. Identifying and characterizing the PEG transition(s) on PBS is, however, important for understanding the behavior of grafted PEG chains at phospholipid surfaces in aqueous media of biological relevance. Hence, our discussion will be focused on identifying the transitions in mixed DPPE-succinyl/DPPEPEG2000 monolayers as well as elucidating the effect of saline on these transitions. We will begin with investigating the effect of saline on the monolayer behavior of the matrix phospholipid, DPPE-succinyl.

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4.1. Effect of Saline on Phase Transition of DPPE-Succinyl. As seen in the Figure 1 inset, DPPE-succinyl bears two C16 aliphatic chains joined via a dual carbonyl linkage to glycerol and a substituted PE headgroup. The substitution of two amine protons of the PE group by succinyl alters the charge distribution on the PE group by leaving a single negative charge on the phosphate group and eliminating the positive charge on the amino group. The succinyl group attaches to the PE group via a carbonyl linkage and contains an ionogenic carboxyl group at its distal end, which can provide another negative charge on the phospholipid headgroup when dissociated. Thus, in the following, we will interpret the monolayer behavior of DPPE-succinyl in terms of headgroup dissociation and electrostatic interactions in the headgroup region, which we believe are relevant to the remarkable difference seen in the isotherms in Figure 1. Observation of the dark LC phase domains coexisting with the fluorescent LE background in images in Figure 4 captured in the plateau region of isotherms in Figure 1 suggests that the plateau corresponds to the LE-LC phase transition;22,23,26 however, the transition on PBS obviously differs from that on water. It occurs at a higher surface pressure and exhibits the formation of much smaller domains, which might be indicative of a higher barrier to the LC phase growth in the monolayer on PBS.29,30 This barrier might be due to the PBS-induced electrostatic interactions in the monolayer headgroup region.29 A more negative value of ψ0,PBS potential inferred from the analysis of ΔV data in Figure 7 points toward a higher negative surface charge density in the DPPEsuccinyl monolayer on PBS compared with that on water. Indeed, for monolayers of anionic phospholipids, the negative value of the ψ0 potential has been shown to scale with the surface charge density.27 The latter (being essentially the surface concentration of ionized carboxyl, -COO- 3 3 3 Hþ, and phosphate, -PO4- 3 3 3 Hþ, groups) is determined by the degree of dissociation of the phospholipid headgroups.27 The more negative value of the ψ0,PBS potential should then be indicative of a headgroup dissociation occurring in the DPPE-succinyl monolayer on PBS on a somewhat larger scale than in the monolayer on water. In fact, the degree of dissociation for carboxyl and phosphate groups is expected to increase with increasing pH and ionic strength of the subphase27,29 when going from water (0 M, pH = 6.2) to PBS (∼0.1 M, pH = 7.4). A higher overall degree of the headgroup dissociation must induce a stronger electrostatic repulsion in the DPPE-succinyl monolayer on PBS.29 Dissociated headgroups also hydrate strongly.27,31 Moreover, the negative charge on dissociated headgroups will attract cations from the PBS subphase, which may result in the formation of outer-sphere complexes as well as charge bridges between carbonyl oxygens.27,32 Altogether, these factors should cause a significant increase in the effective headgroup area and larger lateral separation between DPPE-succinyl molecules. This interpretive scheme is thus in good accord with the observation of a more expanded isotherm for DPPE-succinyl on PBS (Figure 1). A stronger repulsion in the headgroup region can also explain the observation of smaller-diameter LC domains forming at much higher surface pressures on PBS as compared with those on water (cf. images in Figure 4). Indeed, the electrostatic repulsion along the boundary of an LC phase domain formed by charged phospholipids will hinder the condensation of more of such phospholipids into a large domain. Instead, the nucleation of a number of small domains that do not change much upon compression will be favored.29 Eventually, a much higher surface pressure of ∼45 mN/m (compared with ∼20 mN/m on water) 3309


The Journal of Physical Chemistry B is required to overcome the electrostatic repulsion as well as to squeeze out cations associated with PE-succinyl headgroups and hydration water molecules to convert the DPPE-succinyl monolayer on PBS into an entire LC phase. It is noteworthy that the PBS-induced electrostatic interactions in the monolayer headgroup region may affect not only the LE-LC transition of DPPE-succinyl. In mixed DPPE-succinyl/ DPPE-PEG2000 monolayers, the dissociated headgroups and cations from PBS penetrating the monolayer might compete with the grafted PEG2000 chains for hydration water molecules and thus affect their hydration and conformation.3,7 Therefore, the interpretive scheme developed here might be helpful for understanding the effect of saline on transitions in PEG-grafted monolayers discussed below. 4.2. Transitions in Mixed DPPE-Succinyl/DPPE-PEG2000 Monolayers. As can be judged from the isotherms in Figure 3A obtained on water, mixed DPPE-succinyl/DPPE-PEG2000 monolayers undergo two transitions upon compression. The appearance of a plateau with a midpoint at ∼5.5 mN/m reminiscent to that in the isotherm a in Figure1 is suggestive of an LE-LC phase transition of DPPE-succinyl in mixed monolayers. Indeed, EFM has revealed the coexistence of the two phases in the isotherm plateau for all the mixed monolayers (images A-D in Figure 5). The second change in the slope of mixed monolayer isotherms at π ≈ 10 mN/m is likely associated with a conformational transition in grafted PEG2000 chains.12,14 In general terms, this transition can be described as a cooperative change in grafted PEG chains from 2D pancakes to a quasi-3D conformation. The latter has been interpreted in the literature as a mushroom,8,9,11 a pseudobrush,10,15 a cigar,33 and a brush.9,12 Since in this report we are not presenting any experimental data that could enable us to conclude which of the models is the most adequate in describing the conformation of the PEG2000 chains grafted onto mixed DPPE-succinyl/DPPE-PEG2000 monolayers, the term “quasi-3D conformation” will be used throughout the manuscript to refer to the PEG conformation adopted

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upon the transition at ∼10 mN/m. To better identify the transitions, we have analyzed the π-A isotherms obtained on water for DPPE-succinyl (curve a in Figure 1), DPPE-PEG2000 (curve a in Figure 2), and their mixtures (Figure 3A) in terms of the lateral area compressibility, C, as discussed below. The C values were calculated from the slope of the π-A isotherms as given by C ¼ -ð1=AÞðDA=DπÞT

ð4Þ

Equation 4 is taken from ref 21. Being inversely proportional to the first derivative of π with respect to A, the compressibility is extremely sensitive to the changes in the isotherm slope upon compression.8,13,14,17,34 In particular, LE-LC and conformational transitions can be immediately identified in the compressibility plots by clear-cut peaks.8,13,34 Figure 8 shows the C-π compressibility plots obtained by numerical differentiation of π-A isotherm data sets for the monolayers on water. The C-π plots for the monolayers of the pure components, DPPE-succinyl and DPPE-PEG2000, show a single peak. For DPPE-succinyl, the peak is centered at ∼5 mN/m, as displayed by curve a in Figure 8A. The peak appears in the surface pressure range corresponding to the plateau in the DPPE-succinyl isotherm and thus uniquely identifies the LE-LC phase transition in C16 aliphatic chains of DPPE-succinyl at the air/water interface. For DPPE-PEG2000 monolayer, the only peak in its C-π plot f splits into several subpeaks around ∼10 mN/m, as seen in Figure 8B. This peak has been attributed in the previous studies to the conformational transition in grafted PEG2000 chains at the air/water interface.8 The C-π plots for the mixed DPPE-succinyl/DPPEPEG2000 monolayers exhibit two peaks. For the clarity of presentation, the portions of the C-π plots featuring the peaks in the surface pressure range corresponding to the first transition plateau in the isotherms of mixed monolayers are presented in Figure 8A, and peaks appearing at higher surface pressures are

Figure 8. Lateral compressibility, C, as a function of surface pressure for the pure DPPE-succinyl (a); mixed DPPE-succinyl/DPPE-PEG2000 monolayers containing 1 (b), 3 (c), 6 (d), and 9 mol % PEG (e); and pure DPPE-PEG2000 (f) monolayers at the air/water interface. The plots were obtained by numerical differentiation of the π-A isotherms in Figure 4A on the basis of eq 4. For the clarity of presentation, the peaks observed in the C-π plots are shown separately for the surface pressure ranges corresponding to the LE-LC transition (A) and PEG conformational transition (B) (see text for more detail). 3310



Figure 9. Lateral compressibility, C, as a function of surface pressure for the pure DPPE-succinyl (a); mixed DPPE-succinyl/DPPE-PEG2000 monolayers containing 1 (b), 3 (c), 6 (d), and 9 mol % PEG (e); and pure DPPE-PEG2000 (f) monolayers on PBS subphase. The plots were obtained by numerical differentiation of the π-A isotherms in Figure 4B on the basis of eq 4 (see text for more detail).

gathered in Figure 8B. The position of the C-π peaks in Figure 8A coincides with the characteristic DPPE-succinyl peak at 5 mN/m, thus confirming that the first plateau in mixed monolayer isotherms is, indeed, associated with the LE-LC phase transition. With increasing PEG content, the weight of this peak diminishes while the peaks on the higher-pressure side of C-π plots in Figure 8B become more pronounced. Between the two pure components, only DPPE-PEG2000 exhibits a peak in its C-π plot f in the same surface pressure range (Figure 8B). It is therefore safe to conclude that the second transition in mixed monolayers involves predominantly DPPE-PEG2000 molecules and is likely associated with the conformational transition in grafted PEG2000 chains. Although not apparent in the π-A isotherms of monolayers containing 1 and 3 mol % PEG, this transition in fact occurs in all the mixed monolayers, as revealed by the C-π compressibility plots in Figure 8B. Thus, the comparative analysis of π-A isotherms, EFM images, and C-π compressibility plots have enabled us to identify the two transitions as (i) the LE-LC phase transition, followed by (ii) the conformational transition in grafted PEG2000 chains upon compression of mixed DPPE-succinyl/DPPE-PEG2000 monolayers on water. 4.3. Effect of Saline on Transitions in Mixed DPPE-Succinyl/DPPE-PEG2000 Monolayers. In sharp contrast to the isotherms on water in Figure 3A exhibiting two transitions, the isotherms in Figure 3B measured on PBS show only one plateau. At 1 mol % PEG, the isotherm in Figure 3B is virtually identical to that of the pure DPPE-succinyl monolayer (curve b in Figure 1). EFM has also revealed somewhat similar morphology in both monolayers, in particular, the coexistence of small, dark LC domains with the fluorescent LE phase in the isotherm plateau (cf. image B in Figure 4 and image E in Figure 5). Hence, we conclude that the major cause for the appearance of the plateau in the isotherm of the mixed monolayer containing 1 mol % PEG is the LE-LC phase transition. This interpretive scheme, however, may not be valid for the mixed monolayers containing 3-9 mol % PEG. The broadening of the plateau with increasing PEG content points toward the contribution of grafted PEG2000

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chains into this transition. To elucidate the nature of transition(s) in the isotherms in Figure 3B, we used the above analysis of π-A data in terms of lateral area compressibility, C. Figure 9 shows the C-π plots for the monolayers on PBS. As seen in the figure, C-π plot a for the DPPE-succinyl monolayer on PBS exhibits a single narrow peak centered at 25 mN/ m, which correlates well with the midpoint of its isotherm plateau in Figure 1. This peak can thus be attributed to the LELC transition in C16 aliphatic chains of DPPE-succinyl on PBS. Figure 9 displays the same peak for all the mixed DPPEsuccinyl/DPPE-PEG2000 monolayers on PBS. This implies that, similarly to the pure DPPE-succinyl monolayer, the LELC transition occurs in the mixed monolayers at a significantly elevated surface pressure of ∼25 mN/m compared with ∼5 mN/m on water. Therefore, the effect of saline on the LE-LC transition in the mixed DPPE-succinyl/DPPE-PEG2000 monolayers is virtually the same as in the case of DPPEsuccinyl discussed above. With increasing PEG content, the peak in C-π plots in Figure 9 corresponding to the LE-LC transition broadens and develops a shoulder centered at ∼20 mN/m for the monolayer containing 6 mol % PEG. At 9 mol %, the shoulder transforms into a second peak with a maximum at ∼18 mN/m. To determine the possible origin of this second peak, we have referred to the compressibility data for the pure DPPEPEG2000 monolayer on PBS. As seen in curve f in Figure 9, the compressibility plot of the pure DPPE-PEG2000 monolayer exhibits a peak at ∼12 mN/m. This peak coincides with the pseudoplateau region in the DPPE-PEG2000 isotherm and thus can be attributed to the conformational transition in the PEG2000 moiety on PBS. Then, comparison of curves e and f in Figure 8 shows that the second peak in the C-π plot for the mixed monolayer containing 9 mol % PEG (curve e) appears on the higher-pressure side of the PEG2000 conformational transition peak. Furthermore, increasing PEG content in mixed monolayers above 9 mol % causes this peak to rise and become identical to that seen in curve f in Figure 9 (the compressibility plots for the monolayers with higher PEG contents are provided in the Supporting Information). Hence, this peak, as well as the shoulder in the C-π plot for the monolayer containing 6 mol % PEG, is likely to originate from the conformational transition in grafted PEG2000 chains. The broadening of the peak in the C-π plot c in Figure 9 might also indicate some conformational change in grafted PEG2000 chains accompanying the LE-LC transition in the mixed monolayer containing 3 mol % PEG, yet this change is probably insignificant, as in the case of the monolayer containing 1 mol % PEG. Thus, the analysis of C-π plots in Figure 9 impels us to the conclusion that the plateau in the isotherms of mixed monolayers on PBS in fact results from two transitions superimposing on each other: (i) a conformational transition in PEG2000 chains, followed by (ii) the LE-LC phase transition. At this point, however, we cannot describe in detail the PEG2000 conformational transition in mixed monolayers on PBS. Given much higher transition pressure (∼18-20 mN/m) than that observed for the pure DPPE-PEG2000 monolayer on PBS (∼12 mN/m) and reported for PEG2000-grafted monolayers on water (∼8-10 mN/m),8,9,12,14 we can only speculate that the PEG2000 conformational transition in the mixed DPPEsuccinyl/DPPE-PEG2000 monolayers on PBS differs from those previously described in the literature.8-15,18 In fact, the progressive shift of the low-compressibility region of the π-A isotherms 3311


The Journal of Physical Chemistry B in Figure 3B to the right upon increasing PEG content together with the positive Aexc values obtained for mixed monolayers on PBS (Table 2) suggests that PEG2000 chains still contribute to the monolayer mean molecular area above the transition plateau. This implies that some of the -(CH2CH2O)- monomers of grafted PEG2000 chains might remain in the monolayer headgroup region at high surface pressures. 4.4. Effect of Saline on Phase and Conformational Behavior of Binary DPPE-Succinyl/DPPE-PEG2000 Mixtures. As visualized by EFM, DPPE-PEG2000 forms predominantly LE phase monolayers on both water and PBS. This is mainly due to the area mismatch between the bulky PEG2000 moiety and the phospholipid part of the DPPE-PEG2000 molecule. Although it has been suggested that DPPE-PEG2000 molecules can assemble in periodic structures with the LC-type ordering of their aliphatic chains, the steric repulsion between PEG2000 chains limits the scale length of this ordering to several nanometers,35 which evidently prevents the formation of a continuous LC monolayer. When mixed with LC-phase-forming phospholipids, such as DPPE-succinyl, DPPE-PEG2000 molecules are also likely to be excluded from the LC phase domains because otherwise, the steric repulsion between bulky PEG2000 would introduce disorder into the LC phase packing of DPPEsuccinyl.18 DPPE-PEG2000 should then partition preferentially into the LE phase, which is in agreement with the progressive expansion of the area occupied by the fluorescent LE phase in the EFM images in Figure 5 observed upon increasing PEG content in mixed DPPE-succinyl/DPPE-PEG2000 monolayers on water and PBS. Because of large spatial separation introduced by grafted PEG2000 chains, the tilt of C16 aliphatic chains in the LE phase might become significant. By contrast, in the LC phase, the C16 aliphatic chains remain more perpendicular to the interface.36,37 This difference in the aliphatic chain tilt between the LE and LC phases will contribute to a barrier to the LC phase growth and stabilize the separated phases.18,38,39 To overcome this barrier, grafted PEG2000 chains have to submerge into the subphase underneath the monolayer upon compression, which should eliminate the large spatial separation in the LE phase and give way to the formation of an entire LC phase monolayer.18 Indeed, the morphology change from a phase-separated monolayer to a single LC phase has been reported for several PEG-phospholipid monolayers.8,15,18,33 The values for the percent dark domains in Figure 6A indicate that, on water, all the mixed DPPEsuccinyl/DPPE-PEG2000 monolayers also convert to a continuous dark monolayer containing 100% of the LC phase above ∼20 mN/m. Moreover, the isotherms in Figure 3A converge in the low-compressibility region and show negative values of Aexc (Table 1), which means that grafted PEG2000 chains do not contribute to the mean molecular area of mixed monolayers on water at high surface pressures.18 This enables us to conclude that in mixed DPPE-succinyl/DPPE-PEG2000 monolayers on water, grafted PEG2000 chains undergo a conformational transition to a quasi-3D conformation and completely submerge underneath the monolayer upon compression, thus giving way to the formation of a single LC phase. The most striking finding of the present study is that, on PBS, none of the mixed monolayers converts into a single LC phase. This points toward a somewhat enhanced barrier to the LC phase growth in mixed monolayers on PBS, which might be associated with both PBS-induced electrostatic interactions and PEG2000induced disorder in the monolayer headgroup region, although

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curve a in Figure 6B suggests that the effect of PBS-induced electrostatic interactions on the LC phase growth must diminish upon compression because the pure DPPE-succinyl monolayer eventually converts into a 100% LC phase monolayer at ∼45 mN/m. The fact that percent dark domains in mixed monolayers merely attains ∼70%, even at high surface pressures, implies that in these monolayers, grafted PEG2000 chains might contribute to the barrier hindering the LC phase growth by undergoing only a partial transition to a quasi-3D conformation. Actually, the C-π plots in Figures 8B and 9 support this conclusion. Although all the mixed monolayers on water clearly show the PEG2000 conformational transition peak at ∼10-12.5 mN/m in Figure 8B, this peak is not well-defined in Figure 9 for the monolayers on PBS and appears at higher surface pressures (∼1820 mN/m). Hence, the PEG2000 conformational transition in mixed DPPE-succinyl/DPPE-PEG2000 monolayers on PBS either occurs on somewhat smaller scale or differs from that described in the literature transition,8-15,18 as we noted above. Plausibly, when adopting the 2D pancake conformation upon the monolayer spreading, grafted PEG2000 chains interact with PBS ions, in particular with Naþ and Kþ,3-6,40 penetrating the headgroup region of mixed DPPE-succinyl/DPPE-PEG2000 monolayers. Complexed with Naþ or Kþ, ether oxygens of -(CH2CH2 O)- monomers may not be accessible to water molecules that are supposed to cross-link the PEG monomers through hydrogen bonding while packing grafted PEG chains in a quasi-3D conformation.12,13 Interactions with Naþ or Kþ might therefore partially impair the transition in grafted PEG2000 chains to a quasi-3D conformation and keep them entangled in the monolayer. In this case, DPPE-PEG2000 molecules are more likely to form periodic nanostructures35 in the LE phase rather than to participate in the formation of a continuous LC phase. In fact, the specular X-ray reflectivity study by Helm et al.35 has suggested that, at high surface pressures, aliphatic chains of DPPE-PEG2000 molecules tend to pack into LC-type nanoassemblies embedded in grafted PEG chains partially submerged into the subphase. The formation of this periodic nanostructure has been reported for the DPPE-PEG2000 monolayer on water in the surface pressure range corresponding to the high-pressure transition in its π-A isotherm.35 The same transition is clearly seen in the isotherms of two of the mixed monolayers on PBS (Figure 3B), yet at an elevated surface pressure of ∼42 mN/m, as mentioned above. Thus, a somewhat similar phenomenon of periodic DPPE-PEG2000 nanostructure formation might hinder the growth of the LC phase domains in the mixed monolayers on PBS. Eventually, this may result in the collapse of the mixed DPPE-succinyl/DPPE-PEG2000 monolayers on PBS long before a single LC phase is formed.15,16 Further study is necessary, however, to clarify this point. Such a study requires the use of other experimental tools,12,14,15,35 which is beyond the scope of the present report. To better demonstrate the effect of saline on the phase and conformational behavior of the binary mixtures of DPPE-succinyl and DPPE-PEG2000, the above findings are summarized in phase diagrams in Figure 10. The solid curves in the figure correspond to the onset (below 15 mN/m) and completion (above 20 mN/m) of the LC phase formation as inferred from the EFM data in Figure 6. These curves indicate the liquidus and solidus transition,16,41 respectively. The dotted curves are drawn through the starting surface pressures of the PEG peaks in the C-π plots in Figures 8B and 9; they mark the onset of the PEG conformational transition in the binary mixtures. The filled area 3312



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Figure 10. Phase diagrams for the binary DPPE-succinyl/DPPE-PEG2000 mixtures at the air/water interface (A) and on the PBS subphase (B). Solid curves correspond to the liquidus and solidus transitions. Dotted curves indicate the onset of PEG conformational transition. Filled areas show the mixed LC phase, LCmix. LEmix and LEmix þ LCmix denote the mixed LE phase and the coexistence of the two mixed phases, respectively. LEmix þ LCDPPE-suc indicates the coexistence of two “immiscible” phases; that is, the LC phase of DPPE-succinyl and a mixed LE phase, as shown in the schematic in panel B (see text for more detail). The schematics of binary mixtures with the grafted PEG chains completely submerged underneath the monolayer (panel A) and partially embedded between the phospholipid molecules (panel B) are made on the basis of results of previous studies.8,13,35 The DPPE-succinyl molecules are drawn in black; DPPE-PEG2000 molecules are in gray. The dotted gray bracket with arrows indicates a motif of the periodic PEGphospholipid nanostructure.35

shows a single LC phase with grafted PEG2000 chains extending away from the phospholipid surface, as seen in the schematic in Figure 10A. Although the LC phase existed in a wide range of surface pressure and PEG mole percent variations in the binary DPPE-succinyl/DPPE-PEG2000 mixtures on water, it was not typical of the mixtures on PBS. Indeed, the solidus transition is virtually absent from the diagram in Figure 10B, since none of the binary mixtures converted into a single LC phase on PBS as discussed above. Thus, a rather different phase and conformational behavior is depicted in the schematic in Figure 10B for the mixtures on PBS. As seen in the figure, the two “immiscible” phases, the LC phase of predominantly DPPE-succinyl and a mixed LE phase plausibly containing the periodic DPPEPEG2000 nanostructure,35 are most likely to persist in the mixtures on PBS over the entire range of PEG contents studied.

5. CONCLUSIONS Although bearing a close structural resemblance, the two phospholipids, DPPE-succinyl and DPPE-PEG2000, displayed a nonideal miscibility in monolayers on both water and PBS. The latter induced unfavorable interactions between DPPE-succinyl and DPPE-PEG2000, which stabilized immiscible phases in their binary mixtures over the entire range of PEG contents studied. As a result, these monolayers exhibited a somewhat unusual phase behavior, never forming a continuous LC phase. Moreover, transitions in the monolayers on PBS have been found to remarkably differ from those on water. The conformational transition in grafted PEG2000 chains on PBS could not be easily described by the existing interpretive schemes. This led us to a novel suggestion that, in the monolayers on PBS, grafted PEG2000 chains might remain at the interface and undergo only a partial conformational transition to a quasi-3D conformation. Plausibly, interactions with Naþ and Kþ penetrating the

headgroup region partially impair the transition in grafted PEG2000 chains. This may have implications for understanding the phase and conformational behavior of PEG-grafted phospholipid surfaces in aqueous media of biological relevance.

’ ASSOCIATED CONTENT

bS

Supporting Information. The compressibility plots for mixed DPPE-succinyl/DPPE-PEG2000 monolayers on PBS with PEG contents above 9 mol %. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Phone: 1 416 7362100. Fax: 1 416 736 5936. E-mail: valeriat@ yorku.ca.

’ ACKNOWLEDGMENT We thank the Natural Sciences and Engineering Research Council of Canada and the Canada Foundation for Innovation for financial support of this study. ’ REFERENCES (1) Vermette, P.; Meagher, L. Colloids Surf. B 2003, 28, 153. (2) Immordino, M. L.; Dosio, F.; Cattel, L. Int. J. Nanomed. 2006, 1 (3), 297. (3) Heeb, R.; Lee, S.; Venkataraman, N. V.; Spencer, N. D. Appl. Mater. Interfaces 2009, 1 (5), 1105. (4) Ananthapadmanabhan, K. P.; Goddard, E. D. Langmuir 1987, 3, 25. (5) Sindel, J.; Bell, N. S.; Sigmund, W. M. J. Am. Ceram. Soc. 1999, 82, 2953. 3313



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Effect of Saline on Transitions in Poly(ethylene glycol)-Grafted

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