Monitoring of membrane collapse and enzymatic reaction with single giant liposomes embedded in agarose gel

Giant liposomes are often used as models for studies on cell membranes. We embedded giant liposomes in agarose gel to fix them for assays. Giant liposomes of dioleoylphosphatidylcholine were embedded in 1% (w/v) agarose gel with a low melting temperature: While only 20–25% of giant liposomes survived embedment, their size distribution was unaffected. Using a confocal laser scanning microscope, we monitored dynamic changes in individual agarose gel-embedded giant liposomes induced by the addition of a surfactant (Triton X-100). The permeation and collapse could be clearly discriminated from each other. Invaginated buds on liposome membranes could also be captured as intermediate structures. Additionally, an enzymatic (β-glucosidase) reaction encapsulated within the target liposome was triggered by the external addition of a non-fluorescent substrate and successfully monitored. These results suggest that embedment in agarose gel is useful for the simple fixation of giant liposomes for biochemical and biophysical assays.     

Structure of small actin-containing liposomes probed by atomic force microscopy: effect of actin concentration & liposome size

Actin-containing liposomes were prepared via extrusion through 400 and 600 nm pore diameter membranes at different monomeric actin concentrations in low ionic strength buffer (G-buffer). After subjecting the liposome dispersions to high ionic strength polymerization buffer (F-buffer), topological changes in liposome structure were studied using atomic force microscopy (AFM). Paired dumbbell, horseshoelike, and disklike assemblies were observed for actin-containing liposomes extruded through 400 and 600 nm pore diameter membranes. The topology of actin-containing liposomes was found to be highly dependent on both liposome size and actin concentration. At 1 mg/mL actin, the actin-containing liposomes transformed into a disklike shape, whereas, at 5 mg/mL actin, the actin-containing liposomes retained a spherical shape. On the basis of these observations, we hypothesize that actin could either polymerize on the surface of the inner leaflet of the liposome membrane or polymerize in the aqueous core of the liposome. We explain the associated shape changes induced in actin-containing liposomes on the basis of the hypothesized mechanism of actin polymerization inside the liposomes. At higher actin concentrations (5 mg/mL), we observed membrane-induced actin self-assembly in G-buffer, which implies that G-actin is able to interact directly with lipid bilayers at sufficiently high concentrations.     

DNA-controlled assembly of soft nanoparticles

Immobilization of DNA (encoding) on solid nanoparticles requires surface chemistry, which is well established for gold surfaces but often tedious and not generally applicable for many other inorganic surface materials. While substantial effort has been devoted to expanding surface chemistry techniques for solid nanoparticles, considerably less attention has been given to the development of noncovalent attachment of DNA to soft nanoparticles, like liposomes. Here we report a DNA-controlled assembly of liposomes in solution and on solid supported membranes, this process displays remarkably sharp thermal transitions from an assembled to a disassembled state, allowing application of DNA-controlled liposome assembly for the detection of polynucleotides (e.g., DNA) with single mismatch discrimination power. The method is based on a single DNA strand (contains two lipid membrane anchors), which is able to noncovalently attach to a liposome surface. This design enables detection of biological polynucleotide targets as the complementary strand can be unmodified DNA and RNA strands.     

Arraying of intact liposomes into chemically functionalized microwells

Here, we describe a protocol to bind individual, intact phospholipid bilayer liposomes, which are on the order of 1 microm in diameter, in microwells etched in a regular array on a silicon oxide substrate. The diameter of the wells is on the order of the liposome diameter, so only one liposome is located in each well. The background of the silicon oxide surface is functionalized with a PEG oligomer using the contact printing of a PEG silane to present a surface that resists the adsorption of proteins, lipid material, and liposomes. The interiors of the wells are functionalized with an aminosilane to facilitate the conjugation of biotin, which is then bound to Neutravidin. The avidin-coated well interiors bind the liposomes whose surfaces contain biotinylated lipids. The specific binding of the liposomes to the surface using the biotin-avidin linkage, together with the resistant nature of the background and the physical confinement of the wells, allows the liposomes to remain intact and to not unravel, rupture, and fuse onto the surface. We demonstrate this intact arraying using confocal laser scanning microscopy of fluorophores specifically tagging the microwells, the lipid bilayer, and the aqueous interior of the liposome.     

Liposome chromatography: liposomes immobilized in gel beads as a stationary phase for aqueous column chromatography

Liposomes have been used as a stationary phase for column chromatography with an aqueous mobile phase. They were immobilized in the pores of carrier gel beads by two methods: (A) hydrophobic ligands were coupled to the matrix of gel beads, which then were packed into a column and liposomes were applied and became associated with the ligands by hydrophobic interaction; and (B) phospholipids and detergent were dialysed in the presence of gel beads; many of the liposomes that formed in the pores of the beads were sterically immobilized by the gel matrix. Proteoliposomes containing red cell glucose transport protein in the lipid bilayers were immobilized in a column by method A. This column retained D-glucose longer than L-glucose. In contrast to L-glucose, D-glucose was transported into and out of the immobilized liposomes, causing an increased retention. Liposomes with (stearylamine)+ or (phosphatidylserine)- in their lipid bilayers were immobilized by method B and the gel beads were packed into a column. A protein of opposite charge was applied in excess. Under suitable conditions, the protein molecules became close-packed on the liposome surfaces. Ion-exchange chromatographic experiments with proteins showed that these sterically immobilized liposomes were also stable enough to be used as a stationary phase. The loss of lipids was 5-23% in the first run at high protein load and with sodium chloride gradient elution but was lower in subsequent runs. It is proposed that water-soluble molecules can be separated and their interactions with liposome surfaces studied by chromatography on immobilized liposomes in detergent-free aqueous solution. Membrane proteins can be inserted and ligands can be anchored in the lipid bilayers for chromatographic purposes.     

Formation and finite element analysis of tethered bilayer lipid structures

Rapid solvent exchange of an ethanolic solution of diphytanoyl phosphatidylcholine (DPhyPC) in the presence of a mixed self-assembled monolayer (SAM) [thiolipid/β-mercaptoethanol (βME) (3/7 mol/mol) on Au] shows a transition from densely packed tethered bilayer lipid membranes [(dp)tBLMs], to loosely packed tethered bilayer lipid membranes [(lp)tBLMs], and tethered bilayer liposome nanoparticles (tBLNs) with decreasing DPhyPC concentration. The tethered lipidic constructs in the aqueous medium were analyzed by atomic force microscopy (AFM) and electrochemical impedance spectroscopy (EIS). Finite element analysis (FEA) was applied to interpret spectral EIS features without referring to equivalent circuit modeling. Using structural data obtained earlier from neutron reflectometry and dielectric constants of lipid bilayers, we reproduced experimentally observed features of the electrochemical impedance (EI) spectra of complex surface constructs involving small pinhole defects, large membrane-free patches, and bound liposomes. We demonstrated by FEA that highly insulating (dp)tBLMs with low-defect density exhibit EI spectra in the shape of a perfect semicircle with or without low-frequency upward "tails" in the Cole-Cole representation. Such EI spectra were observed at DPhyPC concentrations of >5 × 10(-3) mol L(-1). While AFM was not able to visualize very small lateral defects in such films, EI spectra unambiguously signaled their presence by increased low frequency "tails". Using FEA we demonstrate that films with large diameter visible defects (>25 nm by AFM) produce EI spectral features consisting of two semicircles of comparable size. Such films were typically obtained at DPhyPC concentrations of <5 × 10(-3) mol L(-1). At DPhyPC concentrations of <1.0 × 10(-3) mol L(-1) the planar bilayer structures were replaced by ellipsoidal liposomes with diameters ranging from 50 to 500 nm as observed in AFM images. Despite the distinct surface morphology change, the EI curves exhibited two semicircle spectral features typical for the large size defects in planar tBLMs. FEA revealed that, to account for these EI features for bound liposome systems (50-500 nm diameter), one needs to assume much lower tBLM conductivities of the submembrane space, which separates the electrode surface and the phospholipid bilayer. Alternatively, FEA indicates that such features may also be observed on composite surfaces containing both bound liposomes and patches of planar bilayers. Triple semicircular features, observed in some of the experimental EI curves, were attributed to an increased complexity of the real tBLMs. The modeling demonstrated that such features are typical for heterogeneous tBLM surfaces containing large patches of different defectiveness levels. By integrating AFM, EIS, and FEA data, our work provides diagnostic criteria allowing the precise characterization of the properties and the morphology of surface supported bilayer systems.     

Regulation of CD40 reconstitution into a liposome using different ratios of solubilized LDAO to lipids

The integral membrane protein CD40 was found on the surface of B lymphocytes that interact with CD40L on T cells during the immune response. The hydrophobic transmembrane domains of membrane proteins can be stabilized in detergent or in lipid bilayers such as liposomes. Membrane proteins can be incorporated into the liposome in a similar fashion to the way they are handled in vivo. In this study, a large amount of full-sequence CD40 was produced using a bacterial system that contained a Mistic construct. The CD40 was then reconstituted into liposomes by detergent-mediated reconstitution. All stages in the process of liposome disruption with various detergent ratios were easily observed by monitoring the optical density. The structure of the liposome and the reconstitution of CD40 were confirmed by cryo-TEM. The results of the present study show that the detergent ratio had an effect on the structure of the liposome and the amount of CD40 that was reconstituted into the liposome.     

The effect of added liposomes on the rheological properties of a hydrogel: a systematic study

Rheological characteristics of liposome-containing-hydrogels were studied. Sonicated unilamellar vesicles (SUV), prepared by probe sonication and multilamellar vesicles (MLV) prepared by thin film hydration were loaded in a hydrogel containing carbopol 974 NF and hydroxyethylcellulose (Natrosol 250 HX). Phosphatidylcholine (PC) or hydrogenated-PC (HPC) liposomes, plain or mixed with cholesterol (chol) were used. Static (steady-stress sweep-tests) and dynamic (frequency sweep-tests) rheological measurements were carried out. All gels had a shear thinning behaviour (fitted well by Cross model). Zero-rate shear viscosity and power law index values, revealed that PC liposome addition in the hydrogel had minimum effect on its rheological properties even at the highest lipid concentration used (20 mg/ml). Oppositely, HPC (or HPC/chol) liposome addition resulted in significant modulations of the same rheological characteristics (which increased with increasing lipid concentration). HPC liposomes also caused a significant increase in gel relaxation time, which indicates that the elastic character of the gel strengthens as HPC liposome concentration increases. Concluding, liposome composition (membrane rigidity) and lipid concentration, but not liposome size, seem to be very important factors that determine the rheological modulations caused by liposome addition in gels.     

Micropatterning of polystyrene nanoparticles and its bioapplications

Micropatterning of biomolecules forms the basis of cell culture, biosensor and microarray technology. Currently, the most widely used techniques are photoresist lithography, soft lithography or using robots which all involve multi-step surface modification directly on a planar substrate. Here we report a method to pattern biomolecules through self-assembling polystyrene nanoparticles in arrayed microwells on a solid surface to form well-ordered patterning, followed by attaching biomolecules to the assembled nanoparticles. The formation of colloidal patterns depends on capillary force, surface wettability and physical confinement. This method can be used for micropatterning a variety of biomolecules such as protein and antibody.     

Probing mechanical properties of liposomes using acoustic sensors

Acoustic devices were employed to characterize variations in the mechanical properties (density and viscoelasticity) of liposomes composed of 1-oleoyl-2-palmitoyl- sn-glycero-3-phosphocholine (POPC) and cholesterol. Liposome properties were modified in three ways. In some experiments, the POPC/cholesterol ratio was varied prior to deposition on the device surface. Alternatively, the ratio was changed in situ via either insertion of cholesterol or removal of cholesterol with beta-cyclodextrin. This was done for liposomes adsorbed directly on the device surface and for liposomes attached via a biotin-terminated poly(ethylene glycol) linker. The acoustic measurements make use of two simultaneous time-resolved signals: one signal is related to the velocity of the acoustic wave, while the second is related to dissipation of acoustic energy. Together, they provide information not only about the mass (or density) of the probed medium but also about its viscoelastic properties. The cholesterol-induced increase in the surface density of the lipid bilayer was indeed observed in the acoustic data, but the resulting change in signal was larger than expected from the change in surface density. In addition, increasing the bilayer resistance to stretching was found to lead to a greater dissipation of the acoustic energy. The acoustic response is assessed in terms of the possible distortions of the liposomes and the known effects of cholesterol on the mechanical properties of the lipid bilayer that encloses the aqueous core of the liposome. To aid the interpretation of the acoustic response, it is discussed how the above changes in the lipid bilayer will affect the effective viscoelastic properties of the entire liposome/solvent film on the scale of the acoustic wavelength. It was found that the acoustic device is very sensitive to the mechanical properties of lipid vesicles; the response of the acoustic device is explained, and the basic underlying mechanisms of interaction are identified.     

Young's moduli of surface-bound liposomes by atomic force microscopy force measurements

Mechanical properties of layers of intact liposomes attached by specific interactions on solid surfaces were studied by atomic force microscopy (AFM) force measurements. Force-distance measurements using colloidal probe tips were obtained over liposome layers and used to calculate Young's moduli by using the Hertz contact theory. A classical Hertz model and a modified Hertz one have been used to extract Young's moduli from AFM force curves. The modified model, proposed by Dimitriadis, is correcting for the finite sample thickness since Hertz's classical model is assuming that the sample is infinitely thick. Values for Young's moduli of 40 and 8 kPa have been obtained using the Hertz model for one and three layers of intact liposomes, respectively. Young's moduli of approximately 3 kPa have been obtained using the corrected Hertz model for both one and three layers of surface-bound liposomes. Compression work performed by the colloidal probe to compress these liposome layers has also been calculated.     

Optimization of DNA-tagged dye-encapsulating liposomes for lateral-flow assays based on sandwich hybridization

A novel protocol for the synthesis of dye-encapsulating liposomes tagged with DNA oligonucleotides at their outer surface was developed. These liposomes were optimized for use as signal enhancement agents in lateral-flow sandwich-hybridization assays for the detection of single-stranded RNA and DNA sequences. Liposomes were synthesized using the reverse-phase evaporation method and tagged with oligonucleotides by adding cholesteryl-modified DNA probes to the initial lipid mixture. This resulted in a greatly simplified protocol that provided excellent control of the probe coverage on the liposomes and cut the preparation time from 16 hours to just 6 hours. Liposomes were prepared using probe concentrations ranging from 0.00077 to 0.152 mol% of the total lipid, several hydrophobic and polyethylene glycol-based spacers between the cholesteryl anchor and the probe, and liposome diameters ranging from 208 nm to 365 nm. The liposomes were characterized by dynamic light scattering, visible spectroscopy, and fluorescence spectroscopy. Their signal enhancement functionality was compared by using them in lateral-flow optical biosensors for the detection of single-stranded DNA sequences. In these assays, an optimal reporter probe concentration of 0.013 mol%, liposome diameter of 315 nm, and liposome optical density of 0.4–0.6 at 532 nm were found. The spacer length between the cholesteryl anchor and the probe showed no significant effect on the signals in the lateral-flow assays. The results presented here provide important data for the general use of liposomes as labels in analytical assays, with specific emphasis on nucleic acid detection via lateral flow assays.     

Complexation of polycations to anionic liposomes: composition and structure of the interfacial complexes

Poly(N-ethyl-4-vinylpyridinium bromide) (a polycation with a degree of polymerization of 1100) was adsorbed onto liposomes composed of egg lecithin with a 0.05-0.20 molar fraction (nu) of anionic headgroups provided by cardiolipin (a doubly anionic lipid). According to electrophoretic mobility data, this led to total charge neutralization of the liposomes, whereupon the liposomes adopted a positive charge as additional polymer continued to adsorb. Although the liposomes aggregated at the charge-neutralization point, they disassembled into individual liposomes after becoming positively charged. The degree of polymer adsorption was shown to reach a limit. Thus, by measuring the free polymer content in a liposome suspension, it was possible to determine the polymer concentration at which the liposome surface became saturated with polymer. Beyond this point, an electrostatic/steric barrier at the surface suppressed further adsorption. Dynamic light scattering studies of liposomes with and without adsorbed polymer allowed calculation of the polymer film thickness which ranged from 22 to 35 nm as the molar fraction of cardiolipin (nu) increased from 0.05 to 0.20. The greater the content on the anionic lipid in the bilayer, the thicker the polymer film. The maximum number of polymer molecules adsorbed onto the liposomes was estimated: 1-2 molecules for nu = 0.05; 3 molecules for nu = 0.1; 4- molecules for nu = 0.15; and 6 molecules for nu = 0.2. The polymer appears to lie on the liposome surface, rather than embedding into the bilayer, because addition of NaCl easily dislodges the polymer from the liposome into the bulk water.     

Unilamellar liposomes covalently coupled on silica gel for liquid chromatography

Silica gel was used as a support for the covalent coupling of liposomes, which could overcome drawbacks of soft gel beads in column efficiency and separation speed. The influences of the concentration of added dimethylaminopyridine and reaction time on the chloroformate activation reaction of silica gel were investigated. Temperature and pH for covalent coupling of liposomes on the activated silica gel were also optimized. Experimental results indicated that the stability of the covalently coupled liposome columns was obviously superior to that of the noncovalently coated liposome columns but the selectivity of both columns was basically identical. Separation and analysis of a crude extract of a traditional Chinese medicine Ligusticum Wallichii and a mixture of small peptides on both columns further support this conclusion.     

Mechanical properties and stability measurement of cholesterol-containing liposome on mica by atomic force microscopy

The micromechanical properties of pure and cholesterol modified egg yolk phosphatidylcholine (EggPC) vesicles prepared by sonication were studied by atomic force microscopy (AFM) on mica surface. The force curves between an AFM tip and an unruptured vesicle were obtained by contact mode. During approach, two repulsion regions with two breaks were observed. The slopes of the two repulsive force regimes for the pure EggPC vesicles are determined to be several times lower than that of EggPC/cholesterol vesicles. The elastic properties from force plot analysis based on the Hertzian model showed that Young's modulus (E) and the bending modulus (kc) of cholesterol-modified vesicles increased several-fold compared with pure EggPC vesicles. The significant difference is attributed to the enhanced rigidity of the EggPC vesicles as a result of the incorporation of cholesterol molecules. The behavior of cholesterol-modified vesicles upon adsorption is different from that in solution as revealed by mechanical properties. The results indicate that AFM can provide a direct method to measure the mechanical properties of adsorbed small liposomes and to detect the stability change of liposomes.     

Stable polymeric nanoballoons: lyophilization and rehydration of cross-linked liposomes

The cross-linking of supramolecular assemblies of hydrated lipids is an effective method to stabilize these assemblies to disruption by surfactants or aqueous alcohol. The heterobifunctional lipids, Acryl/DenPC(16,18) and Sorb/DenPC(18,21), are examples of a new class of polymerizable lipid designed for the creation of cross-linked lipid structures. The robust nature of cross-linked liposomes was demonstrated by lyophilization of the liposomes followed by their essentially complete redispersion in water. The resulting liposomes were compared to the original sample by quasi-elastic light scattering and transmission electron microscopy. There was no major change in the size or structure of the cross-linked liposomes after rehydration of the freeze-dried powder of liposomes. Moreover, the rehydrated cross-linked liposomes continued to be resistant to surfactant solubilization. Neutral cross-linked liposomes were predominantly redispersed after freeze-drying with the aid of bath sonication. The small amount of residual liposome aggregation observed with neutral liposomes could be prevented by incorporating a surface charge into the liposome or attaching hydrophilic polymers, for example, poly(ethylene glycol), onto the liposome.     

Immobilization of histidine-tagged proteins on nickel by electrochemical dip pen nanolithography

Dip-pen nanolithography (DPN) is becoming a popular technique to "write" molecules on a surface by using the tip of an atomic force microscope (AFM) coated with the desired molecular "ink". In this work, we demonstrate that poly-histidine-tagged peptides and proteins, and free-base porphyrins coated on AFM probes, can be chelated to ionized regions on a metallic nickel surface by applying an electric potential to the AFM tip in the DPN process. DPN has been accomplished in the Tapping Mode of AFM, which creates many possible applications of positioning and subsequently imaging biomolecules, especially on soft surfaces.     

Construction of supported lipid membrane modified piezoelectric biosensor for sensitive assay of cholera toxin based on surface-agglutination of ganglioside-bearing liposomes

A novel piezoelelctric biosensor has been developed for cholera toxin (CT) detection based on the analyte-mediated surface-agglutination of ganglioside (GM1)-functionalized liposomes. To achieve a CT-specific agglutination at the surface, the gold electrode is modified by a GM1-functionalized supported lipid membrane via spontaneous spread of the liposomes on a self-assembled monolayer of a long-chain alkanethiol. In the presence of CT, the GM1-incorporated liposomes in assay medium will rapidly specifically agglutinate at the electrode surface through the binding of CT to GM1 on the electrode surface and the liposome interface. This results in an enormous mass loading on the piezoelelctric crystal as well as a significant increase of density and viscosity at the interface, thereby generating a decrease in frequency of the piezoelelctric crystal. The combination of mass loading with interfacial change in the surface-agglutination reaction allows the developed piezoelelctric biosensor to show substantial signal amplification in response to the analyte CT. The detection limit can be achieved as low as 25 ng mL⁻¹ CT. This is the first demonstration on CT detection based on specific surface-agglutination of GM1-modified liposomes. The supported lipid layer based sensing interface can be prepared readily and renewably, making the developed technique especially useful for simple, reusable and sensitive determination of proteins.     

Effect of lipid headgroup composition on the interaction between melittin and lipid bilayers

The effect of the lipid polar headgroup on melittin-phospholipid interaction was investigated by cryo-TEM, fluorescence spectroscopy, ellipsometry, circular dichroism, electrophoresis and photon correlation spectroscopy. In particular, focus was placed on the effect of the lipid polar headgroup on peptide adsorption to, and penetration into, the lipid bilayer, as well as on resulting colloidal stability effects for large unilamellar liposomes. The effect of phospholipid headgroup properties on melittin-bilayer interaction was addressed by comparing liposomes containing phosphatidylcholine, -acid, and -inositol at varying ionic strength. Increasing the bilayer negative charge leads to an increased liposome tolerance toward melittin which is due to an electrostatic arrest of melittin at the membrane interface. Balancing the electrostatic attraction between the melittin positive charges and the phospholipid negative charges through a hydration repulsion, caused by inositol, reduced this surface arrest and increased liposome susceptibility to the disruptive actions of melittin. Furthermore, melittin was demonstrated to induce liposome structural destabilization on a colloidal scale which coincided with leakage induction for both anionic and zwitterionic systems. The latter findings thus clearly show that coalescence, aggregation, and fragmentation contribute to melittin-induced liposome leakage, and that detailed molecular analyses of melittin pore formation are incomplete without considering also these colloidal aspects.     

Insight in the role of bovine serum albumin for promoting the in situ surface growth of polyhydroxybutyrate (PHB) on patterned surfaces via enzymatic surface-initiated polymerization

Polyhydroxyalkanoates (PHAs) are a family of aliphatic polyesters produced by a variety of microorganisms as a reserve of carbon and energy. Enzymes involved in the synthesis of PHAs can be utilized to produce polymers in vitro, both in bulk and on solid surfaces. Here, site-specific attachment of the key catalytic enzyme, PHA synthase, on lithographically patterned surfaces and subsequent addition of (R)-3-hydroxybutyryl-CoA substrate allowed us to fabricate spatially ordered polyhydroxybutyrate (PHB) polymeric structures via an in situ enzymatic surface-initiated polymerization (ESIP). By varying the reaction conditions, we enhanced the growth of PHB on solid surfaces and analyzed the resulting structures by fluorescence microscopy, atomic force microscopy (AFM), attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, and gel permeation chromatography (GPC). We found that stabilization of smaller PHB granule structures by an addition of bovine serum albumin (BSA) was the most important factor for a successful synthesis of a PHB layer up to 1mum in thickness, consisting mainly of larger cluster assemblies of PHB granules that cover the entire patterned area. Immunofluorescence detection and surface contact angle analysis revealed that BSA was physically bound to the PHB polymer all through the cluster, and reduced the overall hydrophobicity of the polymer surface. Based on information obtained from AFM, kinetic measurements and various polymer characterization methods, a plausible model for roles of BSA in the enhancement of PHB formation on surfaces is discussed. Furthermore, by using biotinylated BSA conjugates, we were able to incorporate biotin groups into the PHB polymer matrix, thus generating a bioactive surface that can be used for displaying other functional biomolecules through streptavidin-biotin interaction on the PHB structures. Because of its versatility, our fabrication strategy is expected to be a useful surface modification tool for numerous biomedical and biotechnological applications.