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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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

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., (©2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

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 (C 14:0 ) and hexadecyl (C 16:0 ) chains to longer but unsaturated oleyl (C 18:1 ) 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 (C 14:0 ) and hexadecyl (C 16:0 ) chains by unsaturated oleyl (C 18:1 ) 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.

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.

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., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A series of novel malonic acid diamides (second generation) with two long hydrophobic alkyl chains and an alkaline polar head group was synthesised and characterised as a new class of amino-functionalised lipids. These peptide-mimic lipids are suitable for polynucleotide transfer. The lipids bear a novel backbone consisting of a lysine unit and a malonic acid unit. Six different head-group structures, which vary in size and number of amino groups that can be protonated, were attached to the backbone structure. Furthermore, different alkyl chains were used to build the lipophilic part (namely tetradecyl, hexadecyl, and oleyl). Phase transitions of the new compounds in aqueous dispersions at pH 10 were analysed and discussed in terms of head group and alkyl chain variations. The shape and size of the formed aggregates of selected lipid dispersions were investigated by dynamic light scattering and transmission electron microscopy., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In the present work, we describe the successful stabilization of bolalipid nanofibers by sol-gel condensation (silicification) of tetraethoxysilane (TEOS) or 3-mercaptopropyltriethoxysilane (MP-TEOS), respectively, onto the nanofibers. The conditions for an effective and reproducible silicification reaction were determined, and the silicification process was pursued by transmission electron microscopy (TEM). The resulting bolalipid-silica composite nanofibers were characterized by means of differential scanning calorimetry (DSC), TEM, (13)C, and (31)P NMR spectroscopy. Finally, the novel silicified bolalipid nanofibers were used as templates for the fixation of 5 and 2 nm AuNPs, respectively, resulting in one of the rare examples of one-dimensional AuNP arrangements in aqueous suspension.

Cationic liposome/DNA complexes can be used as nonviral vectors for direct delivery of DNA-based biopharmaceuticals to damaged cells and tissues. To obtain more effective and safer liposome-based gene transfection systems, two cationic lipids with identical head groups but different chain structures are investigated with respect to their in vitro gene-transfer activity, their cell-damaging characteristics, and their physicochemical properties. The gene-transfer activities of the two lipids are very different. Differential scanning calorimetry and synchrotron small- and wide-angle X-ray scattering give valuable structural insight. A subgel-like structure with high packing density and high phase-transition temperature from gel to liquid-crystalline state are found for lipid 7 (N'-2-[(2,6-diamino-1-oxohexyl)amino]ethyl-2,N-bis(hexadecyl)propanediamide) containing two saturated chains. Additionally, an ordered head-group lattice based on formation of a hydrogen-bond network is present. In contrast, lipid 8 (N'-2-[(2,6-diamino-1-oxohexyl)amino]ethyl-2-hexadecyl-N-[(9Z)-octadec-9-enyl]propanediamide) with one unsaturated and one saturated chain shows a lower phase-transition temperature and a reduced packing density. These properties enhance incorporation of the helper lipid cholesterol needed for gene transfection. Both lipids, either pure or in mixtures with cholesterol, form lamellar phases, which are preserved after addition of DNA. However, the system separates into phases containing DNA and phases without DNA. On increasing the temperature, DNA is released and only a lipid phase without intercalated DNA strands is observed. The conversion temperatures are very different in the two systems studied. The important parameter seems to be the charge density of the lipid membranes, which is a result of different solubility of cholesterol in the two lipid membranes. Therefore, different binding affinities of the DNA to the lipid mixtures are achieved., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Herein, we report the synthesis of two novel, amino-functionalized single-chain bolalipids and, based on those, a general synthetic approach for the insertion of various carboxylic acids into the bolalipid headgroups, e.g. alpha-lipoic acid for one-dimensional fixation of gold nanoparticles, sorbic acid for polymerization experiments, or lysine for the use in gene delivery systems. The temperature- and pH-dependent self-assembly of amino-functionalized bolalipids into nanofibers and micelles was investigated by differential scanning calorimetry (DSC), transmission electron microscopy (TEM) and dynamic light scattering (DLS). Rheological measurements were used to describe the macroscopic behavior of the formed temperature switchable hydrogels that can be fine-tuned for drug delivery applications. We showed that the viscoelastic properties of the hydrogel strongly depend on ionic interactions between bolalipid headgroups as well as on the ability to form hydrogen bonds.

Gene delivery using nonviral approaches has been extensively studied as a basic tool for intracellular gene transfer. Despite intensive research activity, the aim of creating a vector which meets all necessary demands has still not been reached. One possibility to solve the nonviral vector associated problem of low transfection efficacy is the development of new cationic amphiphiles. Therefore, the non-glycerol-based cationic lipids 1-9 have been synthesized and tested for in vitro gene delivery experiments. The backbone structure of the lipids consists of a malonic acid diamide with two long hydrophobic chains. The degree of saturation of the hydrophobic chains and the structure of the polar cationic headgroup were varied. The preparation follows an easy process and facilitates the trouble-free insertion of different alkyl chains. By studying in vitro gene delivery an increase of transfection efficacy was observed when using at least one unsaturated alkyl chain in the hydrophobic part and lysine or bis(2-aminoethyl)aminoethylamid as hydrophilic headgroup. This leads to cationic lipids exhibiting comparable or even higher transfection efficacies compared to the commercially available LipofectAmine and SuperFect.

The stratum corneum (SC), the outermost layer of the mammalian skin, is the main skin barrier. Ceramides (CERs) as the major constituent of the SC lipid matrix are of particular interest. At the moment, 11 classes of CERs are identified, but the effect of each single ceramide species is still not known. Therefore in this article, the thermotropic behaviour of the long chain omega-acylceramides CER[EOS] and CER[EOP] was studied using X-ray powder diffraction and FT-Raman spectroscopy. It was found that the omega-acylceramides CER[EOS] and CER[EOP] do not show a pronounced polymorphism which is observed for shorter chain ceramides as a significant feature. The phase behaviour of both ceramides is strongly influenced by the extremely long acyl-chain residue. The latter has a much stronger influence compared with the structure of the polar head group, which is discussed as extremely important for the appearance of a rich polymorphism. Despite the strong influence of the long chain, the additional OH-group of the phyto-sphingosine type CER[EOP] influences the lamellar repeat distance and the chain packing. The less polar sphingosine type CER[EOS] is stronger influenced by the long acyl-chain residue. Hydration is necessary for the formation of an extended hydrogen-bonding network between the polar head groups leading to the appearance of a long-periodicity phase (LPP). In contrast, the more polar CER[EOP] forms the LPP with densely packed alkyl chains already in the dry state.

Investigation of DNA interactions with cationic lipids is of particular importance for the fabrication of biosensors and nanodevices. Furthermore, lipid/DNA complexes can be applied for direct delivery of DNA-based biopharmaceuticals to damaged cells as non-viral vectors. To obtain more effective and safer DNA vectors, the new cationic lipids 2-tetradecylhexadecanoic acid-{2-[(2-aminoethyl)amino]ethyl}amide (CI) and 2-tetradecylhexadecanoic acid-2-[bis(2-aminoethyl)amino]ethylamide (CII) were synthesized and characterized. The synthesis, physical-chemical properties and first transfection and toxicity experiments are reported. Special attention was focused on the capability of CI and CII to complex DNA at low and high subphase pH values. Langmuir monolayers at the air/water interface represent a well-defined model system to study the lipid/DNA complexes. Interactions and ordering of DNA under Langmuir monolayers of the new cationic lipids were studied using film balance measurements, grazing incidence X-ray diffraction (GIXD) and X-ray reflectivity (XR). The results obtained demonstrate the ability of these cationic lipids to couple with DNA at low as well as at high pH value. Moreover, the observed DNA structuring seems not to depend on subphase pH conditions. An influence of the chemical structure of the lipid head group on the DNA binding ability was clearly observed. Both compounds show good transfection efficacy and low toxicity in the in vitro experiments indicating that lipids with such structures are promising candidates for successful gene delivery systems.

In order to be used in versatile DNA delivery systems, novel cationic lipids were synthesized. The head groups of the new compounds represented by monoamines or oligoamines can be charged or uncharged depending on the environmental pH. Since their pK values are unknown, the protonation properties of these lipids have been studied in a wide pH range. In our experiments, the amphiphilic molecules were organized as a Langmuir monolayer at the air-water interface. Total reflection X-ray fluorescence (TRXF) was used to determine the 2D concentration of bromide counterions bound to a positively charged (protonated) Langmuir monolayer. The protonation rate of the novel cationic lipids was estimated by comparing the fluorescence intensity with that of dioctadecyldimethylammonium bromide monolayers as a reference. TRXF investigations were supplemented with results of film-balance measurements, grazing incidence X-ray diffraction, and X-ray reflectivity data. The results obtained display that the monolayers of all studied compounds are completely uncharged at pH values above 10. In the investigated pH region, the highest protonation rate of the monolayers is observed at pH 3. The influence of the monolayer packing density on the protonation properties is clearly shown.

Cationic liposomes/DNA complexes can be used as nonviral vectors for direct delivery of DNA-based biopharmaceuticals to damaged cells and tissues. In order to obtain more effective and safer liposome-based gene transfection systems, the new cationic lipid 2-amino-3-hexadecyloxy-2-(hexadecyloxymethyl)propan-1-ol (AHHP) was synthesized. In this paper we report on the synthesis of AHHP and investigations of its physical-chemical properties. Langmuir monolayers of AHHP were studied at the air/buffer interface by film balance measurements, grazing incidence X-ray diffraction (GIXD), and infrared reflection absorption spectroscopy (IRRAS). Structure and thermotropic phase behavior of AHHP in aqueous dispersion were examined by small-angle and wide-angle X-ray scattering (SAXS/WAXS) and differential scanning calorimetry (DSC). The results show clear differences in structure and phase behavior of AHHP, both in the monolayer system and in aqueous dispersions, in dependence on the subphase pH due to protonation or deprotonation of the primary amine in the lipid head group. Thermodynamic data derived from pi-A isotherms provide information about the critical temperature (Tc), which is in rough agreement with the temperature of the lipid phase transition from gel to fluid state (Tm) found by X-ray and calorimetry studies of AHHP aqueous dispersions. The packing properties of the molecules in mono- and bilayer systems are very similar. DNA couples to the monolayer of the new lipid at low as well as at high pH but in different amounts. The DNA coupling leads to an alignment of adsorbed DNA strands indicated by the appearance of a Bragg peak. The distance between aligned DNA strands does not change much with increasing monolayer pressure.

The phase and miscibility behavior of a triple-chain phosphatidylcholine (TPHPC) and a single-chain surfactant (CTAB) were investigated in aqueous dispersions and in monolayers at the air/water interface. CTAB can be incorporated in the TPHPC monolayer because of its complementary molecule shape and reduces the tilt angle of TPHPC. The type of phases and the phase sequence (L2 - LS) are the same in the pure TPHPC monolayer and in the TPHPC/CTAB (80:20 mol:mol) mixture. No indication of any ordering of adsorbed DNA was observed. In the aqueous dispersions, TPHPC exhibits an inverted hexagonal phase above the chain melting. The addition of 30 mol% CTAB leads to the appearance of a lamellar Lalpha phase. The binding of DNA to the mixture is obvious but this is accompanied by a separation of the two lipids what is supported by monolayer experiments. The system has no long-term stability. The main reason seems to be not only the stronger interaction of DNA with CTAB, but also especially the unexpected weak interaction between CTAB and TPHPC. The transfection efficiency is lower compared with lipofectamine. The main disadvantage of this system is the cytotoxicity of CTAB, which could not be lowered by incorporation of CTAB in the TPHPC bilayer.

In order to elucidate the mode of action of the lipophilic penetration enhancer isopropyl myristate (IPM) on a molecular scale, we investigated oriented quaternary stratum corneum (SC) lipid model membranes based on ceramide AP, cholesterol, palmitic acid and cholesterol sulfate containing 10 wt% IPM by means of neutron diffraction. Our results indicate that IPM affects the lamellar lipid assembly in terms of bilayer perturbation and disordering. Phase segregation occurred, indicating that IPM is not likely to mix properly with the other SC lipids due to its branched structure. We used selective deuterium labelling to localize the penetration enhancer, and could successfully prove the presence of IPM in the two coexisting lamellar phases. We conclude that IPM's mode of action as penetration promoter is presumably based on incorporation into the SC lipid matrix, extraction of certain SC lipids into a separate phase and perturbation of the multilamellar lipid assembly. Copyright © 2012 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]