Effects of frequency and power of ultrasound on the size reduction of liposome

The solutions of liposome made of l-α-dilauroyl phosphatidylcholine are sonicated at various powers and frequencies (43-480 kHz), and the resultant change in the size of liposome is measured by the dynamic light scattering method. The ultrasonic power dissipated into the solution is determined by the calorimetric method in order to compare the effects of ultrasound of different frequencies. The faster reduction of the mean size of liposome is achieved at the lower frequency. Comparing at the same frequency and total energy, short-time irradiation of strong ultrasound is more efficient than long-time irradiation of weak ultrasound. These results indicate that the small number of cavitation events with stronger physical disturbance on liposome can reduce the size of the liposome more efficiently than the large number of cavitation events with weaker disturbance.     

Pressure effects on lipid membrane structure and dynamics

The effect of hydrostatic pressure on lipid structure and dynamics is highly important as a tool in biophysics and bio-technology, and in the biology of deep sea organisms. Despite its importance, high hydrostatic pressure remains significantly less utilised than other thermodynamic variables such as temperature and chemical composition. Here, we give an overview of some of the theoretical aspects which determine lipid behaviour under pressure and the techniques and technology available to study these effects. We also summarise several recent experiments which highlight the information available from these approaches.     

Phase behavior of lipid mixtures

Biological membranes are two-dimensional mixtures of an enormous number of different components. Modeling cell membranes as simple bilayer mixtures reveals rich phase behavior, but how can we use the observed phase behavior to understand the real membranes?     

Phase diagrams and lipid domains in multicomponent lipid bilayer mixtures

Understanding the phase behavior of biological membranes is helped by the study of more simple systems. Model membranes that have as few as 3 components exhibit complex phase behavior that can be well described, providing insight for biological membranes. A number of different studies are in agreement on general findings for some compositional phase diagrams, in particular, those that model the outer leaflet of animal cell plasma membranes. These model mixtures include cholesterol, together with one high-melting lipid and one low-melting lipid. An interesting finding is of two categories of such 3-component mixtures, leading to what we term Type I and Type II compositional phase diagrams. The latter have phase regions of macroscopic coexisting domains of {Lα + Lβ + Lo} and of {Lα + Lo}, with domains resolved under the light microscope. Type I mixtures have the same phase coexistence regions, but the domains seem to be nanoscopic. Type I mixtures are likely to be better models for biological membranes.     

Cubic topology in surfactant and lipid mixtures

Ternary systems of palmitoyl-oleoyl-phosphatidylcholine (POPC) and the non-ionic surfactant C₁₂EO₂ (di-ethylene-oxide-mono-dodecyl-ether) in water have been studied with optical microscopy, NMR, DSC and X-rays from ambient temperatures to 45 °C. Below 29 °C the system is in the lamellar liquid crystalline state. Between 30 and 32 °C it transforms into a cubic Ia3d structure which converts into the cubic Pn3m phase at 39 °C. The transitions are fully reversible. An epitaxial relationship between all three phases was found, which is an elegant and convenient way to rearrange molecules from lamellar bilayers to a network of curved surfaces. The la3d (Q²³⁰) to Pn3m (Q²²⁴) transition occurs without measurable enthalpy change. This, together with the metric relation of 1.60 between the cubic lattice constants is strong evidence for a Bonnet transformation, where the structural changes occur without change in curvature. The potential significance of the cubic phases as intermediate structures for biological processes, e. g. transport across a bilayer or fusion of membranes, are discussed.     

A method to evaluate the effect of liposome lipid composition on its interaction with the erythrocyte plasma membrane

Lipid aggregates are considered promising carriers for macromolecules and toxic drugs. In order to fulfill this function, aggregates should have properties that ensure the efficient delivery of their cargo to the desired location. One of these properties is their stability in blood when accumulating in the targeted tissue. This stability may be affected by a number of factors, including enzymatic activity, protein adsorption, and non-specific lipid exchange between the aggregate and morphological blood components. Since blood cells in the majority consist of erythrocytes, their interaction with aggregates should be carefully analyzed. In this paper, we present a method that allows the exchange of lipid between liposomes and the erythrocyte plasma membrane to be evaluated. The extent of this exchange was measured in terms of the toxicity of a cationic lipid (DOTAP) incorporated into the liposome lipid bilayer, evaluated by plasma membrane mechanical properties. After liposomes were formed from DOTAP/PC or DOTAP/PE mixtures, erythrocyte plasma membranes were destabilized in a manner dependent on DOTAP concentration. A constant quantity of DOTAP mixed with various proportions of SM caused no such effect, indicating very limited lipid exchange with the cell membrane for such liposome formulations.     

Determination of liposome size: a tool for protein reconstitution

Reconstitution of proteins into liposomes is a widespread approach to analyzing their biological function. Many protocols exist for this procedure and for the subsequent analysis of proteins. Here, we establish a procedure for preparation and analysis of liposomes with a lipid composition reflecting the outer envelope of chloroplasts. First, the stability of the liposomes in different buffer systems was investigated to provide information for the storage of the reconstituted system. Then, the size of the liposomes created by filtration through a polycarbonate filter dependent on the lipid composition was analyzed. Subsequently, solubilization of the liposomes composed of lipids with the outer envelope composition by dodecylmaltoside and octylglucoside as a preceding step of reconstitution was studied. Finally, we developed a straightforward method to determine the size of liposomes by absorption spectroscopy. The described setup allows the construction of reconstitution protocols, including the final determination of the liposome size.     

The involvement of the lipid phase transition in the plasma-induced dissolution of multilamellar phosphatidylcholine vesicles

Unsonicated liposomes prepared from dimyristoyl phosphatidylcholine were nearly completely dissolved during a 3 h incubation with rat plasma at or close to the phase-transition temperature of 24°C. At 37 or 15°C virtually no liposomal disintegration was observed even after 24 h of incubation. The liposomal solubilization, which was monitored by turbidity measurements or by determination of phospholipid sedimentability, was accompanied by the formation of a phospholipid-protein complex similar or identical to the one we previously reported to be formed from sonicated liposomes of egg phosphatidylcholine (Scherphof, G., Roerdink, F., Waite, M. and Parks, J. (1978) Biochim. Biophys. Acta 542, 296–307). Unsonicated multilamellar liposomes made of egg phosphatidylcholine were completely resistant to the dissolving potency of plasma when incubated at 37°C. Liposomes from equimolar mixtures of dimyristoyl and dipalmitoyl phosphatidylcholine were only degraded by plasma in the temperature range between 30 and 35°C at which temperature this cocrystallizing phospholipid mixture undergoes a phase transition. However, even at these temperatures the rate of dissolution of this mixture was significantly lower than of dimyristoyl phosphatidylcholine at 24°C. In the dissolving process of this mixture a slight preference for the lower-melting component was observed.The ability of cholesterol to completely abolish the susceptibility of dimyristoyl phosphatidylcholine liposomes to plasma at a 1:2 molar ratio of cholesterol to phospholipid substantiates the essential role of the phase transition in the process of liposome solubilization.When liposomes of the monotectic mixtures dimyristoyl and distearoyl phosphatidylcholine or dilauroyl and distearoyl phosphatidylcholine were incubated with plasma at temperatures in between those at which the constituent lipids undergo a phase change in the mixture, the liposomes were slowly disolved. Under those conditions a selective removal of the lipids in the liquid-crystalline phase was observed.It is concluded that for the plasma-induced dissolution of unsonicated liposomes, which is most probably achieved by interaction with (apo)lipoproteins, the presence of phase boundaries is required in much the same way as was first reported for phospholipases by Op den Kamp, J.A.F., de Gier, J. and Van Deenen, L.L.M. (1974) Biochim. Biophys. Acta 345, 253–256).     

The mechanism of liposome accumulation in infarction

This paper explores the mechanism(s) whereby liposomes accumulate in chronically ischaemic myocardium and intestine. Plasma prepared from venous blood obtained at sites of myocardial and intestinal infarction does not promote the lysis of positively and negatively charged liposomes in vitro. Albumin-bound lysophosphatidylcholine (≥ 2 mM) lyses positively and negatively charged liposomes in vitro at similar rates. [^99mTc]Diethylenetriamine pentaacetic acid (DTPA) entrapped in positively charged liposomes was accumulated in ischaemic rat caecum/colon 6 and 24 h after mesenteric ligation. Presumably allied to the accumulation of liposomal components, necrotic caecum/colon showed marked Ca²⁺ accumulation and phospholipid depletion. It is postulated that Ca²⁺ and Ca²⁺-activated membrane phospholipases may be implicated in the mechanism of liposome accumulation in chronic ischaemia.     

Effect of lipid phase transition on the binding of anions to dimyristoylphosphatidylcholine liposomes

Temperature dependence of the electrophoretic mobility of multilamellar liposomes prepared from dimyristoylphosphatidylcholine was measured in the presence of salts with different anions in aqueous solutions. It was established that specific binding of anions to liposome surface induced a pronounced zeta potential (electrostatic potential at the hydrodynamic plane of shear). A combination of Langmuir, Gouy-Chapman, and Boltzmann equations was used to describe the dependence of the zeta potential on the concentration of anions. The values of binding constants (K) and maximum numbers of binding sites per unit area (σ_max) were determined by this method. The sequence for anion affinities to liposome surface was found to be as follows: trinitrophenol >ClO₄⁻ >I⁻ >SCN⁻ >Br⁻ >NO₃⁻ >Cl⁻ SO₄²⁻. A sharp increase in the negative zeta potential was detected at the temperature of phase transition of the lipid from the gel to liquid-crystalline state. It was found that the parameter K did not change at lipid phase transition and the shifts in zeta potential might be due to alterations of σ_max. The binding sites were considered as defects in the package of lipid molecules in membranes.     

Oscillatory reaction of catalase wrapped by liposome

To date we have been studying the enzymatic oscillatory reaction caused by gradual entry of substrate via semi-permeable membrane. It has been found that many enzymes cause oscillatory reaction. Here we present an oscillatory reaction of enzyme wrapped by liposome. We used catalase as an enzyme, since its oscillatory reaction has been already investigated in detail in the absence of liposome. Distinct oscillation with shorter period than without liposome was obtained. On the other hand, it was shown that the presence of liposome facilitated the permeation rate of hydrogen peroxide through semi-permeable membrane. This is thought to be factor of shortening the oscillation period compared with that in the absence of liposome. Oscillation was also temperature dependent. Our finding may provide an important insight into the study of enzyme reaction taking part in rhythms in living systems.     

Phase transitions and cholesterol effects in phospholipid liposomes A new method employing the enhancement of the O-O vibronic transition of pyrene

The enhancement of the weakly allowed O-O vibronic transition in the fluorescence spectrum of the probe pyrene, which we previously showed to result from ground-state complexation with polar groups, has been shown in the present study to offer a new method for determining phase transition temperatures of liposomes and for studying the effects of cholesterol on the structure of their semipolar glycerol backbone. For dipalmitoylphosphatidylcholine it is found that small cholesterol contents (～9 mol%) induce an increase in the polarity of the microenvironment of the probe, whereas contents ?13 mol% induce a decrease in the polarity. The results are discussed in terms of cholesterol effects on the frequency and extent of thermally-induced structural fluctuations which, in turn, affect the penetration of the probe into the bilayer.     

Delivery of liposome membrane-associated sterols through silastic membranes

The transport of sterols incorporated into the lecithin bilayer of small unilamellar liposomes through a model membrane was studied. A two-chamber diffusion cell containing liposomes with incorporated [4-¹⁴C]cholesterol or β-[4-¹⁴C]sitosterol in the donor chamber and liposomes with unlabeled cholesterol in the receiver chamber was used. The permeability coefficients of the sterols through silastic rubber membranes which served as a model membrane were measured. The permeability for cholesterol incorporated into liposomes in a phosphatidyl choline/cholesterol molar ratio of 1 : 1, produced by sonication for 1 h, and subsequent centrifugation at 100000 × g for 1 h, was 1.6 · 10^?8 cm sec^?1. Dilution of the liposome suspension did not change the permeability coefficient significantly. The permeability coefficient of sitosterol incorporated into liposomes was about 4-times smaller than that of cholesterol. These results suggest that the sterols were delivered to the silastic membrane by the intact liposomes and that free solute was not involved in the transport to the membrane to a significant degree. The large differences in the permeability coefficients between cholesterol and sitosterol indicate that an aqueous interfacial barrier was crossed by the sterol during the delivery to the membrane.     

Phase behavior of freeze-dried phospholipid-cholesterol mixtures stabilized with trehalose

A study is presented of the role of cholesterol content on the gel-to-liquid crystalline phase transition of freeze-dried liposomes stabilized with trehalose, a well known lyoprotectant. The phospholipids considered in this work, DPPC and DPPE, belong to the two predominant phospholipid species found in numerous biological membranes. Cholesterol is found in abundance in mammalian plasma membranes. DSC measurements reveal that cholesterol-containing liposomes exhibit multiple phase transitions upon dehydration. Addition of trehalose to these systems lowers the phase transition temperature and limits the phase separation of the lipidic components upon freeze-drying. This work provides strong evidence for the effectiveness of trehalose in stabilizing cholesterol-containing membranes upon lyophilization.     

Early events in calcium-induced liposome fusion

Calcium-induced interaction of liposomes composed of pure phosphatidylserine (PS) has been studied using a rapid-mixing, rapid-freeze device. Freeze-fracture electron microscopy of this material revealed that liposomes react very rapidly after addition of calcium ions. After only 10 ms (the resolution of the technique) vesicle fusion was apparent. At the same time, however, vesicles also collapsed, and appeared as aggregates of flattened membranes. This may explain controversies which have arisen over vesicle fusion studied with more indirect methods.     

Serum-induced leakage of liposome contents

Efflux of contents from small unilamellar vesicles of various compositions, containing a highly quenched fluorescent compound (calcein, 175 mM) was determined as a function of temperature in the presence and absence of human serum. Efflux of calcein from the liposomes was monitored as an increase in fluorescence as calcein became dequenched upon release from the liposomes. The presence of serum significantly increased liposome leakage in all cases. Incorporation of increasing molar ratios of cholesterol into liposomes reduced leakage of calcein from liposomes incubated with buffer and with serum. Leakage was significantly faster from liposomes with an osmotic gradient across the membrane (higher inside) than from equiosmolar liposomes. The leakage of [¹⁴C]sucrose from egg lecithin liposomes at 37°C was also dramatically increased in the presence of serum.     

The phase behaviour of skin lipid mixtures based on synthetic ceramides

The lipid lamellae present in the outermost layer of the skin, the stratum corneum (SC), form the main barrier for diffusion of molecules across the skin. The main lipid classes in SC are cholesterol (CHOL), free fatty acids (FFA) and at least nine classes of ceramides (CER), referred to as CER1 to CER9. In the present study the phase behaviour of four synthetic CER, either single or mixed with CHOL or CHOL and FFA, has been studied using small and wide angle X-ray diffraction. The lipid mixtures showed complex phase behaviour with coexistence of several phases. The results further revealed that the presence of synthetic CER1 as well as a proper composition of the other CER in the mixture were crucial for the formation of a phase with a long periodicity, characteristic for SC lipid phase behaviour. Only a mixture containing synthetic CER1 and CER3, CHOL and FFA showed similar phase behaviour to that of SC.     

Development of metal affinity-immobilized liposome chromatography and its basic characteristics

Metal affinity-immobilized liposome chromatography (MA-ILC) was newly developed as a chromatographic technique to separate and analyze peptides. The MA-ILC matrix gel was first prepared by immobilizing liposomes modified with functional ligands. The functional ligand used to adsorb metal ions was N-hexadecyl iminodiacetic acid (HIDA), which is obtained by attaching a long alkyl chain to an iminodiacetic acid (IDA). Cu(II) ion was first adsorbed on the gel matrix through its complex formation with the HIDA on the surface of the immobilized liposome. Synthetic peptides of various types ranging in size from 5 to 40 residues were then used, and their retention properties on the MA-ILC were evaluated. The retention property of peptides on the MA-ILC by using a usual imidazole elution was compared with the retention property in the case of the immobilized metal affinity chromatography (IMAC) and an immobilized liposome chromatography (ILC). It was found that the retention property of peptides on the MA-ILC has the features of both the IMAC and the ILC; the retention ability of peptides depends on both the number of histidine residues in peptides and the liposome membrane affinity of the peptides. Histidine and tryptophan residues among amino acid residues in peptides indicated a high contribution coefficient for the peptide retention on the MA-ILC, probably due to their metal ion and membrane interaction properties, respectively.