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-<i>sn</i>-glycero-3-phosphoethanolamine (DOPE), 1,2-dimyristoyl-<i>sn</i>-glycero-3-phosphoethanolamine (DMPE), or 1,2-dimyristoyl-<i>sn</i>-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

Functionalization of Bolalipid Nanofibers by Silicification and Subsequent One-Dimensional Fixation of Gold Nanoparticles

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, ¹³C, and ³¹P 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

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 <i>sn</i>-3 position, carrying a hydroxyl group at the ω position. The C16 alkyl chain in the <i>sn</i>-2 position either possesses a racemic methyl branch at the 10 position of the short alkyl chain (lipid <b>II</b>) or does not (lipid <b>I</b>). The <i>sn</i>-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 <b>I</b>, 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

A T-Shaped Amphiphilic Molecule Forms Closed Vesicles in Water and Bicelles in Mixtures with a Membrane Lipid

The T-shaped amphiphilic molecule A6/6 forms a columnar hexagonal liquid-crystalline phase between the crystalline and the isotropic liquid when studied in bulk (Chen et al., 2005). Because of the hydrophilic and flexible oligo­(oxyethylene) side chain terminated by a 1-acylamino-1-deoxy-d-sorbitol moiety attached to a rigid terphenyl core with terminal hexyloxy alkyl chains, it was expected that also formation of lyotropic phases could be possible. We therefore studied the behavior of A6/6 in water and also in mixtures with bilayer-forming phospholipids, such as dipalmitoyl-phosphatidylcholine (DPPC), using differential scanning calorimetry (DSC), transmission electron microscopy (TEM), cryo-transmission electron microscopy (cryo-TEM), dynamic light scattering (DLS), and solid-state nuclear magnetic resonance (ssNMR). DSC showed for the pure A6/6 suspended in water a phase transition at ca. 23 °C. TEM and cryo-TEM showed vesicular as well as layered structures for pure A6/6 in water below and above this phase transition. By atomic force microscopy (AFM), the thickness of the layer was found to be 5–6 nm. This leads to a model for a bilayer formed by A6/6 with the laterally attached polar side chains shielding the hydrophobic layer built up by the terphenyl core with the terminal alkyl chains of the molecules. For DPPC:A6/6 mixtures (10:1), the DSC curves indicated a stabilization of the lamellar gel phase of DPPC. Negative staining TEM and cryo-TEM images showed planar bilayers with hexagonal morphology and diameters between 50 and 200 nm. The hydrodynamic radius of these aggregates in water, investigated by dynamic light scattering (DLS) as a function of time and temperature, did not change indicating a very stable aggregate structure. The findings lead to the proposition of a new bicellar structure formed by A6/6 with DPPC. In this model, the bilayer edges are covered by the T-shaped amphiphilic molecules preventing very effectively the aggregation to larger structures

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

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 <i>sn</i>-3 position, a methyl-branched C16 alkyl chain in the <i>sn</i>-2 position, and a zwitterionic phosphocholine headgroup in the <i>sn</i>-1 position of a glycerol moiety. The long C32 alkyl chain is terminated either by a second phosphocholine (<b>PC-Gly­(2C16Me)­C32-PC</b>) or by a phos­pho­di­methyl­ethan­ol­amine headgroup (<b>PC-Gly­(2C16Me)­C32-Me</b>_<b>2</b><b>PE</b>). 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 <b>PC-Gly­(2C16Me)­C32-PC</b> and <b>PC-Gly­(2C16Me)­C32-Me</b>_<b>2</b><b>PE</b> 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 <b>PC-Gly­(2C16Me)­C32-Me</b>_<b>2</b><b>PE</b> 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

Temperature-Dependent In-Plane Structure Formation of an X‑Shaped Bolapolyphile within Lipid Bilayers

Polyphilic compound B12 is an X-shaped molecule with a stiff aromatic core, flexible aliphatic side chains, and hydrophilic end groups. Forming a thermotropic triangular honeycomb phase in the bulk between 177 and 182 °C but no lyotropic phases, it is designed to fit into DPPC or DMPC lipid bilayers, in which it phase separates at room temperature, as observed in giant unilamellar vesicles (GUVs) by fluorescence microscopy. TEM investigations of bilayer aggregates support the incorporation of B12 into intact membranes. The temperature-dependent behavior of the mixed samples was followed by differential scanning calorimetry (DSC), FT-IR spectroscopy, fluorescence spectroscopy, and X-ray scattering. DSC results support in-membrane phase separation, where a reduced main transition and new B12-related transitions indicate the incorporation of lipids into the B12-rich phase. The phase separation was confirmed by X-ray scattering, where two different lamellar repeat distances are visible over a wide temperature range. Polarized ATR-FTIR and fluorescence anisotropy experiments support the transmembrane orientation of B12, and FT-IR spectra further prove a stepwise “melting” of the lipid chains. The data suggest that in the B12-rich domains the DPPC chains are still rigid and the B12 molecules interact with each other via π–π interactions. All results obtained at temperatures above 75 °C confirm the formation of a single, homogeneously mixed phase with freely mobile B12 molecules

Cryo-Electron Microscopy Snapshots of Eukaryotic Membrane Proteins in Native Lipid-Bilayer Nanodiscs

New technologies for purifying membrane-bound protein complexes in combination with cryo-electron microscopy (EM) have recently allowed the exploration of such complexes under near-native conditions. In particular, polymer-encapsulated nanodiscs enable the study of membrane proteins at high resolution while retaining protein–protein and protein–lipid interactions within a lipid bilayer. However, this powerful technology has not been exploited to address the important question of how endogenousas opposed to overexpressedmembrane proteins are organized within a lipid environment. In this work, we demonstrate that biochemical enrichment protocols for native membrane–protein complexes from Chaetomium thermophilum in combination with polymer-based lipid-bilayer nanodiscs provide a substantial improvement in the quality of recovered endogenous membrane–protein complexes. Mass spectrometry results revealed ∼1123 proteins, while multiple 2D class averages and two 3D reconstructions from cryo-EM data furnished prominent structural signatures. This integrated methodological approach to enriching endogenous membrane–protein complexes provides unprecedented opportunities for a deeper understanding of eukaryotic membrane proteomes

Synthesis, Characterization, and Nanoencapsulation of Tetrathiatriarylmethyl and Tetrachlorotriarylmethyl (Trityl) Radical DerivativesA 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 <b>13</b>) 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