Diameter Of Liposomes Research Articles

Flocculation behavior of egg phosphatidylcholine liposomes caused by Ca 2+ ions

The interaction between bilayer phospholipid membranes has been studied through flocculation phenomena of phospholipid liposomes. Unlike liposomes composed of other phospholipids, liposomes made of egg phosphatidyl-choline (PC) have a strong resistance against aggregation. This phenomenon can be attributed to the hygroscopic nature of the choline group, i.e. the repulsion force that is the so-called “hydration force” or the “steric force” with hydration is the origin of the phenomenon. By changing the diameter of the PC liposomes, we observed a different flocculation behavior of the liposomes when the electrostatic surface potential of the liposomes is zero. For diameters above 320 nm, a weak flocculation occurred, but liposomes with diameters below 200 nm are stable as colloidal dispersions. This diameter dependency of flocculation has been explained by Derjaguin-Landau-Verwey-Overbeek (DLVO) theory together with hydration forces.

Complexation of amphotericin B with egg phosphatidylcholine liposomes

The complexation and physical characteristics of egg phosphatidylcholine (PC) liposome containing amphotericin B (AmB) were investigated through circular dichroism (CD) spectra, the size distribution, the turbidity change, and the calcein release. CD spectra of AmB-containing egg PC mixture exhibited a positive peak around 330 nm indicative of complexation of AmB and four negative peaks. The positive peak increased up to 2.2 millidegree/μg AmB as AmB contents increased up to 12% (w/w), suggesting that AmB-phospholipid complexation was promoted by the antibiotics. The effective diameter of liposomes by dynamic light scattering decreased from 450 nm to 220 nm as the amount of AmB in liposomes increased from 0 to 30% (w/w). The complexation may be responsible for the reduction in size. On the other hand, at around 1 mM deoxycholate (DOC), the relative turbidities of 5 and 10% (w/w) AmB-containing liposome suspension were less than 1 probably due to the solubilization of the complex, while those of pure PC liposome suspension were larger than 1 at the same concentration. Deoxycholate-induced release of calcein entrapped in AmB-containing liposomes was lower than those of pure egg PC liposomes, indicating the intercalation of the drug into the bilayers. Therefore, it is concluded that in AmB/egg PC/water system, AmB-phospholipid complex coexists with AmB-containing liposomes.

Generation of contrast-carrying liposomes of defined size with a new continuous high pressure extrusion method

A novel continuous high pressure extrusion method was evaluated for the generation of radiopaque and paramagnetic liposomes. The magnetic resonance contrast agent gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA) as well as the X-ray contrast agent iopromide were used as water-soluble model substances for liposomal encapsulation. The continuous process, which is introduced here, allows the fast and efficient extrusion of large batches of liposomal preparations with maximum flow rates of 500 ml/min. Applying high pressures up to 10.5 MPa, MLV prepared by the film method were sequentially extruded through polycarbonate membranes of decreasing pore size. Encapsulation efficiency was found to be dependent on lipid composition and concentration, amount of contrast agent in the preparation as well as choice of final pore size for extrusion. Application of freeze-thaw cycles markedly improved the entrapment of iopromide, whereas for Gd-DTPA freeze-thaw surprisingly turned out to have only minor effects. Entrapment values rose with increasing lipid concentration and fell sharply with increasing solute concentration. Mean liposome diameters could be varied using polycarbonate membranes of differing pore sizes. Smaller final pore sizes led to vesicle populations with smaller mean diameters and lower encapsulation efficiencies. Excellent maximum encapsulation efficiencies of more than 50% for iopromide and over 60% for Gd-DTPA were obtained for vesicles with mean diameters of around 100 nm, as determined by photon correlation spectroscopy (PCS) and confirmed by negative-staining electron microscopy. Employing medium contrast (100 mg/g iodine and 180μmol/g Gd) and lipid concentrations (150 mg/g), entrapment values as high as 40% for the X-ray and 50% for the paramagnetic contrast agent could still be achieved. Best results were obtained using a lipid mixture of soy phosphatidylcholine (SPC), cholesterol (Chol) and soy phosphatidylglycerol (SPG) in a molar ratio of 6:3:1 for iopromide and SPC, Chol 7:3 for Gd-DTPA. Liposomal preparations remained stable upon storage at 2–8°C for 6 months. The new continuous high pressure extrusion method proved to be suitable for the generation of large volumes of stable, contrast-carrying liposomes with outstanding encapsulation efficiencies.

Direct Measurement of trans-Golgi pH in Living Cells and Regulation by Second Messengers

In the endocytic compartment, an acidic pH plays a key role in receptor and ligand sorting, vesicular transport, and protein degradation. In the secretory compartment, indirect estimates of trans-Golgi pH based on partitioning of weak bases and following viral infection suggest a mildly acidic pH of > 6.0. We developed a liposome microinjection method to introduce fluorescent indicators into the aqueous compartment of trans-Golgi in living cells. In the presence of ATP and at 37 degrees C, 70-nm diameter liposomes delivered their fluid-phase contents selectively into the trans-Golgi compartment as assessed by colocalization with the trans-Golgi stain N-[6-[(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino]caproyl]-sphingosine (C6-NBD-ceramide). Liposome fusion was ATP- and temperature-dependent and blocked by N-ethylmaleimide but not by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S). trans-Golgi pH in skin fibroblasts was 6.17 +/- 0.02 (S.E., n = 174) as measured by ratio imaging confocal microscopy using fluorescein and rhodamine-based indicators and an in vivo calibration procedure. trans-Golgi pH increased to 6.8 +/- 0.1 by cAMP agonists and to 6.5 +/- 0.1 by protein kinase C activation. These results provide the first direct measurement of trans-Golgi pH in living cells and demonstrate pH regulation by second messengers.

Preparation and evaluation of proliposomes containing propranolol hydrochloride.

Proliposomes were prepared by the penetration of a methanol-chloroform solution of propranolol hydrochloride (PH) and lecithin into microporous sorbitol, with subsequent vacuum drying. They were characterized for surface morphology and flowability, and following the conversion to liposomes upon hydration, size distribution, drug content and in vitro drug release of the reconstituted liposomes were examined. The porous structure of sorbitol was maintained in the proliposomes, affording the proliposomes good flowability at lecithin-to-sorbitol ratios (w/w) of not more than 0.2. Multilamellar liposomes were reconstituted spontaneously from the proliposomes upon hydration. The mean diameter of the resultant liposomes was highly dependent on the PH-to-lecithin ratio, but less dependent on the lecithin-to-sorbitol ratio and sorbitol particle size (105-710 microns). Entrapment efficiency of PH in liposomes showed a maximum 10% at PH-to-lecithin ratio < 0.5 and a lecithin-to-sorbitol ratio > 0.1. Sustained release of propranolol from the proliposomes was achieved when the lecithin-to-PH ratio was > 2, and the lecithin-to-sorbitol ratio was in the range examined (0.067-0.2). In conclusion, granular proliposomes of PH with good flowability and sustained release characteristics could be prepared by controlling the drug/lecithin/sorbitol ratio and sorbitol particle size. PH proliposomes can be potential candidates for the sustained drug delivery of propranolol when applied directly onto the mucosal membranes.

Synthesis of Magnetic Liposomes

Giant magnetic liposomes (diameter up to 40 μm) have been synthesized. They are constituted by an aqueous magnetic fluid at pH 7 encapsulated in phosholipids vesicles. When they are submitted to a magnetic field, even of small intensity (0.01 T), these liposomes become elongated in the direction of the field; the study of this deformation may be a way to measure the elasticity modulus of the membrane.

Spontaneous transfer of viral protein from membrane of influenza virus-infected cells to liposomes is dependent on the diameter of receiver.

Spontaneous transfer of protein from either influenza virus-infected or uninfected cells to dimyristoylphosphatidylcholine (DMPC) liposomes was examined. The amount of transferred protein of liposomes incubated with influenza virus-infected cells (I-lipo) was more than that of liposomes incubated with uninfected cells (U-lipo). The ratio of the amount of spontaneous transferred protein of I-lipo to that of U-lipo increased in proportion to the diameter of liposomes except for small unilamellar vesicles.

5419893 Aminopolycarboxylic acids and derivatives thereof for magnetic resonance imaging

A variety of factors were evaluated in order to establish the characteristics of the liposomes obtained with a high-pressure homogenizer (Microfluidizer 110S). The experimental mean diameter of the liposomes and the width of their size distribution were correlated to surfaces calculated from the responses obtained by the combination of two groups of factors. First, the inlet pressure of the homogenizer, the times that the samples were processed (cycles) and the bulk ionic strength. Second, the phospholipid (P) and ethanol concentrations. The variation of the entrapped liposome volume upon the pressure and cycles was also studied. All the calculated surfaces are statistically significant and have a low S.E. of estimate. Mean liposome diameter decreases with increasing inlet pressure, number of cycles and ethanol concentration, and increases raising ionic strength. No P concentration effect was observed. The variation of the entrapped volume of liposomes upon the cycles and pressure has a similar behavior to the diameter. Within the studied variable range, microfluidization does not increase the P oxidation index, nor does it significantly alter the P concentration.

Changes in Physical Properties of Soybean Phosphatidylcholine-Liposome during Autoxidation

Effects of temperature and change in physical properties, such as membrane fluidity and size of liposome were investigated during the autoxidation of liposomes prepared from soybean PC.The oxidation rates of soybean PC liposomes and soybean PC solution markedly differed between 30 and 35°C. Unoxidized PC liposome diameter decreased from 120nm to 30nm by oxidation. Unoxidized liposome membrane fluidity increased somewhat with temperature while that of the oxidized membrane did so remarkably.Linoleic acid content of the soybean PC liposomes decreased considerably by oxidation. The oxidation products of unsaturated phospholipid were thus considered to alter the physical properties of oxidized liposomes.

Potentiation of ricin A immunotoxin by monoclonal antibody targeted monensin containing small unilamellar vesicles

The carboxylic ionophore monensin could be successfully entrapped in small unilamellar vesicles made by the extruder method. Monensin liposomes of size range 100–150 nm were more potent in potentiating ricin A immunotoxin activity in vitro as compared to monensin liposomes of diameter 500 nm or more. These liposomes were further successfully linked to tumor specific monoclonal antibodies with full retention of immunoreactivity. Monoclonal antibody targeted monensin liposomes were 100 times more potent than monensin liposomes in potentiating the activity of ricin A immunotoxins against various tumor cell lines in vitro.

Formation and vesiculation of biomembranes during spaceflight

Shuttle flight, sounding rocket flight, and parabolic flight experiments demonstrate the formation of bilayer membrane vesicles (liposomes) in reduced gravity, following the dilution of detergent from detergent-phospholipid mixed micelles. The reduction in detergent concentration initiates assembly of bilayer membrane sheets, which are sensitive to solution distrubances. An increase in distrubances by forced dilution results in small diameter liposomes (< 150 nm), in both ground and flight samples. In the absence of forced dilution, liposomes remain small at 1-g, but exhibit much larger diameters at 0-g (1000–2000 nm). Our spaceflight data reveal that membrane assembly and vesiculation are strongly influenced by gravity-induced solution disturbances (e.g., convection currents), which limit vesicle diameter.

Unusual low proton permeability of liposomes prepared from the endogenous myelin lipids.

In contrast with most other lipid substrates, in this article we show that liposomes prepared from the total myelin lipids exhibited a negligible proton permeability. Neither the generation of valinomycin-induced potassium diffusion potentials as high as -177 mV nor the imposition of large pH gradients (up to three units) was able to produce a substantial flux of protons through liposomal membranes, as determined by the distribution of [14C]-methylamine, or the changes in the fluorescence of the probes 9-aminoacridine, acridine orange, and pyranine. The presence of cations (Na+, K+, Ca2+) did not alter this behavior. Voltage clamping did not increase the transmembrane delta pH-driven proton permeability. However, liposome diameter was found to be critical because small unilamellar vesicles displayed a much higher proton permeability than large unilamellar or multilamellar vesicles. This abnormally low proton permeability is interpreted by virtue of the characteristic biochemical composition of myelin lipid matrix, with a high content of cholesterol and sphingolipids and a very low level of free fatty acids. These results could be important for elucidating the role of myelin in the regulation of pH in the brain. In addition, the myelin lipid extract could be useful for reconstituting proteins that participate in the transport of H+ through the membrane.

The active trapping of doxorubicin in liposomes by pH gradient: photon correlation spectroscopy and fluorimetric study

Changes in liposome diameter in response to a pH gradient during uptake of doxorubicin were studied. After an initial increase liposomes underwent shrinkage which was dependent on lipid composition and temperature of incubation. Changes in fluorescence undergone by the drug during the active trapping process were also monitored. The observed self-quenching of doxorubicin was consistent with the kinetics of drug entrapement within the internal aqueous compartment of the vesicles except when incubation was carried out at 37°C in the presence of cholesterol.

Use of two-sided bifunctional liposomes in the study of a hypothalamic Na,K-ATPase inhibitor.

Two-sided bifunctional (ATP-filled) Na,K-ATPase liposomes have been developed as a result of knowledge about the average liposome diameter and volume, the liposome size distribution, the average number of Na,K-ATPase molecules reconstituted per liposome, and the orientation of the reconstituted Na,K-ATPase molecules. The addition of 5-10 microM external 86Rb to the liposomes containing 50 mM encapsulated ATP provoked an impressive 86Rb accumulation by the cell-like-oriented pumps. The successive addition of external ATP activated the pumps in the reversed orientation of the same liposome, leading to total extrusion of the previously accumulated 86Rb. An inhibitor extracted from bovine hypothalamus (hypothalamic inhibitory factor) inhibited the cell-like-oriented population, i.e., acted like an extracellular inhibitor at 30 nM. Conversely, at 75 nM, the reversed pump population was also blocked, indicating that the inhibitor either transversed the membrane or was able to act also at the intracellular enzyme side at a higher concentration. Thus, the side of action as well as the membrane permeability of structurally unknown endogenous Na,K-ATPase inhibitors can be determined simultaneously in a single suspension of two-sided bifunctional Na,K-ATPase liposomes.

Partition of sodium dodecyl sulfate into stratum corneum lipid liposomes.

Synthetic detergents produce deleterious effects on human skin as the result of being taken up by the stratum corneum (SC). The present study aimed to determine to what extent a typical detergent enters the SC lipid lamellae, and what effect this might have on the physical properties of the lipids. These effects were studied in large unilamellar liposomes prepared from SC lipids (50% by weight of epidermal ceramides, 28% cholesterol, 17% free fatty acids, and 5% cholesteryl sulfate) by extrusion through successive polycarbonate filters of decreasing pore size, finally 400 nm. Freeze-fracture electron microscopy and light-scattering particle size analysis indicated a uniform liposome diameter averaging 230 nm. Partitioning of sodium dodecyl sulfate (SDS) into the lipid phase from aqueous buffer solutions was measured using the SC lipid liposomes and [U-14C]SDS. The partition coefficient was 416, 450, and 588 at pH 8.5 and 524, 507, and 807 at pH 7 for three different concentrations (0.1%, 0.02%, and 0.004%) of SDS. This high degree of partitioning into the liposomes is consistent with the high level of SDS partitioning seen in full SC. At the maximum, the SDS represented 18% of the liposomal lipids. Preparation of stable liposomes from SC lipids to which 10% or 20% of SDS had been added confirmed the ability of the liposomes to survive these high concentrations of surfactant. The permeability of the liposomes was enhanced as a result of SDS partitioning into the bilayers, as measured by the increased release of trapped [U-14C]glucose from these vesicles, and by their increased permeability to water in osmotic shock experiments.(ABSTRACT TRUNCATED AT 250 WORDS)

Leakage of a trapped fluorescent marker from liposomes: Effects of eutectic crystallization of NaCl and internal freezing

Leakage of trapped carboxyfluorescein from dl-α-dipalmitoylphosphatidylcholine multilamellar liposomes (diameter 1–2 μm) in NaCl solutions was measured after rapid freezing to temperatures between − 15 and − 55 °C. Leakage was low after freezing between − 15 and − 35 °C, but increased steeply between − 35 and − 45 °C. From DSC measurements it was found that the increase in leakage was associated with two crystallization processes: Eutectic crystallization of NaCl and freezing of undercooled solvent trapped in the interior of the liposomes (“internal freezing”). Damage caused by the former process could effectively be prevented by small amounts of trehalose (1% ⩽ w ⩽ 1.5%). Trehalose in these concentration also decreased damage due to internal freezing, but to a minor degree. In addition to these damaging transitions, a time-dependent process was found to cause leakage from the liposomes at − 25 °C. The association between leakage and thermal activity suggests that DSC supplements cryomicroscopy and leakage measurements in the characterization of cryostability of liposomes.

Effect of liposomes on percutaneous penetration of lipophilic materials

Liposomes were prepared of two lipophilic drugs, dl-α-tocopherol nicotinate (TN) and the anti-inflammatory substance, L440. In the case of dl-α-tocopherol nicotinate liposomes, oleoyl-hydrolyzed animal protein (OHAP) was added in order to control the vesicle size. The skin penetration ability of both drugs from liposomal gels into human stratum corneum was determined in vivo by a stripping method and compared with conventional galenical formulations. The anti-inflammatory effect of the released L440 was examined in the ear edema model in mice. The penetration tests showed significantly higher absorption rates for both drugs after application of the liposomal preparations in comparison to other topical formulations. However, only a slight dependence of the extent of drug permeation into the stratum corneum on liposome diameter was observed. The pharmacological effect of L440 was investigated in the mouse ear edema test (Young et al., J. Invest. Dermatol., 80 (1982) 48–52; and J. Invest. Dermatol., 82 (1984) 367–371). No difference was found between the liposome preparation and an o/w base, whereas a w/o base yielded only a 50% reduction in edema.

Role of liposome size and RES blockade in controlling biodistribution and tumor uptake of GM1-containing liposomes

We have examined the effect of liposome size on liposome circulation time in the blood. Liposomes composed of phosphatidylcholine, cholesterol and ganglioside GM1 were prepared in the various size range. Optimal circulation activity (55% injected dose at 4 h post injection) of GM1-containing liposomes, which correlated with a relatively high uptake of liposomes by EMT6 tumor in mouse, was obtained with a size range of 70 to 200 nm in diameter. Increasing the diameter of liposome to greater than 200 nm resulted in an enhancement of the spleen uptake and decrease of the blood level. For liposomes with a diameter of less than 70 nm, 70% of the injected dose were taken up by the liver, presumably by the parenchymal cells. In contrast, the biodistribution of phosphatidylserine-containing liposomes was relatively insensitive to changes in liposome size; most of the injected dose was found in the liver. The effect of RES blockade on the circulation time of large (d > 300nm), GM1-containing liposomes was also studied. Dextran sulfate 500, a commonly used blockade reagent for Kupffer cells, bad no effect. On the other hand, preinjection of a large dose of liposomes with a diameter greater than 500 nm showed variable results depending on the lipid composition of the blocking liposomes. Preinjection of liposomes containing GM1, phosphatidylinositol or (N-polycthyleneglycol) phosphatidylethanolamine effectively reduced the spleen uptake of the large GM1-containing liposomes, whereas liposomes containing phosphatidic acid showed no effect. These results indicate that only spleen homing liposomes can be used as a blocking reagent to prolong the circulation time of the large GM1-containing liposomes.

Light microscopic localization of silver-enhanced liposome-entrapped colloidal gold in mouse tissues

Silver-enhanced liposome-entrapped colloidal gold was developed for light microscopic localization of liposomes. Preparation of colloidal gold entrapped in liposomes was achieved by a modified method of Hong, et al. (1983) Biochim. Biophys. Acta 732, 320–323). In this report, a gold chloride/citrate solution of low pH (3.4) was used to inhibit the formation of gold granules during the liposome preparation. The diameter of most liposomes ranged from 80 to 100 nm. Following liposome preparation, the pH was adjusted to 6, and the temperature increased to 55°C. The majority of the liposomes contained one to three gold particles. Liposomes were injected into mice via tail vein; 24 h later, tissues were collected. Sections were processed for silver enhancement of the gold particles and examined by light microscopy. Silver-enhanced gold particles were clearly observed in both liver and implanted tumor. Localization was confirmed by electron and fluorescence microscopy. Thus, we have shown that silver enhancement of colloidal gold liposomes is a direct and sensitive method for tracing the fate of liposomes in vivo, providing minimal background interference and a good definition of various cell types.

Large liposomes containing ganglioside GM 1 accumulate effectively in spleen

Large liposomes, with a composition of egg phosphatidylcholine, cholesterol and ganglioside GM 1, prepared by an extrusion method, were injected intravenously into mice. After 24 h, up to 50% of injected dose was accumulated in spleen compared with about 15% in spleen for liposomes containing no GM 1. The effect of GM 1 on spleen accumulation of liposomes was liposome size dependent. Only relatively large liposomes ( d > 300nm) showed high accumulation; smaller liposomes were progressively less accumulated. The spleen accumulation increased with increasing injection dose of the liposomes. It was noted that the enhanced uptake by spleen was accompanied by a decrease in the liver uptake, but the total uptake of liposomes by liver and spleen was not dependent on the diameter of liposome or the presence of the ganglioside GM 1. Autoradiographs of fixed and sectioned spleen using 125I-labeled tyraminylinulin as a content marker for the liposomes, showed that liposomes localized at the reticular meshwork of the red pulp. These results suggest that larger liposomes containing GM 1 are filtered by the spleen during the circulation in blood. The smaller ones with a mean diameter of less than 100 nm are not retained by the filter. The function of GM 1 is to prevent liposomes from a rapid uptake by the liver so that liposomes may circulate through the spleen and be filtered. These results, together with the observation that the liposome-entrapped proteins were degraded by the spleen, suggest the potential use of these liiposomes for specific drug delivery to the spleen.