Intermolecular Interaction between Phosphatidylcholine and


Sep 19, 2016 - We synthesized 1,2-dipalmitoyl-sn-glycero-3-sulfobetaine (DPSB) to represent a reversed-head lipid; 1,2-dipalmitoyl-sn-glycero-3-phosph...
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Article

Intermolecular Interaction between Phosphatidylcholine and Sulfobetaine Lipid: A Combination of Lipids with Antiparallel Arranged Headgroup Charge Tatsuo Aikawa, Keisuke Yokota, Takeshi Kondo, and Makoto Yuasa Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b02563 • Publication Date (Web): 19 Sep 2016 Downloaded from http://pubs.acs.org on September 21, 2016

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Intermolecular Interaction between Phosphatidylcholine and Sulfobetaine Lipid: A Combination of Lipids with Antiparallel Arranged Headgroup Charge

Tatsuo Aikawa†, Keisuke Yokota†, Takeshi Kondo†,‡, Makoto Yuasa†,‡,*



Department of Pure and Applied Chemistry, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan.



Research Institute for Science & Technology (RIST), Tokyo University of Science, 2641 Noda, Chiba 278-8510, Japan.

*Corresponding author: (M.Y.) E-mail address: [email protected]; Tel +81-4-7124-1501 (3607); Fax +81-4-71212439

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Keywords: phosphatidylcholine; sulfobetaine lipid; lipid monolayer; intermolecular interaction; surface pressure-area (π-A) isotherms; differential scanning calorimetry (DSC)

Abstract

Intermolecular interactions between lipid molecules are important when designing lipid bilayer interfaces, which have many biomedical applications such as in drug delivery vehicles and biosensors. Phosphatidylcholine, a naturally occurring lipid, is the most common lipid found in organisms. Its chemical structure has a negatively charged phosphate linkage, adjacent to an ester linkage in a glycerol moiety, and a positively charged choline group, placed at the terminus of the molecule. Recently, several types of synthetic lipids that have headgroups with the opposite charge to that of phosphatidylcholine have emerged, i.e., a positively charged ammonium group is present adjacent to the ester linkage in their glycerol moiety and a negatively charged group is placed at their terminus. These types of lipids constitute a new class of soft material. The aim of this study was to determine how such lipids, with antiparallel arranged headgroup charge, interact with naturally occurring phosphatidylcholines. We synthesized 1,2-dipalmitoyl-sn-glycero-3-sulfobetaine (DPSB) to represent a reversed-head lipid; 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) was used to represent a naturally occurring phospholipid. The intermolecular interaction between these lipids was investigated using surface pressure-area (π-A) isotherms of the lipid monolayer at the air/water interface. We found that the extrapolated area and excess free energy of the mixed monolayer deviated negatively when compared with the ideal values from additivity. Moreover, differential scanning calorimetry of the lipid mixture in aqueous dispersion showed that the gel-to-liquid crystal

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transition temperature increased compared with that of each pure lipid composition. These results clearly indicate that DPSB preferably interacts with DPPC in the mixture. We believe that the attraction between the oppositely charged headgroups of these lipids reinforces the intermolecular interaction. Our results provide insight into the intermolecular interaction between phospholipids and reversed-head lipids, which may prove useful for the design of lipidbased materials in the future.

1. Introduction

Phosphatidylcholines are naturally occurring molecules that constitute the main component of the plasma membrane of biological cells in the form of the lipid bilayer. Lipid-based materials have many potential applications such as the surface modification of biosensors1–3 and microfluidic devices,1,4 drug delivery vehicles in the form of vesicles,5–8 scaffolds for membranebound proteins,9,10 and templates for biomineralization.11 1,2-Dipalmitoyl-sn-glycero-3phosphatidylcholine (DPPC) is one of the most common phospholipids observed in biological organisms, and has been used as a drug carrier. In terms of structural features, DPPC is a waterinsoluble amphiphilic molecule composed of a hydrophilic headgroup and two hydrophobic palmitoyl tailgroups (Fig. 1). The zwitterionic headgroup of DPPC comprises a positively charged trimethyl ammonium moiety and a negatively charged phosphate ester. When designing lipid-based materials, it is important to understand the intermolecular interaction between the lipids, which is greatly affected by the characteristics of their functional groups.



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Recently, a series of lipid molecules with headgroups containing moieties that are oppositely charged to those of the associated phosphatidylcholines have been synthesized.12–16 In this paper, we have called such lipid groups “reversed-head lipids.” A characteristic feature of reversedhead lipids is that the cationic moiety of the headgroup is usually composed of a quaternary ammonium group. However, the anionic moiety may be one of several groups such as phosphate (-PO4--), sulfonate (-SO3-), or carboxylate (-CO2-) groups, which correspond to the headgroups of choline phosphate (CP), sulfobetaine (SB), and carboxybetaine (CB), respectively. For example, Szoka et al. synthesized reversed-head lipids with SB or CB headgroups.12–16 They also reported the thermotropic phase transition behavior of a bilayer composed of SB lipids,12 and described the intermolecular interaction between the lipid molecules. In their report, the gel-to-liquid crystal phase transition temperature (Tc) of the SB lipids was higher than that of DPPC. This increase in Tc for the SB lipids should be attributed to strong ionic headgroup interaction because an increase in Tc normally indicates enhancement of intermolecular interaction between the lipids in the bilayer membrane.12,16,17 However, the intermolecular interaction between phosphatidylcholines and reversed-head lipids with oppositely charged headgroups has not been studied. In this study, we hypothesized that intermolecular interaction between the phosphatidylcholines and the reversed-head lipids is due Coulombic interaction and/or dipole–dipole interaction between the headgroups (Fig. 1). In a closely related study, Schmuck et al. reported that guanidiniocarbonyl pyrrole carboxylate zwitterions undergo molecular self-assembly, even in polar solvents.18–21 The equilibrium constant for the dimerization of zwitterions is estimated to be > 1010 M-1 in dimethyl sulfoxide. Even in pure water, the equilibrium constant is 170 M-1. In general, most intermolecular

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interactions except for hydrophobic interaction are likely to be hindered in polar solvents.22 The ability of zwitterions to form interactions even in polar solvents is expected to be useful in developing biomaterials, because they could be used as “binders” in physiological environments. As another example in polymer aggregates, Muthukumar et al. investigated the self-assembly of a polyzwitterionic diblock polymer comprising side chains of phosphatidylcholine (PC) and nbutyl-substituted choline phosphate (CP).23 The oppositely charged moieties in the PC and CP side chains were arranged adjacent to each other. The researchers anticipated that the driving force for aggregation in the polymers would intermolecular interaction arising from the dipole moments. As described above, there have been reports on intermolecular interactions between zwitterionic polar groups in which oppositely charged groups complement each other. To the best of our knowledge, there have been no reports on the intermolecular interactions between lipids that have polar head groups comprising oppositely charged groups that are arranged adjacent to each other. To clarify the above hypothesis, we employed a combination of lipids, 1,2-dipalmitoyl-snglycero-3-sulfobetaine (DPSB) and 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), which have the same acyl groups but have antiparallel arranged headgroup charges. Figure 1 illustrates the intermolecular interaction we attempted to clarify in this work. Intermolecular interaction between DPPC and DPSB was investigated using the surface pressure-area (π-A) isotherms of the binary lipid monolayer formed at the air/water interface, and by differential scanning calorimetry (DSC) of the lipid dispersion. Because the tailgroups of the lipids have the same structure, the intermolecular association can only be explained by the interaction between their headgroups. This study may provide significant insight into the effect of betaine compounds

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on the structure of molecular aggregates composed of phospholipid bilayers. Especially, for designing lipid-based materials, physicochemical properties of the lipid membrane should be appropriately controlled (e.g., packing density, fluidity, and solute permeability). Therefore, it is important to study how betaine compounds influence on intermolecular interaction in the phospholipid membrane and modulate physicochemical properties of the lipid membrane.

2. Materials and Methods

2.1. Materials

DPPC (> 99%) was purchased from Yuka Sangyo (Tokyo, Japan) and used without further purification. DPSB was synthesized according to a previously published method,12 and the synthesis and structural confirmation information are provided in the Supporting Information. Ultrapure water was obtained using the Milli-Q system (resistivity: 18.2 MΩ·cm at 25°C; Merck-Millipore, USA).

2.2. Acquisition of π-A isotherms

Surface pressure-area (π-A) isotherms were acquired using a trough equipped with a Wilhelmy plate (HBM 700LB; Kyowa Interface Science, Niiza, Japan). To form monolayers at the air/water interface, lipids were first dissolved in chloroform/methanol (8:2 volume ratio), and 70 µL of the lipid solution was then spread on the subphase filled with ultrapure water using a microsyringe. After spreading the lipid, the monolayer was equilibrated on the subphase for 15

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min at 20°C. The isotherms were acquired under the following conditions: the barrier speed was 28 cm2/min and the temperature of the subphase was maintained at 20°C. The extrapolated area (AL) represents the cross-sectional area of a single lipid molecule, which is free from external pressure from surrounding lipids in the condensed lipid monolayer (please see Supporting Information for the definition of AL). The compressibility modulus of the lipid monolayers was calculated using equation (1):24

CS−1 = −A(d π / dA)

(1)

Where: π is the surface pressure of the lipid monolayer at the air/water interface; A is the occupied molecular area per single molecule constituting in the lipid monolayer. The miscibility of lipids in the mixed monolayer and the interactions between lipid components were analyzed quantitatively on the basis of the excess free energy of mixing (∆GExc) calculated for the obtained π-A isotherms, as defined in equation (2):

π

∆G Exc = N A ∫ A12 − ( A1Χ1 + A2 Χ 2 ) d π 0

(2)

Where: A12 is the occupied molecular area for a particular composition of a mixed monolayer at a given surface pressure; A1 and A2 are the occupied molecular areas for pure monolayers of components 1 and 2 at the same surface pressure, respectively; X1 and X2 indicate molar fractions of the components in the mixed monolayer; and NA is Avogadro’s number.25

2.3. Differential scanning calorimetry (DSC)

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To determine the gel-to-liquid crystal phase transition temperature (Tc) of the aqueous dispersion composed of pure or mixed lipids, DSC was carried out using a calorimeter (DSC 1, Mettler Toledo, Switzerland). The lipid dispersions for DSC were prepared as follows. The lipid mixture was dissolved in CHCl3/methanol (8:2 volume ratio) and placed in a 10-mL round bottom flask. The solvent was then rotary evaporated to form a thin lipid layer on the bottom. The obtained lipid film was hydrated in ultrapure water for 2 min at 80°C with gentle agitation. The hydrated lipid was agitated with a bath-type sonicator (AZU-6D, As One, Osaka, Japan) for 1 min at 80°C. The obtained lipid dispersion (40 µL) was added to a 100-µL aluminum pan and sealed tightly with a lid. The scan rate was 2°C/min during the heating and cooling process. Tc was defined as the onset temperature of the endothermic peaks.

2.4. Statistical analysis

Results of AL, Cs-1, ∆GExc, and Tc as a function of molar fraction of DPSB in DPPC (ΧDPSB) were represented as mean ± standard deviation (SD). Statistical analysis of these data was carried out using one-way analysis of variance (ANOVA) using Excel 2016 software (Microsoft). Significant difference was defined as p 250 mN/m.24 Although there was no significant difference in the addition of DPSB slightly increased

Max

Max

Cs-1 values (p > 0.05), the

Cs-1 of the monolayer. The mixed monolayer remained

in the solid phase (Max Cs-1 > 250 mN/m) at the compressed state. Interestingly, pure DPSB monolayer exhibited the lowest Max Cs-1 but the mixing DPSB with DPPC could increase Max Cs-1 of the monolayer, suggesting that there was intermolecular interaction between DPSB and DPPC in the monolayer. This fact can also be characterized by data from the extrapolated area, excess free energy, and a shift in the gel-to-liquid crystal phase transition temperature of the mixed system, as discussed below.



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3.4. Excess free energy of mixing lipids in the monolayer

To evaluate the thermodynamic stability of the mixed monolayer at the air/water interface, we calculated the excess free energy of mixing (∆GExc) between the DPPC and DPSB at a given

ΧDPSB (Fig. 5). The values of ∆GExc for the mixed monolayer became negative at all ranges of ΧDPSB, indicating that mixing DPSB and DPPC in the monolayer is thermodynamically favored regardless of the composition. Moreover, we noticed that the minimum value of ∆GExc occurred at ΧDPSB = 0.5 at any surface pressure range. Thus, the most stable film should be formed at the air/water interface when the film is composed of an equimolar mixture of DPPC and DPSB. In the present system, it was assumed that Coulombic and/or dipole–dipole interactions between the PC and SB headgroups may contribute to the thermodynamically favored mixing and the formation of the stable monolayer. However, there was an indication that binary hydrocarbon mixtures with anti-paralleled dipole moments do not contribute to thermodynamically favored mixing. Petrov et al. reported on the miscibility of a mixed monolayer comprising two hydrocarbon-containing compounds: CH3(CH2)21-O-CH2CH3 and CH3(CH2)21-O-CH2CF3.32 The dipole moments of their headgroups (ethyl ether and trifluoroethyl ether) were directly opposed. In their binary system, the value of ∆GExc for mixing these compounds in the monolayer at the air/water interface was positive. This suggests that these compounds favored phase separation in the monolayer. In contrast to that study, although the dipole moments of the lipids were also directly opposed in our study, ∆GExc was negative, indicating that the lipids could be spontaneously mixed in the monolayer. Therefore, this spontaneous mixing observed in this study may be attributed to Coulombic interaction between the PC and SB headgroups rather than dipole-dipole interaction between the headgroups.

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3.5. Effect of mixing DPSB on gel-to-liquid crystal phase transition temperature of lipid dispersion

The gel-to-liquid crystal phase transition temperature (Tc) reflects the strength of the intermolecular interaction between the lipid molecules in the bilayered membrane. The representative DSC thermograms and mean values of Tc of the DPPC aqueous dispersion containing different mole fractions of DPSB is shown in Figure 6. The Tc of the pure DPPC dispersion was consistent with that previously published.33 For the mixed lipid system, the Tc significantly increased with increasing ΧDPSB until it reached a maximum at ΧDPSB = 0.5 (Fig. 6b). At ΧDPSB > 0.5, the Tc significantly decreased with increasing ΧDPSB. This suggests that the intermolecular interaction between DPSB and DPPC was greatest in an equimolar composition of these lipids. Because the tailgroups of the two lipids in the mixture were the same, the changes in Tc should be attributed to interaction between their headgroups. We assumed that Coulombic interaction and/or dipole–dipole interaction between the headgroups is related to the enhanced lipid interaction. Controversially, the Tc value for pure DPSB in the present study was slightly smaller than that reported by Szoka et al.12 The reason for this reduced Tc has not yet been identified. Although the extrapolated area (AL) of pure DPSB was larger than that of pure DPPC, as discussed in the previous section, the size of the lipid should not affect the Tc.17



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DSC thermograms can also provide information on the mixing state of the lipid bilayer. In Figure 6a, relatively sharp endothermic peaks were observed at 0.4–0.6 of ΧDPSB, suggesting that the lipid molecules were homogeneously mixed in the bilayer. In contrast, at 0.1–0.3 and 0.7–0.9 of ΧDPSB, the endothermic peaks were broadened, indicating that the lipid molecules are heterogeneously mixed in the bilayer when containing 10–30 mol% of either DPPC or DPSB. Considering these results, DPPC and DPSB are likely to form a lipid complex with an equimolar composition. Therefore, when the lipid bilayer contains 10–30 mol% of either DPPC or DPSB, two types of lipid domains might exist; one domain should be a pure component of either DPPC or DPSB and the other should be composed of the equimolar complex of both lipids. Nevertheless, it can be assumed that the heterogeneity of the mixed state is slight. Given that a distinct peak separation was not observed in the thermogram, even at 0.1–0.3 and 0.7–0.9 of

ΧDPSB, remarkable phase separation should not occur in the lipid bilayer. This assumption can also be supported by the negative values of ∆GExc, which negates the possibility of a phase separation in the monolayer (Fig. 5).



As shown in Figure 6a, two endothermic peaks were observed for pure DPSB (ΧDPSB = 1), which was similar to that previously reported by Szoka et al.12 In addition to these endothermic peaks, an exothermic peak was observed just after the first endothermic peak at the lower temperature. Reproducibility of such thermotropic behavior for DPSB was confirmed in an additional experiment independently repeated. Our interpretation about the thermotropic

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behavior for DPSB is illustrated in Scheme 1. The interaction between headgroups contributes to the increased gel-to-liquid crystal phase transition temperature.17 Therefore, the endothermic peaks at higher (56.8°C) and lower (40.8°C) temperatures can be assigned to gel-to-liquid crystal phase transitions of DPSB that have headgroups with and without an intermolecular interaction between the neighboring headgroups, respectively. Notably, the Tc value at the first endothermic peak was close to that of pure DPPC (41.4°C). Thus, it can be assumed that phosphorylcholine does not interact with the neighboring headgroups. Previous literature described that sulfobetaine lipid forms intramolecular salt formation in the headgroup, which is a so-called inner-salt formation.12 However, we assumed that there is little possibility for inner-salt formation to occur in DPSB, because if DPSB formed an inner-salt, the hydrophobic tailgroup packing should be increased, resulting in a reduction of the surface area per single molecule in the lipid monolayer. However, the relatively large AL value for DPSB minimizes the possibility for inner-salt formation (Fig. 3, ΧDPSB = 1). The exothermic peak was also observed just after the first endothermic peak, which was reproducibly exhibited in experiments independently repeated. As shown in Scheme 1 (from state 2 to state 3), DPSB molecules in liquid crystal phase, which are located in a site close to the lipid domain composed of DPSB interacting with the neighboring headgroups, may return to the gel phase with reconstructing an interaction with neighboring headgroups. It was also interesting that the second endothermic peak at the higher temperature disappeared by addition of the small amount of DPPC (Fig. 6a, ΧDPSB =0.9). This fact indicates that SB headgroups dissociate their interaction with the neighboring SB headgroups, and then interact with the PC groups even in the presence of small amounts of DPPC (e.g., 10 mol%). It is predicted that the antiparallel charge arrangement of each headgroup may be contribute to this exchanging of interaction from SB–SB to SB–PC. Although results of the π-A isotherms and the

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DSC in the present study strongly indicate intermolecular interaction between DPPC and DPSB, the detailed mechanism underlying the interaction between both headgroups requires further examination.

4. Conclusions

In the present work, we revealed that a phosphatidylcholine can interact with a sulfobetaine lipid, which has an oppositely charged headgroup to that of the phosphatidylcholine. Intermolecular interaction was indicated by the data obtained from the π-A isotherm and the DSC. The extrapolated area of the mixed monolayer exhibited negative deviation from the ideal additivity, indicating that attractive interaction was taking place between the lipids in the monolayer. Excess free energy of mixing of the lipid also deviated negatively, which indicates that mixing DPPC and DPSB is thermodynamically favored. The DSC of the binary lipid dispersion revealed that the increment of gel-to-liquid crystal phase transition temperature occurred in the equimolar composition. This supports reinforcement of intermolecular interaction by mixing DPPC and DPSB. We supposed that the enhanced intermolecular interaction could be attributed to Coulombic interaction rather than dipole–dipole interaction, caused by antiparallel arranged charges in each headgroup. At this stage, we have not identified the detailed mechanism underlying the intermolecular interaction between DPPC and DPSB. Therefore, further investigation of the mechanism should be needed. To date, several types of reversed-head lipid have been synthesized. Our results give an insight into how reversed-head lipids interact with naturally occurring lipids (phosphatidylcholines), and form the molecular aggregates of the binary lipid system.

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Acknowledgements The authors would like to express their appreciation to Dr. Kenichi Sakai for technical guidance and advice on isotherm acquisition. This work was supported by Foundation, Oil&Fat Industry Kaikan, Terumo Foundation for Life Sciences and Arts, and JSPS KAKENHI Grant Number JP16K21398 (T.A.).

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Figure captions Figure 1. Hypothesized intermolecular interaction between 1,2-dipalmitoyl-sn-glycero-3phosphatidylcholine (DPPC) and the reversed-head lipid (1,2-dipalmitoyl-sn-glycero-3sulfobetaine (DPSB)). As a counterpart to DPSB, DPPC was employed as a typical phospholipid widely observed in physiological environments. Figure 2. Surface pressure-area (π-A) isotherms of lipid monolayers of (1,2-dipalmitoyl-snglycero-3-phosphatidylcholine (DPPC) in the presence of (1,2-dipalmitoyl-sn-glycero-3sulfobetaine (DPSB) with different molar fraction (ΧDPSB). The subphase contained ultrapure water. The temperature of the subphase was 20°C. The isotherms were from twice or more independent measurement. Figure 3. The extrapolated molecular area (AL) of the lipids as a function of ΧDPSB, as determined using the isotherms. The dashed line represents the ideal additivity for the binary system composed of DPPC and DSPB. p < 0.01 (one-way ANOVA, n ≥ 2). Figure 4. Representative plots of compressibility modulus (CS-1) as a function of the occupied molecular area (a). Plots of the maximum CS-1 values as a function of ΧDPSB (b). p > 0.05 (oneway ANOVA, n ≥ 2). Figure 5. The excess free energy of mixing (∆GExc) as a function of ΧDPSB at various surface pressure region; 0–5 (open circles), 0–10 (filled circles), 0–20 (open triangles) , 0–30 (filled triangles), and 0–40 mN/m (diamonds)). The dashed line represents the ideal ∆GExc value for the binary system composed of DPPC and DPSB. P-values for the surface pressure ranges, 0–10, 0–

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20, 0–30, 0–40 mN/m, were < 0.01 (one-way ANOVA, n ≥ 2). For the surface pressure range, 0– 5 mN, p > 0.05 (one-way ANOVA, n ≥ 2). Figure 6. Representative DSC thermograms of an aqueous dispersion of DPPC in the presence of DPSB with various molar fraction (a). The thermograms for pure DPPC and DPSB were shown with downsized intensity (× 0.5). Plots of onset temperature of gel-to-liquid crystal phase transition (Tc) as a function of ΧDPSB (b). p < 0.01 (one-way ANOVA, n ≥ 2). Triangle symbol represents Tc of DPSB having interaction between neighboring headgroups. Scheme 1. Schematic illustration of the thermotropic behavior of a lipid membrane composed of pure DPSB during the heating process in the DSC. The thermogram of pure DPSB in aqueous dispersion, which is classified into four different phase states of the lipid membrane, is depicted at the top of the scheme. In state 1, there are two different domains where the lipids have interaction or non-interaction between the neighboring headgroups. At this state, all DPSB in both domains are in the gel phase. In state 2, DPSB that do not have an intermolecular interaction are in the liquid crystal phase. In contrast, DPSB that have an intermolecular interaction remain in the gel phase. In state 3, DPSB that are located in a site close to the domain composed of the DPSB with the intermolecular interaction return to the gel phase and interact with the DPSB in the headgroup. In state 4, all DPSB are in the liquid crystal phase. Table Table 1. Experimental conditions for isotherm measurement of (1,2-dipalmitoyl-sn-glycero-3phosphatidylcholine (DPPC) Spreading solvent

Compression rate

Chloroform/ethanol

8.2 Å2/molecular × min Ultrapure water

Subphase

Reference 26

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(8:2) Chloroform/methanol (8:2)

28 cm2/min

Ultrapure water

This work

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References (1) Castellana, E. T.; Cremer, P. S. Solid Supported Lipid Bilayers: From Biophysical Studies to Sensor Design. Surf. Sci. Rep. 2006, 61, 429–444. (2) Kataoka-Hamai, C.; Miyahara, Y. Field-Effect Detection Using Phospholipid Membranes. Sci. Technol. Adv. Mater. 2010, 11, 033001. (3) Czolkos, I.; Jesorka, A.; Orwar, O. Molecular Phospholipid Films on Solid Supports. Soft Matter 2011, 7, 4562–4576. (4) Phillips, K. S.; Cheng, Q. Microfluidic Immunoassay for Bacterial Toxins with Supported Phospholipid Bilayer Membranes on Poly(dimethylsiloxane). Anal. Chem. 2005, 77, 327–334. (5) Torchilin, V. P.; Omelyanenko, V. G.; Papisov, M. I.; Bogdanov, A. A.; Trubetskoy, V. S.; Herron, J. N.; Gentry, C. A. Poly(ethylene Glycol) on the Liposome Surface: On the Mechanism of Polymer-Coated Liposome Longevity. Biochim. Biophys. Acta 1994, 1195, 11–20. (6) Allen, T. M.; Cullis, P. R. Liposomal Drug Delivery Systems: From Concept to Clinical Applications. Adv. Drug Delivery. Rev. 2013, 65, 36–48. (7) Bedu-Addo, F. K.; Huang, L. Interaction of PEG-Phospholipid Conjugates with Phospholipid: Implications in Liposomal Drug Delivery. Adv. Drug Deliv. Rev. 1995, 16, 235– 247. (8) Aikawa, T.; Ito, S.; Shinohara, M.; Kaneko, M.; Kondo, T.; Yuasa, M. A Drug Formulation Using an Alginate Hydrogel Matrix for Efficient Oral Delivery of the Manganese PorphyrinBased Superoxide Dismutase Mimic. Biomater. Sci. 2015, 3, 861–869.

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(9) Deems, R. A. Interfacial Enzyme Kinetics at the Phospholipid/ Water Interface: Practical Considerations. Anal. Biochem. 2000, 287, 1–16. (10) Hagn, F.; Etzkorn, M.; Raschle, T.; Wagner, G. Optimized Phospholipid Bilayer Nanodiscs Facilitate High-Resolution Structure Determination of Membrane Proteins. J. Am. Chem. Soc. 2013, 135, 1919–1925. (11) Collier, J. H.; Messersmith, P. B. Phospholipid Strategies In Biomineralization And Biomaterials Research. Annu. Rev. Mater. Res. 2001, 31, 237–263. (12) Perttu, E. K.; Szoka, F. C. Zwitterionic Sulfobetaine Lipids That Form Vesicles with SaltDependent Thermotropic Properties. Chem. Commun. 2011, 47, 12613–12615. (13) Kohli, A. G.; Walsh, C. L.; Szoka, F. C. Synthesis and Characterization of Betaine-like Diacyl Lipids: Zwitterionic Lipids with the Cationic Group at the Bilayer Interface. Chem Phys Lipids 2012, 165, 252–259. (14) Walsh, C. L.; Nguyen, J.; Szoka, F. C. Synthesis and Characterization of Novel Zwitterionic Lipids with pH-Responsive Biophysical Properties. Chem. Commun. 2012, 48, 5575–5577. (15) Perttu, E. K.; Kohli, A. G.; Szoka, F. C. Inverse-Phosphocholine Lipids: A Remix of a Common Phospholipid. J. Am. Chem. Soc. 2012, 134, 4485–4488. (16) Venditto, V. J.; Dolor, A.; Kohli, A.; Salentinig, S.; Boyd, B. J.; Szoka, F. C. Sulfated Quaternary Amine Lipids: A New Class of Inverse Charge Zwitterlipids. Chem. Commun. 2014, 50, 9109–9111.

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(17) Boggs, J. M. Lipid Intermolecular Hydrogen Bonding: Influence on Structural Organization and Membrane Function. Biochim. Biophys. Acta 1987, 906, 353–404. (18) Schmuck, C.; Rehm, T.; Geiger, L.; Schäfer, M. Synthesis and Self-Association Properties of Flexible Guanidiniocarbonylpyrrole-Carboxylate Zwitterions in DMSO: Intra- versus Intermolecular Ion Pairing. J. Org. Chem. 2007, 72, 6162–6170. (19) Rehm, T.; Schmuck, C. How to Achieve Self-Assembly in Polar Solvents Based on Specific Interactions? Some General Guidelines. Chem. Commun. 2008, 801–813. (20) Schmuck, C.; Wienand, W. Highly Stable Self-Assembly in Water: Ion Pair Driven Dimerization of a Guanidiniocarbonyl Pyrrole Carboxylate Zwitterion. J. Am. Chem. Soc. 2003, 125, 452–459. (21) Schmuck, C. Highly Stable Self-Association of 5-(Guanidiniocarbonyl)-1H-Pyrrole-2Carboxylate in DMSO – The Importance of Electrostatic Interactions. European J. Org. Chem. 1999, 2397–2403. (22) Israelachvili, J. N. Intermolecular and Surface Forces, Third Edition; Elsevier Inc.: MA. USA, 2011. (23) Morozova, S.; Hu, G.; Emrick, T.; Muthukumar, M. Influence of Dipole Orientation on Solution Properties of Polyzwitterions. ACS Macro Lett. 2016, 5, 118–122. (24) Davies, J. T.; Rideal, E. K. Interfacial Phenomena, second ed., Academic Press, New York, 1963, p. 265. (25) Gains, G. L. Insoluble Monolayers at Liquid/Gas Interfaces; Wiley-Interscience: New York, 1966.

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(26) Miñones, J.; Rodríguez Patino, J. M.; Conde, O.; Carrera, C.; Seoane, R. The Effect of Polar Groups on Structural Characteristics of Phospholipid Monolayers Spread at the Air-Water Interface. Colloids Surfaces A Physicochem. Eng. Asp. 2002, 203, 273–286. (27) Duncan, S. L.; Larson, R. G. Comparing experimental and simulated pressure-area isotherms for DPPC. Biophys. J. 2008, 94, 2965–2986. (28) Munden, J. W.; Swarbrick, J. Effect of Spreading Solvent on Monolayer Characteristics of Dipalmitoyl Lecithin. J. Colloid Interface Sci. 1973, 42, 657–659. (29) Jyoti, A.; Prokop, R. M.; Li, J.; Vollhardt, D.; Kwok, D. Y.; Miller, R.; Möhwald, H.; Neumann, A. W. An Investigation of the Compression Rate Dependence on the Surface Pressure-Surface Area Isotherm for a Dipalmitoyl Phosphatidylcholine Monolayer at the Air/water Interface. Colloids Surfaces A Physicochem. Eng. Asp. 1996, 116, 173–180. (30) Klopfer, K. J.; Vanderlick, T. K. Isotherms of Dipalmitoylphosphoatidylcholine (DPPC) Monolayers: Features Revealed and Features Obscured. J. Colloid Interface Sci. 1996, 182, 220– 229. (31) Tristram-Nagle, S.; Zhang, R.; Suter, R. M.; Worthington, C. R.; Sun, W. J.; Nagle, J. F. Measurement of Chain Tilt Angle in Fully Hydrated Bilayers of Gel Phase Lecithins. Biophys. J. 1993, 64, 1097–1109. (32) Petrov, J. G.; Andreeva, T. D.;Moehwald, H. Dipolar Interactions and Miscibility in Binary Langmuir Monolayers with Opposite Dipole Moments of the Hydrophilic Heads. Langmuir 2009, 25, 3659–3666.

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(33) Mannock, D. A.; Lee, M. Y. T.; Lewis, R. N. A. H.; McElhaney, R. N. Comparative Calorimetric and Spectroscopic Studies of the Effects of Cholesterol and Epicholesterol on the Thermotropic Phase Behaviour of Dipalmitoylphosphatidylcholine Bilayer Membranes. Biochim. Biophys. Acta 2008, 1778, 2191–2202.

SUPPORTING INFORMATION Synthetic procedure of DPSB, Definition of AL

Synopsis (TOC)

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Figure 1. Hypothesized intermolecular interaction between 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and the reversed head lipid (1,2-dipalmitoyl-sn-glycero-3-sulfobetaine (DPSB)). As a counterpart to DPSB, DPPC was employed as a typical phospholipid widely observed in physiological environments. 73x80mm (300 x 300 DPI)

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Figure 2. Surface pressure-area (-A) isotherms of lipid monolayers of (1,2-dipalmitoyl-sn-glycero-3phosphatidylcholine (DPPC) in the presence of (1,2-dipalmitoyl-sn-glycero-3-sulfobetaine (DPSB) with different molar fraction (DPSB). The subphase contained ultrapure water. The temperature of the subphase was 20°C. The isotherms were from twice or more independent measurement. 219x176mm (300 x 300 DPI)

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Figure 3. The extrapolated molecular area (AL) of the lipids as a function of DPSB, as determined using the isotherms. The dashed line represents the ideal additivity for the binary system composed of DPPC and DSPB. p < 0.01 (one-way ANOVA, n ≥ 2). 84x82mm (300 x 300 DPI)

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Figure 4. Representative plots of compressibility modulus (CS-1) as a function of the occupied molecular area (a). Plots of the maximum CS-1 values as a function of DPSB (b). p > 0.05 (one-way ANOVA, n ≥ 2). 178x234mm (300 x 300 DPI)

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Figure 5. The excess free energy of mixing (∆GExc) as a function of DPSB at various surface pressure region; 0–5 (open circles), 0–10 (filled circles), 0–20 (open triangles) , 0–30 (filled triangles), and 0–40 mN/m (diamonds)). The dashed line represents the ideal ∆GExc value for the binary system composed of DPPC and DPSB. P-values for the surface pressure ranges, 0–10, 0–20, 0–30, 0–40 mN/m, were < 0.01 (one-way ANOVA, n ≥ 2). For the surface pressure range, 0–5 mN, p > 0.05 (one-way ANOVA, n ≥ 2). 85x81mm (300 x 300 DPI)

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Figure 6. Representative DSC thermograms of an aqueous dispersion of DPPC in the presence of DPSB with various molar fraction (a). The thermograms for pure DPPC and DPSB were shown with downsized intensity (× 0.5). Plots of onset temperature of gel-to-liquid crystal phase transition (Tc) as a function of DPSB (b). p < 0.01 (one-way ANOVA, n ≥ 2). Triangle symbol represents Tc of DPSB having interaction between neighboring headgroups. 84x243mm (300 x 300 DPI)

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Scheme 1. Schematic illustration of the thermotropic behavior of a lipid membrane composed of pure DPSB during the heating process in the DSC. The thermogram of pure DPSB in aqueous dispersion, which is classified into four different phase states of the lipid membrane, is depicted at the top of the scheme. In state 1, there are two different domains where the lipids have interaction or non-interaction between the neighboring headgroups. At this state, all DPSB in both domains are in the gel phase. In state 2, DPSB that do not have an intermolecular interaction are in the liquid crystal phase. In contrast, DPSB that have an intermolecular interaction remain in the gel phase. In state 3, DPSB that are located in a site close to the domain composed of the DPSB with the intermolecular interaction return to the gel phase and interact with the DPSB in the headgroup. In state 4, all DPSB are in the liquid crystal phase. 73x165mm (300 x 300 DPI)

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