High Hydrostatic Pressure Induces a Lipid Phase Transition and Molecular Rearrangements in Low-Density Lipoprotein Nanoparticles.

Low-density lipoproteins (LDL) are natural lipid transporter in human plasma whose chemically modified forms contribute to the progression of atherosclerosis and cardiovascular diseases accounting for a vast majority of deaths in westernized civilizations. For the development of new treatment strategies, it is important to have a detailed picture of LDL nanoparticles on a molecular basis. Through the combination of X-ray and neutron small-angle scattering (SAS) techniques with high hydrostatic pressure (HHP) this study describes structural features of normolipidemic, triglyceride-rich and oxidized forms of LDL. Due to the different scattering contrasts for X-rays and neutrons, information on the effects of HHP on the internal structure determined by lipid rearrangements and changes in particle shape becomes accessible. Independent pressure and temperature variations provoke a phase transition in the lipid core domain. With increasing pressure an inter-related anisotropic deformation and flattening of the particle are induced. All LDL nanoparticles maintain their structural integrity even at 3000 bar and show a reversible response toward pressure variations. The present work depicts the complementarity of pressure and temperature as independent thermodynamic parameters and introduces HHP as a tool to study molecular assembling and interaction processes in distinct lipoprotein particles in a nondestructive manner.

Peptide self-assembly into lamellar phases and the formation of lipid-peptide nanostructures.

Lipids exhibit an extraordinary polymorphism in self-assembled mesophases, with lamellar phases as biologically most relevant representative. To mimic lipid lamellar phases with amphiphilic designer peptides, seven systematically varied short peptides were engineered. Indeed, four peptide candidates (V4D, V4WD, V4WD2, I4WD2) readily self-assembled into lamellae in aqueous solution: small-angle X-ray scattering patterns (SAXS) revealed ordered lamellar structures with a repeat distance of ~4-5 nm. Transmission electron microscopy (TEM) images confirmed the presence of stacked sheets. Two derivatives (V3D and V4D2) remained as loose aggregates dispersed in solution; one peptide (L4WD2) formed twisted tapes with internal lamellae and an antiparallel ß-type monomer alignment. To understand the interaction of peptides with lipids they were mixed with phosphatidylcholines. Low peptide concentrations (1.1 mM) induced the formation of a heterogeneous mixture of vesicular structures: large multilamellar vesicles (d-spacing ~6.3 nm) coexisted with oligo- or unilamellar vesicles (~50 nm in diameter) and bicelle-like structures (~45 nm length, ~18 nm width). High peptide concentrations (11 mM) led to unilamellar vesicles (ULV, diameter ~260-280 nm) with a homogeneous mixing of lipids and peptides. SAXS revealed the temperature-dependent fine structure of these ULVs: at 25 °C the bilayer is in a fully interdigitated state (headgroup-to-headgroup distance dhh ~2.9 nm), whereas at 50 °C this interdigitation opens up (dhh ~3.6 nm). Our results highlight the versatility of self-assembled peptide superstructures: subtle changes in the amino acid composition are key design elements in creating peptide- or lipid-peptide nanostructures with the same richness in morphology as known from the lipid-world.

Peptides at the Interface: Self-Assembly of Amphiphilic Designer Peptides and Their Membrane Interaction Propensity.

Self-assembling amphiphilic designer peptides have been successfully applied as nanomaterials in biomedical applications. Understanding molecular interactions at the peptide-membrane interface is crucial, since interactions at this site often determine (in)compatibility. The present study aims to elucidate how model membrane systems of different complexity (in particular single-component phospholipid bilayers and lipoproteins) respond to the presence of amphiphilic designer peptides. We focused on two short anionic peptides, V4WD2 and A6YD, which are structurally similar but showed a different self-assembly behavior. A6YD self-assembled into high aspect ratio nanofibers at low peptide concentrations, as evidenced by synchrotron small-angle X-ray scattering and electron microscopy. These supramolecular assemblies coexisted with membranes without remarkable interference. In contrast, V4WD2 formed only loosely associated assemblies over a large concentration regime, and the peptide promoted concentration-dependent disorder on the membrane arrangement. Perturbation effects were observed on both membrane systems although most likely induced by different modes of action. These results suggest that membrane activity critically depends on the peptide's inherent ability to form highly cohesive supramolecular structures.

Impact of atomization technique on the stability and transport efficiency of nebulized liposomes harboring different surface characteristics.

The objective of this study was to evaluate the impact of nebulization on liposomes with specific surface characteristics by applying three commercially available inhaler systems (air-jet, ultrasonic and vibrating-mesh). Conventional liposome formulations composed of phosphatidylcholine and cholesterol were compared to sterically stabilized PEGylated liposomes and cationic polymer coated liposomes.Liposomes of similar size (between 140 and 165 nm in diameter with polydispersity indices <0.1) were prepared by dry lipid film rehydration followed by size extrusion. Their stability upon nebulization was determined in terms of size, polydispersity index and leakage using a fluorescence quenching system. The transport efficiencies of the nebulizer devices and the influences of both salt and liposomes on the droplet size distribution of the aerosol were investigated. While the droplet size of the aerosol decreased with increasing salt concentration the liposomes had no influence on the droplet size distribution. The output of the nebulizers in terms of liposomal transport efficiencies differed significantly among the nebulizer principles (20­100%, p < 0.05), with the vibrating-mesh nebulizers being the most effective. The integrity of the conventional liposomes was almost unaffected by the atomization process, while polymer coated and especially positively charged liposomes showed enhanced leakage. The release rates for the hydrophilic model drug system were highest for the vibrating-mesh nebulizers regardless of the surface characteristics of the liposomes (increasing from 10% to 20% and 50% for the conventional, PEGylated and positively charged formulations, respectively). In view of surface modified liposomes our data suggest that drug delivery via nebulization necessitates the finding of a compromise between nebulizer efficiency, formulation stability and drug release profile to accomplish the development of tailored formulations suitable for advanced inhalation therapy.

Liposomal nanoparticles encapsulating iloprost exhibit enhanced vasodilation in pulmonary arteries.

Prostacyclin analogues are standard therapeutic options for vasoconstrictive diseases, including pulmonary hypertension and Raynaud's phenomenon. Although effective, these treatment strategies are expensive and have several side effects. To improve drug efficiency, we tested liposomal nanoparticles as carrier systems. In this study, we synthesized liposomal nanoparticles tailored for the prostacyclin analogue iloprost and evaluated their pharmacologic efficacy on mouse intrapulmonary arteries, using a wire myograph. The use of cationic lipids, stearylamine, or 1,2-di-(9Z-octadecenoyl)-3-trimethylammonium-propane (DOTAP) in liposomes promoted iloprost encapsulation to at least 50%. The addition of cholesterol modestly reduced iloprost encapsulation. The liposomal nanoparticle formulations were tested for toxicity and pharmacologic efficacy in vivo and ex vivo, respectively. The liposomes did not affect the viability of human pulmonary artery smooth muscle cells. Compared with an equivalent concentration of free iloprost, four out of the six polymer-coated liposomal formulations exhibited significantly enhanced vasodilation of mouse pulmonary arteries. Iloprost that was encapsulated in liposomes containing the polymer polyethylene glycol exhibited concentration-dependent relaxation of arteries. Strikingly, half the concentration of iloprost in liposomes elicited similar pharmacologic efficacy as nonencapsulated iloprost. Cationic liposomes can encapsulate iloprost with high efficacy and can serve as potential iloprost carriers to improve its therapeutic efficacy.