A Diazirine-Modified Membrane Lipid to Study Peptide/Lipid Interactions - Chances and Challenges.

Although incorporation of photo-activatable lipids into membranes potentially opens up novel avenues for investigating interactions with proteins, the question of whether diazirine-modified lipids are suitable for such studies, remains under debate. Focusing on the potential for studying lipid/peptide interactions by cross-linking mass spectrometry (XL-MS), we developed a diazirine-modified lipid (DiazPC), and examined its behaviour in membranes incorporating the model α-helical peptide LAVA20. We observed an unexpected backfolding of the diazirine-containing stearoyl chain of the lipid. This surprising behaviour challenges the potential application of DiazPC for future XL-MS studies of peptide and protein/lipid interactions. The observations made for DiazPC most likely represent a general phenomenon for any type of membrane lipids with a polar moiety incorporated into the alkyl chain. Our finding is therefore of importance for future protein/lipid interaction studies relying on modified lipid probes.

Influence of a single ether bond on assembly, orientation, and miscibility of phosphocholine lipids at the air-water interface.

How does a small change in the structure of a phospholipid affect its supramolecular assembly? In aqueous suspensions, the substitution of one ester linkage in DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) by an ether linkage alters its phase behaviour completely. To unravel the effect of replacing a phospholipid's ester linkage by an ether linkage in lipid monolayers, we characterized pure monolayers of the model lipid DPPC and its sn-2 ether analogue PHPC (1-palmitoyl-2-O-hexadecyl-sn-glycero-3-phosphocholine) as well as mixtures of both by measurements of surface pressure-molecular area (π-Amol) isotherms. In addition, we used infrared reflection absorption spectroscopy (IRRAS) to study lipid condensation, lipid chain orientation, headgroup hydration, and lipid miscibility in all samples. Mixed monolayers consisting of DPPC and PHPC were studied further using epifluorescence microscopy. Our results indicate a strong influence of the sn-2 ether linkage on headgroup hydration and ordering effects in the regions of the apolar chains and the headgroups. Both effects could originate from changes in glycerol conformation. Furthermore, we observed a second plateau in the π-Amol isotherms of DPPC/PHPC mixtures and analysis of the mixed π-Amol isotherms reveals a non-ideal mixing behaviour of both lipids which may be caused by conformational differences in their headgroups.

An Azidolipid Monolayer - Transitions, Miscibility, and UV Reactivity Studied by Infrared Reflection Absorption Spectroscopy.

In this study, we characterized monolayers of an azide-modified lipid at the air-water interface, pure and in its mixtures with the model lipid DPPC, with the aim of proving its potential to be applied for photo-cross-linking with other molecules. We chose a phospholipid bearing a terminal azide group in one of its hydrophobic tails to study its monolayer characteristics with the Langmuir film balance technique. Furthermore, we performed infrared reflection absorption spectroscopy (IRRAS) to get detailed insights into the organization of those monolayers as well as high-resolution mass spectrometry (HRMS) to see the effects of UV-irradiation on the lipids' chemical structure and organization. Our results suggest that in expanded monolayers of pure azide-modified membrane lipids, the azido-terminated chain folds back toward the air-water interface. Above the LE/LC (liquid-expanded/liquid-condensed) phase transition, the chains stretched, and thus, the azide group detaches from the interface. From temperature-dependent monolayer compressions, we evaluated all relevant thermodynamic parameters of the monolayers, such as the phase transition pressure, the critical temperature, and the triple point, and compare them to those of model lipids. For future applications, we studied the miscibility of the azide-modified lipid with DPPC in monolayers and found at least a certain miscibility over all investigated mixing ratios ranging from 10 to 75% of the azidolipid. Finally, we irradiated the azidolipid monolayer with UV light at 305 nm and measured photodissociation of the azide, leading to chemical cross-linking with other lipids, which shows the potential to be used as a cross-linking agent within self-assembled lipid or lipid/protein layers.

Tuning the Thickness of a Biomembrane by Stapling Diamidophospholipids with Bolalipids.

In a biological membrane, proteins require specific lipids of distinctive length and chain saturation surrounding them. The active tuning of the membrane thickness therefore opens new possibilities in the study and manipulation of membrane proteins. Here, we introduce the concept of stapling phospholipids to different degrees of interdigitation depth by mixing 1,3-diamidophospholipids with single-chain bolalipids. The mixed membranes were studied by calorimetric assays, electron microscopy, X-ray, and infrared measurements to provide a complete biophysical characterization of membrane stapling. The matching between the diamidophospholipids and the bolalipids can be so strong as to completely induce a new phase that is more stable than the gel phase of the individual components.

Synthesis and aggregation behaviour of single-chain, 1,32-alkyl-branched bis(phosphocholines) - part 2: lateral chain length triggers self-assembling from sheets to fibres to vesicles.

Six single-chain, 1,32-alkyl-branched bis(phosphocholines) PC-C32(1,32Cm)-PC have been synthesized as model lipids for naturally occurring archaeal membrane lipids. The preparation of these bipolar amphiphiles bearing lateral alkyl chains of different lengths (C4-C15) was realized using a Cu-catalyzed Grignard bis-coupling reaction of various primary alkyl-branched bromides as side parts and a 1,22-dibromide as the centre part. The aggregation behaviour of these bolalipids in water was initially investigated by differential scanning calorimetry and transmission electron microscopy. As a main result, the types of aggregates found and their stability upon heating were strongly connected to the length of the lateral alkyl chain of the bolalipid: short and long lateral chains led to lamellar structures, whereas side chains of medium length led to fibrous aggregates. In future, these bolalipids could be used to produce tailored and stabilized liposomes for oral drug delivery.

Azide-Modified Membrane Lipids: Miscibility with Saturated Phosphatidylcholines.

In this study, we describe the miscibility of four azide-modified membrane phospholipids (azidolipids) with conventional phospholipids. The azidolipids bear an azide group at different positions of the sn-1 or sn-2 alkyl chain and they further differ in the type of linkage (ester vs ether) of the sn-2 alkyl chain. Investigations regarding the miscibility of the azidolipids with bilayer-forming phosphatidylcholines will evaluate lipid mixtures that are suitable for the production of stable azidolipid-doped liposomes. These vesicles then serve as model membranes for the incorporation of model peptides or proteins in the future. The miscibility of both types of phospholipids was studied by calorimetric assays, electron microscopy, small-angle X-ray scattering, infrared spectroscopy, and dynamic light scattering to provide a complete biophysical characterization of the mixed systems.

Impact of Headgroup Asymmetry and Protonation State on the Aggregation Behavior of a New Type of Glycerol Diether Bolalipid.

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 methyl-branched 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)C32-PC) or by a phosphodimethylethanolamine headgroup (PC-Gly(2C16Me)C32-Me2PE). The temperature- and pH-dependent 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 PC-Gly(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 PC-Gly(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.

Lysine-based amino-functionalized lipids for gene transfection: 3D phase behaviour and transfection performance.

Based on previous work, the influence of the chain composition on the physical-chemical properties of five new transfection lipids (TH10, TT10, OH10, OT10 and OO10) containing the same lysine-based head group has been investigated in aqueous dispersions. For this purpose, the chain composition has been gradually varied from saturated tetradecyl (T, C14:0) and hexadecyl (H, C16:0) chains to longer but unsaturated oleyl (O, C18:1) chains with double bonds in the cis configuration. In this work, the lipid dispersions have been investigated in the absence and presence of the helper lipid DOPE and calf thymus DNA by small-angle and wide-angle X-ray scattering (SAXS/WAXS) supplemented by differential scanning calorimetry (DSC), attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR) and Fourier-transform Raman spectroscopy (FTRS). Lamellar and inverted hexagonal mesophases have been observed in single-component systems. In the binary mixtures, the aggregation behaviour changes with an increasing amount of DOPE from lamellar to cubic. The lipid mixtures with DNA show a panoply of mesophases. Interestingly, TT10 and OT10 form cubic lipoplexes, whereas OO10 complexes the DNA sandwich-like between lipid bilayers in a lamellar lipoplex. Surprisingly, the latter is the most effective lipoplex.

Azide-Modified Membrane Lipids: Synthesis, Properties, and Reactivity.

In the present work, we describe the synthesis and the temperature-dependent behavior of photoreactive membrane lipids as well as their capability to study peptide/lipid interactions. The modified phospholipids contain an azide group either in the middle part or at the end of an alkyl chain and also differ in the linkage (ester vs ether) of the second alkyl chain. The temperature-dependent aggregation behavior of the azidolipids was studied using differential scanning calorimetry (DSC), Fourier-transform infrared (FTIR) spectroscopy, and small-angle X-ray scattering (SAXS). Aggregate structures were visualized by stain and cryo transmission electron microscopy (TEM) and were further characterized by dynamic light scattering (DLS). We show that the position of the azide group and the type of linkage of the alkyl chain at the sn-2 position of the glycerol influences the type of aggregates formed as well as their long-term stability: P10AzSPC and r12AzSHPC show the formation of extrudable liposomes, which are stable in size during storage. In contrast, azidolipids that carry a terminal azido moiety either form extrudable liposomes, which show time-dependent vesicle fusion (P15AzPdPC), or self-assemble in large sheet-like, nonextrudable aggregates (r15AzPdHPC) where the lipid molecules are arranged in an interdigitated orientation at temperatures below Tm (LßI phase). Finally, a P10AzSPC:DMPC mixture was used for photochemically induced cross-linking experiments with a transmembrane peptide (WAL-peptide) to demonstrate the applicability of the azidolipids for the analysis of peptide/lipid interactions. The efficiency of photo-cross-linking was monitored by attenuated total reflection infrared (ATR-IR) spectroscopy and mass spectrometry (MS).

Synthesis and EPR-spectroscopic characterization of the perchlorotriarylmethyl tricarboxylic acid radical (PTMTC) and its 13C labelled analogue (13C-PTMTC).

A hydrophilic tris(tetrachlorotriaryl)methyl (tetrachloro-TAM) radical labelled 50% with 13C at the central carbon atom was prepared. The mixture of isotopologue radicals was characterised by continuous wave and pulsed X-band electron paramagnetic spectroscopy (EPS). For the pharmaceutical and medical applications planned, the quantitative influence of oxygen, viscosity, temperature and pH on EPR line widths was studied in aqueous buffer, DMSO, water-methanol and water-glycerol mixtures. Under in vivo conditions, pH can be disregarded. There is a clear oxygen dependence of the width of the 12C isotopologue single EPR line in aqueous solutions while changes in rotational motion (viscosity) are observable only in the doublet lines of the central carbon of the 13C isotopologue. The tetrachloro-TAM proved to be very stable as a solid. Its thermal decay was determined quantitatively by thermal annealing. Towards ascorbic acid as a reducing agent and towards an oocyte cell extract it had a half-life of approx. 60 and 10 min. Thus for in vivo applications, 50% 13C tetrachloro-TAMs are suitable for selective and simultaneous oxygen and macroviscosity measurements in a formulation, e.g. nanocapsules.

Aggregation behaviour of a single-chain, phenylene-modified bolalipid and its miscibility with classical phospholipids.

In the present work, we describe the synthesis of a single-chain, phenylene-modified bolalipid with two phosphocholine headgroups, PC-C18pPhC18-PC, using a Sonogashira cross-coupling reaction as a key step. The aggregation behaviour was studied as a function of temperature using transmission electron microscopy (TEM), differential scanning calorimetry (DSC), Fourier-transform infrared (FTIR) spectroscopy, and small angle neutron scattering (SANS). We show that our new bolalipid self-assembles into nanofibres, which transform into flexible nanofibres at 27 °C and further to small elongated micelles at 45 °C. Furthermore, the miscibility of the bolalipid with bilayer-forming phosphatidylcholines (DMPC, DPPC, and DSPC) was investigated by means of DSC, TEM, FTIR, and small angle X-ray scattering (SAXS). We could show that the PC-C18pPhC18-PC is partially miscible with saturated phosphatidylcholines; however, closed lipid vesicles with an increased thermal stability were not found. Instead, bilayer fragments and disk-like aggregates are formed.

Investigation of Binary Lipid Mixtures of a Three-Chain Cationic Lipid with Phospholipids Suitable for Gene Delivery.

In the present work, we characterize binary lipid mixtures consisting of a three-chain amino-functionalized cationic lipid (DiTT4) with different phospholipids, namely, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). The mixing behavior was investigated by differential scanning calorimetry (DSC). Additionally, aqueous dispersions of the binary mixtures were characterized by means of dynamic light scattering (DLS), laser Doppler electrophoresis, and transmission electron microscopy (TEM) to get further information about particle size, charge, and shape. The complex formation between different binary lipid mixtures and plasmid DNA (pDNA) was investigated by zeta-(ζ)-potential (laser Doppler electrophoresis) and DLS measurements, and the lipid/DNA complexes (lipoplexes) were screened for efficient DNA transfer (transfection) in cell culture. Finally, efficient lipid compositions were investigated with respect to serum stability. This work provides a detailed characterization of the cationic lipid mixtures as foundation for further research. Efficient gene transfer in the presence of serum was demonstrated for selected lipoplexes showing their capability to be used as high-potency gene delivery vehicles.

Lamellar versus Micellar Structures-Aggregation Behavior of a Three-Chain Cationic Lipid Designed for Nonviral Polynucleotide Transfer.

The front cover artwork is provided by the groups of Prof. Bodo Dobner, Prof. Andreas Langner, and research partners Dr. Gerd Hause, Dr. Simon Drescher, and Dr. Annette Meister (MLU Halle-Wittenberg) as well as the group of Prof. Gerald Brezesinski (MPI of Colloids and Interfaces). The image shows the space-filling model of a three-chain amino-functionalized lipid designed for gene transfer and the preferred pH-dependent aggregates (multilamellar stacks, vesicles, rod-like micelles). The background shows a Cryo-TEM image of rod-like micelles. Read the full text of the article at 10.1002/cphc.201500188.

Lamellar versus Micellar Structures-Aggregation Behavior of a Three-Chain Cationic Lipid Designed for Nonviral Polynucleotide Transfer.

The aggregation behavior of a cationic lipid, N-[6-amino-1-oxo-1-(N-tetradecylamino)hexan-(2S)-2-yl]-N'-{2-[N,N-bis(2-aminoethyl)amino]ethyl}-2,2-ditetradecylpropandiamide (DiTT4), is investigated in aqueous dispersions at different pH values (5, 7.3, and 10). An unusual aggregation behavior is observed whereby DiTT4 forms bilayer structures at pH 10 and 7.3. At pH 5, rod-like micelles are the dominant aggregate form. The thermotropic and lyotropic behavior is studied using differential scanning calorimetry, small-angle X-ray scattering, and FTIR spectroscopy. In addition, investigations at the air-water interface are performed by recording area-pressure-isotherms and infrared reflection-absorption (IRRA) spectra. Complementary dynamic light scattering experiments and transmission electron microscopy (TEM and cryoTEM) are also used. The ability of DiTT4 to complex plasmid DNA is investigated using fluorescence techniques and zeta potential measurements. Cell culture experiments demonstrate the ability of DiTT4 to enhance plasmid transfer in A549 cells.

Highly asymmetrical glycerol diether bolalipids: synthesis and temperature-dependent aggregation behavior.

In the present work, we describe the synthesis and temperature-dependent aggregation behavior of two examples of a new class of highly asymmetrical glycerol diether bolaphospholipids. The bolalipids contain a long alkyl chain (C32) bound to glycerol in the sn-3 position, carrying a hydroxyl group at the ω position. The C16 alkyl chain in the sn-2 position either possesses a racemic methyl branch at the 10 position of the short alkyl chain (lipid II) or does not (lipid I). The sn-1 position of the glycerol is linked to a zwitterionic phosphocholine moiety. The temperature-dependent aggregation behavior of both bolalipids was studied using differential scanning calorimetry (DSC), Fourier-transform infrared (FTIR) spectroscopy, and X-ray scattering. Aggregate structures were visualized by transmission electron microscopy (TEM). We show that both bolalipids self-assemble into large lamellar sheetlike aggregates. Closed lipid vesicles or other aggregate structures such as tubes or nanofibers, as usually found for diglycerol tetraether lipids, were not observed. Within the lamellae the bolalipid molecules are arranged in an antiparallel (interdigitated) orientation. Lipid I, without an additional methyl moiety in the short alkyl chain, shows a lamellar phase with high crystallinity up to a temperature of 34 °C, which was not observed before for other phospholipids.

Synthesis, Characterization, and Nanoencapsulation of Tetrathiatriarylmethyl and Tetrachlorotriarylmethyl (Trityl) Radical Derivatives­A Study To Advance Their Applicability as in Vivo EPR Oxygen Sensors.

Tissue oxygenation plays an important role in the pathophysiology of various diseases and is often a marker of prognosis and therapeutic response. EPR (ESR) is a suitable noninvasive oximetry technique. However, to reliably deploy soluble EPR probes as oxygen sensors in complex biological systems, there is still a need to investigate and improve their specificity, sensitivity, and stability. We reproducibly synthesized various derivatives of tetrathiatriarylmethyl and tetrachlorotriarylmethyl (trityl) radicals. Hydrophilic radicals were investigated in aqueous solution mimicking physiological conditions by, e.g., variation of viscosity and ionic strength. Their specificity was satisfactory, but the oxygen sensitivity was low. To enhance the capability of trityl radicals as oxygen sensors, encapsulation into oily core nanocapsules was performed. Thus, different lipophilic triesters were prepared and characterized in oily solution employing oils typically used in drug formulations, i.e., middle-chain triglycerides and isopropyl myristate. Our screening identified the deuterated ethyl ester of D-TAM (radical 13) to be suitable. It had an extremely narrow single EPR line under anoxic conditions and excellent oxygen sensitivity. After encapsulation, it retained its oxygen responsiveness and was protected against reduction by ascorbic acid. These biocompatible and highly sensitive nanosensors offer great potential for future EPR oximetry applications in preclinical research.

The headgroup (a)symmetry strongly determines the aggregation behavior of single-chain phenylene-modified bolalipids and their miscibility with classical phospholipids.

In the present work, we describe the synthesis of two single-chain phenylene-modified bolalipids, namely PC-C17pPhC17-PC and PC-C17pPhC17-OH, with either symmetrical (phosphocholine) or asymmetrical (phosphocholine and hydroxyl) headgroups using a Sonogashira cross-coupling reaction as key step. The temperature-dependent aggregation behavior of both bolalipids in aqueous suspension was studied using transmission electron microscopy (TEM), differential scanning calorimetry (DSC), Fourier-transform infrared (FTIR) spectroscopy, small angle neutron scattering (SANS), and X-ray scattering. We show that different headgroup symmetries lead to a change in the aggregation behavior: Whereas PC-C17pPhC17-PC forms nanofibers with a diameter of 5.7 nm that transform into small ellipsoidal micelles at 23 °C, the PC-C17pPhC17-OH self-assembles into lamellae with bolalipid molecules in an antiparallel orientation up to high temperatures. Furthermore, the mixing behavior of both bolalipids with bilayer-forming phospholipids (DPPC and DSPC) was studied by means of DSC and TEM. The aim was to stabilize bilayer membranes formed of phospholipids in order to improve these mixed lipid vesicles for drug delivery purposes. We show that the symmetrical PC-C17pPhC17-PC is miscible with DPPC and DSPC; however, closed lipid vesicles are not observed, and elongated micelles and bilayer fragments are found instead. In contrast, the asymmetrical PC-C17pPhC17-OH shows no miscibility with phospholipids at all.

New micellar transfection agents.

Two novel micelle-forming amino-functionalized lipids (OT6 and TT6) bearing two alkyl chains connected to a large positively charged hexavalent headgroup, which might be interesting polynucleotide transferring agents with the advantage of an easy and reproducible production of micelle dispersions, have been characterized. The critical micelle concentration (cmc) of both lipids has been determined by two different methods, namely, isothermal titration calorimetry (ITC) and 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence experiments. In addition, the lipid dispersions were studied as a function of temperature using differential scanning calorimetry (DSC), dynamic light scattering (DLS), Fourier-transform infrared (FT-IR) spectroscopy, and cryo-transmission electron microscopy (cryo-TEM). The OT6 and TT6 micelles effectively complex DNA as determined by ITC and DSC measurements. In addition, DLS and ζ-potential measurements were performed to determine lipoplex formulations that exhibit colloidal stability. Finally, the structures of OT6/DNA complexes were investigated by means of X-ray scattering and TEM.

Structure-property relationships in a series of diglycerol tetraether model lipids and their lyotropic assemblies: the effect of branching topology and chirality.

Three novel diglycerol tetraether lipids with one membrane-spanning chain have been synthesized. These lipids contain only two or four racemic methyl branches at selected positions of the hydrophobic chains in contrast to natural lipids from archaebacterial membranes with an isoprenoid substitution pattern. The insertion of the methyl moieties was realized starting from either (RS)-citronellyl bromide or the inexpensive methyl malonic acid ethyl ester. For chain elongation the Cu-catalysed Grignard coupling reaction was used. The preparation of diglycerol tetraethers was either performed by condensing suitable blocked monoglycerol diethers by Grubbs metathesis or by reaction of the transmembrane C32-chain with blocked glycerols followed by further alkylation steps. Finally, we could show that the resulting lipids can form closed lipid vesicles comparable to the optically pure counterparts. Therefore, these much simpler lipids compared to the natural lipids from archaebacterial membranes are also suitable for preparation of stable tailored liposomes.

The role of phospholipids in drug delivery formulations - Recent advances presented at the Researcher's Day 2023 Conference of the Phospholipid Research Center Heidelberg.

This Focus on Meetings contribution summarizes recent advances in the research on phospholipids and their applications for drug delivery and analytical purposes that have been presented at the hybrid Researcher's Day 2023 Conference of the Phospholipid Research Center (PRC), held on July 3-5, 2023, in Bad Dürkheim, Germany. The PRC is a non-profit organization focused on expanding and sharing scientific and technological knowledge of phospholipids in pharmaceutical and other applications. This is accomplished by, e.g., funding doctoral and postdoctoral research projects. The progress made with these projects is presented at the Researcher's Day Conference every two years. Four main topics were presented and discussed in various lectures: (1) formulation of phospholipid-based nanocarriers, (2) therapeutic applications of phospholipids and phospholipid-based nanocarriers, (3) phospholipids as excipients in oral, dermal, and parenteral dosage forms, and (4) interactions of phospholipids and phospholipid-based vesicles in biological environment and their use as analytical platforms.