Ionizable lipids based on branched fatty acids - An explorative study on Langmuir monolayers.

Ionizable lipids are a class of pharmaceutical excipients with a main application in lipid nanoparticles for nucleic acid delivery. New ionizable lipids are needed to tune characteristics of lipid-based nucleic acid delivery systems, e.g. stability, nucleic acid loading capacity and binding strength, as well as bio-distribution. Herein, we present the synthesis of three novel ionizable lipids as putative excipients for lipid-based nucleic acid delivery systems. Langmuir monolayer experiments with classical surface pressure/area isotherm evaluation were used to understand the self-assembly behavior of the lipids. Additional experiments with surface sensitive techniques, namely grazing incidence x-ray scattering and infrared reflection-absorption spectroscopy (IRRAS), were performed to understand structural characteristics of lipid associates. The latter technique was also used to investigate the nucleic acid binding process between DNA and the ionizable lipids. Finally, first transfection experiments with the novel lipids formulated as cationic liposomes were performed providing first efficacy data. Although the alkyl chain pattern was comparable for all three ionizable lipids, the results demonstrated that with increasing head-group size the DNA binding capacity changed and the alkyl chain fluidity was increased. The lipid with the lowest phase transition temperature and the smallest packing parameter showed the highest DNA transfer efficiency.

Two- and Three-Dimensional Physical-Chemical Characterization of CER[AP]: A Study of Stereochemistry and Chain Symmetry.

The stratum corneum represents the first skin barrier against chemical and physical damage. These unique properties are based on its peculiar lipid composition with ceramides (CERs) as the main protagonists. In this study, the structural and chemical properties of the α-OH phytosphingosine [AP] CER class have been investigated. α-OH CERs are present in the stratum corneum in their d-forms; however, in most model systems the diastereomer mixture with the synthetically produced l-form is used. The d-form is well-known to form a hydrogen bonding network that helps to reduce the permeability of the lipid matrix, while the l-form does not show any hydrogen bonding network formation. In this paper, 2D (monolayers) and 3D (aqueous dispersions) models have been used to thoroughly study the physical-chemical behaviors of CER[AP] diastereomers taking into account how the symmetry of the chain pattern influences the behavior of the molecules. The chains of both diastereomers arrange in an oblique unit cell, but only the d-CER[AP] forms a supramolecular lattice (subgel phase) in both model systems. Interestingly, the chain pattern does not play any role in structure formation since the hydrogen bonding network dictates the packing properties. The 1:1 mixture of the diastereomers phase separates into two domains: one is composed of practically pure d-form and the other one is composed of a mixture of the l-form with a certain amount of d-form molecules.

The Impact of Alkyl-Chain Purity on Lipid-Based Nucleic Acid Delivery Systems - Is the Utilization of Lipid Components with Technical Grade Justified?

The physicochemical properties and transfection efficacies of two samples of a cationic lipid have been investigated and compared in 2D (monolayers at the air/liquid interface) and 3D (aqueous bulk dispersions) model systems using different techniques. The samples differ only in their chain composition due to the purity of the oleylamine (chain precursor). Lipid 8 (using the oleylamine of technical grade for cost-efficient synthesis) shows lateral phase separation in the Langmuir layers. However, the amount of attached DNA, determined by IRRAS, is for both samples the same. In 3D systems, lipid 8 p forms cubic phases, which disappear after addition of DNA. At physiological temperatures, both lipids (alone and in mixture with cholesterol) assemble to lamellar aggregates and exhibit comparable DNA delivery efficiency. This study demonstrates that non-lamellar structures are not compulsory for high transfection rates. The results legitimate the utilization of oleyl chains of technical grade in the synthesis of cationic transfection lipids.

DNA Delivery Systems Based on Peptide-Mimicking Cationic Lipids-The Effect of the Co-Lipid on the Structure and DNA Binding Capacity.

In continuation of previous work, we present a new promising DNA carrier, OO4, a highly effective peptide-mimicking lysine-based cationic lipid. The structural characteristics of the polynucleotide carrier system OO4 mixed with the commonly used co-lipid DOPE and the saturated phospholipid DPPE have been studied in two-dimensional and three-dimensional model systems to understand their influence on the physical-chemical properties. The phase behavior of pure OO4 and its mixtures with DOPE and DPPE was studied at the air-water interface using a Langmuir film balance combined with infrared reflection-absorption spectroscopy. In bulk, the self-assembling structures in the presence and absence of DNA were determined by small-angle and wide-angle X-ray scattering. The amount of adsorbed DNA to cationic lipid bilayers was measured using a quartz crystal microbalance. The choice of the co-lipid has an enormous influence on the structure and capability of binding DNA. DOPE promotes the formation of nonlamellar lipoplexes (cubic and hexagonal structures), whereas DPPE promotes the formation of lamellar lipoplexes. The correlation of the observed structures with the transfection efficiency and serum stability indicates that OO4/DOPE 1:3 lipoplexes with a DNA-containing cubic phase encapsulated in multilamellar structures seem to be most promising.

The long periodicity phase (LPP) controversy part I: The influence of a natural-like ratio of the CER[EOS] analogue [EOS]-br in a CER[NP]/[AP] based stratum corneum modelling system: A neutron diffraction study.

This study used neutron diffraction to investigate a ceramide-[NP] C24/[AP] C24 /[EOS]-br C30/cholesterol/lignoceric acid (0.6: 0.3: 0.1: 0.7: 1) based stratum corneum modelling system. By adding specifically deuterated ceramides-[NP]-D3, [AP]-D3, and [EOS]-br-D3, detailed information on the lamellar and the nanostructure of the system was obtained. For the short periodicity phase a natural-like lamellar repeat distance of 5.47 ±â€¯0.02 nm was observed, similar to the [NP]/[AP] base system without the [EOS]-br. Unlike in this system the ceramides here were slightly tilted, hinting towards a slightly less natural arrangement. Due to the deuteration it was possible to observe that the long ceramide chains were overlapping in the lamellar mid-plane. This is considered to be an important feature for the natural stratum corneum. Despite the presence of a ceramide [EOS] analogue - able to form a long phase arrangement - no distinct long periodicity phase was formed, despite a slightly higher than natural ω-acyl ceramide ratio of 10 mol%. The deuterated variant of this ceramide determined that the very long ceramide was integrated into the short periodicity phase, spanning multiple layers instead. The - compared to the base system - unchanged repeat distance highlights the stability of this structure. Furthermore, the localisation of the very long ceramide in the short periodicity phase indicates the possibility of a crosslinking effect and thus a multilayer stabilizing role for the ceramide [EOS]. It can be concluded, that additionally to the mere presence of ceramide-[EOS] more complex conditions have to be met in order to form this long phase. This has to be further investigated in the future.

Interactions of Cationic Lipids with DNA: A Structural Approach.

Colloidal nucleic acid carrier systems based on cationic lipids are a promising pharmaceutical tool in the implementation of gene therapeutic strategies. This study demonstrates the complex behavior of DNA at the lipid-solvent interface facilitating structural changes of the lyotropic liquid-crystalline phases. For this study, the structural properties of six malonic acid based cationic lipids were determined using small- and wide-angle X-ray scattering (SAXS and WAXS) as well as differential scanning calorimetry (DSC). Selected lipids (lipid 3 and lipid 6) with high nucleic acid transfer activity have been investigated in detail because of the strong influence of the zwitterionic helper lipid 1,2-di(9 Z-octadecenoyl)- sn-glycero-3-phosphoethanolamine (DOPE) on the structural properties as well as of the complex formation of lipid-DNA complexes (lipoplexes). In the case of lipid 3, DNA stabilizes a metastable cubic mesophase with Im3 m symmetry and an Im3 m Qαc lipoplex is formed, which is rarely described for DNA lipoplexes in literature. In the case of lipid 6, a cubic mesophase with Im3 m symmetry turns into a fluid lamellar phase while mixing with DOPE and complexing DNA.

Impact of the ceramide subspecies on the nanostructure of stratum corneum lipids using neutron scattering and molecular dynamics simulations. Part I: impact of CER[NS].

For this study mixtures based on the ceramides [NS] (NS = non-hydroxy-sphingosine) and [AP] (AP = α-hydroxy-phytosphingosine) in a 2:1 and 1:2 ratio, together with cholesterol and lignoceric acid, were investigated. These mixtures are modelling the uppermost skin layer, the stratum corneum. Neutron diffraction, utilizing specifically deuterated ceramide molecules, was used to obtain a maximum amount of experimental detail. Highly detailed molecular dynamics simulations were used to generate even more information from the experimental data. It was possible to observe a single lamellar phase for both systems. They had a lamellar repeat distance of 5.43 ±â€¯0.05 nm for the [NS]/[AP] 2:1 and a slightly shorter one of 5.34 ±â€¯0.05 nm for the 1:2 system. The structure and water content was uninfluenced by excess humidity. Both the experimental and simulation data indicated slightly tilted ceramides, with their C24 chains overlapping in the lamellar mid-plane. This arrangement is well comparable to systems investigated before. The structure of both systems, except for the differing repeat distance, looks similar at first. However, on a smaller scale there were various distinct differences, demonstrating only low redundancy between the different ceramide species, despite only minor chemical differences. The mainly ceramide [AP] determined 1:2 system has a slightly smaller repeat distance. This is a result of a tighter arrangement of the lipids chain along the bilayer normal and increased overlapping of the long chains in the lamellar middle. For the CER[NS] some novel features could be shown, despite it being the overall most investigated ceramide. These include the low adaptability to changed lateral interactions, leading to an increased chain opening. This effect could explain its low miscibility with other lipids. The investigated model systems allows it to directly compare results from the literature which have used ceramide [NS] to the most recent studies using the phytosphingosine ceramides such as ceramide [AP].

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: the influence of the chain composition on 2D properties.

The influence of the chain composition on the physical-chemical properties will be discussed for five transfection lipids containing the same lysine-based head group. For this purpose, the chain composition will be gradually varied from saturated tetradecyl (C14:0) and hexadecyl (C16:0) chains to longer but unsaturated oleyl (C18:1) chains with double bonds in the cis configuration. In this work, we investigated the lipids as Langmuir monolayers at the air-water-interface in the absence and presence of calf thymus DNA applying different techniques such as infrared reflection absorption spectroscopy (IRRAS) and grazing incidence X-ray diffraction (GIXD). The replacement of saturated tetradecyl (C14:0) and hexadecyl (C16:0) chains by unsaturated oleyl (C18:1) chains increases the fluidity of the lipid monolayer: TH10 < TT10 < OH10 < OT10 < OO10 resulting in a smaller packing density. TH10 forms the stiffest and OO10 the most fluid monolayer in this structure-property study. OO10 has a higher protonation degree compared to the saturated lipids TT10 and TH10 as well as to the hybrids OT10 and OH10 because of a better accessibility of the amine groups. Depending on the bulk pH, different scenarios of DNA coupling to the lipid monolayers have been proposed.

Investigation of a CER[NP]- and [AP]-Based Stratum Corneum Modeling Membrane System: Using Specifically Deuterated CER Together with a Neutron Diffraction Approach.

Neutron diffraction was used as a tool to investigate the lamellar as well as molecular nanostructure of ceramide-[NP]/ceramide-[AP]/cholesterol/lignoceric acid model systems with a nativelike 2:1 ratio and a 1:2 ratio to study the influence of the ceramide-[AP]. By using mixtures together with cholesterol and free fatty acids as well as a humidity and temperature chamber while measuring, natural conditions were simulated as closely as possible. Despite its simplicity, the system simulated the native stratum corneum lipid matrix fairly closely, showing a similar lamellar thickness with a repeat distance of 5.45 ± 0.1 nm and a similar arrangement with overlapping long C24 chains. Furthermore, despite the very minor chemical difference between ceramide-[NP] and ceramide-[AP], which is only a single OH group, it was possible to demonstrate substantial differences between the structural influence of the two ceramides. Ceramide-[AP] could be concluded to be arranged in such a way that its C24 chain in both ratios is somehow shorter than that of ceramide-[NP], not overlapping as much with the opposite lamellar leaflet. Furthermore, in the unnatural 1:2 ratio, the higher ceramide-[AP] content causes an increased tilt of the ceramide acyl chains. This leads to even less overlapping within the lamellar midplane, whereas the repeat distance stays the same as for the ceramide-[NP]-rich system. In this nativelike 2:1 ratio, the chains are arranged mostly straight, and the long C24 chains show a broad overlapping region in the lamellar midplane.

Determination of the influence of C24 D/(2R)- and L/(2S)-isomers of the CER[AP] on the lamellar structure of stratum corneum model systems using neutron diffraction.

This study was able to investigate the different influence of the d- and l-ceramide [AP] on the lamellar as well as molecular nanostructure of stratum corneum simulating lipid model mixtures. In this case, neutron diffraction together with specifically deuterated ceramide was used as an effective tool to investigate the lamellar and the molecular nanostructure of the mixtures. It could clearly be demonstrated, that both isomers show distinctly different characteristics, even though the variation between both is only a single differently arranged OH-group. The l-ceramide [AP] promotes a crystalline like phase behaviour even if mixed with ceramide [NP], cholesterol and free fatty acids. The d-ceramide [AP] only shows crystalline-like features if mixed only with cholesterol and free fatty acids but adopts a native-like behaviour if additionally mixed with ceramide [NP]. It furthermore demonstrates that the l-ceramide [AP] should not be used for any applications concerning ceramide substitution. It could however possibly serve its own purpose, if this crystalline like behaviour has some kind of positive influence on the SC or can be utilized for any practical applications. The results obtained in this study demonstrate that the diastereomers of ceramide [AP] are an attractive target for further research because their influence on the lamellar as well as the nanostructure is exceptionally strong. Additionally, the results furthermore show a very strong influence on hydration of the model membrane. With these properties, the d-ceramide [AP] could be effectively used to simulate native like behaviour even in very simple mixtures and could also have a strong impact on the native stratum corneum as well as high relevance for dermal ceramide substitution. The unnatural l-ceramide [AP] on the other hand should be investigated further, to assess its applicability.

Potential application of oat-derived ceramides in improving skin barrier function: Part 1. Isolation and structural characterization.

The impaired epidermal barrier and skin dryness in chronic skin conditions such as atopic dermatitis, psoriasis and aged skin are associated with the depletion of ceramides (CERs) in the stratum corneum. Previously, the beneficial effects of phyto-CERs, mainly from wheat and rice, in replenishing the depleted epidermal CERs and restoring the skin barrier have been shown. However, very few efforts have been made to exploit CERs from other plants for dermal applications. In an attempt to explore alternative plant source of CERs, glucosylceramides (GlcCERs) were isolated from the lipid extract of Ethiopian oat grain (Avena abyssinica). The GlcCER species were separated on a reversed phase HPLC and the structure of individual GlcCERs were identified by tandem MS with atmospheric pressure chemical ionization interface. The glycosidic linkage of the GlcCERs was cleaved by acid treatment and the predominant CERs species were isolated using column chromatography and preparative LC-MS. Further structural characterization of the CERs was made by HR/ESI-MS and NMR analyses. All the detected oat-derived GlcCER species consisted of C18 dihydroxy sphingoid bases amide-linked with α-hydroxylated saturated fatty acids (C16-C24). The two predominant GlcCER species consisted of sphingenine (d18:1) amide-linked to hydroxypalmitic acid (h16:0) and hydroxyarachidic acid (h20:0). The molecular formulae of the two major CERs assigned by HR/ESI-MS were identical to the ones identified by LC/APCI-MS/MS. The structural information was also supported by 1H, 13C, 1H COSY NMR and HMBC spectral analyses. The amount of GlcCERs in oat grain, quantified by HPTLC, was found to be 193.5mg/kg. The results indicated the similarity of oat CERs with commercial plant CERs (with comparable GlcCER content) suggesting its potential as source of CERs for oral (as dietary supplements) as well as topical applications.

Influence of a Novel Dimeric Ceramide Molecule on the Nanostructure and Thermotropic Phase Behavior of a Stratum Corneum Model Mixture.

The stratum corneum (SC) is the outermost layer of the skin and is composed of a multilayered assembly of mostly ceramids (Cer), free fatty acids, cholesterol (Chol), and cholesterol sulfate (Chol-S). Because of the tight packing of these lipids, the SC features unique barrier properties defending the skin from environmental influences. Under pathological conditions, where the skin barrier function is compromised, topical application of molecules that rigidify the SC may lead to a restored barrier function. To this end, molecules are required that incorporate into the SC and bring back the original rigidity of the skin barrier. Here, we investigated the influence of a novel dimeric ceramide (dim-Cer) molecule designed to feature a long, rigid hydrocarbon chain ideally suited to forming an orthorhombic lipid phase. The influence of this molecules on the thermotropic phase behavior of a SC mixture consisting of Cer[AP18] (55 wt %), cholesterol (Chol, 25 wt %), steric acid (SA, 15 wt %), and cholesterol sulfate (Chol-S, 5 wt %) was studied using a combination of neutron diffraction and 2H NMR spectroscopy. These methods provide detailed insights into the packing properties of the lipids in the SC model mixture. Dim-Cer remains in an all-trans state of the membrane-spanning lipid chain at all investigated temperatures, but the influence on the phase behavior of the other lipids in the mixture is marginal. Biophysical experiments are complemented by permeability measurements in model membranes and human skin. The latter, however, indicates that dim-Cer only partially provides the desired effect on membrane permeability, necessitating further optimization of its structure for medical applications.

Lysine-based amino-functionalized lipids for gene transfection: the protonation state in monolayers at the air-liquid interface.

Cationic lipids are considered as non-viral carriers for genetic material used in gene therapy. They have no carcinogenic potential and cause low immune response compared to existing viral systems. The protonation degree of these cationic lipids is a crucial parameter for the binding behavior of polynucleotides (e.g., DNA). Newly synthesized peptide-mimic lysine-based amino-functionalized lipids have been investigated in 2D models as monolayers at the air-liquid interface. Standard surface pressure - area isotherms have been measured to prove the layer stability. Total reflection X-ray fluorescence (TRXF) has been used as a surface sensitive analytical method to estimate the amount of counterions at the head groups. Using a standard sample as a reference, the protonation degree of these cationic lipids can be quantified on buffers with different pH values. It is found that the protonation degree depends linearly on the packing density of the lipid monolayer.

Synthesis of specific deuterated derivatives of the long chained stratum corneum lipids [EOS] and [EOP] and characterization using neutron scattering.

The synthesis of specific deuterated derivatives of the long chained ceramides [EOS] and [EOP] is described. The structural differences with respect to the natural compounds are founded in the substitution of the 2 double bonds containing linoleic acid by a palmitic acid branched with a methyl group in 10-position. The specific deuteration is introduced both in the branched and in the terminal methyl group, which was realized by common methods of successive deuteration of carboxylic groups in 3 steps. These modified fatty acids resp. the corresponding ceramides [EOS] and [EOP] were prepared for neutron scattering investigations. First results of these investigations were presented in this manuscript showing that the deuterated compounds could be detected in the stratum corneum lipid model membranes. The deuterated ceramides [EOS] and [EOP] are valuable tools to investigate the influence of these long chained ceramide species on the nanostructure of stratum corneum lipid model membranes.

Synthesis of specifically deuterated ceramide [AP]-C18 and its biophysical characterization using neutron diffraction.

The very heterogeneous group of ceramides is known to be mandatory for proper barrier functions of the outermost layer of mammalian skin, referred to as stratum corneum (SC). The synthesis of a specifically deuterated ceramide [AP]-C18 variant is described. The synthesized ceramide contains the racemic forms of the α hydroxy fatty acid. For the biophysical implementation, the received diastereomeric ceramide was applied in a neutron diffraction experiment. Therefore, a SC lipid model membrane was prepared containing the described ceramide (CER), cholesterol (CHOL), stearic acid (SA), and cholesterol sulfate (ChS) in a ratio of 55/25/15/5wt%. Thus, we were able to localize the deuterated molecule part within the bilayers. In the process, a short-periodicity phase (SPP) was observed with a unit cell scale of about 44Å. For the first time, we were able to confirm former ideas concerning the arrangement of the CER within this quaternary lipid model membrane.

Phase separation in ceramide[NP] containing lipid model membranes: neutron diffraction and solid-state NMR.

The stratum corneum is the outermost layer of the skin and protects the organism against external influences as well as water loss. It consists of corneocytes embedded in a mixture of ceramides, fatty acids, and cholesterol in a molar ratio of roughly 1 : 1 : 1. The unique structural and compositional arrangement of these stratum corneum lipids is responsible for the skin barrier properties. Many studies investigated the organization of these barrier lipids and, in particular, the exact conformation of ceramides. However, so far no consensus has been reached. In this study, we investigate a model system comprised of N-(non-hydroxy-tetracosanoyl)-phytosphingosine/cholesterol/tetracosanoic acid (CER[NP]-C24/CHOL/TA) at a 1 : 1 : 1 molar ratio using neutron diffraction and 2H solid-state NMR spectroscopy at temperatures from 25 °C to 80 °C. Deuterated variants of all three lipid components of the model system were used to enable their separate investigation in the NMR spectra and quantification of the amount of molecules in each phase. Neutron scattering experiments show the coexistence of two lipid phases at low temperatures with repeat spacings of 54.2 Å and 43.0 Å at a physiological skin temperature of 32 °C. They appear to be indistinguishable in the 2H NMR spectra as both phases are crystalline and ceramide molecules do not rotate around their long axis on a microsecond timescale. The evolution of these phases upon heating is followed and with increasing temperature fluid and even isotropically mobile molecules are observed. A model of the organization of the lamellar phases is proposed in which the thicker phase consists of CER[NP]-C24 in a hairpin conformation mixed with CHOL and TA, while the phase with a repeat spacing of 43.0 Å contains CER[NP]-C24 in a V-shape conformation.

Influence of the penetration enhancer isopropyl myristate on stratum corneum lipid model membranes revealed by neutron diffraction and 2H NMR experiments.

The stratum corneum (SC) provides the main barrier properties in native skin. The barrier function is attributed to the intercellular lipids, forming continuous multilamellar membranes. In this study, SC lipid membranes in model ratios were enriched with deuterated lipids in order to investigate structural and dynamical properties by neutron diffraction and 2H solid-state NMR spectroscopy. Further, the effect of the penetration enhancer isopropyl myristate (IPM) on the structure of a well-known SC lipid model membrane containing synthetically derived methyl-branched ceramide [EOS], ceramide [AP], behenic acid and cholesterol (23/10/33/33wt%) was investigated. IPM supported the formation of a single short-periodicity phase (SPP), in which we determined the molecular organization of CER[AP] and CER[EOS]-br for the first time. Furthermore, the thermotropic phase behavior of the lipid system was analyzed by additional neutron diffraction studies as well as by 2H solid-state NMR spectroscopy, covering temperatures of 32°C (physiological skin temperature), 50°C, and 70°C with a subsequent cooldown back to skin temperature. Both techniques revealed a phase transition and a hysteresis effect. During the cooldown, Bragg peaks corresponding to a long-periodicity phase (LPP) appeared. Additionally, 2H NMR revealed that the IPM molecules are isotopic mobile at all temperatures.

An Asymmetrical Glycerol Diether Bolalipid with Protonable Phosphodimethylethanolamine Headgroup: The Impact of pH on Aggregation Behavior and Miscibility with DPPC.

Investigations regarding the self-assembly of (bola)phospholipids in aqueous media are crucial to understand the complex relationship between chemical structure of lipids and the shape and size of their aggregates in water. Here, we introduce a new asymmetrical glycerol diether bolaphospholipid, the compound Me2PE-Gly(2C16)C32-OH. This bolalipid contains a long (C32) ω-hydroxy alkyl chain bond to glycerol in the sn-3 position, a C16 alkyl chain at the sn-2 position, and a protonable phosphodimethylethanolamine (Me2PE) headgroup at the sn-1 position of the glycerol. The aggregation behavior of this bolalipid was studied as a function of temperature and pH using transmission electron microscopy (TEM), differential scanning calorimetry (DSC), and Fourier transform infrared (FTIR) spectroscopy. We show that this bolalipid aggregates into condensed lamellar sheets in acidic milieu and in large sheet-like aggregates at neutral pH-value. By contrast, at a pH-value of 10, where the Me2PE headgroup is only partially protonated, small lipid disks with diameter 50⁻100 nm were additionally found. Moreover, the miscibility of this asymmetrical bolalipid with the bilayer-forming phosphatidylcholine DPPC was investigated by means of DSC and TEM. The incorporation of bolalipids into phospholipid membranes could result in stabilized liposomes applicable for drug delivery purposes. We show that mixtures of DPPC and Me2PE-Gly(2C16)C32-OH form large lamellar aggregates at pH of 5, 7, and 10. However, closed lipid vesicles (liposomes) with an increased thermal stability were not found.

Synthesis of ceramides NS and NP with perdeuterated and specifically ω deuterated N-acyl residues.

The synthesis of 12 deuterated ceramides with either a deuteration at the last carbon atom of the amide bound fatty acid or a perdeuterated fatty acid chain is described. The ceramides were prepared starting from sphingosine or phytosphingosine and ω deuterated or perdeuterated fatty acids with PyBOP® as activating agent in high yields. For the synthesis of the specifically deuterated fatty acids, dicarboxylic acids were transformed into ω deuterated alkyl bromide, which was chain elongated with blocked ω bromo alcohols by copper catalyzed Grignard coupling. Oxidation of regenerated alcohol function yields the ω deuterated fatty acids.