Reducing Liposome Size with Ultrasound: Bimodal Size Distributions

Sonication is a simple method for reducing the size of liposomes. We report the size distributions of liposomes as a function of sonication time using three different techniques. Liposomes, mildly sonicated for just 30 sec, had bimodal distributions when surface-weighted with modes at about 140 and 750 nm. With extended sonication, the size distribution remains bimodal but the average diameter of each population decreases and the smaller population becomes more numerous. Independent measurements of liposome size using Dynamic Light Scattering (DLS), transmission electron microscopy (TEM), and the nystatin/ergosterol fusion assay all gave consistent results. The bimodal distribution (even when number-weighted) differs from the Weibull distribution commonly observed for liposomes sonicated at high powers over long periods of time and suggests that a different mechanism may be involved in mild sonication. The observations are consistent with the following mechanism for decreasing liposome size. During ultrasonic irradiation, cavitation, caused by oscillating microbubbles, produces shear fields. Large liposomes that enter these fields form long tube-like appendages that can pinch-off into smaller liposomes. This proposed mechanism is consistent with colloidal theory and the observed behavior of liposomes in shear fields.     

Microfluidic preparation of drug-loaded PEGylated liposomes,and the impact of liposome size on tumour retention and penetration

Understanding the effect of liposome size on tendency for accumulation in tumour tissue requires preparation of defined populations of different sized particles. However, controlling the size distributions without changing the lipid composition is difficult, and differences in compositions itself modify distribution behaviour. Here, a commercial microfluidic format as well as traditional methods was used to prepare doxorubicin-loaded liposomes of different size distributions but with the same lipid composition, and drug retention, biodistribution and localization in tumour tissues were evaluated. The small (～50?nm diameter) liposomes prepared by microfluidics and large (～75?nm diameter) liposomes displayed similar drug retention in in vitro release studies, and similar biodistribution patterns in tumour-bearing mice. However, the extent of extravasation was clearly dependent on size of the liposomes, with the small liposomes showing tissue distribution beyond the vascular area compared to the large liposomes. The use of microfluidics to prepare smaller size distribution liposomes compared to sonication methods is demonstrated, and allowed preparation of different size distribution drug carriers from the same lipid composition to enable new understanding of tissue distribution in compositionally consistent materials is demonstrated.     

Particle Sizing of Liposomal Dispersions: A Critical Evaluation of Some Quasi-Elastic Light-Scattering Data-Analysis Software Programs

Abstract

The particle size of four liposomal dispersions was estimated by quasi-elastic light scattering. The data were analyzed by three different software programs. Comparing the estimated values for the average particle size and the particle size distribution, some differences became obvious. First, it was observed that the average values obtained by different calculation methods were hardly comparable, since some systematic deviations occurred. Comparing the estimated particle size distributions, the Malvern Automeasure software was shown to produce overly smoothed, broad monomodal distributions. On the other hand, the results of both the CONTIN and the Brookhaven ISDA package agreed much better with the particle size distributions estimated by image analysis of electron microscopic photographs, revealing that all samples studied were bimodal, containing small liposomes with a rather constant diameter as well as much bigger aggregates that were gradually broken down during sonication. Concerning the ISDA software in detail, more accurate results were obtained by the NNLS-based histogram method compared to the exponential sampling method. Overall, CONTIN seemed to yield the most reliable distributions, since the smaller liposomes were detected even in the unsonicated sample.     

Macromolecular transport in heart valves. III. Experiment and theory for the size distribution of extracellular liposomes in hyperlipidemic rabbits

The heart valve leaflets of 29-day cholesterol-fed rabbits were examined by ultrarapid freezing without conventional chemical fixation/processing, followed by rotary shadow freeze-etching. This procedure images the leaflets' subendothelial extracellular matrix in extraordinary detail, and extracellular lipid liposomes, from 23 to 220 nm in diameter, clearly appear there. These liposomes are linked to matrix filaments and appear in clusters. Their size distribution shows 60.7% with diameters 23-69 nm, 31.7% between 70 and 119 nm, 7.3% between 120 and 169 nm, and 0.3% between 170 and 220 nm (superlarge) and suggests that smaller liposomes can fuse into larger ones. We couple our model from Part II of this series (Zeng Z, Yin Y, Jan KM, Rumschitzki DS. Am J Physiol Heart Circ Physiol 292: H2671-H2686, 2007) for lipid transport into the leaflet to the nucleation-polymerization model hierarchy for liposome formation proposed originally for aortic liposomes to predict liposome formation/growth in heart valves. Simulations show that the simplest such model cannot account for the observed size distribution. However, modifying this model by including liposome fusing/merging, using parameters determined from aortic liposomes, leads to predicted size distributions in excellent agreement with our valve data. Evolutions of both the liposome size distribution and total liposome mass suggest that fusing becomes significant only after 2 wk of high lumen cholesterol. Inclusion of phagocytosis by macrophages limits the otherwise monotonically increasing total liposome mass, while keeping the excellent fit of the liposome size distribution to the data.     

The role of cavitation in liposome formation

Liposome size is a vital parameter of many quantitative biophysical studies. Sonication, or exposure to ultrasound, is used widely to manufacture artificial liposomes, yet little is known about the mechanism by which liposomes are affected by ultrasound. Cavitation, or the oscillation of small gas bubbles in a pressure-varying field, has been shown to be responsible for many biophysical effects of ultrasound on cells. In this study, we correlate the presence and type of cavitation with a decrease in liposome size. Aqueous lipid suspensions surrounding a hydrophone were exposed to various intensities of ultrasound and hydrostatic pressures before measuring their size distribution with dynamic light scattering. As expected, increasing ultrasound intensity at atmospheric pressure decreased the average liposome diameter. The presence of collapse cavitation was manifested in the acoustic spectrum at high ultrasonic intensities. Increasing hydrostatic pressure was shown to inhibit the presence of collapse cavitation. Collapse cavitation, however, did not correlate with decreases in liposome size, as changes in size still occurred when collapse cavitation was inhibited either by lowering ultrasound intensity or by increasing static pressure. We propose a mechanism whereby stable cavitation, another type of cavitation present in sound fields, causes fluid shearing of liposomes and reduction of liposome size. A mathematical model was developed based on the Rayleigh-Plesset equation of bubble dynamics and principles of acoustic microstreaming to estimate the shear field magnitude around an oscillating bubble. This model predicts the ultrasound intensities and pressures needed to create shear fields sufficient to cause liposome size change, and correlates well with our experimental data.     

Size distribution of spontaneously formed liposomes by the alcohol injection method

A dynamic light scattering study of the size distribution of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) liposomes formed by the injection method is presented. By this method, an aliquot of methanol stock solution containing the surfactant is injected into water. The main aim of the present work was to determine under which conditions a monomodal and narrow size distribution could be obtained. The influence of several parameters on the size distribution was investigated. Firstly, we examined the influence of the POPC concentration in the initial stock methanol solution, when the POPC concentration in the final aqueous solution remains constant; secondly, the influence of POPC concentration in the aqueous phase, while the lipid concentration in the stock methanol remains constant. In both cases narrow monomodal size distributions of liposomes, centered between 40 and 70 nm, are obtained at low concentrations of POPC, in the stock methanol solution (相似文献   

Location of ubiquinone-10 (CoQ-10) in phospholipid vesicles

Egg yolk phosphatidylcholine (PC) liposomes were prepared by ultrasonic irradiation. At least 25 mol% of coenzyme Q-10 (CoQ-10) can be incorporated nonstoichiometrically into PC liposomes. Electron microscopy showed no visible influence of CoQ-10 on the membrane structure. Nuclear magnetic resonance spectra of sonicated PC liposomes containing CoQ-10 showed two peaks (3.82 and 3.98 ppm) due to CoQ-10 methoxyl protons and a 'high-field component' (1.52 and 1.58 ppm). The areas of these peaks were inversely related and influenced by the time of ultrasonic irradiation. After short sonication the low-field positions (3.98/1.58 ppm) are favoured, after long sonication the high-field positions (3.82/1.52 ppm). No gradual shift of the two peaks is observed. The 'critical' liposome diameter was found to be between 500 to 700 A. Lanthanide induced pseudocontact shift on CoQ-10 resonances ('high-field component' and methoxyl) does not lead to a split of the peaks and the difference between them remains constant. It is concluded that CoQ-10 is incorporated into the membrane core, beyond C-2 of the PC acyl chains, with two bilayer curvature-dependent resonance positions.     

Hematin- and peroxide-catalyzed peroxidation of phospholipid liposomes

The effect of hydroperoxides on hematin-catalyzed initiation and propagation of lipid peroxidation was examined utilizing soybean phosphatidylcholine liposomes as model membranes. Polarographic and spectrophotometric methods revealed a bimodal pseudocatalytic activity for hematin. A slow initiation phase of peroxidation was observed in the presence of low peroxide concentrations, whereas a fast propagative phase was observed at higher peroxide levels. Peroxide levels were manipulated enzymatically by the combination of phospholipase A2 and lipoxidase or by the direct addition of linoleic acid hydroperoxide, cumene hydroperoxide, or hydrogen peroxide. In addition, the effect of two different techniques for liposome preparation, i.e., sonication and extrusion, were compared on the basis of peroxidation kinetics. High pressure liquid chromatography analysis showed that sonicated liposomes contained higher levels of endogenous peroxides than the extruded ones. These sonicated liposomes also exhibited more rapid peroxidation following hematin addition. Extruded liposomes were more resistant to hematin-catalyzed peroxidation but became better substrates when exogenous hydroperoxides were added. All three peroxides reacted with hematin during which decomposition of peroxide and irreversible oxidation of hematin took place. Spectral analysis of hematin indicated that a higher oxidation state of hematin iron may be transiently formed during reaction with hydroperoxides and accounts for the propagation of lipid peroxidation when reactions proceed in the presence of soybean phosphatidylcholine liposomes. Of the three peroxides studied, linoleic acid hydroperoxide was most efficient in supporting hematin-catalyzed lipid peroxidation. The relevance of our findings is discussed in terms of the concentration dependence for lipid peroxides in determining the rate and extent of radical propagation chain reactions catalyzed by heme-iron catalysts such as hematin. Variation of hematin and linoleic hydroperoxide concentrations may provide an efficient and reproducible method for inducing and manipulating the rates and extent of lipid peroxidation through facilitation of the propagative phase of lipid peroxidation. In addition, we address a problem inherent to in vitro studies of heme-catalyzed lipid peroxidation where preparations of peroxide-free membranes should be of concern.     

Liposome filtration. Dependence on transition temperature

Liposomes formed by vortexing and passed through polycarbonate surface retention membranes showed appreciable differences in filtration behavior depending on the temperature of filtration relative to the liposome gel-liquid crystal transition temperature. Below transition, liposomes were filterable and size distributions could be determined; the cumulative volume distributions were log-normal. Above transition, liposomes were not filterable: smaller liposomes were formed until a limiting size was reached. These results suggest that liquid crystal liposome size distributions cannot be determined by filtration. This filtration behavior is a physical property of liposomes, related to the gel-liquid crystal transition, not previously reported. This property could be exploited as a new method for controlling liposome size distributions, but the implications for lipid membranes, including biological membranes, are general.     

Filter-extruded liposomes revisited: a study into size distributions and morphologies in relation to lipid-composition and process parameters

Filter-extrusion is a widely used technique for down-sizing of phospholipid vesicles. In order to gain a detailed insight into size and size distributions of filter-extruded vesicles composed of egg phosphatidyl-choline (with varying fractions of cholesterol) – in relation to extrusion-parameters (pore-size, number of filter passages, and flow-rate), flow field-flow fractionation in conjunction with multi-angle laser light scattering (AF4-MALLS, Wyatt Technology Corp., Santa Barbara, CA) was employed. Liposome size-distributions determined by AF4-MALLS were compared with those of dynamic light scattering and correlated with cryo-transmission electron microscopy and ³¹P-NMR-analysis of lamellarity. Both the mean size of liposome and the width of size distribution were found to decrease with sequential extrusion through smaller pore size filters, starting at a size range of ≈70–415?nm upon repeated extrusion through 400?nm pore-filters, eventually ending with a size range from ≈30 to 85?nm upon extrusion through 30?nm pore size filters. While for small pores sizes (50?nm), increased flow rates resulted in smaller vesicles, no significant influence of flow rate on mean vesicle size was seen with larger pores. Cholesterol at increasing mol fractions up to 0.45 yielded bigger vesicles (at identical process conditions). For a cholesterol mol fraction of 0.5 in combination with small filter pore size, a bimodal size distribution was seen indicating cholesterol micro-crystallites. Finally, a protocol is suggested to prepare large (～?300?nm) liposomes with rather narrow size distribution, based on the filter extrusion at defined flow-rates in combination with freeze-/thaw-cycling and bench-top centrifugation.     

Entrapment of ovalbumin into liposomes--factors affecting entrapment efficiency, liposome size, and zeta potential

Various amounts of Ovalbumin (OVA) were encapsulated into positively and negatively charged multilamellar liposomes, with the aim to investigate the entrapment efficiency in different buffers and to study their effects on the liposome size and zeta potential. Results showed that the entrapment efficiency of OVA in anionic liposomes was the same in 10 mM Phosphate Buffer (PB) as in Phosphate-Buffered Saline (PBS; PB + 0.15 M NaCl). Also, liposome size was approximately 1200 nm for all anionic liposomes incorporating OVA. The entrapment efficiency of OVA in cationic liposomes was highly dependent on ionic strength. The size of cationic liposomes was approximately 1200 nm in PBS, regardless of protein content, but increased with the amount of the incorporated protein in PB. Aggregation of cationic liposomes in PB was observed when the mass of the protein was 2.5 mg or greater. The zeta potential of anionic liposomes was negative and of cationic liposomes positive in the whole range of protein mass tested. These results show how different compositions of lipid and aqueous phases can be used to vary the entrapment efficiency, liposome size, and zeta potential--the factors that are of great importance for the use of liposomes as drug carriers.     

Formation of unilamellar liposomes from total polar lipid extracts of methanogens.

Unilamellar liposomes were formed by controlled detergent dialysis of mixed micelles consisting of acetone-insoluble total polar lipids extracted from various methanogens and the detergent n-octyl-beta-D-glucopyranoside. The final liposome populations were studied by dynamic light scattering and electron microscopy. Unilamellar liposomes with mean diameters smaller than 100 nm were obtained with lipid extracts of Methanococcus voltae, Methanosarcina mazei, Methanosaeta concilii, and Methanococcus jannaschii (grown at 50 degrees C), whereas larger (greater than 100-nm) unilamellar liposomes were obtained with lipid extracts of M. jannaschii grown at 65 degrees C. These liposomes were shown to be closed intact vesicles capable of retaining entrapped [14C]sucrose for extended periods of time. With the exception of Methanospirillum hungatei liposomes, all size distributions of the different liposome populations were fairly homogeneous.     

Assessing the accuracy of routine photon correlation spectroscopy analysis of heterogeneous size distributions

The aim of the current study was to investigate the ability of a fixed-angle routine photon correlation spectrometer (PCS) to resolve bimodal size distributions. The focus was on dispersions consisting of a majority of smaller and a minority of bigger particles. Monodisperse latex beads of sizes from 21 to 269 nm were measured first as single-size dispersions and then with various binary blends. For single-size dispersions, the mean diameters obtained were as indicated by the manufacturer, except for 21- and 34-nm particles, which were somewhat smaller. PCS analysis of blends of 21+102-nm and 34+102-nm particles resulted in bimodal distributions with particle diameters of the 2 peaks in the expected magnitude down to critical blending ratios of 0.002% and 0.08% of bigger particles, respectively. At these ratios, PCS results became inconsistent, and an increased number of monomodal results and/or high residuals were seen. For 21+102-nm blends, at even smaller ratios (0.001%), more consistent results were obtained again with predominantly monomodal distributions in the size range of the smaller particles (ie, the bigger particles were neglected). PCS analysis of blends of 21+269-nm particles yielded bimodal distributions with diameters within the expected magnitude as long as the content of bigger particles did not exceed 0.005%. Above this ratio, predominantly monomodal results with mean diameters in the magnitude of the bigger particles were obtained (ie, the smaller particles were neglected). In conclusion, a routine PCS instrument can resolve bimodal size distributions of colloidal dispersions only at certain ratios of the 2 subpopulations. Both low and high ratios lead to 1 of the 2 subpopulations being neglected. Phone: Not Forwarded; Fax: Not Forwarded;     

Transmission electron microscopy observations of sonication-induced changes in liposome structure.

Freeze-fracture Transmission Electron Microscopy (TEM) was used to show that sonication does not homogeneously disrupt liposome dispersions to form vesicles. Many large multilamellar particles remain intact after sonication and small, unilamellar vesicles are present after just 10 s of exposure. Small vesicles appear to coexist with large liposomes even before sonication. The mechanical and thermal stresses induced by sonication nucleate liquid crystalline defects in the liposomes, including edge and screw dislocations and +1 disclinations, but the Dupin cyclide structure of unsonicated liposomes is still recognizable in the larger particles after sonication. Defects in the bilayer organization may provide pathways for enhanced transport within the liposome, as well as from the liposome interior to exterior. A screw dislocation-catalyzed mechanism of liposome-to-vesicle conversion is proposed that accounts for the TEM observations.     

Preparation of submicron liposomes exhibiting efficient entrapment of drugs by freeze-drying water-in-oil emulsions

A novel liposome preparation method is described as freeze-drying of water-in-oil emulsions containing sucrose in the aqueous phase (W) and phospholipids and poly(ethylene glycol)₁₅₀₀ (PEG) in the oil phase (O). The water-in-oil emulsions were prepared by sonication and then lyophilized to obtain dry products. Upon rehydration, the dry products formed liposomes with a size smaller than 200 nm and an encapsulation efficiency (EE) higher than 60% for model drugs. The presence of lyoprotectant and PEG was found to be a prerequisite for the formation of liposomes with desirable properties, such as a small particle size and high EE. The lyophilates were stable and could be rehydrated to form liposomes without any change in size or EE even after a storage period of 6 months. Also, the lipophilic drug-containing FWE liposomes were stable and could be stored for at least 6 months although the liposomes containing hydrophilic drugs showed significant leakage. Based on the vesicle size and EEs of the model drugs, as well as the scanning electron micrograph (SEM) and small angle X-ray scattering (SAXS) pattern of the lyophilates, a possible mechanism for the liposome formation is proposed.     

Study and formation of vesicle systems with low polydispersity index by ultrasound method

The formation of liposomes with low polydispersity index by application of ultrasounds was investigated considering methodology specifications such as sonication time and sonication power. Phosphatidylcholine (PC) liposomes were formed by the evaporation–hydration method. The vesicles were sonicated using several sonication conditions. The liposomes were then characterized by dynamic light scattering (DLS) and freeze-fracture electron microscopy (FFEM). Correlation functions from DLS were treated by cumulants method and GENDIST to obtain the mean radius and polydispersity index. These calculations allowed to fix an optimal sonication time (3000 s) and a useful interval of ultrasound power between 39 and 91 W. DLS and FFEM results confirmed that vesicle size, lamellarity and the polydispersity index decreased with the increase of sonication power. Thus, we propose a systematic method to form liposomes in which the physical characteristics of the vesicles may be controlled as a function of sonication time and power.     

Selective partitioning of cholesterol and a model drug into liposomes of varying size

The resistance of a lipid bilayer with respect to a bending deformation generally depends on the presence of membrane additives such as sterols, cosurfactants, peptides, and drugs. As a consequence, the partitioning of membrane additives into liposomes becomes selective with respect to liposome size; i.e., membrane rigidification depletes the membrane additives in the smaller (more strongly curved) liposomes. We have measured this liposome size-selective partitioning for two membrane additives - cholesterol and the porphyrin-based photosensitizer temoporfin - using asymmetrical flow field-flow fractionation (AF4) of liposomes and radioactive labeling of the membrane additive and lipid. The method yields either the molar cholesterol-to-lipid or the temoporfin-to-lipid ratio as a function of liposome size, from which we calculate the corresponding change of the membrane bending stiffness. For small unilamellar fluid-phase liposomes composed of palmitoyloleoylphosphatidylcholine (POPC) and palmitoyloleoylphosphatidylglycerol (POPG), we find that cholesterol rigidifies the host membrane in a manner consistent with previously reported measurements. In contrast, temoporfin softens this membrane. Partitioning results for gel-phase liposomes composed of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) are also curvature-sensitive but cannot be interpreted on the basis of the bending stiffness alone.     

Differential effects of prion particle size on infectivity in vivo and in vitro

The conversion of cellular prion protein to the disease-associated isoform (PrP^Sc) has been suggested to follow a mechanism of seeded aggregation. Here, we show that fragmentation of PrP^Sc aggregates by sonication increases converting activity in cell culture in a way similar to in vitro conversion assays. In contrast, under the same conditions the infectious titer of sonicated samples in vivo was reduced. We modified the size distribution of PrP^Sc by adsorption to nitrocellulose, which resulted in a reduction of the infectious titer in non-sonicated samples and an increase in sonicated samples. Our results indicate that NC-adsorption can (i) block some active sites of PrP^Sc aggregates and (ii) reduce the rate of clearance from the brain. For large particles with low clearance the effect of NC-particles on the number of available active sites may dominate, whereas for smaller particles (i.e. sonicated samples) the effect of NC-adsorption on clearance dominates resulting in increased infectivity.     

Preparation of DPPC liposomes using probe-tip sonication: Investigating intrinsic factors affecting temperature phase transitions

Liposomes are an important tool and have gained much attention for their promise as an effective means of delivering small therapeutic compounds to targeted sites. In an effort to establish an effective method to produce liposomes from the lipid, dipalmitoyl-phosphatidylcholine or DPPC, we have found important aspects that must be taken into consideration. Here, we used probe-tip sonication to prepare liposomes on a batch scale. During this process we uncovered interesting steps in their preparation that altered the thermodynamic properties and phase transitions of the resulting liposome mixtures. Using differential scanning calorimetry to assess this we found that increasing the sonication time had the most dramatic effect on our sample, producing almost an entirely separate phase transition relative to the main phase transition. This result is consistent with reports from the current literature. We also highlight a smaller transition, which we attribute to traces of unincorporated lipid that seems to gradually disappear as the total lipid concentration decreases. Overall, sonication is an effective means of producing liposomes, but we cannot assert this method is optimal in producing them with precise physical properties. Here we highlight the physical effects at play during this process.     

The effect of bilayer order and fluidity on detergent-induced liposome fusion

Surfactants induce fusion (or increase in size) of sonicated liposomes. This phenomenon is enhanced by cholesterol and inhibited by the intrinsic polypeptide gramicidin A. By comparison with previous physical studies we conclude that liposome 'fusion' is facilitated when both fluidity and static order of the bilayer are high.