Langmuir 1987, 3, 592-595
592
Letters Liquid Crystal Morphology and Defects in in Vivo Human and Mammalian Phosphatidylcholine Lung Surfactant Joseph A. N. Zasadzinski* Department of Chemical and Nuclear Engineering, University of California, Santa Barbara, California 93106
C. J. Stratton and D. Heetderks Department of Anatomy, University of Nevada School of Medicine, Reno, Nevada 89557 Received November 6 , 1986 The lung great alveolar cell in mammals, including man, produces phosphatidylcholine aggregates known as lung multilamellar bodies (LMB) that act as a surfactant to provide the critical surface tension properties of the alveolus. In an aqueous environment, phosphatidylcholine exists as lyotropic lamellar liquid crystalline bilayers organized as spherical or toroidal aggregates up to about 100 bilayers in diameter. The lung multilamellar bodies appear morphologically identical with in vitro natural and synthetic dispersions of lyotropic lamellar liposomes and include a population of liquid crystalline defects such as disclinations and edge dislocations. The morphology and interactions of the defects are explained by the continuum theory of liquid crystals. We report the first observations with molecular resolution of the structure and interactions of disclinations and edge dislocations in lamellar phases. 1. Introduction
The lung great alveolar cell in mammals, including man, produces phospholipid aggregates known as lung multilamellar bodies (LMB) that act as a surfactant to provide the critical surface tension properties of the a1veolus.l Lung multilamellar bodies contain about 90% saturated phosphatidylcholine (PC), the bulk of the remainder being protein.2 Premature infants lack sufficient quantities of lung PC, an often fatal condition known as hyaline membrane disease. In the aqueous environment of the cell, PC forms bilayers organized in spherical or toroidal aggregates of about 100 bilayers in diameter.2 Mammal (rat) and human LMB were observed by transmission electron microscopy of thin sections of lung tissue preserved by a lipid retaining fixation and embedment procedure free of the usual artifacts of such technique^.^ The configuration and morphology of the bilayers in the LMB are predicted by using continuum theories of thermotropic and lyotropic liquid crystals4 and are shown to be morphologically identical to in vitro multilamellar liposomes of natural and synthetic s u r f a ~ t a n t . ~These , ~ results indicate that the same forces and interactions controlling bilayer organization in vitro are operating in vivo and could lead to a better understanding of the nature and mechanisms of natural surfactancy . P C is an amphiphilic molecule consisting of two hydrophobic acyl chains covalently linked to a hydrophilic zwitterionic head group. Therefore, one end of the molecule is strongly polar, the other strongly apolar. Organization into bilayers reduces the contact between PC heads and tails, and, in an aqueous environment, between water and the tails. The bilayers separate the environment into polar and nonpolar regions; the zwitterionic heads are
solvated by and are in contact with water, but the hydrocarbon tails contact only other tails. The molecular organization within each bilayer a t physiological temperatures is liquid-like; there are no long-range correlations in the plane of the layer. However, the equilibrium bilayer separation, which is set by a balance of competing forces such as van der Waals attraction, double layer repulsion, hydration, and undulation forces, is crystal-like in its regularity and requires a large energy to change at a given temperature and composition. This liquid crystalline anisotropy of the bilayer structure induces an anisotropy in the the deformation energy of the bilayers. A stack of bilayers can bend easily providing that the bilayer separation remains ons st ant.^ Minimizing both the large elastic energy involved in changing the equilibrium bilayer separation and the repulsive hydrophobic-hydrophilic interaction between the hydrocarbon tails of PC and the aqueous phase requires that the bulk of the surfactant in the LMB is organized in continuous, parallel, and closed bilayer surface^.^ Deviation from this ideal configuration occurs only at a limited number of defects called disclinations and edge dislocations a t which the bilayer organization is locally disrupted. The structure and interactions of the defects are predicted by minimal energy solutions of the continuum theory of liquid crystal^.^-^ Edge dislocations and disclinations have important effects on the physical properties of smectic and lamellar (1) Stratton, C. J. In Pulmonary Surfactant; Robertson, B., Van Golde, L. M. G., Batenburg, J. J., Eds.; Elsevier: Amsterdam, 1984; Chapter 3. (2) Stratton, C. J.; Zasadzinski, J. A,; Heetderks, D. Anat. Rec., in press. (3) Stratton, C. J., Erickson, T. B.; Wetzstein, H. Y. Tissue Cell 1982, 14, 13.
Author to whom correspondence should be addressed ((805)961-4769).
0743-7463/87/2403-0592$01.50/0
(4) Zasadzinski, J. A.; Scriven, L. E.; Davis, H. T. 1985, Philos. Mag., [Part]A 1985 51, 287. Zasadzinski, J. A. N.; Kerins, J.; Scriven, L. E.; Davis, H. T. J . Electron. Microsc. T e c h . 1986, 3, 385.
0 1987 American Chemical Society
Langrnuir, Vol. 3, No. 4, 1987 593
Letters
phases:+l0 they act as pathways for enhanced transport or segregation of large molecules solubilized in bilayers,' mediate phase transitions,8 promote interlayer transport? and act as sites for membrane fusion or disruption.8J0 Dislocations might also be important in the swelling of multilamellar bodies to form tubular myelin surfactant, an essential intermediary in coating the lung surfaces.'J Previow investigators have used freezefracture techniques to visualize bilayer organization and defect structure in natural and svnthetic linosomes."s6 However. the limited resolution (>$nm)~offreezefractureprecludes observation of defect cores, which are of special interest as the assumptions of continuum theories break down in these regions. The chemical fixation-thin sectioning techniques used in this study can resolve individual halves of the bilayer and approach molecular resolution. We report here the first direct observations of disclinations and edge dislocations and their cores in lamellar phases. ~~~
~
~
~~
.~~~
2. Materials and Methods Rat (Figure la,b) and human (Figure IC)lung multilamellar bodies were prepared by a glutaraldehydmmium tetroxidetannic acid fixation and then dehydrated through Epon 812 resin. Ultrathin sections were taken by using glass or diamond knives on a Sorvall MT-2h ultramicrotome. The thin sections were collected on Formvar-coatedgrids to stabilizethe sections against mechanical and electron beam damage. The sections were post-stained with uranyl acetate-lead citrate to enhance the contrast between the dark, electron-densebead group regions and the light, electron-transparenttail group regions. A Hitatchi 125-E transmission electron microscope operated at 100 kV was used to record images. Details of the procedures are published else~here.2.~ Previous work has shown that this fixation procedure extracts a minimal amount of lipid and is the best available procedure for preserving LMB for high-resolution electron microscopy? The bilayer repeat distance measured from the micrographs is 6.6 nm, in good agreement with X-ray diffraction measurements on other PC bilayers," indicating that minimal structural rearrangements have occurred in the fixation process.
8-
3. Results and Discussion
Figure la shows the bilayer c o n f v a t i o n in a rat LMB. The dark lines are caused by electron scattering from the heavy metal stains, uranyl acetate and lead citrate, that are known to congregate in the polar head group regions of the bilayers.2J The tail groups, which consist of mainly carbon and hydrogen, do not scatter electrons well and appear light in the micrographs. In the bulk of the LMB, the bilayers are smooth, parallel, and continuous. The orderly progression of the bilayers is broken at a limited number of edge dislocations (arrowed). A high magnification view of the edge dislocation at 1is shown in Figure lb. The bilayer configuration is that of a large Burgers vedor edge dislocation (b = 16 bilayers) dissociated into two paired *l/* disclinations (see Figure 2). Experimental observations of in vitro lamellar and smectic phases show ( 5 ) Psrshan, P. S. J . Appl. Phys. 1974,45, 1590.
(6) KIPman, M.: W i U i s , C. E.; Caswllo. M. J.: Gulik-Krzywicki, T. Phtloa. Mog 1971,35,33. Kleman, M.Points. Linea, and W a l k Wiley Chirhcster. 198% pp 139-150. (71 Schneider, M.B.;Chan. W. K.; Webb, W. W. Rioph,s. J . 198443,
(10) Zessdzinski, J. Bmphjs. J . 1586.49, 1119. 111) Jsniak, M. J.: Small, D. M.: Shipley, C. G. J . B i d . Chern. 1979, 254.6068 Stamatoff. J.: Feuer. 6.:Gummheim. H.. J.: Tellez. G.. Vs. _. mane, T. Biophys. J. 1982,38, 11.
Figure 1. (a) Lung multilamellar body from rat. Edge dislodisclination of the cations are at 1 and 2. Arrows mark illa dislocations. (b) Edge dislocation of Burgers vector 16 bilayers dissociated into +'I2disclination (arrow) and -lI2disclination (asterisk). ill2disclination core is located between head groups of opposing bilayers. (c) Human multilamellar body with several interacting dislocations. See text. that edge dislocations are always dissociated into paired dis~linations."~The strain energy per unit length of an edge dislocation in a lamellar phase is6"
K is the Frank splay constant (==lo*dyn for PC),B is the
elastic constant for bilayer compression (=lo7 dyn/cm*),
594 Langmuir, Vol. 3, No. 4, 1987
Q
8 Figure 2. (a) Allowable core structures of +lI2 disclinations. The solid lines represent bilayer head groups. Top: disclination core is located between head groups. Bottom: disclination core is located between tail groups. (b) Allowable core structures of -l/* disclinations. Note the difference at the defect core, in particular the path ABCDEFGA. Top: disclination core is located between head groups. Bottom: disclination core is located between tail groups. (c) Edge dislocations dissociated into a combination of and -lI2 disclinations (after Kleman6). Top: +'Izdiscliiation is on the right, -lI2 on the left. Bottom: -'I2 disclination is on the left, +1/2 on the right. Burgers vector, b, equals four layers, the change in number of layers from left to right.
b is the Burgers vector of the dislocation (by convention, positive for an increase in the number of bilayers from left to right), d is the bilayer thickness, and 7,is a core energy independent of b. X is a length, on the order of the bilayer thickness, over which elastic and bend energies are comparable. Equation 1 shows that E(2b) < 2E(b);hence a single-edge dislocation of large Burgers vector is of lower energy that two (or more) edge dislocations of the same total Burgers vector.6 This is the opposite of what is true for solid crystals. Figure 2 shows the possible configurations of (Figure 2a) and -lI2 (Figure 2b) disclination cores. As in the micrographs, dark lines signify PC head groups. Symmetry dictates that the disclination core be located either between the head groups (top row) or between the tail groups (bottom row) of the bilayers. The + l I 2 disclination on the right side of the dislocation in Figure l b has its core between the PC head groups. The preservation of the core of the -'I2disclination on the left side of Figure
Letters l b is not adequate to describe the core. Most of the stress is localized in the region of the - l J 2disclination core: and the poor preservation of the bilayers might be a reflection of the stress; the PC molecules might not be as well aligned in these areas, and the fixative could not link them together as well as in the less strained areas. The smaller edge dislocation a t 2 in Figure l a (b = 10) is also dissociated into paired disclinations. The core of the -ll2 disclination on the left side of the arrow is located between the head groups of the bilayers, as is the core of the +lI2 disclination to the right of the arrow. The bilayers around the -ll2disclination appear more distorted; they are wavy and irregularly spaced as compared to the bilayers surrounding the + l I 2 disclination. This reflects the larger stress associated with this portion of the edge dislocation. Figure ICshows several interacting defects in a human lung multilamellar body. The sample was taken from fresh normal biopsy material, in compliance with the requirements of the United States Department of Health, Education and Welfare Regulations of Protection of Human Subjects. A large edge dislocation of Burgers vector b = 19 changes the number of bilayers from 7 (lower left) to 26 (center). A -lI2 disclination with a core of tails is visible a t D in Figure IC. At A is a disclination with a core of heads. At the dark points are disclinations with cores of heads. The arrow marks the disclination of the dislocation. Surprisingly, both types of cores are commonly seen, suggesting that there is little, if any, energy difference between the core of heads and the core of tails. Segregation of different length PCs or proteins into the disclination cores might stabilize them and negate any difference between their structural energy. Between the small open arrows is one of the few elementary edge dislocations we observed. As mentioned earlier, fewer edge dislocations of larger Burgers vector are of lower energy than many dislocations of small Burgers vector. However, edge dislocations of similar sign repel each other.jI6 If one of the dislocations is at ( x , , ~ , )(x being parallel to the bilayers, z being perpendicular) and the other at (x2,z2),the interaction energy and forces between dislocations are5b6
Fi = -ViE
i = 1, 2
The energy is large near x1 - x 2 = 0 and small for z1 - z 2 = 0. Entropy also contributes to the free energy of the dislocation in the amount k T l d per length of disl~cation.~ In the regions near z1 - z 2 = 0, the interaction forces are weaker than the thermal forces (entropy), which have no preferred direction. Hence, dislocations of the same sign tend to gather along this line to anneal and form fewer dislocations of larger Burgers vector, thereby lowering the overall strain energy. The elementary edge dislocation a t the white arrows is along the same line as the large edge dislocation on the right. Eventually this and other small dislocations are absorbed by a few large dislocations. The asterisk in Figure 1 marks the limiting membrane of the lung multilamellar body. At the free boundary, the stresses are zero. This is equivalent to placing the image of an edge dislocation, one of opposite sign, a t a position symmetrical to the first one outside of the boundary. The edge dislocation within the body is attracted by this image dislocation and hence to the free ~ u r f a c e .As ~ can be seen in Figure IC,most of the edge dislocations we observed were near the free surface of the lung multilamellar body.
Langmuir 1987,3, 595-591 4. Conclusions It is possible to understand the structure and morphology of phospholipid LMB with the basic principles of liquid Physics. The in mammalian and 'Onand human LMB are tinuous, a configuration that minimizes the large elastic strain energy associated with changing the equilibrium bilayer separation and the hydrophobic-hydrophilic repulsion between the hydrocarbon tails of PC and the aqueous phase. This ideal behavior is disrupted at a limited population of large Burgers vector edge dislocations dissociated into paired *'/2 disclination pairs. Defect cores
595
are located between both head groups and tail groups in human LMB, suggesting that both types of core are similar in energy. This may be the result of partitioning of proteins or other non-lipid impurities in the LMB to the defect cores. The edge dislocation defects interact in ways too minimize the overall strain energy. Such defects are likely to be important in the conversion of multilamellar bodies into tubular myelin
Acknowledgment. We would like to acknowledge the technical assistance of Chris Bugas and Robert Rudolphi. C.J.S. was supported by a grant from the American Cancer Society through the Reno Cancer Center.
Reversible Redox of 2,5-Dihydroxythiophenol Chemisorbed on Gold and Platinum Electrodes: Evidence for Substrate-Mediated Adsorbate-Adsorbate Interactions Beatriz G. Bravo, Thomas Mebrahtu, and Manuel P. Soriaga* Department of Chemistry, Texas A&M University, College Station, Texas 77843
Donald C. Zapien and Arthur T. Hubbard Department of Chemistry, University of Cincinnati, Cincinnati, Ohio 45221
John L. Stickney Department of Chemistry, University of Georgia, Athens, Georgia 30602 Received February 27, 1987. I n Final Form: April 27, 1987 The reversible quinone/diphenol redox of 2,5-dihydroxythiophenol (DHTP) chemisorbed on smooth polycrystalline platinum and gold electrodes has been studied by thin-layer electrochemistry. The packing density and mode of binding (through the SH moiety) of the subject compound were found to be identical on both surfaces. However, the width of the redox peak of the pendant diphenolic group, an indicator of intermolecular quinone-diphenol interactions, is at least twice as large on Pt as it is on Au. In the absence of the SH moiety, hydroquinone itself is irreversibly adsorbed on platinum but not on gold. These results suggest that the adsorbate-adsorbate interactions occurring on Pt are principally substrate mediated.
Introduction The oxidation-reduction properties of chemisorbed molecules is of fundamental importance in electrochemical surface science. Chemisorption-induced alterations in redox potentials, for example, provide valuable insights into the nature of the metal-adsorbate bond. Numerous investigations have been done on the reversible reactions of redox centers anchored indirectly to a metal surface by an electroinactive moiety;' in these cases, the redox potentials are generally not highly perturbed by surface attachment, indicating that the interaction between the pendant electroactive moiety and the electrode surface is not sufficiently strong so as to alter the relative stabilities of the oxidized and reduced forms of the redox center on going from the solution to the adsorbed state. In the event that the electroactive center is itself strongly and directly bonded to the surface, dramatic changes in the redox potentials have been observed.2 While the redox potential shift gives a measure of the strength of the substrate-adsorbate bond, the width of a voltammetric peak provides an indicator of the strength of adsorbate-adsorbate interaction^.^ It is known that *Towhom correspondence should be addressed.
intermolecular interactions within an adsorbed layer can occur directly (through space) or indirectly (through the metal): and it is of fundamental importance to be able to distinguish between the two cases. It is in this context that the present study on the reversible quinone/diphenol redox behavior of 2,5-dihydoxythiophenol (DHTP) chemisorbed on smooth polycrystalline platinum and gold electrodes has been undertaken. On platinum, organic mercapto compounds tend to bind preferentially through the SH moiety, even if the organic group by itself is reactive toward platinum; an identical mode of binding is expected on gold. The close packing of the pendant diphenol redox centers in S-chemisorbed DHTP would create conditions favorable toward adsorbate-adsorbate (1) (a) Lane, R. F.; Hubbard, A. T.J.Phys. Chem. 1973, 77,1401. (b) Lenhard, J. R.; Rocklin, R.; Abruna, H.; Willman, K.; Kuo, K.; Nowak, R.; Murray, R. W. J. Am. Chem. SOC.1978, 100, 5213. (c) Sharp, M.; Petersson, M.; Edstrom, K. J. Electroanal. Chem. 1979, 95, 123. (d) Soriaga, M.P.; Hubbard, A. T. J. Electroanal. Chem. 1983,159, 101. (e) Weaver, M. J.; Tomi, T. T. L. J. Phys. Chem. 1986, 90, 3823. (2) Bravo, B. G.; Rodriguez, J. F.;Mebrahtu, T.; Soriaga, M. P. J. Phys. Chem., in press. (3) Laviron, E. J. Electroanal. Chem. 1974,52, 395. (4) Somorjai, G.A. Chemitry in Two Dimensions: Surfaces.; Cornel1 University Press: Ithaca, NY, 1981.
0743-7463/87/2403-0595$01.50/00 1987 American Chemical Society
