Azide- and diazirine-modified membrane lipids: Physicochemistry and applicability to study peptide/lipid interactions via cross-linking/mass spectrometry.

Although the incorporation of photo-activatable lipids into membranes potentially opens new avenues for studying interactions with peptides and proteins, the question of whether azide- or diazirine-modified lipids are suitable for such studies remains controversial. We have recently shown that diazirine-modified lipids can indeed form cross-links to membrane peptides after UV activation and that these cross-links can be precisely determined in their position by mass spectrometry (MS). However, we also observed an unexpected backfolding of the lipid's diazirine-containing stearoyl chain to the membrane interface challenging the potential application of this modified lipid for future cross-linking (XL)-MS studies of protein/lipid interactions. In this work, we compared an azide- (AzidoPC) and a diazirine-modified (DiazPC) membrane lipid regarding their self-assembly properties, their mixing behavior with saturated bilayer-forming phospholipids, and their reactivity upon UV activation using differential scanning calorimetry (DSC), dynamic light scattering (DLS), small-angle X-ray scattering (SAXS), transmission electron microscopy (TEM), and MS. Mixtures of both modified lipids with DMPC were further used for photo-chemically induced XL experiments with a transmembrane model peptide (KLAW23) to elucidate similarities and differences between the azide and the diazirine moiety. We showed that both photo-reactive lipids can be used to study lipid/peptide and lipid/protein interactions. The AzidoPC proved easier to handle, whereas the DiazPC had fewer degradation products and a higher cross-linking yield. However, the problem of backfolding occurs in both lipids; thus, it seems to be a general phenomenon.

Investigating bolalipids as solubilizing agents for poorly soluble drugs: Effects of alkyl chain length on solubilization and cytotoxicity.

Synthetic single-chain bolalipids with symmetrical headgroups have shown potential in various pharmaceutical applications, such as the stabilization of liposome bilayers. Despite their amphiphilic character, synthetic bolalipids have not yet been investigated for their suitability as solubilizing agents for poorly soluble drug compounds. In this study, three synthetic single-chain bolalipids with increasing alkyl chain lengths (C22, C24 and C26) were investigated. All three bolalipids were able to achieve an increased solubility of the model drug, mefenamic acid, by approximately 180% in a pH 7.4 buffer compared to only a 102-105% increase achieved by sodium dodecyl sulfate (SDS) or the non-ionic surfactant pegylated hydroxystearate (PEG-HS). Subsequently, interfacial activity of bolalipids and their ability to destabilize liposomal bilayers were investigated. The C22 bolalipid exhibited a consistently lower interfacial activity, which was consistent with its significantly lower cytotoxicity in the macrophage-like cell line, J774. A1, compared to C24 and C26 counterparts. The mean IC50 values of the bolalipids tested (0.035-0.093 mM) were approximately 4-100-fold lower than that of SDS (0.401 mM) or PEG-HS (0.922 mM), with the mechanism of toxicity linked to increased cell membrane permeability, as is expected for surfactants. In summary, evidence from this study shows that decreasing the length of the bolalipid alkyl linker from C26 to C22 resulted in a significantly decreased cytotoxicity with no loss in drug solubilization efficiency.

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.

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.

Bolalipid-Doped Liposomes: Can Bolalipids Increase the Integrity of Liposomes Exposed to Gastrointestinal Fluids?

The use of archaeal lipids and their artificial analogues, also known as bolalipids, represents a promising approach for the stabilization of classical lipid vesicles for oral application. In a previous study, we investigated the mixing behavior of three single-chain alkyl-branched bolalipids PC-C32(1,32Cn)-PC (n = 3, 6, 9) with either saturated or unsaturated phosphatidyl-cholines. We proved, that the bolalipids PC-C32(1,32C6)-PC and PC-C32(1,32C9)-PC show miscibility with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). In the present work, we extended our vesicle system to natural lipid mixtures using phosphatidylcholine from soy beans, and we investigated the effect of incorporated bolalipids on the integrity of these mixed liposomes (bolasomes) in different gastrointestinal fluids using a dithionite assay and a calcein release assay in combination with particle size measurements. Finally, we also studied the retention of calcein within the bolasomes during freeze-drying. As a main result, we could show that in particular PC-C32(1,32C6)-PC is able to increase the stability of bolasomes in simulated gastric fluid-a prerequisite for the further use of liposomes as oral drug delivery vehicles.

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.

Mixing behaviour of bilayer-forming phosphatidylcholines with single-chain alkyl-branched bolalipids: effect of lateral chain length.

Liposomes are a promising class of drug delivery vehicles. However, no liposomal formulation has been approved for an oral application so far, due to stability issues of the liposomes in the gastrointestinal tract. Herein, we investigate the miscibility of three novel single-chain alkyl-branched bolalipids PC-C32(1,32Cn)-PC (n = 3, 6, 9) with either saturated or unsaturated phosphatidylcholines by means of differential scanning calorimetry (DSC), transmission electron microscopy (TEM) of stained samples, vitrified specimens, or replica of freeze-fractured samples, and dynamic light scattering (DLS). The novel bolalipids contain lateral alkyl chains of different length in 1- and 32-position of the long membrane-spanning C32 alkyl chain. We will show for the first time that these single-chain alkyl-branched bolalipids show a miscibility with bilayer-forming phospholipids-by maintaining the vesicular aggregate structure-due to the lateral alkyl substituents located next to the phosphocholine headgroup of the bolalipid. We are convinced that these alkyl side chains are able to fill the void volume, which is created when unmodified single-chain bolalipids are inserted in a transmembrane fashion into a phospholipid bilayer. Consequently, the miscibility of our alkyl-chained bolalipids with bilayer-forming phospholipids rose with increasing lengths of the lateral alkyl chain of the bolalipid. Finally, we were successful in preparing liposomes from various bolalipid/phospholipid mixtures, which were stable in size upon storage for at least 21 days. These mixed liposomes (bolasomes) could be used as oral drug delivery systems in the near future.

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).