9 research outputs found
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, <sup>13</sup>C, and <sup>31</sup>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
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
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><sub><b>2</b></sub><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><sub><b>2</b></sub><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><sub><b>2</b></sub><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 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 <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