Immobilization of liposomes onto quartz crystal microbalance to detect interaction between liposomes and proteins

To study the interaction between liposomes and proteins, intact liposomes were immobilized on a metal planar support by chemical binding and/or bioaffinity using a quartz crystal microbalance (QCM). A large decrease in the resonance frequency of quartz crystal was observed when the QCM, modified by a self-assembled monolayer (SAM) of carboxythiol, was added to liposome solutions. The stable chemical immobilization of intact liposomes onto SAM was judged according to the degree with which adsorbed mass depended on the prepared size of liposomes, as well as on the activation time of SAMs when amino-coupling was introduced, where the liposome coverage of electrodes was 69+/-8% in optimal conditions. When avidin-biotin binding was used on amino-coupling liposome layers, liposome immobilization finally reached 168% coverage of the electrode surface. Denatured protein was also successfully detected according to the change in the frequency of the liposome-immobilized QCM. The adsorbed mass of denatured carbonic anhydrase from bovine onto immobilized liposomes showed a characteristic peak at a concentration of guanidine hydrochloride that corresponded to a molten globule-like state of the protein, although the mass adsorbed onto deactivated SAM increased monotonously.     

Azide-reactive liposome for chemoselective and biocompatible liposomal surface functionalization and glyco-liposomal microarray fabrication

Chemically selective liposomal surface functionalization and liposomal microarray fabrication using azide-reactive liposomes are described. First, liposome carrying PEG-triphenylphosphine was prepared for Staudinger ligation with azide-containing biotin, which was conducted in PBS buffer (pH 7.4) at room temperature without a catalyst. Then, immobilization and microarray fabrication of the biotinylated liposome onto a streptavidin-modified glass slide via the specific streptavidin/biotin interaction were investigated by comparing with directly formed biotin-liposome, which was prepared by the conventional liposome formulation of lipid-biotin with all other lipid components. Next, the covalent microarray fabrication of liposome carrying triphenylphosphine onto an azide-modified glass slide and its further glyco-modification with azide-containing carbohydrate were demonstrated for glyco-liposomal microarray fabrication via Staudinger ligation. Fluorescence imaging confirmed the successful immobilization and protein binding of the intact immobilized liposomes and arrayed glyco-liposomes. The azide-reactive liposome provides a facile strategy for membrane-mimetic glyco-array fabrication, which may find important biological and biomedical applications such as studying carbohydrate-protein interactions and toxin and antibody screening.     

Liposomes as signal amplification reagents for bioassays in microfluidic channels

Liposomes with encapsulated carboxyfluorescein were used in an affinity-based assay to provide signal amplification for small-volume fluorescence measurements. Microfluidic channels were fabricated by imprinting in a plastic substrate material, poly(ethylene terephthalate glycol) (PETG), using a silicon template imprinting tool. Streptavidin was linked to the surface through biotinylated-protein for effective immobilization with minimal nonspecific adsorption of the liposome reagent. Lipids derivatized with biotin were incorporated into the liposome membrane to make the liposomes reactive for affinity assays. Specific binding of the liposomes to microchannel walls, dependence of binding on incubation time, and nonspecific adsorption of the liposome reagent were evaluated. The results of a competitive assay employing liposomes in the microchannels are presented.     

Liposomal glyco-microarray for studying glycolipid–protein interactions

A microarray enables high-throughput interaction screening of numerous biomolecules; however, fabrication of a microarray composed of cellular membrane components has proven difficult. We report fabrication of a liposomal glyco-microarray by using an azide-reactive liposome that carries synthetic and natural glycolipids via chemically selective and biocompatible liposome immobilization chemistry. Briefly, liposomes carrying anchor lipid dipalmitoylphosphatidylethanolamine (DPPE)-PEG(2000)-triphenylphosphine and ganglioside (GM1 or GM3) were prepared first and were then printed onto an azide-modified glass slide so as to afford a liposomal glyco-microarray via Staudinger ligation. Fluorescent dye release kinetics and fluorescence imaging confirmed successful liposome immobilization and specific protein binding to the intact arrayed glycoliposomes. The liposomal glyco-microarray with different gangliosides showed their specific lectin and toxin binding with different binding affinity. The azide-reactive liposome provides a facile strategy for fabrication of either a natural or a synthetic glycolipid-based membrane-mimetic glycoarray. This liposomal glyco-microarray is simple and broadly applicable and thus will find important biomedical applications, such as studying glycolipid-protein interactions and toxin screening applications.     

Spontaneous immobilization of liposomes on electron-beam exposed resist surfaces

We have found an interesting immobilization technique of liposomes on electron-beam exposed resist surfaces. The immobilized liposomes have been visualized by the atomic force microscope and have shown well-organized three-dimensional physical structures, in which the liposomes maintain their shapes and sizes similar to those of the original design in prepared solution. The immobilization is based on a strong static charge interaction between the resist surface and the liposomes. Further experiments show that very strong charge in the surfaces produces the firm immobilization of the liposome. We believe these findings can be related to various liposome applications such as drug delivery system, electrochemical or biosensors, and nanoscale membrane function studies.     

Liposome layers characterized by quartz crystal microbalance measurements and multirelease delivery

Intact liposomes have been immobilized onto solid surfaces by a NeutrAvidin-biotin link. The construction of these layers has been followed up by X-ray photoelectron spectroscopy (XPS) and quartz crystal microbalance (QCM) measurements with energy dissipation monitoring. Also, the simultaneous release of two fluorescent probes from these liposome layers has been investigated with the aim to validate this method in multirelease delivery systems. XPS showed the successful immobilization of the different layers. XPS results also point out the importance of the deactivation method used to reveal the presence of the specific NeutrAvidin-biotin attachment. QCM measurements allowed the buildup of the different layers to be followed in real time and in situ and suggest that biotinylated liposomes stay intact upon surface attachment on NeutrAvidin-covered surfaces and had viscoelastic behavior. QCM experiments also demonstrated that surface-immobilized liposomes were able to resist irreversible adsorption from fetal bovine serum. Release kinetic profiles were studied by monitoring the release of two different fluorescent probes, namely, carboxyfluorescein and levofloxacin, from these liposome layers. These studies showed that it was possible to modulate to some extent the release rates of the two molecules by using different configurations of liposome layers.     

Flat hydrogel substrate for atomic force microscopy to observe liposomes and lipid membranes

In order to avoid denaturation of biomolecules due to strong adsorption on solid surfaces, a soft substrate has to be used for atomic force microscopy (AFM) observation. We propose a hydrophilic agarose gel surface as a soft substrate for AFM to observe liposomes and lipid membranes. Although our simple method does not require any delicate control at the molecular level, an agarose gel surface can be simply flattened to 0.3 nm in roughness using an atomically flat solid surface during gelation. The AFM images revealed that liposomes were unruptured on the gel surface at low liposome density, whereas an unruptured state was difficult to obtain on a solid surface like mica. This indicates that the weak interaction between the liposome and the soft surface inhibits the liposome from rupturing, and also that the surface rougher than the solid surface prevents lateral diffusion of the liposomes along the surface to be fused. Increasing the liposome density resulted in a lipid membrane at various thicknesses forming on the hydrogel surface by the fusion and rupture of liposomes. Using the soft substrate, it can be expected to promote investigations of structures and functions of biomolecules at the nanometer scale under physiological conditions with AFM.     

Phospholipid-lysozyme coating for chiral separation in capillary electrophoresis

A phospholipid coating with lysozyme as chiral recognition reagent permeated into the phospholipid membrane was developed for the chiral capillary electrophoretic (CE) separation of D- and L-tryptophan. As a kind of carriers, coated as phospholipid membranes onto the inner wall of a fused-silica capillary, liposomes are able to interact with basic proteins such as lysozyme, which may reside on the surface of the phospholipid membrane or permeate into the middle of the membrane. The interaction results in strong immobilization of lysozyme in the capillary. Coatings prepared with liposomes alone did not allow stable immobilization of lysozyme into the phospholipid membranes, as seen from the poor repeatability of the chiral separation. When 1-(4-iodobutyl)-1,4-dimethylpiperazin-1-ium iodide (M1C4) was applied as a first coating layer in the capillary, the electroosmotic flow (EOF) was effectively suppressed, the phospholipid coating was stabilized, and the lysozyme immobilization was much improved. The liposome composition, the running buffer, and the capillary inner diameter all affected the chiral separation of D- and L-tryptophan. Coating with 4 mM M1C4 and then 1 mM phosphatidylcholine (PC)/phosphatidylserine (PS) (80:20 mol%), with 20 mM (ionic strength) Tris at pH 7.4 as the running buffer, resulted in optimal chiral separation with good separation efficiency and resolution. Since lysozyme was strongly permeated into the membrane of the phospholipids on the capillary surface, the chiral separation of D- and L-tryptophan was achieved without lysozyme in the running buffer. The effects of different coating procedures and separation conditions on separation were evaluated, and the M1C4-liposome and liposome-lysozyme interactions were elucidated. The usefulness of protein immobilized into phospholipid membranes as a chiral selector in CE is demonstrated for the first time.     

Cyclic Disulfide Liposomes for Membrane Functionalization and Cellular Delivery

Liposomes are effective therapeutic delivery nanocarriers due to their ability to encapsulate and enhance the pharmacokinetic properties of a wide range of therapeutics. Two primary areas in which improvement is needed for liposomal drug delivery is to enhance the ability to infiltrate cells and to facilitate derivatization of the liposome surface. Herein, we report a liposome platform incorporating a cyclic disulfide lipid (CDL) for the dual purpose of enhancing cell entry and functionalizing the liposome membrane through thiol-disulfide exchange. In order to accomplish this, CDL-1 and CDL-2 , composed of lipoic acid (LA) or asparagusic acid (AA) appended to a lipid scaffold, were designed and synthesized. A fluorescence-based microplate immobilization assay was implemented to show that these compounds enable convenient membrane decoration through reaction with thiol-functionalized small molecules. Additionally, fluorescence microscopy experiments indicated dramatic enhancements in cellular delivery when CDLs were incorporated within liposomes. These results demonstrate that multifunctional CDLs serve as an exciting liposome system for surface decoration and enhanced cellular delivery.     

A reusable liposome array and its application to assay of growth-hormone-related peptides

We describe a reusable liposome array based on the formation of cleavable disulfide cross-links between liposomes and the surface of a glass slip. The N-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP)-modified liposomes encapsulating a pH-sensitive fluorescence dye were immobilized on a 3-mercaptopropyltrimethoxysilane (MTS)-modified glass slip through the formation of disulfide bonds. The regeneration of a used slip was performed by the lysis of immobilized liposomes with Triton X-100 and the cleavage of disulfide bonds by reduction with TCEP, followed by immobilization of SPDP-modified liposomes. The regeneration steps did not affect the fluorescence intensity of re-immobilized liposomes. The liposome array was applied to simultaneous quantification of growth hormone related peptides, i.e., GHRF and somatostatin, in a mixture. After optimizing the assay condition, the method allowed quantification of GHRF and somatostatin in concentration ranges from 0.5 × 10⁻⁹ to 0.5 × 10⁻⁷ g/mL with detection limits of 2 × 10⁻¹⁰ and 3 × 10⁻¹⁰ g/mL, respectively.     

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.     

Electric impedance method for evaluation of the release property of calcein-encapsulated liposomes

This paper is concerned with the study on development of a novel method for evaluation of the liposomes release property by measuring the electric impedance changes of liposome suspensions. Calcein/NaOH encapsulated liposomes (calcein-liposomes) were prepared with deionized water and were treated with ultrasonic irradiation in order to investigate the release property of the liposomes. To validate the proposed impedance measuring method, the calcein release rates were evaluated both by the impedance changes and the fluorescence intensity changes in calcein-liposome suspensions. With the comparison of these results obtained by the two methods, it is shown that the impedance method has much wider detecting concentration range than the fluorescence one. Furthermore, the impedance method can be efficiently used for evaluation of the release property on various ionic substances encapsulated within liposomes.     

Attenuated total reflectance infrared studies of liposome adsorption at the solid-liquid interface

Interfacial interactions between liposomes and the solid–liquid interface (i.e. a ZnSe internal reflection element, modified to mimic a biological surface) were studied by Fourier transform infrared (FTIR) spectroscopy in attenuated total reflectance (ATR) mode. Both conventional liposomes, containing lecithin and cholesterol and Stealth^® liposomes containing poly(ethylene)glycol (PEG)5000- or PEG2000-lipids were investigated. IR bands due to the liposome components were observed to increase with time and enabled the liposome adsorption kinetics and thermodynamics to be quantified. The liposome solution conditions, surface properties and compositions have all been shown to influence liposome adsorption. Free energies of adsorption were determined to be in the range from −10.0 to −11.0 kJ mol⁻¹ and slightly reduced by PEG incorporation. The adsorption rate constant is decreased with increased solution pH and decreased ionic strength; this reflects the importance of electrostatics in controlling liposome adsorption. Increasing the level and molecular weight of PEG incorporation in the liposomes significantly reduced both the rate and extent of liposome adsorption; steric hindrance is considered to play a key role. Findings from this research will improve the understanding of liposome interaction during drug delivery, give insight into the actions of liposomes in the body and may form the basis for improved liposome formulations.     

Detachable immobilization of liposomes on polymer gel particles

Immobilization of liposomes on hydrophobized Sephacryl gel and controlled detachment of the liposomes from the gel were examined. The gel was chemically modified and bore octyl, hexadecyl or cholesteryl moiety via disulfide linkage as anchors to liposomal bilayer membrane. Upon interaction with the gel, egg phosphatidylcholine liposomes were successfully immobilized onto the gel. The gel with cholesteryl moiety showed 1.7 times higher liposome immobilization per anchor moiety than the gels with the alkyl moieties. The immobilization of liposomes on the gel was stable, and no significant spontaneous detachment of phospholipid or leakage of fluorescein isothiocyanate-conjugated dextran encapsulated in the immobilized liposomes was observed in 24h. Reductive cleavage of the disulfide linkage by dithiothreitol resulted in detachment of the liposomes from the gel. The majority of the detached liposomes were found encapsulating the dextran derivative, and these liposomes should have kept their structural integrity throughout the immobilization and the detachment processes. The release of the liposomes was insignificant until the ratio of the dithiothreitol to the hydrophobic anchor reached a threshold. The presence of the threshold suggests that the immobilization of liposomes should require a certain minimum number of the hydrophobic moieties anchored in the liposomal membrane. By applying the present immobilization-detachment system, preparation of liposomes encapsulating the dextran derivative without using costly gel filtration or ultracentrifugation procedure was successfully demonstrated.     

The Influence of the Chain Length of Polycations on their Complexation with Anionic Liposomes

A series of strong polycations is synthesized through the anionic polymerization of 2‐vinylpyridine, followed by subsequent quaternization of the resulting polymer. Polycations based on quaternized 2‐vinylpyridine (PVPQs) with degrees of polymerization (DP) from 20 to 440 are adsorbed on the surface of small anionic liposomes. Liposome/PVPQ complexes are characterized by using a number of physicochemical methods. All PVPQs are totally adsorbed onto the liposome surface up to a certain concentration at which saturation is reached (which is specific for each PVPQ). The integrity of the adsorbed liposomes remains intact. Short PVPQs interact with anionic lipids localized on the outer membrane leaflet, whereas long PVPQs extract anionic lipids from the inner to outer leaflet. Complexes tend to aggregate, and the largest aggregates are formed when the initial charge of the liposomes is fully neutralized by the charge of the PVPQ. PVPQs with intermediate DPs demonstrate behavioral features of both short and long PVPQs. These results are important for the interpretation of the biological effects of cationic polymers and the selection of cationic polymers for biomedical applications.     

Liposomes Prepared from Inverse Micelles

A method for the preparation of liposome is introduced, which contains two experimental steps: (a) inverse micelles of lecithin are formed in water-in-oil system by sonication; (b) the micelles are spread on the water surface, passed Through the oil-water interface, and transformed into liposomes in the water phase. The main advantage of this method is that the inner aqueous solution encapsulated by liposomes could be different from their enviromental medium. The liposome size is less than 0.5 μm in diameter by atomic force microscope. Comparison of activities of urease with and without liposome encapsulation suggested that urease was well entraped into liposomes.     

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.     

Fabrication of nano-scale liposomes containing doxorubicin using Shirasu porous glass membrane

Nano-scale liposomes were successfully produced using a Shirasu porous glass (SPG) membrane emulsification technique. Primary liposomes prepared by a film-hydration method were treated using SPG membranes with different pore sizes (2.0, 1.0, 0.7, 0.5, and 0.2 μm) for control over the liposome size. The liposome sizes were evaluated using a dynamic light scattering method and their morphologies were observed by optical microscopy and transmission electron microscopy. As the passage number of liposomes through SPG membrane increased, the size and its distribution of the liposomes gradually decreased. A smaller pore size of the SPG membrane and a higher applied pressure resulted in liposomes with a smaller size. After the preparation of nano-scale liposomes containing ammonium sulfate (AS), doxorubicin (DOX) was encapsulated in the liposomes by a remote loading method, where AS served as a precipitant for DOX. The encapsulation efficiency of the DOX was maximized up to 94% when the concentrations of AS and DOX were 250 and 0.045 mM, respectively. We have obtained the release profiles of the liposomes with different sizes. As shown below, liposomes with smaller size exhibited a faster release profile of drug due to the large surface area. These nano-scale liposomes encapsulating an anti-cancer drug can potentially be employed as drug delivery vehicles for intravenous injection.     

Analysis of liposomes using asymmetrical flow field-flow fractionation: Separation conditions and drug/lipid recovery

Liposomes composed of dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylglycerol were analyzed by asymmetrical flow field-flow fractionation coupled with multi-angle laser light scattering. In addition to evaluation of fractionation conditions (flow conditions, sample mass, carrier liquid), radiolabeled drug-loaded liposomes were used to determine the liposome recovery and a potential loss of incorporated drug during fractionation. Neither sample concentration nor the cross-flow gradient distinctly affected the size results but at very low sample concentration (injected mass 5 μg) the fraction of larger vesicles was underestimated. Imbalance in the osmolality between the inner and outer aqueous phase resulted in liposome swelling after dilution in hypoosmotic carrier liquids. In contrast, liposome shrinking under hyperosmotic conditions was barely visible. The liposomes themselves eluted completely (lipid recoveries were close to 100%) but there was a loss of incorporated drugs during separation with a strong dependence on the octanol-water partition coefficient of the drug. Whereas corticosterone (partition coefficient ～2) was washed out more or less completely (recovery about 2%), loss of temoporfin (partition coefficient ～9) was only minor (recovery about 80%). All fractionations were well repeatable under the experimental conditions applied in the present study.     

Effect of water-soluble polymers on fusion of egg yolk and soybean phospholipid liposomes

The efficiencies of polyelectrolytes, i.e., polycations and polyanions, and several kinds of water-soluble polymers as fusogens on soybean phospholipid liposome (SL) and egg yolk phospholipid liposome (EL) were investigated by the fluorescence quenching method. There were optimal concentrations for the induction of fusion in every system. Polycations induced fusion of liposomes at very low concentration in comparison with other polymers. Poly(carboxylic acid)s induced fusion at relatively high concentration. A strong acidic polyanion with high molecular weight also induced fusion of liposomes. The induction efficiency of poly(ethylene glycol) on fusion was higher than other nonionic polymers. The efficiency of fusion of EL was lower than that of SL in all systems because of the higher stability of EL membrane. It was found that electrostatic interactions, hydrogen bonding and/or hydrophobic interaction between these water-soluble polymers and liposomal membranes played an important role on aggregation and fusion of liposomes.