Flipper Probes for the Community.

This article describes four fluorescent membrane tension probes that have been designed, synthesized, evaluated, commercialized and applied to current biology challenges in the context of the NCCR Chemical Biology. Their names are Flipper-TR®, ER Flipper-TR®, Lyso Flipper-TR®, and Mito Flipper-TR®. They are available from Spirochrome.

Improvement of DNA Vector Delivery of DOTAP Lipoplexes by Short-Chain Aminolipids.

Cellular delivery of DNA vectors for the expression of therapeutic proteins is a promising approach to treat monogenic disorders or cancer. Significant efforts in a preclinical and clinical setting have been made to develop potent nonviral gene delivery systems based on lipoplexes composed of permanently cationic lipids. However, transfection efficiency and tolerability of such systems are in most cases not satisfactory. Here, we present a one-pot combinatorial method based on double-reductive amination for the synthesis of short-chain aminolipids. These lipids can be used to maximize the DNA vector delivery when combined with the cationic lipid 1,2-dioleoyl-3-trimethylammonium propane (DOTAP). We incorporated various aminolipids into such lipoplexes to complex minicircle DNA and screened these systems in a human liver-derived cell line (HuH7) for gene expression and cytotoxicity. The lead aminolipid AL-A12 showed twofold enhanced gene delivery and reduced toxicity compared to the native DOTAP:cholesterol lipoplexes. Moreover, AL-A12-containing lipoplexes enabled enhanced transgene expression in vivo in the zebrafish embryo model.

Tuning the Thickness of a Biomembrane by Stapling Diamidophospholipids with Bolalipids.

In a biological membrane, proteins require specific lipids of distinctive length and chain saturation surrounding them. The active tuning of the membrane thickness therefore opens new possibilities in the study and manipulation of membrane proteins. Here, we introduce the concept of stapling phospholipids to different degrees of interdigitation depth by mixing 1,3-diamidophospholipids with single-chain bolalipids. The mixed membranes were studied by calorimetric assays, electron microscopy, X-ray, and infrared measurements to provide a complete biophysical characterization of membrane stapling. The matching between the diamidophospholipids and the bolalipids can be so strong as to completely induce a new phase that is more stable than the gel phase of the individual components.

Near-Infrared Light Triggered-Release in Deep Brain Regions Using Ultra-photosensitive Nanovesicles.

Remote and minimally-invasive modulation of biological systems with light has transformed modern biology and neuroscience. However, light absorption and scattering significantly prevents penetration to deep brain regions. Herein, we describe the use of gold-coated mechanoresponsive nanovesicles, which consist of liposomes made from the artificial phospholipid Rad-PC-Rad as a tool for the delivery of bioactive molecules into brain tissue. Near-infrared picosecond laser pulses activated the gold-coating on the surface of nanovesicles, creating nanomechanical stress and leading to near-complete vesicle cargo release in sub-seconds. Compared to natural phospholipid liposomes, the photo-release was possible at 40 times lower laser energy. This high photosensitivity enables photorelease of molecules down to a depth of 4 mm in mouse brain. This promising tool provides a versatile platform to optically release functional molecules to modulate brain circuits.

Activity of the Gramicidin A Ion Channel in a Lipid Membrane with Switchable Physical Properties.

Lipid bilayer membranes formed from the artificial 1,3-diamidophospholipid Pad-PC-Pad have the remarkable property that their hydrophobic thickness can be modified in situ: the particular arrangement of the fatty acid chains in Pad-PC-Pad allows them to fully interdigitate below 37 °C, substantially thinning the membrane with respect to the noninterdigitated state. Two stimuli, traversing the main phase transition temperature of the lipid or addition of cholesterol, have previously been shown to disable the interdigitated state. Both manipulations cause an increase in hydrophobic thickness of about 25 Å due to enhanced conformational entropy of the lipids. Here, we characterize the interdigitated state using electrophysiological recordings from free-standing lipid-membranes formed on micro structured electrode cavity arrays. Compared to standard membranes made from 1,2-diphytanoyl-sn-glycero-3-phosphocholin (DPhPC), pure Pad-PC-Pad membranes at room temperature had lowered electroporation threshold and higher capacitance. Ion channel formation by the peptide Gramicidin A was clearly facilitated in pure Pad-PC-Pad membranes at room temperature, with activity occurring at significantly lower peptide concentrations and channel dwell times increased by 2 orders of magnitude with respect to DPhPC-membranes. Both elevation of temperature beyond the phase transition and addition of cholesterol reduced channel dwell times, as expected if the reduced membrane thickness stabilized channel formation due to decreased hydrophobic mismatch.

Small-Angle Neutron Scattering Study of Temperature-Induced Structural Changes in Liposomes.

Liposomes of specific artificial phospholipids, such as Pad-PC-Pad and Rad-PC-Rad, are mechanically responsive. They can release encapsulated therapeutics via physical stimuli, as naturally present in blood flow of constricted vessel segments. The question is how these synthetic liposomes change their structure in the medically relevant temperature range from 22 to 42 °C. In the present study, small-angle neutron scattering (SANS) was employed to evaluate the temperature-induced structural changes of selected artificial liposomes. For Rad-PC-Rad, Pad-Pad-PC, Sur-PC-Sur, and Sad-PC-Sad liposomes, the SANS data have remained constant because the phase transition temperatures are above 42 °C. For Pad-PC-Pad and Pes-PC-Pes liposomes, whose phase transitions are below 42 °C, the q-plots have revealed temperature-dependent structural changes. The average diameter of Pad-PC-Pad liposomes remained almost constant, whereas the eccentricity decreased by an order of magnitude. Related measurements using transmission electron microscopy at cryogenic temperatures, as well as dynamic light scattering before and after the heating cycles, underpin the fact that the non-spherical liposomes flatten out. The SANS data further indicated that, as a consequence of the thermal loop, the mean bilayer thickness increased by 20%, associated with the loss of lipid membrane interdigitation. Therefore, Pad-PC-Pad liposomes are unsuitable for local drug delivery in the atherosclerotic human blood vessel system. In contrast, Rad-PC-Rad liposomes are thermally stable for applications within the human body.

1-Deoxydihydroceramide causes anoxic death by impairing chaperonin-mediated protein folding.

Ischaemic heart disease and stroke are the most common causes of death worldwide. Anoxia, defined as the lack of oxygen, is commonly seen in both these pathologies and triggers profound metabolic and cellular changes. Sphingolipids have been implicated in anoxia injury, but the pathomechanism is unknown. Here we show that anoxia-associated injury causes accumulation of the non-canonical sphingolipid 1-deoxydihydroceramide (DoxDHCer). Anoxia causes an imbalance between serine and alanine resulting in a switch from normal serine-derived sphinganine biosynthesis to non-canonical alanine-derived 1-deoxysphinganine. 1-Deoxysphinganine is incorporated into DoxDHCer, which impairs actin folding via the cytosolic chaperonin TRiC, leading to growth arrest in yeast, increased cell death upon anoxia-reoxygenation in worms and ischaemia-reperfusion injury in mouse hearts. Prevention of DoxDHCer accumulation in worms and in mouse hearts resulted in decreased anoxia-induced injury. These findings unravel key metabolic changes during oxygen deprivation and point to novel strategies to avoid tissue damage and death.

Artificial Phospholipids and Their Vesicles.

Phospholipids are at the heart and origin of life on this planet. The possibilities in terms of phospholipid self-assembly and biological functions seem limitless. Nonetheless, nature exploits only a small fraction of the available chemical space of phospholipids. Using chemical synthesis, artificial phospholipid structures become accessible, and the study of their biophysics may reveal unprecedented properties. In this article, the recent advances by our work group in the field of chemical lipidology are summarized. The family of diamidophospholipids is discussed in detail from monolayer characterization to the formation of faceted vesicles, culminating in the template-free self-assembly of phospholipid cubes and the possible applications of vesicle origami in modern personalized medicine.

Spatially resolved small-angle X-ray scattering for characterizing mechanoresponsive liposomes using microfluidics.

Atherosclerosis gives rise to blood vessel occlusion associated with blood flow alteration and substantial increase of average wall shear stress. This modification was proved acting as a purely physical trigger for targeted vasodilator release from a particular type of liposomes composed of 1,3-diaminophospholipids (Pad-PC-Pad). The flow-induced structural changes of these faceted liposomes, however, are completely unknown. Therefore, spatially resolved small-angle X-ray scattering was combined with microfluidics to uniquely study the purely physical mechanisms, which give rise to the highly efficient drug release from mechanoresponsive liposomes of nanometer size. The microfluidic device, designed to mimic a stenotic blood vessel, consisted of a 1-mm-wide channel with a constriction, 125 â€‹µm in diameter. Here, the changes of the average bilayer thickness and the mean size of the mechanoresponsive liposomes have been locally detected under flow conditions. Overall shape and bilayer thickness do change already near the constriction inlet, but the alteration is dominant near the outlet. At a flow rate of 0.2 â€‹µL/s, the liposome's bilayer thickness increased by 30 % compared to the situation well before the constriction and under static condition. The detected bilayer thickness increase of the faceted liposomes is in line with the mechanically induced loss of interdigitation between the phospholipid amide chains. These results imply that rather the gradient force than the wall shear stress provokes structural changes of Pad-PC-Pad liposomes and the related drug release at stenoses. The approach, i.e. the combination of microfluidics and spatially resolved small-angle X-ray scattering, paves the way to design highly efficient and specific systems for the targeted drug delivery at constrictions with predefined morphology.

Against the rules: pressure induced transition from high to reduced order.

Envisioning the next generation of drug delivery nanocontainers requires more in-depth information on the fundamental physical forces at play in bilayer membranes. In order to achieve this, we combine chemical synthesis with physical-chemical analytical methods and probe the relationship between a molecular structure and its biophysical properties. With the aim of increasing the number of hydrogen bond donors compared to natural phospholipids, a phospholipid compound bearing urea moieties has been synthesized. The new molecules form interdigitated bilayers in aqueous dispersions and self-assemble at soft interfaces in thin layers with distinctive structural order. At lower temperatures, endothermic and exothermic transitions are observed during compression. The LC1 phase is dominated by an intermolecular hydrogen bond network of the urea moieties leading to a very high chain tilt of 52°. During compression and at higher temperatures, presumably this hydrogen bond network is broken allowing a much lower chain tilt of 35°. The extremely different monolayer thicknesses violate the two-dimensional Clausius-Clapeyron equation.

Synthesis and Biophysical Characterization of an Odd-Numbered 1,3-Diamidophospholipid.

Nanomedicine suffers from low drug delivery efficiencies. Mechanoresponsive vesicles could provide an alternative way to release active compounds triggered by the basic physics of the human body. 1,3-Diamidophospholipids with C16 tails proved to be an effective building block for mechanoresponsive vesicles, but their low main phase transition temperature prevents an effective application in humans. As the main phase transition temperature of a membrane depends on the fatty acyl chain length, we synthesized a C17 homologue of a 1,3-diamidophospholipid: Rad-PC-Rad. The elevated main phase transition temperature of Rad-PC-Rad allows mechanoresponsive drug delivery at body temperature. Herein, we report the biophysical properties of Rad-PC-Rad monolayer and bilayer membranes. Rad-PC-Rad is an ideal candidate for advancing the concept of physically triggered drug release.

Facile and Rapid Formation of Giant Vesicles from Glass Beads.

Giant vesicles (GVs) are widely-used model systems for biological membranes. The formulation of these vesicles, however, can be problematic and artifacts, such as degraded molecules or left-over oil, may be present in the final liposomes. The rapid formulation of a high number of artifact-free vesicles of uniform size using standard laboratory equipment is, therefore, highly desirable. Here, the gentle hydration method of glass bead-supported thin lipid films has been enhanced by adding a vortexing step. This led to the formulation of a uniform population of giant vesicles. Batches of glass beads coated with different lipids can be combined to produce vesicles of hybrid lipid compositions. This method represents a stable approach to rapidly generate giant vesicles.

Immunological response to nitroglycerin-loaded shear-responsive liposomes in vitro and in vivo.

Liposomes formulated from the 1,3-diamidophospholipid Pad-PC-Pad are shear-responsive and thus promising nano-containers to specifically release a vasodilator at stenotic arteries. The recommended preclinical safety tests for therapeutic liposomes of nanometer size include the in vitro assessment of complement activation and the evaluation of the associated risk of complement activation-related pseudo-allergy (CARPA) in vivo. For this reason, we measured complement activation by Pad-PC-Pad formulations in human and porcine sera, along with the nanopharmaceutical-mediated cardiopulmonary responses in pigs. The evaluated formulations comprised of Pad-PC-Pad liposomes, with and without polyethylene glycol on the surface of the liposomes, and nitroglycerin as a model vasodilator. The nitroglycerin incorporation efficiency ranged from 25% to 50%. In human sera, liposome formulations with 20mg/mL phospholipid gave rise to complement activation, mainly via the alternative pathway, as reflected by the rises in SC5b-9 and Bb protein complex concentrations. Formulations having a factor of ten lower phospholipid content did not result in measurable complement activation. The weak complement activation induced by Pad-PC-Pad liposomal formulations was confirmed by the results obtained by performing an in vivo study in a porcine model, where hemodynamic parameters were monitored continuously. Our study suggests that, compared to FDA-approved liposomal drugs, Pad-PC-Pad exhibits less or similar risks of CARPA.

Correlation of surface pressure and hue of planarizable push-pull chromophores at the air/water interface.

It is currently not possible to directly measure the lateral pressure of a biomembrane. Mechanoresponsive fluorescent probes are an elegant solution to this problem but it requires first the establishment of a direct correlation between the membrane surface pressure and the induced color change of the probe. Here, we analyze planarizable dithienothiophene push-pull probes in a monolayer at the air/water interface using fluorescence microscopy, grazing-incidence angle X-ray diffraction, and infrared reflection-absorption spectroscopy. An increase of the lateral membrane pressure leads to a well-packed layer of the 'flipper' mechanophores and a clear change in hue above 18 mN/m. The fluorescent probes had no influence on the measured isotherm of the natural phospholipid DPPC suggesting that the flippers probe the lateral membrane pressure without physically changing it. This makes the flipper probes a truly useful addition to the membrane probe toolbox.

Vesicle Origami: Cuboid Phospholipid Vesicles Formed by Template-Free Self-Assembly.

Phospholipid liposomes are archetypical self-assembled structures. To minimize the surface tension, the vesicles typically are spherical. Deciphering the bilayer code, the basic physical interactions between phospholipids would allow these molecules to be utilized as building blocks for novel, non-spherical structures. A 1,2-diamidophospholipid is presented that self-assembles into a cuboid structure. Owing to intermolecular hydrogen bonding, the bilayer membranes form an exceptionally tight subgel packing, leading to a maximization of flat structural elements and a minimization of any edges. These conditions are optimized in the geometrical structure of a cube. Surprisingly, the lateral surface pressure in the membrane is only one third of the value typically assumed for a bilayer membrane, questioning a long-standing rule-of-thumb.

Structure and conserved function of iso-branched sphingoid bases from the nematode Caenorhabditis elegans.

Sphingolipids are bio-active metabolites that show structural diversity among eukaryotes. They are essential for growth of all eukaryotic cells but when produced in an uncontrolled manner can lead to cell death and pathologies including auto-immune reactions, cancer, diabetes and neurodegeneration. Caenorhabditis elegans is an important genetic model organism both to find new drug-targets against parasitic nematodes and to study the conserved roles of sphingolipids in animals like their essential functions in very basic cellular processes ranging from maintenance of cell polarity and mitochondrial repair to growth and survival. C. elegans produces sphingoid bases which are structurally distinct from those of other animals as both iso- and anteiso-branched species have been reported. Using metabolic labeling we show that most worm sphingoid bases are iso-branched. We have synthesized the nematode-specific C17 iso-branched sphinganine and its 1-deoxy analogue and could show that both the iso-branch and the 1-hydroxyl group are essential to form functional nematode sphingolipids which are needed to maintain intestinal function. The organism specificity was examined by complementation experiments in Saccharomyces cerevisiae yeast cells lacking sphingoid base synthesis. We found that iso-branched sphingoid base did not support growth of mutant cells and was toxic to wild type yeast. 1-Deoxy sphingolipids have been linked to the hereditary disease HSAN1A and other metabolic disorders including diabetes. We found that in C. elegans the 1-deoxy analogue cannot rescue the intestinal phenotype caused by sphingoid base depletion. In fact, in wild-type animals with normal sphingoid base biosynthesis, exogenous 1-deoxy analogue had a disruptive effect on apical cytoskeletal organization of intestinal cells indicating that atypical bases can interfere with normal sphingolipid function.

Vesicle Origami and the Influence of Cholesterol on Lipid Packing.

The artificial phospholipid Pad-PC-Pad was analyzed in 2D (monolayers at the air/water interface) and 3D (aqueous lipid dispersions) systems. In the gel phase, the two leaflets of a Pad-PC-Pad bilayer interdigitate completely, and the hydrophobic bilayer region has a thickness comparable to the length of a single phospholipid acyl chain. This leads to a stiff membrane with no spontaneous curvature. Forced into a vesicular structure, Pad-PC-Pad has faceted geometry, and in its extreme form, tetrahedral vesicles were found as predicted a decade ago. Above the main transition temperature, a noninterdigitated Lα phase with fluid chains has been observed. The addition of cholesterol leads to a slight decrease of the main transition temperature and a gradual decrease in the transition enthalpy until the transition vanishes at 40 mol % cholesterol in the mixture. Additionally, cholesterol pulls the chains apart, and a noninterdigitated gel phase is observed. In monolayers, cholesterol has an ordering effect on liquid-expanded phases and disorders condensed phases. The wavenumbers of the methylene stretching vibration indicate the formation of a liquid-ordered phase in mixtures with 40 mol % cholesterol.

Surprising lack of liposome-induced complement activation by artificial 1,3-diamidophospholipids in vitro.

Cardio-vascular diseases are the main cause of death, emphasizing the need to improve patient treatment and survival. One therapeutic approach is a liposome-based drug carrier system specifically targeting constricted arteries. The recently discovered mechano-sensitive liposomes use hemodynamic shear-stress differences between healthy and constricted blood vessels as trigger for drug release. Liposomes are promising delivery containers but are being recognized as foreign by the immune system. Complement activation as essential factor of the recognition leads to adverse effects. Here, we tested complement activation by liposomes formulated from the artificial phospholipid Pad-PC-Pad in vitro. Surprisingly no complement activation was detected in human sera and porcine plasma. In in vivo experiments with three pigs, neither anaphylactic reactions nor other significant hemodynamic changes were observed even at comparably high liposome doses. The pilot study holds promise for an absence of complement-mediated adverse effects of Pad-PC-Pad liposomes in human. FROM THE CLINICAL EDITOR: A lot of research has been done on new treatment for cardiovascular diseases. Liposome-based carrier systems have also shown promises. In this article, the authors studied the potential risks of complement activation by liposomes in in-vivo experiments. The absence of complement activation by Pad-PC-Pad liposomes may indicate its use in humans.

Bilayer properties of 1,3-diamidophospholipids.

A series of 1,3-diamido phosphocholines was synthesized, and their potential to form stable bilayers was investigated. Large and giant unilamellar vesicles produced from these new lipids form a wide variety of faceted liposomes. Factors such as cooling rates and the careful choice of the liposome preparation method influence the formation of facets. Interdigitation was hypothesized as a main factor for the stabilization of facets and effectively monitored by small-angle X-ray scattering measurements.

Rigid urea and self-healing thiourea ethanolamine monolayers.

A series of long-tail alkyl ethanolamine analogs containing amide-, urea-, and thiourea moieties was synthesized and the behavior of the corresponding monolayers was assessed on the Langmuir-Pockels trough combined with grazing incidence X-ray diffraction experiments and complemented by computer simulations. All compounds form stable monolayers at the soft air/water interface. The phase behavior is dominated by strong intermolecular headgroup hydrogen bond networks. While the amide analog forms well-defined monolayer structures, the stronger hydrogen bonds in the urea analogs lead to the formation of small three-dimensional crystallites already during spreading due to concentration fluctuations. The hydrogen bonds in the thiourea case form a two-dimensional network, which ruptures temporarily during compression and is recovered in a self-healing process, while in the urea clusters the hydrogen bonds form a more planar framework with gliding planes keeping the structure intact during compression. Because the thiourea analogs are able to self-heal after rupture, such compounds could have interesting properties as tight, ordered, and self-healing monolayers.