Spontaneous formation of nanometer scale tubular vesicles in aqueous mixtures of lipid and block copolymer amphiphiles.

Many common amphiphiles self-assemble in water to produce heterogeneous populations of discrete and symmetric but polydisperse and multilamellar vesicles isolating the encapsulated aqueous core from the surrounding bulk. But when mixtures of amphiphiles of vastly different elastic properties co-assemble, their non-uniform molecular organization can stabilize lower symmetries and produce novel shapes. Here, using high resolution electron cryomicroscopy and tomography, we identify the spontaneous formation of a membrane morphology consisting of unilamellar tubular vesicles in dilute aqueous solutions of binary mixtures of two different amphiphiles of vastly different origins. Our results show that aqueous phase mixtures of a fluid-phase phospholipid and an amphiphilic block copolymer spontaneously assume a bimodal polymorphic character in a composition dependent manner: over a broad range of compositions (15-85 mol% polymer component), a tubular morphology co-exists with spherical vesicles. Strikingly, in the vicinity of equimolar compositions, an exclusively tubular morphology (Lt; diameter, ∼15 nm; length, >1 µm; core, ∼2.0 nm; wall, ∼5-6 nm) emerges in an apparent steady state. Theory suggests that the spontaneous stabilization of cylindrical vesicles, unaided by extraneous forces, requires a significant spontaneous bilayer curvature, which in turn necessitates a strongly asymmetric membrane composition. We confirm that such dramatic compositional asymmetry is indeed produced spontaneously in aqueous mixtures of a lipid and polymer through two independent biochemical assays - (1) reduction in the quenching of fluorophore-labeled lipids and (2) inhibition in the activity of externally added lipid-hydrolyzing phospholipase A2, resulting in a significant enrichment of the polymer component in the outer leaflet. Taken together, these results illustrate the coupling of the membrane shape with local composition through spontaneous curvature generation under conditions of asymmetric distribution of mixtures of disparate amphiphiles.

Peptide-assembled graphene oxide as a fluorescent turn-on sensor for lipopolysaccharide (endotoxin) detection.

Lipopolysaccharide (LPS) is a toxic inflammatory stimulator released from the outer cell membrane of Gram-negative bacteria, known to be directly related to, for example, septic shock, that causes millions of casualties annually. This number could potentially be lowered significantly if specific, sensitive, and more simply applicable LPS biosensors existed. In this work, we present a facile, sensitive and selective LPS sensor, developed by assembling tetramethylrhodamine-labeled LPS-binding peptides on graphene oxide (GO). The fluorescence of the dye-labeled peptide is quenched upon interaction with GO. Specific binding to LPS triggers the release of the peptide-LPS complex from GO, resulting in fluorescence recovery. This fluorescent turn-on sensor offers an estimated limit of detection of 130 pM, which is the lowest ever reported among all synthetic LPS sensors to date. Importantly, this sensor is applicable for detection of LPS in commonly used clinical injectable fluids, and it enables selective detection of LPS from different bacterial strains as well as LPS on the membrane of living E. coli.

Reporter-encapsulated liposomes on graphene field effect transistors for signal enhanced detection of physiological enzymes.

A novel approach for enzymatic assay using reporter-encapsulated liposomes on graphene field effect transistors (FET) is proposed. This approach involves real time monitoring of drain current (Id) of reduced graphene oxide (rGO) upon rupture of reporter-encapsulated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) liposomes triggered by enzymes. For validation of the proposed approach, 2,4,6-trinitrophenol (TNP) is used as the reporter for specific detection of phospholipase A2 (PLA2), a key enzyme in various membrane related physiological processes. Experimental results revealed that Id increased with PLA2 concentration, which is attributed to the interaction between released TNP and rGO. The limit of detection (LOD) achieved by the proposed approach was 80 pM, which is superior to most assays reported previously and much lower than the cut-off level of circulating secretory PLA2 (2.07 nM). Besides the high accuracy of the electronic detection methodology, the signal enhancement effect realized by the excess concentration of TNP (approximately 1 mM) in liposomes is believed to be the main reason for the significantly enhanced sensitivity of the proposed assay, indicating great potential for further improvement in the sensitivity by increasing the concentration of TNP. In addition, the proposed approach is rapid (incubation time ≤ 10 min) and label-free, thus showing great potential for practical applications in the future.

Tuning Liposome Membrane Permeability by Competitive Peptide Dimerization and Partitioning-Folding Interactions Regulated by Proteolytic Activity.

Membrane active peptides are of large interest for development of drug delivery vehicles and therapeutics for treatment of multiple drug resistant infections. Lack of specificity can be detrimental and finding routes to tune specificity and activity of membrane active peptides is vital for improving their therapeutic efficacy and minimize harmful side effects. We describe a de novo designed membrane active peptide that partition into lipid membranes only when specifically and covalently anchored to the membrane, resulting in pore-formation. Dimerization with a complementary peptide efficiently inhibits formation of pores. The effect can be regulated by proteolytic digestion of the inhibitory peptide by the matrix metalloproteinase MMP-7, an enzyme upregulated in many malignant tumors. This system thus provides a precise and specific route for tuning the permeability of lipid membranes and a novel strategy for development of recognition based membrane active peptides and indirect enzymatically controlled release of liposomal cargo.