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.     

Effect of amphiphilic drugs on the stability and zeta-potential of their liposome formulations: a study with prednisolone, diazepam, and griseofulvin

Multilamellar liposomes consisting of phosphatidylcholine and incorporating prednisolone (PZ), diazepam (DZ), or griseofulvin (GF) were prepared and characterized. Liposome size, surface charge, and stability (in buffer and serum proteins) were measured for drug-incorporating liposomes and empty liposomes for comparison. The results reveal that for all drugs studied drug incorporation has a substantial effect on the vesicle zeta-potential and stability. Drug-incorporating liposomes have a negative surface charge, while their membrane integrity is significantly higher when compared with that of empty liposomes. Release of DZ from liposomes is induced by dilution. Summarizing, the results of this study demonstrate that the presence of PZ, DZ, or GF in liposome membranes has a significant effect on main vesicle properties and correlates well with those obtained previously for hydrochlorothiazide and chlorothiazide. Thereby, we may conclude that the previously demonstrated effects of the thiazides on liposome properties are not solely related to their structure.     

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.     

Polyion-induced liposomal vesicle aggregation: a radiowave dielectric relaxation study

The radiowave dielectric properties of aqueous heterogeneous systems during the complexation of charged polyions and oppositely charged liposomal particles have been measured in a wide frequency range, between 100 Hz and 2 GHz. The formation of a polyion-liposome complex driven by the correlated polyion adsorption at the particle surface implies two concomitant effects referred to as reentrant condensation and charge inversion. Both of them are governed by electrostatic interactions and there is now strong evidence, based on experiments and simulations, that counterion release is the driving force of the aggregation process. From this point of view, dielectric technique may offer a suitable tool in the investigation of the structural properties of these aggregates. In spite of the fact that interaction of polyions with oppositely charged surfaces was extensively experimentally investigated, there are no papers concerning the dielectric properties during the polyion-induced aggregation. To get an insight into this important topic, the authors present here an extensive set of radiowave dielectric measurements of liposomal vesicle aqueous suspensions where the liposome aggregation was induced by an oppositely charged polyion. The aggregation was followed from the beginning, when most of the isolated liposomes predominate, up to the formation of polyion-coated liposomes of inverted charge, crossing the isoelectric condition, where large, almost neutral, aggregates appear. The authors describe the observed dielectric dispersions as due to counterion polarization in the adjacency of the liposome and liposome aggregate surface, primarily governed by the zeta potential, according to the standard electrokinetic model.     

Nanoparticle-assisted surface immobilization of phospholipid liposomes

Phospholipid liposomes (100-200 nm diameter) are deposited onto solid substrates after stabilizing them against fusion with the solid by allowing charged nanoparticles to adsorb at approximately 25% surface coverage. The immobilized vesicles remain stable over a period of days. Epifluorescence imaging shows that they diffuse freely over surfaces with the same charge but adsorb tightly onto surfaces with opposite charge. Nanoparticle adsorption to surface patterns of opposite charge provides a facile method to create large-scale surface-supported arrays of intact liposomes. This surface attachment method is simple chemically and applies generally for solid surfaces that can be hydrophobic or hydrophilic. Offering routes to localize proteins and other vesicle-contained objects at surfaces in tailored spatial patterns, these immobilized liposome arrays may find diverse applications in the emerging field of nanobiotechnology.     

膜材性质及制备方法调控下的脂质体负载干扰素的研究

依据干扰素(IFN)分子、磷脂分子本身的理化性质和结构特点, 分别用三种制备方法, 以四种脂质体为膜材, 制备IFN脂质体, 考察了不同膜材、不同制备方法对脂质体粒径及包封率的影响. 结果表明, 以二肉豆蔻酰胆碱和二棕榈酰磷脂酰胆碱复合材料为主要膜材, 采用薄膜蒸发法制备的IFN脂质体有良好的稳定性, 60 d内其粒径可以保持在200~350 nm, 包封率可保持30%~40%.     

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.     

Sterically Stabilized Vesicles

After significant developments in liquid crystal and polymer research, scientists became interested in lyotropic systems containing polymers. These studies investigated, for instance, phase behavior and stability characteristics of suspensions of colloidal particles containing water-soluble or surface-adsorbed polymers or block copolymers. The most frequently studied were micelles, latex prticles, and lipid vesicles (liposomes). Liposomes aggregate and fuse in the presence of hydrophilic polymers but their properties were difficult to explain when block copolymers were adsorbed or surfactants with larger polymeric polar heads were inserted into the liposome membrane, because such systems are inherently ill defined. Liposomes containing diacyl surfactants with covalently linked, longer polymer chains display many new properties with very important consequences for both basic and applied research. They stimulate fundamental studies on phase behavior and polymer conformation, scaling laws, colloidal and surface properties, and cell function: applications deal predominantly with liposomes as drug delivery systems. While in basic research theory is currently more advanced than experiment, in medical applications theoretical understanding lags behind experimental achievements. It was discovered only relatively late that liposomes with an appropriate polymer coating are significantly more stable in a biological milieu, a necessary condition for their utility as drug carriers. In particular in medical applications, this practice has rejuvenated the field of anticancer therapy and targeted drug deliviery. All these advances were made possible by an effective and synergistic overlap of many different disciplines.     

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.     

124-kDa PHYTOCHROME IN MODEL MEMBRANE SYSTEMS: ASSOCIATION STUDIES AND COVALENT BINDING TO PREFORMED LIPOSOMES

Abstract— 124-kDa Phytochrome from oat has been covalently bound to the surface of preformed unilamellar liposomes doped with functionalized lipids. The extent of phytochrome binding varied from 100% (to soybean lecithin and dioleoyl phosphatidylcholine liposomes) to (90 ± 10)% (to dimyristoyl phosphatidylcholine liposomes) and (50 ± 10)% to dipalmitoyl phosphatidylcholine liposomes). The photochromic properties of phytochrome were fully retained in the liposome-bound systems. Attempts to bring about spontaneous incorporation and binding of the phytochrome to neutral and positively charged liposomes failed. These results were independent of liposome size, the presence of cholesterol, and whether phytochrome was added prior to or after the liposome formation.     

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.     

Carbazole as an excited state proton transfer fluorescent probe for lipid bilayers in alkaline medium

In our effort to look for novel excited state proton transfer (ESPT) fluorescent probes in alkaline pH range, we have examined carbazole as a possible candidate because of its high extinction coefficient, high quantum yield and a larger difference in ionization constant between the ground and excited state (pKa - pKa*). The photodissociation of carbazole was studied in liposome membrane by steady state fluorescence measurements at alkaline pH ranges. The neutral form and the anionic form of carbazole emit at 362 and 417 nm, respectively. This large shift in emission makes it convenient to monitor the physical properties of liposomes. The neutral form fluorescence intensity of carbazole is sensitive to phase changes in the membrane and also shows a maximum at phase transition temperature. This variation of intensity can be explained in terms of redistribution of probe between the surface and interior of the liposomes. Cholesterol induced phase changes of liposomes were also sensed by the ESPT of carbazole.     

Intrinsic heterogeneity in liposome suspensions caused by the dynamic spontaneous formation of hydrophobic active sites in lipid membranes

The spontaneous, dynamic formation of hydrophobic active sites in lipid bilayer membranes is studied and characterized. It is shown that the rates of formation and consumption of these active sites control at least two important properties of liposomes: their affinity for hydrophobic surfaces and the rate by which they spontaneously release encapsulated molecules. The adhesion and spreading of liposomes onto hydrophobic polystyrene nanoparticles and the spontaneous leakage of an encapsulated fluorescent dye were monitored for different liposome compositions employing Cryo-TEM, DLS, and fluorescence measurements. It was observed that an apparently homogeneous, monodisperse liposome suspension behaves as if composed by two different populations: a fast leaking population that presents affinity for the hydrophobic substrate employed, and a slow leaking population that does not attach immediately to it. The results reported here suggest that the proportion of liposomes in each population changes over time until a dynamic equilibrium is reached. It is shown that this phenomenon can lead to irreproducibility in, for example, spontaneous leakage experiments, as extruded liposomes leak much faster just after preparation than 24 h afterward. Our findings account for discrepancies in several experimental results reported in the literature. To our knowledge, this is the first systematic study addressing the issue of an existing intrinsic heterogeneity of liposome suspensions.     

Size Dependence of Protein-Induced Flocculation of Phosphatidylcholine Liposomes

The adsorption of lysozyme and cytochrome C on phosphatidylcholine liposomes essentially changes the physical properties of the phospholipid membranes and under certain circumstances greatly affects the stability of the colloid dispersion by inducing bridging liposome flocculation. This study was designed to examine experimentally the influence of liposome size on two kinetic parameters of the flocculation, its rate constant and activation energy. As the liposome radius increased in the range 50-500 nm, the activation energy tended to decrease, resulting in an increased flocculation rate, except for the flocculation of 400-nm liposomes, which was greatly impeded. The pronounced influence of the liposome size on the flocculation rate constant was evident, since a well-defined minimum in the kinetic rate of flocculation of 400-nm liposomes was detected experimentally. The obtained nonlinear radius dependencies of the flocculation rates and activation energies are interpreted in terms of the bridging mechanism of the protein-induced liposome flocculation and the supplementary concept of the stability of thin liquid films formed between approaching protein-adsorbed liposomes. Copyright 2000 Academic Press.     

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.     

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.     

Covalent immobilization of liposomes on plasma functionalized metallic surfaces

A method was developed to functionalize biomedical metals with liposomes. The novelty of the method includes the plasma-functionalization of the metal surface with proper chemical groups to be used as anchor sites for the covalent immobilization of the liposomes. Stainless steel (SS-316) disks were processed in radiofrequency glow discharges fed with vapors of acrylic acid to coat them with thin adherent films characterized by surface carboxylic groups, where liposomes were covalently bound through the formation of amide bonds. For this, liposomes decorated with polyethylene glycol molecules bearing terminal amine-groups were prepared. After ensuring that the liposomes remain intact, under the conditions applying for immobilization; different attachment conditions were evaluated (incubation time, concentration of liposome dispersion) for optimization of the technique. Immobilization of calcein-entrapping liposomes was evaluated by monitoring the percent of calcein attached on the surfaces. Best results were obtained when liposome dispersions with 5mg/ml (liposomal lipid) concentration were incubated on each disk for 24h at 37°C. The method is proposed for developing drug-eluting biomedical materials or devices by using liposomes that have appropriate membrane compositions and are loaded with drugs or other bioactive agents.     

Encapsulation of drugs by lyophilized empty dipalmitoylphosphatidylcholine liposomes: effect of calcium ion

The effects of divalent cation (Ca2+) on the characteristics of dipalmitoylphosphatidylcholine (DPPC) liposomes regenerated from lyophilized empty liposomes by rehydration and warming were investigated. The results showed that the volume (ml) of internal aqueous compartment per g lipid (captured volume; Vcap) has a maximum at a certain concentration range of calcium chloride and the maximal value is more than ten times the minimal value. This phenomenon can be explained by considering that binding of Ca2+ to phosphate groups in DPPC molecules induces an increase in the distance (r) between adjacent bilayer membranes in multilamellar liposomes through electrostatic force and causes an increase in Vcap. The dynamic properties of lyophilized liposomes in the rehydration process were examined using a multilamellar vesicle model. The results of simulation suggested that a repulsive force induced between the adjacent bilayer membranes causes rearrangement of the constituent lipid molecules in a liposome followed by an increase in the distance r, a decrease in the internal lamellar number, a smaller increase in liposome size and finally a significant increase in Vcap.     

Liposome Capillary Electrophoresis of Peptides and Proteins

Liposome capillary electrophoresis (LCE) using unilamellar liposomes composed of the zwitterionic phospholipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) as a suspended pseudo stationary phase has been investigated for its capability at separating peptides and proteins in bare fused-silica capillaries. The study has explored different strategies for allowing the liposome suspension to act as a disperse pseudo stationary phase with the ability of modulating selectivity, resolution and separation performance of peptides and proteins in bare-fused silica capillaries. Such strategies comprise the use of capillaries either partially or totally filled with the liposome suspension, whereas the electrolyte solution is liposome-free, or the incorporation of the liposomes into the buffer solution employed for rinsing the capillary and as the background electrolyte. Three synthetic peptides of similar amino acid sequence and four basic standard proteins have been employed as test analytes. Varying the volume of the liposome suspension introduced in the capillary promoted differentiated variations in the migration velocity of the three peptides reflecting their selective interactions with the liposomes. Efficient separation of basic proteins was obtained at pH 7.4 in a bare fused-silica capillary with the electrolyte solution containing 60 μM POPC.