Fusion and lipid exchange in vesicles containing lipophilic spin labels

Lipophilic non-electrolyte spin labels greatly accelerate the fusion of unilamellar vesicles of dipalmitoylphosphatidylcholine when the system is maintained below the lipid phase transition. Differential scanning calorimetry and centrifugation measurements show that the transformed vesicles are large and probably unilamellar. Differential scanning calorimetry and fluorescence depolarization measurements were also carried out on mixtures of labeled dipalmitoylphosphatidylcholine vesicles and of vesicles composed of pure dimyristoylphosphatidylcholine. A mixing of the membrane components is observed when the vesicles are incubated above the transition temperature of the two constituent lipids. However, the process does not involve a real fusion of the entire vesicles. An exchange of lipid and label monomers between the two lipid phases seems to occur. These observations are discussed in view of the molecular organization of the spin label within the dipalmitoylphosphatidylcholine matrix below and above the lipid transition temperature.     

Pigment containing lipid vesicles

Summary It was shown previously (Walz, 1976) that chlorophylla incorporated into the membrane of lecithin vesicles is a probe which detects the aggregational state of the lipids. This phenomenon is interpreted in terms of a solvatochromism, i.e., the effect of various solvents on the absorption spectrum of the solute. The sensor characteristics can be expressed by a set of solvatochromic coefficients, which are pertinent to the electronic transitions occuring in chlorophylla on excitation with light, and by means of the absorption bands associated with these transitions. An unambiguous resolution of spectra into absorption bands is not yet practicable, but at least part of the bands can be approximated by, Gaussian components which then allows us to estimate the solvatochromic coefficients From these data and based on the currently available theoretical and experimental information about, solvatochromism, it is concluded that the chromophore, i.e., the porphyrin ring of chlorophylla, is located adjacent to the glycerol-ester moieties of the lecithin molecules in the membrane, and that the sensor ability relies on different orientations of the chromophore for lecithin in different states of aggregation.     

Pigment containing lipid vesicles

Summary Vesicles obtained by sonication of chlorophylla-lecithin mixtures dispersed in an aqueous medium closely resemble the well-characterized vesicles similarly prepared from pure lipids. They are bounded by one spherical lipid bilayer which contains the chlorophylla. Appropriate conditions for sonication prevent substantial degradation of the membrane constituents. Up to one chlorophylla molecule per 55 lecithins can be incorporated into the membranes. The average Stokes' radius of the vesicles determined by analytical sieve chromatography is 102±5 Å and independent of the chlorophylla content. The membrane is visible in the electron-microscope when the vesicles are treated with osmium tetroxide prior to negative staining. The osmium fixation is, however, not strong enough to allow for a preparation of the vesicles for thin sectioning (dehydration, embedding in epoxide).     

Synthesis and biological properties of the lipophilic technetium-99m complex 99mTc(acac)3

In the development of technetium-99m radiopharmaceuticals for the evaluation of regional cerebral perfusion, one series of complexes that has remained unexplored is the neutral lipophilic tris complexes formed with β-diketonato ligands. The prototype complex of this series, tris(2,4-pentanedionato) technetium(III), has been prepared via a new synthetic route and chemically characterized using ⁹⁹Tc and the biodistribution of the no-carrier-added ^99mTc complex has been determined. The ^99mTc complex was found to be distributed throughout the body with persistant high blood levels indicative of a high degree of protein binding. The primary route of excretion was the hepatobiliary system as indicated by the appearance of ^99mTc in the gut and feces at longer sample times post-injection. Although this complex was not retained by the brain, it does provide a starting point from which a more effective agent might be developed.     

Nanosized self-emulsifying lipid vesicles of diacylglycerol-PEG lipid conjugates: Biophysical characterization and inclusion of lipophilic dietary supplements

Hydrated diacylglycerol-PEG lipid conjugates, glyceryl dioleate-PEG12 (GDO-PEG12) and glyceryl dipalmitate-PEG23 (GDP-PEG23), spontaneously form uni- or oligolamellar liposomes in their liquid crystalline phase, in distinct difference from the PEGylated phospholipids which form micelles. GDP-PEG23 exhibits peculiar hysteretic phase behavior and can arrange into a long-living hexagonal phase at ambient and physiological temperatures. Liposomes of GDO-PEG12 and its mixture with soy lecithin exchange lipids with the membranes much more actively than common lecithin liposomes; such an active lipid exchange might facilitate the discharging of the liposome cargo upon uptake and internalization, and can thus be important in drug delivery applications. Diacylglycerol-PEG lipid liposome formulations can encapsulate up to 20-30 wt.% lipophilic dietary supplements such as fish oil, coenzyme Q10, and vitamins D and E. The encapsulation is feasible by way of dry mixing, avoiding the use of organic solvent.     

Interaction of the polyene antibiotics with lipid bilayer vesicles containing cholesterol

The interaction of the polyene antibiotics, amphotericin B, nystatin and filipin with cholesterol-containing single bilayer lipid vesicles has been characterized using gel permeation chromatography and proton magnetic resonance. All three antibiotics bind to vesicles at low concentrations without causing a large amount of vesicle destruction. The strength of binding as determined by gel permeation studies is greater for filipin and amphotericin than for nystatin. Nystatin and amphotericin B at these low concentrations induce a rapid loss of internal vesicle contents consistent with pore formation. Filipin induces no leakage beyond that expected from partial vesicle destruction or general detergent action.At antibiotic levels above 1 : 1 antibiotic : cholesterol ratios the NMR results show all three antibiotics to cause extensive vesicle destruction. The onset of this behavior, which appears to be independent of the total antibiotic concentration, indicates a well defined antibiotic : cholesterol interaction stoichiometry. Despite the fact that cholesterol is required for antibiotic activity, the NMR spectra prior to vesicle destruction show no changes indicative of an antibiotic-induced reversal of cholesterol restriction of phosphatidylcholine mobility. The contrast with polyene antibiotic behavior in more extended bilayers is discussed.     

Interaction of the polyene antibiotics with lipid bilayer vesicles containing cholesterol.

The interaction of the polyene antibiotics, amphotericin B, nystatin and filipin with cholesterol-containing single bilayer lipid vesicles has been characterized using gel permeation chromatography and proton magnetic resonance. All three antibiotics bind to vesicles at low concentrations without causing a large amount of vesicle destruction. The strength of binding as determined by gel permeation studies is greater for filipin and amphotericin than for nystatin. Nystatin and amphotericin B at these low concentrations induce a rapid loss of internal vesicle contents consistents consistent with pore formation. Filipin induces no leakage beyond that expected from partial vesicle destruction or general detergent action. At antibiotic levels above 1:1 antibiotic: cholesterol ratios the NMR results show all three antibiotics to cause extensive vesicle destruction. The onset of this behavior, which appears to be independent of the total antibiotic concentraion, indicates a well defined antibiotic : cholesterol interaction stoichiometry. Despite the fact that cholesterol is required for antibiotic activity, the NMR spectra prior to vesicle destruction show no changes indicative of an antibiotic-induced reversal of cholesterol restriction of phosphatidylcholine mobility. The contrast with polyene antibiotic behavior in more extended bilayers is discussed.     

Pigment containing lipid vesicles. I. Preparation and characterization of chlorophyll a-lecithin vesicles.

Vesicles obtained by sonication of chlorophyll a-lecithin mixtures dispersed in anaqueous medium closely resemble the well-characterized vesicles similarly prepared from pure lipids. They are bounded by one spherical lipid bilayer which contains the chlorophyll a. Appropriate conditions for sonication prevent substantial degradation of the membrane constituents. Up to one chlorophyll a molecule per 55 lecithins can be incorporated into membranes. The average Stokes' radius of the vesicles determined by analytical sieve chromatography is 102 +/- 5 A and independent of the chloropyll a content. The membrane is visible in the electron-microscope when the vesicles are treated with osmium tetroxide prior to negative staining. The osmium fixation is, however, not strong enough to allow for a preparation of the vesicles for thin sectioning (dehydration, embedding in epoxide).     

Stability and fusion of lipid vesicles containing headgroup-modified analogues of phosphatidylethanolamine

We have used lipid mixing, contents mixing and contents-leakage assays to characterize the divalent cation-mediated interactions of vesicles composed of various headgroup-modified analogues of phosphatidylethanolamine, PE (N- and C-2-alkylated derivatives, and analogues with increased separations of the phosphoryl and amino groups) together with a low mole percentage of phosphatidylserine (PS). Vesicles containing different structural analogues of PE exhibit marked differences, both in the threshold divalent cation concentrations that are required to initiate vesicle-vesicle interactions and in the rates of contents mixing and leakage observed at suprathresholds divalent cation concentrations. The efficiencies of divalent cation-promoted contents leakage, and to a slightly lesser extent those of contents mixing, for PS/PE (analogue) vesicles show a marked inverse correlation with the lamellar-to-hexagonal II transition temperature (TH) of the PE (analogue) component. However, the destabilization kinetics for such vesicles show no abrupt changes over the temperature range around the equilibrium TH value measured for the vesicle lipids. Vesicles combining PS with different PE analogues exhibit divalent cation thresholds for aggregation that are not correlated with the TH values of the PE (analogue) components but appear instead to be correlated with the equilibrium interbilayer separations measured in multilamellar dispersions of these species. We have identified headgroup-modified analogues of PE that can be used to prepare vesicles that fuse more rapidly under a given set of conditions, or that show a bette ratio of fusion-to-contents-leakage rates, than do PE-containing vesicles. These results may be useful both for understanding better the bases for the high fusion-supporting ability of PE and for the preparation of lipid vesicles 'tailored' for particular practical applications.     

Existence of lipid vesicles containing platelet-activating factor in endothelial cell lysate

Platelet aggregation activity due to platelet-activating factor (PAF) was detected at high molecular weight (HMW) and low molecular weight fractions after gel-filtration chromatography of cell lysate of endothelial cells. [³H]PAF added to the cell lysate was similarly distributed after chromatography. The radioactivity associated with HMW fraction was not reduced by digesting the lysate with trypsin, suggesting that PAF was not making complexes with proteins but was included in lipid vesicles in cell lysate. Further evidence showed that an unknown specific factor(s) was needed to form these PAF-containing lipid vesicles. Radioactivity was not found in HMW fraction when [³H]PAF was mixed with cell lysate of vascular smooth muscle cells. When monomeric PAF was added to endothelial cell lysate, the specific activity of aggregation decreased to the level exerted by endogenous PAF-containing lipid vesicles due to incorporation into lipid vesicles. PAF in the form of lipid vesicles was more stable in plasma than monomeric form.     

Inhibition of serum complement haemolytic activity by lipid vesicles containing phosphatidylserine

The effect of artificial model membranes on the complement system was investigated. Incubation of the model membranes with human serum resulted in consumption of complement haemolytic activity when phosphatidylserine-containing vesicles were used. The activation of the complement system appeared to proceed through the alternative pathway. This conclusion was supported by the failure of [125I]Clq to bind to the membranes suggesting that the classical pathway was not involved. Although always obtained when phosphatidylserine was present in the model membranes, the activation of complement was enhanced by the contemporaneous presence of phosphatidylethanolamine. Liposomes prepared from lipid extracts of red blood cells were also able to stimulate a concentration-dependent activation of complement. Fresh, intact erythrocytes, however, could not initiate the same effects unless opsonized by antibodies. When artificially aged in vitro, red blood cells were lysed if incubated with normal human serum or with Clq-depleted serum. However, no lysis was obtained if the 'aged' erythrocytes were incubated with serum pretreated with ammonia to destroy the C3 component of complement. It is suggested that one of the mechanisms of macrophage recognition of senescent erythrocytes might be provided by the activation of the alternative pathway of complement if phosphatidylserine becomes exposed on the surface of the aging cells.     

Low pH induced membrane fusion of lipid vesicles containing proton-sensitive polymer

For the purpose of cytoplasmic delivery of aqueous content in liposomes through endosomes, we synthesized a pH-sensitive polymer, cetylacetyl(imidazol-4-ylmethyl)polyethylenimine (CAIPEI), which generates polycations at acidic pH. CAIPEI in its aqueous phase caused aggregation of sonicated vesicles composed of phosphatidylserine (PS) and phosphatidylcholine (PC) (molar ratio 1:4) when the pH of the solution was lowered. The polymer also induced membrane intermixing as measured by resonance energy transfer between vesicles containing N-(7-nitro-2,1,3-benz[d]oxadiazol-4-yl)phosphatidylethanolamine and those containing N-Rhodamine phosphatidylethanolamine at pH 4-5, while the addition of CAIPEI caused neither aggregation of PC vesicles nor the intermixing of liposomal membranes between PC and PC/PS vesicles at any pH. The CAIPEI-induced membrane intermixing was dependent on the polymer/vesicle ratio rather than on the polymer concentration. Then the polymer was incorporated into the bilayers of PC vesicles. These CAIPEI vesicles also caused membrane intermixing with liposomes containing PS under acidic conditions. The reconstituted CAIPEI did not reduce the trapping efficiency of vesicles or increase their permeability to glucose even at low pH. The vesicles caused the low pH induced aggregation and membrane intermixing with other negatively charged liposomes containing phosphatidic acid or phosphatidylglycerol. These results suggest that the protonation of the polymer at acidic pH endows the CAIPEI vesicles with the activity to fuse with negatively charged liposomes.     

Biological evaluation of 99mTc labelled cis-platinum containing benzyliminodiacetic acids as potential tumour imaging agents

The synthesis of three new potential tumour-imaging radiopharmaceuticals in which a cis-platin derivative is attached to benzyl iminodiacetic acid, a ligand capable of forming a stable complex with ^99mTc, has previously been reported by us [Awaluddin et al. Appl. Radiat, Isot. 38, 671–674 (1987)]. We have now carried out extensive biodistribution studies on these compounds as well as on two fragments of their structures which do not contain platinum. The results suggest that the presence of platinum is not essential for the tumour-localizing properties of the radiopharmaceuticals.     

Gene transfection into HeLa cells by vesicles containing cationic peptide lipid

Lipid vesicles are potentially useful as microcapsules for drug and/or gene delivery. We developed cationic lipid vesicles consisting mainly of sorbitan monooleate (Span 80) and cationic peptide lipid (CPL), and evaluated the CPL vesicles as gene transfection vectors. The optimum CPL concentration for gene transfection into HeLa cells was found to be 20 wt % of total lipid, and such CPL vesicles did not exhibit significant cytotoxicity. Co-culture of Poly-L-lysine and plasmids prior to making CPL vesicle-plasmid complexes was effective. Lipofection using LipofectAMINE was suppressed in 10% serum-supplemented medium. The transfection efficiency of 20 wt % CPL vesicles, however, was not affected by serum in the medium when plasmids were treated with poly-L-lysine.     

Preparation of lipid vesicles containing high levels of entrapped radioactive cations.

The ionophore A23187 was incorporated into the lipid bilayer of unilamellar vesicles and used to earry externally added indium ions to a chelator, nitrilotriacetic acid, which was previously entrapped in the vesicles. The extent of vesicle loading was assayed by Sephadex chromatography and γ-ray perturbed angular correlation spectroscopy. Incubation conditions were developed which allow rapid loading of more than 90% of added ¹²¹In³⁺ with specific activities of 200–300 μCi/mg lipid.     

Direct labeling of proteins with 99mTc

The direct labeling of antibodies and antibody fragments to form a highly stable bond between technetium and the sulfide groups of proteins is now well established. To optimize this reaction, the antibody protein must have sufficient reactive sulfides available to accept that technetium metal ions that are formed by the reduction of pertechnetate in the presence of a weak complexing agent. The reactive sulfide groups are provided by first reducing a small fraction of the disulfide bridges in the antibody protein or by starting with Fab′ fragments, which already have reactive sulfide groups. When the antibody protein has been appropriately reduced, and the reactive sulfide groups protected by a metal ion with a lower binding affinity than technetium, such as tin or zinc, very high labeling yields of high-affinity-bonded ^99mTc can be achieved. This can be accomplished without loss of immunoreactivity, measured as either affinity or immunoreactive fraction.Side reactions can produce radiochemical impurities such as low-affinity, bound ^99mTc; ^99mTc colloids; ^99mTc peptides or antibody aggregates; or ^99mTc-complexes. Also, pertechnetate ions may be an impurity if the sodium pertechnetate solution added to the reduced antibodies is not completely reduced. The specifics of minimizing these side reactions have not been extensively discussed in the prior literature; however, it is clear that appropriate reduction of the protein prior to labeling and complete removal of the reducing agent, particularly if it contains reactive sulfide groups or is toxic, are critical.One- or two-step ^99mTc-labeling kits for preparing ^99mTc-labeled antibody or antibody fragments are rapidly being introduced for use in clinical nuclear medicine studies. These direct labeling methods employ a common sequence of chemical reactions, although the reducing agents for both the antibody and the [^99mTc]pertechnetate may vary. Different ^99mTc transfer agents may be used, but all transfer agents have the common feature of quickly forming weak to moderately strong complexes with reduced technetium. Most use Sn(II) to reduce the pertechnetate, although other reducing agents can be used.     

Effects of amyloid beta-peptides on the lysis tension of lipid bilayer vesicles containing oxysterols

Amyloid β-peptides (Aβ) applied directly from solution to model lipid membranes produced dramatic changes in the material properties of the bilayer when certain oxysterols were present in the bilayer. These effects were dependent on both lipid and peptide composition, and occurred at peptide concentrations as low as 100 nM. Using micropipette manipulation of giant unilamellar vesicles, we directly measured the lysis tension of lipid bilayers of various compositions. The glycerophospholipid 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) constituted the main lipid component at 70 mol %. The remaining 30 mol % was composed of the following pure or mixed sterols: cholesterol (CHOL), 7-ketocholesterol (KETO), or 7β-hydroxycholesterol (OHCHOL). SOPC/CHOL bilayers did not exhibit significant changes in mechanical properties after exposure to either Aβ(1-42) or Aβ(1-40). Partial substitution of CHOL with KETO (5 mol %), however, caused a drastic reduction of the lysis tension after exposure to Aβ(1-42) but not to Aβ(1-40). Partial substitution of CHOL with OHCHOL (5 mol %) caused a drastic reduction of the lysis tension after exposure to Aβ(1-40) and to Aβ(1-42). We attribute these effects to the reduction in intermolecular cohesive interactions caused by the presence of the second dipole of oxysterols, which reduces the energetic barrier for Aβ insertion into the bilayer.     

Spent 99Mo/99mTc generator as an economical source of 99Mo

An improved method for utilization and purification of ⁹⁹Mo from spent ⁹⁹Mo/^99mTc generators has been described. After washing the generator with saline to remove the generated ^99mTc, followed by 2 mL 5 M NaOH containing a few drops of H₂O₂, the ⁹⁹Mo was quantitatively eluted from the generator with 5 mL 5 M NaOH. The alkaline eluate containing ⁹⁹Mo was contaminated with partially dissolved alumina. In the present method, an anion-exchange resin Dowex 1 × 8 column was used for purification of ⁹⁹Mo from the contaminating alumina. The resultant ⁹⁹Mo was of high purity and contained < 1 μg Al³⁺/mL ⁹⁹Mo solution, as estimated by atomic absorption. The eluted ⁹⁹Mo was used for radiolabeling of monoclonal antibodies (Mab) to verify the theoretical modeling of an in vivo generator for radioimmunotherapy, and the results of this study will be published elsewhere.