Martin Loew

Reduction-Sensitive Liposomes from a Multifunctional Lipid Conjugate and Natural Phospholipids: Reduction and Release Kinetics and Cellular Uptake

The development of targeted and triggerable delivery systems is of high relevance for anticancer therapies. We report here on reduction-sensitive liposomes composed of a novel multifunctional lipidlike conjugate, containing a disulfide bond and a biotin moiety, and natural phospholipids. The incorporation of the disulfide conjugate into vesicles and the kinetics of their reduction were studied using dansyl-labeled conjugate 1 in using the dansyl fluorescence environmental sensitivity and the Förster resonance energy transfer from dansyl to rhodamine-labeled phospholipids. Cleavage of the disulfide bridge (e.g., by tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT), l-cysteine, or glutathione (GSH)) removed the hydrophilic headgroup of the conjugate and thus changed the membrane organization leading to the release of entrapped molecules. Upon nonspecific uptake of vesicles by macrophages, calcein release from reduction-sensitive liposomes consisting of the disulfide conjugate and phospholipids was more efficient than from reduction-insensitive liposomes composed only of phospholipids. The binding of streptavidin to the conjugates did not interfere with either the subsequent reduction of the disulfide bond of the conjugate or the release of entrapped molecules. Breast cancer cell line BT-474, overexpressing the HER2 receptor, showed a high uptake of the reduction-sensitive doxorubicin-loaded liposomes functionalized with the biotin-tagged anti-HER2 antibody. The release of the entrapped cargo inside the cells was observed, implying the potential of using our system for active targeting and delivery.

Lipid Domain Specific Recruitment of Lipophilic Nucleic Acids: A Key for Switchable Functionalization of Membranes

Lipid domains in mammalian plasma membranes serve as platforms for specific recruitment or separation of proteins involved in various functions. Here, we have applied this natural strategy of lateral separation to functionalize lipid membranes at micrometer scale in a switchable and reversible manner. Membrane-anchored peptide nucleic acid and DNA, differing in their lipophilic moieties, partition into different lipid domains in model and biological membranes. Separation was visualized by hybridization with the respective complementary fluorescently labeled DNA strands. Upon heating, domains vanished, and both lipophilic nucleic acid structures intermixed with each other. Reformation of the lipid domains by cooling led again to separation of membrane-anchored nucleic acids. By linking appropriate structures/functions to complementary strands, this approach offers a reversible tool for triggering interactions among the structures and for the arrangement of reactions and signaling cascades on biomimetic surfaces.

Controlled Assembly of Vesicle‐Based Nanocontainers on Layer‐by‐Layer Particles via DNA Hybridization

Layer-by-layer (LbL) particles represent versatile microstructures for numerous applications such as encapsulation and controlled release of proteins, sensors for biomolecules, and chemical reactors on a micrometer scale. Advantages of these structures are the simple functionalization of the capsule wall, high stability, large size range (from 100 nm to 15mm), monodispersity, tunable permeability, and biodegradability. Successful incorporation of various polymers, and even intact lipid vesicles, into LbL assemblies have been demonstrated. LbL particles carrying nanocontainers with reactants to be released on demand would offer novel attractive applications. LbL particles coated with different types of vesicles containing specific reactants could be used to generate microenvironments, in which distinct reactions at a specific time and place could be triggered.

Lipid nature and their influence on opening of redox-active liposomes.

The pathway for content release from reduction-sensitive liposomes based on a quinone-dioleoylphosphatidylethanolamine lipid conjugate (Q-DOPE) is outlined using results from fluorescent dye content release assays as well as single- and multiple-angle light scattering. Experimental observations are consistent with a shape/size change of the reduced liposomes prior to their aggregation, with subsequent near-quantitative content release achieved only when the lipid membrane experiences conditions favorable to a lamellar to an inverted hexagonal phase transition. Addition of poly(ethyleneglycol)-modified DOPE (PEG-DOPE) to the Q-DOPE liposomal formulation results in stabilization of the lipid bilayer, whereas incorporation of DOPE yields faster content release. At high DOPE concentrations, DOPE/PEG-DOPE/Q-DOPE liposomes exhibit larger content release, indicating a change in pathway for content release. The outcomes here provide a better understanding of the underlying principles of triggered liposomal content release and the potential utility of specific lipid properties for the rational design of drug delivery systems based on the novel Q-DOPE lipid.

Nucleic Acid Diagnostic FRET Particles Based on Layer-by-Layer Technology

Due to the fast growing needs in rapid disease diagnosis, gene sequencing, food and environmental analysis, specifi c detection of nucleic acids has drawn great attention from researchers worldwide. [ 1 ] Nucleic acid detection is usually based on hybridization of a target to the probe DNA immobilized on a solid support, e.g. microarray slides, gold nanoparticles, quantum dots (QDs) or other particle-based systems. [ 2,3 ] The detection of the hybridization event is mainly performed by optical or electrochemical methods. [ 4 ] One widely used optical method is based on Förster Resonance Energy Transfer (FRET). [ 5 ] When an excited fl uorescent dye molecule comes close to a dye of lower excitation energy in a few nanometer range, the energy is transferred to the latter one, which start to show fl uorescence while the fl uorescence of the originally excited molecule is quenched. This effect is used as molecular ruler for structure and conformational changes of nucleic acids and proteins and for several sensing applications. [ 6 ] Some FRET systems for DNA detection are applied in solution, such as the Molecular Beacon method [ 7 ]

Characterization of lipid bilayers adsorbed on spherical LbL-support

Structural and dynamic properties of membranes composed of phosphatidylcholine (PC) and phosphatidylserine (PS) on layer-by-layer (LbL) polyelectrolyte coated particles were investigated using solid-state nuclear magnetic resonance (NMR) and fluorescence methods. These spherically supported membranes showed structural, dynamic, and elastic properties similar to free-standing membranes as proved by 31P and 2H NMR. Small differences between behaviour of PC and PS on LbL support due to interaction with the polyelectrolyte were observed. Fluorescence lifetime imaging microscopy (FLIM) using 7-nitro-2-1,3-benzoxadiazol (NBD) labeled PC and PS showed a stronger impact of the outermost polyelectrolyte (PAH) on the fluorescence lifetimes of NBD-PS compared to NBD-PC. Although small defects in nm range allowing passage of Mn2+ to both layers of the membrane coat were present, a rather homogeneous coating observed by fluorescence microscopy, complete fluorescence recovery after photobleaching, and NMR results reveal that somewhat continuous lipid bilayers were formed around the LbL particles.

Release Rates of Liposomal Contents Are Controlled by Kosmotropes and Chaotropes

Contents release from redox-responsive liposomes is anion-specific. Liposomal contents release is initiated by the contact of apposed liposome bilayers having in their outer leaflet 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), whose presence is due to the redox-stimulated removal of a quinone propionic acid protecting group (Q) from Q-DOPE lipids. Contents release occurs upon the phase transition of DOPE from its lamellar liquid-crystalline phase (Lα) to its hexagonal-II inverted micelle (HII) phase. Contents release is slower in the presence of weakly hydrated chaotropic anions versus highly hydrated kosmotropic anions and is attributed to ion accumulation near the zwitterionic DOPE headgroups, in turn altering the headgroup hydration, as indicated by the Lα → HII phase transition temperature, TH, for DOPE. The results are significant, not only for mechanistic aspects of liposome contents release in DOPE-based systems but also for drug delivery applications wherein exist at drug targeting sites variations in the type and concentration of ions and neutral species.