The effect of surface curvature on the head-group structure and phase transition properties of phospholipid bilayer vesicles

Proton nuclear magnetic resonance spectra at 360 MHz of small sonicated distearoyl phosphatidylcholine vesicles show easily distinguishable resonances due to choline N-methyl head-group protons located in the inner and outer bilayer halves. A study of the chemical shift of these resonances as a function of temperature reveals that the splitting between them increases below the phase transition. This occurs as a result of an upfield shift of the inner layer resonance at the phase transition. Consideration of the possible causes of this effect results in the conclusion that, at the phase transition, there is a change in the organization of the inner layer head-groups which does not occur for the outer layer head-groups.     

Effect of hydrophobic structure on the catalysis of nitric oxide release from zwitterionic diazeniumdiolates in surfactant and liposome media.

The effect of phospholipid liposomes and surfactant micelles on the rate of nitric oxide release from zwitterionic diazeniumdiolates, R1R2N[N(O)NO]-, with significant hydrophobic structure, has been explored. The acid-catalyzed dissociation of NO has been examined in phosphate-buffered solutions of sodium dodecylsulfate (SDS) micelles and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl-sn-glycero-3-[phospho-(1-glycerol)] sodium salt (DPPG) phospholipid liposomes. The reaction behavior of dibenzylamine-, monobenzylamine-, and dibutylamine-derived substrates [1]: R1 = C6H5CH2, R2 = C6H5CH2 NH2+(CH2)2, 2: R1 = C6H5CH2, R2 = NH3+(CH2)2, and 3: R1 = n-butyl, R2 = n-butyl-NH2+(CH2)6] has been compared with that of SPER/NO, 4: R1 = H2N(CH2)3, R2 = H2N(CH2) 3NH2+(CH2)4]. Catalysis of NO release is observed in both micellar and liposome media. Hydrophobic interactions contribute to micellar binding for 1-3 and appear to be the main factor facilitating catalysis by charge neutral DPPC liposomes. Binding constants for the association of 1 and 3 with SDS micelles were 3-fold larger than those previously obtained with comparable zwitterionic substrates lacking their hydrophobic structure. Anionic DPPG liposomes were much more effective in catalyzing NO release than either DPPC liposomes or SDS micelles. DPPG liposomes (at 10 mM total lipid) induced a 30-fold increase in the NO dissociation rate of SPER/NO compared to 12- and 14-fold increases in that of 1 and 3.     

Proton nuclear magnetic resonance spectroscopy of isolated rabbit fundic glands

1H-nuclear magnetic resonance spectroscopy (NMR) was adapted to isolated rabbit fundic glands and identification made of compounds responsible for several observed spectral resonances. A minimum gland concentration of 0.5 mg dry weight or 5 mg wet weight per 0.5 ml was needed for adequate signal-to-noise ratio. At physiological temperature and pH, the glands demonstrated reproducible spectra, stability for accumulation times greater than 30 min and responsiveness to histamine stimulation, as measured by oxygen consumption and aminopyrine uptake. The relatively anaerobic conditions favored use of proton compared to phosphorus NMR, since 1H-NMR allowed significantly shorter spectral accumulation times and therefore did not compromise glandular viability to the same extent as 31P-NMR. The most conspicuous resonance in the gland spectrum was assigned to the -N+(CH3)3 protons of choline and related compounds. In membrane-free lysates, several components of the signal were resolvable and assigned to choline, phosphatidylcholine, phosphocholine and L-alpha-glycerophosphocholine. Thin-layer chromatography verified that phosphatidylcholine and phosphatidylethanolamine were the major phospholipids present in gland lipid. Presumably, they represent the source of the surface-active phospholipids present in gastric juice, which may play a role in gastric cytoprotection.     

Comparison of steady-state fluorescence polarization and urea permeability of phosphatidylcholine and phosphatidylsulfocholine liposomes as a function of sterol structure

The well-known reduction in the permeability properties of liposomes of dimyristoylphosphatidylcholine (DMPC) by sterols has also been demonstrated for its sulfonium analog (DMPSC) in which the N+(CH3)3 group of choline is replaced by S+(CH3)2. We have now compared the effects of 25 mol% 24-methylenecholesterol and cholesterol on the initial rates of urea permeation into dipalmitoyl-PC (DPPC) and dipalmitoyl-PSC (DPPSC) liposomes above the gel-to-liquid-crystalline phase transition temperature and found a greater reduction with 24-methylenecholesterol/DPPSC than with cholesterol/DPPSC liposomes but little difference between the two sterols in DPPC liposomes. Fluorescence polarization studies, using diphenylhexatriene as a probe, show that polarization (P) values are considerably higher in DMPSC liposomes containing 20 and 30 mol% 24-methylenecholesterol than in DMPC liposomes containing 20 and 30 mol% cholesterol. Higher P values were also obtained in DMPSC liposomes containing other 24-alkyl-substituted sterols (beta-sitosterol, ergosterol and campesterol) than in DMPC liposomes containing the same sterols. Reduced permeability rates in PSC liposomes containing 24-alkyl-substituted sterols are correlated with higher polarization values, reflecting an increased degree of order and/or motion in these liposomes compared with liposomes from the corresponding PC. These results suggest that alkyl substitution at C-24 of the sterol molecule results in tighter interactions with the sulfonium analog of PC than with PC.     

Interaction of N,N,N-trialkylammonioundecahydro-closo-dodecaborates with dipalmitoyl phosphatidylcholine liposomes

N,N,N-Trialkylammonioundecahydrododecaborates (1-), a novel class of compounds of interest for use as anions in ionic liquids, interact with DPPC liposomes. Increasing compound concentration causes an increasing negative ζ potential. Dissociation constants demonstrate that the binding capacity increases strongly with longer chain length. N,N,N-Trialkylammonioundecahydrododecaborates with longer alkyl chains show a detergent-like behavior: the compounds incorporate into the liposome membrane and differential scanning calorimetric experiment show already low concentrations cause a complete disappearance of the peak representing the gel-to-liquid crystalline phase transition. In contrast, compounds with shorter alkyl chains only interact with the headgroups of the lipids. Investigations by means of cryo-TEM reveal that all derivatives induce significant morphological changes of the liposomes. N,N,N-Trialkylammonioundecahydrododecaborates with short alkyl chains produce large bilayer sheets, whereas those with longer alkyl chains tend to induce the formation of open or multi-layered liposomes. We propose that the binding of N,N,N-trialkylammonioundecahydrododecaborates is mainly due to electrostatic interactions between the doubly negatively charged cluster unit and the positively charged choline headgroup; the positively charged ammonium group might be in contact with the deeper-lying negatively charged phosphate. For N,N,N-trialkylammonioundecahydrododecaborates with longer alkyl chains hydrophobic interactions with the non-polar hydrocarbon part of the membrane constitute an additional important driving force for the association of the compounds to the lipid bilayer.     

1H-NMR of phosphatidylcholine liposomes at low p2H in the presence of a paramagnetic shift reagent

1H-NMR spectra of egg phosphatidylcholine liposomes in 2H2O were obtained at several p2H values in the presence of 10 mM PrCl3 added after sonication of the phospholipid. It has been found that as the p2H is lowered below 2, the two distinct signals corresponding to the outer and inner phospholipid trimethylammonium groups which arise by the shifting effect of the paramagnetic cation on the external surface of vesicles, tend to coalesce into a single, high-field peak, at the position corresponding to the internal, non-shifted -N+ (CH3)3 protons. These results can be interpreted to mean that the shifting effect of Pr3+ on phosphatidylcholine NMR spectra, is due to electrostatic interaction between the lanthanide and the ionized group of the lipid. At low p2H, as the phosphodiester becomes protonated, the paramagnetic cation is no longer attracted by the liposome surface and its shifting effect on the phospholipid NMR signals disappears. The plot of the p2H dependence of the chemical shift of the outer trimethylammonium resonance of phosphatidylcholine liposomes with praseodymium ions present only on the outside of vesicles, results in a sigmoidal titration curve with its midpoint at p2H 1.5. In contrast, the inner signal is not affected by p2H. If coalescence of signals is considered as indicative of complete protonation of the phosphate moiety, the value of 1.5 can be taken as the apparent pK for the ionization of that group under the experimental conditions employed, i.e., 10 mM PrCl3 in 2H2O. That the low p2H-induced merging of the signals is reversible, is shown by the reappearance of the two peaks when the p2H of the phospholipid dispersion is raised from 1 to 5.7. Since the recovery of the trimethylammonium signal splitting indicates that Pr3+ has remained excluded from the liposome inner compartment, these experiments also demonstrate that the vesicles have not been disrupted by exposure to such an extreme acidic condition as p2H 1.     

The interaction of 1-anilino-8-naphthalenesulfonate with lipids: A nuclear magnetic resonance study

The proton magnetic resonance (PMR) and phosphorus magnetic resonance (PhMR) spectra of egg phosphatidylcholine in the presence of 1-anilino-8-naphthalenesulfonate (ANS) have been studied. At low ratios of ANS to phospholipid, the spectra indicate that ANS molecules are in the lipid interface region where they interact with the head-group protons. ANS also penetrates into the hydrocarbon region to some extent. As the ANS/phospholipid ratio approaches one, a significant splitting of the head-group signal occurs. This splitting is associated with head-group signals from inner and outer molecules of the phospholipid vesicles. As the ANS/phospholipid ratio is further increased, a gel phase often occurs. The spectra for this gel phase suggest a highly mobile head-group. Further ANS addition results in a PMR spectrum suggestive of ANS—phospholipid micelle formation. The results for a phospholipid—cholesterol complex and for the total lipid extract from a cell membrane show that the ANS effect is more complicated in these cases.     

Fusion of liposomes containing a novel cationic lipid, N-[2,3-(dioleyloxy)propyl]-N,N,N-trimethylammonium: induction by multivalent anions and asymmetric fusion with acidic phospholipid vesicles

The fusion behavior of large unilamellar liposomes composed of N-[2,3-(dioleyloxy)propyl]-N,N,N-trimethylammonium (DOTMA) and either phosphatidylcholine (PC) or phosphatidylethanolamine (PE) has been investigated by a fluorescence resonance energy transfer assay for lipid mixing, dynamic light scattering, and electron microscopy. Polyvalent anions induced the fusion of DOTMA/PE (1:1) liposomes with the following sequence of effectiveness: citrate greater than EDTA greater than phosphate, in the presence 100 mM NaCl, pH 7.4. Sulfate, dipicolinate, and acetate were ineffective. DOTMA/PC (1:1) vesicles were completely refractory to fusion in the presence of multivalent anions in the concentration range studied, consistent with the inhibitory effect of PC in divalent cation induced fusion of negatively charged vesicles. DOTMA/PE vesicles could fuse with DOTMA/PC vesicles in the presence of high concentrations of citrate, but not of phosphate. Mixing of DOTMA/PE liposomes with negatively charged phosphatidylserine (PS)/PE or PS/PC (1:1) vesicles resulted in membrane fusion in the absence of multivalent anions. DOTMA/PC liposomes also fused with PS/PE liposomes and, to a limited extent, with PS/PC liposomes. These observations suggest that the interaction of the negatively charged PS polar group with the positively charged trimethylammonium of DOTMA is sufficient to mediate fusion between the two membranes containing these lipids and that the nature of the zwitterionic phospholipid component of these vesicles is an additional determinant of membrane fusion.     

Diazeniumdiolate reactivity in model membrane systems.

The effect of small unilamellar phospholipid vesicles on the acid-catalyzed dissociation of nitric oxide from diazeniumdiolate ions, R(1)R(2)N[N(O)NO](-), [1: R(1)=H(2)N(CH(2))(3)-, R(2)=H(2)N(CH(2))(3)NH(CH(2))(4)-; 2: R(1)=R(2)=H(2)N(CH(2))(3)-; 3: R(1)=n-butyl-, R(2)=n-butyl-NH2+(CH(2))(6)-; 4: R(1)=R(2)=nPr-] has been examined at pH 7.4 and 37 degrees C. NO release was catalyzed by anionic liposomes (DPPG, DOPG, DMPS, POPS and DOPA) and by mixed phosphatidylglycerol/phosphatidylcholine (DPPG/DPPC and DOPG/DPPC) covesicles, while cationic liposomes derived from 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and the zwitterionic liposome DMPC did not significantly affect the dissociation rates of the substrates examined. Enhancement of the dissociation rate constant in DPPG liposome media (0.010M phosphate buffer, pH 7.4, 37 degrees C) at 10mM phosphoglycerol levels, ranged from 37 for 1 to 1.2 for the anionic diazeniumdiolate 4, while DOPA effected the greatest rate enhancement, achieving 49-fold rate increases with 1 under similar conditions. The observed catalysis decreases with increase in the bulk concentration of electrolytes in the reaction media. Quantitative analysis of catalytic effects has been obtained through the application of pseudo-phase kinetic models and equilibrium binding constants at different liposome interfaces are compared. The stoichiometry of nitric oxide release from 1 and 2 in DPPG/DPPC liposome media has been obtained through oxyhemoglobin assay. DPPG=1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)], DOPG=1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)], DMPS=1,2-dimyristoyl-sn-glycero-3-[phospho-l-serine], POPS=1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-l-serine], DOPA=1,2-dioleoyl-sn-glycero-3-phosphate; DPPC=1,2-dipalmitoyl-sn-glycero-3-phosphocholine, DMPC=1,2-dimyristoyl-sn-glycero-3-phosphocholine, DOTAP=1,2-dioleoyl-3-trimethylammonium-propane.     

Osmotic behavior of liposomes of phosphatidylcholine and phosphatidylsulfocholine as a function of lipid concentration

A relationship between the initial rate of liposome swelling, d(1/A)/dt and the reciprocal of the lipid concentration of the liposomes has been derived and then utilized to describe the osmotic swelling behavior of serially diluted liposomes and chloroplasts exposed to hypertonic urea solutions. The slopes of plots of d(1/A)/dt vs. the reciprocal of the lipid concentration of liposomes were not affected by differences in the initial absorbance of phosphatidylcholine-sterol bilayers, and were used to assess the ability of sterols to reduce the initial rates of urea permeation through dimyristoylphosphatidylcholine (DMPC) bilayers in the liquid-crystalline state. Multilamellar liposomes and sonicated vesicles were prepared from dimyristoylphosphatidylsulfocholine (DMPSC), in which the quaternary ammonium group of choline is replaced by -S⁺(CH₃)₂. Cholesterol reduced the initial rate of osmotic urea penetration into liposomes and the rate of 6-carboxyfluorescein efflux from vesicles at 35°C. The effect of cholesterol on bilayers of phosphatidylsulfocholine and phosphatidylcholine was very similar, suggesting that no strict structural requirements need be met in the choline moiety for lecithin-cholesterol interaction. The sulfonium analog could thus functionally replace phosphatidylcholine in natural membranes.     

Use of phospholipid exchange protein to measure inside-outside transposition in phosphatidylcholine liposomes

The exchange of phosphatidylcholine between [³²P]phosphatidylcholine liposomes and unlabeled mitochondria was catalyzed by a purified phospholipid exchange protein from bovine heart cytosol. The loss of [³²P]phosphatidylcholine from the liposomes appeared to proceed in two stages: with 100 units of phospholipid exchange protein per ml the half-time of initial stage was about 10 min and that of the final stage 4 days or greater. Agarose-gel chromatography of the liposomes showed an elution compatible with a homogeneous pool of small single walled vesicles. Treatment of phosphatidyl [¹⁴C]choline liposomes with phospholipase D (phosphatidylcholine phosphatidohydrolase) showed that labeled phospholipid removable during the rapid exchange phase was subject to hydrolysis by the phospholipase, but that the labeled phospholipid left after the rapid exchange was completed could not be hydrolyzed by phospholipase D. It is proposed that the rapidly exchanging phosphatidylcholine constitutes the outer layer of the liposome bilayer. The long half-lives of 4 days or more probably represent the transposition of Phosphatidylcholine from the inner to the outer layer of the liposome bilayer.     

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

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

Tissue distribution of EDTA encapsulated within liposomes of varying surface properties

Liposomes containing ethylenediaminetetraacetic acid (EDTA) were prepared with different surface properties by varying the liposomal lipid constituents. Positively charged liposomes were prepared with a mixture of phosphatidylcholine, cholesterol, and stearylamine. Negatively charged liposomes were prepared with a mixture of phosphatidylcholine, cholesterol, and phosphatidylserine. Neutral liposomes were prepared with phosphatidylcholine alone, dipalmitoyl phosphatidylcholine alone, or with a mixture of phosphatidylcholine and cholesterol. Distributions of ¹⁴C-labeled EDTA were determined in mouse tissues from 5 min to 24 h after a single intravenous injection of liposome preparation. Differences in tissue distribution were produced by the different liposomal lipid compositions. Uptake of EDTA by spleen and marrow was highest from negatively charged liposomes. Uptake of EDTA by lungs was highest from positively charged liposomes; lungs and brain retained relatively high levels of EDTA from these liposomes between 1 and 6 h after injection. Liver uptake of EDTA from positively or negatively charged liposomes was similar; the highest EDTA uptake by liver was from the neutral liposomes composed of a mixture of phosphatidylcholine and cholesterol. Liposomes composed of dipalmitoyl phosphatidylcholine produced the lowest liposomal EDTA uptake observed in liver and marrow but modrate uptake by lungs. Tissue uptake and retention of EDTA from all of the liposome preparations were greater than those of non-encapsulated EDTA. The results presented demonstrate that the tissue distribution of a molecule can be modified by encapsulation of that substance into liposomes of different surface properties. Selective delivery of liposome-encapsulated drugs to specific tissues could be effectively used in chemotherapy and membrane biochemistry.     

The metabolism of the phosphonium analogue of choline in cultured cells. A useful nuclear-magnetic-resonance probe for membrane phosphatidylcholine.

1. Replacement of choline by the phosphonium analogue does not affect the growth rate of P815Y, NIL, 3T3, and SV40/3T3 cells in culture. 2. The fatty acid composition of the resulting phosphonium phosphatidylcholine is similar to that of phosphatidylcholine. 3. The rate of synthesis and degradation of phosphatidylcholine and of the phosphonium analogue are similar. 4. Phospholipid-exchange protein does not distinguish between phosphatidylcholine and the phosphonium analogue. 5. By contrast, incorporation of phosphonium choline into sphingomyelin occurs to only a minor extent. 6. It is concluded that, since the enzymes involved in the turnover of phosphatidylcholine do not discriminate between quaternary N and quaternary P in the polar head-group region, phosphonium choline should prove to be a useful probe for 31P nuclear-magnetic-resonance (n.m.r.) studies of natural membranes.     

Kinetics of the interaction of hemin liposomes with heme binding proteins

As a model for the transport of hemin across biological membranes, sonicated phosphatidylcholine liposomes with incorporated hemin were characterized. The interaction of the hemin liposomes with the heme binding proteins albumin, apomyoglobin, and hemopexin was examined as a function of liposome charge and cholesterol content. In all cases, there was an almost complete transfer of hemin from liposome to protein; a rapid phase and a slow phase were observed for the transfer. For negatively charged liposomes (with 11% dicetyl phosphate), the rapid and slow phases showed observed rates of transfer of ca. 2 and 0.01 s-1, respectively, for all three proteins. The presence of cholesterol in the liposomes decreased the observed rates by a factor of 2, and positively charged liposomes (with 11% stearylamine) showed about one-fifth the observed rates of negatively charged liposomes. The observed rates were independent of protein concentration, indicating that the rate-determining step is hemin efflux from the lipid bilayer. The hemin interaction with the phospholipid bilayer is suggested to be primarily hydrophobic with some electrostatic character. The two phases are suggested to arise from two different populations of hemin within the liposomes and are interpreted as arising from two different orientations of hemin within the bilayer.     

Interaction of alkylphospholipid liposomes with MT-3 breast-cancer cells depends critically on cholesterol concentration

We have investigated interaction of alkyphospholipid (APL) liposomes consisting of 1,1-dimethylpiperidin-1-ium-4-yl) octadecyl phosphate (OPP) and different concentrations of cholesterol (CH) with human MT-3 breast-cancer cells using electron paramagnetic resonance method (EPR) with advanced characterization of EPR spectra of spin labeled liposome membranes. After incubation of OPP liposomes with MT-3 cells, a reduction of liposome entrapped, water soluble spin-probe tempocholine (ASL) was observed, indicating that ASL is released from liposomes and is reduced by oxy-redoxy systems inside the cells. This process is fast if cholesterol content in the bilayer was 29 or 45 mol%, whereas at 56 mol% cholesterol the process is almost stopped. The rate of spin-probe reduction in first 10 min after incubation with cells is even faster as for the free ASL, indicating that liposomes with low amount of cholesterol accelerate penetration of ASL into the cells. A faster release of hydrophilic material from liposomes with low cholesterol content coincides with the presence of domains with highly disordered alkyl chain motion that disappears at 50 mol% of cholesterol. We propose that these highly fluid domains are responsible for interaction of OPP liposomes with cells and fast release of the entrapped material into the cells. These results suggest that micelles are not the only reason for cytotoxic effect of OPP liposome formulations, as it was suggested before. OPP in liposomes, containing 45 mol% cholesterol or less, also contributes to the cytotoxic effect, due to their fast interaction with breast-cancer cells.     

The Effect of Cholesterol in the Liposome Bilayer on the Stabilization of Incorporated Retinol

The effect of cholesterol in the liposome bilayer on the stability of incorporated retinol was studied. Retinol was incorporated into liposomes containing soybean phosphatidylcholine (PC) and cholesterol (CH) at various ratios, and the liposomes were prepared as multilamellar vesicles by the dehydration–rehydration method. Retinol readily incorporated into liposomes at a ratio of 0.01:1 (w/w) retinol:lipid, with over 94.52% being incorporated in all conditions studied. The incorporation efficiency of retinol increased slightly with increasing CH content in the liposome and with increasing pH of the hydration buffer. Average particle size increased as the CH content increased, and mean particle sizes at pH 5, 7, and 9 were 30.27, 89.53, and 41.42 µm, respectively. The time course of retinol degradation in aqueous solution in liposomes with various ratios of PC to CH was determined under a variety of pH conditions (pH 5, 7, and 9), and temperatures (4, 25, 37, and 50°C). The stability of incorporated retinol was enhanced by increasing the CH content. At pH 7.0 and 4°C, for example, 90.17% of the retinol in liposomes containing 50:50 (PC:CH) remained after 10 days of storage, whereas 51.46% remained at 100:0 (PC:CH). These results indicate that CH in liposomes greatly increases the incorporation efficiency of retinol and the stability of incorporated retinol.     

The effect of cholesterol in the liposome bilayer on the stabilization of incorporated Retinol

The effect of cholesterol in the liposome bilayer on the stability of incorporated retinol was studied. Retinol was incorporated into liposomes containing soybean phosphatidylcholine (PC) and cholesterol (CH) at various ratios, and the liposomes were prepared as multilamellar vesicles by the dehydration-rehydration method. Retinol readily incorporated into liposomes at a ratio of 0.01:1 (w/w) retinol:lipid, with over 94.52% being incorporated in all conditions studied. The incorporation efficiency of retinol increased slightly with increasing CH content in the liposome and with increasing pH of the hydration buffer. Average particle size increased as the CH content increased, and mean particle sizes at pH 5, 7, and 9 were 30.27, 89.53, and 41.42 microm, respectively. The time course of retinol degradation in aqueous solution in liposomes with various ratios of PC to CH was determined under a variety of pH conditions (pH 5, 7, and 9), and temperatures (4, 25, 37, and 50 degrees C). The stability of incorporated retinol was enhanced by increasing the CH content. At pH 7.0 and 4 degrees C, for example, 90.17% of the retinol in liposomes containing 50:50 (PC:CH) remained after 10 days of storage, whereas 51.46% remained at 100:0 (PC:CH). These results indicate that CH in liposomes greatly increases the incorporation efficiency of retinol and the stability of incorporated retinol.     

Influence of liposome charge on the association of liposomes with Kupffer cells in vitro. Effects of divalent cations and competition with latex particles

We studied the interaction of large unilamellar liposomes carrying different surface charges with rat Kupffer cells in maintenance culture. In addition to 14C-labeled phosphatidylcholine, all liposome preparations contained either 3H-labeled inulin or 125I-labeled bovine serum albumin as a non-degradable or a degradable aqueous space marker, respectively. With vesicles carrying no net charge, intracellular processing of internalized liposomes caused nearly complete release of protein label into the medium in acid-soluble form, while phospholipid label was predominantly retained by the cells, only about one third being released. The presence of the lysosomotropic agent, ammonia, inhibited the release of both labels from the cells. At 4 degrees C, the association and degradation of the vesicles were strongly reduced. These results are very similar to what we reported on negatively charged liposomes (Dijkstra, J., Van Galen, W.J.M., Hulstaert, C.E., Kalicharan, D., Roerdink, F.H. and Scherphof, G.L. (1984) Exp. Cell Res. 150, 161-176). The interaction of both types of vesicles apparently proceeds by adsorption to the cell surface followed by virtually complete internalization by endocytosis. Similar experiments with positively charged vesicles indicated that only about half of the liposomes were taken up by the endocytic route, the other half remaining adsorbed to the cell-surface. Attachment of all types of liposomes to the cells was strongly dependent on the presence of divalent cations; Ca2+ appeared to be required for optimal binding. Neutral liposomes only slightly competed with the uptake of negatively charged vesicles, both at 4 degrees and 37 degrees C, whereas negatively charged small unilamellar vesicles and negatively charged latex beads were found to compete very effectively with the large negatively charged liposomes. Neutral vesicles competed effectively for uptake with positively charged ones. These results suggest that neutral and positively charged liposomes are largely bound by the same cell-surface binding sites, while negatively charged vesicles attach mainly to other binding sites.     

Analysis of 1H NMR-detectable mobile lipid domains for assessment of apoptosis induced by inhibitors of DNA synthesis and replication

Cell membrane rearrangements coincident with apoptosis may contribute to the increase in the ratio of methylene (CH(2) at 1.3 ppm) to methyl (CH(3) at 0.9 ppm) resonance signal intensity as observed by proton nuclear magnetic resonance ((1)H NMR). We studied CH(2) and CH(3) resonances in cultured cell lines treated with etoposide and fludarabine or bioflavonoid quercetin. Etoposide treatment (10 microM, 18 h) resulted in 3.3 fold increase of the CH(2)/CH(3) signal intensity ratio and 6.4 fold decrease in choline signal of MT4 cells. Incubation of Namalwa cells with fludarabine (3 microM, 72 h) increased the CH(2)/CH(3) signal intensity ratio by 2.4 fold and choline resonance intensity was unchanged. Quercetin treatment (30 microM, 1.5 month) increased CH(2)/CH(3) ratio by 2.1 fold. Necrotic cell death upon ethanol (20%) or DMSO (30%) treatment did not change the CH(2)/CH(3) signal intensity ratio. (1)H NMR-based study of mobile lipid domains is sensitive for detection of early engagement into apoptosis.