Effect of pH on the control release of microencapsulated dye in lecithin liposomes. II

The objective of our work has been the microencapsulation of dyes with lecithin from soybean, with the formation of liposomes, as a substitute for synthetic auxiliaries so as to improve the quality of the effluent. Current scenarios promote the disintegration and leakage of the liposomes, such as, changes in temperature, pH, and the use of surfactants. Since dyeing process is a mix of all these parameters, we pretended to study each one separately. Changes in pH at constant temperature induce a release of dye similar with changes in temperature. In acid conditions, we found a very fast initial dye release which doesn't occur in basic conditions. Using carboxyfluorescein, as a pH fluorescence probe, we concluded that the liposome membrane doesn't protect the liposome interior from changes on the external pH.     

Study of the release of a microencapsulated acid dye in polyamide dyeing using mixed cationic liposomes

The main objective of this work was to increase the retarding effect of the acid dye Telon^® Blue RR (C.I. Acid Blue 62; DyStar, Frankfurt, Germany) release on polyamide fibres dyeing by encapsulation of the dye in liposomes as an alternative to synthetic auxiliaries, in order to reduce effluent pollution. The retarding effect achieved with the use of mixed cationic liposomes of dioctadecyldimethylammonium bromide (DODAB)/soybean lecithin (containing a 10% molar fraction of DODAB) was better in comparison with either pure soybean lecithin liposomes or synthetic auxiliaries. The retarding effect of liposomes on the dye release was analysed through changes in the absorption and fluorescence spectra of the acid dye at different conditions. The effect of temperature (in the range of 25 °C - 70 °C) on the spectroscopic behaviour of the dye in the absence and in presence of polyamide was also studied, in order to simulate the dyeing conditions. Exhaustion curves obtained in dyeing experiments showed that, below 45 °C, the retarding effect of the mixed liposomes (lecithin/DODAB (9:1)) was similar to that of the auxiliaries, but better than the one of pure lecithin liposomes. At higher temperatures (above 45 °C), the system lecithin/DODAB presents a better performance, achieving a higher final exhaustion level when compared with the commercial leveling agent without losing the smoothing effect of lecithin.     

Effect of cholesterol on sensitivity of lecithin liposomes to surface-active substances

The effect of cholesterol incorporation into multilamellar egg lecithin liposomes on the liposomes sensitivity toward N-acyl derivatives of amino acids was examined. Free energy of intermolecular interaction between lecithin head groups in the bilayer is estimated as 3.8 ± 0.1 kcal/mol.     

Effect of alpha-tocopherol incorporation of glucose permeability and phase transition of lecithin liposomes.

Liposomes were prepared from dipalmitoyllecithin, dimyristoyllecithin, dioleoyllecithin, egg lecithin, and soybean lecithin, and the effects of incorporation of various quantities of alpha-tocopherol or its analogs on permeability of the liposomes to glucose were studied at various temperatures (4--40 degrees C). Results showed that increase in the quantity of alpha-tocopherol incorporated into dipalmitoyllecithin and dimyristoyllecithin liposomes lowered the transition temperature for marked release of glucose and also decreased the maximum rate of temperature-dependent permeability, alpha-Tocopherol also had similar but less marked effects on the permeability of dioleoyllecithin and egg lecithin liposomes, but little effect on those of soybean lecithin, which has a higher degree of unsaturation. In dipalmitoyllecithin liposomes phytol showed a similar effect of permeability to that of alpha-tocopherol, but phytanic acid caused a different pattern of temperature-dependent permeability. With analogs of alpha-tocopherol, the regulatory effect on permeability decreased with shortening and disappearance of the isoprenoid side chain. The significance of these observations is discussed in relation to the physiological functions of tocopherols in natural membranes.     

Action of surfactants on egg lecithin liposomes

The lytic action of several homologous series of surfactants including N-acyl derivatives of the Na-salt of amino acids on the egg lecithin multilamellar liposomes was examined. The affinity for the lipid membrane and the solubilising capacity of the agents were estimated. The contribution of a CH₂ group and that of the polar head group of surfactants to the free energy of the agent's binding to the membrane were evaluated. The results obtained indicate that the contribution of a CH₂ group to the free binding energy depends on the nature of the surfactants' head group. This dependence is attributed to either various localisation of the agent's molecules in the lipid bilayer or to different properties of the agent's hydrocarbon tails. The contributions of the head groups of the surfactants are assumed to reflect the affinity of these head groups for the lecithin polar head group at the membrane interface. The results obtained indicate some degree of specificity involved in the interactions of the head groups.     

Interactions between flavonoids and hemoglobin in lecithin liposomes

In this paper, the binding of flavonoids (quercetin and rutin) to hemoglobin (Hb) have been investigated by fluorescence, absorption spectroscopy and circular dichroism (CD) spectroscopy. The binding parameters and binding mode between flavonoids and Hb are determined and the results of CD and synchronous fluorescence spectra indicate a conformational change of Hb with addition of flavonoids. The effects of lecithin liposomes on the binding parameter of quercetin and rutin to Hb are also studied. When incorporated into liposome, flavonoids can reduce the fluorescence of tryptophanyl residues of Hb to a lesser extent. The difference of the structure characteristics between quercetin and rutin has a significant effect on their binding affinity for Hb.     

Quenching of fluorescence of pyrene-substituted lecithin by tetracyanoquinodimethane in liposomes.

In this work we have applied a kinetic scheme derived from fluorescence kinetics of pyrene-labeled phosphatidylcholine in phosphatidylcholine membrane to explain the fluorescence quenching of 1-palmitoyl-2-(10-[pyrenl-yl]-sn-glycerol-3-phosphatidylchol ine (PPDPC) liposomes by tetracyanoquinodimethane (TCNQ). The scheme was also found to be applicable to neat PPDPC and the effect of the quencher could be attributed to certain steps of the proposed mechanism. The TCNQ molecules influence the fluorescence of pyrene moieties in PPDPC liposome in two ways. Firstly, an interaction between the quencher molecule and the pyrene monomer in the excited state quenches monomer fluorescence and effectively prevents the diffusional formation of the excimer. Secondly, an interaction between the quencher molecule and the excited dimer quenches the excimer fluorescence. The TCNQ molecule does not prevent the formation of the excimer in pyrene moieties aggregated in such a way that they require only a small rotational motion to attain excimer configuration. The diffusional quenching rate constant is calculated to be 1.0 x 10(8) M-1 s-1 for the pyrene monomer quenching and 1.3 x 10(7) M-1 s-1 for the pyrene excimer quenching. The diffusion constant of TCNQ is 1.5 x 10(-7) cm2 s-1 for the interaction radii of 0.8-0.9 nm. The TCNQ molecules are practically totally partitioned in the membrane phase.     

Fluorescence quenching in lecithin and lecithin/cholesterol liposomes by parmagenetic lipid analogues. Introduction of a new probe approach.

1. Perylene, whether incorporated into lecithin or lecithin/cholesterol (1:1) liposomes, exhibits identical fluorescence spectra, but fluorescence in the presence of cholesterol is enhanced by 30-50%. 2. The fluorescence of perylene in pure dipalmitoyllecithin vesicles increases sharply at the transition temperature (Tt equals 41 degrees C). No such fluorescence jump is observed in lecithin/cholesterol (1:1) micelles. 3. In lecithin liposomes maximal quenching of perylene fluorescence at 25 degrees C is effected by cholestane spin label (80%) followed by androstane spin label (70%), 5-nitroxide stearate (60%) and 16-nitroxide stearate (50%). 4. In liposomes containing 5 mol % cholesterol these differences are reduced; however, the sequence of quenching efficiencies is the same except for the nitroxide stearates, which interchange their positions. 5. 5. Paramagnetic quenching of perylene fluorescence is stable below 35 degrees C and above 45 degrees C, but decreases sharply about the phase-transition temperature of dipalmitoyllecithin. 6. In lecithin/cholesterol (1:1, molar ratio) lipsomes fluorescence quenching diminishes linearly, but only slightly, with increasing temperature. 7. Cholestane spin label and androstane spin label at concentrations of greater than 20 mol % themselves suppress the quenching discontinuity at Tt, indicating a cholesterol-like structural effect. 8. The quenching phenomena observed are attributed to a non-random accommodation of fluorophore and quencher molecules (co-clustering) below the phase transition and a statistical distribution of both impurities above Tt. 9. In the presence of cholesterol the clustering tendencies are reduced or even eliminated; this is compatible with the concept that cholesterol fluidizes the phosphatide acyl chains below the transtion temperature.     

beta-Carotene or chlorophyll alpha incorporated in lecithin liposomes. Analysis by ultracentrifugation.

Ultracentrifugal analyses show that β-carotene and chlorophyll a are incorporated into the membranes of lecithin liposomes without any increase of the particle size. The sedimentation coefficient and particle weight of the liposomes increase up to a maximum value (4.3 S and 3.1 × 10⁶ respectively) for a molar ratio of pigment to lecithin of about 0.02, due to an increasing number of lecithin molecules per particle. The pigments lower the average surface area of the polar heads of lecithin down to a minimum of 79 A²/molecule. When the molar ratio of chlorophyll a to lecithin is above 0.02, the characteristics of the vesicles do not change, indicating a different type of incorporation of the chlorophyll molecules in the membranes.     

Interaction of mitochondrial aspartate aminotransferase with negatively charged lecithin liposomes.

Several kinds of hydrophilic proteins were examined to determine their interaction with artificial liposomes. Mitochondrial aspartate aminotransferase (m-GOT) [EC 2.6.1.1], as well as cytochrome c, was found to interact strongly with negatively charged liposomes. In each case, an appreciable amount of the protein bound to liposomes remained unreleased after raising the salt concentration in the medium. The m-GOT tightly bound to the liposomes was also found to become latent in its enzymatic activity, and could be reversibly activated by solubilization of the liposomes with detergent. This is also the case for cytochrome c, which ceases to be reducible by external reductant, such as dithionite. Furthermore, the tightly bound m-GOT was not susceptible to the proteolytic action of trypsin, or that of Nagarse. From these observations it can be inferred that these basic proteins interact with acidic liposomes not only electrostatically but also hydrophobically. This kind of hydrophobic interaction was not observed in the combination of positively charged liposomes and acidic proteins, including s-GOT. Mitochondrial GOT was shown to be bound to isolated intact mitochondrial, but the bound enzyme was fully active, in contrast to the case of acidic liposomes. The hydrophobic interaction of water-soluble protein with liposomes is discussed in connection with the penetration of matrix enzyme through mitochondrial membranes.     

Comparison of dynamics of lecithin liposomes and brain total lipid liposomes with synaptosomal membranes. An EPR spectroscopy study.

Dynamics and/or order of the hydrophobic part of phosphatidylcholine (PC) liposomes and rat brain total lipid (TL) liposomes and synaptosomes were studied and compared by EPR spectroscopy using the spin probes 5 or 16-doxyl stearic acid and 14-doxyl phosphatidylcholine. The dynamics and/or order of the hydrophobic part of TL liposomes or synaptosomes were similar but differed largely from those of PC liposomes. The dynamics of the hydrophobic part of the liposomes decreased gradually with the increasing TL/PC ratio in the sample. To obtain in TL liposomes or synaptosomes the same EPR spectrum parameters as in PC liposomes at 37 degrees C, the formers have to be heated to temperatures of approximately 50-60 degrees C. The dynamics and/or order of the hydrophobic part of lecithin liposomes at 5-10 degrees C were comparable with those of TL liposomes or synaptosomes at 37 degrees C. The results emphasize the role of the lipid composition in studies concerning drug-lipid and protein-lipid interactions in model and biological membranes.     

Microviscosity in lecithin liposomes: effect of nicotinic acid

We have studied the effect of nicotinic acid, a drug commonly used as a vasodilatory agent and also for the treatment of hypercholesterolemia, on the fluidity profile of liposomes of egg lecithin and dipalmitoyl lecithin, using a fluorescent polarization probe. In both cases the drug decreases the membrane fluidity and for cholesterol-probed liposomes, it disrupts the "intermediate fluid condition" induced by cholesterol. The drug also affects the activation energy for diffusion in the hydrophobic region of the liposomes.     

Phospholipases. III. Effects of ionic surfactants on the phospholipase-catalyzed hydrolysis of unsonicated egg lecithin liposomes.

Apparent values of Km and Vmax have been measured for catalysis of hydrolysis of unsonicated egg lecithin liposomes, activated through addition of 0.4 M n-hexanol, by phospholipases A2 from bee and snake venoms and by phospholipase C from Clostridium welchii as a function of the concentration of three surfactants: hexadecylamine, hexadecyltrimethylammonium bromide, and dihexadecyl phosphate. For all three enzymes, values of Km and Vmax show little or no dependence on the concentration of these ionic surfactants, demonstrating that the liposomal surface charge is not a crucial factor in determining susceptibility to phospholipase-catalyzed hydrolysis.     

Solubilizing effects caused by the nonionic surfactant dodecylmaltoside in phosphatidylcholine liposomes.

The interaction of the nonionic surfactant dodecylmaltoside (DM) with phosphatidylcholine liposomes was investigated. Permeability alterations were detected as a change in 5(6)-carboxyfluorescein released from the interior of vesicles and bilayer solubilization as a decrease in the static light scattered by liposome suspensions. This surfactant showed higher capacity to saturate and solubilize PC liposomes and greater affinity with these structures than those reported for the octyl glucoside. At subsolubilizing level an initial maximum in the bilayer/water partitioning (K) followed by an abrupt decrease of this parameter occurred as the effective molar ratio of surfactant to phospholipid in bilayers (Re) rose. However, at solubilizing level a direct dependence was established between both parameters. A direct correlation took place in the initial interaction steps (Re up to 0.28) between the growth of vesicles, their fluidity, and Re. A similar direct dependence was established during solubilization (Re range from 0.9 to 1.7) between the decrease in both the surfactant-PC aggregate size, the light scattering of the system, and Re (composition of aggregates). The fact that the free DM concentration at subsolubilizing and solubilizing levels showed values lower than and similar to its critical micelle concentration indicates that permeability alterations and solubilization were determined, respectively, by the action of surfactant monomer and by the formation of mixed micelles.