Egg Lecithin Vesicles Research Articles

Lipid Membrane Flexibility in Protic Ionic Liquids.

Here, we determine by neutron spin echo spectrometry (NSE) how the flexibility of egg lecithin vesicles depends on solvent composition in two protic ionic liquids (PILs) and their aqueous mixtures. In combination with small-angle neutron scattering (SANS), dynamic light scattering (DLS), and fluorescent probe microscopy, we show that the bending modulus is up to an order of magnitude lower than in water but with no change in bilayer thickness or nonpolar chain composition. This effect is attributed to the dynamic association and exchange of the IL cation between the membrane and bulk liquid, which has the same origin as the underlying amphiphilic nanostructure of the IL solvent itself. This provides a new mechanism by which to tune and control lipid membrane behavior.

PH-responsive polymer core-shell nanospheres for drug delivery

Stimuli-responsive polymer nanoparticles are playing an increasingly more important role in drug delivery applications. However, limited knowledge has been accumulated about processes which use stimuli-responsive polymer nanospheres (matrix nanoparticles whose entire mass is solid) to carry and deliver hydrophobic therapeutics in aqueous solution. In this research, pyrene was selected as a model hydrophobic drug and a pyrene-loaded core-shell structured nanosphere named poly(DEAEMA)-poly(PEGMA) was designed as a drug carrier where DEAEMA and PEGMA represent 2-(diethylamino)ethyl methacrylate and poly(ethylene glycol) methacrylate, respectively. The pyrene-loaded core-shell nanospheres were prepared via an in situ two-step semibatch emulsion polymerization method. The particle size of the core-shell nanosphere can be well controlled through adjusting the level of surfactant used in the polymerization where an average particle diameter of below 100 nm was readily achieved. The surfactant was removed via a dialysis operation after polymerization. Egg lecithin vesicles (liposome) were prepared to mimic the membrane of a cell and to receive the released pyrene from the nanosphere carriers. The in vitro release profiles of pyrene toward different pH liposome vesicles were recorded as a function of time at 37 °C. It was found that release of pyrene from the core-shell polymer matrix can be triggered by a change in the environmental pH. In particular the pyrene-loaded nanospheres are capable of responding to a narrow window of pH change from pH = 5, 6, to 7 and can achieve a significant pyrene release of above 80% within 90 h. The rate of release increased with a decrease in pH. A first-order kinetic model was proposed to describe the rate of release with respect to the concentration of pyrene in the polymer matrix. The first-order rate constant of release k was thus determined as 0.049 h−1 for pH = 5; 0.043 h−1 for pH = 6; and 0.035 h−1 for pH = 7 at 37 °C. The release of pyrene was considered to follow a diffusion-controlled mechanism. The synthesis and encapsulation process developed herein provides a new approach to prepare smart nanoparticles for efficient delivery of hydrophobic drugs. © 2013 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2013, 51, 4440–4450

Liposomes for Topical Use: A Physico-Chemical Comparison of Vesicles Prepared from Egg or Soy Lecithin

Developments in nanotechnology and in the formulation of liposomal systems provide the opportunity for cosmetic dermatology to design novel delivery systems. Determination of their physico-chemical parameters has importance when developing a nano-delivery system. The present study highlights some technological aspects/characteristics of liposomes formulated from egg or soy lecithins for topical use. Alterations in the pH, viscosity, surface tension, and microscopic/macroscopic appearance of these vesicular systems were investigated. The chemical composition of the two types of lecithin was checked by mass spectrometry. Caffeine, as a model molecule, was encapsulated into multilamellar vesicles prepared from the two types of lecithin: then zeta potential, membrane fluidity, and encapsulation efficiency were compared. According to our observations, samples prepared from the two lecithins altered the pH in opposite directions: egg lecithin increased it while soy lecithin decreased it with increased lipid concentration. Our EPR spectroscopic results showed that the binding of caffeine did not change the membrane fluidity in the temperature range of possible topical use (measured between 2 and 50 °C). Combining our results on encapsulation efficiency for caffeine (about 30% for both lecithins) with those on membrane fluidity data, we concluded that the interaction of caffeine with the liposomal membrane does not change the rotational motion of the lipid molecules close to the head group region. In conclusion, topical use of egg lecithin for liposomal formulations can be preferred if there are no differences in the physico-chemical properties due to the encapsulated drugs, because the physiological effects of egg lecithin vesicles on skin are significantly better than that of soy lecithin liposomes.

Combined Interaction of Phospholipase C and Apolipoprotein A-I with Small Unilamellar Lecithin-Cholesterol Vesicles: Influence of Apolipoprotein A-I Concentration and Vesicle Composition

We report the combined effects of phospholipase C (PLC), a pronucleating factor, and apolipoprotein A-I (apo A-I), an antinucleating factor, in solutions of model bile. Results indicate that apo A-I inhibits cholesterol nucleation from unilamellar lecithin vesicles by two mechanisms. Initially, inhibition is achieved by apo A-I shielding of hydrophobic diacylglycerol (DAG) moieties so as to prevent vesicle aggregation. Protection via shielding is temporary. It is lost when the DAG/apo A-I molar ratio exceeds a critical value. Subsequently, apo A-I forms small ( approximately 5-15 nm) complexes with lecithin and cholesterol that coexist with lipid-stabilized (400-800 nm) DAG oil droplets. This microstructural transition from vesicles to complexes avoids nucleation of cholesterol crystals and is a newly discovered mechanism by which apo A-I serves as an antinucleating agent in bile. The critical value at which a microstructural transition occurs depends on binding of apo A-I and so varies with the cholesterol mole fraction of vesicles. Aggregation of small, unilamellar, egg lecithin vesicles (SUVs) with varying cholesterol composition (0-60 mol %) was monitored for a range of apo A-I concentrations (2 to 89 microg/mL). Suppression of aggregation persists so long as the DAG-to-bound-apo A-I molar ratio is less than 100. A fluorescence assay involving dansylated lecithin shows that the suppression is an indirect effect of apo A-I rather than a direct inhibition of PLC enzyme activity. The DAG-to-total apo A-I molar ratio at which suppression is lost increases with cholesterol because of differences in apo A-I binding. Above this value, a microstructural transition to DAG droplets and lecithin/cholesterol A-I complexes occurs, as evidenced by sudden increases in turbidity and size and enhancement of Forster resonance energy transfer; structures are confirmed by cryo TEM.

Membrane transport of a polyacid-tied doxorubicin.

A model, based on fluorescence data, is developed for the poly(acrylic acid)-assisted transport of doxorubicin, a cationic antitumor drug, through a bilayer membrane. Accordingly, the doxorubicin binds to the poly(acrylic acid) via electrostatic polymer-drug interactions plus drug-drug stacking within the complex. This complex associates with neutral egg lecithin vesicles by means of hydrophobic attraction between the doxorubicin and the vesicle bilayers. In the process, the doxorubicin "destacks", providing a fluorescence change that can be monitored. Finally, the doxorubicin enters the vesicle interior which has been imparted with an acidic pH to protonate the doxorubicin and thus, in a second stage, yield an additional fluorescence change that can also be detected. A portion of the poly(acrylic acid), now devoid of doxorubicin, then leaves the outer vesicle surface and enters to external solution.

Interaction of a cationic polymer with negatively charged proteoliposomes

Proteoliposomes were prepared by making bilayer vesicles from neutral egg yolk lecithin and negatively charged α-chymotrypsin that had been previously stearoylated. Interaction of these proteoliposomes with a cationic polymer, poly-( N-ethyl-4-vinylpryidinium bromide) (PEVP) was examined. For comparison purposes, interaction of PEVP with egg lecithin vesicles containing an anionic phospholipid, cardiolipin, was also examined. Binding of PEVP to both types of vesicles was electrostatic in nature with the polymer manifesting a higher affinity to the cardiolipin relative to the enzyme. PEVP had no effect on the permeability of the bilayer membranes to sodium chloride. On the other hand, PEVP increased the transmembrane permeability of the nonionic anti-tumor drug, doxorubicin. The greater the negatively charged component in the membrane, the greater the PEVP effect. Polycation binding to the vesicles was accompanied by clustering of the stearoylated chymotrypsin (sCT) molecules within the membrane. This protein clustering is most likely responsible for the increase in the doxorubicin permeation. Enzymatic activity of the membrane-associated sCT remained unchanged upon PEVP binding. These findings seem relevant to the effects of polyelectrolytes on cellular membranes.

A study of the adsorption of bile salts onto model lecithin membranes

Bile salts play a central role in the promotion of cytotoxicity or cytoprotection. In this study, we examined the interaction of different bile salts with egg lecithin vesicles using 31P NMR spectroscopy. The effects of taurochenodeoxycholate (TCDC or 3α,7α,-dihydroxy-5β-cholanoyl taurine, of tauroursodeoxycholate (TUDC) or 3α,7β,-dihydroxy-5β-cholanoyl taurine) and of tauroβmuricholate (TβMC or 3α,6β,7β,-trihydroxy-5β-cholanoyl taurine), at various bile salt/lecithin ratios, were evaluated. From the percent 31P present in vesicles, the micellar capacity of bile salts to dissolve lecithin was determined. TCDC was incorporated into vesicles for bile salt/lecithin molar ratios lower than 0.62 while for TUDC and TβMC, the critical ratios were 0.94 and 1.1, respectively. The 31P chemical shift change was markedly larger with TCDC than that found with TUDC and TβMC. In order to specify the low interactions observed between hydrophilic bile salts and lecithin, we determined the intermixed micellar/vesicular bile salt concentrations (IMVC) of bile salt/lecithin solutions using rapid ultrafiltration–centrifugation for TUDC and lecithin solubility measurements for TUDC, TβMC and TCDC. The low IMVC obtained indicate that even hydrophilic bile salts were bound mostly to the mixed aggregates. In conclusion, the low disturbance in the arrangement of lecithin induced by TUDC and TβMC appears to be due to the interfacial location of these bile salts. TCDC (7α OH) penetrates more deeply in the membrane than the 7β hydroxylated bile salts that may partly explain the distinct damaging effects of these bile salts.

Lecithin Protects against Plasma Membrane Disruption by Bile Salts

Introduction.Detergent disruption of epithelial plasma membranes by bile salts may contribute to pathogenesis of cholestasis and gastroesophageal reflux disease. Bile, despite containing high concentrations of bile salts, normally is not toxic to biliary or intestinal epithelia. We hypothesize that lecithin in bile may protect cell membranes from disruption by bile salts.Methods.We studied the interactions of taurine conjugates of ursodeoxycholate (TUDCA), cholate (TCA), chenodeoxycholate (TCDCA), and deoxycholate (TDCA) with erythrocyte plasma membranes with or without large unilamellar egg lecithin vesicles for various times at 23°C. Release of hemoglobin was quantified spectrophotometrically. The concentration of bile salt monomers and simple micelles in the intermixed micellar aqueous phase (IMMC) was determined by centrifugal ultrafiltration.Results.The degree of hemolysis depended on the hydrophobicity of the bile salts and was progressive over time. Addition of lecithin reduced the hemolytic effects of 20 mM TCA or 2 mM TDCA in a concentration-dependent manner at both 30 min and 4 h. Increasing the concentration of lecithin progressively reduced the IMMC of TDCA. Hemolysis following addition of lecithin to 2 mM TDCA was comparable to hemolysis produced by lecithin-free TDCA solutions when diluted to similar IMMC values.Conclusion.We conclude that lecithin reduces plasma membrane disruption by hydrophobic bile salts. This protection may be attributable to association of bile salts with vesicles and mixed micelles, reducing the concentration of bile salt monomers and simple micelles available to interact with cell membranes. Lecithin may play a key role in preventing bile salt injury of biliary and gastrointestinal epithelia.

Cryo-Transmission Electron Microscopy Confirms Controlled Synthesis of Cadmium Sulfide Nanocrystals within Lecithin Vesicles

Size-quantized CdS nanocrystals of controlled dimensions are synthesized within monodisperse egg lecithin vesicles made by detergent depletion. Cryo-transmission electron microscopy (cryo-TEM) enables imaging in situ both of the vesicle wall and of the encapsulated nanocrystal. Cryo-TEM images confirm that only one particle per vesicle forms and that nanocrystal diameter is determined by the number of Cd2+ ions initially encapsulated. These micrographs further show that the particles adsorb to the inner surface of the vesicle membrane, which suggests that crystal growth and particle core structure could be controlled through manipulation of vesicle bilayer chemistry.

Influence of vesicle curvature on fluorescence relaxation kinetics of fluorophores

The effect of membrane curvature on the fluorescence decay of 2- p-toluidinyl-naphthalene-6-sulfonic acid (TNS), 2-(9-anthroyloxy) stearic acid (2-AS) and 12-(9-anthroyloxy)-stearic acid (12-AS) was investigated for egg lecithin vesicles of average diameter d m = 22 nm and 250 nm. The biexponential fluorescence decay of TNS at the red edge of the emission spectrum was analysed according to the model of Gonzalo and Montoro [1]. Over the entire temperature range (1–40°C) the small TNS labelled vesicles showed significantly shorter solvent relaxation times τ r than their larger counterparts (e.g. 1.3 ns compared with 2.1 ns at 5°C), indicating a higher mobility of the hydrated headgroups in the highly curved, small vesciles. The fluorescence decay of both AS derivatives is also biexponential. While the shorter decay times (1–3 ns) are practically identical for small and large vesicles, the longer decay times (5–14 ns) are identical only for 12-AS but not for 2-AS. This indicates that the microenvironment is similar in small and large vesicles deep in the membrane in spite of the differences in curvature.

Induction of Ca 2+ transport in liposomes by insulin

The requirement of extracellular Ca 2+ for insulin action has been indicated by past studies. With a view to understand the interaction of insulin with Ca 2+ in the vicinity of the cell membrane, we have examined the ability of insulin and its constituent polypeptide chains A and B to translocate Ca 2+ and Mg 2+ across the lipid bilayer in two sets of synthetic liposomes. The first were unilamellar vesicles made of dimyristoylphosphatidylcholine and contained the Ca 2+ sensor dye arsenazo III. Peptide-mediated Ca 2+ and Mg 2+ transport in these vesicles was monitored at 37°C in a neutral buffer containing CaCl 2 or MgCl 2 using a difference absorbance method. In the second set, multilamellar vesicles of egg lecithin containing trapped fura-2 were employed and the cation transport was followed at 20°C by fluorescence changes in the dye. Control experiments indicated that the hormonal peptides caused no appreciable perturbation of the vesicles leading to leakage of contents or membrane fusion. In both liposome systems, substantial Ca 2+ and Mg 2+ transport was observed with insulin and the B chain; the A chain was less effective as an ionophore. Quantitative analysis of the transport kinetic data on the B chain showed a 1:1 peptide-Ca 2+ complex formed inside the membrane. In light of the available structural data on Ca 2+ binding by insulin and insulin receptor, our results suggest the possibility of the hormone interacting with the receptor with the bound Ca 2+.

Mechanism of the spontaneous transfer of unconjugated bilirubin between small unilamellar phosphatidylcholine vesicles.

Unconjugated bilirubin (bilirubin-IX alpha), the hydrophobic end product of heme degradation, is esterified in the hepatocyte endoplasmic reticulum to water-soluble conjugates prior to excretion in bile. To characterize the process of intracellular bilirubin transport, the kinetic and thermodynamic activation parameters for the spontaneous transfer of bilirubin between small unilamellar egg lecithin vesicles were determined. Bilirubin-IX alpha was added to donor vesicles labeled with the fluorescent phospholipid probe, (5-(dimethylamino)naphthalene-1-sulfonyl) dipalmitoyl-L-alpha-phosphatidylethanolamine (dansyl-PE). When bound to the donor vesicles, bilirubin quenches the dansyl probe fluorescence through resonance energy transfer. The movement of bilirubin from dansyl-labeled donor vesicles to unlabeled acceptor vesicles was monitored directly by the reemergence of dansyl fluorescence over time. Vesicle fusion and intervesicle transfer of the dansyl-PE probe were excluded by quasielastic light scattering and fluorescence resonance energy transfer studies. Stopped-flow analysis demonstrated that the transfer of bilirubin was described by a single-exponential function with a mean half-time of 2.0 +/- 0.1 ms (+/- SD) at 37 degrees C. The rate of bilirubin transfer was independent of acceptor vesicle concentration and decreased with increasing buffer ionic strength, indicating that intermembrane transfer occurred via aqueous diffusion, rather than vesicle collisions. The free energy of activation (delta G++) for the dissociation of bilirubin from donor vesicles was 14.2 kcal.mol-1. These studies suggest that bilirubin is associated with phospholipid bilayers at the membrane-water interface. We postulate that the movement of unconjugated bilirubin between intracellular membranes occurs via spontaneous transfer through the aqueous phase.

Anesthetic steroid mobility in model membrane preparations examined by high-resolution proton and deuterium NMR spectroscopy

A series of structurally related pregnane analogues which exhibit a wide range of anesthetic potencies were incorporated into unilamellar egg lecithin vesicles and their relative mobilities examined with 1H and 2H high-resolution NMR spectroscopy. The data from this study reveal a trend suggesting a relationship between the motional properties of a steroid and its anesthetic potency. The data are congruent with the idea that anesthetic activity is associated with perturbation of the membrane bilayer by the steroid molecule; the degree to which the membrane is perturbed is apparently dependent upon the specific structural and stereochemical features of the steroid. This study supports the hypothesis that lipid bilayers are capable of a high degree of structural discrimination.

The Role of the Side Chain in the Antioxidant Activity of Ubiquinones

The question has been addressed whether the side chain contributes to the antioxidant activity of Ubiquinones. The length, the chemical composition and structure of the chain have been considered. The effect of the actual concentration of the quinone in egg lecithin vesicles has been investigated by means of both UV spectroscopy and time resolved fluorescence quenching experiments of 12-AS. The results indicate that the antioxidant properties of the quinone do not seem to depend on the side chain.

Evidence from e.s.r. studies for virtual immobility in niosomes derived from steroidal lariat ethers

Nitroxide radical probes exhibit correlation times which are 300 times longer in steroidal lariat ether-derived niosomes than are observed in egg lecithin vesicles and the membranes are so rigid that the spectra are almost identical to spectra observed for frozen systems.

Hydrogen peroxide permeation across liposomal membranes: a novel method to assess structural flaws in liposomes.

Hydrogen peroxide permeation across large multilamellar vesicles of defined and complex lipid composition was shown to obey precise kinetic relationships for the activity of the occluded catalase. Careful assay conditions precluded simultaneous peroxidative damage to the lipids. The kinetic data was consistent with a barrier role for the bilayer for hydrogen peroxide permeation. More interestingly, hydrogen peroxide permeation across liposomes of complex lipid mixtures exhibited osmotic inhibition of permeation of hydrogen peroxide. On the other hand, purified egg lecithin vesicles did not exhibit any effect of external osmolality on hydrogen peroxide permeation in an experimentally defined non-lytic zone of external osmolarity. These results argue in favour of a heterogeneous, heteroporous structure of bilayers with complex lipid composition.

Photochemical properties of 1,2-di-(1-naphthyl)ethylene in micellar solutions

1. Photochemical transformations of dinaphthylethylenes in micellar solutions have a number of features which are caused by the high viscosity and polarity of the medium of the micellar nucleus. 2. The rate of formation of picene increases significantly in Triton X-100 and egg lecithin vesicles.

Site-specific epsilon-NH2 monoacylation of pancreatic phospholipase A2. 2. Transformation of soluble phospholipase A2 into a highly penetrating "membrane-bound" form.

Long-chain lecithins present in bilayer structures like vesicles or membranes are only very poor substrates for pancreatic phospholipases A2. This is probably due to the fact that pancreatic phospholipases A2 cannot penetrate into the densely packed bilayer structures. To improve the weak penetrating properties of pancreatic phospholipases A2, we prepared and characterized a number of pancreatic phospholipase A2 mutants that have various long acyl chains linked covalently to Lys116 in porcine and to Lys10 in bovine phospholipase A2 [Van der Wiele, F.C., Atsma, W., Dijkman, R., Schreurs, A.M.M., Slotboom, A.J., & De Haas, G.H. (1988) Biochemistry (preceding paper in this issue)]. When monomolecular surface layers of L- and D-didecanoyllecithin were used, it was found that the introduction of caprinic, lauric, palmitic, and oleic acid at Lys116 in the porcine enzyme increases its penetrating power from 13 to about 17, 20, 32, and 22 dyn/cm, respectively, before long lag periods were obtained. Incorporation of a palmitoyl moiety at Lys10 in the bovine enzyme shifted the penetrating power from 11 to about 25 dyn/cm. Only the best penetrating mutant, viz., porcine phospholipase A2 having a palmitoyl moiety at Lys116, was able to cause complete leakage of 6-carboxyfluorescein entrapped in small unilamellar vesicles of egg lecithin under nonhydrolytic conditions. Similarly, only this latter palmitoylphospholipase A2 completely hydrolyzed all lecithin in the outer monolayer of the human erythrocyte at a rate much faster than Naja naja phospholipase A2, the most powerful penetrating snake venom enzyme presently known.

Comparison of the effects of inserted C 40- and C 50-terminally dihydroxylated carotenoids on the mechanical properties of various phospholipid vesicles

We have measured the extent of incorporation of zeaxanthin (C 40) and decaprenozeaxanthin (C 50) in unilamellar vesicles of dimyristoylphosphatidylcholine ( n-C 14) and dipalmitoylphosphatidylcholine ( n-C 16). The incorporation is larger when the molecular length of the carotenoid corresponds to the thickness of the phospholipid bilayer. Stereochemically pure 2,3-di- O-phytanyl- sn-glycero-1-phosphocholine was prepared by modification of the polar heads of the phospholipids of Halobacterium halobium. Vesicles of this branched-chain ether phospholipid incorporate poorly the carotenoids, whereas egg lecithin vesicles incorporate them better. Osmotic swelling and water permeability of vesicles, with or without carotenoids, were measured in a stopped-flow, light-scattering system. The reinforcing effect (lower permeability and higher rigidity) of carotenoids at 1.5 mol% incorporation into diphytanylphosphatidylcholine vesicles is comparable to that of 5 mol% cholesterol; however, carotenoids have no measurable effect on the egg lecithin vesicles. These results imply that the reinforcement of the membrane depends on a subtle adjustment of the phospholipid-carotenoid system.

Elastic Torques about Membrane Edges: A Study of Pierced Egg Lecithin Vesicles

The shape of mechanically pierced giant vesicles is studied to obtain the elastic modulus of Gaussian curvature of egg lecithin bilayers. It is argued that such experiments are governed by an apparent modulus, kappa(app), not the true modulus of Gaussian curvature, kappa. A theory of kappa(app) is proposed, regarding the pierced bilayer vesicle as a closed monolayer vesicle. The quantity measured, i.e. kappa(app)/kappa, where kappa is the rigidity, agrees satisfactorily with the theory. We find kappa(app) = -(1.9 +/- 0.3) . 10(-12) erg (on the basis of kappa = (2.3 +/- 0.3) . 10(-12) erg). The result may have implications for bilayer fusion.