Molecular Packing in Langmuir Monolayers Composed of a

Article pubs.acs.org/JPCB

Molecular Packing in Langmuir Monolayers Composed of a Phosphatidylcholine and a Pyrene Lipid Denise Gradella Villalva,† Marco Diociaiuti,‡ Luisa Giansanti,*,§ Manuela Petaccia,§ Neva Bešker,⊥ and Giovanna Mancini# †

Dipartimento di Chimica, Università degli Studi di Roma “Sapienza”, P.le Aldo Moro 5, 00185 Roma, Italy Dipartimento di Tecnologia e Salute, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Roma, Italy § Dipartimento di Scienze Fisiche e Chimiche, Università degli Studi dell’Aquila, Via Vetoio, 67100 Coppito, AQ, Italy ⊥ CINECA, SCAISuper Computing Applications and Innovation Department, Via dei Tizii, 6, 00185, Rome, Italy # CNRIstituto di Metodologie Chimiche, Via Salaria km 29.300, 00016 Monterotondo Scalo, Roma, Italy ‡

ABSTRACT: Pyrene lipids are useful tools to investigate membrane organization and intracellular lipid trafficking. The molecular interactions controlling the organization of lipid monolayers composed of a cationic amphiphile tagged with a pyrene residue and a saturated or unsaturated phospholipid, namely, 1,2-dimyristoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-phosphocholine, were investigated by Langmuir trough isotherms to understand how the molecular structure of the components and their relative amount affect the physicochemical properties of lipid monolayers. The obtained results show that the cationic headgroups and unsaturation of hydrophobic chains strongly affect the organization of the lipid monolayer as a function of the amount of components. On the other hand, the presence of the pyrene moiety does not seem to have a marked influence on the interaction within lipid assembly.

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INTRODUCTION Lipid molecules containing pyrene (Pyr-Ls) in their molecular structure are powerful probes largely used in membrane biophysics, biochemistry, and cell biology to study lateral arrangement, fluidity, and phase transitions of lipid bilayers, membrane fusion, lipid conformation, and lipid trafficking in living cells, because of the versatility of pyrene as a chromophore and a fluorophore.1−4 It is well-known that Pyr-Ls form excimers in a concentration-dependent manner; however, phase separation, in terms of both lipid composition and coexistence of solid and fluid phases, can affect this phenomenon because a fraction of the pyrene probe can be isolated and not available to the formation of excimers.5 As a consequence, the determination of lateral miscibility of Pyr-L in lipid bilayers is crucial information to identify the effective fraction of pyrene probe that actually can form excimers. It is often assumed that the lateral distribution of lipid is not affected by the presence of a pyrene residue in the lipid skeleton. However, this is not always true because even subtle changes in the structure of hydrophobic chains can have a significant effect on lipid organization in membranes.1 Actually, the polycyclic aromatic ring of pyrene is much more rigid and bulky with respect to an alkyl chain;1 thus its presence can drastically alter the organization of the lipid bilayer in which it is embedded. Besides the steric perturbation in the hydrophobic region, the pyrene moiety might tend to remain at the lipid/water interface due to specific interactions © 2016 American Chemical Society

with lipid headgroups, such as cation−π interaction, thus affecting the headgroup hydration level.6 In its turn the hydration level and the interactions between lipid polar headgroups significantly affect the properties and the thermodynamics of lipid bilayer/water interface.7 With these premises, it is obvious that in the case of lipid assemblies containing Pyr-Ls, the spectroscopic evidence has to be interpreted with caution. Herein we report on the investigation of mixed lipid monolayers composed of the cationic amphiphile 1, characterized by a pyrrolidinium headgroup and a hydrocarbon tail tagged with a pyrene moiety,6 and either a saturated or unsaturated phospholipid, namely, 1,2-dimyristoyl-sn-glycero-3phosphocholine (DMPC) or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) (Chart 1). The lipid monolayers, which can be considered as two-dimension membrane models, were investigated for mixed systems at different molar ratios by Langmuir compression isotherms to determine how and to what extent the organization and the properties of lipid bilayer are influenced by the molecular structure and the amount of lipid components. Received: December 3, 2015 Revised: January 21, 2016 Published: January 22, 2016 1126

DOI: 10.1021/acs.jpcb.5b11836 J. Phys. Chem. B 2016, 120, 1126−1133

The Journal of Physical Chemistry B

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RESULTS AND DISCUSSION One-Component Langmuir Monolayers. The π−A isotherms of DMPC and DOPC monolayers reported in Figure 1 exhibit higher collapse pressure (πc) and steeper slope

Chart 1. Molecular Structure of Lipid Components

Figure 1. Isotherms of DMPC, DOPC, and 1.

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with respect to the π−A isotherm of pyr-A 1, indicating that DMPC and DOPC form a Langmuir monolayer more elastic than 1. DMPC and DOPC monolayers show a typical behavior; i.e., they remain in the liquid-expanded (LE) phase up to their collapse and they do not show any visible plateau, suggesting the lack of domains or lack of phase transitions, in agreement with literature reports.8,9 Compression starts at a large molecular area (more than 130 Å2) where the lipid molecules are not forced together. In the LE phase the molecules behave like in a two-dimension liquid, their mobility is reduced with respect to the initial gaseous phase,10 the phospholipid hydrocarbon chains are randomly oriented, and the hydrophobic chains are mostly in syn conformation because the phospholipid density on water surface is still relatively low. As the Langmuir film is compressed, a slow and gradual increase in π is observed and the Ah varies considerably. For DMPC isotherm the π rises rapidly indicating that the molecules are very close to each other and the acyl chains assume mostly an anti conformation because of the reduction of the surface area available for each molecule due their higher compaction. The lipid film collapses at πc = 55.0 mN/m and Ah ≈ 37.5 Å2. The small value of Ah corresponds to a tightly packed and highly stable monolayer that is characteristic of two alkyl-chain phospholipids.11−13 The shape of DOPC isotherm and the fact that the π rises more rapidly than in DMPC isotherm reveal that the presence of cis double bond on the acyl chains disturbs lipid film packing and increases the fluidity of lipid molecules in the monolayer.14−16 The lower πc of DOPC (πc = 48.0 mN/m) with respect to DMPC (πc = 55.0 mN/m) indicates a lower stability of DOPC film under compression at high π. Literature reports claim that polyunsaturation of acyl chains, together with their length,17,18 leads to increased Ah 19 and decreases the mechanical stability of lipid bilayers. As a consequence, the membrane is less rigid20 and lower energy is required for elastic membrane deformation. Even if the isotherm of pyr-A 1 features a similar shape with respect to those of DMPC and DOPC, it exhibits a significantly different compression profile characterized by reduced slope and πc. These differences could be ascribed to the fact that the pyrrolidinium headgroup is characterized by a lower conformational freedom6 with respect to the glycerol backbone, thus

EXPERIMENTAL SECTION

Materials. DMPC and DOPC were purchased from Avanti Polar Lipids (Alabaster, AL) and used as such (99% purity). The pyrenyl amphiphile (pyr-A) 1 was prepared as previously described.3 Phosphate-buffered saline (PBS) tablets (0.01 M phosphate buffer; 0.0027 M KCl; 0.137 M NaCl; pH 7.4) were purchased from Sigma-Aldrich. Methods. Langmuir Trough Measurements. Aqueous PBS was used as subphase. Surface pressure (π) measurements were carried out by means of a Wilhelmy plate (39.24 mm of perimeter) technique using a Langmuir Minitrough, KSV Instruments Ltd., Helsinki in Teflon with 325 mm of length and 75 mm width and total area of 24 380 mm2 enclosed in a plexiglass box to reduce surface contamination. All measurements were performed at 21 °C ± 0.5 °C. An amount of 25 μL of 1.0 mg/mL CHCl3 solution containing a mixture of DOPC/ 1 or DMPC/1 was spread over the aqueous subphase using a Hamilton microsyringe. After the deposition, the solvent was allowed to evaporate for 5 min before beginning three cycles of precompression, up to π = 4.0 mN/m, and expansion allowing monolayer formation and stabilization. The symmetric compression/expansion was achieved by means of two moving nylon barriers at a constant rate of 10 mm/min. The experimental error for the final concentration of each sample was estimated by means of the error propagation, being of the order of ±2.5% for the reported values. For the calculation, we considered the error of the analytical balance to be ±0.005% and the error of Hamilton syringe of ±1.25% of nominal volume. As precaution to prevent DOPC oxidation and to avoid changes in surface pressure due to air exposure, we have optimized the acquisition of each isotherm, completing it in about 10 min; therefore all the samples were exposed to air for the same length of time and no changes were observed. The reported isotherms represent the average values of at least three different and independent runs of the same sample for each concentration. The standard error (σa) was used to compare the variability of area per molecule (Ah) of the samples at different π. 1127

DOI: 10.1021/acs.jpcb.5b11836 J. Phys. Chem. B 2016, 120, 1126−1133

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The Journal of Physical Chemistry B conferring a different geometry to the whole molecule. The shape of π−A isotherm of 1 is characterized by a continuous strong increase of π while the mean molecular area value decreases, showing a typical LE phase.21 Under further compression, close to 70.0 Å2, even though a plateau region is not observed,20 the absence of a steep profile of the curve suggests the possibility of a LE−LC phase transition.22,23 A larger Ah with respect to those of DMPC or DOPC is observed in the entire range of compression of the isotherm, and the monolayer exhibits a significantly lower πc (36.5 mN/m) with respect to DMPC (55.0 mN/m) and DOPC (48.0 mN/m). These results indicate that the headgroup cohesion in the case of pyr-A 1 is significantly reduced with respect to DMPC and DOPC, probably because of the repulsion between the charged pyrrolidinium headgroups and of their low conformational freedom. As suggested in the case of other pyrrolidinium based surfactants,24 the pyrrolidinium ring of 1 may assume a conformation that exposes the charged nitrogen to the bulk while the ring lies at the air/water interface, thus occupying a large area and hindering a good compaction of the monolayer as depicted in Scheme 1. This conformation can affect water

Figure 2. π−A isotherms of DMPC (black curve), 1 (yellow curve), and mixed DMPC/1 monolayers. The inset reports the variation of the πc as a function of X1.

of pyr-A 1 involves a higher packing of lipid molecules in the mixed film. Probably the cationic headgroups of 1 induce lateral polarization of the close PC headgroups; i.e., they cause significant dipole orientation of the P−N dipole vector of the PC headgroup.27 This phenomenon would induce monolayer compression near the polarized region, decreasing the local Ah in DMPC/1 mixtures.24 In liposomes, which are lipid bilayers vesicles, the lipid packing effect can also be ascribed to the different dimensions of headgroups of liposomes components. In bilayers composed only of lipids in which the cross section of the headgroup is larger than the hydrophobic portion, like in PCs, the acyl chains can bend and/or the headgroups partially overlap to fill/avoid empty spaces in the hydrocarbon region; thus neither the headgroups nor the chains adopt their most favorable packing conformation. Literature reports show that the inclusion of a lipid with a small headgroup in PC bilayers involves the decrease of the mean cross section occupied by the headgroups and of the volume occupied by the acyl chains, thus reducing the voids by favoring the packing of lipids and maximizing van der Waals interactions.28 In the case of our lipid monolayers it is possible that the conformation of 1 headgroups changes by involving a decrease of their cross section and a higher packing of component chains in anti conformation. Other reports suggest that higher chain packing in phospholipid monolayers containing a Pyr-L is due to the pyrene residue because phospholipid molecules surrounding a Pyr-L in mixed monolayers are more tightly packed, and the distribution of the area per PC is shifted toward lower values when next to the pyrenil groups.29 At increasing X1 the lift-off value of Ah increases and the πc decreases indicating that the molecular order of the lipid film at the air/water interface decreases. The π−A isotherms of mixed DOPC/1 monolayers are reported in Figure 3. Up to X1 = 0.6 the curves are shifted to smaller Ah with respect to the isotherms of pure components, whereas for X1 > 0.6 the curves are shifted to larger Ah values and are more similar to pyr-A 1 isotherm. Different from that observed in the case of DMPC containing monolayers, for high X1 the curves are shifted to higher values with respect to those of the pure components and the Ah is larger compared to DMPC/1 monolayers at the same pressure. This result clearly indicates that at high X1 the effect of the unsaturated hydrophobic chains on the organization prevails. On the other hand, at small X1

Scheme 1. Illustration of Topology of 1 at the Air/Water Interface

penetration and, as a consequence, lipid packing. In fact, it is known that for phospholipids featuring two hydrocarbon chains, the size of hydrophilic headgroup and its ability to interact with water and with neighboring headgroups influence the hydrocarbon chain packing and thus the molecular aggregation state in condensed monolayer.11 The presence of the bulky pyrene moiety in the alkyl chains might further reduce the cohesion between the lipid molecules and contribute to the large molecular area values observed in the case of 1.25 Two-Component Langmuir Monolayers. Compression Isotherms. The π−A isotherms of mixed DMPC/1 monolayers at different molar fraction (X1) were measured at the air/water interface at 21.0 °C and are reported in Figure 2. All mixed DMPC/1 curves are shifted to smaller values of area per molecule with respect to the isotherm of pure 1, indicating that DMPC has a condensing effect on 1. DMPC has a zwitterionic headgroup that may shield the electrostatic repulsion between the cationic headgroups of pyr-A 1, thus allowing molecules to arrange tighter in the mixed monolayers.26 Further, in the isotherms corresponding to DMPC enriched monolayers (i.e., containing less than 50% of 1) the values of Ah at the same π are lower if compared with the isotherm of pure DMPC, thus revealing that also the presence 1128

DOI: 10.1021/acs.jpcb.5b11836 J. Phys. Chem. B 2016, 120, 1126−1133

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The Journal of Physical Chemistry B

When two components are miscible, in correspondence of a phase transition or at πc two uniform phases are present in equilibrium in the monolayer, with PS = 2. Thus, the degree of freedom of the system is F = 1, which implies a first-order relation, i.e., a direct relation between the transition pressure (or πc) and the film composition (X). On the other hand, if two components are immiscible, three surface phases coexist in the monolayer at a phase transition (or πc), the system being characterized by F = 0. This means that the system exhibits constant π during the phase transition of the films. If the binary system does not have a clear phase transition, the plot of πc versus X1 might be used to determine the miscibility of the components. Thus, in the range of immiscibility, πc is invariant of the composition and the (πc − X) curve must be flat over this region.33 The adapted interface phase rule cannot be applied for systems containing small domains because they do not correspond exactly to a binary system but are suggested to be one phase monolayer containing microscopic heterogeneities.34 If the two lipid components are miscible at the air/water interface, πc of the mixed monolayers will decrease linearly; i.e., the film collapses at lower π as a function of the molar fraction of the less stable component in the mixture. The inset in Figure 2 reports the experimental results of πc as a function of X1 for DMPC/1 monolayers. As a whole, πc decreases with a trend close to linearity, suggesting a good miscibility. Similar findings were also reported for other mixed monolayers containing DMPC and a cationic component.6,35 On the other hand, the πc versus X1 results are reported in the inset of Figure 3 for DOPC/1, showing an almost constant value of πc up to X1 ≈ 0.3 that suggests low miscibility of components according to eq 1; thus it is a partial miscible system.. At increasing X1 the systems has a first order trend and πc decreases as a function of increasing X1, suggesting an increased miscibility of the components. If the components are at least partially miscible, a more detailed thermodynamic investigation of the system can provide further information on the energetics of the miscibility process and upon possible specific interactions between the two components though Ah and ΔG analysis.33 Mean Molecular Area (Ah) and Gibbs Free Energy of Mixing (ΔG) Analysis. Values of Ah at a certain surface pressure (π) taken from the experimental isotherms were used to elucidate the molecular interactions in monolayers of binary systems. For ideal two-component monolayers, considered ideally miscible mixtures, the Ah value, A12, follows a linear behavior according to eq 2 where X1, X2 are the molar fraction

Figure 3. π−A isotherms for DOPC (black curve), 1 (yellow curve), and mixed DOPC/1 monolayers. The inset reports the variation of the πc as a function of the molar percentage of 1.

both the attractive interactions between the charged pyrrolidinium headgroups and the zwitterionc headgroups of DOPC as well as the effect of the pyrene moiety on the neighbor lipid chains involve a higher compaction of the monolayer. Miscibility Analysis. The molecular miscibility of a multicomponent lipid monolayer can be predicted by an adaptation of the interface phase rule to monolayers systems if they are in equilibrium conditions.30−32 A number of experiments in which the investigated monolayers were compressed at slower compression rate showed very similar πc (data not shown) indicating that the isotherms were obtained near equilibrium conditions. The phase rule is given by eq 1, where F is the number of degrees of freedom, CB is the number of components in bulk equilibrated in the system, CS is the number of components restricted to the surface, PB is the number of bulk phases, PS is the number of monolayer phases in equilibrium with each other, and 3 is the number of variables corresponding to surface tension (π), temperature, and external pressure. F = CB + CS − P B − PS + 3

(1) B

S

For binary mixed monolayers C = 2 (water, air), C = 2 (lipid components, i.e., phospholipid and pyr-A 1), and PB = 2 (liquid, gaseous), and considering only π as variable, i.e., external pressure and temperature constant, eq 1 is given by F = 3 − PS. This adaptation of the interface phase rule predicts the miscibility at a transition between two states (at πc or at LE− LC transition in cases where the πc is not easily measurable).

Figure 4. Mean molecular area (Ah) reported as a function of composition for DMPC/1 and DOPC/1 monolayers, respectively. The black straight lines were obtained from eq 2 for ideal mixing. All systems were investigated at 21 °C and at 10, 20, and 30 mN/m. 1129

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Figure 5. Excess free energy of mixing, ΔG, as a function of composition for DMPC/1 and DOPC/1 monolayers, respectively. All systems were investigated at 21 °C and at 10, 20, and 30 mN/m.

and A1, A2 are the area occupied by the pure components 1 and 2, respectively. A12 = X1A1 + X 2A 2

reduces their cross section with respect to pure 1 monolayer) counterbalance the disturbing effect of chain unsaturation. At higher X1 the repulsive electrostatic interactions between the charged headgroups of pyr-A prevail thus yielding a partial lateral segregation. The only exception occurs at X1 = 0.05 where an A12 value close to Ah (thus an ideal mixing behavior) is observed. The thermodynamic of the mixed monolayers can be analyzed in terms of ΔG for two-component monolayers. ΔG is calculated by integrating the π−A isotherms (the effective area A12), with π ranging from zero up to a given surface pressure according to eq 3. The two terms on the right contain the integration value of the average π−A isotherms measured for pure components 1 and 2, and X1 and X2 are the molar fractions of each component in the two-component isotherms.30

(2)

Nonideal mixtures present deviations from the ideality that can lead to an effective larger mean molecular area (positive area variation, Ah > A12) or smaller mean molecular area (negative area variation, Ah < A12) with respect to the linear behavior of ideal mixtures. These deviations offer valuable information about the repulsive or attractive interaction forces between the components in the monolayer. Positive deviations from the linearity suggest the formation of two-dimensional aggregates and/or domains characterized by repulsive forces, whereas negative deviations indicate stronger and attractive interactions between components 1 and 2, in some cases leading to the formation of complexes arrangements.36 Thus, to investigate quantitatively the miscibility and interaction between pyr-A 1 and DMPC or DOPC in a monolayer, interfacial thermodynamic characteristics were evaluated. Figure 4 shows Ah versus X1 plots for mixed DMPC/1 monolayers at three π values at different compression in the LE phase, π = 10 mN/m, π = 20 mN/m, and π = 30 mN/m. In particular, π = 30 mN/m corresponds to a more ordered LE phase typical of lipid vesicle membranes.37 All mixed DMPC/1 monolayers exhibit negative area deviations from ideality (Ah < A12), confirming the occurrence of condensing effect in mixed DMPC/1 monolayers due both to the electrostatic interactions between the headgroups and to the van der Waals interactions between the hydrophobic chains. By increasing π, the interfacial molecules are pushed into a more compact state with a consequent reduction of Ah. A similar phenomenon was reported for mixed monolayers composed of a saturated phospholipid and a cationic twin tail surfactant investigated by trough experiments26 and by atomistic molecular dynamics simulation of lipid bilayers.38,39 The minima Ah values (maximum ordering) were found at X1 = 0.4. The Ah versus X1 plots for mixed DOPC/1 monolayers, reported in Figure 4, exhibit negative area deviations from ideality up to X1 = 0.4 at π = 30 mN/m, up to X1 = 0.5 at π = 20 mN/m, and up to X1 = 0.6 at π = 10 mN/m and, different from DMPC/1, positive deviations for higher X1. The most significant negative deviation was found at X1 = 0.3 and the most significant positive deviation at X1 = 0.9 with similar trends at the three evaluated π. These results indicate that up to a certain amount of pyr-A 1 into a DOPC monolayer, the attractive electrostatic interactions of the headgroups and a possible change of the conformation of 1 headgroups (which

ΔG =

∫π

π 0

A12 dπ − X1

∫π

π 0

A1 dπ − X 2

∫π

π 0

A 2 dπ

(3)

Equation 3 was used to calculate the excess free energy of mixing. For an ideally miscible mixture, the ΔG is zero, meaning there are no interactions between the components. If ΔG < 0, there are attractive interactions between the components 1 and 2, whereas if ΔG > 0, there are repulsive interactions. The calculation of ΔG gives information on the thermodynamic stability of a mixed monolayer with respect to the monolayers of the pure components.30 The ΔG was calculated from eq 3 ranging from π0 = 0 to π = 10 mN/m, 20 mN/m, or 30 mN/m. The analysis of data from the π−A isotherms gave the ΔG of mixed DMPC/1 and DOPC/1 monolayers as a function of composition at three π. In mixed DMPC/1 monolayers the ΔG values for all compositions are negative as shown in Figure 5. The fact that the negative deviations increase linearly with π confirms that the presence of DMPC minimizes the repulsive electrostatic interactions of the cationic headgroups of 1 and that 1 has a packing effect on the empty spaces of the phospholipids matrix. The presence of pyrene, in on one of the alkyl tails of 1, does not disturb lipid packing at any composition. In the case of mixed DOPC/1 monolayers (Figure 5) the values of excess free energy change of mixing are negative at low X1 (X1 < 0.5) and, on the other hand, are positive at X1 > 0.5. These results suggest when pyr-A 1 is the major component of the mixture phase separation, partial miscibility or domains formation between the two components occurs. Morover, it is confirmed that the presence of both the bulky pyrene moiety in one of 1 the alkyl chains and the unsaturation on the DOPC acyl chains interfere with lipid packing. It is 1130

DOI: 10.1021/acs.jpcb.5b11836 J. Phys. Chem. B 2016, 120, 1126−1133

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The Journal of Physical Chemistry B

system loses the capacity of filling the empty spaces of the phospholipids matrix in cis configuration. Compressibility Modulus Analysis. To deepen our knowledge and elucidate the details of the transition from LE to LC, two-dimensional compressibility of the monolayers was investigated using the following equation:

reported that in systems containing a cationic lipid and a neutral phospholipid, the occurrence of positive or negative deviations of ΔG depends on the component molar ratio.29,40 On the other hand, in the case of the more ordered DMPC monolayer, the presence of the pyrene moiety does not disturb lipid miscibility and lipid packing but, as described for other systems, has an ordering effect.27,41 The prevalence of repulsive or attractive interactions as a function of the composition was observed also in mixed monolayers composed of two unsaturated isomeric lipids, i.e., elaidic acid, which behaves like a straight-chained fatty acid because its double bond is in trans configuration, and oleic-acid whose double bond is in cis configuration.15 In pure films of both lipids the presence of unsaturation involves an expanded phase. On the other hand, when they are included in two-component monolayers with other lipids featuring saturated chains, the elaidic acid (trans configuration) leads to the compaction of lipids while the oleic acid (cis configuration) is arranged with difficulty in the monolayer. In mixed films containing both oleic acid and elaidic acid the resultant monolayer arrangements might be a balance between the double-bond interactions and the steric hindrance of chains depending on the relative amounts of the components and their lateral distributions. As a whole, our results put in evidence that the presence of pyrene tagging of an acyl chain of 1 does not strongly disturb lipid cohesion in saturated phospholipids (Figure 6A, Figure

CS−1 = −Aπ (dπ /dAπ )T

(4)

where CS−1 is the compression modulus, Aπ is the area per molecule at the corresponding π. The plots of CS−1 obtained as a function of π for DMPC/1 and DOPC/1 mixed systems, which reflect the fluidity/elasticity of the monolayers, are reported in Figure 7. The maximum in each curve corresponds to the state at which the monolayer shows the minimum compressibility (i.e., the maximum packing of the lipid film) and the minimum fluidity.42,43 The lower compressibility for all mixed systems is observed at π ≈ 40 mN/m, and as expected, in the case of DOPC/1 monolayers the compressibility is lower with respect to the corresponding DMPC/1 monolayers. This result is in good agreement with the highest slope of the isotherms of the DOPC/1 monolayers that, as explained above, is associated with the presence of kink structures induced by the presence of double bonds in the cis-conformation of DOPC alkyl chains. Moreover, it can be noticed that the monolayer of pure pyr-A 1 shows the highest compressibility and fluidity, parameters that in the mixed systems diminish linearly at increasing molar fraction of phospholipid in the monolayer. The electrostatic repulsion among the cationic polar headgroups of pyr-A 1, inducing a lateral expansion of the lipid monolayer, accounts for this result. Obviously, the higher is X1, the more intense this effect is as indicated by the increase of the maximum CS−1 values. The asymmetry of the peak suggests that the LE−LC phase transition consists of at least two steps, indicating that the phase transition involves two or more kinds of molecular reorientation, probably in the lipid headgroup region.44

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CONCLUSIONS DMPC/1 and DOPC/1 mixtures were investigated as monolayers at the air−water interface by Langmuir trough technique. The influence of the structure and the amount of components on their organization and miscibility, both in the polar and in the hydrophobic regions, was investigated. Pyr-A 1, characterized by a less flexible pyrrolidinium headgroup, exhibits a slightly higher Ah and a lower πc with respect to the PC. This indicates that lipid cohesion in the monolayer of pyr-A 1 is smaller with respect to PC because of the rigidity of

Figure 6. Schematic representation of mixed monolayers. X1 = 0.2: (A) DMPC/1 and (B) DOPC/1. X1 = 0.5: (C) DMPC/1 and (D) DOPC/1. X1 = 0.8: (E) DMPC/1 and (F) DOPC/1.

6C, Figure 6E) and in unsaturated phospholipids at X1 < 0.5 (Figure 6B, Figure 6D). However, in the case of DOPC at high concentration of 1, the repulsive electrostatic interactions have a disordering effect as illustrated in Figure 6F. In this case the

Figure 7. Compressibility modulus as a function of composition for DMPC/1 (A) and DOPC/1 monolayers (B). 1131

DOI: 10.1021/acs.jpcb.5b11836 J. Phys. Chem. B 2016, 120, 1126−1133

Article

The Journal of Physical Chemistry B

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the pyrrolidinium ring and due to tilts of the ring at the monolayer/water surface. On the other hand, in the mixed systems, when favorable electrostatic interactions prevail, the presence of 1 leads to a compaction of lipid monolayer. The different behavior of DMPC/1 and DOPC/1 monolayers confirms that, besides the interactions between lipid headgroups, other parameters such as surface hydration25 and the nature of the alkyl chains (number of unsaturation) have to be considered to reach a complete understanding of these mixed systems. Morover, the presence of pyrene moiety linked to the hydrophobic portion of the amphiphilic molecules does not affect the organization of lipid in the monolayer except at a high concentration of pyr-A in mixed DOPC/1 monolayers. In conclusion, when Pyr-Ls are used at very low concentration, they do not affect the organization of lipid assemblies and can be employed to monitor the interior of micelles, bilayers and biomembranes.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful to the FIRB Project 2012 RBFR12BGHO, the PRIN project Multinanoita (Project 2010JMAZML), and the Program “Ciência sem Fronteiras” from CAPES Foundation by and Ministry of Education of Brazil for financial support and to Mrs. Giuliana Gigli, Mr. Marco Pastore, Mr. Enrico Rossi, and Mrs. Aurelia Stella for technical and administrative support.

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REFERENCES

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DOI: 10.1021/acs.jpcb.5b11836 J. Phys. Chem. B 2016, 120, 1126−1133

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DOI: 10.1021/acs.jpcb.5b11836 J. Phys. Chem. B 2016, 120, 1126−1133