Changes In Vesicle Size Research Articles

198. In Vitro and In Vivo Gene-Transferring Characteristics of Novel Cationic Lipids, DMKD(O,O′-dimyristyl-N-lysyl aspartate) and DMKE(O,O′-dimyristyl-N-lysyl Glutamate)

Previously, we introduced a novel cationic lipid consisting of three biocompatible components, lysine, aspartic acid, and two C-14 hydrocarbon chains, named as O,O′-dimyristyl-N-lysyl aspartate (DMKD) as a potent gene transfection reagent. Recently, we have chemically synthesized another gene-transferring cationic lipid consisting of a core of glutamic acid and lysine with two C-14 hydrocarbon chains, assigned the acronym DMKE (O,O′-dimyristyl-N-lysyl glutamate). Biophysical characteristics of the cationic liposomes made of DMKE or DMKD were compared with those of conventional DOTAP liposomes. The DMKE- and DMKD-liposomes exhibited similarly stronger DNA-binding affinity than the DOTAP liposomes. Interestingly, vesicle size changes of DMKE- and DMKD-liposomes by lipoplex formation were far smaller than that of the DOTAP liposomes. Their gene-transferring capabilities were also tested in various cell-lines and compared with the control DOTAP liposomes. Under the same transfection conditions, the order of in vitro gene transfection efficiency was DMKE=DMKD>DOTAP. The pH changes (pH 5.0 – 7.4) in culture media little affected fusigenic activities of all the cationic liposomes. However, cell surface-binding affinities of the DMKE- and DMKE-lipoplexes were similar to each other, but higher than the DOTAP liposomes. In mouse studies, intravenously administered DMKE- and DMKD-lipoplexes did not exhibited superior gene expression in lungs to the DOTAP lipoplexes. Interestingly, they were more favorably transferred to the liver, not to the lungs. Particularly, under the same experimental conditions of intratumoral transfection, the DMKE lipoplexes showed more efficient gene expression in the tumor tissues than the DOTAP- and DMKD-lipoplexes. This study suggests that a small difference in the backbone structure of cationic lipid significantly affects their gene transfection capabilities. The DMKE- and DMKD-liposomes can be utilized as an efficient gene-transferring vector for hepatic or intratumoral transgene expression.

Polystyrene-b-poly(acrylic acid) Vesicle Size Control Using Solution Properties and Hydrophilic Block Length

Polymeric vesicles have attracted considerable attention in recent years, since they are a model for biological membranes and have versatile structures with several practical applications. In this study, we prepare vesicles from polystyrene-b-poly(acrylic acid) block copolymer in dioxane/water and dioxane/THF/water mixtures. We then examine the ability of additives (such as NaCl, HCl, or NaOH), solvent composition, and hydrophilic block length to control vesicle size. Using turbidity measurements and transmission electron microscopy (TEM) we show that larger vesicles can be prepared from a given copolymer by adding NaCl or HCl, while adding NaOH yields smaller vesicles. The solvent composition (ratio of dioxane to THF, as well as the water content) can also determine the vesicle size. From a given copolymer, smaller vesicles can be prepared by increasing the THF content in the THF/dioxane solvent mixture. In a given solvent mixture, vesicle size increases with water content, but such an increase is most pronounced when dioxane is used as the solvent. In THF-rich solutions, on the other hand, vesicle size changes only slightly with the water concentration. As to the effect of the acrylic acid block length, the results show that block copolymers with shorter hydrophilic blocks assemble into larger vesicles. The effect of additives and solvent composition on vesicle size is related to their influence on chain repulsion and aggregation number, whereas the effect of acrylic acid block length occurs because of the relationship among the block length, the width of the molecular weight distribution, and the stabilization of the vesicle curvature.

Interaction of mucin with cholesterol enriched vesicles: role of mucin structural domains.

We utilized fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS) to examine the role of gallbladder mucin (GBM) in promoting the aggregation and/or fusion of cholesterol enriched vesicles. By fluorescent labeling either the vesicle or the mucin, we could examine the change in vesicle size as well as changes in mucin's diffusion constant. Both FRAP and FCS show that GBM has a profound effect in inducing vesicles to aggregate/fuse, particularly after overnight incubation. GBM mucin domains (either protease digested or reduced GBM) are not as effective as native GBM. Intact GBM alone was able to shorten crystal appearance time and increase the number of crystals nucleated by polarized optical microscopy. In summary, our findings would suggest that both glycosylated and nonglycosylated domains of GBM are involved in early aggregation of cholesterol enriched vesicles but that this effect is reversible in the absence of nonglycosylated domains.

Biopharmaceutical characterization of topical liposome formulations bearing 5-fluorouracil

Liposome dispersions and liposome gel formulations for topical administration were evaluated as modified release delivery systems for 5-fluorouracil. Drug substance has been entrapped in the internal aqueous compartment of liposomes during the preparation. The concentration of 5-fluorouracil in the hydration medium was varied and the effect on the liposome characteristics was considered. Liposome gel formulations were prepared by incorporation of liophylized liposomes into a structured vehicle of chitosan. The decrease of the amount of aqueous phase bearing total drug quantity (drug/aqueous phase ratio from 1:100, 1:60, 1:40) led to an increase of the percentage of liposome-entrapped drug, and decreased percentage of drug release, while particle size analysis showed no changes in vesicle size. Liposome gel formulations showed initially a higher drug release rate in comparision with liposome dispersions, which could be related to the release of “free” 5-fluorouracil, leaked from liposomes due to the process of liophylization. This was followed by slower release (after 1.5 hour) as a result of the influence of the viscosity of the gel matrix.

Ultradeformable lipid vesicles can penetrate the skin and other semi-permeable barriers unfragmented. Evidence from double label CLSM experiments and direct size measurements

The stability of various aggregates in the form of lipid bilayer vesicles was tested by three different methods before and after crossing different semi-permeable barriers. First, polymer membranes with pores significantly smaller than the average aggregate diameter were used as the skin barrier model; dynamic light scattering was employed to monitor vesicle size changes after barrier passage for several lipid mixtures with different bilayer elasticities. This revealed that vesicles must adapt their size and/or shape, dependent on bilayer stability and elasto-mechanics, to overcome an otherwise confining pore. For the mixed lipid aggregates with highly flexible bilayers (Transfersomes®), the change is transient and only involves vesicle shape and volume adaptation. The constancy of ultradeformable vesicle size before and after pores penetration proves this. This is remarkable in light of the very strong aggregate deformation during an enforced barrier passage. Simple phosphatidylcholine vesicles, with less flexible bilayers, lack such capability and stability. Conventional liposomes are therefore fractured during transport through a semi-permeable barrier; as reported by other researchers, liposomes are fragmented to the size of a narrow pore if sufficient pressure is applied across the barrier; otherwise, liposomes clog the pores. The precise outcome depends on trans-barrier flux and/or on relative vesicle vs. pore size. Lipid vesicles applied on the skin behave accordingly. Mixed lipid vesicles penetrate the skin if they are sufficiently deformable. If this is the case, they cross inter-cellular constrictions in the organ without significant composition or size modification. To prove this, we labelled vesicles with two different fluorescent markers and applied the suspension on intact murine skin without occlusion. The confocal laser scanning microscopy (CLSM) of the skin then revealed a practically indistinguishable distribution of both labels in the stratum corneum, corroborating the first assumption. To confirm the second postulate, we compared vesicle size in the starting suspension and in the blood after non-invasive transcutaneous aggregate delivery. Size exclusion chromatograms of sera from the mice that received ultradeformable vesicles on the skin were undistinguishable from the results measured with the original vesicle suspension. Taken together, the results support our previous postulate that ultradeformable vesicles penetrate the skin intact, that is, without permanent disintegration.

Reverse Biomembrane Vesicles for Effective Controlled Delivery of Doxorubicin HCl

A new derivative of erythrocyte ghosts--reverse biomembrane vesicles (RBVs)--was developed, loaded with doxorubicin HCl (Dox RBVs), and extensively characterized both in vivo and in vitro. The formulation of reverse biomembrane is based on budding of membrane into the ghost interiors (endocytosis) leading to accumulation of small vesicles within each parent ghost. The system was evaluated in vitro for entrapment efficiency, membrane orientation, vesicle size, release profile, and centrifugal stress. The stability studies were performed at 4 degrees C by assessing the effect on vesicle size and leakage after 7, 15, and 21 days. The stability study showed negligible leakage and no appreciable change in vesicle size after storage for several days--an indication of good stability of RBVs formulation. The formulation and process variables were optimized to obtain vesicles of 1.2-1.8 microm. The amount of doxorubicin entrapped in RBVs was 0.75 mg/ml of packed vesicles. The in vitro release profile showed 52.86% of drug release in 16 hr. A membrane orientation study revealed inversion of membrane surface because only 10% of sialic acid was accessible on trypsin treatment. The plasma clearance data revealed an increase in half-life and bioavailability of drug. Tissue concentration data suggest that liver and spleen remain the major organs for clearance of these drug-loaded RBVs, which suggests preferential uptake by the reticuloendothelial system. On the whole, this vesicle system proved to be of potential application in cancer chemotherapy.

Lyophilization and rehydration of polymer-coated lipid vesicles containing a lipophilic chelator in the presence of sucrose: Labeling with 99mTc and biodistribution studies

In this paper we describe studies of the effect of lyophilization and rehydration of polymer-coated lipid vesicles bearing a lipophilic surface chelator upon subsequent labeling with technetium-99m and in vivo biodistribution behavior. Unilamellar vesicles of average diameter 100 nm were prepared containing 2 mol% of the lipophilic chelator phosphatidylethanolamine-diethylenetriaminetetraacetic acid (PE-DTTA) and either 0 or 3 mol% of the lipid-polymer conjugate, dipalmitoyl-phosphatidylethanolamine-monomethoxy polyethylene glycol 5000 (PE-MPEG) in 0.9% sodium chloride to which was added varying amounts of sucrose to a final concentration of 100–500 mM. The size of the vesicles in sucrose was determined before lyophilization and after rehydration and the effect of sucrose on the ability to label the vesicles with pertechnetate in the presence of stannous chloride was determined. Biodistribution studies were done in rabbits with vesicles before lyophilization and after rehydration in order to determine whether the rate of clearance from the blood pool was affected by the lyophilization/rehydration procedure. Results demonstrate that vesicles containing PE-DTTA and without PE-MPEG can be lyophilized and rehydrated with no change in average size or size distribution so long as the external sucrose concentration is greater than approx. 250 mM. When PE-MPEG is also present in the membrane the average vesicle size increases from approx. 140 to 200 nm, consistent with vesicle fusion. However, this small change in vesicle size makes no difference to the resulting circulation half-life (~2h at a lipid dose of ~10 μmol/kg). In no case does the presence of sucrose on the exterior of the vesicle interfere with technetium labeling of the vesicle surface.

Physical stability of different liposome compositions obtained by extrusion method.

Six different liposome compositions were evaluated according their physical stability. The compositions used were (in mole ratio): dipalmitoylphosphatidylcholine (DPPC): DPPC:cholesterol (CHOL) (1:1), DPPC:CHOL (1:6:1), DPPC:CHOL:sphingomyelin (SFM) (7:2:1), DPPC:CHOL:stearylamine (STE) (8:5:1) and DPPC:CHOL:dicetyl phosphate (DCP) (8:5:1). The liposomes were obtained by the extrusion method, through polycarbonate membranes of 0.1 micron pore size. The captured volume, number of lamellae, size and polydispersity of the vesicle populations were determined for all compositions. The physical stability was checked at -20 degrees C, room temperature, 4 degrees C and 50 degrees C by determining the changes in vesicle size over a maximum of 40 days. The process of aggregation and/or fusion was observed by photon correlation spectroscopy. From the results, we can establish that the above compositions are metastable at a temperature of 50 degrees C. On the other hand, values of captured volume were smaller than predicted by theory. This fact can be explained by the non-sphericity of extruded vesicles. In relation to the stability, the introduction of CHOL in the formulation allows keeping the vesicles at 4 degrees C. In contrast, liposomes containing only DPPC are very stable at room temperature. Compositions with a high stability are those that have present SFM or STE. The latter keeps the structural bilayer at temperatures < 0 degrees C without cryoprotectors. Both the lipids, STE and DCP, form vesicles with a higher number of lamellae.

Polymer-derivatized technetium [formula omitted] liposomal blood pool agents for nuclear medicine applications

By using the lipophilic chelator, dipalmitoylphosphatidylethanolamine-diethylenetriaminetetraacetic acid (DPPE-DTTA), lipid vesicles may be prepared labeled on their surface with technetium 99m. When technetium-labeled vesicles were injected intravenously into rabbits, the half-life for clearance of the label from the circulation was less than 30 min. By further incorporating a synthetic phosphatidylethanolamine-monomethoxypoly(ethylene glycol) 5000 conjugate (PE-MPEG) the circulation half-life of the radiolabel was increased, liver uptake decreased and exchange of technetium from the vesicle surface suppressed, depending upon both the DPPE-DTTA and PE-MPEG content. For vesicles containing 20 mol% DPPE-DTTA, incorporation of PE-MPEG had no effect upon the circulation half-life of the radiolabel, however, for vesicles containing 2 mol% DPPE-DTTA, incorporation of more than 4 mol% PE-MPEG increased the circulation half-life of the label to more than 12 h. Less than 2 mol% PE-MPEG or 8 mol% ganglioside GM1 were, however, ineffective at increasing the circulation half-life of surface-bound technetium. It was shown that unilamellar lipid with DPPE-DTTA can be lyophilized in the presence of external sucrose, subsequently rehydrated with no change in vesicle size and labeled with technetium. It is suggested that polymer-derivatized, technetium-labeled vesicles may prove a useful substitute for technetium-labeled red blood cells as a vascular marker in various nuclear medicine procedures and that lyophilization/rehydration provides a possible route to realization of such vesicles in a pharmaceutically useful form.

Mechanical properties of vesicles. II. A model for osmotic swelling and lysis

Vesicle polydispersity and leakage of solutes from the vesicle lumen influence the measurement and analysis of osmotically induced vesicle swelling and lysis, but their effects have not been considered in previous studies of these processes. In this study, a model is developed which expressly includes polydispersity and leakage effects. The companion paper demonstrated the preparation and characterization of large unilamellar lipid vesicles. A dye release technique was employed to indicate the leakage of solutes from the vesicles during osmotic swelling. Changes in vesicle size were monitored by dynamic light scattering (DLS). In explaining the results, the model identifies three stages. The first phase involves differential increases in membrane tension with strain increasing in larger vesicles before smaller ones. In the second phase, the yield point for lysis (leakage) is reached sequentially from large sizes to small sizes. In the final phase, the lumen contents and the external medium partially equilibrate under conditions of constant membrane tension. When fit to the data, the model yields information on polydispersity-corrected values for membrane area compressibility, Young's modulus, and yield point for lysis.

The CRF neurosecretory vesicle: vasopressin-dependent changes in vesicle size after adrenalectomy

Neurosecretory vesicles of paraventricular corticotropin releasing factor (CRF) neurons increase in size after adrenalectomy (ADX) in parallel with an augmentation of their content in vasopressin (VP) immunoreactive sites. We have investigated in the Brattleboro rat model whether changes in vesicle size are related to elevated intravesicular concentrations of VP. Using quantitative immunelectron microscopy, VP and CRF immunoreactive sites were assessed in the dense core vesicle compartment off CRF axon terminals in the median eminence. In heterozygous (control) rats, CRF was co-packaged with VP, and ADX induced a 3-fold increase in VP labeling intensity and produced a significant increase in the vesicle diameter. In homozygous rats lacking VP, only CRF immunoreactivity was detectable, and ADX was not accompanied by an increase in vesicle size. These observations suggest that the presence of VP is necessary for the ADX induced increase in vesicle size and that this increase might be needed to accomodate the excess of VP in the vesicle space.

Bilayer curvature and certain amphipaths promote poly(ethylene glycol)-induced fusion of dipalmitoylphosphatidylcholine unilamellar vesicles.

Unilamellar vesicles of varying and reasonably uniform size were prepared from 1,2-dipalmitoyl-3-sn-phosphatidylcholine (DPPC) by the extrusion procedure and sonication. Quasi-elastic light scattering was used to show that different vesicle preparations had mean (Z-averaged) diameters of 1340, 900, 770, 630, and 358 A (sonicated). Bilayer-phase behavior as detected by differential scanning calorimetry was consistent with the existence of essentially uniform vesicle populations of different sizes. The response of these different vesicles to treatment with poly(ethylene glycol) (PEG) was monitored using fluorescence assays for lipid transfer, contents leakage, and contents mixing, as well as quasi-elastic light scattering. No fusion, as judged by vesicle contents mixing and change in vesicle size, was detected for vesicles of diameter greater than 770 A. The diameters of smaller vesicles increased dramatically when treated with high concentrations of PEG, although mixing of their contents could not be detected both because of their small trapped volumes and because of the extensive leakage induced in small vesicles by high concentrations of PEG. Lipid transfer was detected between vesicles of all sizes. We conclude the high bilayer curvature does encourage fusion of closely juxtaposed membrane bilayers but that highly curved vesicles appear also to rupture and form larger structures when diluted from high PEG concentration, a process that can be confused with fusion. Despite the failure of PEG to induce fusion of large, uncurved vesicles composed of a single phosphatidylcholine, these vesicles can be induced to fuse when they contain small amounts of certain amphiphathic compounds thought to play a role in cellular fusion processes. Thus, vesicles which contained 0.5 mol % L-alpha-lysopalmitoylphosphatidylcholine, 5 mol % platelet activating factor, or 0.5 mol % palmitic acid fused in the presence of 30%, 25%, and 20% (w/w) PEG, respectively. However, vesicles containing 1,2-dipalmitoyl-sn-glycerol, 1,2-dioleoyl-sn-glycerol, 1-oleoyl-2-acetyl-sn-glycerol, or monooleoyl-rac-glycerol at surface concentrations up to 5 mol % did not fuse in the presence or absence of PEG. There was no correlation between the abilities of these amphipaths to induce phase separation or nonlamellar phases and their abilities to support fusion of pure DPPC unilamellar vesicles in the presence of high concentrations of PEG. The results are discussed in terms of the type of disrupted lipid packing that could be expected to favor PEG-mediated fusion.

Dopamine inhibits Na(+)-H+ exchanger activity in renal BBMV by stimulation of adenylate cyclase

To determine a renal tubular mechanism for the natriuretic effect of dopamine (DA) and DA-1 agonists, we measured Na(+)-H+ exchange activity (amiloride sensitive) in rat renal cortical brush-border membrane vesicles (BBMV). Renal cortical tissues were preincubated with ligands before BBMV preparation to study Na(+)-H+ exchange activity in the absence of the added ligands that may compete for ion binding sites of the exchanger. DA and DA-1 agonist-inhibited Na(+)-H+ exchange activity was concentration and time dependent. The inhibitory effect was not due to increased permeability, collapse of the proton gradient, or change in vesicle size and did not extend to Na(+)-glucose symport. DA-2 agonists had no effect, whereas alpha-adrenergic agonists increased Na(+)-H+ exchange activity. Kinetic analysis revealed that the DA-1 agonist inhibited Na(+)-H+ exchange activity by a noncompetitive process. 2',5'-Dideoxyadenosine inhibited adenylate cyclase activity and reversed the inhibitory effect of DA-1 agonist on the exchanger. H4, an isoquinoline sulfonamide, which inhibits protein kinase A, also reversed the inhibitory effect of DA-1 agonist on the exchanger. Thus the DA-1 agonist-mediated inhibition of Na(+)-H+ exchange activity in BBMV is a receptor-mediated adenylate cyclase-linked process.

Sedimentation field flow fractionation of fused unilamellar vesicles: Comparison with electron microscopy and gel filtration

Sedimentation field flow fractionation (SF 3), a relatively new instrumentation methodology for separating particles according to size, has been used to monitor changes in vesicle size during the formation of fused unilamellar vesicles of dipalmitoylphosphatidylcholine. The fusion of 500-Å small unilamellar vesicles to 700-Å large unilamellar vesicles (LUVs) and the slower conversion to 950-Å LUVs at 4°C was readily monitored by SF 3 over the time course of a month. Changes in the physical state of these vesicles as a result of structural reorganizations were characterized by high sensitivity differential scanning calorimetry. The advantages and limitations of SF 3 are discussed and correlated with electron microscopy and gel filtration.

Cimetidine transport in isolated luminal membrane vesicles from rabbit kidney.

Experiments were conducted to study the transport of the histamine H2-receptor antagonist, cimetidine, in luminal membrane vesicles prepared from rabbit renal cortex. Cimetidine accumulated in the vesicles with time. Cimetidine uptake was sensitive to changes in vesicle size, suggesting that the compound is transported into an osmotically reactive intravesicular space. Its rate of uptake could be described by both a saturable and a nonsaturable process. The Km was 4.6 +/- 4.0 microM and the Vmax was 6.8 +/- 2.3 pmol X s-1 X mg protein-1 (mean +/- SD, n = 4). N1-methylnicotinamide (NMN), cimetidine, cimetidine sulfoxide, and ranitidine inhibited the uptake of cimetidine. Cimetidine uptake in the presence of an outwardly directed proton gradient was enhanced in vesicles preloaded with a higher concentration of unlabeled cimetidine (2.4 X 10(-4) M). An outwardly directed proton gradient enhanced the uptake of cimetidine to values exceeding its equilibrium accumulation. Uptake stimulated in this way could be inhibited by the cation, NMN, the bases, ranitidine, and cimetidine sulfoxide, and interestingly, by the anion, probenecid. The effect of probenecid did not appear to be due to nonspecific effects on membrane binding, membrane potential, or vesicle size. These data are consistent with data obtained in isolated perfused proximal tubules, demonstrating that probenecid inhibits cimetidine transport. The data in this study suggest that the effect of probenecid on cimetidine transport specifically involves the transporter in the luminal membrane.

Interaction of liver clathrin coat protein with lipid model membranes.

Coated vesicles were prepared from rat liver homogenates by sucrose density gradient centrifugation and the clathrin coat, dissociated by mild treatment with Tris-Cl, was characterized by sodium dodecyl sulfate gel electrophoresis. At pH 6.5 and below, the soluble clathrin preparations induced a voltage-dependent increase in ion conductance across a black lipid membrane of oxidized cholesterol under conditions where the intact coated vesicles were inert. Further evidence for the interaction of clathrin with the lipid bilayer was provided by the demonstration that the fluorescent dye, carboxyfluorescein, was released from the internal aqueous space of small unilamellar dioleoyl and dipalmitoyl phosphatidylcholine vesicles upon the addition of the soluble protein. Dye release was shown to be a function of both calcium and pH. In the presence of calcium, release occurred at or above pH 8.0; in the absence of calcium, release was most prominent at pH 5.0-6.5. Formation of stable protein-phospholipid complexes was shown by KBr density gradient centrifugation. Stability of the complexes in high salt concentration suggested that electrostatic interactions were not essential to maintain the recombinants. Here, as with the black lipid membrane, incubation of the phospholipid vesicles with intact coated vesicles failed to induce release of the dye at pH 6.5 or below. Interaction of the clathrin coat protein with unilamellar vesicles at low pH resulted in a change in vesicle size and morphology as shown by electron microscopy.

Fusion kinetics of phosphatidylcholine-phosphatidic acid mixed lipid vesicles: A proton nuclear magnetic resonance study

The kinetics of Ca 2+-induced fusion of phosphatidylcholine-phosphatidic acid vesicles has been studied using the dependence of proton nuclear magnetic resonance linewidths on vesicle size. The linewidth of the lipid acyl chain methylene resonance been shown to be sensitive to changes in vesicle size but insensitive to vesicle aggregation. For vesicle systems with the same lipid composition, the linewidth increases in a linear fashion with vesicle radius over the range 125–300 Å. This dependence has been used to determine quantitatively fusion rates and the dependence of such rates on Ca 2+ as well as an vesicle concentration. For vesicle concentrations in the range of 3 · 10 −6–10 −5 M and Ca 2+ concentration at a level approaching 1 : 1 with respect to phosphatidic acid, the initial fusion rates have been found to be fast, with half-times of 1–10 min. An order of reaction of 2.7 with respect to vesicle concentration has been observed. Mechanisms of vesicle fusion are discussed in view of these observations.

Ion specificity in fusion of phosphatidic acid-phosphatidylcholine mixed lipid vesicles

Fusion of phosphatidylcholine-phosphatidic acid mixed lipid vesicles has been studied under the influence of the divalent Ca 2+, Mg 2+, Cd 2+, and Ba 2+, which range in size from much smaller to much larger, than Ca 2+. Fusion has also been studied under the influence of Eu 3+, which has a similar radius but different charge. The effects of these ions are reflected in the different degrees of fusion determined by changes in vesicle size, and in the varying fusion rates monitored by 1H-NMR spectroscopy. Cd 2+, which has an ionic radius similar to Ca 2+, exhibits the same effect on fusion as Ca 2+, while other ions show lower efficiencies. This suggests that the vesicle fusion intermediate has a geometry ideally suited to the binding of Ca 2+ or Cd 2+.

High resolution proton relaxation studies of lecithins

1. 1. Spin-lattice ( T 1) proton relaxation data have been obtained for lecithins in solution in methanol, in micelles in [ 2H]chloroform, and in bilayers in 2H 2O. The relaxation times are characteristic of the type of structure formed, and reflect molecular motion within the lecithin molecule in each structure. 2. 2. The data are not consistent with a single T 1 for all the protons in the lecithin bilayer in 2H 2O and spin diffusion is not a dominant relaxation mechanism. 3. 3. Sonicated aqueous suspensions of dipalmitoyl lecithin show a striking set of spectral changes through the transition temperature at 43° in which the fatty acid chain resonances disappear completely and the −N(CH 3) 3+ resonance undergoes a sharp increase in linewidth. These spectral changes through the transition are entirely reversible and do not involve a significant change in vesicle size. 4. 4. The T 1 values for the major resonances of lecithin in bilayers are consistent with a structure, based on 13C T 1 measurements, in which the bilayer is most tightly packed at the glycerol region of the lecithin molecules, with increasing freedom of motion both towards the terminal methyl of the alkyl chain and the −N(CH 3) 3+ group at the surface of the bilayer. 5. 5. Cholesterol perturbs the bilayer structure and produces changes in the T 1 values of the lecithin resonances, consistent with the effect on the permeability of the lecithin vesicle.