Evaluation of in vitro stability of small unilamellar vesicles coated with collagen and chitosan

To improve the in vitro stability of small unilamellar vesicles (SUV), the permeability of SUV, made from soyabean phosphatidylcholine (PC) and coated with collagen and chitosan, was studied using 5(6)-carboxyfluorescein (5(6)-CF) as a fluorescence probe. The results showed that the coating with a collagen/PC weight ratio of 2, or a chitosan/PC weight ratio of 8, significantly decreased the permeability of liposomal membranes. In addition, the fluorescence polarization method was used to study the influence of a coating with collagen and chitosan at the above ratios on the fluidity of liposomal membranes, employing an intramolecular charge transfer compound, a 3-methoxy-4′-N ,N-dimethylaminoflavone derivative (DMMF), as a fluorescence probe. The fluidity of a liposomal membrane coated with either of the two macromolecules showed no obvious changes, indicating that SUV coated with collagen and chitosan at appropriate weight ratios, can significantly improve the in vitro stability of liposomal membranes without disturbing their fluidity. © 1999 Society of Chemical Industry     

Comparative Benzene-Induced Fatty Acid Changes in a Rhodococcus Species and Its Benzene-Sensitive Mutant: Possible Role of Myristic and Oleic Acids in Tolerance

A Gram positive bacterium of the genus Rhodococcus was isolated from a contaminated site in Sydney, Australia, for its ability to tolerate and degrade high concentrations of benzene. To identify fatty acids that may impart this Rhodococcus sp. with tolerance to toxic solvents, a benzene-sensitive strain, labeled M2b, was isolated using EMS mutagenesis. A comparative analysis of fatty acid profiles showed that strain M2b was unable to increase its saturated:unsaturated ratio of fatty acids to the level achieved by the w-t strain when both strains were challenged with benzene. This was due to M2b's increased abundance of myristic acid, and decreased abundance of oleic acid. In addition, by measuring the generalized polarization of the fluorescent membrane probe laurdan using fluorescence spectroscopy, we have shown for the first time the effects of an aromatic hydrocarbon on the membrane fluidity of a Rhodococcus sp. The fluidity of the membranes increased after only 0.5 hr of exposure to benzene, thus suggesting the partitioning of benzene within the lipid bilayer. The response of this Rhodococcus sp. to benzene may suggest a mechanism for how other microorganisms survive when toxic solvents are released within the vicinity of their environment.     

Destabilizing effects of fructose-1,6-bisphosphate on membrane bilayers

Fructose-1,6-bisphosphate (FBP) is a high-energy glycolytic intermediate that decreases the effects of ischemia; it has been used successfully in organ perfusion and preservation. How the cells utilize external FBP to increase energy production and the mechanism by which the molecule crosses the membrane bilayer are unclear. This study examined the effects of FBP on membrane bilayer permeability, membrane fluidity, phospholipid packing, and membrane potential to determine how FBP crosses the membrane bilayer. Large unilamellar vesicles composed of egg phosphatidylcholine (Egg PC) were made and incubated with 50 mM FBP spiked with ¹⁴C-FBP at 30°C. Uptake of FBP was significant (P<0.05) and dependent on the lipid concentration, suggesting that FBP affects membrane, bilayer permeability. With added calcium (10 mM), FBP uptake by lipid vesicles decreased significantly (P<0.05). Addition of either 5 or 50 mM FBP led to a significant increase (P<0.05) in Egg PC carboxyfluorescein leakage. We hypothesized that the membrane-permeabilizing effects of FBP may be due to a destabilization of the membrane bilayer. Small unilamellar vesicles composed of dipalmitoyl pC (DPPC) were made containing either diphenyl-1,3,5-hexatriene (DPH) or trimethylammmonia-DPH (TMA-DPH) and the effects of FBP on the fluorescence anisotropy (FA) of the fluorescent labels examined. FBP caused a significant decrease in the FA of DPH in the liquid crystalline state of DPPC (P<0.05), had no effect on FA of TMA-DPH in the liquid crystalline state of DPPC, but increased the FA of TMA-DPH in the gel state of DPPC. From phase transition measurements with DPPC/DPH or TMA-DPH, we calculated the slope of the phase transition as an indicator of the cooperativity of the DPPC molecules. FBP significantly decreased the slope, suggesting a decrease in fatty acyl chain interaction (P<0.05). The addition of 50 mM FBP caused a significant decrease (P<0.05) in the liquid crystalline/gel state fluorescence ratio of merocyanine 540, indicating increased head-group packing. To determine what effects these changes would have on cellular membranes, we labeled human endothelial cells with the membrane potential probe 3,3′-dipropylthiacarbocyanine iodide (DiSC₃) and then added FBP. FBP caused a significant, dose-dependent decrease in DiSC₃ fluorescence, indicating membrane depolarization. We suggest that FBP destabilizes membrane bilayers by decreasing fatty acyl chain interaction, leading to significant increases in membrane permeability that allow FBP to diffuse into the cell where it can be used as a glycolytic intermediate.     

Lipid molecular order in liver mitochondrial outer membranes, and sensitivity of carnitine palmitoyltransferase I to malonyl-CoA

Mitochondrial outer membranes were prepared from livers of rats that were in the normal fed state, starved for 48 h, or made diabetic by injection of streptozotocin. Membranes were also prepared from starved late-pregnant rats. The latter three conditions have previously been shown to induce varying degrees of desensitization of mitochondrial overt carnitine palmitoyltransferase (CPT I) to malonyl-CoA inhibition. We measured the fluorescence polarization anisotropy of two probes, 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene-p-toluenesulfonate (TMA-DPH) which, when incorporated into membranes, report on the hydrophobic core and on the peripheral regions of the bilayer, respectively. The corresponding polarization indices (r _DPH and r _TMA-DPH) were calculated. In membranes of all three conditions characterized by CPT I desensitization to malonyl-CoA, r _DPH was decreased, whereas there was no change in r _TMA-DPH, indicating that CPT I is sensitive to changes in membrane core, rather than peripheral, lipid order. The major lipid components of the membranes were analyzed. Although significant changes with physiological state were observed, there was no consistent pattern of changes in gross lipid composition accompanying the changes to membrane fluidity and CPT I sensitivity to malonyl-CoA. We conclude that CPT I kinetic characteristics are sensitive to changes in lipid composition that are localized to specific membrane microdomains.     

Thermal adaptation in biological membranes: Functional significance of changes in phospholipid molecular species composition

Quantities of 1-palmitoyl 2-docosahexaenoyl phosphatidylcholine (16∶0/22∶6-PC or PDPC) increase from 24 to 40 weight percent as a consequence of cold acclimation in mitochondrial membranes of rainbow trout liver (J. Comp. Physiol. 156, 665–674, 1986). The present study was undertaken to assess the impact of such a large change in the proportions of a single molecular species on the fluidity, lateral packing (as sensed by phospholipase A₂), and permeability of biological membranes. These properties were examined in multilamellar liposomes prepared from binary mixtures of dipalmitoyl phosphatidylcholine (DPPC) and PDPC in proportions increasing from 10 to 40 mole% PDPC. Glucose permeability was positively correlated with both assay temperature and PDPC content. The temperature dependence of Na⁺ permeability declined steadily as the mole fraction of PDPC increased; consequently, sodium permeability was positively correlated with PDPC content at 5°C, but inversely correlated at 20°C. Phospholipase A₂ activity was independent of both assay temperature and vesicle composition. Vesicles of all compositions displayed a single transition in the temperature dependence of 1,6 diphenyl-1,3,5-hexatriene (DPH) fluorescence polarization, which shifted to lower temperature and broadened as proportions of PDPC increased. At temperatures belowthe transition, fluidity was positively correlated with the mole fraction of PDPC, but interfacial and deeper regions of the bilayer were affected differently by variations in PDPC content. Nonelectrolyte permeability was the only index of membrane structure or function to be significantly correlated with the fluidity of the bilayer interior. The tendencies of PDPC to both fluidize the membrane and to reduce the temperature sensitivity of electrolyte permeation may promote the adaptation of membrane function to low temperature.     

Non-Polar Lipids as Regulators of Membrane Properties in Archaeal Lipid Bilayer Mimics

The modification of archaeal lipid bilayer properties by the insertion of apolar molecules in the lipid bilayer midplane has been proposed to support cell membrane adaptation to extreme environmental conditions of temperature and hydrostatic pressure. In this work, we characterize the insertion effects of the apolar polyisoprenoid squalane on the permeability and fluidity of archaeal model membrane bilayers, composed of lipid analogues. We have monitored large molecule and proton permeability and Laurdan generalized polarization from lipid vesicles as a function of temperature and hydrostatic pressure. Even at low concentration, squalane (1 mol%) is able to enhance solute permeation by increasing membrane fluidity, but at the same time, to decrease proton permeability of the lipid bilayer. The squalane physicochemical impact on membrane properties are congruent with a possible role of apolar intercalants on the adaptation of Archaea to extreme conditions. In addition, such intercalant might be used to cheaply create or modify chemically resistant liposomes (archeaosomes) for drug delivery.     

Effect of Sonication on the Thermotropic Behavior of DODAB Vesicles Studied by Fluorescence Probe Solubilization

The effect of sonication on fluorescence probe solubilization in cationic vesicles of dioctadecyldimethylammonium bromide (DODAB) was investigated by steady-state fluorescence of pyrene (Py), trans-diphenylpolyenes—diphenylbutadiene (DPB), diphenylhexatriene (DPH), and their corresponding 4,4′-dialkyl derivatives 4B4A and 4H4A fluorescence probes. The data indicate that sonication affects the bilayer polarity, the melting temperature (T _m), and the cooperativity of the melting process due to changes in vesicle morphology. The effect of temperature on the fluorescence intensity and yielding Φ_f and anisotropy <r> shows that the ionizable probes 4B4A and 4H4A are solubilized close to the vesicle interfaces, whereas the non-ionizable DPH and DPB are deeper in the bilayers. Py solubilization indicates that sonicated vesicles exhibit less densely packed bilayers.     

Biophysical studies of a synthetic mimic of the apoptosis-detecting protein annexin v

A Zn²⁺-dipicolylamine coordination compound is shown to sense the presence of anionic phospholipids in a membrane bilayer. The sensor contains two dipicolylamine subunits attached to an anthracene scaffold, which exhibits a maximum absorbance at 380 nm, and undergoes an enhancement in fluorescence intensity when exposed to membranes enriched in phosphatidylserine. For these reasons, the compound is referred to as PSS-380 (Phosphatidylserine Sensor, 380 nm). The fluorescence emission of PSS-380 is enhanced up to tenfold by the presence of vesicles containing the anionic phospholipids phosphatidylserine, phosphatidylglycerol, or phosphatidic acid. No enhancement in fluorescence is observed upon exposure to vesicles containing only zwitterionic phosphatidylcholine, or exposure to monodispersed (non-aggregated) anionic phospholipids. The sensing effect is cooperative; not only does association to the vesicles increase if the vesicles have raised levels of anionic phospholipid, but the maximum fluorescence at sensor saturation is also enhanced. It appears that sensing is triggered by the three-component self-assembly of sensor, Zn²⁺, and the anionic membrane surface, which leads to diminished photo-induced electron transfer (PET) quenching. The utility of PSS-380 in flow cytometry and fluorescence microscopy is demonstrated by using the molecule to detect the appearance of phosphatidylserine on the plasma membrane surface of various cell lines. Thus, PSS-380 can identify apoptotic cells in the same way as the commonly used protein reagent annexin V.     

Incorporation oftrans fatty acids into submandibular salivary gland lipids

Three groups of rats were fed diets containing 20% corn oil, 20% margarine stock (MS) or 19% MS +1% corn oil. Diets were fed for 12 weeks, 1 week of pregnancy, 3 weeks of lactation and 8 weeks post-weaning. The incorporation oftrans-octadecenoate into various lipids of the submandibular salivary gland (SMSG) homogenates and plasma membranes was studied.Trans octadecenoate was incorporated into all the lipid fractions studied. Its levels were the highest in phosphatidylethanolamine. The double bond index of phospholipid fatty acids in the plasma membranes of the SMSG was substantially lower in the group fed 20% MS. The fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) was generally higher in the membranes of SMSG from rats fed MS than that of the other two groups, thus indicating lower fluidity. Also, the breakpoints in fluorescence polarization were at a higher temperature in the membranes from rats fed MS as compared with those fed corn oil. Lower fluidity of plasma membranes of SMSG observed in rats fed 20% MS may result in modification of the activities of membrane-bound enzymes. Part of this work was presented at the Federation of American Societies for Experimental Biology (FASEB) 68th Annual Meeting, St. Louis, Missouri, April 1984. Alam, S.Q., Alam, B.S., and Banerji, A. Fed. proc. 43,317 (1984).     

Composition and physical state of phospholipids in calanoid copepods from India and Norway

The fatty acid composition and physical state of isolated phospholipids obtained from marine copepods collected on the Southwest coast of India (Calanus ssp.) and the West coast of Norway (Calanus finmarchicus) were investigated to compare the adaptation of membrane lipids with seawater temperatures. Phospholipid vesicles obtained from the tropic copepods proved more rigid than those fromC. finmarchicus, as assessed by diphenylhexatriene fluorescence polarization techniques. In each case, there were two breaks present on the fluorescence polarization vs 1/T plots, suggesting that the onset and completion of phase separation to occurred above 0 C. For the tropic copepods, the onset of phase separation roughly corresponded to the ambient water temperature, while forC. finmarchicus some discrepancies were observed, depending on the time of the year. Phospholipids in copepods from both habitats contained more than 50% unsaturated fatty acids, the animals from Norway containing slightly higher amounts. The data indicate an adaptation of membranes to temperature.     

Surface modification of vesicles with methylol urea

Surface-modified vesicles were prepared using N-[3-(dimethylamino)propyl]-octadecanamide and stearic acid as bilayer-forming lipids, and N-methylol urea-dodecylamine conjugated (MU-DOA) as a surface modifier. The conjugation of MU to DOA was confirmed by FTIR spectra. MU-DOA was incorporated into the vesicles by co-homogenization of the lipids and MU-DOA, and the incorporated MU-DOA was then reacted with MU in aqueous bulk phase through a self-condensation reaction between the methylols under an acidic condition at 70°C. On a scanning electron microscope, the vesicles were spherical and multilamellar, and they exhibited thin polymer films on their surfaces. The incorporation of MU-DOA into the bilayer and the surface coating of the vesicles did not significantly influence the transition temperature of the vesicles. The absolute values of zeta potentials of the surface-modified vesicles were smaller than those of the unmodified vesicles, and the point of zero charge was shifted from ca. pH 9.5 to ca pH 6.5 by the surface modification.     

包覆NIPAM—ODA双亲共聚物的脂质体的温控释放行为

为了研究对温度敏感的双亲性共聚物包覆的脂质体的温控释放行为，合成了Ｎ－异丙基丙烯酰胺（ＮＩＰＡＭ）和丙烯酸十八酯（ＯＤＡ）的共聚物。利用荧光探针法研究了共聚物水溶液随温度升高时出现的ＬＣＳＴ现象，表明该高分子在温度升高到３０℃以上时存在着明显的相分离行为。     

Effect of docosahexaenoic acid on mouse mitochondrial membrane properties

Long-chain polyunsaturated (n-3) fatty acids have been proposed to be involved in a wide variety of biological activities. In this study, mitochondrial docosahexaenoic acid (DHA) levels were increased by either dietary manipulation or by fusing the mitochondria with phospholipid vesicles made from 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (18:0/22:6 PC). The fused mitochondria exhibited a DHA-induced decrease in respiratory control index (RCI) and membrane potential and an increase in proton movement. The modified mitochondria also demonstrated an increase in fluidity (as detected by 1,6-diphenyl-1,3,5-hexatriene anisotropy) and changes in membrane structure detected by the fluorescence probes MC540 and pyrene decanoate. Proton movement in lipid vesicles made from mitochondrial lipid extracts was shown to be enhanced by incorporated 18:0/22:6 PC. Mitochondria were isolated from young (5-mon) and old (24-mon) mice which were maintained on either a diet rich in saturated fats (hydrogenated coconut oil) or rich in n−3 polyunsaturated fats (menhaden oil). Mitochondrial bioenergetic function was followed by RCI, state 3 respiration, ATP level, and phosphate uptake. In addition, lipid composition, phospholipid area/molecule, and extent of lipid peroxidation were also determined. Decreases in RCI for the menhaden oil diet-modified mitochondria paralleled those in which DHA levels were enhanced by fusion with phospholipid vesicles. RCI reductions are attributed to DHA-induced increases in H⁺ movement, producing diminished mitochondrial membrane potentials. One purpose of this project was to determine if the deleterious effects of aging on mitochondrial bioenergetic function could be reversed by addition of n−3 fatty acids. The experiments reported here indicate that incorporation of long-chain polyunsaturated n−3 fatty acids into mitochondrial membranes does not appear likely to reverse the effects of age on mitochondrial function.     

Fluorescence Anisotropy,FT-IR Spectroscopy and 31-P NMR Studies on the Interaction of Paclitaxel with Lipid Bilayers

To understand the bilayer interaction with paclitaxel, fluorescence polarization, Fourier transform infrared spectroscopy (FT-IR) and 31-phosphorus nuclear magnetic resonance (31P-NMR) studies were performed on paclitaxel bearing liposomes. Fluorescence anisotropy of three probes namely, 1,6-diphenyl-1,3,5-hexatriene (DPH), 12-(9-anthroyloxy) stearic acid (12AS) and 8-anilino-1-naphthalene sulfonate (ANS) were monitored as a function of paclitaxel concentration in the unsaturated bilayers. The incorporation of paclitaxel decreased anisotropy of 12AS and ANS probes, while slightly increased anisotropy of DPH. Paclitaxel has a fluidizing effect in the upper region of the bilayer whereas the hydrophobic core is slightly rigidized. FT-IR spectroscopy showed an increase in the asymmetric and symmetric methylene stretching frequencies, splitting of methylene scissoring band and broadening of carbonyl stretching mode. These studies collectively ascertained that paclitaxel mainly occupies the cooperativity region and interact with the interfacial region of unsaturated bilayers and induces fluidity in the headgroup region of bilayer. At higher loadings (>3 mol%), paclitaxel might gradually tend to accumulate at the interface and eventually partition out of bilayer as a result of solute exclusion phenomenon, resulting in crystallization; seed non-bilayer phases, as revealed by 31P-NMR, thereby destabilizing liposomal formulations. In general, any membrane component which has a rigidization effect will decrease, while that with a fluidizing effect will increase, with a bearing on headgroup interactions, partitioning of paclitaxel into bilayers and stability of the liposomes.     

A role for phospholipid polyunsaturation in modulating membrane protein function

Visual transduction is one of the best characterized G protein—coupled signalling systems. In addition, about 50% of the disk membrane phospholipid acyl chains are 22:6n-3, making this system ideal for determining the role of polyunsaturation in modulating membrane-signalling systems. The extent of formation of metarhodopsin II (MII), the G protein—activating photointermediate of rhodopsin, was studied in phospholipid vesicles composed of a variety of phosphatidylcholines, differing in their acyl chain composition at thesn-2 position. The amount of MII formed increased progressively with the level of acyl chain unsaturation at thesn-2 position. The effect of added cholesterol was to reduce the amount of MII formed. The acyl chain packing free volume of the rhodopsin containing lipid vesicles was characterized by a fractional volume parameter f_v derived from measurements of the time-resolved fluorescence anisotropy decay of the hydrophobic membrane probe 1,6-diphenyl-1,3,5-hexatriene. The relationship amongsn-2 acyl chain unsaturation, cholesterol content, and MII formation is explained on the basis of variation in f_v with bilayer lipid composition and a novel model for the packing of phospholipids containing polyenoic acyl chains, such as 22:6n-3.     

DPH标记法研究自由基对活细胞膜流动性的影响

以组织培养的肝活细胞Ｌ－０２为材料,对荧光探剂ＤＰＨ标记活细胞膜脂的荧光光谱进行了研究,同时探讨了Ｆｅ（Ⅱ）诱发的活性氧自由基对人肝细胞膜脂流动性的影响及茶多酚的调节。结果表明,ＤＰＨ标记Ｌ－０２细胞后,荧光峰值蓝移,同时标记时间在３０－９０分钟间荧光偏振度P值基本稳定在 ０． ２７７－０． ２８０。 ０－５０ μ ｇ／ｍｌ的茶多酚对 Ｌ－０２细胞膜脂的流动性影响不大, Ｆｅ（Ⅱ）诱发的活性氧自由基则明显降低细胞膜的流动性, １０、 ２０、 ５０ μ ｓ／ｍｌ的 ＴＰ能保护细胞膜脂的流动性,且呈量效关系。     

Synthesis and biological evaluation of some new coumarinyl thiazolopyrimidinones

Two new series of coumarin linked, linear and angularly fused thiazolo-[3,2-a]-pyrimidinones have been synthesized from 3-bromoacetyl coumarins by azole and azine approaches. Regioisomeric 5H and 7H thiazolo-[3,2-a]-pyrimidinones have been clearly distinguished by their IR and UV fluorescence spectral data. All the compounds have been characterized by analytical and spectroscopic methods. Rate and yield enhancements have been achieved using microwave irradiation. The results of in vivo diuretic activity indicate that substituents on coumarin do not enhance the activity. In vitro antimicrobial activities have shown that the compounds are specifically active against Gram-positive but are inactive against Gram-negative bacterial strains. Moderate fungal activity was observed against Candida albicans and Penicillium chrysogenum and all the compounds were found to be inactive against Aspergillus niger.     

An Artificial Channel Type Ionophore

A polymer of an isocyanide (R–N=C<)_n has been synthesized in which R contains a benzo-18-crown-6 group. The crown ether rings are on top of each other and form channels which bind metal ions. The channel compound can be incorporated in vesicles and is able to transport ions across the bilayer membrane.     

Solubilization of phospholipid vesicular structures into mixed micelles of zwitterionic surfactants

Interactions between the binary combination of dimethyltetradecylammoniopropanesulfonate (TPS) and l-α-phosphatidylcholine (PC), 1,2-didecanoyl-sn-glycero-3-phosphocholine, and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine in the aqueous bulk phase were evaluated with the help of pyrene fluorescence (l ₁/l ₃) measurements by studying the aggregation processes of TPS in pure water and in the presence of 7–36 μM of fixed concentrations of each lipid. The fluorescence measurements showed that TPS monomers undergo two kinds of aggregation process, which were identified by the three breaks. The first break, C₁, and the second, C₂, indicated the onset and completion of bilayer solubilization, respectively, on the incorporation of TPS monomers into the bilayer assemblies, which led to bilayer solubilization in the form of mixed micelles. This process was not clearly visible in the presence of PC, whereas some kinds of structure transitions were observed upon the incorporation of surfactant monomers. The partition coefficient (K), which defines the degree of partitioning of surfactant monomers into the bilayers with respect to the aqueous medium, was evaluated. A high K value of TPS-lipid aggregates indicated stronger interactions between surfactant and bilayer assemblies of lipid. The K values determined for the three phospholipids are close to each other, which indicates that K values do not depend on the hydrocarbon chain length of the phospholipid but of the surfactant used.     

Lipid exchanges between dioctadecyldimethylammonium bromide monolayer and vesicles in the subphase

Studies on the interaction of lipid monolayer with bilayer structures, such as vesicles, are relatively scarce in the literature. In order to ascertain whether these structures interact for cationic dioctadecyldimethylammonium bromide (DODAB) monolayers at the aqueous surfaces of 0, 0.05, and 0.5 mmol L⁻¹ DODAB vesicle dispersions, differential scanning calorimetry (DSC) and surface film balance experiments were carried out. DSC results confirmed the presence of unilamellar vesicles in the subphase, while changes in the monolayer surface pressure–area per molecule (π–A) isotherm profile yielded by the presence of DODAB vesicle in the subphase revealed monolayer-vesicle interactions as a result of monomer exchanges between the monolayer and the vesicles with stronger effects at the higher vesicle concentration investigated.