Impact of Headgroup Asymmetry and Protonation State on the

Article Cite This: Langmuir 2018, 34, 4360−4373

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Impact of Headgroup Asymmetry and Protonation State on the Aggregation Behavior of a New Type of Glycerol Diether Bolalipid Simon Drescher,*,† Christian Otto,‡ Sindy Müller,† Vasil M. Garamus,§ Christopher J. Garvey,∥ Susanne Grünert,‡ Anke Lischka,‡ Annette Meister,⊥ Alfred Blume,# and Bodo Dobner*,‡ †

Institute of PharmacyBiophysical Pharmacy and ‡Institute of PharmacyBiochemical Pharmacy, Martin Luther University (MLU) Halle-Wittenberg, Wolfgang-Langenbeck-Strasse 4, 06120 Halle (Saale), Germany § Helmholtz-Zentrum Geesthacht: Centre for Materials and Coastal Research (HZG), Max-Planck-Strasse 1, 21502 Geesthacht, Germany ∥ Australian Nuclear Science and Technology Organisation (ANSTO), Kirrawee DC, NSW Australia ⊥ Institute of Biochemistry and Biotechnology, MLU Halle-Wittenberg, Kurt-Mothes-Strasse 3, 06120 Halle (Saale), Germany # Institute of Chemistry, MLU Halle-Wittenberg, von-Danckelmann-Platz 4, 06120 Halle (Saale), Germany S Supporting Information *

ABSTRACT: In the present work, we describe the synthesis and the temperature-dependent aggregation behavior of a new class of asymmetrical glycerol diether bolalipids. These bolalipids are composed of a membrane-spanning alkyl chain with 32 carbon atoms (C32) in the sn-3 position, a methylbranched C16 alkyl chain in the sn-2 position, and a zwitterionic phosphocholine headgroup in the sn-1 position of a glycerol moiety. The long C32 alkyl chain is terminated either by a second phosphocholine (PC-Gly(2C16Me)C32PC) or by a phosphodimethylethanolamine headgroup (PCGly(2C16Me)C32-Me2PE). The temperature- and pHdependent aggregation behavior of both lipids was studied using differential scanning calorimetry (DSC), Fourier transform infrared (FTIR) spectroscopy, small-angle X-ray scattering (SAXS), and small-angle neutron scattering (SANS) experiments. The morphology of the formed aggregates in an aqueous suspension was visualized by transmission electron microscopy (TEM). We show that PC-Gly(2C16Me)C32-PC and PCGly(2C16Me)C32-Me2PE at pH 5 self-assemble into large lamellar aggregates and large lipid vesicles. Within these structures, the bolalipid molecules are probably assembled in a monolayer with fully interdigitated chains. The lipid molecules seem to be tilted with respect to the layer normal to ensure a dense packing of the alkyl chains. A temperature increase leads to a transition from a lamellar gel phase to the liquid-crystalline phase at about 28−30 °C for both bolalipids. The lamellar aggregates of PCGly(2C16Me)C32-Me2PE started to transform into nanofibers when the pH value of the suspension was increased to above 11. At pH 12, these nanofibers were the dominant aggregates.

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INTRODUCTION Bipolar amphiphiles, which are also called bolaamphiphiles or bolalipids, are composed of two hydrophilic headgroups connected to both ends of one or two hydrophobic alkyl spacers.1 This special class of phospholipids originates in membrane lipids of certain types of archaea, where they are responsible for the survival of these organisms under inhospitable conditions, such as high temperatures and low pH values.2−6 The reasons for this outstanding stability can be found in the chemical structure of archaea-type membrane lipids. Whereas the membrane lipids of eukaryotes and bacteria contain fatty acid residues in the sn-1,2 configuration of the glycerol backbone, the membrane lipids of archaea possess ether-bounded fatty alcohols in the inverse sn-2,3 configuration. Therefore, the term tetraether lipid (TEL) is also used for this class of lipids. Furthermore, the fatty alcohol residues can © 2018 American Chemical Society

contain a variable number of cyclopentane rings as well as methyl branches in an isoprenoid substitution pattern.7,8 Archaeal membrane lipids can generally be divided into monopolar archaeol- and bipolar caldarchaeol-type lipids. The latter ones, which are found in some methanogens and thermoacidophiles, consist of two membrane-spanning (transmembrane) alkyl chains of 32 carbon atoms forming a macrocycle.9 Because of the stability of archaeal membranes against high temperatures, strong acidic milieu, and esterhydrolyzing enzymes, the use of archaeal membrane lipids in biotechnology, material sciences, and pharmacy is very attractive and promising.10−18 Liposomes composed of bipolar Received: February 15, 2018 Revised: March 19, 2018 Published: March 20, 2018 4360

DOI: 10.1021/acs.langmuir.8b00527 Langmuir 2018, 34, 4360−4373

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Table 1. Schematic Structures of Symmetrical (Type A and B) and Asymmetrical (Type C, D, and E) Bolalipids and Their Preferred Aggregate Structures in Aqueous Suspensions

lipids (called archaeosomes19) are especially suitable as longcirculating vehicles for drug-delivery purposes.16,20−24 However, the main problem for application is to obtain sufficient amounts of lipid material from natural sources. The isolation of archaeal lipids is very expensive and often leads to mixtures of lipids including different alkyl chain patterns. In addition, the total synthesis of naturally occurring archaeal membrane lipids, published by Kakinuma and co-workers,25−30 is highly elaborate and therefore not applicable to production on a large scale. A more practical way is to imitate the properties of natural archaeal membrane lipids by preparing simpler model compounds. Several groups have used this strategy of simplification,17,31−35 leading to model lipids that are mostly characterized by one membrane-spanning (C32) alkyl chain bound to two glycerol moieties and two shorter (C16) alkyl

chains, in many cases a phytanyl chain, attached to each of the glycerol backbones. We have shown recently that only two or four methyl branches in the “sensitive” 10 and 10′ position of the membrane-spanning C32 alkyl chain and/or in the 10 position of the C16 alkyl chain (Table 1, type A) are sufficient to mimic the behavior of natural archaeal membrane lipids with respect to liposome formation and phase-transition temperatures. This is independent of the absolute configuration of the methyl groups.36,37 An even more simplified archaeal model lipid is the singlechain dotriacontane-1,32-diylbis[2-(trimethylammonio)ethylphosphate] (PC-C32-PC; Table 1, type B) consisting of two phosphocholine (PC) headgroups connected by a long, unmodified alkyl chain of 32 carbon atoms without a glycerol moiety.38 This bolalipid shows unexpected aggregation 4361


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Figure 1. Schematic representation (top) and chemical structure (bottom) of asymmetrical glycerol diether lipids.

behavior. It self-assembles in water into long, flexible nanofibers, leading to the formation of a clear and transparent hydrogel.38 These nanofibers, which have a thickness of about 6 nm corresponding to the length of a PC-C32-PC molecule, reversibly transform into small micelles upon heating, and the gel character is lost. This gel/sol transformation is accompanied by a cooperative endothermic transition at Tm = 48 °C.38 Alterations in the alkyl chain lengths39 as well as modifications in the headgroup structures of the single-chain bolalipids lead to changes in the transition temperature and/or the occurrence of an additional fiber/fiber transition. Moreover, other aggregate structures, such as sheetlike structures or square lamellae, were found.40,41 The insertion of a phosphodimethylethanolamine (Me2PE) headgroup, where one methyl group of the PC headgroup is substituted by a proton, leads to pHsensitive aggregation behavior of these single-chain bolalipids.42,43 Besides symmetrical single-chain bolalipids, with PC-C32-PC as the lead structure, and symmetrical TELs, we recently focused on the synthesis and characterization of asymmetrical bolalipids. Here, the term “asymmetry” is used to illustrate that the two different headgroups bound to the membrane-spanning alkyl chain have different sizes, polarities, and/or (de)protonation abilities. The general question that we aim to answer is, How does the chemical structure of the bolalipid influences its aggregation and phase behavior? The structural asymmetry could lead to different orientations of the bolalipid molecules within the aggregate and hence to different aggregate structures. A parallel orientation, where headgroups of the same type are arranged on one side of the aggregate, could force the self-assembly of rods,50 nanotubes,50−52 nanotapes,53 or liposomes with different curvature of the inner and outer leaflet. By contrast, an antiparallel (interdigitated) orientation of the bolalipid molecules could trigger the formation of large sheetlike structures, where the thickness of the lipid monolayer is basically determined by the length of the lipid molecule.51,54 Within the last few years, we have investigated two different types of asymmetrical bolalipids: glycerol diether bolalipids and single-chain bolalipids. For example, the single-chain DMAPPC-C32-OH, which bears a large pH-sensitive 2(dimethylaminopropyl)-PC (DMAPPC) headgroup and a small hydroxy group at the ends of a C32 alkyl chain (Table 1, type C), self-assembles into small elongated micelles at pH 5.44 At pH 10, this bolalipid forms lamellar aggregates with an interdigitated orientation of the DMAPPC-C32-OH molecules.44 The same chain interdigitation was found or postulated for other single-chain bolalipids that are composed of one PC and one hydroxy moiety attached to an unmodified alkyl chain of different lengths (PC-C32-OH46 or PC-C22-OH47) or a phenyl-modified alkyl chain (PC-C17pPhC17-OH45). The glycerol diether bolalipids are characterized by a glycerol

backbone that carries a long C32 alkyl chain in the sn-3 position, a short C16 alkyl chain in the sn-2 position, and a zwitterionic PC headgroup in the sn-1 position (Table 1, type D).48 The membrane-spanning C32 chain further contains a small hydroxy group at the other end, which makes the lipid bipolar (PC-Gly(2C16)C32-OH and PC-Gly(2C16Me)C32OH). We could show that this cone-shaped glycerol diether bolalipid self-assembles into large sheetlike aggregates, whereas closed lipid vesicles (as found for TELs) or nanofibers (as shown for symmetrical single-chain bolalipids) were not observed. Again, the glycerol diether bolalipid molecules are arranged in a fully interdigitated fashion.48 Recently, we have investigated a glycerol diether bolalipid of type D bearing a protonatable Me2PE instead of a PC headgroup (Me2PEGly(2C16)C32-OH).49 This bolalipid self-assembles into large sheetlike aggregates at acidic and neutral pH values, respectively. At pH 10, where the Me2PE headgroup is only partially protonated, small lipid disks with a diameter of 50− 100 nm were additionally found. Again, closed lipid vesicles were not observed at all pH values investigated. With this study, we intend to close the gap between the symmetrical TELs (type A) and the highly asymmetrical glycerol diether bolalipids (type D). We therefore synthesized two model compounds (bolalipids of type E) that are based on the type D bolalipid mentioned above,48 where the free hydroxy group is replaced by a PC (PC-Gly(2C16Me)C32PC) or a Me2PE (PC-Gly(2C16Me)C32-Me2PE) headgroup (Figure 1). With the introduction of a second phosphate headgroup, the overall asymmetry of both bolalipids is decreased to some extent. Both novel glycerol diether bolalipids further contain a racemic methyl group in the 10 position of the short C16 alkyl chain because previous investigations revealed that without this perturbation a lamellar phase with very high crystallinity up to a temperature of 34 °C is formed.48 In our study, we want to address the question of whether the second large phosphate headgroup has an influence on the aggregation behavior in aqueous suspensions. Moreover, with the insertion of the Me2PE headgroup the question arose as to whether the aggregation behavior of glycerol diether bolalipids can be tuned by changes in the pH value. The bolalipid systems were investigated using transmission electron microscopy (TEM), differential scanning calorimetry (DSC), Fourier transform infrared (FTIR) spectroscopy, small-angle X-ray scattering (SAXS), and small-angle neutron scattering (SANS).

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EXPERIMENTAL SECTION

Syntheses. The synthesis procedures and the analytical data of the two new glycerol diether lipids (PC-Gly(2C16Me)C32-PC and PCGly(2C16Me)C32-Me2PE) are described in detail in the Supporting Information (SI). 4362


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Langmuir Scheme 1. Synthesis Pathway for the Preparation of Glycerol Diether Lipids PC-Gly(2C16Me)C32-PC and PCGly(2C16Me)C32-Me2PE with Two Phosphate-Containing Headgroups

2048 pixels × 2048 pixels) was used. The sample-to-detector distance was 108.3 cm, and the accessible q range was from 0.1 to 2.5 nm−1. Samples were used to fill glass capillaries of 2 mm diameter. The temperature of the capillaries (T = 10, 45, or 50 °C) was controlled using a temperature control unit (ΔT = 0.1 K). The raw scattering data were corrected for the background from the solvent measured in a capillary with the same diameter and then converted to absolute units using the scattering of pure water measured in a capillary with the same thickness and at the same temperatures compared to those of the sample (program SuperSAXS, Prof. C. L. P. Oliveira and Prof. J. S. Pedersen). Small-Angle Neutron Scattering (SANS). In a manner similar to the procedure described previously,56 SANS measurements were made on fixed-wavelength reactor-based pinhole SANS instrument QUOKKA,57 which is found on cold neutron guide CG1 at the Australian Nuclear Science and Technology Organisation’s (ANSTO) research reactor OPAL (Lucas Heights, Australia). A sample of PCGly(2C16Me)C32-Me2PE (c = 1 mg mL−1 in D2O; a pD value of ≈12 was adjusted using NaOD solution) was placed in cylindrical quartz cuvettes with a path length of 2 mm. SANS spectra were recorded on a position-sensitive detector consisting of 192 pixels × 192 pixels (5 × 5 mm2) at three sample-to-detector distances (2, 14, and 20 m) using neutrons of wavelengths λ = 5.0 Å for the first two camera lengths and λ = 8.1 Å (Δλ/λ = 0.1) with lens optics for the longest camera length. After correcting the raw data for the sensitivity of each detector pixel, masking the beam stop shadow, and subtracting backgrounds consisting of the sample cells and the dark current, we normalized the scattering intensity to the empty beam intensity. The isotropic scattering patterns were then radially averaged using the measurement geometry. The radially averaged scattering data from each sample-to-detector distance were joined to produce a continuous q range of 0.0009 to 0.4 Å−1, where q = 4π × sin(2θ)/λ and θ is the scattering angle. Data reduction was achieved using macros specifically modified for Quokka from those macros written for the NIST Center for Neutron Research (Gaithersburg, MD, USA) SANS instruments58 and the IgorPro program (version 6.34, WaveMetrics Inc., 2013).

Methods. Sample Preparation. An appropriate amount of the bolalipid was suspended in H2O (Milli-Q Millipore water), D2O, acetate buffer (DSC: c = 10 mM, pH 5.0. FTIR: c = 300 mM, pH 4.7), phosphate buffer (c = 10 mM, pH 7.6), or carbonate buffer (c = 10 mM, pH 10.0). For DSC measurements using buffer solutions at pH 11 and 12, carbonate buffer was used to dissolve the bolalipid, and the final pH was adjusted with conc NaOH solution. Homogeneous suspensions were obtained by heating and vortex mixing. Differential Scanning Calorimetry (DSC). DSC measurements were performed using a MicroCal VP-DSC differential scanning calorimeter (MicroCal Inc. Northampton, MA, USA). Before the measurements, the sample suspension (c = 1 mg mL−1) and the water or buffer reference were degassed under vacuum while stirring. A heating rate of 60 K h−1 was used, and the measurements were performed in the temperature interval from 5 to 75/95 °C. To check the reproducibility, three consecutive scans were recorded for each sample. The water/water or buffer/buffer baseline was subtracted from the thermogram of the sample, and the DSC scans were evaluated using MicroCal Origin 8.0 software. Fourier Transform Infrared Spectroscopy (FTIR). Infrared spectra were collected on a Bruker Vector 22 Fourier transform spectrometer (Bruker Optics GmbH, Karlsruhe, Germany) with a DTGS detector operating at 2 cm−1 resolution. The sample suspension (c = 100 mg mL−1 in H2O for PC-Gly(2C16Me)C32-PC or in acetate buffer at pH 4.7, 300 mM for PC-Gly(2C16Me)C32-Me2PE) was placed between two CaF2 windows, separated by a 6 μm spacer. IR spectra were recorded in steps of 1 K in the temperature range from 9 to 69 °C. After an equilibration time of 8 min, 128 scans were accumulated. The corresponding spectra of the solvent (H2O or acetate buffer) were subtracted from the sample spectra using the OPUS software supplied by Bruker. For data presentation, Origin 2016 software was used. Transmission Electron Microscopy (TEM). The samples were prepared by spreading 5 μL of the bolalipid suspension (c = 0.05 mg mL−1) onto a copper grid coated with a Formvar film. After 1 min, excess liquid was blotted off with filter paper and 5 μL of a 1% aqueous uranyl acetate solution was placed onto the grid and drained off after 1 min. Specimens prepared below ambient temperature (T ≈ 7 °C) were dried for at least 1 day at this temperature and kept in a desiccator at room temperature. Specimens, which were prepared in a modified drying oven above ambient temperature, were further dried for 1 h at the appropriate temperature and finally kept in a desiccator at room temperature. All specimens were examined with a Zeiss EM 900 transmission electron microscope (Carl Zeiss Microscopy GmbH, Jena, Germany). Small-Angle X-ray Scattering (SAXS). According to the procedure described previously,55 SAXS measurements were performed with a laboratory SAXS instrument (Nanostar, Bruker AXS GmbH, Karlsruhe, Germany). The instrument includes an IμS microfocus Xray source with a power of 30 W using the wavelength of the Cu Kα line. As the detector, a VÅNTEC-2000 detector (14 × 14 cm2 and

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RESULTS Synthesis of Bolalipids. The preparation of our novel glycerol diether bolalipids bearing two phosphor-containing headgroups is mainly based on the synthesis procedures published previously.48 In brief, the synthesis starts with the alkylation of commercially available (S)-1,2-O-isopropylidenglycerol using benzyl-protected 32-bromodotriacontan-1-ol. After cleavage of the isopropylidene protection group and selective tritylation of the sn-1 position of the glycerol, the second alkyl chain was introduced into the sn-2 position. After deprotection and insertion of the PC headgroup in the sn-1 position of the glycerol, the remaining benzyl blocking group 4363


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endothermic and cooperative transition at Tm = 27.9 °C and a broad and less cooperative transition between 31 and 41 °C (Figure 2A, red line). In the cooling curve (Figure 2A, blue

was removed by hydrogenation.48 The resulting glycerol diether bolalipid bearing a PC headgroup on one side (the glycerol side) and a small hydroxy moiety on the other side of the molecule (see Scheme 1, top) acts now as starting material for the final preparation step. The introduction of the second headgroup was carried out by the same procedure as for the first headgroup, a method first described by Eibl and coworkers59 using 2-bromoethylphosphoric acid dichloride60 as the phosphorylating reagent. For the quarternization step, a solvent mixture of CHCl3, acetonitrile, and an ethanolic solution of trimethylamine (PC-Gly(2C16Me)C32-PC) and dimethylamine (PC-Gly(2C16Me)C32-Me2PE), respectively, was used (Scheme 1). Both asymmetrical bolalipids, [3-O-(32{2-[(trimethylammonio)ethyl]oxyphosphoryloxy}dotriacontyl)-2-O-[(10RS)-10-methylhexacedyl]-sn-glycer-1-yl]-2[(trimethylammonio)ethylphosphate] (PC-Gly(2C16Me)C32-PC) and [3-O-(32-{2-[(dimethylammonio)ethyl]oxyphosphoryloxy}dotriacontyl)-2-O-[(10RS)-10-methylhexacedyl]-sn-glycer-1-yl]-2-[(trimethylammonio)ethylphosphate] (PC-Gly(2C16Me)C32-Me2PE), were obtained in 52−61% yield after purification via column chromatography. As shown here, the free hydroxy moiety of the precursor lipid (Scheme 1, top) offers the possibility for subsequent modifications of asymmetrical bolalipids. Besides the introduction of a second (pH-sensitive) headgroup, also fluorescent or EPR spin probes, sugar moieties as well as small peptide fragments can be attached at this position. Temperature-Dependent Aggregation Behavior in an Aqueous Suspension. To clarify the impact of the second headgroup on the temperature-dependent aggregation behavior of our new glycerol diether bolalipids in an aqueous suspension, we performed several physicochemical investigations. In recent years, it has proven an advantage to start with differential scanning calorimetry (DSC) to detect possible endo- and/or exothermic transitions between different aggregation states. If such transitions are observed, then imaging techniques such as transmission electron microscopy (TEM) of negative stained samples or vitrified specimens can be used to visualize the shape of aggregates below and above this transition. In addition, Fourier transform infrared (FTIR) spectroscopy and different scattering techniques such as X-ray scattering in the small-angle (SAXS) or wide-angle (WAXS) range and small-angle neutron scattering (SANS) can be used to get more information about the nature of this transition and to identify different phase states or dimensions of aggregates below and above the transitions. DSC Measurements. Both bolalipids, PC-Gly(2C16Me)C32-PC in water and PC-Gly(2C16Me)C32-Me2PE in acetate buffer at pH 5, are difficult to disperse. An opalescent suspension (c = 1 mg mL−1) can be formed only after several cycles of heating to 90 °C and vortex mixing. This observation is a first indication of the formation of larger aggregate structures, such as multilamellar vesicles and sheetlike aggregates, since the self-assembly into very small structures (micelles) or nanofibers should result in a virtually clear and transparent suspension. Moreover, attempts to extrude the suspension at room temperature through a polycarbonate membrane of 100 nm pore size failed. Only particles with large hydrodynamic radii and a high polydispersity index (PdI), measured by means of dynamic light scattering (DLS), were found (data not shown). The DSC heating curve of an aqueous suspension of PCGly(2C16Me)C32-PC (c = 1 mg mL−1 in water) shows an

Figure 2. (A) DSC heating (red line) and cooling (blue line) curves of an aqueous suspension (c = 1 mg mL−1) of PC-Gly(2C16Me)C32PC. The heating rate was 60 K h−1. (B) DSC heating curves of aqueous suspensions (c = 1 mg mL−1) of PC-Gly(2C16Me)C32Me2PE at different pH values: acetate buffer at pH 5.0 (red line); phosphate buffer at pH 7.6 (green line); and carbonate buffer at pH 10 (cyan line), pH 11 (blue line), and pH 12 (dark blue line). The heating rate was 60 K h−1. The curves are shifted vertically for clarity.

line), the main transition appears at Tm = 25.6 °C, i.e., only small hysteresis of ΔT = 2.3 K occurs. The DSC heating curve of a suspension of PC-Gly(2C16Me)C32-Me2PE (c = 1 mg mL−1) in acetate buffer at pH 5 shows an endothermic transition at Tm = 30.5 °C, including a small low-temperature shoulder at 26.5 °C. In addition, a very broad transition between 33 and 43 °C can be observed (Figure 2B, red line). The increase in Tm of about 3 K, when PC-Gly(2C16Me)C32Me2PE is compared to PC-Gly(2C16Me)C32-PC, can be attributed to the stabilizing effect of additional hydrogen bonds formed between the protonated Me2PE headgroups. The corresponding cooling curve reveals virtually the same peak pattern, including a very narrow cooperative transition at Tm = 27.0 °C with a small hysteresis of ΔT = 3.5 K (Figure S1, Supporting Information). Furthermore, a broad peak between 42 and 30 °C and a small peak at T = 23.7 °C are observed. When the pH is increased, i.e., the Me2PE headgroups become more and more negatively charged, the transition 4364


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Langmuir temperatures decrease gradually. In phosphate buffer at pH 7.6 (Figure 2B, green line), the sequence of the DSC heating curve is similar to the curve at pH 5.0 with a small peak at T = 26.3 °C, Tm at 29.3 °C, and a plateau until a temperature of 43 °C. In carbonate buffer at pH 10.0, the DSC curve shows only one transition at Tm = 25.7 °C (Figure 2B, cyan line) without the additional plateau or broad transition at higher temperatures. If the pH value is further increased by adding small amounts of NaOH, then the transition temperature stays virtually the same: Tm = 25.4 °C (pH 11, Figure 2B, blue line) and Tm = 25.0 °C (pH 12, Figure 2B, dark-blue line). The corresponding cooling curves at pH 7.6, 10.0, 11.0, and 12.0 reveal a similar peak pattern (Figure S1, Supporting Information). FTIR Measurements. To get more information on the nature of the thermotropic transitions observed in the DSC scan, temperature-dependent FTIR experiments of an aqueous suspension of PC-Gly(2C16Me)C32-PC (c = 100 mg mL−1 in water) and PC-Gly(2C16Me)C32-Me2PE (c = 100 mg mL−1 in acetate buffer, pH 4.7, 300 mM) were performed. The wavenumbers of both methylene stretching vibrational bands, the symmetric (νs(CH2)) and the antisymmetric (νas(CH2)) bands, provide information about the conformational order of the alkyl chain within the lipid aggregates.61,62 In addition, the position of the methylene scissoring vibrational band (δ(CH2)) was also analyzed, which offers information about the alkyl chain packing mode, i.e., whether orthorhombic, hexagonal, triclinic, or other phases are formed.62 For PC-Gly(2C16Me)C32-PC, the frequencies of the bands at low temperatures are located at 2849.6 cm−1 for νs(CH2) (red symbols in Figure 3A, top) and 2919.2 cm−1 for νas(CH2) (Figure S2, Supporting Information). These values are comparable to the wavenumbers of the CH2 stretching vibrational bands of other asymmetrical but single-chained bolalipids, e.g., PC-C17pPhC17-OH45 (2849.6 and 2917.0 cm−1), and also symmetrical single-chained PC-C32-PC38 (νs(CH2) 2849.7 cm−1), indicating that the alkyl chains of PC-Gly(2C16Me)C32-PC are in an all-trans conformation and are well ordered. However, these frequencies are slightly higher than those observed for the glycerol diether bolalipid bearing a hydroxy group instead of a second PC headgroup (νs(CH2) 2848.7 cm−1, νas(CH2) 2916.7 cm−1).48 This is probably caused by the second PC headgroup, which prevents a denser packing of the alkyl chains. With increasing temperature, the wavenumbers of both bands stay nearly constant up to Tm as observed in DSC and further jump to 2851.4 and 2920.8 cm−1 (at T = 27.0 °C) for the νs(CH2) and νas(CH2) bands, respectively. This increase in wavenumbers is caused by an increasing number of gauche conformers within the alkyl chains; i.e., PC-Gly(2C16Me)C32-PC is in the fluid phase at this temperature (above Tm). With further heating, both frequency values increase. Within the temperature range of the second, broad transition observed in DSC (T = 31−41 °C), a sigmoidal increase in frequency is seen. The corresponding cooling curves (blue symbols in Figure 3A, top, and Figure S2) follow the same pattern except for a small hysteresis of ΔT = 2 K, which was also observed in the DSC measurements. To get more information about the chain-packing geometry, the position of the δ(CH2) band was analyzed (Figure 3A, bottom). At low temperature, one observes only a narrow band located at 1471.6 cm−1, indicating triclinic chain packing. Other packing modes would lead to either lower wavenumbers of the δ(CH2) band between 1467 and 1469 cm−1 if the chains are arranged in a hexagonal lattice or to a splitting of the δ(CH2)

Figure 3. Wavenumber (heating, red; cooling, blue) of the symmetric methylene stretching vibrational band (upper parts) and the methylene scissoring vibrational band (lower parts) as a function of temperature for suspensions of (A) PC-Gly(2C16Me)C32-PC (c = 100 mg mL−1 in water) and (B) PC-Gly(2C16Me)C32-Me2PE (c = 100 mg mL−1 in acetate buffer, pH 4.7, 300 mM).

band (1465 and 1473 cm−1) if orthorhombic geometry is preferred.61−63 At the main transition, a pronounced jump to 1468.1 cm−1 (at T = 27.0 °C) is seen, accompanied by an intensity loss and a broadening of the band. This jump indicates a change in the molecular packing at Tm. Further heating of the sample leads to an almost linear decrease in the δ(CH2) band to 1466.7 cm−1 at T = 58.8 °C. Comparable behavior was observed for the symmetrical single-chain PC-C32-PC38 (type B) and the asymmetrical single-chain PC-C17pPhC17-OH (type C).45 By contrast, another type C bolalipid that bears a larger difference in the headgroup size (DMAPPC-C32-OH) shows an orthorhombic chain-packing pattern at low temperatures,44 whereas the precursor lipid of PC-Gly(2C16Me)C32PC, the corresponding type D bolalipid (PC-Gly(2C16Me)C32-OH, lipid II in ref 48), reveals a hexagonal chain-packing mode over the whole temperature range investigated.48 The temperature-dependent FTIR spectroscopic data of PCGly(2C16Me)C32-Me2PE (c = 100 mg mL−1 in acetate buffer, pH 4.7, 300 mM) are shown in Figure 3B and Figure S3. In principle, the temperature dependence of both methylene stretching vibrational bands (νas(CH2), see Figure S3; νs(CH2), see Figure 3B, top) and the methylene scissoring vibrational band (Figure 3B, bottom) exhibits the same behavior compared to that of PC-Gly(2C16Me)C32-PC. 4365


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Langmuir Transmission Electron Microscopy (TEM). To visualize the aggregate shapes formed in an aqueous suspension of PCGly(2C16Me)C32-PC (c = 0.05 mg mL−1) at different temperatures, i.e., below and above Tm, negatively stained samples were prepared for TEM (Figure 4). Below Tm, PC-

PC-Gly(2C16Me)C32-Me2PE self-assembles in acetate buffer at pH 5 into large sheetlike aggregates of several micrometers in size (Figure 5A). These large lamellae, which are sometimes folded due to the drying process during EM sample preparation, are stacked onto each other (Figure 5B). The type of aggregates is the same at neutral pH values: in phosphate buffer at pH 7.6, similar large lamellar aggregates are found (Figure 5C). However, the existence of large, closed lipid vesicles cannot be excluded since in some cases a characteristic folding of collapsed vesicles is found in TEM images. When the pH value of the sample suspension is increased (Figure 6), a change in the aggregation structure is seen. In

Figure 4. TEM images of an aqueous suspension of PC-Gly(2C16Me)C32-PC (c = 0.05 mg mL−1). The samples were prepared at about 7 °C (A and B), at ambient temperature (C), and at about 50 °C (D) and were stained with uranyl acetate before drying.

Gly(2C16Me)C32-PC self-assembles into large lamellar sheets of several micrometers in size (Figure 4A), which appear folded due to the drying process during sample preparation. In addition, large collapsed vesicles can also be observed (Figure 4B). With increasing temperature, the shape of the aggregates remains nearly unchanged: At ambient temperature, i.e., in the temperatures range of the first transition observed in DSC, large vesicles and nearly rounded sheets can be seen (Figure 4C). Above the second DSC transition temperature (at T ≈ 50 °C), again large lamellar aggregates and collapsed vesicles are found (Figure 4D).

Figure 6. TEM images at different magnifications of an aqueous suspension of PC-Gly(2C16Me)C32-Me2PE (c = 0.05 mg mL−1) in carbonate buffer at different pH values: pH 10.0 (A and B) and pH 12.0 (C and D). The samples were prepared at T ≈ 7 °C (below Tm) and were stained with uranyl acetate before drying. In A and B, the white arrows point to sheetlike aggregates, whereas the white arrowheads point to vesicular structures. In D, the black arrows point to single fibers whereas the black arrowheads point to bundles of fibers (fiber strands).

Figure 5. TEM images of an aqueous suspension of PC-Gly(2C16Me)C32-Me2PE (c = 0.05 mg mL−1) at different pH values: in acetate buffer at pH 5.0 (A and B) and in phosphate buffer at pH 7.6 (C). The samples were prepared at ambient temperature (T ≈ 22 °C) and were stained with uranyl acetate before drying. 4366


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Langmuir carbonate buffer at pH 10.0 and at low temperature (7 °C), i.e., below Tm, PC-Gly(2C16Me)C32-Me2PE self-assembles into lamellae that are smaller compared to aggregates found at pH 5.0 and 7.6 (Figure 6A, white arrows). In addition, some collapsed vesicular structures (liposomes), which show a characteristic folding, are found, as indicated by the white arrowheads in Figure 6A,B. The lamellar structure of PCGly(2C16Me)C32-Me2PE aggregates is stable for at least 1 week at pH 10, which was proven by preparing EM samples from a PC-Gly(2C16Me)C32-Me2PE suspension in carbonate buffer after different times of storage at 4 °C (see Figure S4, Supporting Information). A drastic change in the aggregate structure can be found at pH 12. Here, self-assembly into long nanofibers is observed (Figure 6C,D). These fibers appear either as single fiber strands with a thickness of about 5 to 6 nm (black arrows in Figure 6D) or as a bundle of two or three fibers (black arrowheads in Figure 6D). The thickness of a single fiber corresponds to the length of a PC-Gly(2C16Me)C32-Me2PE molecule. Additionally, also some very small lamellar aggregates can be found in the sample at pH 12 (see Figure S5, Supporting Information). The change from lamella to fibers by increasing the pH value of the suspension is also macroscopically visible. The opalescent suspension turned into a clear, transparent suspension when the pH was increased from 10 to 12. As the transformation from lamellar to fiber aggregates occurs between pH 10 and 12, we prepared additional samples for EM investigations using carbonate-buffered bolalipid suspensions at four different pH values between 9.6 and 11.1. The EM images of these negatively stained samples are shown in Figure 7. Starting at a pH value of 9.6, PC-Gly(2C16Me)C32-Me2PE aggregates have a sheetlike structure with sizes of between 200 and 500 nm (Figure 7A,B). Closed vesicular structures (liposomes) are only rarely seen. A closer inspection reveals a relative smooth surface of these lamellae (Figure 7B) without any structuring. At pH 10, sheetlike aggregates and some collapsed liposomes without a distinct surface structure are found again (Figure 7C,D). The structures are similar to those shown before in Figure 6A,B. At pH 10.5, the size of the lamellar aggregates decreases to 150−250 nm (Figure 7E). At this pH, 50% of the Me2PE headgroups are now charged, assuming a pKa of 10.5. The negatively charged bolalipid molecules could possibly accumulate at the rim of the aggregates, stabilizing the sheets against fusion to larger aggregates. The TEM images show that the surfaces of these lamellae seem to have a regular structure, which appears either as lamellar fine structure (see black arrows in Figure 7F) or as a kind of reticulated structure of crossed fibers (see asterisks in Figure 7F,H). An increase in pH to 11.1 leads to the formation of fibrous aggregates growing from the small lamellar sheets (Figure 7G,H). These fibers have a thickness of about 7−24 nm, and it seems that they are composed of either single fibers or associated fiber strands, which appear more like a tape. That means that the lamellae → fiber transformation appears at about pH 11, i.e., in the region of the pKa value. In this context, the question often arises as to whether the uranyl counterion could have an effect on the aggregation behavior of the bolalipid molecules. The problem of creating artifacts during the staining and drying procedure is well known. However, we have found in our previous experiments using other bolalipids of similar chemical structure that uranyl acetate staining does not introduce artifacts into these systems,

Figure 7. TEM images at different magnifications of an aqueous suspension of PC-Gly(2C16Me)C32-Me2PE (c = 0.05 mg mL−1) in carbonate buffer at different pH values: pH 9.6 (A and B), pH 10.0 (C and D), pH 10.5 (E and F), and pH 11.1 (G and H). The black arrows point to lamellar fine structure in the small sheets, whereas the asterisks indicate a reticulated structure. The samples were prepared at T ≈ 7 °C (below Tm) and were stained with uranyl acetate before drying.

as the corresponding cryo-TEM images showed comparable aggregate structures.64 Small-Angle X-ray Scattering (SAXS). To gain more information about the structural organization of the bolalipid molecules within the layered aggregates and to investigate structural changes upon heating, aqueous suspensions of PCGly(2C16Me)C32-PC and PC-Gly(2C16Me)C32-Me2PE (acetate buffer at pH 5) were investigated using small-angle X-ray diffraction. The results are shown in Figure 8. 4367


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at q1 = 0.936 nm−1 and the third peak at q2 = 1.853 nm−1 correspond to a lamellar repeat distance of d = 6.71 nm. With increasing temperature, both peaks are shifted to lower q values, again indicating an increase in the repeat distance (Figure 8B, open green circles). At T = 45 °C, equidistant reflections at q1 = 0.764 nm−1 and q2 = 1.507 nm−1 can be found, indicating a repeat distance of d = 8.22 nm. At both temperatures, a third peak can be seen in the diffractograms, which does not change its position considerably upon heating: q = 1.206 and 1.180 nm−1 at T = 10 and 45 °C, respectively. In a similar manner to PC-Gly(2C16Me)C32-PC, this additional reflection could correspond to second lamellar repeat distances of d = 5.21 and 5.32 nm, respectively. Small-Angle Neutron Scattering (SANS). To gain further insight into the structure and dimensions of the nanofibers built of PC-Gly(2C16Me)C32-Me2PE at pH 12 at a temperature below Tm and to characterize the aggregates formed above Tm, small-angle neutron scattering (SANS) experiments were conducted. The results are shown in Figure 9 and Figure S6.

Figure 8. SAXS diffractograms of aqueous suspensions of bolalipids (c = 40 mg mL−1) at different temperatures: (A) PC-Gly(2C16Me)C32PC in water at 10 °C (filled blue squares) and at 50 °C (open red circles) and (B) PC-Gly(2C16Me)C32-Me2PE in acetate buffer (pH 5) at 10 °C (filled black squares) and at 45 °C (open green circles).

The SAXS curve of an aqueous suspension of PCGly(2C16Me)C32-PC (c = 40 mg mL−1) at T = 10 °C, i.e., below Tm, shows several reflections (Figure 8A, filled blue squares). The two most pronounced reflections at q1 = 0.881 nm−1 and q2 = 1.764 nm−1 are equidistant and could correspond to a lamellar repeat distance (membrane thickness plus interlamellar water layer thickness) of d = 7.13 nm. A third, less intense reflection at q = 1.11 nm−1 could correspond to a second set of equidistant peaks indicating a second lamellar repeat distance of d = 5.66 nm. Heating the PC-Gly(2C16Me)C32-PC sample to above Tm leads to a change in the SAXS diffraction pattern (Figure 8A, red open circles). Now, three reflections at q = 0.813, 1.133, and 1.636 nm−1 are visible. The reflections are less intense compared to the diffractogram at 10 °C. The first and third peaks are again equidistant and correspond to a lamellar repeat distance of d = 7.72 nm, indicating an increase in the repeat distance (layer thickness) with increasing temperature. The second reflection at q = 1.133 nm−1 could again correspond to a second lamellar repeat distance of d = 5.55 nm. The Me2PE analog of PC-Gly(2C16Me)C32-PC reveals quite similar behavior. The SAXS curve of PC-Gly(2C16Me)C32-Me2PE (c = 40 mg mL−1 in acetate buffer at pH 5) at T = 10 °C, i.e., below Tm, depicts three reflections of nearly the same intensity (Figure 8B, filled black squares). The first peak

Figure 9. SANS data of a suspension of PC-Gly(2C16Me)C32Me2PE (c = 1 mg mL−1 in D2O at pD ≈ 12; scattered data) at T = 10 °C (A) and at T = 40 °C (B) with IFT analysis (solid black lines).

Since we did not observe nearly constant scattering intensities in the log−log plot in the Guinier region at the lowest q values, the size of the studied objects is larger than the observation window of present SANS experiments (approximately 600 nm), and we can determine only the flexibility, cross-sectional parameters, or interface of studied objects.65 4368


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Langmuir Data were first analyzed by the slope approach, i.e., the determination of parameter α in the approximation I(q) ∼ q−α, which can give a hint of the possible shape of the objects. The detected slopes α are schematically shown in Figure 9. At T = 10 °C (Figure 9A), a slope of −2 has been detected in the lowest q range, which changes to a slope of −(1 + δ) in the intermediate q range and further to −(4 − δ) for the largest scattering vectors. The δ value points to small deviations and is always positive. The change from −2 to −(1 + δ) is connected with the scattering behavior of flexible fibers, where at the lowest q values (scale above a few tens of nanometers) the system behaves like a Gaussian chain with a rigid-rod-like part on a length scale of up to several nanometers.66 The crossover between flexible and rigid properties at around q = 0.06 nm−1 can be connected with a persistent length of fibers of around 30 nm. At the highest scattering vector, the magnitude of the slope is 0.1 nm−1 were analyzed using the cross-sectional pair distance distribution function pCS(r) of rodlike aggregates, which is expressed as a sum of N b-splines evenly distributed over the interval [0, Dmax], with Dmax as the maximal size or cross-section of aggregates. The values of the coefficients are calculated numerically by a least-squares fitting of the IFT model curve to the experimental data. Within the q range analyzed, the experimental data and the IFT fitting curve coincide very well (Figure 9, solid black lines). From the maximal distance of the pCS(r) function, we get a first estimation of the cross-sectional diameter of approximately 9 nm (Figure S6). However, the shape of the pCS(r) function suggests that a core/shell structure (Figure S6) exists. The shape of pCS(r) can also possibly be caused by the fact that some of the fibers are sticking together, which was seen in the EM image (see black arrowheads in Figure 6D). After the determination of the pair distance distribution function, the mass per unit length of the aggregate (ML) and the diameter of the fiber were calculated. For PC-Gly(2C16Me)C32-Me2PE fibers, we obtained the following values: radius of gyration of the cross section, Rg,CS = 2.6 ± 0.1 nm, which corresponds to a homogeneous circular crosssectional diameter of the nanofibers of 7.3 ± 0.2 nm, and ML = (3.7 ± 0.1) × 10−13 g cm−1 giving an aggregation number Nagg of about 20 bolalipid molecules per 1 nm of fiber length. (See the Supporting Information for further explanations.) For the bolalipid suspension at T = 40 °C, the interval of q from 0.04 to 0.2 nm−1 was analyzed by the IFT method on the basis of a disklike approximation. The scattering data were described using the thickness pair distance distribution function pT(r). After the determination of pT(r), the thickness of the

bilayer and the mass per surface unit (MS) were calculated from pT(r) (details in Supporting Information). For PC-Gly(2C16Me)C32-Me2PE aggregates at T = 40 °C, we obtained the following values: radius of gyration of thickness, Rg,T = 2.3 ± 0.1 nm, which corresponds to the thickness of a homogeneous bilayer of 8.0 ± 0.2 nm, and MS = (3.5 ± 0.5) × 10−7 g cm−2 resulting in an area of 0.55 nm2 for one bolalipid molecule in the surface of the bilayer.

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DISCUSSION The self-assembly of long-chain bolalipids in aqueous suspension is mainly driven by hydrophobic interactions of the long alkyl chain. Packing frustrations resulting from different cross-sectional areas of the headgroup and the attached alkyl chain lead to the formation of various types of aggregates (see Table 1). Symmetrical single-chain bolalipids mostly form nanofibers or small spherical particles,41 whereas the asymmetrical counterparts mainly self-assemble into lamellar structures with an interdigitated orientation of the bolalipid molecules.44,45 The symmetrical double-chain diglycerol tetraether lipids (TELs) aggregate into closed lipid vesicles (liposomes),36,37 whereas the asymmetrical glycerol diether bolalipids bearing a pronounced asymmetry in the headgroup size form large sheetlike structures, again with an interdigitated arrangement of the bolalipid molecules and a high crystallinity of the alkyl chains.48 With the introduction of a second phosphate headgroup into a type D bolalipid (see Table 1), we synthesized a new class of asymmetrical glycerol diether bolalipids (type E). Both lipids, PC-Gly(2C16Me)C32-PC in water and PC-Gly(2C16Me)C32-Me2PE in acetate buffer at pH 5, show the formation of large lamellar and also vesicular aggregates. A temperature increase led to a transition from a lamellar gel phase to a liquidcrystalline phase at Tm between 28 and 30 °C for both bolalipids. The corresponding symmetrical TEL bearing the same methylation pattern, i.e., no methylation in the membrane-spanning alkyl chain and a racemic methyl group in the C16 chain, shows only one broad transition at Tm = 19.1 °C (lipid I from ref 37).37 By contrast, the asymmetrical glycerol diether bolalipid with a small hydroxy group at the membrane-spanning C32 alkyl chain (PC-Gly(2C16Me)C32OH; lipid II from ref 48)48 depicts a cooperative transition at Tm = 63.7 °C, where the transition from the LβI to the Lα phase takes place.48 That means that the replacement of the hydroxy group with a phosphate headgroup led to a drastic decrease in Tm of about 35 K. In contrast to PC-Gly(2C16Me)C32-PC, PC-Gly(2C16Me)C32-Me2PE is able to form intermolecular hydrogen bonds at low pH due to the presence of a hydrogen atom in the dimethylammonium group. In the case of single-chain bolalipid Me2PE-C32-Me2PE forming fibers, this additional stabilization leads to an increased fiber/micelle transition temperature of T = 69.5 °C42,68 at pH 5 compared to that for PC-C32-PC (T = 48 °C) having two PC headgroups.38 At higher pH values, the dissociation of the proton from the Me2PE group leads to a bolalipid with two negatively charged headgroups. Consequently, the fiber/micelle transition temperature of Me2PE-C32-Me2PE in basic milieu is drastically decreased to T = 12 °C (pH 11) and T = 6 °C (pH 12), respectively.43 On the basis of the results of these observations, we assumed an apparent pKa value for the protonated dimethylammonium group of about 10.0−10.5. At higher pH values, PC-Gly(2C16Me)C32-Me2PE shows a lamellae → 4369


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Langmuir fiber transformation. The increase in pH from 10 to 12 leads to an almost complete deprotonation of the Me2PE headgroups. This results in a negative charge of the one of the headgroups in the bolalipid and an increased repulsion between neighboring headgroups in the layer. As a consequence, lamellar structures become more and more unfavorable and fibers are energetically more stable. That means the lamellae → fiber transformation of PC-Gly(2C16Me)C32-Me2PE is a consequence of increased charge repulsion at high pH values. It is not a kinetic effect since the lamellar aggregates are stable during storage. A timedependent transformation in the opposite direction, i.e., a change from nanofibers to square lamellae, was previously observed for long, single-chain bolalipids composed of two Me2PE headgroups connected by a C34 or a C36 alkyl chain (Me2PE-C34/36-Me2PE).40 Inner Structure of Lamellar Aggregates. On the basis of our results from EM and SAXS investigations and the proposed lamellar structure of PC-Gly(2C16Me)C32-OH,48 we suggest a similar arrangement of the molecules within the lamellar aggregates for compounds PC-Gly(2C16Me)C32-PC and PCGly(2C16Me)C32-Me2PE, respectively. The molecules are probably arranged in a monolayer with fully interdigitated alkyl chains, where the ends of the short C16 alkyl chains join each other (see Figure 10, left). A partial interdigitation, where the

enough to fully hydrate the PC headgroups. PC-Gly(2C16Me)C32-Me2PE self-assembles at pH 5.0 into lamellae with a d value of 6.7 nm; i.e., a slightly thinner water layer of l = 1.2 nm is present in that case if a comparable, tilted arrangement of the bolalipid molecules within the monolayer is assumed (see Figure 10). The thinner water layer of PCGly(2C16Me)C32-Me2PE lamella, when compared to PCGly(2C16Me)C32-PC, is obviously due to the exchange of one PC by a Me2PE headgroup. Since the Me2PE group contains a free proton capable of the formation of intermolecular hydrogen bonds, the Me2PE headgroup is less hydrated compared to the PC headgroup, resulting in a smaller repeat distance. An increase in temperature to above Tm leads to an increase in the repeat distance to d = 7.7 and 8.2 nm for PCGly(2C16Me)C32-PC and PC-Gly(2C16Me)C32-Me2PE, respectively. One would expect a decrease in layer thickness with increasing temperature due to the overall shorter alkyl chains caused by an increased number of gauche conformers (see the IR experiments). However, the bolalipid molecules are probably tilted in the ordered lamellar phase and may be not tilted in the liquid-crystalline phase. This reorientation can overcompensate for the shortening of the alkyl chains due to the formation of gauche conformers. In the SAXS diffractograms, an intermediate reflection can be observed for both bolalipids at both temperatures investigated. If this reflection corresponds to a lamellar repeat distance, i.e., a second type of lamella, then d values ranging from 5.2 to 5.7 nm can be calculated, which would finally result in a large tilt angle of the bolalipid molecules with respect to the monolayer normal. Since the suspension consists of a pure bolalipid substance, the coexistence of two lamellar phases (with different repeat distances) could only be a temporal phenomenon, according to the Gibbs phase rule. One phase should transform into the other, thermodynamically stable phase with time. However, we could also observe this additional intermediate reflection after various heating cycles, which means that the two lamellar phases coexist for at least 1 day. Up to now, we cannot say more about the origin and the structure of this second lamellar phase and further investigations are part of ongoing research. Inner Structure of the Nanofibers. An aqueous suspension of PC-Gly(2C16Me)C32-Me2PE shows different aggregate structures depending on the pH value. Starting from large lamellar sheets in acidic and neutral media, smaller disklike assemblies and vesicles are found at pH 10, which further transform into long nanofibers at pH 12 (see Figures 5−7). These fibers reveal a diameter of about 7.3 nm, which is larger than the length of the bolalipid molecule of roughly 6 nm. The dimension of the PC-Gly(2C16Me)C32-Me2PE fibers is also larger with respect to fibrous aggregates of symmetrical, single-chained bolalipids investigated previously: PC-C32-PC fibers reveal a diameter of 4.3 nm, whereas Me2PEC32-Me2PE self-assembles at pH 5 into fibers with a diameter of 5.2 nm.68 In both cases, the bolalipid molecules are tilted with respect to the fiber normal, giving a smaller thickness of fibers compared to the length of the bolalipid molecule. Moreover, the fibers of PC-C32-PC and Me2PE-C32-Me2PE are composed of 11−12 bolalipid molecules per 1 nm of fiber length.68 For PC-Gly(2C16Me)C32-Me2PE, an aggregation number of 20 molecules per 1 nm of fiber length was calculated, which means that the PC-Gly(2C16Me)C32Me2PE molecules are densely packed within the nanofiber. Probably, the bolalipid molecules are arranged side by side and

Figure 10. (Left) Scheme of a lamellar phase consisting of fully interdigitated PC-Gly(2C16Me)C32-PC molecules in a monolayer arrangement with a tilt of 30° of the bolalipid molecules relative to the layer normal. (Right) CPK model of PC-Gly(2C16Me)C32-PC molecules in a monolayer arrangement.

PC headgroup of the long C32 chain of one PC-Gly(2C16Me)C32-PC molecule is positioned next to the end of the C16 chain of another bolalipid molecule, is very unlikely because in this case the hydrophilic PC headgroups will be located in the middle of the hydrophobic alkyl chain region. Moreover, the PC-Gly(2C16Me)C32-PC molecules, which have a length of about 6 nm, are likely tilted by about 30° with respect to the layer normal (Figure 10, right), resulting in a thickness of the bolalipid monolayer of about 5.5 nm. The FTIR results shown above indicate the presence of a monoclinic packing pattern of the alkyl chains, supporting the model of Figure 10. Without this tilt, i.e., if the bolalipid molecules were oriented perpendicular to the layer normal, empty space would be created in the middle part of the monolayer (see Figure S7, Supporting Information), which is energetically unfavorable. The results from our SAXS measurements support this model. At temperatures below Tm, a lamellar repeat distance of d = 7.1 nm was found for PC-Gly(2C16Me)C32-PC, resulting in an interlamellar water layer thickness of l = 1.6 nm. This is 4370


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Langmuir not tilted with respect to the fiber axis but are only slightly twisted relatively to each other within the nanofiber. This twist should be not as pronounced as proposed for PC-C32-PC or Me2PE-C32-Me2PE fibers, since the PC-Gly(2C16Me)C32Me2PE molecule carries a glycerol moiety and a second C16 alkyl chain. The latter could minimize the packing frustration caused by the size difference between the large PC headgroup and the small cross-sectional area of a single alkyl chain.41 The proposed perpendicular orientation of the PC-Gly(2C16Me)C32-Me2PE molecules with respect to the fiber normal is supported by SANS data, which indicate a fiber diameter of 7.3 nm, a value very close to the distance of the headgroups of two molecules in an antiparallel orientation of bolalipid molecules in the fiber (see Figure S7).

the small sheetlike aggregates. When the pH value is further increased to 12, these nanofibers become the dominant aggregate form. The reason for this lamella → nanofiber transformation remain unclear at this time, but it seems to be conceivable that the negatively charged and repulsive Me2PE headgroups prevent a densely packed two-dimensional assembly of bolalipid molecules and a one-dimensional aggregate (nanofiber) is formed instead. Upon heating, these nanofibers transform into lamellar, disklike assemblies as shown by SANS experiments. The results demonstrate that a delicate balance among the chemical structure of the bolalipid molecules (molecule asymmetry), the steric requirements of the headgroups, and the possibility for attractive intermolecular hydrogen bonds or repulsive charge interactions determines the aggregation behavior of asymmetrical glycerol diether bolalipids in an aqueous suspension. With the introduction of a new type of glycerol diether bolalipid including two phosphate headgroups, we close the gap among the symmetrical tetraether lipids (TELs), the highly asymmetrical glycerol diether bolalipids including only one phosphate headgroup, and the asymmetrical single-chain bolalipids. PC-Gly(2C16Me)C32-Me2PE is of especially great interest since the aggregate structure found for this bolalipid in an aqueous suspension can be tuned by changes in the pH value for large lamellar aggregates to vesicles to long nanofibers. The question of whether this novel class of bolalipids is miscible with conventional phospholipids, e.g., dipalmitoylphosphatidylcholine (DPPC) and dioleoylphosphatidylcholine (DOPC), which can lead to stabilized liposomes applicable for drug delivery purposes, is part of ongoing research.

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SUMMARY In this work, we investigated the temperature-dependent aggregation behavior of a new kind of glycerol diether bolalipid. The bolalipids are composed of a long C32 alkyl chain bond to the sn-3 position, a short methyl-branched C16 alkyl chain in the sn-2 position, and a zwitterionic phosphocholine headgroup in the sn-1 position of the glycerol. The membrane-spanning C32 chain further carries at its end a second phosphocholine (PC) headgroup (PC-Gly(2C16Me)C32-PC) or a protonatable phosphodimethylethanolamine (Me2PE) headgroup (PCGly(2C16Me)C32-Me2PE), leading to a bolalipid structure. With this work, we would like to answer the following questions: What is the influence of the second phosphate headgroup on the self-assembly properties in comparison to other bolalipids? Can the aggregation behavior of PCGly(2C16Me)C32-Me2PE be tuned by changes in the pH value? PC-Gly(2C16Me)C32-PC forms lamellar structures in an aqueous suspension at all temperatures investigated. The lamellae seem to have a high bending stiffness as only very rarely closed vesicles are observed. In the TEM images, these vesicles seem to have very large diameters and collapse and rupture during the drying and staining procedure. The bolalipid molecules in the lamellae are probably arranged in a monolayer with fully interdigitated alkyl chains. The molecules seem to be tilted with respect to the layer normal to ensure a dense packing of the alkyl chains. At low temperatures, the alkyl chains are in an all-trans conformation, well ordered, and arranged in a triclinic packing mode indicated by the δ(CH2) vibrational band (FTIR). With increasing temperature, a transition from the lamellar gel phase with interdigitated alkyl chains (Lβ’I) to the liquid-crystalline phase (Lα) takes place at Tm = 28 °C. The analog bolalipid bearing a protonatable headgroup (PCGly(2C16Me)C32-Me 2 PE) shows at pH 5 the same aggregation behavior compared to that of PC-Gly(2C16Me)C32-PC. The protonated Me2PE headgroup behaves quite similarly to a PC headgroup despite the fact that additional intermolecular hydrogen bonds lead to a less-hydrated headgroup, a stabilization of the aggregate structure, and, hence, a slightly increased transition temperature. These large sheetlike structures of PC-Gly(2C16Me)C32-Me2PE are stable up to a pH value of 9. Between pH 9.5 and 10.5, the aggregates become smaller and they appear more rounded and disklike. This change is obviously due to an increased number of negatively charged bolalipid molecules, which probably accumulate at the rim of the aggregates and further prevent the fusion of these disks. At pH 11, nanofibers start growing out of

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.8b00527. Synthesis procedures, analytical data of prepared compounds, further DSC and FTIR measurements, and additional TEM images and SANS data (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: +49345-5525196. Fax: +49-345-5527026. *E-mail: [email protected]. Phone: +49345-5525120. Fax: +49-345-5527018. ORCID

Simon Drescher: 0000-0002-3272-1721 Alfred Blume: 0000-0002-8416-7953 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by grants from Deutsche Forschungsgemeinschaft (DFG) project DR 1024/1-1 (to S.D.) and by grants from the Phospholipid Research Center Heidelberg (to S.M.). The support of Dr. Gerd Hause (Biocenter, Martin Luther University Halle-Wittenberg) by providing us access to the electron microscope facility is greatly appreciated. S.D. thanks Dr. Bob-Dan Lechner (University of 4371


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Langmuir

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Exeter, U.K.) for his help with the analysis of SAXS results and Annekatrin Rother (Institute of Biochemistry, MLU, Germany) for her great support in collecting EM images.

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REFERENCES

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Impact of Headgroup Asymmetry and Protonation State on the

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