Interaction of Dipalmitoyl Phosphatidylcholine Monolayers with a

Aug 28, 2013 - Department of Pharmaceutical Sciences and Experimental Therapeutics, The University of Iowa, 115 South Grand Avenue, S215 Pharmacy Buil...
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Interaction of Dipalmitoyl Phosphatidylcholine Monolayers with a Particle-Laden Subphase Amir M. Farnoud† and Jennifer Fiegel*,†,‡ †

Department of Chemical and Biochemical Engineering, The University of Iowa, 4133 Seamans Center, Iowa City, Iowa 52242, United States ‡ Department of Pharmaceutical Sciences and Experimental Therapeutics, The University of Iowa, 115 South Grand Avenue, S215 Pharmacy Building, Iowa City, Iowa 52242, United States ABSTRACT: Recent interest in using submicrometer particles for industrial and therapeutic purposes has led to concerns about their interactions with biological membranes. The mechanisms of particle−membrane interactions are not well understood resulting in contradictory reports on the effects of particles on membrane interfacial properties. In this study, the interactions between negatively charged polystyrene particles (200 nm) and monolayers of dipalmitoyl phosphatidylcholine (DPPC) were investigated. Surface pressure, surface potential, and surfactant microstructure studies were conducted to monitor the interfacial properties of DPPC monolayers spread on a subphase in which particles were dispersed. At a concentration of 0.1 g/L, particles caused a partial collapse of the monolayer. DPPC monolayers spread on a particle-laden subphase also exhibited higher surface potential and increased ratio of ordered domains supporting the presence of a more compact monolayer. These results suggest that particles penetrated the air−water interface thereby altering monolayer packing at the interface. These findings are contrary to our previous work where particles injected into the subphase beneath a DPPC monolayer did not penetrate the interface confirming that the sequence of particle and monolayer addition can alter particle−monolayer interactions. These studies may partially explain the varying results reported in previous studies.

1. INTRODUCTION

Langmuir monolayers containing surface-active lipids are one of the most commonly used systems in studies of biomembrane−particle interactions.6,7,9,10,13,14,16,18−21 The lateral packing of the monolayer molecules can be precisely controlled facilitating examination of monolayer interfacial properties.25 Two of the primary protocols used to expose Langmuir monolayers to colloidal particles are the injection of particles into a subphase upon which a monolayer is already spread9,10 and spreading a monolayer on top of a subphase in which the particles have been dispersed.7,14,16−19,21 The potential role of these particle introduction protocols on particle−surfactant interactions has not been previously studied. However, studies of protein−surfactant interactions have shown that switching the protocol of protein introduction to a surfactant monolayer resulted in different interfacial behavior of the monolayer because of the ability of the protein to compete for space at the interface.26 In light of the recent interest in understanding the interactions between colloidal particles and biomembranes, it is important to understand whether changes in particle introduction protocol affect the interfacial properties of model membrane monolayers. Such understanding is particularly helpful for negatively charged polymeric particles because

Small colloidal particles are increasingly being added to the products we use and are exposed to every day. This has led to a growing interest in how these particles interact with biological components, including cells and biomolecules. Previous studies have shown that exposure of biomembranes to environmental, therapeutic, and model colloids induces significant changes in the interfacial properties of the membranes.1−4 These include changes in dynamic surface tension,5−13 surface potential,14,15 phase behavior,16 and microstructure.17−23 The ability of colloids to interact with biomembranes and to change their interfacial properties can have significant implications for membrane function emphasizing the need for a better understanding of how these interactions occur. Despite the existence of a growing body of literature, the exact mechanisms of interaction between particles and biomembranes are still not well understood. Results from different studies are not always easy to interpret or to compare given the differences in study design, experimental protocol, and particle properties. An interesting example is the studies of polystyrene submicrometer particles and biomembrane models where negatively charged polystyrene particles have been shown to significantly alter membrane interfacial properties in two reports11,24 but show little to no effect in two other studies.9,17 This discrepancy in reports clearly shows the need for more fundamental studies. © 2013 American Chemical Society

Received: June 15, 2013 Revised: August 25, 2013 Published: August 28, 2013 12124

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of their promise for drug delivery applications.27 Nondegradable negatively charged particles such as polystyrene provide a simple model to enhance fundamental understanding on biomembrane−particle interactions without having the complexity of degradable systems such as poly(lactic-co-glycolic acid). We have previously investigated the interactions between monolayers of dipalmitoyl phosphatidylcholine (DPPC), a zwitterionic surfactant commonly used as a biomembrane and pulmonary surfactant model,1−6,14,18−21,26,28 and negatively charged polystyrene particles (200 nm) when the particles were injected into subphase after spreading the monolayer.17 Using the particle injection protocol, it was observed that the particles significantly altered the microstructure of the DPPC monolayers but did not change the surface tension behavior of the monolayers.17 In the current study, particle−monolayer interactions are examined when the monolayer is spread on a subphase in which the particles have been already dispersed. Tensiometric experiments during dynamic compression− expansion cycles or monolayer collapse, surface potential, and surfactant microstructure studies were performed to elucidate the mechanism by which the particles alter the interfacial properties of the monolayer.

Emmett, Teller (BET) adsorption method. Approximately 100 mg of particles was washed and lyophilized for each experiment. Surface area was determined by nitrogen adsorption at 77.4 K using an automated surface area analyzer (Quantachrome BET Nova 4200e). The surface chemical composition of washed and lyophilized particles was determined using a Kratos XPS UltraAxis instrument under ultrahigh vacuum (∼10−9 Torr). A monochromatic aluminum Al Kα (1486.6 eV) was used to eject the electrons from the sample, and a hemispherical sector analyzer was used to determine the kinetic energy of electrons. Survey scans were performed in the range of −5 to 1200 eV with a step size of 1 eV, and high resolution scans were performed at regions of interest with a step size of 0.1 eV. CasaXPS software was used for X-ray photoelectron spectroscopy (XPS) data analysis, and spectra were calibrated using the carbon C 1s peak at 285 eV. 2.3. Surface Pressure versus Surface Area Isotherms. Tensiometric studies were conducted using a Langmuir− Blodgett apparatus (Minitrough System 4, KSV Instruments Ltd., Finland). The apparatus consisted of a Teflon coated trough (782 mm × 75 mm × 5 mm, surface area = 558 cm2, subphase volume = 250 mL), equipped with two hydrophilic Delrin barriers for symmetric compression, and a force transducer connected to a platinum Wilhelmy plate (perimeter = 39.24 mm, width = 19.62 mm, height = 10 mm). The Delrin barriers limit leakage of the surfactant.29 The trough was filled with a freshly made subphase solution, which was allowed to equilibrate to room temperature (23.3 ± 0.6 °C) for 30 min and which then was aspirated to remove any surface impurities. The Wilhelmy plate was placed perpendicular to the surface and approximately 1 mm into the subphase. The surface tension of pure water was confirmed at room temperature (72.7 mN/m), and then DPPC monolayers were formed on the surface by dropping 50 μL of surfactant solution onto the interface using a Hamilton microsyringe. For particle− surfactant interaction studies, a mixture of the subphase containing suspended particles at the desired concentration was added to the trough, and then the DPPC monolayer was spread on the surface. Chloroform was allowed to evaporate for 20 min prior to the start of experiments. For dynamic compression−expansion experiments, the surface of the trough was compressed to 200 cm2 (mean molecular area of 40 Å2/molecule) and was expanded to the fully expanded area of 558 cm2 (mean molecular area of 111.5 Å2/molecule) three times with a barrier speed of 10 mm/min (1.5 Å2/molecule·min). The area between the compression and the expansion curves (hysteresis area) was calculated using KaleidaGraph v. 3.6. In addition, the compression modulus, Cs−1, was calculated from the first compression cycle of the isotherm using the following equation:

2. EXPERIMENTAL METHODS 2.1. Commercial Reagents,. R-DPPC was purchased from Genzyme Pharmaceuticals (Cambridge, MA). Fresh solutions were made by dissolving DPPC in HPLC-grade chloroform (Sigma-Aldrich, St. Louis, MO) at a concentration of 1.22 g/L. Sodium chloride and calcium chloride were purchased from Sigma-Aldrich and were dissolved in purified water for subphase preparation. All water used in experiments was obtained from a NANOpure II system (Barnstead International, Dubuque, IA) and had a resistivity of 18.2 MΩ·cm. Carboxyl modified polystyrene particles were purchased from Invitrogen (Carlsbad, CA). Particles had a nominal size of 200 nm and a carboxyl group content of 2 × 105 groups per particle according to the certificate of analysis issued by the manufacturer. 2.2. Particle Characterization. 2.2.1. Zeta Potential and Size Distribution. Particles were characterized in a solution of 150 mM NaCl and 1.5 mM CaCl2, which was adjusted to a pH of 7 with NaOH (henceforth referred to as the subphase solution). Particle size distribution was determined by dynamic light scattering (DLS), and zeta potential was determined by laser Doppler anemometry using a Zetasizer Nano ZS (Malvern Instruments, Worcestershire, United Kingdom). For these measurements, particles were diluted to approximately 0.01 g/L in the subphase solution, were vortexed for 90 s, and were sonicated for 10 min by bath sonication three times. One milliliter of the suspension was loaded into clear disposable folded capillary cells (DTS 1060C cuvettes) for characterization. The nominal diameter of particles was verified using transmission electron microscopy (TEM). TEM samples were prepared by suspending washed and lyophilized particles in methanol. One drop of this suspension was dropped onto a Formvar and carbon coated 400 mesh copper TEM grid using a Pasteur pipet. Imaging was performed after the evaporation of methanol using a JEOL JEM-1230 (Peabody, MA) transmission electron microscope. Images were analyzed using the ImageJ software. The diameters of at least 100 particles were measured to determine the average particle size. 2.2.3. Surface Area and Chemical Composition. The surface area of particles was measured using the Brunauer,

⎛ dπ ⎞ Cs−1 = −A⎜ ⎟ ⎝ dA ⎠

where π is the surface pressure (mN/m) and A is the surface area (cm2). For monolayer collapse experiments, the surface of the trough was compressed to 100 cm2 (mean molecular area of 20.6 Å2/molecule) with no subsequent expansion. The surface area of the first linear increase in surface pressure and the surface area of the final plateau in surface pressure (i.e., surface pressure ∼ 72 mN/m) were recorded as the lift-off and the final collapse surface areas, respectively. Data in all experiments were acquired approximately every second during surface compres12125

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m2/g was measured using the BET adsorption method, which was close to the theoretical size of spherical particles with a diameter of 200 nm (29 m2/g). 3.2. Tensiometric Studies. 3.2.1. Collapse Experiments. Monolayer collapse experiments were performed by compressing DPPC monolayers beyond their limiting mean molecular area in a single compression cycle. Surface pressure isotherms are presented primarily as a function of trough surface area though mean molecular areas are also provided for the pure DPPC monolayer (Figure 1). Because the presence of particles

sion and expansion and were recorded using the LayerBuilder software. 2.4. Surface Potential Measurements. Surface potential measurements of the air−water interface during monolayer compression were conducted using a surface potential sensor (SPOT, KSV-NIMA, Finland), which is based on the vibrating plate (Kelvin) method.30,31 The sensor consists of a probe head with a plate which vibrates at 140 Hz and is connected to an electrode which is submerged in the subphase. The vibration of the plate results in an electric current because of the difference in the potential of water surface and the vibrating plate. A DC voltage in the opposite direction compensates the difference in potential and reduces the current to zero. At this point, the voltage is equal to interfacial potential and is recorded. Surface potential measurements were performed after spreading DPPC solution on the subphase with and without suspended particles. For measurements, the probe head was maintained 1 to 2 mm above the surface, and surface potential values were recorded as the surface was being compressed from an initial surface area of 558 cm2 (124 Å2) to a final area of 180 cm2 (40 Å2). Surface potential of the monolayer during compression was measured with respect to the potential of the expanded monolayer which was taken as zero. Surface potential data upon compression were recorded approximately every second using the Layer Builder software. The conventional Helmholtz equation was used to estimate surfactant dipole moment, Δμ, from the surface potential data: Δμ = εε0ΔVa

where ε is the relative permittivity of the monolayer, ε0 is the relative permittivity of free space, ΔV is the surface potential (in units of Volts), and α is the mean molecular area (in units of m2/molecule). 2.5. Surfactant Microstructure Imaging. Studies of surfactant microstructure were performed using an Olympus BX-51 fluorescent microscope (Olympus, Center Valley, PA) as explained previously.17 Briefly, the trough was washed and was mounted on the microscope stage. The trough was filled with subphase in which different concentrations of washed and lyophilized particles were dispersed. The fluorescent probe Texas-Red DHPE was added at approximately 1 mol % to the DPPC solution. Texas-Red DHPE preferentially partitions into the fluid phases at the surface. A filter with an emission range of 573−648 nm was used to enable visualization of the Texas-Red probe. Images of the microstructure were acquired for DPPC monolayer spread on pure subphase and subphase including 0.001, 0.01, and 0.1 g/L of particles.

Figure 1. Surface pressure versus surface area (bottom axis) or mean molecular area (top axis) of DPPC monolayers on a clean or particleladen subphase performed with surface compression from an initial surface area of 558 cm2 (111.5 Å2/molecule) to a final area of 100 cm2 (20 Å2/molecule). All monolayers collapsed at a surface pressure of ∼72 mN/m. A second, partial collapse is observed for the DPPC monolayer on a subphase containing 0.1 g/L of particles at a surface area of 233 ± 9 cm2.

may affect the area between the DPPC molecules at the surface, the mean molecular areas presented are only valid for the DPPC surface pressure isotherm performed on the subphase with no particles. Surface compression reduced the area between DPPC molecules at the air−water interface and led to distinct phases in the surface pressure isotherm (Figure 1, solid line). At high surface areas (above 500 cm2), the gas phase was observed where no significant change in surface pressure occurred. Further compression led to the liquid-expanded (LE) phase (between 500 cm2 and 400 cm2) where the surface pressure increased almost linearly. The liquid expanded−liquid condensed (LE−LC) phase was observed between 400 cm2 and 300 cm2 as noted by the plateau in the surface pressure isotherm. Reduction of surface area to less than 300 cm2 resulted in the liquid-condensed (LC) phase which continued until 200 cm2 and was marked by an exponential increase in surface pressure. This phase was followed by a plateau in surface pressure at a surface area of 174 ± 3 cm2 denoting the collapse of the monolayer. Further compression after the collapse point did not cause a significant change in surface pressure. The surface pressure isotherm of DPPC on pure subphase solution and the onset of different phases were in

3. RESULTS 3.1. Particle Characterization. A mean particle diameter of 218 ± 18 nm was measured by TEM, and a hydrodynamic diameter of 236 ± 5 nm was measured by DLS. The hydrodynamic diameter, which measures the diameter of hydrated particles, was expected to be larger than the diameter of dry particles.32 The presence of the carboxyl group on the particle surface was verified by XPS analysis which indicated 5.45% carboxyl group (peak at 289.48 eV). The presence of the polystyrene core was confirmed by the presence of a strong peak at 285.04 eV, indicating the saturated carbon bond, and a peak at 291.59 eV, indicating the pi−pi* bond.33,34 The zeta potential of the particles was measured to be −28.4 ± 2.9 mV confirming the negative charge due to the presence of carboxyl groups on the particle surface. Finally, a surface area of 27 ± 3 12126

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agreement with previous reports.35,36 DPPC monolayers compressed on a pure subphase collapsed at a surface pressure of 72.3 ± 0.1 mN/m as expected. When spread on a subphase containing particles, no significant change in the maximum surface pressure of DPPC monolayers was observed (Table 1). However, in the presence

concentration of 0.01 g/L was observed. However, at the highest particle concentration (0.1 g/L), significant changes in the surface pressure isotherm and hysteresis area of the monolayer were observed (Figure 2e). The lift-off area, the area at which the first nonzero surface pressure is obtained upon compression, significantly increased in the presence of 0.1 g/L of particles compared to the pure subphase (550 ± 12 cm2 vs 507 ± 16 cm2) marking a faster transition from the gas phase to the LE phase. A partial monolayer collapse in the LC phase was observed upon compression to a surface area of 233 ± 9 cm2. The presence of the particles also affected the surfactant respreadability as demonstrated by changes in the hysteresis area between the compression and expansion cycles of the isotherm. The hysteresis area of the first cycle of the DPPC surface pressure isotherm was significantly increased from 750.2 ± 118.7 to 2109 ± 334.9 (mN/m·cm2) in the presence of 0.1 g/L of particles in the subphase (Figure 2, Table 2). Finally, the presence of particles reduced the maximum surface pressure obtainable at the end of the third compression cycle from 54.6 ± 1.7 mN/m (pure subphase) to 51.2 ± 1.8 mN/m (particleladen subphase) (Table 2). The effects of particles on surfactant function were also examined by comparing the compression modulus of the monolayer with and without 0.1 g/L of particles in the subphase. The compression modulus (Cs−1) is a rheological quantity related to the monolayer rigidity and is a measure of the elastic energy stored in the monolayer upon compressive deformation of the surface.37 The plot of compression modulus versus surface area for DPPC on a pure subphase showed two maxima at surface areas of 437 ± 21 cm2 and 214 ± 4 cm2 which correspond to the LE and LC phases, respectively (Figure 3). The higher peak in the LC phase denotes a more rigid monolayer because of the space constraints and alignment of the surfactant molecules in this phase. Similar values have been previously reported for DPPC films.20,37,38 The presence of 0.1 g/L of particles in the subphase caused a shift in the LC phase peak to a lower surface area (244 ± 3 cm2 with suspended particles in subphase compared to 214 ± 4 cm2 with pure subphase) but did not significantly change the location of the LE phase peak. In addition, the presence of particles significantly increased the maximum value of Cs−1 (296.0 ± 16.8 mN/m compared to 239.6 ± 39.1 mN/m on pure subphase) denoting the presence of a more rigid monolayer when particles were present. A sharp drop in the compression modulus of the DPPC film on a particle-laden subphase was observed at low surface areas (with a minimum at 225 cm2) because of the premature collapse of the DPPC monolayer. 3.3. Surface Potential Studies. The surface potential of the air−water interface in the presence of the DPPC monolayer is correlated with the sum of DPPC dipoles in the normal direction and increases as the surface is compressed and the molecules become vertically aligned. The main contributor to the surface potential of DPPC is the carbonyl group.39 The carbonyl group is a strongly polar moiety in which the oxygen molecule is directed toward water40 causing a positive potential in the normal direction as the molecules are compressed. Compared to surface pressure, changes in surface potential occur at higher mean molecular areas for DPPC monolayers41,42 making surface potential a sensitive method to changes in molecular packing and alignment at high surface areas.

Table 1. Collapse Surface Pressures and Surface Areas Determined from π−A Isotherms after Compressing Pure DPPC Films (Control) and DPPC Films on a Subphase Containing Carboxyl Modified Polystyrene Particles at Concentrations of 0.001, 0.01, and 0.1 g/La

pure DPPC NP concn: 0.001 g/L NP concn: 0.01 g/L NP concn: 0.1 g/L

final collapse surface pressure ± SD (mN/m)

surface area at final collapse ± SD (cm2)

72.3 ± 0.1 72.1 ± 0.4

174 ± 3 166 ± 4

71.7 ± 0.4

162 ± 14

72.6 ± 0.5

173 ± 3

partial collapse surface pressure ± SD (mN/m)

surface area at partial collapse ± SD (cm2)

53.2 ± 5.0

233 ± 9

a

All isotherms were generated by compressing the monolayer from an initial area of 558 cm2 to 100 cm2.

of particles at the highest concentration of 0.1 g/L, an increase in the surface pressure of the LC phase of the π−A isotherm was noticed. This effect was followed by the partial collapse of the monolayer at a surface pressure of 53.2 ± 5.0 mN/m (Figure 1, dashed line). These effects were concentration dependent and were not observed at lower particle concentrations (Figure 1, dotted line and dashed−dotted line). 3.2.2. Compression−Expansion Cycles. During cycles of monolayer compression and expansion, DPPC monolayers were compressed to a surface area of 200 cm2 so that collapse did not occur, and then they were fully expanded, and the cycle was repeated. Preliminary experiments were performed with a subphase including 200 nm carboxyl modified particles at a concentration of 0.1 g/L and no DPPC monolayer at the air− water interface. These experiments proved that particles show no surface activity at the air−water interface. The surface tension of the particle-laden solution was measured to be 73.5 ± 0.4 mN/m (compared to 72.7 mN/m for water at 20 °C); upon compression to a surface area of 200 cm2, the surface pressure changed by only 0.2 ± 0.2 mN/m (Figure 2a). Compression expansion cycles with a DPPC monolayer spread at the air−water interface showed the same surface pressure regimes (gas, LE, LE-LC, and LC phases) that were apparent in the collapse experiments (Figure 2b). However, lower surface pressures were observed during expansion leading to a hysteresis effect. This hysteresis is due to nonefficient respreading of DPPC molecules at the air−water interface and has been previously used as a measure of surfactant respreadability.17,20 The surface pressure isotherms of DPPC monolayers spread on a subphase containing 200 nm carboxyl modified particles are presented in Figure 2c−e. Changes in the surface pressure isotherms were observed as a function of particle concentration. At the lower particle concentrations of 0.001 g/L and 0.01 g/L, only minor changes were observed in the hysteresis area or maximum surface pressure of the DPPC monolayer obtained upon cycling (Table 2). A slight increase in the maximum surface pressure of the first compression cycle at a particle 12127

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Figure 2. (a) Surface pressure isotherm of the subphase containing 0.1 g/L of 200 nm carboxyl modified polystyrene particles with no DPPC monolayer spread at the air−water interface and DPPC surface pressure isotherms on the subphase containing (b) no particles or (c) 0.001 g/L, (d) 0.01 g/L, and (e) 0.1 g/L of particles. All isotherms were performed with consecutive compression and expansion between surface areas of 558 cm2 and 200 cm2 (first cycle is shown).

the surface potential for a fixed dipole moment according to the Helmholtz equation. Following this jump, a plateau in the surface potential isotherm is observed until the start of the LC region where compression of highly ordered molecules results in increase in surface potential until the collapse of the monolayer. The surfactant dipole moment of DPPC during surface compression was estimated from the surface potential data

The surface potential versus surface area isotherm of DPPC on pure subphase showed a typical reverse sigmoid shape observed in previous reports (Figure 4a).15,38,39 The sudden jump in surface potential at a surface area of ∼520 cm2 has been suggested to be the result of hydrogen bonding between DPPC headgroups and water molecules.43 This hydrogen bonding reduces the local permittivity as the previously free water molecules will now be bound to the monolayer and increases 12128

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Table 2. Summary of Hysteresis Areas and Maximum Surface Pressures Obtained during Sequential Cycling of DPPC Films on Clean Subphase (Control) and a Subphase Including 200 nm Carboxyl Modified Polystyrene Particles at Concentrations of 0.001, 0.01, and 0.1 g/La hysteresis area (mN/m·cm2) 1st cycle pure subphase 0.001 g/L of NP 0.01 g/L of NP 0.1 g/L of NP

750.2 475.6 986 2109

± ± ± ±

118.7 423.2 339.4 334.9b

2nd cycle 539.9 378.1 556.9 554.0

± ± ± ±

195.9 154.3 40.9 108.6

maximum surface pressure (mN/m) 3rd cycle 543.1 331.2 519.5 539.8

± ± ± ±

214.7 173.2 65.3 112.1

1st cycle 58.3 62.0 63.2 58.5

± ± ± ±

2.2 2.1 2.0b 2.0

2nd cycle 56.2 57.0 59.5 54.6

± ± ± ±

1.4 2.6 2.3 1.6

3rd cycle 54.6 55.5 57.1 51.2

± ± ± ±

1.7 1.4 2.5 1.8b

a

All isotherms were generated by consecutive compression and expansion of the monolayer from an initial area of 558 cm2 to 200 cm2 for three cycles. bSignificantly different from pure subphase.

pure DPPC monolayer as observed by the large standard deviation in surface potential (Figure 4b). At surface areas higher than 500 cm2 (gas phase), a significant increase in the monolayer surface potential was observed in the presence of particles. For example, at a surface area of 530 cm2, the surface potential was increased from 10 ± 1 mV on pure subphase to 97 ± 25 mV in the presence of particles. The same trend was also observed in comparing the dipole moments (Figure 4b inset) providing evidence that the monolayer is either more compressed or vertically aligned in the gas phase when particles are present. 3.4. Surfactant Microstructure Studies. The microstructure of DPPC monolayers during transition from liquidexpanded to liquid-condensed phase was studied using fluorescent microscopy. In these studies, DPPC solution was doped with a fluorescent probe (Texas-Red DHPE) that partitions into the liquid-expanded phase leaving the condensed phases (commonly known as lipid domains) as black spots within a background of red, expanded phase (Figure 5a). The presence of the probe in the monolayer did not significantly affect the surface pressure isotherm of DPPC (data not shown). The morphology of the lipid domains was in agreement with previous studies of DPPC lateral structure performed on a subphase including salts.17,45,46 The presence of particles in the subphase significantly affected the microstructure of the surfactant. Particles appeared to facilitate the fusion of lipid domains with more connected and large blocks of domains observed at lower particle concentrations (Figure 5b and c). At the highest concentration

Figure 3. Plot of compression modulus versus surface area calculated from the compression cycle of the surface pressure isotherms presented in Figure 2.

using the conventional Helmholtz model (Figure 4a inset) and was also in agreement with previous reports.14,44 No statistical difference was observed between surface potential values of the DPPC monolayer with and without particles below a surface area of 500 cm2 (LE and LC phases). However, the presence of 0.1 g/L of particles in the subphase resulted in larger fluctuations in monolayer surface potential compared to a

Figure 4. Surface potential (⧫) and dipole moment (●) of DPPC monolayers as a function of surface area performed on (a) pure subphase and (b) subphase containing 0.1 g/L of particles. Monolayers were compressed from an initial area of 558 cm2 to an area of 200 cm2. 12129

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Figure 5. Fluorescent microscopy images of DPPC films doped with Texas Red-DHPE during surface compression on a subphase containing (a) no particles and (b) 0.001 g/L, (c) 0.01 g/L, and (d) 0.1 g/L of 0.2 μm CML particles. All images were acquired at the same surface area corresponding to a surface pressure of 5.2 ± 1.8 mN/m for pure DPPC, scale bar = 100 μm.

particle adsorption at the air−water interface is likely facilitated by the formation of partially hydrophobic particle-surfactant complexes. Such complexes can form due to the electrostatic interactions between negatively charged carboxyl group on particle surfaces and positively charged ammonium group of DPPC headgroups. The formation of similar complexes has been previously suggested between negatively charged silica nanoparticles and DPPC monolayers.18,19 Partial monolayer collapse has been observed with mixtures of immiscible components in a Langmuir monolayer, with the first collapse attributed to loss of one of the components from the interface.4,48 In the current study, a partial collapse of the DPPC monolayer was observed at a surface pressure of 53.2 mN/m in the presence of 0.1 g/L particles and led to a more loosely packed monolayer,1,20 suggesting that a significant portion of particles absorbed at the air−water interface were squeezed out at this surface pressure. Partial collapse was not observed at lower particle concentrations. A similar concentration dependency on collapse has been noted for DPPC monolayers exposed to endohedral metallofullerene particles.23 Final monolayer collapse was observed at a surface pressure of about 72 mN/m for all particle concentrations studied. Such high surface pressures can only be obtained with a pure DPPC monolayer49 confirming that the particles were squeezed out from the monolayer before the end of compression. Particle squeeze-out from surface-active monolayers has previously been reported for polystyrene particles.9 Compression−expansion cycling experiments allow analysis of the hysteresis behavior of the surface pressure isotherms. The presence of 0.1 g/L of particles in the subphase resulted in a considerably higher hysteresis area in the first cycle compared to the isotherm performed on pure subphase (Figure 2e). Hysteresis is created when surfactant molecules are expelled

of 0.1 g/L, the surface was almost completely covered with lipid domains, and only a small fraction of expanded phase was observed (Figure 5d). At this concentration, the surface showed the properties of a very crystalline monolayer with the domains showing very restricted movement at the air−water interface even at the low surface pressure of 5.2 ± 1.8 mN/m (data not shown). The observation of more connected lipid domains and increased ratio of the domains compared to the expanded phase, especially at the highest particle concentration, provides further evidence that the monolayer was more compressed in the presence of particles. This observation is in agreement with the results from surface pressure and surface potential studies.

4. DISCUSSION The results of this study demonstrate that carboxyl modified polystyrene particles disrupt the function of DPPC monolayers when incorporated into the subphase at a concentration of 0.1 g/L but not at lower concentrations. Tensiometric experiments in which the monolayer was compressed from a fully expanded state to collapse indicated significant changes in the surface pressure isotherm in the presence of particles at the highest concentration. Higher surface pressures in all phases of the π− A isotherm led to an increase in the surface area at which both lift-off and the highest compression modulus was observed. The earlier lift-off during compression on a subphase containing 0.1 g/L of particles suggests the presence of a more compact monolayer compared to that spread on a clean subphase. The higher compression modulus, which is a measure of monolayer rigidity, indicates a more rigid monolayer in the LC region.1,2,19,20,22,23,38,39,47 These results suggest the presence of particles at the air−water interface which reduced the area available for the surfactant molecules. Since the particles in the absence of surfactant were not surface-active (Figure 2a), 12130

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compression effect on the monolayer,57 which is in agreement with the observations in the study. On the basis of the results from this study, we propose the following interaction mechanism: At large surface areas, particles adsorb to the air−water interface likely as particle− lipid complexes. Particle incorporation into the monolayer results in a more compact DPPC film compared to the films on pure subphase solution (Figure 6a). This compact surfactant

from the surface at reduced surface areas and do not return to the surface fast enough to participate in the following compression−expansion cycles.50−52 The substantial increase in the hysteresis area observed in this study suggests that in the presence of particles either more molecules are ejected from the interface upon collapse or the ejected molecules take a longer time to readsorb from the subphase. Surface potential experiments provided insight into the effect of particles on monolayer packing. In the presence of 0.1 g/L of particles in the subphase, the jump in surface potential of the DPPC monolayer occurred at significantly higher surface areas compared to the pure subphase. It has been shown that this jump in surface potential is caused by hydrogen bonding between phospholipid headgroups and water molecules that occurs at a critical mean molecular area.43 The observation that this jump in surface potential is shifted to higher surface areas in the presence of particles suggests the presence of a more compact DPPC monolayer at higher surface areas which is in agreement with the surface pressure isotherms (Figures 1 and 2). This phenomenon further supports particle penetration to the air−water interface and increasing the packing or the alignment of the monolayer, which causes artificial compression of the monolayer. Surfactant microstructure studies provided information on the effects of particles on surfactant phases. Larger and more connected lipid domains could be observed at low particle concentrations, and the surface was almost completely covered with the domains at the highest particle concentration. These observations confirm the existence of a more packed monolayer in the presence of particles especially at the highest particle concentration. It is known that the size of lipid domains is controlled by a balance between line tension and electrostatic repulsion between DPPC dipoles.53−56 Keller et al.53 have divided the electrostatic repulsive forces into two categories: the repulsive forces within the domains that favor domain elongation and the forces between the domains that oppose elongation. In the current study, the microstructure images in the presence of particles show larger and connected domains; thus, it is plausible that the repulsive forces within the domains have been boosted by the presence of negatively charged particles. We have previously shown that negatively charged particles are associated with DPPC domains.17 Particle association with the domains can result in excess negative charge and increased repulsive forces within the domains which increases domain size and results in more surface area coverage by the domains. A few previous studies have focused on the effects of submicrometer particles on DPPC microstructure.6,17,19,21−23 In all of these studies, a reduction in the size of DPPC domains has been reported following the introduction of particles in the subphase with hindrance in domain nucleation6,22 and formation of lipid−particle complexes19 proposed as the mechanisms for reduction in domain size. An increase in the fraction of ordered phases after the addition of particles is observed for the first time in the current study. Interestingly, a similar phenomenon has been observed after the addition of nonoxynol-9 (a nonionic surfactant) to the monolayer in a study by McConlogue et al.57 The addition of this molecule also increased the surface pressure at fixed surface areas in all phases of the DPPC surface pressure isotherm similar to the present study, and the authors suggested that this molecule penetrates the air−water interface showing an artificial

Figure 6. A schematic of particle−monolayer interactions during surface compression: at the start of compression, the presence of particles reduces the area between DPPC molecules resulting in higher surface potential and surface pressure and a more crystalline microstructure (upper panel); further compression of the surface results in ejection of particles along with some DPPC molecules from the interface reducing the slope of surface pressure increase at the end of the LC phase (middle panel). This process leaves a pure DPPC monolayer at the surface which can reach near zero surface tension at collapse (lower panel). Figure not to scale.

film generates a higher initial surface potential and a more crystalline microstructure compared to pure DPPC and with compression results in higher surface pressures at all surface areas. At lower surface areas, particle−surfactant complexes are squeezed out of the air−water interface causing a partial collapse in the surface pressure isotherm (Figure 6b). As a result of this squeeze-out, the air−water interface becomes free of particles and the DPPC molecules at the interface show an interfacial behavior similar to a pure DPPC monolayer (Figure 6c) as evidenced by monolayer collapse at a surface pressure of ∼72 mN/m. This type of interaction mechanism has been suggested previously for interactions between DPPC and biofuel combustion emissions and endohedral metallofullerenes.7,23 It is worthwhile to mention the role of particle introduction protocols on the effects of particles on surfactant function. Particle effects on DPPC monolayers reported in Figures 1− 5 were observed by first dispersing the particles in the subphase and then spreading DPPC monolayer on top. We have previously shown that no significant effect is induced on the surface pressure isotherm of DPPC by the particles when the DPPC monolayer is spread on top of the subphase and when the particles are subsequently injected into the subphase.17 This observation held true even when the particle concentration was 12131

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The Journal of Physical Chemistry B at 0.1 g/L (data not shown). This is in contrast to the significant effect observed in the current study at 0.1 g/L suggesting that particle effects on surfactant function have a strong dependence on the particle introduction protocols. Interestingly, a similar effect has been observed in the binary systems of surfactant and proteins previously.26 In this study, when bovine serum albumin (BSA) was injected into the subphase after spreading the surfactant monolayer, BSA was unable to penetrate the air−water interface, and the surface pressure isotherm reflected only the presence of the surfactant. However, when BSA was introduced to the subphase before the surfactant was spread on the surface, the protein absorbed to the air−water interface and the resulting film showed interfacial properties similar to that of a pure protein film. A similar effect may occur for other particle−surfactant systems studied in literature. For example, submicrometer gelatin particles caused a decrease in the surface pressure of DPPC monolayers in one study28 but induced an increase in the surface pressure of DPPC monolayers in another study by the same researchers.21 While not directly compared by the researchers, a review of the experimental details of these studies suggests that the observed differences in surface pressure isotherms between the two studies are due to differences in the particle introduction protocol. These observed changes based on the particle introduction protocol are important in understanding the mechanism of interaction between surfactant monolayers and foreign particles or molecules.

ACKNOWLEDGMENTS

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REFERENCES

We gratefully acknowledge support from the University of Iowa Center for Health Effects of Environmental Contamination (J.F.) and the Executive Council for Graduate and Professional Students at the University of Iowa (A.F.). The authors thank Dr. Vicki Grassian for the use of the surface area analyzer and Biolin Scientific for the use of the surface potentiometer. We also acknowledge The University of Iowa’s Central Microscopy Research Facility and the Office for the Vice President of Research for access to TEM and XPS equipment.

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5. CONCLUSIONS Negatively charged colloidal particles (200 nm) dispersed in an aqueous subphase at a concentration of 0.1 g/L disrupted the interfacial properties of DPPC monolayers at an air−water interface. DPPC monolayers were more compact in the presence of particles at a concentration of 0.1 g/L and partially collapsed at relatively large surface areas (well before the limiting mean molecular area of the DPPC molecules). The increased packing or alignment of the monolayers was evident in the surface pressure and surface potential isotherms. Surfactant microstructure studies also proved the presence of a highly condensed, crystalline monolayer at a particle concentration of 0.1 g/L further supporting the presence of a more compact DPPC monolayer in the presence of particles. Particles also affected the dynamic behavior of the monolayer by increasing the hysteresis area between the compression and the expansion cycles and by lowering the maximum surface pressure attainable in subsequent cycles. Taken together, these results suggest that particles were absorbed to the interface during DPPC spreading and were squeezed into the subphase upon compression. These results are important in understanding the influence of small particles on phospholipid monolayers and in interpreting the mechanisms of particle− monolayer interaction.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 319-335-8830. Fax: 319-335-9349. E-mail: jenniferfi[email protected]. Notes

The authors declare no competing financial interest. 12132

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