Perfluorinated Moieties Increase the Interaction of ... - ACS Publications

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Perfluorinated Moieties Increase the Interaction of Amphiphilic Block Copolymers with Lipid Monolayers Christian Schwieger,* Jacob Blaffert, Zheng Li, Jörg Kressler, and Alfred Blume Institute of Chemistry, Martin Luther University Halle-Wittenberg, D-06099 Halle (Saale), Germany S Supporting Information *

ABSTRACT: The interaction of amphiphilic and triphilic block copolymers with lipid monolayers has been studied. Amphiphilic triblock copolymer PGMA20-PPO34-PGMA20 (GP) is composed of a hydrophobic poly(propylene oxide) (PPO) middle block that is flanked by two hydrophilic poly(glycerol monomethacrylate) (PGMA) side blocks. The attachment of a perfluoro-n-nonyl residue (F9) to either end of GP yields a triphilic polymer with the sequence F9-PGMA20PPO34-PGMA20-F9 (F-GP). The F9 chains are fluorophilic, i.e., they have a tendency to demix in hydrophilic as well as in lipophilic environments. We investigated (i) the adsorption of both polymers to differently composed lipid monolayers and (ii) the compression behavior of mixed polymer/lipid monolayers. The lipid monolayers are composed of phospholipids with PC or PE headgroups and acyl chains of different length and saturation. Both polymers interact with lipid monolayers by inserting their hydrophobic moieties (PPO, F9). The interaction is markedly enhanced in the presence of F9 chains, which act as membrane anchors. GP inserts into lipid monolayers up to a surface pressure of 30 mN/m, whereas F-GP inserts into monolayers at up to 45 mN/m, suggesting that F-GP also inserts into lipid bilayer membranes. The adsorption of both polymers to lipid monolayers with short acyl chains is favored. Upon compression, a two-step squeeze-out of F-GP occurs, with PPO blocks being released into the aqueous subphase at 28 mN/m and the F9 chains being squeezed out at 48 mN/m. GP is squeezed out in one step at 28 mN/m because of the lack of F9 anchor groups. The liquid expanded (LE) to liquid condensed (LC) phase transition of DPPC and DMPE is maintained in the presence of the polymers, indicating that the polymers can be accommodated in LE- and LC-phase monolayers. These results show how fluorinated moieties can be included in the rational design of membrane-binding polymers.

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they interact only marginally with the target membranes17 and reduce the polymers’ insertion propensity. To overcome this ambiguity, a new class of amphiphilic block copolymers was introduced, where the PEO blocks are substituted by poly(glycerol monomethacrylate) (PGMA) blocks.18 The ability of PGMA to act as a hydrogen bond donor and acceptor should increase their interaction propensity with the lipid headgroups. The self-assembly of these PGMAPPO-PGMA triblock copolymers in aqueous solution19 as well as at the air/water interface20 was studied previously. It was also shown that they insert into lipid monolayers and effect the organization of the lipids.21 To further increase the interaction propensity of the polymers with lipid membranes, we additionally modified the polymer by attaching perfluoroalkyl residues (F9) to either end of the PGMA blocks.22 This modification results in triphilic block copolymers23 with the structure F9-PGMA-PPO-PGMAF9, with a lipophilic middle block, hydrophilic side blocks, and

INTRODUCTION Interactions of amphiphilic polymers with lipid membranes are widely studied for their potential use in technical and pharmaceutical applications.1−3 The most extensively studied polymers are poloxamers (also known under the trade name Pluronics or Synperonics). These block copolymers have a symmetric ABA architecture and consist of a hydrophophic poly(propylene oxide) (PPO) middle block flanked by two hydrophilic poly(ethylene oxide) (PEO) blocks on either side. The amphiphilicity of these polymers leads to complex selfaggregation behavior in solution 4 and at interfaces. 5,6 Simultaneously, their architecture allows them to interact with lipid membranes7−9 and monolayers10−13 by inserting their hydrophobic PPO middle block into the lipid acyl chain region. The PPO block length14 and the hydrophilic/hydrophobic balance (HLB)15 determine the strength of interaction, the mode of insertion, and the effect on membrane integrity. Thus, poloxamers may behave membrane sealing1 on one hand or membrane disrupting16,17 on the other hand. An increased PPO block length enhances the interaction with the lipid membranes. Conversely, the PEO blocks supply sufficient hydrophilicity to render the polymers water-soluble. However, © 2016 American Chemical Society

Received: April 26, 2016 Revised: June 17, 2016 Published: July 21, 2016 8102

DOI: 10.1021/acs.langmuir.6b01574 Langmuir 2016, 32, 8102−8115

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Figure 1. Chemical structure, nomenclature, and schematic drawing of the amphiphilic (GP) and triphilic (F-GP) polymers used in this study. Approximate block lengths are calculated for fully extended chain conformations.

fluorophilic end groups. The fluorophillic end groups are at the same time hydrophobic and lipophobic,24 i.e., they have a tendency to separate into their own fluorophilic phase. The triphilicity of the new polymer leads to the formation of multicompartment micelles in aqueous solution.22 In a recent study, we investigated the amphiphilic and triphilic polymers with respect to their interaction with different lipid bilayers, with the aim of elucidating the role of the perfluorinated end groups in membrane binding.25 We could show that both polymers insert their hydrophobic PPO blocks into lipid bilayers and postulate that the terminal F9 chains act as an additional membrane anchor. Differential scanning calorimetry (DSC) experiments revealed an influence of polymer binding on the lipids’ main phase-transition temperature, which was slightly different for the amphiphilic and triphilic polymers. The influence of the F9 terminal groups became evident when investigating the effects of polymer binding on the membrane permeability. The triphilic polymer markedly increased the membrane porosity and was able to translocate through a lipid bilayer, whereas the amphiphilic one was not. However, these results remain quite phenomenological, and the impact of the perfluorocarbon end groups on the binding affinity of the polymers could not be quantified. Therefore, we now present a systematic study on the interaction of both polymers with lipid monolayers, where the monolayers are a model for half a bilayer. The advantage of this simplified model system is that many parameters, such as lipid density, surface pressure, and the monolayer phase state and composition are easily controllable and can be varied in a systematic way.26−28 Furthermore, the lateral monolayer pressure is an easily accessible observable that allows conclusions about the amount of bound polymer. Two types of experiments were performed. On the one hand, we examined polymer adsorption to preformed monolayers of different composition and initial lateral pressure. This provides information on the amount of adsorbed polymer as well as on the limiting lipid packing density that allows the insertion of the polymers. On the other hand, we studied compression isotherms of preformed mixed lipid/polymer monolayers. This reveals the influence of the polymers on the lipid phase

transition and allows us to examine polymer reorganization and polymer squeeze-out from lipid monolayers as well as lipid/ polymer miscibility. The lipids used in this study varied in acyl chain length, saturation, and headgroup structure, which allowed us to identify the key parameters of the interaction. Prior to the lipid/polymer interaction, we studied the interaction of the polymers with the bare air/water interface, i.e., the formation and compression behavior of a pure polymer film. Combining the conclusions drawn from these experiments, we are able to develop a clear picture of the way the polymers interact with lipid monolayers and to elucidate the effect of the perfluorinated end-caps in a quantitative manner. The polymers used in this study are depicted in Figure 1 along with their approximate contour block lengths. Both polymers were synthesized in one batch, such that PPO and PGMA block lengths and polydispersity are absolutely identical and all differences in binding behavior can unambiguously be attributed to the presence or absence of terminal perfluoroalkyl chains. Throughout this study, amphiphilic block copolymer PGMA20-PPO34-PGMA20 is abbreviated with GP, and triphilic block copolymer F9-PGMA20-PPO34-PGMA20-F9 is abbreviated with F-GP.

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MATERIALS AND METHODS

Materials. Lipids. 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), and 1,2dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) were purchased from Genyzme (Neu-Isenburg, Germany). 1,2-Distearoyl-snglycero-3-phosphocholine (DSPC) was a gift from Nattermann Phospholipid GmbH (Cologne, Germany), and 1-palmitoyl-2-oleoylsn-glycero-3-phosphocholine (POPC) was received from SigmaAldrich (Steinheim, Germany). 1-Palmitoyl-2-oleoyl-sn-glycero-3phosphoethanolamine (POPE) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, US). All lipids were used without further purification. Polymers. Triblock copolymer PGMA20-PPO34-PGMA20 (GP) and perfluoro-n-nonyl end-capped triblock copolymer F9-PGMA20-PPO34PGMA20-F9 (F-GP) were synthesized as described elsewhere.19 Their number-average molar mass (MGP = 8555 g/mol; MF‑GP = 9830 g/ mol), degree of polymerization (given by the subscripts), and 8103


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Figure 2. (A) Equilibrium surface pressures of GP (black circles) and F-GP (red squares) solutions as a function of their subphase concentrations measured after the injection of respective amounts of polymer stem solution into pure water. (B) Compression of pure GP (black) and F-GP (red) layers on the air/water interface and (C) elastic modulus (solid lines) and compressibility (dotted lines) of the polymer layers. Experiments were performed at 20 °C. polydispersity (PDI = 1.2), were determined by 1H NMR spectroscopy and GPC. Both polymers were synthesized from the same batch, i.e., the block lengths and polydispersities of the PPO and PGMA blocks are identical. Other Compounds. HPLC-grade organic solvents were used. All experiments were performed in ultrapure water (MiliQ Advantage A10, Merck Millipore, Billerica, MA) with a conductivity lower than 0.55 μS·cm−1. Methods. Monolayer Adsorption. Adsorption experiments were performed in two identical home-built circular PTFE troughs that hold a volume of 8 mL and have a diameter of 6 cm. The troughs were thermostated at 20 °C by an external circulating water bath and covered with PMMA hoods to protect the surface from dust and reduce the evaporation of water. The gas phase inside the hoods is saturated with water vapor. The troughs were filled with ultrapure water. The surface pressure was measured with a Wilhelmy pressure sensor (Riegler and Kirstein GmbH, Berlin, Germany) equipped with filter paper as the Wilhelmy plate. The instrumental error of the pressure sensor is ±0.1 mN/m. Lipids were spread at the air/water interface by depositing lipid solutions in CHCl3/methanol dropwise until the desired initial surface pressure (π0) was reached. The solvent was allowed to evaporate, and the lipid film was allowed to equilibrate for at least 30 min. Ten microliters of an aqueous polymer solution of a concentration appropriate to give the final subphase concentration was injected directly into the subphase through injection holes within the PTFE troughs, i.e., the lipid film was not disturbed during injection. The subphase was stirred by small magnetic stirrers for the rapid mixing of injected compounds. After injection, the surface pressure was recorded as a function of time until a constant value (πeq) was reached (typically 5−10 h). The surface pressure increase was calculated as Δπ = πeq − π0. In addition, the polymer solutions were injected in various concentrations into pure water in order to determine their surface activity and underneath a DPPC film of 15 mN/m initial surface pressure. Compression Isotherms. Surface pressure vs area compression isotherms were recorded using a film balance equipped with two moveable barriers and a Wilhelmy pressure sensor from Riegler and Kirstein GmbH (Berlin, Germany). The instrumental error in the pressure sensor is ±0.1 mN/m. The surface area of the film balance was 550 cm2, its subphase volume was 250 mL, and the compression ratio (completely expanded/completely compressed) was 10. The film balance was thermostated to 20 °C by an external water bath and placed within a hood to reduce dust deposition and evaporation. Ultrapure water was used as the subphase. Lipid, polymer, or mixed lipid/polymer films were deposited dropwise on the water surface from a CHCl3/methanol solution using a microdosing syringe with a precision of 0.33 μL. The solvent was allowed to evaporate for at least 15 min before the start of the compression. The pressure sensor was calibrated to 0 and 72.6 mN/m in pure water and air, respectively. All films were compressed at a speed of 2 Å2 molecule−1 min−1. Data were recorded with a time resolution of 2 s with RUK trough control

software (Riegler and Kirstein GmbH, Berlin, Germany). Raw data were further processed to calculate compressibilities κ, elastic moduli ε, and transition pressures πtrans.

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RESULTS AND DISCUSSION Behavior of Pure Polymers at the Air/Water Interface. Prior to the lipid polymer interaction studies, the pure polymers were investigated with respect to their surface activity and compression behavior. Interfacial properties of the amphiphilic polymer without perfluoroalkyl end-caps (GP) were reported by Amado et al.21 However, the block lengths and polydispersity are not the same as for the polymer (GP) used in this study. Therefore, it was necessary to reevaluate its properties as a reference. This allows us to study the influence of perfluorinated end-caps on the polymers’ surface activities and their interactions with lipid monolayers. Figure 2A shows the equilibrium surface pressure πeq of aqueous GP and F-GP solutions at different concentrations. Both curves increase steeply in the range of 50−300 nM. The air/water interface saturates at a concentration of 1−2 μM, which is the critical aggregation concentration (cac) of GP and of F-GP. Both polymers have been shown to form micelles above their cac in aqueous solutions.19,22 The shape of the adsorption isotherms is typical for the formation of Gibbs films at the interface; i.e., both polymers are surface-active. GP and FGP show adsorption isotherms of the same shape but different amplitudes. This means that the surface energy is reduced to different extents by the two polymers. The amphiphilic GP decreases the surface tension of water by 20 to 25 mN/m in saturation, whereas the triphilic F-GP decreases it by up to 35− 40 mN/m. This shows that perfluorinated end-caps lead to a strong increase in the surface activity of the polymer, reflecting the strong hydrophobicity of the perfluoro-n-nonyl chains (F9).24,29 In Figure 2B, we present the surface pressure/area compression isotherms of GP and F-GP after direct deposition at the air/water interface. Compressibility κ and elastic modulus ε are calculated from the isotherms and depicted in Figure 2C, where κ=

1 1 dA =− ε A dπ

(1)

The surface pressure of the F-GP film is higher than that of the GP film in all compression states, reflecting the higher surface activity of F-GP. The compression isotherm deviates from π = 0 at high molecular areas of 2500−3000 Å2. This coincides with 8104


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Figure 3. Development of surface pressure (π) with time after the injection of (A) GP and (B) F-GP into the aqueous subphase underneath preequilibrated DPPC monolayers of different initial surface pressures (π0) in the LE phase (red) or LC phase (black) or underneath a bare water surface (blue) at 20 °C. The amount of polymer injected at t = 0 s (dashed vertical lines) results in a 100 nM subphase concentration. Note the different time and pressure scales in A and B.

transition of the PGMA blocks. However, the increasing compressibility of F-GP films at pressures above 35 mN/m implies that F-GP molecules also can be completely desorbed from the air/water interface at these higher pressures, which makes an unambiguous interpretation of the measured area values difficult. Adsorption of Polymers to Lipid Monolayers. To investigate the interaction of the polymers with lipid monolayers, we injected polymer solutions into the aqueous subphase underneath preformed lipid monolayers prepared at different initial surface pressures (π0). After injection, the adsorption of the polymers to the lipid monolayers can be followed by the increase in surface pressure. The adsorption process typically takes several hours before the equilibrium surface pressure (πeq) is reached. The surface pressure increase (Δπ = πeq − π0) is a measure of the amount of adsorbed polymers.32 It was shown that a surface pressure increase or decrease is possible upon adsorption of soluble molecules to lipid monolayers.33,34 A positive Δπ indicates the insertion of hydrophobic moieties into the monolayer, whereas a negative Δπ is a consequence of lipid condensation due to electrostatic or hydrogen-bonding interactions between hydrophilic polymer moieties and the lipid headgroups. Figure 3 shows the adsorption isotherms of GP (panel A) and F-GP (panel B) to DPPC monolayers of different initial surface pressures (π0) as well as the adsorption of GP and F-GP to bare air/water interfaces for comparison. Several observations can be made: (i) Adsorption of the polymers leads to a surface pressure increase in all investigated cases. This reveals hydrophobic interactions between the polymers and the lipid monolayer and shows that the polymer inserts in between the lipid molecules. (ii) πeq values after the adsorption of F-GP are always higher than πeq values after the adsorption of GP. This indicates that F-GP interacts more strongly with the lipid monolayers than does GP. (iii) πeq values are higher than the equilibrium surface pressure of the polymer solutions in the absence of lipid monolayers, which shows that there is an attractive interaction between lipids and polymers. (iv) The surface pressure increase (Δπ) becomes smaller when the polymers adsorb to monolayers with higher π0, i.e., monolayers with a higher lipid packing density. This shows that it becomes less favorable for the hydrophobic polymer moieties (PPO and F9) to insert in between the lipid chains as a result of increasing van der Waal interactions between adjacent lipid acyl chains.

the area one polymer chain would cover if each block performs a self-avoiding walk (SAW) under good solvent conditions according to5,30,31 π A = πR g 2 = RF 2 and RF = lN ν (2) 4 where l is the length of one monomer unit, N is the number of monomers, and ν is the Flory exponent. With lPPO = 3.6 Å, lPGMA = 2.55 Å,18,19 and ν = 0.75 for 2D good solvent conditions,30 this gives AGP = 2930 Å2, which indicates that both PPO and PGMA blocks are adsorbed at the interface at the beginning of the compression. Upon compression, the PGMA blocks will be pushed into the aqueous subphase, whereas the PPO blocks and the F9 chains remain at the interface. Upon further compression, both PGMA and PPO blocks will adopt a mushroom conformation in the aqueous subphase and air, respectively. In this conformation, the polymer is compressed until a surface pressure of about 25 mN/m is reached, with a maximum in the elastic modulus at 15 mN/m for both polymers (Figure 2C). The elastic modulus ε of this phase is lower, i.e., the compressibility is higher, for GP than for F-GP, indicating that the F-GP film is stiffer in this state. Upon compression to pressures higher than 15 mN/m, GP is gradually released into the subphase, as can be seen from the constantly decreasing elastic modulus and the constantly increasing compressibility. In contrast, F-GP exhibits different behavior at high compression. An additional phase transition occurs at 27 mN/m and a corresponding molecular area of 325 Å2. This can be seen from the plateau in the compression isotherm (Figure 2B) as well as from the local maximum in compressibility and the minimum in elastic modulus (Figure 2C). This transition corresponds to the release of the PPO units into the aqueous subphase. Therefore, the behavior of GP differs from that of FGP at this point in the isotherm: while GP is completely dispersed in the subphase, F-GP is still anchored to the surface by the F9 chains and the polymer monolayer can be further compressed. Consequently, another maximum in the elastic modulus can be observed at 34 mN/m, indicating the formation of a new phase. This phase spans from A ≈ 350 to 60 Å2, where the larger area corresponds to two PGMA blocks under 3D good solvents conditions (ν = 0.6) and the smaller area corresponds to the limiting cross-sectional area of two perfluorinated alkyl chains,29 respectively. The compression of this phase is apparently concomitant with a mushroom-to-brush 8105


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Figure 4. Change in surface pressure (Δπ) upon adsorption of GP (black squares) and F-GP (red circles) from a 100 nM bulk solution to lipid monolayers of various initial surface pressure (π0). Monolayers are formed from (left panels) lipids with a PC headgroup and (right panels) lipids with a PE headgroup. The lipids acyl chains are (top to bottom) PO, DM, DP, and DS. The dotted lines are linear fits of the data, which are calculated with an error of ±0.1 mN/m in each data point in the x and y directions. Their intercept with the x axis is used to determine the maximum insertion pressure (MIP). Slopes of the fits (together with their standard errors) and MIPs are given in the figure. Lipids written in blue letters form LE-state monolayers; lipids written in black letters form LC-state monolayers. DPPC and DMPE are in the LE state at π < 6 mN/m and in the LC state at π > 6 mN/m.

monolayers.6 Moreover, F-GP adsorption is slower than GP adsorption, indicating that F-GP has to undergo more complex conformational changes upon adsorption to the lipid monolayer or upon demicellization of the injected stem solution than does GP.

There is a limiting lipid packing density above which the polymers are no longer adsorbed to the lipid monolayers. (v) The adsorption kinetics is much slower for polymer adsorption to lipid monolayers than to the bare air/water interface. This implies that the polymers have to undergo slow conformational transitions upon adsorption or insertion into the lipid 8106


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Langmuir It is common practice to plot the determined Δπ values as a function of the initial surface pressure π0. The resulting plots are depicted in Figure 4 for all examined lipid monolayers. It can be seen that Δπ values are linearly related to π0. The linear fitting functions are characterized by a negative slope and an intersection with the x and y axes. The intercept with the x axis denotes the so-called maximum inserting pressure (MIP),35−37 above which no more polymer is inserted into the monolayer. The MIP values characterize the insertion propensity of the polymers into the monolayers. GP inserts into DPPC monolayers up to a π0 of 28 mN/m, whereas F-GP inserts into DPPC monolayers up to 40 mN/m (Figure 4, third panel, left). The large difference in MIP values (12 mN/m) is evidence of the higher propensity of F-GP to interact with lipid monolayers as compared to GP. Also, the absolute MIP values are interesting when compared to the so-called monolayer− bilayer equivalence pressure, which is reported to be approximately 30−35 mN/m.32,38,39 The MIP of GP is below this value, whereas the MIP of F-GP is well above it. This implies that GP would not insert into gel-phase DPPC bilayers under the chosen conditions (temperature, polymer concentration), whereas F-GP would easily insert. The MIP of 42 mN/m is high compared to known values for membraneinteracting proteins and low-molecular-weight amphiphiles (typically in the range of 30−35 mN/m) and is similar to values determined for antimicrobial peptides. (For an overview of MIP values, see the review of Calvez et al.36) This underlines the high insertion propensity of F-GP into lipid monolayers. Only Δπ values determined in the liquid condensed (LC) state of the DPPC monolayer (π0 > 5 mN/m) were included in the regression. Data points measured at lower pressure deviate from the linear relationship. This is due to the fact that the proportionality between Δπ and the amount of adsorbed polymer is given only for a constant compressibility of the monolayer.32 In addition, the interaction strength of the polymers might depend on the monolayer phase state as well as the nature of the lipids. To test for lipid and phase specificities of the interaction, we repeated the described experiment for a set of different lipids. We used PC lipids with shorter chains (DMPC) and unsaturated chains (POPC) that form LE-phase monolayers in the probed temperature and pressure range. Furthermore, we tested LC-phase lipids with longer acyl chains (DSPC) to evaluate the interaction strength as a function of the hydrophobic thickness of the monolayers. Additionally, we investigated the lipid headgroup specificity by repeating all experiments with lipids with the same acyl chain composition but a phosphoethanolamine (PE) headgroup. Both the hydrophobic thickness and nature of the headgroup were shown to modulate the interaction of the polymers with lipid bilayers.25 The results are summarized in Figure 4. The corresponding adsorption isotherms can be found in the SI (Figure S3−S9). In all tested systems, F-GP adsorption leads to higher Δπ values than GP adsorption. Similarly, all MIP values determined for F-GP are 12−15 mN/m higher than for GP. This shows that F-GP always inserts to a greater extent than GP, independent of the nature of the lipid. Simultaneously, all MIPs for F-GP are higher than the monolayer−bilayer equivalence pressure,32,38,39 indicating that F-GP would also penetrate the corresponding bilayers. Besides these common features, differences between the studied lipid systems shall be discussed in the following paragraphs.

Phase State. The phase state of the lipid monolayers influences the slope of the regression lines. The slopes determined for LE-phase monolayers (POPC, DMPC, and POPE) are significantly lower than those determined for LCphase monolayers (DPPC and DMPE above the transition pressure, DSPC, DPPE, and DSPE). This is true for GP as well as F-GP adsorption. Interestingly, the slopes for GP and F-GP adsorption are always very similar. In contrast, the MIP values seem not to be influenced by the lipid phase state. It was suggested by Calvez et al.37 that the slope is a measure of the interaction strength between adsorbed amphiphiles and the lipids. According to this theory, a slope of −1 indicates that both components interact with the interface but not with each other, whereas an attractive interaction between both components should lead to a slope closer to zero. This theory would suggest a more attractive interaction of the polymers with LE-state monolayers. However, this is contradicted by the similar MIP values. Moreover, the similarity in slopes for GP and F-GP adsorption and the concomitant difference in MIP suggests that the slope itself cannot be taken as a measure of the interaction strength in the systems under investigation. Rather, the smaller slope is a result of the higher compressibility of LE monolayers. This is due to the fact that Δπ is not only caused by the surface activity of the adsorbed polymer (πP) but also contains a contribution of the compression of the lipids (ΔπL). The compression is accomplished by the insertion of the polymer at constant total area of the system and results in an increase in the partial surface pressure of the lipid component. Δπ = πP + ΔπL

(3)

The contribution of ΔπL is greater the lower the compressibility of the lipid monolayer and vice versa. This can be seen by substituting ΔπL by eq 1, where dA becomes the negative area of the adsorbed polymer (−Ap) and A = A0, i.e., the total surface area:

Δπ = πP +

AP κLA 0

(4)

Ap/A0 can further be expressed as the area fraction occupied by the polymer ap: Δπ = πP +

aP κL

(5)

Therefore, the lipid contribution to Δπ is low in a highly compressible LE monolayer, and Δπ is consequently dominated by the surface activity of the polymers themselves. Adsorption to the less-compressible LC phase leads to an increase in Δπ by lipid compression. This also explains the Δπ0 values (extrapolated intersection of the regression lines with the y axis) being higher than πeq,p of the pure polymers for LC phase adsorption. For LE-phase adsorption, Δπ0 is in the range of πeq,p. This phase-state dependency also explains the unusual case of F-GP adsorption to DPPC monolayers, which are in the LE state below 5 mN/m and in the LC state above 5 mN/m. Because of the different compressibilities of the two phases and thus the different contributions of the second term in eqs 3−5, Δπ values are lower in the range of π0 = 0−5 mN/m than at 10 mN/m. The degree of saturation of the lipid acyl chains does not have an impact on the determined slopes, which is deduced 8107


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contact area for van der Waals interactions between adjacent lipids in the monolayer. Therefore, the penalty of inserting other hydrophobic moieties with a lower contact area for van der Waals interactions is higher for longer-chain lipids than for shorter-chain lipids. This argument explains the lower MIP values for insertion into monolayers that are better stabilized by van der Waals interactions, i.e., monolayers with longer acyl chains. The dependence of the MIP values on the lipid acyl chain length seems not to be influenced by the lipid phase state because the relationship includes LE-phase (DMPC) and LCphase (DPPC and DSPC) lipids. The same dependence can be observed for PE lipids, which supports this argument. The MIP values determined for POPC and POPE are similar to those determined for the corresponding dipalmitoyl (DP) lipids. Given the fact that POPC and POPE have a hydrophobic thickness similar to that of DPPC and DPPE, this is consistent with the conclusion that the MIP values are manly influenced by the hydrophobic thickness of the monolayers. Furthermore, these results indicate that at least a partial mixing of polymers and lipids occurs within the monolayer because only in the case of mixing would a reduction in van der Waals interactions occur and could a chain-length dependency be explained. Lipid Headgroup. GP and F-GP adsorption to PC and PE membranes is quite similar, and the nature of the lipid headgroup plays only a minor role in modulating the interactions. However, the MIP values of PE membranes are in general slightly below those for PC membranes. This indicates that the insertion of the polymers into PE membranes is slightly less favorable than insertion into PC membranes. This might be due to the hydrogen bonding network within the PE headgroup region.42 The insertion of polymer moieties would lead to the loss of hydrogen bonds between adjacent PE headgroups and is thus more unfavorable than insertion between PC lipids, which do not form hydrogen bond networks. In addition, the smaller PE headgroup leads to a tighter packing of the acyl chain. PE acyl chains are less tilted than PC acyl chains. Above 30 mN/m, PE acyl chains are oriented perpendicular to the interface.43 This tighter packing leads on one hand to a higher lipid area density at a given pressure and on the other hand to an increase in the van der Waals contact area between the acyl chains. Both effects would reduce the insertion propensity of the polymers. Polymer Concentration. The experiments were done with a polymer bulk concentration of 100 nM, which is below the saturation concentration of the pure polymers (Figure 2A). To

from the similar the slopes for POPC and DMPC measurements. Acyl Chain Length. Saturated PC lipids were measured with three different acyl chain lengths: DMPC, DPPC, and DSPC (14, 16, and 18 C atoms per acyl chain, respectively). DMPC forms an LE-phase monolayer, whereas DPPC and DSPC form LC-phase monolayers at 20 °C. The same series of acyl chains is tested with PE headgroups, where all lipids form LC-phase monolayers.40,41 With this series of experiments, we test whether the hydrophobic interactions between the lipids and the PPO and F9 units of the polymers can be modulated by the hydrophobic thickness of the monolayer. As an overview, all determined MIP values are summarized in Figure 5. The MIP

Figure 5. Maximum insertion pressure (MIP) for the adsorption of GP (black symbols) and F-GP (red symbols) into lipid monolayers composed of PC (filled symbols) and PE (open symbols) lipids. The error bars correspond to the x standard errors from the linear fits shown in Figure 4. The acyl chain length of a lipid is indicated by its two-letter abbreviation on the bottom x axis and by its number of carbon atoms : degree of saturation in sn-1 and sn-2 positions on the top x axis. The vertical dotted line separates unsaturated and saturated phospholipids on the x axis. The horizontal dotted line indicates the monolayer−bilayer equivalence pressure.

of F-GP in PC monolayers decreases from 45 over 40 to 38 mN/m for DMPC, DPPC, and DSPC, respectively. The MIP of GP decreases from 30 over 28 to 25 mN/m for the same lipids. This decrease in the MIP signifies that the polymers are less readily accommodated by monolayers with increasing acyl chain length. The longer the lipid acyl chains, the higher the

Figure 6. Equilibrium surface pressure after adsorption of GP (A) and F-GP (B) from an aqueous subphase of different concentrations to the air/ water interface (opens symbols) and to a DPPC monolayer of 15 mN/m (filled symbols) at 20 °C. 8108


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Figure 7. Compression isotherms of monolayers of DPPC containing different amounts of (A) GP and (B) F-GP and derived film compressibilities of (C) DPPC/GP and (D) DPPC/F-GP). (E) LE → LC phase-transition pressures as a function of the copolymer molar fraction xpolymer for DPPC monolayers containing different amounts of GP (black squares) and F-GP (red circles), determined from the low-pressure maxima in the compressibility traces. Experiments were performed at 20 °C. The area per lipid in A and B is calculated as the total area occupied by the mixed lipid/polymer monolayer divided by the number of lipid molecules.

lipid film. To test if the MIP values are influenced by the slightly higher Δπ values at increased polymer bulk concentrations, we injected F-GP at three different concentrations underneath DPPC monolayers of different initial surface pressures (Figure S2). These experiments show that the MIP value does not depend on the polymer bulk concentration. However, the slopes of the Δπ vs π0 regression lines increase slightly with increasing polymer subphase concentration. The difference between the Δπ0 values and the equilibrium surface pressures of the pure polymers becomes smaller but is still present even at high polymer concentrations. Compression of Mixed Lipid/Polymer Monolayers. To gain further insight into the influence of the polymers on the lipid monolayers and to test for the stability of the polymers at

test for the influence of the polymer bulk concentration, we injected GP and F-GP in a concentration-dependent manner underneath DPPC monolayers at a constant initial surface pressure π0 of 15 mN/m (Figure 6). It can be seen that the saturation concentration of the polymer decreases in the presence of a lipid monolayer as compared to the pure air/ water interface. A 100 nM polymer subphase concentration is, therefore, already close to saturation, and the Δπ values increase only slightly for a higher polymer concentration. The surface pressure after polymer adsorption to the DPPC monolayer is always higher than the surface pressure of the pure polymer film formed at the same bulk concentration and higher than the surface pressure of the initial lipid monolayer. This confirms the attractive interaction of polymers and the 8109


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molecules to bend back to the interface, whereas the nonterminated PGMA blocks of GP can freely dangle into the subphase. This interpretation is further supported by the fact that πtrans of F-GP-containing monolayers is always slightly below πtrans of GP-containing monolayers, indicating that hydrophilic interactions always play a stronger role in compensating for the increase in πtrans in the former system. The compression behavior above the LE → LC phasetransition pressure is different for DPPC/GP and DPPC/F-GP films. Directly above the phase-transition pressure, the area per lipid is still significantly increased when compared to the pure DPPC isotherms. In the case of GP-containing monolayers, this additional area is reduced progressively upon further compression, indicating that the area occupied by GP decreases. This reduction in polymer area is visible as a smaller slope in the isotherms or, more clearly, as an increased compressibility of the mixed monolayer when compared to that of the pure DPPC monolayer (Figure 7 C). These effects can be interpreted as a gradual release of polymer molecules into the aqueous subphase. At about 38 mN/m, all isotherms coincide in one point, independent of the GP concentration. At higher pressures, all isotherms show the same shape and the same low compressibility, which is typical for pure LC-phase lipid monolayers, showing that no more polymer is located at the interface. This is interpreted as the complete squeeze-out of GP from the monolayer. The compressibility plots reveal a second maximum at 27−28 mN/m for all monolayers containing GP. This indicates that a transition in the polymer component of the mixed monolayer takes place before the polymer completely vanishes from the interface. This transition was discussed before for the compression of pure polymer films (Figure 2B) and results from the squeeze-out of the PPO blocks. For GP, this is equivalent to the release of the complete polymer from the interface. Similar squeeze-out pressures have been found for the PPO blocks of the structurally related poloxamers.10 F-GP-containing monolayers behave differently in some aspects. Upon compression beyond the LE → LC phase transition, the isotherms increase with a higher slope than in the GP case. Equivalently, the compressibilities of DPPC/F-GP films are lower than those of DPPC/GP films in the pressure regime of 10−20 mN/m while still being higher than those of pure LC-phase DPPC films. This indicates that F-GP is better anchored to the interface and less prone to squeeze-out than GP. The PPO squeeze-out is visible as a peak in the compressibility plots and as a pseudoplateau in the isotherms. This squeeze-out occurs at 28 mN/m, similar to the case of DPPC/GP monolayers and of pure F-GP monolayers. This indicates that the stability of the PPO blocks at the interface is not influenced by the presence of the perfluoroalkyl chains or the embedding of the polymer into a lipid environment. However, after the PPO squeeze-out the film does not behave like a pure DPPC film. This is in contrast to the GP-containing films and indicates that F-GP is still inserted into the DPPC monolayer by its perfluoroalkyl chains. Interestingly, the area per lipid is still higher than the sum of the molecular areas of DPPC and the F9 chains being embedded within the film. This implies that PPO and/or PGMA steric repulsions still contribute to the total film area. Although the PGMA parts may be stretched in a brushlike conformation, the hydrophobic PPO blocks will be collapsed into a dense coil. The PPO blocks are probably shielded from the aqueous phase by the PGMA blocks. In this conformation, they still have an area demand

the interface, we studied the compression behavior of mixed lipid/polymer monolayers that were directly formed at the interface. Therefore, we recorded pressure/area isotherms of DPPC and DMPE in mixtures with GP and F-GP. Lipid and polymer components were premixed in organic solvent and cospread at the interface. The isotherms contain information about the influence of the polymers on the phase state of lipids within the monolayers, their transition pressures, the monolayer compressibility, and the amount of polymer present at the interface. Figure 7 shows the compression isotherms of mixed DPPC/ GP and DPPC/F-GP films with varying polymer contents from 1 to 20 mol %. This is equivalent to a hypothetical polymer subphase concentration of 1−25 nM assuming complete solubility of the polymer. The DPPC isotherms show LE phase behavior at low surface pressures (high areas), a phase transition plateau at 5.5 mN/m, LC phase behavior at higher pressures, and no further transition up to 55 mN/m. This is completely consistent with the data reported in the literature.40,44−47 The addition of the polymers markedly influences the shape of the isotherms. The lift-off area (i.e., the area where the surface pressure deviates from 0 mN/m) is shifted to a considerably higher area per lipid. The higher the polymer content, the higher the lift-off area, showing that the additional area is occupied by the polymer molecules. This area increase is higher for F-GP than for GP, indicating that F-GP molecules occupy more area at the interface than do GP molecules. This reflects the compression behavior of pure polymer film (Figure 2B). Upon compression, the surface pressure increases up to 5−8 mN/m, where a kink in the isotherms, followed by a plateau, indicates that the lipid LE → LC transition is maintained in the presence of the polymers. The transition plateaus are less defined in the mixed lipid/ polymer films, indicating that the LE/LC coexistence region extends over a certain pressure range. The transition pressures can be determined from the maxima in compressibility of the films that are calculated according to eq 1 and presented in Figure 7C,D as a function of the surface pressure. The determined transition pressures πtrans are depicted in Figure 7E as a function of polymer content. Both GP and F-GP addition increase the LE → LC transition pressure of DPPC up to 8 mN/m in a concentration-dependent manner. The higher the polymer content of the film, the higher the πtrans. The increase in πtrans can be interpreted as being analogous to the freezingpoint depression of the lipids resulting from the decreased lipid activity in the lipid/polymer mixture in the LE phase. This is further proof of the miscibility of the components in the LE phase. However, at low F-GP contents (1−2 mol %) F-GP decreases the transition pressure slightly, whereas GP does not show this effect. Obviously, the reduction of the LE-phase chemical potential is in this case overcompensated for by an effect that stabilizes the LC phase, i.e., an effect that induces lipid order. This could be the dehydration of the lipid headgroups as a consequence of polar interactions with the hydrophilic blocks of the polymers. Because the PGMA blocks have a potential for hydrogen bonding via their lateral glycerol moieties, they might replace water of hydration from the lipid headgroup region. The fact that a πtrans reduction occurs only for F-GP and not for GP would indicate that the PGMA blocks of F-GP are in closer contact with the interface, whereas the GP PGMA blocks are located in the aqueous subphase. This interpretation is reasonable because the terminally attached hydrophobic F9 chains force the PGMA blocks of F-GP 8110


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Figure 8. Compression isotherms of monolayers of DMPE containing different amounts of (A) GP and (B) F-GP as well as derived film compressibilities for (C) DMPE/GP and (D) DMPE/F-GP) and the LE → LC phase-transition pressures as a function of the copolymer molar fraction xpolymer for DMPE monolayers containing different amounts of GP (black squares) and F-GP (red circles), as determined from the lowpressure maxima in the compressibility traces (E). All measurements were performed at 20 °C. The area per lipid in A and B is calculated as the total area occupied by the mixed lipid/polymer monolayer divided by the number of lipid molecules.

that exceeds the diameters of the stretched chains. The presence of F-GP at the interface and its contribution to the film properties can also be judged from the compressibility curves (Figure 7D). The film compressibility does not decrease to the low value of LC-phase DPPC after the PPO squeeze-out peak but remains at a higher level, which indicates the presence of “soft” polymeric material at the interface. A third maximum can be seen in the compressibility plots for DPPC/F-GP mixed monolayers at 46−48 mN/m. This feature is visible as well in the compression isotherms as a small pseudoplateau. Upon compression beyond this plateau, all isotherms coincide at about 54 mN/m, and the film compressibilities decrease to a

low value (similar to that of pure DPPC), indicating that the contribution of F-GP to the film properties vanishes. This behavior reveals the squeeze-out of the F9 anchors at 46−48 mN/m. Thus, the perfluoroalkyl-terminated polymers are released into the aqueous subphase in a two-step mechanism: first, the PPO block is squeezed out at 28 mN/m, and second, the F9 chains are squeezed out at ca. 48 mN/m. The different squeeze-out pressures for the complete GP and F-GP molecules are also reflected by a different reversibility of the π−A isotherms after the first compression to 40 mN/m (Figure S10). GP is completely desorbed from the lipid monolayer at this pressure and diffuses away from the interface. 8111


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Figure 9. Change in transition temperature (ΔTm) of PC membranes upon addition of GP (black squares) and F-GP (red circles) as a function of (A) lipid acyl chain length (DM, 14:0/14:0; DP, 16:0/16:0; DS, 18:0/18:0; DA, 20:0/20:0) and (B) polymer molar fraction (xpolymer). The polymer concentration used in A is 50 μM. Transition temperatures are determined as the midpoint of the Δcp integrals with temperature, i.e., the point where half of the transition enthalpy is consumed. The lipid used in B is DPPC, and transition temperatures are determined from the maxima of the Δcp curves. Polymers were added to the aqueous phase after vesicle preparation and before starting the DSC experiments. The lipid concentration was always 1 mM.

destabilizing effects. By the evaluation of ΔTm, we showed that both the insertion of hydrophobic polymer moieties into the hydrophobic layer of the membrane and the adsorption of the PGMA groups into the headgroup layer occur upon binding of GP and F-GP. The more negative ΔTm is, the higher the effect of insertion of hydrophobic moieties into the membrane, whereas a positive contribution to ΔTm is a consequence of headgroup interactions. Figure 9 summarizes some of the previously reported results.25 Panel A shows the dependence of ΔTm on the hydrophobic thickness of the membrane. It can be seen that the effect of insertion decreases with increasing acyl chain length, i.e., that the polymers insert less readily into membranes with higher hydrophobic thickness. This result nicely correlates with the dependency of the MIP values on the acyl chain length shown here (Figure 5). The transition temperature of a bilayer correlates with the LE → LC phase-transition pressure (πtrans) of the respective monolayer. Both quantities denote the thermodynamic equilibrium between an ordered (Lβ′, LC) and a disordered (Lα, LE) phase.32 A decrease in Tm correlates with an increase in πtrans because both indicate a decrease in the chemical potential (stabilization) in the disordered phase. An increase in Tm correlates with a decrease in Δπtrans and indicates a stabilization of the more condensed phase. Therefore, we can compare the Δπtrans values of mixed DPPC/polymer monolayers discussed above (Figure 7E) with ΔTm values of the corresponding bilayer systems reported before.25 Figure 9B shows the dependence of ΔTm of DPPC bilayers on the polymer molar fraction added to the system. It indicates that ΔTm is slightly positive for low polymer contents, which is ascribed to a dominating gel-phase stabilizing effect due to headgroup interactions with the polymers’ PGMA blocks. At higher polymer concentrations, these effects are overcompensated for by the insertion of the hydrophobic polymer moieties into the hydrophobic membrane layer, leading to a negative ΔTm. This downshift in Tm is more pronounced as more polymer is added. This correlates well with the πtrans shift of polymer-containing DPPC monolayers shown in Figure 7E. Low F-GP contents lead to a slight decrease in πtrans, whereas higher polymer contents (GP and F-GP) lead to positive Δπtrans values as a result of the insertion of polymer moieties into the monolayer. Analogous to the bilayer case, this effect increases

Therefore, only a minor amount of GP is readsorbed upon expansion, and a consecutive second compression isotherm is shifted to lower areas per lipid, indicating an irreversible loss of GP from the interface. In contrast, F-GP is still anchored by the F9 groups to the lipid monolayer after the first compression to 40 mN/m. Consequently, the reinsertion of the PPO groups upon expansion is facilitated, and expansion and second compression isotherms indicate only a slightly reduced F-GP content at the interface. At 40 mN/m, the area per lipid is perfectly reproduced in the second compression isotherm. The compression experiments were repeated with DMPE (Figure 8) to check for the lipid headgroup specificities that were postulated from the monolayer adsorption experiments (see above) as well as from bilayer experiments.25 DMPE was chosen to be compared with DPPC because their transition pressures are in the same range. It can be seen that the polymers have very similar effects on DMPE monolayers compared to those exerted on DPPC monolayers: the DMPE LE → LC phase transition is preserved but slightly increased in the presence of the polymers up to a content of 10 mol %. The PPO blocks are squeezed-out at ca. 28 mN/m, resulting in a complete desorption of GP. The F9 chains are squeezed out at 48 mN/m, leading to the desorption of F-GP. These squeezeout pressures are identical to those determined for DPPC/ polymer mixed monolayers, indicating that headgroup-specific interactions do not play a role in the release of the polymers from lipid monolayers upon compression. The PPO squeeze-out pressure corresponds completely to the MIP values of GP in DPPC and DMPE monolayers, indicating that the lipid/GP systems are in thermodynamic equilibrium and adsorption and desorption processes lead to the same final state. On the contrary, the MIP values of F-GP are 5−8 mN/m lower than the F9 squeeze-out pressures, showing that the mixed F9/phospholipid monolayer is also kinetically stabilized. Comparison to Polymer−Lipid Bilayer Interactions. In a recent paper, we reported the influence of GP and its perfluoroalkyl-terminated analogue F-GP on lipid bilayers in liposomes.25 Systematic studies were done using differential scanning calorimetry (DSC) to evaluate the gel-to-fluid transition temperature (Tm) of pure or mixed membranes. The shift in Tm is a measure of membrane stabilizing or 8112


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the complete polymer molecule, whereas the triphilic polymer is still anchored by the F9 groups to the monolayer between transitions ii and iii. Therefore, transition ii correlates with the MIP values of GP, whereas transition iii correlates with the MIP values of F-GP. The findings discussed above corroborate the conclusion drawn previously from interaction experiments with lipid bilayers.25 However, the information achieved from the herein-described monolayer experiments is more versatile and more quantitative. This is due to the fact that the physical state of a monolayer can be easily controlled and monitored and allows measurements away from the equilibrium state of a selfassembled bilayer. Thus, the study presented shows the value of systematic monolayer studies for the characterization of amphiphile interactions. The introduction of perfluorinated moieties to increase the stability of membrane-bound polymers is a new means for the rational design of membrane-interacting polymers.

with increasing polymer molar fraction. In both bilayer and monolayer cases, the effects on the lipid phase transition depend only marginally on the nature of the polymer, i.e., GP and F-GP influence the phase-transition temperature in a similar way. These good correlations between bilayer and monolayer measurements show that monolayers are an appropriate model system to mimic bilayer behavior, even at surface pressures below the bilayer−monolayer equivalence pressure.

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CONCLUSIONS We studied a novel amphiphilic triblock copolymer with respect to its interaction with different lipid monolayers and the influence of attaching perfluorinated terminal alkyl chains to the polymer. The amphiphilic block copolymer has a hydrophobic PPO middle block with hydrophilic PGMA blocks attached on either side. This yields a polymer that is lipophilic, hydrophilic, and fluorophilic at the same time. The major aim of this study was to elucidate the influence of the terminal F9 groups on the interaction of the polymers with lipid monolayers. Two different kinds of experiments were conducted: adsorption of the polymers from the aqueous subphase to preformed lipid monolayers and compression of mixed lipid/polymer monolayers. Both experiments were performed with a set of different lipids varying in acyl chain length, acyl chain saturation, chemical nature of the headgroup, and physical state of the monolayer. All experiments yield one major piece of information: (i) The perfluoroalkylation of the polymer termini increases its surface activity and its interaction with all kinds of lipid monolayers. The hydrophobicity of the F9 chains is an important driving force for polymer incorporation into the monolayers and anchors the polymers in the monolayers. This can be concluded from the high maximum insertion pressures (MIP) as well as from the high squeeze-out pressures. Both values are well above the monolayer−bilayer equivalence pressure. We conclude that the introduction of fluorinated moieties into amphiphilic polymers is an effective means to increase their interactions with lipid membranes. Besides this main finding, a variety of conclusion could be drawn from the systematic variation of the lipids: (ii) Both polymers insert more effectively into monolayers with shorter lipid acyl chains. (iii) Interactions between the hydrophilic PGMA groups of the polymers and the lipid headgroups contribute to the polymer−lipid interaction. This interaction is stronger for PE headgroups than for PC headgroups. Simultaneously, it is stronger for the triphilic than for the amphiphilic polymers. (iv) Various findings suggest a partial miscibility of polymers and lipids within the monolayer. On one hand, typical features of the pure components are maintained in the compression isotherms of the mixed monolayers, suggesting a partial demixing of both components. On the other hand, the shift in lipid transition pressure and the influence of the acyl chain length indicate that this demixing is not complete. (v) Neither the lipid phase state nor the acyl chain saturation has a large influence on the maximum insertion pressure of the polymers. Furthermore, we could explain the complete compression isotherm of mixed polymer/lipid monolayers. Four different states separated by three distinct transitions were identified. The transitions could be attributed to (i) the LE→ LC transition of the lipid component, (ii) the PPO block squeezeout, and (iii) the F9 chain squeeze-out. For the nonfluorinated polymer, the second transition is identical to the squeeze-out of

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.6b01574. Concentration-dependent Δπ vs π0 curves for F-GP injection underneath DPPC, all time-dependent surface pressure traces recorded after polymer insertion underneath different lipid monolayers, and DPPC/polymer compression and expansion isotherms to test for reversibility (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the Deutsche Forschungsgemeinschaft (DFG) for financial support (FOR 1145, TP 4 and 5). REFERENCES

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