Controlling the Miscibility of X-shaped Bolapolyphiles in Lipid

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B: Biomaterials and Membranes

Controlling the Miscibility of X-shaped Bolapolyphiles in Lipid Membranes by Varying the Chemical Structure and Size of the Polyphile Polar Headgroup Bob-Dan Lechner, Philip Biehl, Helgard Ebert, Stefan Werner, Annette Meister, Gerd Hause, Kirsten Bacia, Carsten Tschierske, and Alfred Blume J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b08582 • Publication Date (Web): 08 Nov 2018 Downloaded from http://pubs.acs.org on November 12, 2018

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The Journal of Physical Chemistry

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Controlling the Miscibility of X-shaped Bolapolyphiles in Lipid Membranes by Varying the Chemical Structure and Size of the Polyphile Polar Headgroup

Bob-Dan Lechner,1 Philip Biehl,1 Helgard Ebert,2 Stefan Werner,1 Annette Meister,3 Gerd Hause,4 Kirsten Bacia,1 Carsten Tschierske,2 Alfred Blume1*

1Institute

of Chemistry - Physical Chemistry, Martin-Luther-Universität Halle-Wittenberg, D-

06120 Halle (Saale), Germany 2Institute

of Chemistry - Organic Chemistry, Martin-Luther-Universität Halle-Wittenberg, D-

06120 Halle (Saale), Germany 3HALOmem

and Institute of Biochemistry and Biotechnology, Martin-Luther-Universität

Halle-Wittenberg, D-06120 Halle (Saale), Germany 4Biocenter,

Martin-Luther-Universität Halle-Wittenberg, D-06120 Halle (Saale), Germany

*Correspondence: [email protected]; Tel.: +49-345-5525850

ACS Paragon Plus Environment

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ABSTRACT: Bolaamphiphiles are well known naturally occurring structures that can increase the thermal and mechanical stability of phospholipid membrane by incorporation in a trans-membrane manner. Modifications of bolaamphiphiles to introduce particular structural elements like a conjugated aromatic backbone and lateral side chains in the hydrophobic region lead to bolapolyphiles (BPs). We investigated the ability of BPs to form lyotropic phases in water. The BPs had identical backbone and side chains, but different headgroup structures leading to different abilities to act as hydrogen bond donors and acceptors. BPs with hydrophilic headgroups capable of acting as hydrogen bond donors as well as acceptors did not form lyotropic phases and were insoluble in water, independent whether the polar groups were small or large. The extended lipophilic core structure and the multiple intermolecular hydrogen bonds between the headgroups prevented the formation of wellhydrated lyotropic aggregates. A BP with two large hydrophilic headgroups of several ethylene oxide moieties terminated by methyl groups formed sheet- and vesicle-like aggregates in water. These headgroups act only as hydrogen bond acceptors and cannot form hydrogen bonds in the absence of water. The miscibility of BPs with vesicles of 1,2dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in water and the resulting aggregate structures were also investigated. For BPs with headgroups acting as donors and acceptors of hydrogen bonds, macroscopic phase separation occurred in the mixed membranes, and two different membrane domains, a DPPC-rich one containing only little polyphile, and a BP-rich one containing varying amounts of lipid were formed. For headgroups without the ability to act as hydrogen bond donors, small BP-aggregates were formed that were homogeneously distributed over the membrane. The lateral organization of BPs in lipid membranes can thus be controlled by the nature of the BP headgroup.



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1. INTRODUCTION Self-assembly into supramolecular structures is one of the most important features of biological materials.1-4 The association of single amphiphilic molecules like lipids or surfactants to form structures like micelles, bilayer membranes5 or lyotropic cubic phases creates materials with new dimensions, astounding supramolecular properties, and a complexity beyond the one found on the molecular level. Supramolecular self-assembled systems nowadays are often used in material science, like photonics or metamaterials.6 In modern medicine, supramolecular assemblies in the form of polymers or nanoparticle systems are used as solubilizers for drugs and nano-sized delivery systems.7-10 The ability of amphiphiles to form supramolecular structures similar to biological systems and combining this property with strategies of material science can result in precisely controlled selfassembled structures with defined functionality. In this way new hybrid materials with purposefully designed properties can be created.4,

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A special molecular setup or affinity

profiles are necessary to form supramolecular structures. For the self-assembly of amphiphilic molecules the driving force is the hydrophobic effect, i.e. the strong tendency of hydrophobic moieties to not be in contact with water but rather to be in contact with other hydrophobic moieties. Other interactions, such as polar and electrostatic interactions, as well as hydrogen bonding modify the self-assembly process in different ways. A single hydrogen bond is rather weak, but multiple hydrogen bonds can lead to exceptional stability of the supramolecular structures. As is well known, hydrogen bonding is one of the most important interactions for self-assembly in biological systems. Lipids are one of the most remarkable building blocks for supramolecular structures on earth. Every living organism depends on their ability to form bilayer membranes that not only allow for compartmentation of space to form cells, but those membranes are also able to


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host guest molecules to enable material exchange, signalling and the regulation of vital parameters. Bolaamphiphiles are lipid-like molecules that can be found as part of the lipid membrane of several organisms that mostly live in places considered as hostile.12 The structures of bolaamphiphiles with two hydrophilic headgroups connected by a lipophilic moiety are well-known in biology and their unique structure and affinity profile allow those molecules to span a whole lipid membrane to add extra strength to the lipid membrane and makes them withstand higher thermal or mechanical stress.12-15 Modification of the bolaamphiphiles structure, like headgroup size and chemical nature or the structure, steric demand and rigidity of the lipophilic moiety, can lead to a new group of polyphilic molecules16, the bolapolyphiles (BPs).17-18 The X-shaped molecules investigated in this study feature a similar structure whereby the lipophilic central part is represented by a rigid oligo(pphenylene ethynylene) (OPE) unit forming the molecule backbone (see Figure 1). To extend the concept of bolaamphiphilic molecules, two additional flexible aliphatic alkyl chains are attached to the central phenyl ring of the backbone giving the molecules an X-like shape. Either end of the backbone is framed by a hydrophilic headgroup of variable size and ability to function as hydrogen bond donor. Those X-shaped BP show liquid crystalline behaviour in the bulk phase and thus possess a strong tendency to self-assemble into supramolecular structures, involving a wide variety of new modes of soft self-assembly with unprecedented complexity. This led to new liquid crystalline (LC) phases, including LC honeycombs, new types of cubic and lamellar LC phases, zeolite-like LC structures and numerous others.18-21 Besides the general importance for the understanding of the development of structural complexity in soft self-assembly, polyphilicity allows the directed organization of conjugated molecules into well-defined nano-structures.22-24 These are of potential interest for soft sub-5 nm nano-lithography,25 light harvesting and organic electronics applications.26-27 Though these complex structures were mainly found for the bulk materials, due to the



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involved polar groups, the self-assembly of these compounds can be affected by polar solvents, leading to solvent stabilized, solvent induced and eventually to lyotropic systems, especially if the size of the polar head groups is enlarged.23 In several publications we reported on the lyotropic self-assembly of the X-shaped bolapolyphile B12, consisting of a OPE core involving 5 phenyl rings alternated by ethynylene groups as backbone, two rather small terminal glycerol headgroups and two lateral C12-alkyl chains.17,

22-23, 28-30

This

molecule could be incorporated into phospholipid membranes and showed a temperature dependent miscibility. The molecular backbone of these molecules enables  stacking to form aggregates inside the bilayer. The glycerol headgroups contribute to this interaction by forming intermolecular hydrogen bonds, which are key structural features to stabilize this type of aggregation. Fluorescence imaging of electroformed giant unilamellar vesicles (GUVs) of B12 and DPPC below the main phase transition revealed phase separated membranes and the presence of six-armed fractal branched star-shaped domains (hexagonal honeycombs) consisting of staggered aggregates of parallel aligned polyphiles with 3-5 lipid molecules per polyphile.17, 28 For the B12-DPPC system, the phase separation into two types of lamellar structures with different thickness and electron density profile could be proven by X-ray reflectivity using synchrotron radiation.22 The thermotropic phase behaviour of the lipid membrane becomes more sophisticated by the interaction with the B12 and several additional transitions could be identified with calorimetric methods (DSC). Some of the lipid molecules are constrained in the polyphile domains, whereas other lipids are still in the gel state, even for temperatures above the main transition temperature Tm. Using temperature dependent infrared and fluorescence spectroscopy, it could be shown that the lipid rich phase undergoes a thermal transition at Tm while the polyphile remains aggregated. These aggregates break up at higher temperatures with concomitant additional thermal transitions to give a homogeneously mixed


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high temperature phase above 75 °C, which could be shown by solid state NMR, grazing incidence wide angle X-ray scattering and fluorescence imaging and spectroscopy. From all the experimental data, a tentative structural model for the phase separated state was derived. The polyphiles adopt a transmembrane orientation and are - staggered, supported by intermolecular headgroup hydrogen bonds, to form filaments along the edge of honeycomb walls. The lateral alkyl chains of the polyphile and a confined fraction of the lipid are located in the centre of the hexagonal honeycomb cells. This confined lipid as part of the polyphile rich phase does not undergo the typical Lβ’ – Lα phase transition at Tm. In the work presented here, we focus on the influence of the headgroup size and structure of the bolaamphiphiles on their miscibility with phospholipids in membranes. The influence of size is addressed by a variation of the overall length of the headgroups. For this, oligo(ethylene oxide) chains with varying length k (EOk) were introduced (Figure 1). These EOk units can only act as H-bond acceptors, but not as H-bond donors. In the case of the molecule E12/7, a methyl group terminates the EOk chain and the headgroup is hydrophilic but hydrogen bonding with adjacent polyphiles cannot occur. Only with water, a hydration shell is formed by hydrogen-bonding with the EO groups. In this way, the influence of the effects of size/length of the hydrophilic group can be studied. If the EOk chain is terminated by a glycerol moiety with 2 OH-groups (molecule D12/3) or a glucopyranoside moiety with 5 OH-groups (molecule F12/4), a network of intermolecular hydrogen bonds with neighbouring polyphile molecules is likely. The influence of the hydrogen bond formation on the aggregation of the polyphiles within the membranes as templates and the phase behaviour of the resulting structures could therefore be studied. For the molecule C12, the headgroups consist only of one OH group at either side of the backbone and thus, the hydrophilic part is very small. The OH groups are capable of hydrogen bonding, but in the lipid layers where only a side-by-side organization of molecules is possible, the molecular geometry would not



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allow for this without adapting angles unusual for hydrogen bonds. C12 was thus used as the most hydrophobic reference compound. The size and structure of the backbone and the length and position of the lateral alkyl chains are exactly the same for all polyphiles investigated here. The only differences are the size and structure of the hydrophilic headgroups, which, as we will show, have a severe influence on the miscibility with phosphatidylcholine (PC) in membranes as well as on the morphology and thermal phase behaviour of resulting structures.

backbone: lipophilic / rigid O R

O

O

R

O

R=

hydrophilic

hydrophilic lipophilic / flexible length ~ 3 - 4 nm

H

C12

(EO)7 Me

E12/7

(EO)3

OH

HO O (EO)4

OH OH

D12/3 OH OH

F12/4

Figure 1. Chemical structure of the X-shaped polyphiles with rigid backbone and lateral alkyl chains (left) as well as the different hydrophilic headgroup structures (R, middle) and names of the molecules (right, yellow shaded); in each case flexible C12 alkyl chains are attached via ether oxygen atoms to the central phenyl ring.

2. MATERIALS AND METHODS 2.1. Materials. The phospholipid DPPC (1,2-dipalmitoyl-sn-3-phosphocholin), its derivative with perdeuterated alkyl chains DPPC-d62 and the fluorescently labelled Rh-DHPE (lissamine rhodamine B sulfonyl 1,2-dihexadecanoyl-sn-3-phosphoethanolamine ammonium salt) were purchased from Avanti Polar Lipids Inc. (Alabaster, USA). The X-shaped polyphiles C12, D12/3 and E12/7 were synthesized as described elsewhere.17 The X-shaped


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polyphile with ß-D-glucopyranosyl-terminated oligo(ethylene oxide) headgroups F12/4 was prepared from C12 as shown in scheme S1 in the supporting information along with detailed procedures of synthesis and analytical data. 2.2. Sample Preparation and Data Collection. Samples were prepared by premixing the polyphile and the lipid in HPLC grade chloroform (Carl Roth GmbH, Karlsruhe, Germany) to achieve the desired molar ratio. The chloroform was evaporated in a N2 stream and the samples were kept in a vacuum furnace to remove remaining CHCl3 for at least 3 hours and were then rehydrated with ultrapure water (Merck Millipore, Billerica, USA). The procedures to perform differential scanning calorimetry (DSC), transmission electron microscopy (TEM), confocal fluorescence microscopy (CFM), fluorescence depolarization measurements, and Fourier transform attenuated total reflection infrared spectroscopy (ATR-FTIR), as well as the respective specific sample preparation techniques have been described before22 and can also be found in the supporting information (SI 1.1 to 1.4).

3. Results and Discussion 3.1. Aggregation of BPs in Water and Lyotropic Behavior. As bulk substances, all polyphiles show at least one thermotropic liquid crystalline phase before they melt. The molecules are of intensive yellow colour and insoluble in water but soluble in organic solvents like CHCl3 or THF. In order to study their potential of forming lyotropic aggregates, a form of supramolecular assembly mediated by interactions of the BP molecules with the solvent, the polyphilic molecules were dispersed in water and treated with ultrasound for 30 min at 60 °C.



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For the molecule with only very small hydrophilic headgroups (C12), no aggregate formation in water was observed as expected. This compound can only be dispersed in water in crystalline form, as the headgroups are too small and not hydrophilic enough to form lyotropic aggregates. An increase of the headgroup size while keeping the core and lateral alkyl chain structure/size unchanged should lead to molecules with a large enough hydrophilic moiety to enable the formation of lyotropic aggregates in water. However, as reported before,22 the polyphile B12 with glycerol headgroups is still unable to form lyotropic aggregates in water, showing that these headgroups are still too small. E12/7 features EO7-Me headgroups that are about twice the size of the glycerol headgroups of B12. The EO7 moiety can only act as hydrogen bond acceptor for water but cannot act as donor. Apparently, this headgroup is large enough so that lyotropic E12/7 aggregates in water can be formed. The stable suspensions (c = 1 mM) show an intense yellow color and a blue opalescence. Cryo-transmission electron microscopy (cryo-TEM) images reveal structures, which could be interpreted as wavy lamellae (Figure 2A, triangle 1). Besides those undulated lamellae, round and elliptical vesicle-like structures with smooth surfaces (Figure 2B and C, triangle 2) occur alongside with planar layers (Figure 2A, triangle 3). It is likely that the E12/7 molecules are packed parallel to form the lamellar phase with the thickness of the length of the molecule as shown in Figure 2E. If the backbones are parallel, the phenyl rings will be able to stabilize the structure by  interactions. The backbone and lateral alkyl chains form the lipophilic inner part of the lamellae with about the same cross sectional area as the headgroups.


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D 34,6°C

160 E12/7

-1

120 -1

Cp / cal mol K

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


-1

H = 690 cal mol

80 40 0 10

20

30

40

50

o

Temperature / C

Figure 2. A-C: Cryo-TEM images of a vitrified aqueous suspension of E12/7 (prepared at 20 °C, c = 1 mM) featuring lamellae (triangle 1), vesicles (triangle 2) and planar layer-like structures (triangle 3). Black arrows mark ethane crystals which occur as preparation artefacts. Scale bars: 200 nm (A) and 500 nm (B, C). D: DSC thermogram of the respective suspension. E: model for a lamellar arrangement of the E12/7 molecules. Scale bars = 200 nm. The aqueous suspension of E12/7 aggregates was investigated by DSC for possible thermotropic transitions. Only a weak transition peak located at 34.6 °C (Figure 2D) could be



12 found. The endothermic transition has only a small transition enthalpy of 0.69 kcal mol-1. The aqueous suspension was also investigated by ATR-FTIR spectroscopy at different temperatures between 10 °C and 60 °C. Analysis of the CH2-stretching vibrations ῦ(CH2) of the two C12-alkyl chains (Figure S1) shows almost no shift of the peak position with changing temperature. The position of the symmetric CH2-stretch is located at ῦs(CH2) 2849 cm-1 indicating slightly ordered alkyl chains in the entire temperature range.31-32 No significant change in wavenumber was observed at the transition temperature. A reorganization of the lateral alkyl chain order as an origin of the transition can therefore be excluded. The nature of the endothermic transition remains an open question. The other investigated compounds with larger headgroup size were also investigated for their ability to form lyotropic aggregates. Surprisingly, for the compounds D12/3 with glycerol terminated EO3-headgroups (headgroup size similar to E12/7), and F12/4, with even bigger headgroups consisting of an EO4-chain terminated by a sugar moiety, no lyotropic aggregates could be observed. The headgroups of both, D12/3 and F12/4, are capable of acting as hydrogen bond donors as well as acceptors and are able to form intermolecular hydrogen bonds between the headgroups. Obviously, the size of the headgroup is not the only factor that determines the ability to form lyotropic aggregates. When the headgroups are very large but allow for intermolecular hydrogen bonding between the molecules themselves, the formation of dispersed aggregates seems to be unfavourable. Self-aggregation of the polyphiles in crystalline form seems to be preferred in these cases. 3.2. Structure and Morphology of BP-lipid Mixtures at Room Temperature. The X-shaped polyphile C12 is the most hydrophobic compound investigated here, as it has only OH-headgroups. Cryo-TEM images of aqueous suspensions of a mixture C12:DPPC = 1:10 show facetted vesicles with diameters between 20 nm and 300 nm (Figure 3A). Apparently, if C12 is incorporated into DPPC membranes, it does so without destruction of the membrane.


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This finding was confirmed by CFM (Figure 4). Two-channel laser scanning images were recorded of electroformed33 giant unilamellar vesicles (GUVs) of the same mixture containing additionally the fluorescence labelled lipid Rh-DHPE (red channel, 0.5 mol- %, excitation at ex(Rh) = 561 nm and emission at em(Rh) = 566 - 681 nm). The autofluorescence of C12 was visualized in the green channel (excitation at ex(C12) = 405 nm and emission at em(C12) = 412 - 545 nm). The fluorescence microscopy images reveal mostly facetted GUVs (Figure 4A) of 10 -100 µm in diameter and some multilamellar vesicles. The images of the vesicles show separated domains with a slightly higher intensity of the C12fluorescence signal and a strongly decreased Rh-DHPE fluorescence as well as domains with a pronounced Rh-fluorescence intensity. Reconstructions of a GUV hemisphere from a series of confocal slices obtained by slight variations of the focal plane reveal C12-rich Rhodaminefluorescence depleted domains that form six-armed, fractal-branched stars.17 Star domains also occurred with a previously studied similar mixture of DPPC with the X-shaped polyphile B12 that bears glycerol headgroups and can be explained by the honeycomb model.17,

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Extensive excitation of the C12 fluorophore leads to irreversible bleaching of the C12. For this reason, the 3D reconstructed GUV hemisphere image was obtained by only exciting the Rhodamine dye.17 The dark domains (no Rhodamine fluorescence) are C12-rich and clearly reveal the hexagonal snowflake symmetry.



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Figure 3. TEM images of negatively stained samples with UO2Ac2 (without frame) and cryoTEM (red frame) images of polyphile-lipid aggregates; A: C12 : DPPC = 1:10; B/C: D12/3 : DPPC = 1:10; D: F12/4 : DPPC = 1:10; yellow arrows mark ethane crystals, an artefact of the


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preparation procedure; black arrow in 3C marks planar layer-like structure; white triangles mark facetted vesicles. Scale bars = 200 nm in A, B, D and 100 nm in C. Despite the small hydrophilic moiety in C12 (only 2 OH-headgroups), it is possible to incorporate this molecule into lipid membranes. The dopant C12 molecules are distributed throughout the whole membrane and in some regions are clustered by - stacking, forming domains of hexagonal symmetry, including six-armed stars. OH-hydrogen bonding among parallel aligned polyphile backbones is unlikely because the distance and the bond angle between the OH-groups is too large. Thus, the dominating driving force for cluster formation is presumably the - stacking interaction of the backbones aromatic system. Electron micrographs of the mixture D12/3:DPPC = 1:10 were obtained from negatively stained as well as from vitrified samples. Whereas the drying process of stained samples leads to collapsed vesicles (Figure 3B) or vesicle fragments, cryo-TEM images of vitrified samples clearly indicate the presence of mainly facetted vesicles with sizes of 40 nm140 nm (Figure 3C). The CFM images of GUVs of this mixture (vesicle diameter 10 µm - 100 µm, Figure 4B) show facetted and also round vesicles with smooth membranes in good agreement with the TEM results. The GUVs show areas with mainly polyphile fluorescence and others with predominantly Rh-fluorescence due to phase separation. The D12/3 domains are boomerang-shaped with no particular higher symmetry.17 Notably, the areas showing polyphile fluorescence represent only a very small portion of a GUV’s total membrane surface.



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Figure 4. Series of two-channel fluorescence microscopy images of C12:DPPC = 1:10 (A), D12/3:DPPC = 1:10 (B), E12/7:DPPC = 1:10 (C), each with 0.5 mol- % Rh-DHPE. The green channel (left) shows the polyphile fluorescence and the red channel (right) the Rhodamine fluorescence. (A) shows a single confocal slice through the GUVs. Non-uniform fluorescence due to domain formation is seen along the GUV perimeter. The inset on the right shows a maximum intensity projection of an axial series of slice images of a GUV of the same composition. The polyphile fluorescence (green channel) could not be imaged in the axial


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series due to photobleaching of the C12 compound. (B) and (C) show maximum intensity projections of axial series. Scale bars = 20 µm. The mixture F12/4:DPPC = 1:10 was only studied using TEM. Exclusively vesicles with exceptionally large diameters of several micrometers were found in negatively stained samples (Figure 3D). Often, significantly smaller vesicles were attached to the bigger ones. The mixture E12/7:DPPC = 1:10, investigated by CFM, forms mostly perfectly round very large GUVs with diameters up to around 80 µm and only a few facetted ones (Figure 4C). Pure DPPC usually features facetted GUVs at 20 °C. The incorporation of E12/7 causes a stabilization of the smooth round vesicle geometry. Both fluorescing molecules, the dye RhDHPE and the polyphile E12/7 itself, appear to be homogenously distributed in the GUV membrane given the spatial resolution of CFM.17,

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Using polarised excitation light, a

position-dependent periodic variation of the E12/7 fluorescence signal along the GUV’s membrane occurs.17 This can be interpreted as a preferential orientation of the polyphile molecules with respect to the membrane normal.34 By solid state NMR studies, a limited mobility of the E12/7 molecules was found indicating the presence of phase separated E12/7 domains.28 The domain size must be below the resolution limit of the CFM. A phase separation and presence of sub-resolution E12/7-rich domains is therefore likely. The reason for the special behavior of E12/7 is the fact that the end of the EO7 moiety is methylated so that the headgroup cannot act as a hydrogen-bond donor and can only accept hydrogen bonds from water molecules. Therefore, no driving force for intermolecular hydrogen bonding between the headgroups themselves is present and additional water molecules are required to mediate the hydrogen bonding between them. The size of the polyphile domains results from a competition of attractive forces by intermolecular - stacking and hydrogen bonding interactions and the steric repulsion between the well



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hydrated EO7 segments of the headgroups. Apparently, the steric repulsion of the headgroups is dominating and lead to a decrease of the cluster sizes. 3.3. Thermotropic Behavior of Mixed Polyphile-lipid Membranes. DSC thermograms of pure DPPC vesicles normally show the so-called pre-transition peak at Tpr = 37 °C, indicating the transition from a gel phase (L’) to a rippled gel phase (P’)35 and the main transition peak at Tm = 42 °C, where the transition into the liquid-crystalline phase (L) occurs. Addition of the polyphiles dramatically changes the thermotropic phase behaviour of DPPC. For the previously characterized mixture of B12 (glycerol headgroups) with DPPC, three additional transition peaks above the main phase transition were observed and a honeycomb model for phase separation was developed.17, 22 Since the miscibility of BP and lipid as well as the phase and domain structure strongly depend on the nature of the added BP, the changes in thermotropic phase behavior will be discussed separately for each of the Xshaped molecules. 3.3.1 C12:DPPC Mixtures.

The thermotropic phase transition behavior of four

different mixtures of the X-shaped BP C12 - with only OH headgroups - and DPPC were investigated using DSC. In all of these DSC thermograms a phase transition peak at Tm = 42 °C (Figure 5A) is seen. For xC12 = 0.05 (C12 : DPPC = 1:50), two well separated peaks at 52 °C and 56 °C occur, whereas for higher C12 content the additional peaks are broad and less separated. The thermogram for xC12 = 0.2 (C12 : DPPC = 1:4) shows 4 additional peaks that are shifted to even higher transition temperatures. The addition of the C12 leads to a highly complex thermal phase behaviour. The presence of additional peaks is due to a phase separation in a lipid-rich phase that has its gel-to-fluid-phase transition at Tm and at least one C12-rich phase with transitions at higher temperatures. The peak intensity and integrated area under the peak at Tm decrease strongly with increasing C12 content (Figure 5B). For the 1:10 mixture, the transition enthalpy for the main


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transition ΔHm amounts to only 20 % of the value of pure DPPC vesicles. Thus, 80 mol- % of the DPPC seems to be located in C12-enriched domains. The rest seems to be either forming pure DPPC domains or is possibly mixed with some C12 not forming the star-like domains seen in fluorescence images (see Figure 4A, right). The polyphile becomes bleached during the imaging process for a 3D image, so that the stars appear black on a red background arising from the lipid rhodamine dye.17 For the 1:4 mixture, only 8 % of total transition enthalpy is due to the melting of almost pure DPPC. Thus, only a small percentage of DPPC is forming pure DPPC domains, most of the phospholipid is mixed with C12. The sum of all enthalpies remains almost the same regardless of the mixing ratio showing that even in the C12/DPPC domains the lipid eventually melts at higher temperature. 3.3.2 D12/3:DPPC Mixtures. All mixtures of D12/3 with DPPC again show the main transition peak at Tm = 42 °C for pure DPPC indicating that the system is not completely mixed, but phase separated (Figure 5C). With increasing concentration of D12/3, two additional peaks at 56 °C and 68 °C appear indicating a thermotropic transition of complexes of D12/3 with DPPC. The heights of the additional peaks increase with the D12/3 content at the expense of the height of the main transition peak (Figure 5C). However, the transition enthalpy ΔHm for the main peak decreases to only ca. 65% of the original value (Figure 5D). This shows that only very small amounts of DPPC are mixed in the residual areas rich in D12/3. It appears as if an almost pure DPPC phase is separated from domains where D12/3 is present. This assumption is supported by the observation that at low D12/3 concentration, an additional peak at the position of the pre-transition of DPPC is visible. Whether a pretransition is observed depends very sensitively on the presence of foreign molecules in the bilayer. It usually disappears when more than 2-3 mol- % of foreign lipids are present. The polyphile rich phase, on the other hand, seems to be heterogeneous in its composition. The fluorescence images (see Figure 4B) show that areas with very high fluorescence intensity of



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the polyphile appear where almost no Rh-DHPE is present. In addition, the merged image of the two fluorescence intensities display almost no yellow areas, supporting the finding that D12/3 and DPPC are mostly demixed. 3.3.3 F12/4:DPPC Mixtures.

The bolapolyphile F12/4 has four EO units in its

headgroup and an additional sugar moiety connected to the end of the EO chain. The headgroup is therefore much larger than the headgroup of D12/3. Again, in all mixtures the main transition peak of almost pure DPPC is seen at Tm = 42 °C, regardless of the F12/4 content (Figure 5E). For xF12/4 ≥ 0.05 (F12/4 : DPPC = 1:n with n ≥ 50), very weak additional transitions occur that are shifted to higher temperatures with increasing F12/4 content. This indicates that a phase separation is occurring, as seen for the other mixtures described above. At higher F12/4 concentration, two high temperature peaks appear at 54 °C and 61 °C. Already for the 1:50 mixture, ΔHm of the main transition is drastically decreased to 42%, although there is no additional transition visible in the thermograms (Figure 5F). For higher F12/4 content, the main transition enthalpy remains relatively constant. Obviously, a large fraction of the lipid is confined in the BP-rich phase and does not undergo a phase transition at Tm. Also, the overall enthalpy including both, main and additional transitions, is decreased for the mixture with the lowest BP content. It slightly increases again with increasing F12/4 content. The DSC behavior indicates that even at very low content of F12/4, a mixed phase of the bolapolyphile with DPPC exists, which undergoes no thermotropic transition in the investigated temperature range. With increasing BP content, the almost pure DPPC phase remains unaffected and F12/4 is solubilized in the BP enriched phase. The TEM images show that the vesicles remain stable. Unfortunately, no CFM images are available for this system. 3.3.4 E12/7:DPPC Mixtures. The headgroup of E12/7 is different in that it cannot act as a hydrogen bond donor. Because of the 7 EO units, the headgroup is well hydrated and also relatively large. Regardless of the E12/7 content, always a sharp transition peak at 42 °C is


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21

visible in the thermograms (Figure 5G). In contrast to the other mixtures, no additional transitions at higher temperature appear. However, the transition enthalpy decreases very strongly with increasing content of E12/7 (Figure 5H). Although not directly obvious from the thermograms, a phase separation might be present, with more transitions that are outside the studied temperature range. The change in ΔHm is most pronounced for small amounts of BP and does not change much for a higher E12/7 content. Obviously, the phase separated domains form already at low BP content. E12/7 was the only bolapolyphile, which showed lyotropic and thermotropic behavior with a transition at 35 °C (Figure 2D) as a pure compound. The thermograms of mixtures with high BP concentration show this very weak peak between 34 °C and 36 °C. This indicates that the transition of a BP-rich phase is strongly dominated by the thermotropic behaviour of the E12/7 component and may contain only marginal amounts of DPPC. The CFM images in Figure 4C show that the phase separated domains must be smaller than the resolution of the confocal microscopy, because a more or less homogeneous distribution of the fluorescence intensities of the lipid dye as well as of the BP is seen.



22 20

B Htrans / kcal mol DPPC

C12 : DPPC

15

DPPC

-1

-1

10 1:50 1:20 5

1:10

1:4 0 20

20

50

60

70

5 1:10 1:4

60

70

80

20

-1

0

1:50 1:10 1:4 1:1 30

40

50

60

70

Htrans / kcal mol DPPC

-1

-1

1 : 10 1:4 E12/7

0 20

30

40 50 60 o Temperature / C

70

80

0.5

4 Hm

2

Hadd

0

Htot

0.1

0.2

0.3

0.4

0.5

xD12/3 Hm

F12/4 : DPPC

Hadd

8

Htot

6 4 2 0 0.1

0.2

xF12/4

0.3

0.4

0.5

E12/7 : DPPC

10

1 : 50

0.4

6

H

10 1 : 20

0.3

D12/3 : DPPC

0.0

E12/7 : DPPC

15 DPPC

5

0.2

8

80

o

Temperature / C

G 20

0.1

xC12

10

1:20

0 20

Htot

2

0.0

-1

-1

Cp / kcal mol K

5

Hadd

F

F12/4 : DPPC

DPPC 10

Hm

4

Temperature / °C

15

-1

6

0.0

-1

10 1:50 1:20

50

8

10

DPPC

40

10

1:1

1:4

C12 : DPPC

D

15

30

1:50 1:20 1:10

80

D12/3 : DPPC

1:1 0 20

E

40

Temperature / °C


-1

Cp / kcal mol K

-1

C

30


Cp / kcal mol k

-1

A

Cp / kcal mol K

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 39

8 6 4 2 0

Hm

0.0

0.1

0.2

xE12/7

0.3

0.4

0.5

Figure 5. Left: DSC thermograms of aqueous suspensions of C12:DPPC, D12/3:DPPC, F12/4:DPPC, and E12/7:DPPC, the molar ratios are given in the graph. Right: Transition enthalpies for main transition (Hm, black), sum of additional transitions (Hadd, red) and sum of all enthalpies (H, blue) as a function of the BP molar fraction xBP (cDPPC = 2 mM). In Figure 5B, the molar ratios are indicated in the top axis. The error in determination of the


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23

total transition enthalpy is around ± 5%. The thermogram of pure E12/7 in water (from Figure 2D) is given in G (red line), the transition peak is not visible in this scaling. The position of the main transition of E12/7 is therefore indicated by the vertical dashed line. 3.4 Temperature Dependent Fluorescence Depolarization. All of the studied mixtures are phase separated at low temperatures (below the main transition of DPPC) and the BP molecules are organized in aggregates that still contain considerable amounts of lipid. To gain information on the mobility of the BP molecules in those aggregates, temperature dependent fluorescence depolarization experiments were performed. Usually, a fluorescent probe is added to the lipid membrane system, for instance, a molecule such as DPH (1,3,5diphenyl-hexatriene).36-37 In our case we do not need an additional probe, as the BP molecules themselves are fluorescent (λex = 342 nm, λem = 466 nm) so that the experiments provide direct information about the BP mobility within the lipid membranes.22 3.4.1 C12:DPPC Mixtures. For the mixture C12:DPPC = 1:10, the fluorescence anisotropy r has a value of ca. 0.23 at room temperature (Figure 6A, top). This r-value is rather low for a molecule incorporated into a gel phase bilayer. However, additional depolarization of the emitted fluorescence signal by light scattering can occur. When the samples are highly turbid, then r-values are usually lower due to this additional depolarization. This is apparently true for the C12:DPPC sample. Therefore, only the change in r with temperature is significant. Heating the sample leads to a decrease of r to approx. 0.12 in the temperature range between 45 and 58 °C. The lower r-value at high temperature indicates that the C12 molecules have now more freedom for reorientational motions within the fluid DPPC bilayers. For the mixture C12:DPPC = 1:4, the decrease in r occurs between 50 °C (r = 0.18) and 60 °C (r = 0.08, Figure 6A, bottom). For both mixtures, no change in fluorescence anisotropy is seen at the thermotropic main transition at Tm, but the decrease in r is in the temperature range where the additional high temperature transitions are seen by DSC.



24

This is clear evidence that almost no C12 is incorporated in the DPPC domains melting at Tm,and that the C12 molecules in the mixed domains become more mobile when the rest of the DPPC molecules become disordered at higher temperature. It should be noted, that the anisotropy for the 1:4 mixture at low temperatures is already distinctly lower than for the 1:10 mixture. The samples are considerably more turbid so the additional depolarization due to scattering of the fluorescent light is increased. 3.4.2 D12/3:DPPC Mixtures. The behavior of D12/3:DPPC mixtures is similar compared to C12:DPPC mixtures. The fluorescence anisotropy for both mixtures, D12/3:DPPC = 1:10 and 1:4, remains constant for T  50 °C. With increasing temperature, r decreases again within the temperature range in which the additional thermotropic transitions occur. Similar to the analogous mixture with C12, the membranes are demixed at low temperature with almost pure DPPC domains melting at Tm and D12/3:DPPC domains. The BP molecules do not become mobile at the main transition temperature Tm, but become disordered at higher temperature together with the residual DPPC molecules in the mixed domains.. 3.4.3 F12/4:DPPC Mixtures. F12/4 has only little effect on the thermotropic behavior of mixtures with DPPC. Only very weak additional high temperature transitions appear and the major transition peak decreases but stays unchanged with increasing F12/4 content (see Figure 5E). The broad decrease of fluorescence anisotropy r occurs over a wide temperature range and is correlated with the thermotropic main transition as well as the range of the additional transitions. In this case, all the thermal transitions gradually lead to the fluidization of the membrane and the increase of the mobility of the F12/4 molecules in the mixed domains.


Page 24 of 39

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25

C12 : DPPC

1:10

0.3

1.5 1.0

)

)

A

,

0.2 -1

0.0

1:4

Tm

0.3

1.5 1.0

0.2 0.5 0.1

-1

Anisotropy r (

Cp / kcal mol K (

0.5 0.1

0.0 30

40

50

60

70

80

Temperature / °C

D12/3 : DPPC

1:10

)

0.3

1.5 1.0

)

B 0.2

Tm

0.3

1.5 1.0

0.2

0.5 0.1 30

40

50

60

70

-1 -1

Anisotropy r (

0.0

1:4

Cp / kcal mol K (

,

0.5 0.1

0.0 80

Temperature / °C

.

F12/4 : DPPC

1:10

1.5

)

0.3

0.1

0.0

1:4 1.5

Tm

0.3

1.0 0.2 0.5 0.1

-1

,

0.5

-1

0.2

Cp / kcal mol K (

1.0

)

C

Anisotropy r (

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.0 30

40

50

60

70

80

Temperature / °C

Figure 6: Fluorescence anisotropy as a function of the temperature (symbols) and correlation with the DSC thermograms (lines, data from Figure 5) of the mixtures C12:DPPC (A), D12/3:DPPC (B) and F12/4:DPPC (C), each 1:10 and 1:4. The BP fluorescence is used as the probe signal with excitation at λex = 342 nm and detection at λem = 466 nm; ctot = 0.5 mM.



Page 26 of 39

26

3.5 ATR-IR Spectroscopy. Attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) was used to examine the thermotropic transition of the lipid acyl chains from L' phase into Lα phase. In mixtures with other compounds also featuring alkyl chains it is advantageous to use a phospholipid with deuterated chains to be able to distinguish between CH2 vibrational modes and CD2 vibrations arising from the lipid.31 Therefore, DPPC-d62 with perdeuterated alkyl chains was used as before.22 The alkyl stretching vibrations of the lipid (ῦs(CD2)) and the BP (ῦs(CH2)) are well separated and can be studied independently. When studying mixtures with DPPC-d62 one has to take into account that the thermotropic main transition is shifted to slightly lower temperature (37 °C) compared to normal non-deuterated DPPC. 3.5.1 C12:DPPC-d62 Mixtures. For both mixtures C12:DPPC-d62 = 1:10 and 1:4, the wavenumber of the symmetric CD2- stretching band (ῦs(CD2)) is constant for T < 40 °C (2088.6 cm-1 for C12:DPPC-d62 = 1:10 and 2088.4 cm-1 for 1:4, Figure 7A). This value is typical for phospholipids in the L' phase.31-32, 38 With increasing temperature, the position of this band shifts to higher values and remains constant above 55 °C at 2093.6 cm-1 (1:10 mixture) and 2093.8 cm-1 (1:4 mixture), typical values for fluid lipid phases.31-32,

38

The

change of frequency of the vibrational bands extends over the temperature range of all thermotropic transitions. A distinct first increase of the wavenumber is seen in the region of the temperature of the main transition of DPPC-d62 in both mixtures. This indicates the fluidization of a fraction of the DPPC-d62 molecules not mixed with C12. The rest of the lipid molecules become disordered at higher temperature, where also C12 itself becomes more mobile. The wavenumber of the ῦs(CH2) band of the lateral alkyl chains of C12 remains constant at 2850.5 cm-1 over the whole temperature range (data not shown). Due to their short length, these chains are fluid within the whole temperature range.


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27

3.5.2 D12/3:DPPC-D62 Mixtures. The analysis of the IR-spectra of these mixtures lead to similar results as described above. The ῦs(CD2) band of the lipid of both mixture, 1:10 and 1:4, is constant for T < 35 °C at about 2088.5 cm-1 (L' phase, Figure 7B) and shifts to 2094.0 cm-1 with increasing temperature. The band shift is mainly correlated with the thermotropic main transition of the lipid. Only marginal changes are seen at high temperature in the range of the additional thermotropic transitions. The symmetric stretching vibration of the D12/3 lateral alkyl chains remains constant again at 2851.1 cm-1 indicating fluid alkyl chains of D12/3 (data not shown). For the D12/3:DPPC-d62 mixture we also determined the orientation of the BP with respect to the membrane normal by using oriented multilayers on an ATR crystal as described before for mixtures with the compound B12.22, 39 The two prominent and well separated inplane ring vibrations parallel to the long axis of the backbone to ῦip1(ring) = 1518 cm-1 and

ῦip2(ring) = 1607 cm-1 were used for this purpose.22 Figure S2 shows spectra of a D12/3:DPPC-d62 = 1:4 mixture obtained with s- and p-polarized light. Both bands are much more intense in the spectrum with p-polarization whereas they are hardly visible in the spectrum with s-polarization. The dichroic ratio for both bands is RATR > 10 indicating that the BP is highly oriented with an order parameter S = 0.78 and thus an average tilt angle of less than 25 °. As the D12/3 molecules are not homogeneously distributed in the lipid membrane in the gel phase, the tilt angle determined applies mostly to D12/3 molecules in the BP-rich phase. Similarly, polarization dependent intensity and integral ratios can be seen of the DPPC. The dichroic ratio for the ῦs(CD2) band is much smaller with a value of RATR = 1.18, leading to an order parameter S = 0.63 and an average tilt angle of 30 ° for the lipid chains. This value is typical for pure DPPC membranes in the gel phase and shows again that an almost pure phase of DPPC-d62 exists.



28

3.5.3 E12/7:DPPC Mixtures. A sample of E12/7:DPPC = 1:10, i.e. without using deuterated DPPC-d62, was also studied by ATR-FTIR. The CH2 stretching vibrations of the lipid and the side chain of E12/7 therefore overlap. However, for a 1:10 mixture the band intensity is almost solely due to DPPC resulting from the ten-fold excess of the lipid. Below 38 °C the ῦs(CH2) band position remains approximately constant at 2850.2 cm-1, a typical value for all-trans acyl chain in the gel phase (Figure 7C). In good correlation with the thermotropic main transition, the band position shifts to higher values and remains constant again above 45 °C at 2855.0 cm-1, a value typical for fluid chains in the L-phase. As for mixtures of E12/7 with DPPC, no significant additional thermotropic transitions are seen, the fluidization of the lipid chains occurs at the temperature of the lipid main transition.


Page 28 of 39

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29

C12 : DPPC-d62 2096 1:10

1.5

2094

1.0

) 0.5

2088

0.0

2096 1:4

1.5

2094

1.0

2092

0.5

2090 2088 20

40

50

60

70

0.0 80

Temperature / °C D12/3 : DPPC-d62 2096 1:10

0.5

2088

0.0

2096 1:4

1.5

-1

2090

2094

1.0

2092

0.5

2090 2088 20

30

40

50

60

70

-1

) -1

s(CD2) / cm (

1.0

2092

)

1.5

2094

Cp / kcal mol K (

B

30

-1

2090

-1

-1

s(CD2) / cm (

)

2092

Cp / kcal mol K (

A

0.0 80

Temperature / °C

E12/7:DPPC

4

1:10

2854

-1

-1

2 2852 1 2850

20

-1

)

3

)

2856

Cp / kcal mol K (

C

s(CH2) / cm (

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0 30

40

50

60

70

80

Temperature / °C

Figure 7: Overlay of the band positions of the ῦs(CD2) vibration (symbols) with the DSC thermograms (full lines, data from Figure 5) of BP-DPPC mixtures. A: C12 : DPPC-d62, B: D12/3:DPPC-d62,, and C: E12/7:DPPCHorizontal dotted lines mark typical literature values of the band position for L’ and L phase,31-32, 38 vertical dashed lines mark Tm of DPPC as a reference (clipid = 2 mM).



30

4. CONCLUSIONS In all BP-DPPC systems phase separation occurred at low temperatures. The DPPC-rich phase contains almost no polyphile molecules whereas the BP-rich phase can have different amounts of lipid, depending on the headgroup size and structure of the BP. For mixtures with C12 and F12/4, a significant amount of DPPC is incorporated into the BP-rich phase, whereas for mixtures with D12/3 the DPPC concentration in this segregated BP phase is very low. In all cases, the main thermotropic transition of DPPC is visible in the DSC thermograms indicating that an almost pure DPPC phase is present in all mixtures. The phase separation is still present directly above Tm and a fluid lipid rich phase coexists with at least one BP-rich phase that contains gel phase DPPC. The additional transitions at Tadd are probably caused by a break-up of the BP aggregates and a fluidization of the residual DPPC molecules leading to a homogeneously mixed L phase with highly mobile BP molecules that can diffuse freely within the membrane. For the DPPC-mixtures of C12 and potentially F12/4, CFM images reveal hexagonally patterned phases that are rich in BP, thus, the phase separation can be explained with the honeycomb model analogue to the DPPC-B12 mixtures.22 For DPPC mixtures with E12/7, an almost homogeneous distribution of the lipid dye as well as the BP is observed in CFM. However, using solid state NMR it could be shown, that small aggregates are present over the whole investigated temperature range.28 Therefore, the size of the presumed aggregates must be much smaller than the optical resolution. A transition peak for pure DPPC domains remains visible even at high BP content. At room temperature, the system consists probably of small pure DPPC domains inside a mixed DPPC:E12/7-phase. With increasing temperature, the DPPC domains melt and disperse in the surrounding matrix. Thus, the E12/7-DPPC superstructures are much more (thermally) stable than those for all the other polyphiles.


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31

In the case of the system D12/3-DPPC, phase separated GUVs without a 6-fold symmetric domain shape and only a very small fraction of BP-rich domains are formed at room temperature. Also, the majority of the DPPC molecules undergo the L’-L transition at Tm, which indicates that only little DPPC is located in the BP-rich phase and vice versa. In this case, the honeycomb model is probably not applicable. Due to their larger headgroups, the D12/3 molecules form a lamellar phase that contains virtually only BP with little embedded DPPC, being surrounded by almost pure DPPC. The BP molecules are likely to adapt a trans-membrane orientation and can form intermolecular hydrogen bonds with adjacent BP molecules in addition to --interactions between the conjugated backbones (Figure 8). By binding water molecules via hydrogen bonding, the effective headgroup (hydrophilic moiety) size is further increased, leading to a favourable ratio of the crosssectional area of the headgroups compared to the rigid core. The lipid membrane can thus serve as a template for the aggregation of the BPs molecules into a lamellar arrangement.

Figure 8. Model of phase separated membranes for the system D12/3:DPPC = 1:10 at low temperature, with the lipid in the gel phase. DPPC acyl chains exhibit all-trans conformation (L' phase); the shorter alkyl chains of D12/3 are disordered. Multiple interactions by hydrogen bonds are one of the most important driving forces for the formation of supramolecular aggregates. This is also the case for the behavior of mixtures from BPs and lipids. The BPs with headgroups capable of acting as hydrogen bond donors (C12, D12/3, F12/4) do not form lyotropic phases in water. They are almost insoluble



32

due to the stabilization of the crystalline state by multiple intermolecular hydrogen bonding networks. The same trend holds for mixtures with DPPC bilayers. All BPs with the headgroups capable of acting as H-bond donors (independent of their size) tend to phase separate within the lipid membrane and form large polyphile-rich domains. The exception is E12/7, which cannot form hydrogen bonds on its own. This compound is able to form aggregates with water as H-bond donor, leading to lyotropic systems in the form of multilamellar sheets and vesicle structures. In mixtures with DPPC, CFM of GUVs shows an almost homogeneous distribution of lipids and E12/7 molecules within the membrane. Probably only small aggregates are formed, not resolvable by conventional fluorescence imaging techniques. At higher temperature, the mixed membranes are fluid, but clustering of BP molecules probably persists. Thus by changing the size and the chemical structure of the BP headgroups, structure formation within lipid membranes as templates can be controlled. Overall, this work provides an in depth understanding of the effect of polar headgroup structure on lyotropic self-assembly of -conjugated rods in phospholipid bilayer membranes. The present study reveals vital information on the influence of size and structure of the BP headgroups in order to achieve a successful incorporation into phospholipid membranes. With tailor-made headgroups the miscibility or phase separation over a wide temperature range can be controlled and so the lipid membrane can be utilized as template for BP structure formation and the BP aggregates, although not water soluble can be dispersed in water. This can be a step towards solubilisation of water insoluble large organic compounds in order to enable transport of those molecules in systems akin to drug carriers. Furthermore, ordered organizations of -conjugated systems in lipid membranes and other well defined superstructures are of potential interest for the development of artificial light harvesting systems.40


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33

Supporting Information: Sample preparations and experimental methods TEM, CFM, DSC, ATR-FTIR, detailed synthesis of compound F12/4 with NMR data, infrared spectra of E12/7 in water and plot of the ῦs(CH2) of E12/7 as function of T and ATR-FTIR spectra of D12/3 with p-/s-polarisation, Acknowledgments This work was supported by the Deutsche Forschungsgemeinschaft (DFG) for the research group FOR1145 to C.T. (Ts 39/21-2), A.B. (Bl 182/23-2)), and K.B. (Ba 4887/1-1)



34

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Controlling the Miscibility of X-shaped Bolapolyphiles in Lipid

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