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Free Vertical Growth of Myelin Figures
Abstract
Myelin figures which grow from a floating egg-yolk lecithin lump on a surface of water/glycerin mixture have been found to grow only vertically towards the bottom of a glass cell and never to grow horizontally along the medium surface nor radially. The structure of the growing myelin figures is basically simple rod-like or a bundle of a few simple rods. Helixing, twisting or coiling myelin figures were never observed under the present experimental condition.Taking the results obtained from the reported growth behaviour of myelin figures into consideration, it is proposed that one of the driving forces of helixing. coiling and twisting growth of myelin figures is thought to be due to a chemical potential difference caused by a concentration gradient along the radial direction perpendicular to the long axis of a myelin figure. The hydrocarbon chains in the fully hydrated stacked bilayer of myelin figures are proposed to be in a more ordered state than reported so far. The arrangement of hydrocarbon chains, both in myelin figures and in an egg-yolk lecithin/water complex, and the density of them also will be discussed.
... Myelin figures are finger like extrusions (Fig. 7) having concentrically arranged alternating aqueous layers and surfactant bilayers around a central core of water. The growth of myelin figures is due to a chemical potential difference caused by a concentration gradient along the radial direction 161 . ...
Role of self assembled structures as a vehicle is significant over the years. Their applications have been found for all routes of drug delivery. These micro and nano structures are containers loaded with drugs, ideal for targeted and sustained release of the drug. Drug efficacy depends on the drug loaded into the vehicle, temperature, drug solubility, pH, release characteristics, additives and most significantly, the vehicle morphology. This in turn suggests that the same vehicle cannot be used with high efficiency for all types of drugs and locations where the drug delivery has to take place. The status of various self assembled structures and their applications in drug delivery is reviewed in this communication.
We showthat nanodiscs stabilized with polymers order and pile up on a surface upon drying. The resulting surface films with an average thickness of one micron are made of collapsed cohesive layers with smectic long-range order. This occurs with and without plastifying stabilizing polymer and produces crevasses. The stacked discs undergo a two-to-three-dimensional crystallization while bottom layers close to the surface fuse and produce infinite bilayers. Small angle X-ray scattering experiments demonstrate that excess polymer is segregated from the crystalline stack. Water adsorption isotherms show that reversible swelling of the excess polymer does not destroy the compact stack of partially fused nanodiscs collapsed parallel to the surface. In the absence of chemical binding, the stacks of layered nanodiscs can be removed by simple washing with pure water. AFM, TEM and SEM experiments demonstrate that presence of crevasses is quenched by the presence of a plastifying polymer.
Myelin figures with unusual surface morphology were observed on contacting Tween85 with water. Myelins, which are normally smooth rodlike forms in other surfactants, are in this system found to be with an irregular, rough surface with vesiclelike structures adhered to the myelin tubes. Besides these, smooth myelin figures were also observed. We term the myelin figures with a rough surface eroded myelin figures. The same myelin could show a coexistence of smooth and rough areas with a sharp boundary between the smooth surface at one end whereas the other end shows a rough texture. The transformation of smooth myelins into eroded forms were observed often whereas the reverse is quite rare. In the later stage, the tip of the eroded myelin figures transforms into tentacles and acts as a source for new myelins and the growth of vesiclelike structures which were expelled into the surrounding medium. The eroded myelin figures are stable for a longer period in comparison to simple, smooth rodlike forms. By studying the myelin growth at different temperatures, it was found that eroded myelin figures were stable in the temperature range of 22-42 degrees C and at > 42 degrees C only smooth myelin figures were observed.
The behavior of purified egg lecithin in water has been investigated in relation to the quantity of water present and the temperature. The complete binary phase diagram of egg lecithin-water is presented as well as X-ray diffraction data on selected mixtures. Dry egg lecithin is present in at least partially crystalline form until about 40°C. Above this temperature it forms a ``wax-like'' phase up to about 88°C. From 88 to 109°C it forms a viscous isotropic phase which gives face-centered cubic spacings by X-ray analysis. Above 110°C its texture is ``neat'' and the structure is assumed to be lamellar until its final melting point at 231°C .
Hydrated lecithin forms (except for a small zone of cubic phase at low water concentrations and high temperature) a lamellar liquid crystalline phase. This phase contains up to 45% water at 20°C. Mixtures containing more water separate into two phases, the lamellar liquid crystalline phase and water. In the melting curve of hydrated lecithin a eutectic is noted at about 16% water and the cubic phase seen when less water is present disappears at this composition of the mixture.
These facts, along with previous vapor pressure measurements, suggest that there is a structural change at about 16% water. X-ray diffraction studies of lecithin at 24°C and calculations from these data suggest that the reason for this may be the presence of a ``free water layer'' when more than 16% water is present.
We describe in this paper measurements of the partial specific volumes of lipid (upsilon L) and water (upsilon W) in mechanical mixtures of egg yolk lecithin and water over a range of hydrations (5-55 wt% water) that includes the region at which excess water appears. upsilon L and upsilon W are found to be very nearly 1 cm3/g over the entire range. The water activities of the mixtures were also determined and found to be the same as for lipid deposited as oriented multilayers on solid substrates.
X-Ray diffraction studies have been carried out on some lipids of various chemical compositions: two monoglycerides (glyceryl-monodecanoate and glyceryl-monododecanoate), several phospholipids (synthetic di-palmitoyllecithin, egg lecithin, lysolecithin derived from the latter, egg phosphatidylethanolamine) and sphingolipids (sphingomyelin and cerebrosides isolated from beef brain) have been studied. The structure of these lipid-water systems depends on concentration and temperature: crystalline phases are formed at low temperatures, whereas higher temperatures and high lipid content promote the formation of liquid-crystalline phases. At low lipid content and high temperature, only lysolecithin forms a micellar solution in water; for the other lipids, a suspension of a liquid-crystalline phase in excess water is observed.Four liquid-crystalline phases, already described in other soap or lipid systems, have been observed here. 1.(a) A lamellar phase occurs in the following systems: water-monoglyceride (monodecanoate: 25 °C; monododecanoate: 45 °C); water-sphingomyelin (45 °C); water-cerebrosides (72 °C); water-phosphatidylethanolamine (20 °C); water-egg lecithin (5 to 35 °C) and anhydrous lecithin at high temperature.2.(b) A hexagonal phase, made of a regular array of lipid cylinders, is observed in the water-lysolecithin system at 35 °C.3.(c) An inverted hexagonal phase (cylinders made of water) occurs in the water-phosphatidylethanolamine system at 55 °C.4.(d) A cubic phase is observed in anhydrous lecithin at high temperature.The structural parameters of these phases and the polymorphism of the various lipids are discussed.
Growth behaviour and morphological features of myelin figures in egg-yolk lecithin/hydrophilic liquid systems were investigated by microscopic observation at room temperature. The morphological features were conveniently classified into three steps according to the time of growth.In the first step, myelin figures with a simple rod-like form rapidly grew all together perpendicularly to the circumference of a lecithin block into the medium. In the second step, more complicated forms, such as helical and coiling ones, occurred. In the third step, the parts where a contact between surfaces of neighbouring rods had occurred wcre associated, fused and transformed into a mosaic structure. The representative growth mechanism of each of these three steps was dependent mainly on a diffusion of aggregates of lecithin molecules towards myelin figures near their roots, on a lateral diffusion in the bilayer along the long axis of a myelin figure and on a lateral diffusion in the bilayer around the long axis, respectively.
The existence of a concentration gradient along the long axis of, i.e. along the growth direction of, growing myelin figures was found by measurements of transmitted-light intensity through the myelin figures at different places along the axis during growth. The observed transmitted-light intensities under crossed-Nicols were stronger at a micro-area near the root of the growing myelin figures than near the top of it. The difference of the intensities between the two micro-areas decreased with the growth time. Since the intensity measured is nearly proportional to the number of the ordered phosphatidylcholine molecules in the selected area, the present results should prove our recent report that the initial growth process of myelin figures is diffusion-limited (Sakurai, I. and Kawamura, Y. (1984) Biochim. Biophys. Acta 777, 347–351).
Cross-sections of myelin figures from an egg-yolk phosphatidylcholine/water system were observed directly by use of a scanning electron microscope (SEM) equipped with a cryo-stage system, for the first time. Observations were made at a temperature of about −140°C and at a relatively low magnification so as to obtain an accurate view of the entire structures of myelin figures, as well as the growth behavior of them. The observed internal structures of myelin figures were partly consistent with those in previous reports based upon optical experiments.
The initial growth process of myelin figures, rod-like lyotropic liquid-crystalline structures, formed by phosphatidylcholine in water, ethylene glycol or glycerin, is suggested to be diffusion-limited with an apparent diffusion coefficient D of approx. 10(-6) cm2/s. D can be expressed by the sum of two processes. One is considered to describe the diffusion of an aggregate of phosphatidylcholine molecules and the other mainly to describe a lateral diffusion in the bilayer membranes which constitute myelin figures.
In a magnetic field of up to 7 kG, myelin figures of the egg-yolk phosphatidylcholine/water system were oriented because of the diamagnetic anisotropy of the molecules through bending at the roots with the long axes parallel to the field. The time interval for an angular change of their axes was nearly in proportion to H-2. From the orientational behaviour, a curvature-elastic modulus K of lipid-bilayer could be roughly evaluated as 4 X 10(-13) erg.
For the purpose of clarifying the magneto-orientation mechanism of several biological membrane systems, xylene-suspended thin-layered single crystals of lecithin, which is a major component of membranes, were subjected to magnetic fields of various strengths, and the orientation behavior of the crystals was analyzed. Lecithin crystals oriented in such a way that the directions of both the hydrocarbon chains and the phosphorylcholine group of the lecithin molecule were perpendicular to the magnetic field, suggesting that the crystals were diamagnetically biaxial. In contrast, hydrocarbon chains of phospholipids in many biological membranes are known to orient parallel to magnetic fields. The value of the volume diamagnetic susceptibility anisotropy, delta chi (i.e. the difference between susceptibilities parallel and perpendicular to the direction of the hydrocarbon chain in the lecithin crystals), estimated from the magneto-orientation behavior was about -9 X 10(-8) cgs. This is distinctly larger than that of vesicles of egg yolk lecithin measured by Boroske and Helfrich [Boroske, E. & Helfrich, W. (1978) Biophys. J. 24, 863-868] and also larger than that of single crystals of polyethylene, which was measured for comparison by the same technique as that used for lecithin in this study. The differences between these values of delta chi seem to result from the contribution of the polar head (i.e., the part of the lecithin molecule other than the two hydrocarbon chains), which is ordered, less ordered, or deleted in the respective cases above, although mainly from the contribution of the hydrocarbon chains, which are ordered in crystals and disordered in vesicles.
Extensive freeze-fracture electron microscopic studies of
Ca2+-containing mixtures of an acidic phospholipid
(cardiolipin) and phosphatidylcholines1-5, have revealed
certain replica features which have been interpreted as regions of
membrane-membrane attachment3-5. Spin-label paramagnetic
resonance spectra of mixtures of another acidic phospholipid
(phosphatidylserine) and phosphatidylcholines have provided evidence for
Ca2+-mediated lateral phase separations of the acidic
phospholipids6-8, and their involvement in intermembrane
binding7. We report here that in the presence of
Ca2+, binary mixtures of cardiolipin and phosphatidylcholines
readily form tightly folded single- and double-helical liposomes; we
invoke membrane-membrane attachment to explain this phenomenon.
Chromatographically homogeneous egg lecithin, as determined by TLC on Silica Gel G, has been isolated from crude egg phosphatides
by column chromatography on alumina through modification of existing, lengthy methods. The modified method involved application
of crude egg phosphatides to a column of alumina in the proportion of 1 g phosphatide/25 g alumina, and elution of the lecithin
fraction with the 2-component solvent system chloroform:methanol, 9:1 by vol. This method of purification separated lecithin
from other choline and non-choline components of crude phosphatides, avoided overloading of the alumina column, and made unnecessary
the need for a second chromatographic fractionation of partially purified lecithin on silicic acid, which is needed in existing
methods of purification of lecithin.
The use of fresh yolks permitted easier removal of pigment from the final product than was possible with commercially dried
yolks.
Phosphatides extracted from dried yolks were much more highly colored than were the phosphatides extracted from fresh yolks
and the color presisted through chromatography on alumina.
The fatty acid/phosphorus molar ratio of the purified lecithin was 2.00, which is the theoretical FA/P molar ratio of phosphatidylcholine;
other materials with this ratio were not present.