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.

Cholesterol-based lipophilic oligonucleotides incorporated into lipid membranes were studied using solid-state NMR, differential scanning calorimetry, and fluorescence methods. Lipophilic oligonucleotides can be used to build nanotechnological structures on membrane surfaces, taking advantage of the specific Watson-Crick base pairing. We used a cholesteryl-TEG anchor first described by Pfeiffer and Hook (J. Am. Chem. Soc. 2004, 126, 10224-10225). The cholesterol-based anchor molecules were found to incorporate well into lipid membranes without disturbing the bilayer structure and dynamics. In contrast to cholesterol, which is known to induce significant condensation of the membrane lipids, the cholesteryl-TEG anchor does not display this property. When the cholesteryl-TEG moiety was covalently bound to an oligonucleotide, the resulting lipophilic DNA molecules inserted spontaneously into lipid membranes without altering their structure. The duplex formed by two complementary cholesteryl-TEG oligonucleotides had increased thermodynamic stability compared to the same oligonucleotides without the anchor, both in solution and incorporated into lipid membranes. Since the cholesteryl-TEG anchor lacks the characteristic properties of cholesterol, oligonucleotides modified with this anchor are equally distributed between liquid-disordered and liquid-ordered domains in "raft" forming membranes. As an example of an application of these lipophilic oligonucleotides, cholesteryl-TEG-DNA was incorporated into supported lipid bilayers formed on polyelectrolyte-coated silica microparticles. The modified oligonucleotides were stably inserted into the lipid membrane and retained their recognition properties, therefore enabling further functionalization of the particles.

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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.

Microscopic colloidal particles allow a precise regulation of chemical reactions in time and place. A controlled assembly of multiple layers of intact lipid vesicles on a solid support provided by layer‐by‐layer particles functionalized by a covalent attachment of DNA oligonucleotides is reported (see image). Lipophilic complementary oligonucleotides are incorporated into lipid vesicles. Fusion of liposomes and release can be triggered.

The pathway for contents release from reduction-sensitive liposomes based on a quinone-dioleoylphosphatidylethanolamine lipid conjugate (Q-DOPE) was outlined using results from fluorescent dye contents 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 contents 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 contents release. At high DOPE concentrations, DOPE/PEG-DOPE/Q-DOPE liposomes exhibit larger contents release, indicating a change in pathway for contents release. The outcomes here provide a better understanding of the underlying principles of triggered liposomal contents release and the potential utility of specific lipid properties for the rational design of drug delivery systems based on the novel Q-DOPE lipid.

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 31 P and 2 H 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 Mn 2+ 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.

An advanced system based on layer‐by‐layer (LbL) technology and fluorescence resonance energy transfer (FRET) for the detection of small amounts of DNA has been developed. Several advantages over conventional particle systems due to nanoroughness, flexibility and specific surface properties of LbL films were determined, making LbL‐oligonucleotide particles a first choice for homogeneous diagnostic assays.