Reverse-phase Evaporation Vesicles Research Articles

Preparation, characterization, therapeutic efficacy and toxicity of liposomes, containing the antitumour drug cis-dichlorodiamineplatinum(II).

Cis-dichlorodiamineplatinum(II) (cis-DDP) was encapsulated in reverse phase evaporation vesicles (REV, 0.6 mg cis-DDP/ml lipid solution) and multilayered liposomes (MLV, 0.3 mg cis-DDP/ml lipid solution) with different cholesterol content. The identity of cis-DDP in free and encapsulated form was checked by various techniques. Particle size, homogeneity of liposomes and distribution of cis-DDP in REVs were shown by electron microscopy. The examination of entrapped cis-DDP in REVs relating to buffer and serum stability, in vitro and in vivo antitumour activity and nephrotoxicity proved that all points are strongly influenced by the cholesterol (CH) content. Enclosed cis-DDP in phosphatidyl (PC)-REV has the same, and in PC: CH-REV, a lower effect in vitro and in vivo compared to treatment with the free drug. Irrespective of the application of tumour cells and substance (i.v., i.p.) in optimal therapeutic doses, an equal increase in life-span (ILS) was registered with the free drug and with PC-cis-DDP-REV, while cis-DDP in PC: CH-REV had a significantly reduced effectiveness. Liposomal encapsulation of cis-DDP also influenced body weight change and leucocyte counts. The blood urea nitrogen (BUN) level, as an indicator of renal toxicity, was only moderately increased after very high doses of cis-DDP (24 mg/kg) in PC: CH-REV.

Influence of lipid membranes on the conformational transitions of nucleic acids.

The conformational transitions of nucleic acids which were enclosed in reverse phase evaporation vesicles (REV) were studied by thermal denaturation with optical recording. Cloned fragments of double-stranded DNA containing 179 base pairs and 187 base pairs, respectively, and polyA.polyU were enclosed in REV with a yield up to every vesicle containing 50 nucleic acid molecules. With the 179 base pairs DNA enclosed in the vesicle from egg lecithin two well resolved helix-coil transitions could be measured; one is very similar in the midpoint-temperature Tm and halfwidth delta T1/2 to the transition of the free nucleic acid, and the other transition occurs stabilized at a 3.5 degrees C higher Tm-value and with a broader delta T1/2, 2.7 degrees C instead of 0.6 degree C. Both transitions are from nucleic acids inside the vesicles. Varying the surface charge of the lipid membrane by adding the negatively charged phosphatidylserine or phosphatidylglycerol, an optimum in the yield of enclosure and a maximum in the increase in Tm (4.5 degrees C) and delta T1/2 (5.5 degrees C instead of 1.0 degrees C) was obtained at 20% phosphatidylserine or phosphatidylglycerol. In vesicles from pure negatively charged lipids no second population of nucleic acids was observed. Qualitatively, similar effects were observed with polyA.polyU. Stabilization and broadening of the second transition is higher for nucleic acids inside vesicles from lipids with unsaturated fatty acids, as dioleoyl-phosphatidylcholine, than with saturated fatty acids, dipalmitoyl-phosphatidylcholine. Stabilization and broadening decrease with increasing ionic strength, whereas the relative contributions of both transitions to the total hypochromicity remain unchanged; the second transition coincides with the first at 90 mM Na+. From the experimental results it was concluded that the interaction of nucleic acids and lipid membranes is mainly of electrostatic nature. The nucleic acids exist inside the vesicles in two populations, one behaving like nucleic acid free in solution and one influenced by the contact with the membrane. All results are in accordance with a model in which the interaction between the nucleic acid and the membrane is in competition with the dipole-dipole interaction inside the membrane surface.

Potato spindle tube̊r viroid (PSTV) RNA oligomers of (+) and (−) polarity are synthesized in potato protoplasts after liposome-mediated infection with PSTV

Potato protoplasts prepared from suspension culture cells were inoculated with potato spindle tuber viroid (PSTV) and with cloned PSTVcDNA, respectively, encapsulated in reverse-phase evaporation vesicle (REV) liposomes. PSTV-infection was achieved by incubation of 10 6 protoplasts with 100 ng of encapsulated PSTV. The replication of PSTV in protoplasts was analyzed by monitoring the synthesis of PSTV(+) and (−)RNA by Northern blot hybridization. In addition to monomeric circular PSTV(+)RNA, i.e. the viroid proper as the main product of replication, linear oligomers of PSTV(+) and (−)RNA could be detected in infected protoplasts. Time course experiments revealed that the synthesis of oligomeric linear PSTV(−)RNA preceded that of the accumulating circular PSTV(+)RNA, suggesting that the oligomeric PSTV(−)RNA molecules represent the transient linear intermediates of replication. A normal PSTV replication and accumulation was also initiated, when liposomes were used to deliver cloned double-stranded DNA containing an infectious multimeric PSTVcDNA-sequence to potato protoplasts.

42] Liposome encapsulation of muramyl peptides for activation of macrophage cytotoxic properties

Publisher Summary This chapter describes a microcytoxicity assay that measures the tumoricidal activity of macrophages activated by liposome-encapsulated muramyl peptides against tumor target cells prelabeled with [ 125 I]iododeoxyuridine ([ 125 I]IUdR). It discusses the advantages and disadvantages of hydrophilic and lipophilic muramyl peptide derivatives for the activation of macrophage cytotoxic properties and the techniques employed to generate appropriate phospholipid vesicles for achieving macrophage activation. The procedure to assess macrophage activation is also presented in the chapter. The activation of macrophages by liposomes containing muramyl peptides can be achieved using three different types of liposomes—namely, (1) multilamellar vesicles (MLVs), small unilamellar vesicles (SUVs), and large unilamellar/oligolamellar vesicles produced by reverse-phase evaporation (REVs). The standard method for generating MLVs involves hydrating a dry lipid film on a glass test tube or a lyophilized phospholipid mixture by vortex mixing above the gel-to-liquid crystalline transition temperature of the lipid. An extensive sonication of MLVs results in the formation of smaller unilamellar vesicles (SUVs). The reverse-phase evaporation vesicles (REVs) are formed from water-in-oil emulsions of phospholipids and buffer in an excess organic phase, followed by removal of the organic phase under reduced pressure.

Studies on the uptake mechanism of liposomes by perfused rat liver. I. An investigation of effluent profiles with perfusate containing no blood component.

Uptake of liposomes by perfused rat liver was examined, and the effluent profiles are discussed. Reverse-phase evaporation vesicles (REV, about 0.1-0.2 μm in diameter) were able to pass through the liver without any interaction or interference and there was little uptake by the liver during single perfusion with phosphate-buffered saline. The transit time of REV was shorter than that of inulin simultaneously injected as a flow marker. These results suggest that the uptake of REV by the liver requires opsonization by blood components, and if REV can escape opsonization, they may be able to pass through the liver freely. It is also clear that the distribution volume of the REV is smaller than that of inulin.On the other hand, small unilamellar vesicles (SUV, about 0.06 μm in diameter) showed uptake corresponding to about 0.25 μmol of total lipid without any participation of blood components. This result suggsts that the uptake mechanism of SUV may be different from that of REV, and opsonization may not be essential for the uptake of SUV by the liver. A comparison of the results for REV and SUV suggests that the functional pore size of the fenestration of liver sinusoids is about 0.06 μm, and about half of the SUV prepared in this study could pass through the fenestration and reach the hepatocytes, while the other part of the SUV drained through the liver as did REV.The uptake of SUV by the liver seemed to be limited in capacity, and it was influenced by temperature and inhibited by predosing with liposomes containing sodium azide or cytochalasin B.

Chloride leakage measurement for the evaluation of the plasma-induced instability of liposomal membrane.

The plasma-induced instability of reverse-phase evaporation vesicles (REV) was successfully evaluated by measuring chloride leakage. The chloride efflux could be followed by using a silver chloride electrode without interference from coexisting substances such as liposomal lipid, plasma or bovine serum albumin (BSA). The permeability of REV was increased by incubation with human plasma or BSA ; the effect of plasma was estimated to be approximately 50 times larger than that of a corresponding amount of albumin. The difference of protein-lipid interaction in the cases of plasma and isolated albumin is discussed on the basis of the leakage pattern and the activation energy of the permeation process.

Infection of tobacco protoplasts with liposome-encapsulated potyviral RNA

The RNA of the potyvirus, tobacco vein mottling virus (TVMV), was encapsulated in reverse-phase evaporation vesicle liposomes containing phosphatidylserine and cholesterol. The liposomes were used to inoculate tobacco mesophyll protoplasts. Analysis of extracts of protoplasts, by enzyme-linked immunosorbent assay (ELISA), hybridization with labeled cDNA, electron microscopy and infectivity assays, indicated that significant multiplication of the virus had occurred after incubation of the protoplasts for 2–3 days. It was estimated that approx. 15% of the protoplasts had become infected.

Interaction of nucleic acids with lipid membranes.

The thermodynamics of nucleic acids which were enclosed in reverse-phase evaporation vesicles was studied by thermal denaturation with optical recording. The denaturation curves were recorded with a dual wavelength spectrophotometer. The sum of the hypochromicity of the nucleic acid and of the change in turbidity of the vesicles was measured at 260 nm and was corrected for the change in turbidity at 320 nm. Cloned fragments of double-stranded DNA containing 180 base pairs and poly A:poly U were enclosed in REV with a yield up to every vesicle containing five nucleic acid molecules. Vesicles were prepared from egg-lecithin, and the surface charge of the vesicles was varied by addition of stearic acid, phosphatidyl-glycerol and phosphatidyl-serine. The helix-coil transition of the nucleic acid enclosed in the vesicle could be resolved from that of the free nucleic acid. Due to the enclosure into the egg-lecithin REV the transition is stabilized from 70.5 degrees to 74 degrees C, the transition is broadened from 0.7 degrees C to 2.7 degrees C. Varying the phosphatidyl-serine-lecithin-ratio from 0-100%, an optimum in the yield of enclosure at 20% was obtained, a further broadening of the transition to 5.5 degrees C and a decrease of the stabilization down to a small destabilization at 100% phosphatidyl serine was observed. Qualitatively, similar effects were observed with poly A:poly U. Variation of the ionic strength led to the conclusion that the replacement of the counterions of the phosphate backbone by the surface charge of the membrane, as well as a direct contact between the nucleic acid and the membrane have to be assumed.(ABSTRACT TRUNCATED AT 250 WORDS)

Stability of liposomes on storage: freeze dried, frozen or as an aqueous dispersion.

For various types of liposomes carboxyfluorescein (CF) latency and physical stability on storage were investigated. Three regimens were compared: storage at 4-6°C in an iso-osmotic aqueous buffer, freezing of the dispersions at -5 or -30°C, or freeze drying of the dispersions. Reverse phase evaporation vesicles (REV) were used with mean diameters between 0.2 and 0.3 µm. Liposomes consisted of egg phosphatidylcholine (PC) and phophatidylserine (PS) without or with cholesterol (chol) (9/1 and 10/1/4, respectively) or of distearoylphosphatidylcholine (DSPC) and dipalmitoylphosphatidylglycerol (DPPG) without or with chol (10/l/[5]). PC/PS liposome dispersions lost 25% of the entrapped CF within 10 days. The leakage rate decreased with the inclusion of cholesterol in the bilayer. Both after a freezing/thawing cycle and after freeze-drying no acceptable CF latency could be obtained. The cryoprotectants that were tested failed to raise CF latency significantly. However, the physical integrity of the liposomes could be maintained by proper choice of the cryoprotectant. When stored at 4-6°C, DSPC/DPPG/(chol) dispersions were stable for at least 6 months. Upon freezing/thawing less than 10 % CF was lost. Freeze drying without cryoprotectants reduced CF latency dramatically on rehydration. The physical structure was maintained and maximum latencies of 70 % could be obtained with the use of lactose as a cryoprotectant.

Preparation of liposome-encapsulated anti-tumor drugs; Relationship between lipophilicity of drugs and in vitro drug release.

Liposomal encapsulation of drugs by a reverse-phase evaporation vesicle (REV) method was carried out, and the relation between the in vitro release rates and the lipophilicity of drugs was investigated. Anti-tumor drugs such as carboquone (CQ), nimustine (ACNU), nimustine hydrochloride (ACNU-HCl), cytosine arabinoside (Ara-C), 5-fluorouracil (5-FU) and related compounds were used as model drugs. Encapsulation of the drugs in liposomes was concluded to have no relation with the partition coefficients of the drugs, whereas the in vitro release rate was highly correlated to the partition coefficient and it was evident that the drug was released more slowly when the partition coefficient was lower. Therefore, to achieve sustained release of a drug by means of liposomal encapsulation, it would be necessary to decrease the partition coefficient of the drug by converting it into the hydrochloride form, as in the case of ACNU, or by forming the sodium salt of the drug, if the drug has a carboxylic acid group.

Reversible depression of the reticuloendothelial system by liposomes

The effect of various doses of different types (reverse phase evaporation vesicles and small unilamellar vesicles) of intravenously injected liposomes on reticuloendothelial activity, as measured by the blood clearance rate of intravenously injected carbon, was investigated. Also the effect of pretreatment with reverse phase evaporation vesicles on blood clearance and tissue distribution of a second dose of similar vesicles was determined. For all concentrations used reverse phase evaporation vesicles caused a reduction in reticuloendothelial activity at least up to 4 h after injection. 24 h after administration the rate of carbon clearance returned to the control level. On the contrary small unilamellar vesicles did not block reticuloendothelial activity. Pretreatment with reverse phase evaporation vesicles (250 mumol/kg) caused an increased blood level and a decreased hepatic uptake of a second dose of the vesicles, injected 1 h after the first dose. This seems to be due to a depression of reticuloendothelial activity and not to a depletion of opsonins. Pretreatment with small unilamellar vesicles (250 mumol/kg) had no significant influence on the tissue distribution of a second dose of vesicles. Our results clearly indicate that reverse phase evaporation vesicles cause a reversible depression of reticuloendothelial activity and this depression seems to be induced by a saturation of reticuloendothelial cells with liposomes.

Altered pharmacological properties of liposome-associated human interferon-alpha.

Human interferon-alpha was associated in different ways with positively (stearylamine) and negatively (phosphatidylserine) charged phosphatidylcholine multilamellar vesicles, depending on the presence or absence of a cholesterol component. Inclusion of cholesterol resulted in interferon that was significantly (P = 0.0001) more deeply internalized within the liposomes, such that detergent disruption was necessary before most of the interferon activity was expressed. Interferon was stably associated with stearylamine-containing liposomes, both with and without a cholesterol component. However, inclusion of cholesterol in the phosphatidylserine-containing liposomes was necessary for stable association of the interferon for more than 2 days at 4 degrees C or for more than 24 h at 37 degrees C. After intramuscular injection into mice, liposome-associated interferon in reverse-phase evaporation vesicles was retained at the local site of injection significantly longer than free interferon. Even 3 days after intramuscular injection, stearylamine-containing liposomes with or without cholesterol resulted in local interferon levels that were comparable to the peak levels obtained 2 to 4 h after free interferon was injected. In contrast, free interferon was not detectable in the local muscles 24 h after injection of 10(4.6) U. Liposomes containing phosphatidylserine and cholesterol resulted in intermediate levels of local interferon retention; without a cholesterol component, phosphatidylserine-containing liposomes resulted in no increased local interferon retention compared with the results when free interferon was injected.

Liposome-encapsulated carboquone. I. Method of preparation and carboquone release.

Encapsulation of carboquone (CQ) in liposomes was investigated by applying a reverse-phase evaporation vesicle (REV) method, and the in vitro release of CQ from the liposomes was also studied. The mean diameter of the liposomes was 0.2μm. The encapsulation efficiency in liposomes was greatly affected by the materials used for the membrane of the liposomes. The efficiency increased with a rise in the phase transition temperature of the phospholipids, the maximum encapsulation of CQ in liposomes being obtained with distearoyl phosphatidylcholine. The results of drug release showed that CQ remained in CQ-liposomes for a long time at 5°C but was released considerably faster at around the phase transition temperature (58°C) of distearoyl phosphatidylcholine.

Altered pharmacologic properties of liposome-associated human interferon-alpha. II.

Association of human interferon-alpha with multilamellar vesicles and reverse-phase evaporation vesicles resulted in altered retention and distribution of the interferon after injection into mice. Intramuscular injection of liposomal-interferon, after extrusion through 0.2 mu filters, resulted in significantly increased localized retention of interferon. Liposomal-interferon was retained locally from one to three days, depending on liposome composition, whereas an equivalent injection of free interferon was undetectable after 24 hrs. Intravenous injection of the liposomal-interferon preparations resulted in increased interferon levels in lung, spleen, and liver as compared to injection of free interferon. Preparation of small unilamellar vesicles in the presence of interferon revealed differences in modes of association of different species with the liposomes, based on differential titers on human vs. bovine cells.

Delivery of tobacco mosaic virus RNA into plant protoplasts mediated by reverse-phase evaporation vesicles (Liposomes)

Protoplasts were isolated from tobacco suspension cultures using a new cellulase preparation. Tobacco mosaic virus (TMV) RNA was encapsulated in reverse-phase evaporation vesicle (REV) liposomes of phosphatidylserine and cholesterol, and was successfully introduced into tobacco protoplasts by treatment of the REV/protoplast mixture with polyvinyl alcohol or polyethylene glycol followed by washing with high pH-high Ca buffer. Delivery of TMV-RNA was monitored by determining the number of infected protoplasts using the immunofluorescence technique. Production of TMV particles in the infected protoplasts was also confirmed by electron microscopy. Because of the high encapsulation efficiency of REV liposomes the amount of TMV-RNA necessary to cause infection in the majority of protoplasts could be reduced to 1/10 to 1/5 that required in the previous study (Fukunaga et al. 1981). The usefulness of the REV-mediated delivery of nucleic acids for genetic manipulation of plant protoplasts is discussed.

Phase transition release, a new approach to the interaction of proteins with lipid vesicles Application to lipoproteins

To study the interaction of proteins with lipid bilayers, we have developed a new experimental approach based on the release of a water-soluble fluorescent dye from liposomes during scans through the lipid phase transition temperature. The fluorescence of carboxyfluorescein is quenched at high dye concentrations inside the vesicles but appears when the dye is released and diluted into the external medium. This new approach, phase transition release, is here applied to the interaction of serum lipoproteins and apolipoproteins with liposomes (for the most part, small unilamellar vesicles of dipalmitoylphosphatidylcholine). The major findings are these: (i) All of the lipoproteins and apolipoproteins tested induce a smooth, rapid release of carboxyfluorescein, essentially complete within a few seconds. HDL apolipoprotein induces 50% carboxyfluorescein release at a lipid/protein molar ratio of 3 400 : 1, whereas a ratio of 160 : 1 is required for native HDL. (ii) The interaction is all-or-none and irreversible. It involves a sufficient perturbation of bilayer structure to permit equal release of carboxyfluorescein ( M r 373 ) and inulin ( M r 5 500 ). In the case of HDL apolipoprotein, this release accompanies formation of a relatively homogeneous population of vesicular recombinant structures. Only at much higher protein/lipid ratios are the often-studied small, disc-shaped recombinants formed. (iii) Much more dye is released if the transition temperature is approached from below than if it is approached from above. (iv) Phase transition release is seen with multilamellar and reverse phase evaporation vesicles, though with patterns different from those seen with small unilamellar vesicles. (v) A large number of proteins are found not to induce phase transition release, even at concentrations of at least 1000-times those required for the HDL apolipoprotein effect. These include trypsin, chymotrypsin, pronase, bovine serum albumin (crystalline), ovalbumin, rabbit immunoglobulin G (and its F(ab)′ 2 and F c fragments), rabbit immunoglobulin M, hemoglobin, hen lysozyme, synexin, ankyrin, myosin, and microtubule-associated proteins. Tubulin and actin, on the other hand, do induce phase transition release. In addition to its use for analysis of protein-bilayer interaction, phase transition release provides a way of reconstituting relatively water-soluble proteins into vesicles, under quantitative control and without detergent.

In vivo fate of large unilamellar sphingomyelin-cholesterol liposomes after intraperitoneal and intravenous injection into rats

We investigated the fate of intraperitoneally and intravenously injected reverse phase evaporation vesicles of fairly uniform size (100–200 m) with respect to blood celarance, tissue distribution and integrity in vivo. The vesicles are composed of sphingomyelin and cholesterol in a molar ratio 3 : 2 and contain 125I-labeled poly(vinyl pyrrolidone) in the aqueous compartment. It is shown that following an intrapersoneal injection the vesicles are transported intact, and not associated with cells, from the peritoneal activity to the blood and are subsequently taken up mainly by liver and spleen, where, particularly in liver, the phospholipid is partially metabolized. After an intraperitoneal injection the rate of vesicle-uptake by liver and spleen is reduced by a factor of 2–3 compared to the rate of vesicle-uptake by liver and spleen following an intravenous injection. The peritoneal cavity functions as a reservior of vesicles for some hours. The rates of blood clearance and uptake of the vesicles by liver and spleen appear to be slower than that found for vesicles of different lipid composition.