Microfluidic directed formation of liposomes of controlled size

A new method to tailor liposome size and size distribution in a microfluidic format is presented. Liposomes are spherical structures formed from lipid bilayers that are from tens of nanometers to several micrometers in diameter. Liposome size and size distribution are tailored for a particular application and are inherently important for in vivo applications such as drug delivery and transfection across nuclear membranes in gene therapy. Traditional laboratory methods for liposome preparation require postprocessing steps, such as sonication or membrane extrusion, to yield formulations of appropriate size. Here we describe a method to engineer liposomes of a particular size and size distribution by changing the flow conditions in a microfluidic channel, obviating the need for postprocessing. A stream of lipids dissolved in alcohol is hydrodynamically focused between two sheathed aqueous streams in a microfluidic channel. The laminar flow in the microchannel enables controlled diffusive mixing at the two liquid interfaces where the lipids self-assemble into vesicles. The liposomes formed by this self-assembly process are characterized using asymmetric flow field-flow fractionation combined with quasi-elastic light scattering and multiangle laser-light scattering. We observe that the vesicle size and size distribution are tunable over a mean diameter from 50 to 150 nm by adjusting the ratio of the alcohol-to-aqueous volumetric flow rate. We also observe that liposome formation depends more strongly on the focused alcohol stream width and its diffusive mixing with the aqueous stream than on the sheer forces at the solvent-buffer interface.     

Rapid liposome quality assessment using a lab-on-a-chip

Although liposomes have many outstanding features such as biocompatibility, biodegradability, low toxicity and structural diversity, and are successfully applied in many areas of chemistry and biotechnology, a lack of characterization standards and quality control tools are still inhibiting the translation of liposome technology into clinical routine. The greatest obstacle to clinical scale commercialization is the inability to ensure liposome formulation stability because small size variations or altered surface chemistries can significantly influence in vivo distribution and excretion kinetics that could in turn lead to unpredictable therapy outcomes. To enhance the product development process we have developed a microfluidic biochip containing embedded dielectric microsensors capable of providing quantitative results on formulation composition and stability based on the monitoring of the unique electric properties of liposomes. Computational fluid dynamic (CFD) simulations confirmed that microfluidics offer reproducible and well-defined measurement conditions where a moving liposome suspension within a microchannel behaves like a bulk material. Results of this study demonstrate the ability of microfluidics, in combination with dielectric spectroscopy and multivariate data analysis methods, to identify nine different liposomes. We also show that various liposome modifications such as membrane-bound surface proteins, lipid bilayer soluble drugs, as well as protein and dye encapsulations, can be detected in the absence of any labels or indicators. Since shelf-life stability of a liposome formulation is regarded of prime importance for regulatory approval and clinical application, we further provide a possible practical application of the developed liposome analysis platform as a high-throughput tool for industrial quality insurance purposes.     

Kinetics of the adhesion of DMPC liposomes on a mercury electrode. Effect of lamellarity, phase composition, size and curvature of liposomes, and presence of the pore forming peptide mastoparan X

Liposomes suspended in aqueous electrolyte solutions can adhere at mercury electrodes. The adhesion is a complex process that starts with the docking and opening and leads to a spreading, finally resulting in the formation of islands of adsorbed lecithin molecules. The adhesion process can be followed by chronoamperometry, and a detailed analysis of the macroscopic and microscopic kinetics can be performed yielding rate constants and activation parameters. By using giant unilamellar liposomes and multilamellar liposomes, the effect of lamellarity and liposome size could be elucidated for liposomes in the liquid crystalline, gel, and superlattice phase states. Below the phase transition temperature, the time constant of opening of the liposomes (i.e., the irreversible binding of the lecithin molecules on the preliminary contact interface liposome|mercury and the therewith associated disintegration of the liposome membrane on that spot) is shown to be strongly size dependent. The activation energy, however, of that process is size independent with the exception of very small liposomes. That size dependence of time constants is a result of the size dependence of the initial contact area. The time constant and the activation energies of the spreading step exhibit a strong size dependence, which could be shown to be due to the size dependence of rate and activation energy of pore formation. Pore formation is necessary to release the solution included in the liposomes. This understanding was corroborated by addition of the pore inducing peptide Mastoparan X to the liposome suspension. The obtained results show that electrochemical studies of liposome adhesion on mercury electrodes can be used as a biomimetic tool to understand the effect of membrane properties on vesicle fusion.     

Liposome solubilization induced by surfactant molecules in a microchip

The dynamics of liposome solubilization was monitored by dynamic light scattering and optical microscopy. A newly designed Y-shape microchannel connected to a room was incorporated into a microchip and the reaction processes of the liposome suspension and surfactant solution were observed in the room after mixing the two fluids and stopping the flow. By using this microchip, we succeeded in real-time monitoring of liposome solubilization and the following dynamic processes of solubilization were proposed: 1) Deformed liposomes become spherical. 2) The liposome size increases until the surfactant/liposome ratio in the liposome membrane reaches a threshold value. 3) Mixed micelles of surfactants and phospholipids are released and the liposomes collapse.     

A study on the characterization and stability of rhenium(III) chloride-incorporated liposomes

Nanoliposomes are important carriers capable of packaging drugs for various delivery applications through passive targeting tumor sites by enhancing permeability and retention effect. Radiolabeled liposomes have potential applications in radiotherapy and diagnostic imaging. However, the physico-chemical instability of liposomes during manufacturing and storage limits their extensive application. Therefore, considerable numbers of studies have been made on the stability of liposomes over the last few years in order to overcome this problem. In this study, we attempted to prepare polymer-coated liposomes using water-soluble chitosan in order to enhance the stability of rhenium(III) chloride-incorporated liposomes. They were characterized by an electrophoretic light-scattering spectrophotometer, Fourier transform infrared spectroscopy (FT-IR), UV–Vis spectrometer, and phase-contrast microscopy. The chitosan-coated liposomes are spherical and the particle size is about 800–850 nm. Incorporation of chitosan into the liposome bilayer decreased rhenium(III) chloride release from the liposome due to an increased rigidity of the liposome membrane structure. Chitosan-coated liposomes showed a higher stability compared with the stability of non-coated liposomes. The release characteristics of rhenium(III) chloride encapsulated in the liposome were taken as a measure of stability of the liposome membrane.     

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.     

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.     

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.     

Singlet-Oxygen Generation from Liposomes: A Comparison of 6β-Cholesterol Hydroperoxide Formation with Predictions from a One-Dimensional Model of Singlet-Oxygen Diffusion and Quenching

Abstract— We measured 6β-cholesterol hydroperoxide (6β-CHP), a specific singlet-oxygen (O₂(δg)) product, during irradiation of unilamellar dimyristoyl 1-α-phosphatidylcholine liposomes containing cholesterol and zinc phthalocyanine (ZnPC). The effects of liposome size, the hydrophobic (O₂(¹δ_g)) quencher, β-carotene, and hydrophilic O₂(¹δ_g) quenchers upon the amount of 6β-CHP formed were determined and interpreted in terms of a one dimensional model of ₂(¹δ_g) quenching and diffusion. The model correctly predicted (1) that the amount of 6β-CHP was increased with increasing liposome size, (2) that P-carotene was more effective at reducing 6β-CHP formation in 400 nm diameter liposomes than 100 nm diameter liposomes and (3) that the hydrophilic quencher, water, was also more effective in large liposomes than in small liposomes.
The hydrophobic quencher, β-carotene, was more effective at reducing the formation of 6β-CHP than at reducing the 1270 nm O₂(¹δ_g) emission. This difference was found to be due to the size distribution present in the liposome preparations because the difference between the 6β-CHP data and the 1270 nm emission data was much smaller in liposome preparations with a narrow size distribution. When a significant size distribution was present, the 6β-CHP data were weighted more heavily with large-diameter liposomes, while the 1270 nm emission data were weighted more heavily with small-diameter liposomes.     

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.     

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.     

Interaction of copper nanoparticles with liposomes

The reduction of copper(II) ions in an aqueous dispersion of positively charged liposomes results in the formation of stable sols of a complex of copper nanoparticles with the surface of liposomes. The mean size (7 nm) and the narrow size distribution of metal nanoparticles are similar to those observed in the case of metal sol formation in polymer solutions. The labile character of bonds between nanoparticles and liposomes makes the latter able to compete with a linear polymer (poly-N-vinylpyrrolidone) in binding to nanoparticles. This ability is manifested in the independence of an almost even distribution of nanoparticles between these competitors from the sol preparation mode in a system including both poly(N-vinylpyrrolidone) macromolecules and liposomes. The evenness of the distribution indicates an approximately identical stability of complexes of copper nanoparticles with both competitors. The replacement of liposomes with poly(N-vinylpyrrolidone) macromolecules in the protective shields of nanoparticles is accompanied by the detachment of the nanoparticles from the surface, thereby allowing the measurement of their size and size distribution in the case where such measurements are impossible because of a high density of nanoparticles on the liposome surface.     

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.     

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.     

Time evolution of the formation of different size cationic liposome-polyelectrolyte complexes

We report on the time evolution of the aggregation behaviour of cationic liposome-polyelectrolyte complexes studied by means of dynamic light scattering technique. Pure dioleoyltrimethilammoniumpropane (DOTAP) and mixed DOTAP-dipalmitoylphosphatidylcholine (DPPC) liposomes in polyacrylate sodium salt aqueous solutions in a wide concentration range have been investigated and the size and size distributions of the resulting aggregates evaluated from the intensity autocorrelation function of the scattered light. Under appropriate conditions, we found two discrete aggregation regimes, resulting in two different structural arrangements, whose time evolution depends on the charge ratio and the polyelectrolyte molecular weight. A first small component of average size in the 100-500 range nm coexists with a larger component, whose typical size increases with time, up to some micrometers. The cluster growth from a single liposome, 70 nm in diameter, to the formation of polymer-coated liposome aggregates has been briefly discussed in the light of steric stabilization of colloids. Moreover, it has been found that the kinetics of aggregation of the larger, time-dependent, component follows a dynamical scaling within the diffusion-limited cluster aggregation (DLCA) regime. The understanding of structures resulting from interactions between polyelectrolytes with oppositely charged liposomes may help towards formulation of "lipoplexes" (cationic lipid-DNA complexes) to use as non-viral gene carriers.     

Influence of lipid composition on the thermotropic behavior and size distribution of mixed cationic liposomes

Cationic liposomes are studied mainly as nonviral nucleic acid delivery systems and to a lesser extent as carriers/adjuvants of vaccines and as low-molecular-weight drug carriers. It is well established that the performance and the biological activity of liposomes in general are strongly related to their physicochemical properties. We investigated the thermotropic behavior and the size distribution of mixed cationic liposomes formulated with different percentages of 1,2 dimyristoyl-sn-glycero-3-phosphatidylcholine and one of four cationic amphiphiles characterized by a pyrrolidinium headgroup with the aim of achieving a better understanding of how the molecular structure of the cationic amphiphile and its mole percentage affect the physicochemical properties of the liposomes. Multilamellar vesicles and large unilamellar vesicles were studied by differential scanning calorimetry and turbidity, respectively, to characterize the thermotropic behavior and lipid phase, whereas dynamic light scattering was used to determine size distribution. This study shows that subtle modifications in the cationic amphiphile's molecular structure and in liposome composition may have dramatic effects on the organization of the liposome bilayer and hence on the morphological and physicochemical features of the liposomes, thus being highly relevant to the biological features investigated previously.     

Investigation of Single‐Drug‐Encapsulating Liposomes using the Nano‐Impact Method

Encapsulating liposomes are widely used for controlled drug delivery. We report the use of nano‐impact experiments for the electrochemical attomolar quantification of the liposome load, uniquely at the single liposome level, using vitamin C encapsulated liposomes as a model. The size of the liposomes and their picomolar concentration are also determined in biological buffer in real time.     

Shape-tunable polymer nodules grown from liposomes via ring-opening metathesis polymerization

A new inisurf (acting as surfactant and initiator) molecule for ring-opening metathesis polymerization (ROMP) was synthesized and used in aqueous solution in order to control the size and shape of polymer nodules grown from liposomes. Nodules were observed to grow in size with conversion of monomer, and depending on the monomer used, they adopted either a spherical or comet-like shape. Here, we investigate polymer production from a liposome surface. We use a hydrophobic derivative of the Grubbs catalyst positioned at the liposome surface to allow for ROMP of monomers dissolved in the aqueous outer phase. We obtain nodules of polymer that can grow up to tens of micrometers, unveiling new efficient possibilities of polymerization from a membrane in an aqueous solution.