| Micheletto, Y. M. S., Marques, C. M., da Silveira, N. P., & Schroder, A. P. (2016). Electroformation of Giant Unilamellar Vesicles: Investigating Vesicle Fusion versus Bulge Merging. Langmuir, 32(32), 8123–8130.|
Abstract: Partially ordered stacks of phospholipid bilayers on.a flat substrate can be obtained by the evaporation of a spread droplet of phospholipid-in-chloroform solution. When exposed to an aqueous buffer, numerous micrometric buds populate the bilayers, grow in size over minutes, and eventually detach, forming-the so-called liposomes or vesicles. While observation of vesicle growth from a hydrated lipid film under an optical microscope suggests numerous events of vesicle fusion, there is little experimental evidence for discriminating between merging of connected buds, i.e., a shape transformation that does not imply bilayer fusion and real membrane fusion. Here, we use electroformation to grow giant unilamellar vesicles (GUVs) from a stack of lipids in a buffer containing either (i) nanometric liposomes or (ii) previously prepared GUVs. By combining different fluorescent labels of the lipids in the substrate and in the solution, and by performing a fluorescence analysis of the resulting GUVs, we clearly demonstrate that merging of bulges is the essential pathway for vesicle growth in electroformation.
| Micheletto, Y. M. S., Marques, C. M., Silveira, N. P. da, & Schroder, A. P. (2016). Electroformation of Giant Unilamellar Vesicles: Investigating Vesicle Fusion versus Bulge Merging. Langmuir, 32(32), 8123–8130.|
Abstract: Partially ordered stacks of phospholipid bilayers on a flat substrate can be obtained by the evaporation of a spread droplet of phospholipid-in-chloroform solution. When exposed to an aqueous buffer, numerous micrometric buds populate the bilayers, grow in size over minutes, and eventually detach, forming the so-called liposomes or vesicles. While observation of vesicle growth from a hydrated lipid film under an optical microscope suggests numerous events of vesicle fusion, there is little experimental evidence for discriminating between merging of connected buds, i.e., a shape transformation that does not imply bilayer fusion and real membrane fusion. Here, we use electroformation to grow giant unilamellar vesicles (GUVs) from a stack of lipids in a buffer containing either (i) nanometric liposomes or (ii) previously prepared GUVs. By combining different fluorescent labels of the lipids in the substrate and in the solution, and by performing a fluorescence analysis of the resulting GUVs, we clearly demonstrate that merging of bulges is the essential pathway for vesicle growth in electroformation.
| Micheletto, Y. M. S., Moro, C. F., Lopes, F. C., Ligabue-Braun, R., Martinelli, A. H. S., Marques, C. M., et al. (2016). Interaction of jack bean (Canavalia ensiformis) urease and a derived peptide with lipid vesicles. Colloids Surf B Biointerfaces, 145, 576–585.|
Abstract: Ureases are metalloenzymes that catalyze the hydrolysis of urea to ammonia and carbon dioxide. Jack bean (Canavalia ensiformis) produces three isoforms of urease (Canatoxin, JBU and JBURE-II). Canatoxin and JBU display several biological properties independent of their ureolytic activity, such as neurotoxicity, exocytosis-inducing and pro-inflammatory effects, blood platelets activation, insecticidal and antifungal activities. The Canatoxin entomotoxic activity is mostly due to an internal peptide, named pepcanatox, released upon the hydrolysis of the protein by insect cathepsin-like digestive enzymes. Based on pepcanatox sequence, Jaburetox-2Ec was produced in Escherichia coli. JBU and its peptides were shown to permeabilize membranes through an ion channel-based mechanism. Here we studied the JBU and Jaburetox-2Ec interaction with platelet-like multilamellar liposomes (PML) using Dynamic Light Scattering and Small Angle X-ray Scattering techniques. We also analyzed the interaction of JBU with giant unilamellar vesicles (GUVs) using Fluorescence Microscopy. The interaction of vesicles with JBU led to a slight reduction of hydrodynamic radius, and caused an increase in the lamellar repeat distance of PML, suggesting a membrane disordering effect. In contrast, Jaburetox-2Ec decreased the lamellar repeat distance of PML membranes, while also diminishing their hydrodynamic radius. Fluorescence microscopy showed that the interaction of GUVs with JBU caused membrane perturbation with formation of tethers. In conclusion, JBU can interact with PML, probably by inserting its Jaburetox “domain” into the PML external membrane. Additionally, the interaction of Jaburetox-2Ec affects the vesicle's internal bilayers and hence causes more drastic changes in the PML membrane organization in comparison with JBU.
Keywords: Jaburetox-2Ec; Jack bean urease; Light scattering; Liposomes; Saxs
| Aoki, P. H. B., Schroder, A. P., Constantino, C. J. L., & Marques, C. M. (2015). Bioadhesive giant vesicles for monitoring hydroperoxidation in lipid membranes. Soft Matter, 11(30), 5995–5998.|
Abstract: Osmotic stresses, protein insertion or lipid oxidation lead to area increase of self-assembled lipid membranes. However, methods to measure membrane expansion are scarce. Challenged by recent progress on the control of phospholipid hydroperoxidation, we introduce a method to quantitatively evaluate membrane area increase based on the bio-adhesion of Giant Unilamellar Vesicles.
| Arranja, A., Schroder, A. P., Schmutz, M., Waton, G., Schosseler, F., & Mendes, E. (2014). Cytotoxicity and internalization of Pluronic micelles stabilized by core cross-linking. Journal of Controlled Release, 196, 87–95.|
Abstract: 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 degrees 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. (C) 2014 Elsevier B.V. All rights reserved.
| Marques, C., Weinberger, A., MacEwan, S., Schmatko, T., Schroder, A., & Chilkoti, A. (2014). Cargo Self-Assembly Controls Affinity Of Cell-Penetrating Peptides To Lipid Membranes. Journal Of Peptide Science, 20, S282.|
Keywords: Cell Penetrating Peptides; Elastin-like Polypetides; Giant Unilamellar Vesicles
| Sun, Y., Marques, C. M., & Schroder, A. P. (2014). Adhesion of giant unilamellar vesicles on double-end grafted DNA carpets. European Physical Journal-Special Topics, 223(9), 1755–1769.|
Abstract: We have recently shown that the bio-mimetic adhesion of Giant Unilamellar Vesicles on carpets of lambda-phage DNAs, grafted by one end to the substrate, leads to DNA scraping and stapling. As the lipid adhesion patch is built, outward forces stretch the DNA, while adhesion patch formation staples the chains into frozen conformations, trapped between the GUV membrane and the substrate. Analysis of the scraped and stapled DNA conformations provides a wealth of information about the membrane/polymer interactions at play during the formation of a bio-adhesive contact zone. In this paper we report new phenomena revealed by scraping and stapling phenomena associated with the bio-mimetic adhesion of Giant Unilamellar Vesicles on carpets of lambda-phage DNAs that were grafted to the substrate by both ends. In particular, the peculiar shapes of stapled DNA observed in this case, suggest that the membrane exerces not only outward radial forces during patch formation, but is is also able to confine the DNA molecules in the orthoradial direction.
| Weber, G., Charitat, T., Baptista, M. S., Uchoa, A. F., Pavani, C., Junqueira, H. C., et al. (2014). Lipid oxidation induces structural changes in biomimetic membranes. Soft Matter, 10(24), 4241–4247.|
Abstract: Oxidation can intimately influence and structurally compromise the levels of biological self-assembly embodied by intracellular and plasma membranes. Lipid peroxidation, a natural metabolic outcome of life with oxygen under light, is also a salient oxidation reaction in photomedicine treatments. However, the effect of peroxidation on the fate of lipid membranes remains elusive. Here we use a new photosensitizer that anchors and disperses in the membrane to achieve spatial control of the oxidizing species. We find, surprisingly, that the integrity of unsaturated unilamellar vesicles is preserved even for fully oxidized membranes. Membrane survival allows for the quantification of the transformations of the peroxidized bilayers, providing key physical and chemical information to understand the effect of lipid oxidation on protein insertion and on other mechanisms of cell function. We anticipate that spatially controlled oxidation will emerge as a new powerful strategy for tuning and evaluating lipid membranes in biomimetic media under oxidative stress.
| Weinberger, A., Tsai, F. - C., Koenderink, G. H., Schmidt, T. F., Itri, R., Meier, W., et al. (2013). Gel-Assisted Formation of Giant Unilamellar Vesicles. Biophysical Journal, 105(1), 154–164.|
Abstract: 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.
| Franco, I. E., Lorchat, P., Lamps, J. - P., Schmutz, M., Schroder, A., Catala, J. - M., et al. (2012). From chain collapse to new structures: spectroscopic properties of poly(3-thiophene acetic acid) upon binding by alkyl trimethylammonium bromide surfactants. Langmuir, 28(10), 4815–4828.|
Abstract: The binding of cationic surfactants with varying alkyl chain length to a regiorandom conjugated polyanion, poly(3-thiophene acetic acid) (PTAA), is studied in an aqueous buffer by using absorption and emission spectroscopies, photon correlation spectroscopy, isothermal titration calorimetry, and cryogenic transmission electron microscopy. We study the mixed solutions as a function of composition ratio R of surfactant molecules to monomer units molar concentrations, at low polymer concentration and in a very wide composition range (10(-6) < R < 10(2)) below the critical micellar concentration. Upon surfactant binding, the molecularly dispersed chains first collapse progressively and then form new structures as the mixed aggregates get enriched in surfactant. The collapse leads to a strong decrease of the conjugation length and to a blue shift of the absorption spectra by 30 to 50 nm. The new structures are responsible for a new intense emission band at about 600 nm, red-shifted by nearly 130 nm from the initial emission maximum of the polymer (~472 nm). As the surfactant tail becomes shorter, the blue shift of the absorption spectra and the intensity raise of the new emission are delayed to larger composition ratios while their variations become smoother functions of the surfactant concentration. These particular spectroscopic properties of PTAA seem related to its unique combination of a strongly hydrophobic backbone, a large ratio of contour length to persistence length, and an overall good aqueous solubility. Our results show that such features are well suited to design a colorimetric biosensor at small composition ratio, and a fluorescent biomarker at large composition ratio.
Keywords: Acetates/*chemistry; Biophysical Phenomena; *Biosensing Techniques; Calorimetry; Cryoelectron Microscopy; Hydrodynamics; Macromolecular Substances/chemistry; Molecular Structure; Quaternary Ammonium Compounds/chemistry; Spectrum Analysis; Surface-Active Agents/*chemistry; Thermodynamics; Thiophenes/*chemistry; Trimethyl Ammonium Compounds/*chemistry
| Haluska, C. K., Baptista, M. S., Fernandes, A. U., Schroder, A. P., Marques, C. M., & Itri, R. (2012). Photo-activated phase separation in giant vesicles made from different lipid mixtures. Biochimica Et Biophysica Acta-Biomembranes, 1818(3), 666–672.|
Abstract: 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. (C) 2011 Elsevier B.V. All rights reserved.
| Nam, G., Hisette, M. L., Sun, Y. L., Gisler, T., Johner, A., Thalmann, F., et al. (2010). Scraping and Stapling of End-Grafted DNA Chains by a Bioadhesive Spreading Vesicle to Reveal Chain Internal Friction and Topological Complexity. Prl, 105(8), 088101.|
Abstract: Stained end-grafted DNA molecules about 20  μm long are scraped away and stretched out by the spreading front of a bioadhesive vesicle. Tethered biotin ligands bind the vesicle bilayer to a streptavidin substrate, stapling the DNAs into frozen confinement paths. Image analysis of the stapled DNA gives access, within optical resolution, to the local stretching values of individual DNA molecules swept by the spreading front, and provides evidence of self-entanglements.
| Campillo, C. C., Schroder, A. P., Marques, C. M., & Pepin-Donat, B. (2009). Composite gel-filled giant vesicles: Membrane homogeneity and mechanical properties. Materials Science & Engineering C-Biomimetic And Supramolecular Systems, 29(2), 393–397.|
Abstract: We further investigate the properties of composite Poly(NIPAM) poly(N-isopropylacrylamide)) gel-filled giant vesicles, focusing here on i) the homogeneity of the membrane, ii) its coupling to the inner gel under strong suction pressures, and iii) the relation between the final elastic modulus of the vesicles and the amount of crosslinker in the pre-gel medium. We show that whereas the photo-polymerization process induces a decrease of the membrane homogeneity at the micrometer size range. the membrane still remains strongly coupled to the internal gel network. The vesicles studied here display average moduli in the range [0.5-25] kPa. confirming their potential as biomimetic mechanical systems. (C) 2008 Elsevier B.V. All rights reserved.
| Mertins, O., da Silveira, N. P., Pohlmann, A. R., Schroder, A. P., & Marques, C. M. (2009). Electroformation of Giant Vesicles from an Inverse Phase Precursor. Biophysical Journal, 96(7), 2719–2726.|
Abstract: 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.
| Riske, K. A., Sudbrack, T. P., Archilha, N. L., Uchoa, A. F., Schroder, A. P., Marques, C. M., et al. (2009). Giant Vesicles under Oxidative Stress Induced by a Membrane-Anchored Photosensitizer. Biophysical Journal, 97(5), 1362–1370.|
Abstract: We have synthesized the amphiphile photosensitizer PE-porph consisting of a porphyrin bound to a lipid head-group. 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.
| Tauk, L., Schroder, A. P., Decher, G., & Giuseppone, N. (2009). Hierarchical functional gradients of pH-responsive self-assembled monolayers using dynamic covalent chemistry on surfaces. Nature Chemistry, 1(8), 649–656.|
Abstract: Surface chemistry is an important field of research, especially for the study and design of (bio)nanostructures in which nearly every atom lies at an interface. Here we show that dynamic covalent chemistry is an efficient tool for functionalizing surfaces in such a way that their interfacial properties can be varied controllably in space and time. Modulation of pH is used to tune the fast, selective and reversible attachment of functional amines (with different pK(a) values) to an aldehyde-coated surface. To illustrate the potential of this technique, we developed dynamic self-assembled monolayers ('DynaSAMs'), which enable the hierarchical construction of mixed gradients comprising either small functional molecules or proteins. Control of the (bio)chemical composition at any point on the surface potentially provides a simple bottom-up method to access numerous surface patterns with a broad range of functionalities.
| Campillo, C. C., Schroder, A. P., Marques, C. M., & Pepin-Donat, B. (2008). Volume transition in composite poly(NIPAM)-giant unilamellar vesicles. Soft Matter, 4(12), 2486–2491.|
Abstract: 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 phi(NIPAM) of encapsulated NIPAM, the precursor monomer for poly(NIPAM). For phi(NIPAM) <= 0.06, we observe a frustrated demixing of the poly(NIPAM) chains in the internal medium; for phi(NIPAM) >= 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 (phi(NIPAM) = 0.09) achieved by osmotic deflation of low volume fraction (phi(NIPAM) = 0.03) sol-GUVs, we observe a full demixing of the internal medium into two well-separated phases.
| Haluska, C. K., Schroder, A. P., Didier, P., Heissler, D., Duportail, G., Mely, Y., et al. (2008). Combining Fluorescence Lifetime and Polarization Microscopy to Discriminate Phase Separated Domains in Giant Unilamellar Vesicles. Biophysical Journal, 95(12), 5737–5747.|
Abstract: 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.
| Hisette, M. L., Haddad, P., Gisler, T., Marques, C. M., & Schroder, A. P. (2008). Spreading of bio-adhesive vesicles on DNA carpets. Soft Matter, 4(4), 828–832.|
Abstract: Cell-adhesion events involve often the formation of a contact region between phospholipid membranes decorated with a variety of bio-macromolecular species. We mimic here such hairy bio-adhesive contact zones by spreading phospholipid vesicles onto surfaces carpeted with end-grafted lambda-phage DNA. Our study reveals that the spreading front acts as a scraper that strongly stretches the DNA molecules, and that the multiple bonds created during vesicle spreading effectively staple the stretched chains in the gap between the membrane and the substrate. The scraping and stapling mechanisms revealed here for the long DNA molecules are expected to also play a role in actual bio-adhesion events of cell walls and tissues.
| Caetano, W., Haddad, P. S., Itri, R., Severino, D., Vieira, V. C., Baptista, M. S., et al. (2007). Photo-induced destruction of giant vesicles in methylene blue solutions. Langmuir, 23(3), 1307–1314.|
Abstract: We study the photodecomposition of phospholipid bilayers in aqueous solutions of methylene blue. Observation of giant unilamellar vesicles under an optical microscope reveals a consistent pattern of membrane disruption as a function of methylene blue concentration and photon density for different substrates supporting the vesicles.
| Durian, D. J., Bideaud, H., Duringer, P., Schroder, A. P., & Marques, C. M. (2007). Shape and erosion of pebbles. Physical Review E, 75(2), 021301.|
Abstract: The shapes of flat pebbles may be characterized in terms of the statistical distribution of curvatures measured along their contours. We illustrate this method for clay pebbles eroded in a controlled laboratory apparatus, and also for naturally occurring rip-up clasts formed and eroded in the Mont St.-Michel bay. We find that the curvature distribution allows finer discrimination than traditional measures of aspect ratios. Furthermore, it connects to the microscopic action of erosion processes that are typically faster at protruding regions of high curvature. We discuss in detail how the curvature may be reliably deduced from digital photographs.
| Durian, D. J., Bideaud, H., Duringer, P., Schroder, A., Thalmann, F., & Marques, C. M. (2006). What is in a pebble shape? Phys. Rev. Lett., 97(2), 4 pp.|
Abstract: 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.
| Fa, N., Marques, C. M., Mendes, E., & Schroder, A. P. (2004). Rheology of giant vesicles: A micropipette study. Phys. Rev. Lett., 92(10), 4 pp.|
Abstract: We develop a micropipette rheometer to study the effect of oscillatory shear flow on the spontaneous fluctuations of phospholipid bilayers. Our results on giant vesicles show that oscillatory shear flow leads to a suppression of membrane fluctuations. They also imply that the Helfrich equation is modified in the presence of the flow. This equation, a fundamental constitutive relation between the amount of area stored in the fluctuations and the membrane tension, must be supplemented under oscillatory shear by a flow excess function that we determine.