Electroformation and collection of giant liposomes on an integrated microchip

In this work, a microchip was developed for the electroformation and collection of giant liposomes. On the chip, a reactor chamber array was expressly designed so that the influences of multiple parameters could be explored rapidly and efficiently to screen for a suitable condition. Furthermore, in order to overcome the difficulty in the collection of giant liposomes, a tailor-made collection chamber was also integrated on this chip.     

Dynamics of liposomes constructed from phytanyl lipids

Liposomes constructed from phytanyl lipids3-F/3-NF exhibit very facile transbilayer (“flip-flop”) lipid migration.     

The highly conducting carbon electrodes derived from spin-coated polyacrylonitrile films

Carbon films prepared from pyrolyzation of spin-casted polyacrylonitrile (PAN) thin films display high electrical conductivity (600 S/cm, at 1000 °C carbonization), low sheet resistance (about 100 Ω/square at the PAN film thickness of 70 nm) and partial transmittance. These pyrolyzed PAN (PPAN) films were patterned as bottom electrodes by photolithography, and utilized as drain and source electrodes to fabricate organic field-effect transistor (OFET) devices with a p-type semiconductor (P3HT) and an n-type semiconductor (DPP-containing quinoidal small molecule) through a spin-coating procedure. The results showed that the devices with the PAN electrodes exhibited almost the same excellent performance without any further modification compared to those devices with traditional Au electrodes. Since these PPAN films had the advantages of low-cost, high performance, easier for large-area fabrication, thermal and chemical stability, it should be a promising electrode material for organic electrodes.     

Elaboration of spin-coated cellulose-xyloglucan multilayered thin films

In the context of developing a biomimetic model of the primary cell wall, our aim was to produce multilayered thin films composed of cellulose nanocrystals (CN) and xyloglucan (XG). We investigated the effect of XG concentrations ranging from 0.5 g/L to 10 g/L. The choice of concentration was based on rheological investigation of the XG solutions which indicated that the two lower concentrations (0.5 and 1 g/L) correspond to a semidilute regime where the polymer chains are not entangled, whereas they are entangled at the highest concentrations (5 and 10 g/L). Several processes of film preparation were tested (dipping or spin-coating, with or without a rinsing step). The film growth profiles obtained for different XG concentrations by mechanical profilometry showed that spin-coating without rinsing was the most efficient process. Results showed that at high XG concentrations (XG = 5 g/L and XG = 10 g/L) plateau values were reached after the formation of 3 or 4 bilayers, whereas growth of the multilayer structure was linear at the lower XG concentrations (XG = 0.5 g/L and XG = 1 g/L). The thickness of one CN/XG bilayer corresponded to a single layer of CN covered by a thin XG layer, despite the absence of a rinsing step between successive coatings. The importance of the XG concentration was confirmed by determining by neutron reflectivity the film architecture obtained from four XG solutions after eight successive paired coatings. The results are discussed in relation to the role of XG in the plant cell wall.     

Characteristics of thin cellulose ester films spin-coated from acetone and ethyl acetate solutions

Spin-coated films of cellulose acetate (CA), cellulose acetate propionate (CAP), cellulose acetate butyrate (CAB) and carboxymethylcellulose acetate butyrate (CMCAB) have been characterized by ellipsometry, atomic force microscopy (AFM) and contact angle measurements. The films were spin-coated onto silicon wafers, a polar surface. Mean thickness values were determined by means of ellipsometry and AFM as a function of polymer concentration in solutions prepared either in acetone or in ethyl acetate (EA), both are good solvents for the cellulose esters. The results were discussed in the light of solvent evaporation rate and interaction energy between substrate and solvent. The effects of annealing and type of cellulose ester on film thickness, film morphology, surface roughness and surface wettability were also investigated. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

AFM characterization of spin-coated multilayered dry lipid films prepared from aqueous vesicle suspensions

We present a detailed AFM study on multilayered dry lipid films prepared from aqueous vesicle suspensions. Different preparation techniques were applied in order to optimize the preparation of homogeneous lipid films of various film thicknesses. Suspensions of preformed DOPC/DPPC vesicles were adsorbed onto indium tin oxide-coated glass coverslips, a substrate also commonly employed for the formation of giant liposomes. We found that the homogeneity of the lipid films could substantially be improved when applying a spin-coating step during the film preparation. These films were much more homogeneous than those prepared by conventional drop-casting and in addition the film thickness could be controlled. When using a combination of vesicle adsorption and spin-coating the quality and thickness of the films depended crucially on the lipid concentration of the vesicle suspension, the adsorption temperature and the adsorption time. For lipid films prepared by direct spin-coating the lipid concentration and the applied spin-coating sequence were critical parameters for the quality and thickness of the deposited lipid films.     

Efficient formation of giant liposomes through the gentle hydration of phosphatidylcholine films doped with sugar

Giant liposomes, or giant vesicles, are cell-size (approximately 5-100 microm) compartments enclosed with phospholipid bilayers, and have often been used in biological research. They are usually generated using hydration methods, "electroformation" and "gentle hydration (or natural swelling)", in which dry lamellar films of phospholipids are hydrated with aqueous solutions. In gentle hydration, however, giant liposomes are difficult to prepare from an electrostatically neutral phospholipid because lipid lamellae cannot repel each other. In this study, we demonstrate the efficient formation of giant liposomes using the gentle hydration of neutral phospholipid (dioleoyl phosphatidylcholine, DOPC) dry films doped with nonelectrolytic monosaccharides (glucose, mannose, and fructose). A mixture of DOPC and such a sugar in an organic solvent (chloroform/methanol) was evaporated to form the films, which were then hydrated with distilled water or Tris buffers containing sodium chloride. Under these conditions, giant liposomes spontaneously formed rapidly and assumed a swollen cell-sized spherical shape with low lamellarity, whereas giant liposomes from pure DOPC films had multilamellar lipid layers, miscellaneous shapes and smaller sizes. This observation indicates that giant unilamellar vesicles (GUVs) of DOPC can be obtained efficiently through the gentle hydration of sugar-containing lipid dry films because repulsion between lipid lamellae is enhanced by the osmosis induced by dissolved sugar.     

Electroformation of giant phospholipid vesicles on a silicon substrate: advantages of controllable surface properties

We introduce the use of silicon (Si) as a substrate for the electroformation of giant phospholipid vesicles. By taking advantage of the tunability of silicon surface properties, we varied the organization of the phospholipid film on the electrode and studied the consequences on vesicle formation. In particular, we investigated the effects of Si surface chemistry and microtopology on the organization of the phospholipid film and the properties of the final vesicles. We established correlations between chemical homogeneity, film defects, and resulting vesicle size distribution. By considering phospholipid films that are artificially fragmented by electrode microstructures, we showed that the characteristic size of vesicles decreases with a decrease in microstructure dimensions. We finally proposed a way to control the vesicle size distribution by using a micropatterned silicon dioxide layer on a Si substrate.     

Surface characterizations of spin-coated films of ethylcellulose and hydroxypropyl methylcellulose blends

Films of pure ethylcellulose (EC) and hydroxypropyl methylcellulose (HPMC) polymers and EC/HPMC blends were prepared from solutions by spin coating where isopropyl alcohol (IPA), water, and IPA/water cosolvent were used as solvents. Surface structures of the films were investigated using optical microscopy, atomic force microscopy (AFM), and Raman mapping and spectroscopy. For the films prepared from EC/HPMC blend solutions using the IPA/water cosolvent, different domain structures such as islands or pits and phase separation between EC and HPMC were observed by optical microscopy and AFM. The nature of the polymer components on the surface of the films was identified by Raman mapping and spectroscopy. Experimental results also indicated that polymer composition, solvent, and temperature during spin coating had significant impacts on surface structures of the films.     

Surface forces measurements of spin-coated cellulose thin films with different crystallinity

A systematic study of the surface forces between a cellulose sphere and cellulose thin films of varying crystallinity has been conducted as a function of ionic strength and pH. Semicrystalline cellulose II surfaces and amorphous cellulose films were prepared by spin-coating of the precursor cellulose solutions onto oxidized silicon wafers before regeneration in water. Crystalline cellulose I surfaces were prepared by spin-coating wafers with aqueous suspensions of sulfate-stabilized cellulose I nanocrystals. These preparation methods produced thin, smooth films suitable for surface forces measurements. The interaction with the cellulose I was monotonically repulsive at pH 3.5, 5.8, and 8.5 and at 0.1, 1, and 10 mM ionic strengths. This was attributed to the presence of strongly ionizable sulfur-containing groups on the cellulose nanocrystal surfaces. The amorphous film typically showed a steric interaction up to 100 nm away from the interface that was independent of the solution conditions. A range of surface forces were successfully measured on the semicrystalline cellulose II films; attractive and repulsive regimes were observed, depending on pH and ionic strength, and were interpreted in terms of van der Waals and electrostatic interactions. Clearly, the forces acting near cellulose surfaces are very dependent on the way the cellulose surface has been prepared.     

Polymerization of liposomes composed of diene-containing lipids by radical initiators. II. Polymerization of monodiene-type lipids as liposomes

1-Palmitoyl-2(2,4-octadecadienoyl)-sn-glycero-3-phosphocholine (POPC), a polymerizable lipid that contains one diene group in only a 2-acyl chain, was polymerized as liposome in an aqueous medium. Polymerization was initiated by water-insoluble azobisisobutyronitrile (AIBN), or water-soluble azobis(2-amidinopropane) dihydrochloride (AAPD). AIBN was mixed with monomeric lipids, and the mixture was dispersed in an aqueous medium by sonication to prepare AIBN-containing monomeric lipid liposomes. On the other hand, AAPD was simply added to the liposome suspension. The POPC liposomes were easily polymerized by the addition of AAPD, a water-soluble radical initiator, but few were polymerized by AIBN. The results suggested that the diene group in the 2-acyl chain was in an aqueous phase and, therefore, easily polymerized by a water-soluble radical initiator. The polymerized POPC liposomes were revealed to be more stable than those of monomeric ones because the scattered-light intensity from the polymerized POPC liposome suspension changed a little by the addition of Triton X-100. For only the polymerized ones, the liposome structure was confirmed by TEM after addition of an excess amount of Triton X-100.     

Patterned monomolecular films from polymerizable and fluorinated lipids for the presentation of glycosylated lipids

This paper deals with pattern formation in Langmuir monolayers of two sets of lipid mixtures that include (1) a fluorinated lipid for phase separation, (2) a polymerizable lipid for stabilization of the patterned structure, and (3) a unit for the presentation of biological recognition units. Differences in the distribution of these functionalities allow a polymerization of dispersed or continuous phase and a placement of the recognition units in crystalline or solid analogue phase. Also, a ternary mixture including a lipid modified with the tandem repeat domain of MUC1 plus a T_N-antigen was studied. Based on the biphasic pattern obtained (starlike crystals of up to 50 μm with a fine structure of some micrometers), we also verified the potential of the laterally patterned monolayer to stimulate the immune system (quartz crystal microbalance). The second set of lipids combines a highly fluorinated itaconic ester (polymerizable unit) with the natural phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine.     

Electroformation in a flow chamber with solution exchange as a means of preparation of flaccid giant vesicles

A recently described technique [Estes and Mayer, Biochim. Biophys. Acta 1712 (2005) 152-160] for the preparation of giant unilamellar vesicles (GUVs) in solutions with high ionic strength is examined. By observing a series of osmotic swellings followed by vesicle bursts upon a micropipette transfer of a single POPC GUV from a sucrose solution into an iso-osmolar glycerol solution, a value for the permeability of POPC membrane for glycerol, P=(2.09+/-0.82) x 10(-8)m/s, has been obtained. Based on this result, an alternative mechanism is proposed for the observed exchange of vesicle interior. With modifications, the method of Estes and Mayer is then applied to preparation of flaccid GUVs.     

Nucleating pattern formation in spin-coated polymer blend films with nanoscale surface templates

We use Dip-Pen Nanolithography (DPN) to generate monolayer surface templates for guiding pattern formation in spin-coated polymer blend films. We study template-directed pattern formation in blends of polystyrene/poly(2-vinylpyridine) (PS/P2VP) as well as blends of PS and the semiconducting conjugated polymer poly(3-hexylthiophene) (P3HT). We show that acid-terminated monolayers can be used to template pattern formation in PS/P3HT blends, while hydrophobic monolayers can be used to template pattern formation in PS/P2VP blends. In both blends, the polymer patterns comprise laterally-phase separated regions surrounded by vertically separated bilayers. We hypothesize that the observed patterns are formed by template-induced dewetting of the bottom layer of a polymer bilayer during the spin-coating process. We compare the effects of template feature size and spacing on the resulting polymer patterns with predictions from published models of template-directed dewetting in thin films and find the data in good agreement. For both blends we observe that a minimum feature size is required to nucleate dewetting/phase separation. We find this minimum template diameter to be approximately 180 nm in 50/50 PS/P2VP blends, and approximately 100 nm in 50/50 PS/P3HT blends. For larger template diameters, PS/P2VP blends show evidence for pattern formation beginning at the template boundaries, while PS/P3HT blends rupture randomly across the template features.     

Block liposomes from curvature-stabilizing lipids: connected nanotubes, -rods, or -spheres

We report on the discovery of block liposomes, a new class of chain-melted (liquid) vesicles, with membranes comprised of mixtures of the membrane-curvature-stabilizing multivalent lipid MVLBG2 of colossal charge +16 e and neutral 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC). In a narrow MVLBG2 composition range (8-10 mol%), cryo-TEM revealed block liposomes consisting of distinctly shaped, yet connected, nanoscale spheres, pears, tubes, or rods. Unlike typical liposome systems, where spherical vesicles, tubular vesicles, and cylindrical micelles are separated on the macroscopic scale, within a block liposome, shapes are separated on the nanometer scale. Diblock (pear-tube) and triblock (pear-tube-pear) liposomes contain nanotubes with inner lumen diameter of 10-50 nm. Diblock (sphere-rod) liposomes were found to contain micellar nanorods approximately 4 nm in diameter and several micrometers in length, analogous to cytoskeletal filaments of eukaryotic cells. Block liposomes may find a range of applications in chemical and nucleic acid delivery and as building blocks in the design of templates for hierarchical structures.     

Flat-on lamellae in spin-coated, stable films of poly(propylene azelate)

Thin films (5-500 nm thick) of the linear aliphatic polyester (3,7) poly(propylene azelate) (PPAz) were prepared by spin-coating of CHCl(3) polymer solutions with different polymer concentrations. The morphology and structure of the spin-coated thin films were investigated by atomic force microscopy (AFM) and by grazing incidence wide-angle X-ray scattering (GIWAXS) techniques. AFM revealed the continuous nature of the flat, spherulitic films which are stable against dewetting even for polymer coatings as thin as 15 nm. GIWAXS patterns revealed a high crystal orientation of the films. A sharp reflection on the meridian whose spacing is related to the polymer chain unit length (c-axis) supports the presence of flat-on lamellae morphology in the whole range of film thicknesses investigated. The flat-on lamellae morphology is also supported by AFM images. A triclinic unit cell with the c*-axis perpendicular to the substrate is proposed for PPAz. The repulsion of the long aliphatic spacer by the Si-substrate is invoked as the main reason for the flat-on morphology observed.     

A novel technique for preparation of monodisperse giant liposomes

A novel technique for the preparation of monodisperse giant liposomes has been developed based on a combination of micro-patterning of ITO glass slides with lipid solution and electroformation. The average diameter of the produced liposomes is determined by size of the micro-pattern features.     

Glass transition behavior of spin-coated thin films of a hydrophilic polymer on supported substrates

Using ellipsometry, it is found the glass transition temperature of the spin-coated polyacrylamide(PAL) thin films on the supported silicon(Si) substrates with an oxide layer decreases with decreasing the film thickness. But Tgs of the asprepared thin films are much higher than that of the bulk sample. Such observations can be attributed to the combined result of the "surface effect" and increased hydrogen bonding interaction between PAL chains due to spin coating/thin film confinement.     

Structure of spin-coated lipid films and domain formation in supported membranes formed by hydration

An atomic force and fluorescence microscopy investigation of the structure of spin-coated lipid films is presented. In the surface of the dry film, lipids are found to orient in a conformation where acyl chains are pointing outward while laterally the individual layers of the multilamellar film exhibit a dewetting pattern similar to what is found in polymer thin films. Hydration of the film in liquid water promotes detachment of bilayers from the surface while a single membrane remains on the mica substrate. This supported membrane is highly uniform and defect-free as compared to supported membranes prepared by conventional methods. It is further demonstrated that supported membranes of binary lipid mixtures prepared by this method exhibit gel-fluid domain coexistence in accordance with expectations from the phase diagrams.