André P. Schroder

Gel-Assisted Formation of Giant Unilamellar Vesicles

Giant unilamellar vesicles or GUVs are systems of choice as biomimetic models of cellular membranes. Although a variety of procedures exist for making single walled vesicles of tens of microns in size, the range of lipid compositions that can be used to grow GUVs by the conventional methods is quite limited, and many of the available methods involve energy input that can damage the lipids or other molecules present in the growing solution for embedment in the membrane or in the vesicle interior. Here, we show that a wide variety of lipids or lipid mixtures can grow into GUVs by swelling lipid precursor films on top of a dried polyvinyl alcohol gel surface in a swelling buffer that can contain diverse biorelevant molecules. Moreover, we show that the encapsulation potential of this method can be enhanced by combining polyvinyl alcohol-mediated growth with inverse-phase methods, which allow (bio)molecule complexation with the lipids.

Giant vesicles under oxidative stress induced by a membrane-anchored photosensitizer.

We have synthesized the amphiphile photosensitizer PE-porph consisting of a porphyrin bound to a lipid headgroup. We studied by optical microscopy the response to light irradiation of giant unilamellar vesicles of mixtures of unsaturated phosphatidylcholine lipids and PE-porph. In this configuration, singlet oxygen is produced at the bilayer surface by the anchored porphyrin. Under irradiation, the PE-porph decorated giant unilamellar vesicles exhibit a rapid increase in surface area with concomitant morphological changes. We quantify the surface area increase of the bilayers as a function of time and photosensitizer molar fraction. We attribute this expansion to hydroperoxide formation by the reaction of the singlet oxygen with the unsaturated bonds. Considering data from numeric simulations of relative area increase per phospholipid oxidized (15%), we measure the efficiency of the oxidative reactions. We conclude that for every 270 singlet oxygen molecules produced by the layer of anchored porphyrins, one eventually reacts to generate a hydroperoxide species. Remarkably, the integrity of the membrane is preserved in the full experimental range explored here, up to a hydroperoxide content of 60%, inducing an 8% relative area expansion.

Combining fluorescence lifetime and polarization microscopy to discriminate phase separated domains in giant unilamellar vesicles.

Using fluorescence lifetime microscopy we study the structure of lipid domains in giant unilamellar vesicles made from sphingomyelin, 1,2-dioleoyl-sn-glycero-3-phosphocholine, and cholesterol. Lifetimes and orientation of a derivative of the fluorescent probe DPH embedded in the membrane were measured for binary and ternary lipid mixtures incorporating up to 42 mol % of cholesterol. The results show that adding cholesterol always increases the lifetime of the probe studied. In addition, the analysis of the probe orientation indicates that cholesterol has little influence on the ordering of the sphingomyelin alkyl chains whereas it has a noticeable effect on the structure of the 1,2-dioleoyl-sn-glycero-3-phosphocholine chains. The measurements made on the orientation and lifetime of the probe show the structure of the membrane in its liquid ordered and liquid disordered domains.

Contactless measurement of the elastic Young's modulus of paper by an ultrasonic technique

Abstract The use of a non-contact ultrasonic technique for the characterization of the in-plane elastic Youngs moduli of paper is considered. The determination of these moduli from A0 (fundamental antisymmetric plate wave mode) measurements, using the empirical technique introduced by Luukkala et al. (1971), is discussed: it is shown that the common low frequency approximation is not valid for paper, owing to its orthotropic structure. Numerical simulations are used to evaluate the validity domain of Luukkalas method. A new device was developed, and Youngs moduli of various paper sheets were determined from A0 measurements. These moduli values were found to be in good agreement with those measured using a common laboratory contact ultrasonic technique.

Photo-activated phase separation in giant vesicles made from different lipid mixtures

Using giant unilamellar vesicles (GUVs) made from POPC, DPPC, cholesterol and a small amount of a porphyrin-based photosensitizer that we name PE-porph, we investigated the response of the lipid bilayer under visible light, focusing in the formation of domains during the lipid oxidation induced by singlet oxygen. This reactive species is generated by light excitation of PE-porf in the vicinity of the membrane, and thus promotes formation of hydroperoxides when unsaturated lipids and cholesterol are present. Using optical microscopy we determined the lipid compositions under which GUVs initially in the homogeneous phase displayed Lo-Ld phase separation following irradiation. Such an effect is attributed to the in situ formation of both hydroperoxized POPC and cholesterol. The boundary line separating homogeneous Lo phase and phase coexistence regions in the phase diagram is displaced vertically towards the higher cholesterol content in respect to ternary diagram of POPC:DPPC:cholesterol mixtures in the absence of oxidized species. Phase separated domains emerge from sub-micrometer initial sizes to evolve over hours into large Lo-Ld domains completely separated in the lipid membrane. This study provides not only a new tool to explore the kinetics of domain formation in mixtures of lipid membranes, but may also have implications in biological signaling of redox misbalance.

Electroformation of Giant Vesicles from an Inverse Phase Precursor

We discuss a simple modification of the well-known method of giant vesicle electroformation that allows for a direct addition of water-soluble species to the phospholipid bilayers. Using this modified method, we prepare phospholipid vesicles decorated with chitosan, a water-soluble polysaccharide currently investigated for potential pharmacological applications. We find that the method allows this polysaccharide with primary amino groups on every glucose subunit to be tightly bound to the membrane, rather than simply being encapsulated.

What Is in a Pebble Shape

We propose to characterize the shapes of flat pebbles in terms of the statistical distribution of curvatures measured along the pebble contour. This is demonstrated for the erosion of clay pebbles in a controlled laboratory apparatus. Photographs at various stages of erosion are analyzed, and compared with two models. We find that the curvature distribution complements the usual measurement of aspect ratio, and connects naturally to erosion processes that are typically faster at protruding regions of high curvature.

Volume transition in composite poly(NIPAM)–giant unilamellar vesicles

We have recently reported on the formation of composite gel vesicles prepared by the photopolymerization and crosslinking of poly(N-isopropyl-acrylamide) [poly(NIPAM)] inside phospholipid giant unilamellar vesicles (GUVs). Here we present a detailed study of the thermo-responsive behaviour of such composite vesicles. Giant vesicles filled with a poly(NIPAM) gel (gel–GUVs) exhibit a global volume phase transition, revealing a strong interaction between the gel and the phospholipid bilayer. Fluorescence studies show that the lipid membrane is not destroyed during the volume transition. The behaviour of giant vesicles filled with a poly(NIPAM) solution (sol–GUVs) depends on the volume fraction ΦNIPAM of encapsulated NIPAM, the precursor monomer for poly(NIPAM). For ΦNIPAM ≤ 0.06, we observe a frustrated demixing of the poly(NIPAM) chains in the internal medium; for ΦNIPAM ≥ 0.07, sol–GUVs behave like homogeneous spheres and undergo a global volume phase transition similar to the one observed in gel–GUVs. For high volume fractions (ΦNIPAM = 0.09) achieved by osmotic deflation of low volume fraction (ΦNIPAM = 0.03) sol–GUVs, we observe a full demixing of the internal medium into two well-separated phases.

Cytotoxicity and internalization of Pluronic micelles stabilized by core cross-linking

A UV-cross-linkable agent was incorporated and polymerized in Pluronic micelle core to create an interpenetrating polymer network (IPN) of poly(pentaerythritol tetraacrylate). This stabilization prevented micelle disruption below the critical micelle temperature (CMT) and concentration (CMC), while maintaining the integrity of the PEO corona and the hydrophobic properties of the PPO core. The prepared stabilized spherical micelles of Pluronic P94 and F127 presented hydrodynamic diameters ranging from 40 to 50 nm. The stability of cross-linked Pluronic micelles at 37 °C in the presence of serum proteins was studied and no aggregation of the micelles was observed, revealing the colloidal stability of the system. Cytotoxicity experiments in NIH/3T3 mouse fibroblasts revealed that the presence of the cross-linking agent did not induce any further toxicity in comparison to the respective pure polymer solutions. Furthermore, stabilized micelles of Pluronic P94 were shown to be less toxic than the polymer itself. A hydrophobic fluorescent probe (Nile red) was absorbed in the cross-linked core of pre-stabilized micelles to mimic the incorporation of a poorly water-soluble drug, and the internalization and intracellular localization of Nile red was studied by confocal microscopy at different incubation times. Overall, the results indicate that Pluronic micelles stabilized by core cross-linking are capable of delivering hydrophobic components physically entrapped in the micelles, thus making them a potential candidate as a delivery platform for imaging or therapy of cancer.

Insight into the gelation habit of oligo(para-phenylene vinylene) derivatives: effect of end-groups

This paper reports a study on the gelation habits of organogels prepared in benzyl alcohol from derivatives of oligo(p-phenylene vinylene) (OPV) bearing C16H33 aliphatic wings and a series of end groups on the backbone. Significant differences are observed based on whether or not these end groups can establish hydrogen bonds with adjacent OPV molecules or with the solvent. It is particularly shown that the fibril structure, the morphology and the molecular organization not only depend upon the aliphatic wings and/or the π–π interactions of the backbones but equally on the nature of these end groups. Evidence highlighting a homogeneous nucleation process for gel formation are also presented and discussed. Discrepancies with thermoreversible gels of covalent polymers are also stressed.