Biomembranes and new hemocompatible materials - American

Langmuir 1993,9, 39-45

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The Langmuir Lectures Biomembranes and New Hemocompatible Materials Dennis Chapman Department of Protein & Molecular Biology, Royal Free Hospital School of Medicine, Rowland Hill Street, London NW3 2PF, U.K. Received September 11,1992 Biomembranesare complex structuresof lipids, proteins, and carbohydrates. Many biophysical studies have been made of the amphiphilic lipid molecules and some of the many studies which have led to our present understanding of the lipid bilayer matrix w i l l be presented. This will include the application of monolayer as well as other biophysical techniques such as NMR and FTIR spectroscopy. The structures of biomembrane proteins are more difficult to study because of the requirement that they should be retained in a hydrophobic environment and are difficult to crystallize. We will describe some of our recent studies, using spectroscopictechniques, of various membrane proteins as well as synthesized polypeptide domains. Based upon biomembrane mimicry, new biomaterials, phospholipid polymers, which are hemocompatible and protein resistant have been developed. The testing and application of these new biomateriale will be shown. Introduction Langmuir in late 1916 and 1917 began to extend the earlier work of Rayleigh, Pockels, Devaux, and Marcelin on the behavior of oil films on water surfaces. He demonstrated that organic molecules containing polar groups such as COOH or OH groups become oriented at a water surface with the hydrophobic hydrocarbon portion of the molecule extending upward from the surface. Later Langmuir, in collaboration with Dr. Wrinch, studied protein and other biological multilayer systems using the Langmuir-Blodgett method. As we will show, these techniques continue to be useful for studies of biomembranes and biomaterial design. Biomembrane Structures All cells have an outer plasma membrane which retains the contents of the cell. Some cells have additional membranes associated with the functional units inside the cell such as the mitochondrial membranes, nuclear membranes, etc.' Gorter and GrendePusedthe Langmuir surface balance to study the lipids of biomembranes.These workers extracted the lipid from erythrocyte membranes, compressed it at an air-water interface, and found that it occupied a surface area equal to double the external area of the cells. This work led to the view that erythrocyte plaemamembranesare built upon a lipid bilayer structure. More recent studies have led to the suggestion that there were compensatingerrors in this derivation and that too low a pressure of compression had been used. When the correct effective pressure is used, the actual area occupied by the membrane lipids is a little less than that required for a two molecule thick layer.3 Nevertheless, the concept of alipid bilayer structure deducedby Gorter and Grendel became an important one for directing our thinking of biomembrane structures. Later studies by Danielli and Davson' interpreted surface tension experiments to indicate that the lipid head groups are covered by layers of proteins. This view however is now not accepted as being correct. (1) Chapman,D., Ed.BiobgicalMembmne8;Academic Preee: London, 1968; V0l:l-v. (2) Gorter, E.; Grendel, F. J . Exp. Med. 1925, 41,439. (3) Engelman, D. M. Nature 1969,223,1279. (4) D a m n , H.; Danielli, J. F. J . Cell. Comp. Physiol. 1936,5, 483.

The present consensus view of biomembrane structures retains the concept that all biomembranes are built upon a lipid matrix (usually in the form of a bilayer structure) into which the membrane proteins (including glycoproteins) are embedded (see Figure 1). The earlier static view of biomembranes based upon electron microscopy hae now been replaced by a more dynamic picture. In many cases the lipids and proteins are able to undergo rotational and lateral diffusion within the plane of the lipid matrix and signaling via transduction processes takes place across the lipid matrix. At the present time, there is particular emphasis on understanding biomembrane protein structures. Other themes of interest concern how protein translocation (involvingsignal or leader sequences)across biomembranestakes place. The process of biomembrane transduction is also attracting much attention. How protons, molecules (e.g. glucose), and ions cross membrane structures are also topics of great current interest. In the present communication I will indicate some of our own studies of the important lipid molecules making up the lipid matrix, and the relationship between monolayer and multilayer lamellar systems, and our present approach to the problem of obtaining information on membrane protein structures while present in the lipid matrix. Finally I will showhow invention via biomembrane mimicry has led us to develop new biomateriale which have important and useful hemocompatible and protein resistant characteristics. The Lipid Matrix When we examine the lipids which make up the biomembrane lipid matrix of erythrocyte and platelet cells, we see that they are based upon glycerol and sphingosine (see Figure 2). It has been shown that the lipids are asymmetrically arranged in the lipid matrix6 so that phosphatidylcholine (lecithin) and sphingomyelin make up some 90% of the outer lipid surface while the lipids which make up the inner surface are mainly phosphatidylserine, phosphatidylethanolamine, and smaller amounta of phosphatidylcholine, sphingomyelin, and phosphatidylinositol (Figure 3). In our own original studies of these lipids molecules we examined pure synthesized phosphatidylethanolamines (5) Zwaal,R. F. A.; Hemker, H. C. Hoemostasis 1982, 11, 12.

0743-7463/9312409-0039804.00/0 Q 1993 American Chemical Society

40 Langmuir, Vol. 9, No. 1, 1993

and later phosphatidylcholines, e.g. dimyristoylphosphatidylcholine and dipaimitoylphosphatidylcholineas simple model systems. We examined first the anhydrous lipids and showed that these lipids in the dry state undergo a major melting of the lipid chains at a transition temperature many tens of degrees below their capillary melting point. This was shown by calorimetry and also by using wide line NMR spectro~copy.e-~ The latter technique revealed the considerable molecular motion of the hydrocarbon which occurred at this transition temperature. We had earlier shown, using infrared spectroscopy, a similar behavior with anhydroussoap systems.8At this temperature water flows in among the polar groups and causes the lipids to swell. The lecithins in water then form multilammelar bilayers structures. Other phospholipids can form different structures, e.g. hexagonal structures in water (seeFigure 4). Some workers have suggested that such nonlamellar structures may be important in membrane fusion and protein translocation proces~es.~ The multilamellar bilayer structuresare essentiallymodel membrane systems. On lowering the temperature, the lipid chains in these bilayers can crystallize,while on raising the temperature, chain melting occurs at a temperature characteristic of the chain length and unsaturation which is present in the lipid chains. The hydrocarbon chains in this melted condition are not in a chaotic random arrangement but show increasing disorder'o toward the center of the bilayer. Further studies of mixed lipid systems led to an appreciation of phase separation characteristics which can also take place within the lipid matrix. Ladbrooke and Chapman (1969) reported studies of binary mixtures of lecithins using calorimetry.ll These authors examined mixtures of distearoyl and dipalmitoyl phosphatidylcholine (lecithin) (DSL-DPL) and also distearoyl lecithin and dimyristoyl lecithin (DSL-DML). With the DSL-DPL mixtures the phase diagram shows a continuous series of solid solutions are formed below the T,line. It was concluded that compound formation does not occur and that with this pair of molecules having only a small difference in chain length cocrystallization occurs. With the system DSL-DML monotectic behavior is observed with limited solid solution formation. Here the difference in chain length is already too great for cocrystallization to occur, so that as the system is cooled, migration of lecithin molecules occurs within the bilayer to give crystalline regions corresponding to the two compounds. Examination of a series of fully saturated lecithins with dioleoyl lecithin gave similar results with phase separation of the individual components taking place.I2 Other workers used spin-labeled lipids to study such lipid phase separation characteristica.13 These phase separation characteristics are of importance when we envisage cooling a cell (as in cryobiology), so that lipid crystallization within the lipid matrix can take place. (6) Chapman, D.; Byme, P.; Shipley, G. G. R o c . R.SOC.London 1966, A290, 115. (7) Salebury, N. J.; Chapman, D. Biochim. Biophys. Acta 1968, 163,

314. (8) Chapman, D. J. Chem. SOC. 1958,152,784. (9) De Kruiiff, B.; Cullii,P. R. Biochim. Biophye. Acta 1980,602,477. (10) Chapman, D.; Salsbury, N. J. Tram Faraday SOC.1966,62,2607. (11) Ladbrooke, B. D.; Chapman, D. Chem. Phys. Lipids 1969,3,304. (12) PhilliDs.M. C.: Ladbro0ke.B. . D.:.ChaDman. . . D. Biochim.BioDhvs. . Acta 1970,198; 35. . (13) Shimshick, E. J.; McConnell,H. M. Biochemistry 1973,12,2351. (14) Phillips, M. C.; Chapman, D. Biochim. Biophys. Acta 1968,162, 301.

The Langmuir Lectures

Monolayer Studies Many attempts had been made in the 1960s by biochemists attempting to relate the lipid monolayer properties at the air-water interface with biomembrane structure. However, these attempts were unsatisfactory, mainly because at that time, surprisingly, no clear view was available as to the molecular details of the monolayer, e.g. what was the molecular organization or dynamics underlying the "expanded state of a monolayer"? Further confusion existed over the meaning of "condensation effects" caused by cholesterol. Phillips and Chapman (1968) pointed out that a correlation exists between the lipid monolayer properties at the air-water interface and the properties of the multilamellar lipid bilayers formed in aqueous dispersions.16 The "condensed monolayer" correlates with the crystalline or gel phase while the expanded state of the monolayer correlateswith the 'fluid" or melted state which occurs above the lipid translation temperature. Similar thermotropic phase changeg occur with both the monolayers and the lipid bilayers.le The isotherme observed at different temperatures with dipalmitoyl phosphatidylcholine are shown in (Figure 6). All monolayer states were shown to be possible with the saturated lecithin and phosphatidylethanolaminehomologues. It is apparent that if the hydrocarbon chains are sufficiently long, condensed monolayers are formed, whereas with shorter chains liquid-expanded f h occur. These two limiting states are sufficiently well defined so that at any particular temperature only one of the homologoues studied exhibits the transition state. The data indicate that variations in hydrocarbonchain length which do not give rise to change in monolayer state do not have a significant effect on the F A curves. Temperature changes can also give rise to the condensed and expanded states for a monolayer of a single homologue. Cholesterol and CondensationEffects. Cholesterol is present in appreciable amountsin plasma biomembranes and there have been many studies attempting to ascertain its role in biomembranes. For some years it was known that cholesterol in the presence of unsaturated phospholipids (at the air-water interface) could cause a condensation effect. The meaning and interpretation of this were however obscure and controversial. Some workersbelieved that a cis double bond in the 9:lO position was essential for this condensation effect to O C C U T . ~ ~These workers invoked unusual structures and complexesto exist between the lipid and cholesterol molecules. We showed in our monolayer studies that a double bond in the 910 position was not essential and further that phospholipidscontaining trans double bonds, e.g. dielaidoyl lecithin could exhibit the condensation effect. Later we showed that even fully saturated phospholipids, when in an expanded state, could exhibit this condensation e f f e ~ t . ~ ~ J ~ Studies of multilamellar lipid systems helped to clarify the meaning of this condensation process. Ladbrooke et al.(1968)described studies20on lecithin-cholesterol-water interactions by differential scanning calorimetry (DSC) and X-ray diffraction. The 1,2-dipalmitoyl-~-phosphati(15) Chapman, D.; Williams, R. M.; Ladbrooke, B. D. Chem. Phys. Lipids, 1967, 1, 445. (16) Albrecht, 0.;Gruler, H.;S a c k " , E. J. Phy8. (Paris) 1978,39, 301. (17) Finean, J. B. Experientia 1959, 9, 17. (18) Chapman, D.; Walker, D. A.; Owens, N. F. Biochim. Biophys. Acta 1966,120, 148. (19) Chapman, D.; Owen, N. F.; Phillips, M. C.; Walker,D. A. Biochim. Biophys. Acta 1969, 183,458. (20) Ladbmoke,B. D.; Willinms,R.M.;Chapman, D.Biochim.Biophy8. Acta 1968,150, 333.


Langmuir, Vol. 9, No. 1, 1993 41

.

Figure 1. A schematic diagram of a biological membrane showing the lipid bilayer matrix with associated proteins and carbohydrates. 0

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distribution of the major phospholipidsin the plasma membranes of human platelets and erythrocytes; PC, phosphatidylcholine; SM, sphinogmyelin; PE, phosphatidylethanolamine; PS, phosphatidylserine; PI, phosphatidylinositol.

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Figure 2. Structures of common lipid molecules: (a) a phosphatidylcholine (lecithin); (b) sphingomyelin; (c) the phosphorylcholine polar group.

dylcholine (DPPC)-cholesterol-water system was studied as a function of both temperature and concentration of components. This particular lecithin was used because it exhibits the thermotropic phase change in the presence

of water at a convenienttemperature (41 "C). The addition of cholesterol to the lecithin in water lowers the transition temperature between the gel and the lamellar fluid crystalline phase and decreases the heat absorbed at the main transition. No transition is observed with an equimolar ratio of lecithin with cholesterol in water. This ratio corresponds to the maximum amount of cholesterol that can be introduced into the lipid bilayer before cholesterol precipitation occurs. Below the lipid T,transition temperature, calorimetric studies show that the main lipid endotherm is removed with increasing amount of cholesterol. The presence of the cholesterol molecules is to modulate the lipid fluidity above and below the transition temperature of the lipid. The first studies of Ladbrooke et al. (1968)suggested that the enthalpy was totally removed at 50 mol %; later



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Reprinted with permission from ref 16. Copyright 1978 SPPlF.

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Figure 4. (a,top) Lipid bilayers which can spontaneously form in water with lecithin molecules. The chain organizationsshowing tilted, vertical, and “melted” arrangements are indicated. (b, bottom)Hexagonalarrangementswhich form in water with some lipid molecules (I, normal; 11, inverse).

studies21suggested that this occurred at 33 mol % . The latter conclusions led to the concept that cholesterolexisted as a 2:l lipid-cholesterol complex. Studies using sensitive scanning calorimeters confirmed22 the early conclusion that the enthalpy is removed at 50 mol % of cholesterol to lipid as originally envisaged. Very recent studiesB have been made of cholesterollphospholipid interactions in mixed monolayers a t an air-water interface. At a monolayer lateral surface pressure of 10 mN/m at 22 “C cholesterol oxidase was used to reveal the stoichiometry at which free cholesterol disappears. These studies show that a clear brake occurs at a 1:l stoichiometry with phosphatidylcholine mixed monolayers while a 2:l stoichiometry occurs with sphinogmyelin mixed monolayers.

wavenumber (cm“

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Figure 6. FTIR spectra showing the amide I region of two membraneproteins: (a) bacteriorhodopsin (b)porin (fromHaris and Chapman, 1989). Studies using other techniques e.g. deuterium NMR24v25 with model biomembranes containing various amounts of cholesterol helped to clarify the meaning of the “condensation effect”. These studies showed that addition of cholesterol at the equimolar level (about 33 wt % ) results in an increase in deuterium quadrupole splitting from 3.6 to 7.8 kHz corresponding to an increase in molecular order parameter (from Smol = 0.18 to S m o l = 0.41). Cooling the sample to a temperature some 5 OC below that of the gel to liquid-crystal phase transition temperature (T,= 23 “C) has little effect on the quadrupole splitting. In summary the condensing effect of cholesterol can be understood in terms of the ordering effect exerted by the rigid planar ring structure of cholesterol on the “melted” lipid chains. Furthermore a t a temperature where the lipid chains would be expected to crystallize, the interdigitated cholesterolmolecules prevent this from occurring. ~

(21)Hinz, H. J.; Sturtevant, J. M. J. Biol. Chem. 1972,247, 3697. (22) Mabrey, S.; Mateo, P. L.; Sturtevant, J. M. Biochemistry 1978, 17, 2464. (23) Slotte, J. P. Biochemistry 1992, 31, 5472.

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(24) Rice, D. M.; Meadows, M. d.;Scheiman, A. 0.; Goni, F. M.; GomezFernandez, J. C.; Moacarello, M. A.; Chapman, D.; Oldfield, E. Biochemistry 1979,18, 5893. (25) Oldfield, E.; Gilmore, R.; Glaser, M.; Gutowsky, H. S.; Hehung, J. C.; Kang, S. U.; King, T. E.; Meadows, M.; Rice, D. h o c . Natl. Acad. Sci. U.S.A. 1978, 73, 4657,



Studies of the various lipid water systems have been valuable in providing an insight into the dynamics of the biomembrane lipid matrix including lateral and rotational diffusion26p27of the lipids and proteins.

Membrane Proteins The structure of membraneproteins is more difficult to study than water-soluble proteins because they require a hydrophobic environment and are particularly difficult to crystallize. Only a few membrane proteins present as a protein-detergent crystal have been as yet examined using X-ray diffraction techniques. Electron diffraction techniques have been applied to some membrane proteins in their native lipid bilayer, e.g. bacteriorhodopsin,28but as yet the resolution obtained does not match what in principle can be achieved using the X-ray single crystal approach. NMR spectroscopy is severly restricted for the study of membrane proteins because of the nonisotropic motion of the proteins within the lipid bilayer matrix. A few studies have been made with some small membrane associated polypeptides and proteins in membrane mimetic environments such as in micellesB and in organic solvents, but these studies do not necessarily provide an accurate picture of the arrangement of the protein within the lipid bilayer membrane. Much progress is being made with the determination of polypeptide sequences often based upon cDNA studies. This enables hydropathic plots to be made with predicted structural models. These are however models and structural data is required. We have been using Fourier transform spectroscopy for studying the secondary structure of membrane proteins. With this technique we are able to study the secondary structure of membrane proteins within the lipid bilayer matrix. We make use of the amide I and I1 bands in the spectra and use deconvolution and second derivative techniques to obtain band narrowing and enhanced resolution. We have studied a large number of membrane proteins. These include, bacteriorhodopsin, rhodopsin, Ca2+ATPase, Na+/K+ ATPase, H+/K+ ATPase, photosynthetic reaction centers, cytochrome c oxidase, acetylcholine particular feature of these receptor, and p ~ r i n . ~ A+ ~ ~ membrane systems is the remarkable consistency in the frequency of the main amide I band associated with the a-helical structure present, except for the membrane proteins bacteriorhodopsin and porin. The latter is known to differ from other membrane proteins in having a &barrel structure.33 Bacteriorhodopsin is an integral membrane protein found in the purple membrane of Halobacterium halobium. It is a light-driven proton pump consisting of 248 amino acid residues with an all-trans retinal chromophore. FTIR spectroscopic studies conducted in our laboratory as well as other laboratories have shown that the amide I frequency of this membrane protein is unusually high compared with what is exDected for normal a-helical- s t ~ u c t ~ r e : This ~ - ~ ~high frequency band for (26)Singer, S.J.; Nicholson, G. L. Science 1972,175,720. (27)Devaux, P.;McConnell, H. M. J. Am. Chem. SOC. 1972,94,4475. (28)Henderson, R.; Unwin, P. N. T. Nature 1975,257,28. (29)Shon, K.J.; Kim, Y.; Colnago, L. A.; Opella, S.J. Science 1991, 252,1303. (30)Jackson, M.; Haris, P. I.; Chapman, D. J. Mol. Struct. 1989,214, 329. (31)Susi, H.;Byler, D. M. Methods Enzymol. 1986,130,290. (32)Haris, P.1.; Chapman, D. Biochem. SOC.Trans. 1989,17,161. (33)Kreutz, A.: Weiss, M. S.: Welte, W.: Weckesser, J.: Schulz. G. E. J. Mol. Bioi. 1991,217,9. (34)Rothschild, K. J.; Clark, N. A. Biophys. J. 1979,25,473. (35)Krimm, S.;Dwivedi, A. M. Science 1982,216,407.

bacteriorhodopsin has been interpreted as arising from the presence of a 1-helicesin the protein%or the presence of short 310-helic regions in addition to normala-helices.M Hydrogen-deuteriumexchange of membrane proteins studied, using FTIR spectroscopy, has also been valuable for understanding membrane protein structure. A large number of proteins have been examined. These include bacteriorhodopsin,rhodopsin,glucosetransporter, etc. The glucosetransporter protein was found to have a remarkably high rate of hydrogen-deuteriumexchange.37 Almost 90 9% of its amide protons are exchanged within 1h. This was explained in terms of an aqueous pore present in this an interpretation supported by other techniques.38 We have also begun to synthesize polypeptide segments or domains of a number of membrane proteins. K+ Ion Channel Peptides. K+-ionchannel proteins have the ability to distinguish between various ions such as sodium, potassium, and calcium. The structural basis for this selectivity has been the subject of research for some time. Recently workers have identified39-4'"the amino acid sequence that constitutes the ion selective pore in the voltage gated K+ channels. It has been speculated that this stretch of amino acids may form two antiparallel &strands traversing the membrane. This pore polypeptide sequence is not what might have been expected. It contains no charge residues and is not particularly hydrophilic. We have chemically synthesized the pore amino polypeptide sequence. Preliminary FTIR and CD studies of this pore polypeptide show that it possesses an a-helical structure and not the 8-sheet structure previously proposed. We are also studying the conformation of other membrane spanning segmentsof the K+ channel protein, e.g. the 52, S4,and 55 polypeptides. The Pfl Coat Protein. In further studies we have also been determining the structure of the Pfl coat protein in the phage and in a membrane environment. We have also synthesized this protein and examined its structure in a lipid membranesystem. This protein exhibits an a-helical arrangement in the virus and also in the lipid matrix. Mitochondrial Transit Peptide. Many proteins are synthesized in the cytoplasm of eukaryotic cells and then translocated to their final destinations in subcellular organelles, for example the mitochondria, crossing one or more membranes in the process. The information which a protein needs to select one intracellular membranefrom several is containedin the initially-translated protein. The preprotein consists of the mature protein plus an N-termind extension,referred to as a signal or transit sequence, of between 17 and 60 residues in length. The primary structureof mitochondrial transit peptides have secondary and tertiary structural features in common. They are relatively rich in positively-charged (basic) amino acids (mainly Arg), lack acidic residues, have a high content of hydroxylated residues and small groups of adjacent hydrophobic residues. Hydropathyanalysis has suggested that these peptides form amphiphilic helices. In recent studies we have been using a combination of FTIR, CD, and NMR spectroscopy to understand the structural

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(36)Haris, P.I.; Chapman, D. Biochim. Biophys. Acta 1988,943,375. (37) Alvarez,J.; Lee, D. C.; Baldwin, S. A.; Chapman, D. J.Biol. Chem. 1987,262,3502. (38)Fischbarg, J.; Kuang, K.; Vera, J. C.; Arant, S.; et al. Roc. Natl. Acad. Sci. U S A . 1990,87,3244. (39)Yool, A. J.; Schwarz, T. L. Nature 1991,349,700. (40)Yellen, G.:Jurman, M. E.: Abramson, T.: MacKinnon. R. Science 1991,251,939. (41)Hartmann. H. A.: Kirsch. G. E.: Drewe,. J. A.:. Tadialatela. M.: et al. Science 1991,kl,942.



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h lvm) Figure 7. (a, top) Formation of a polyconjugated phospholipid polymer from diacetylene phosphatidylcholine monomer. (b, bottom) Visible spectra of 86 Langmuir-Blodgett layers (43 on each side of a quartz slide) of diacetylene phosphatidylcholine a t various irradiation times. Reprinted with permission from ref 43. Copyright 1982 Elsevier.

features of some of these systems and to determine the secondary and tertiary structure of this signal sequence.

New Biomaterials Based upon Biomembrane Mimicry With our growing knowledgeof biomembrane structures, the behavior of the lipids, e.g. their phase behavior and monolayer characteristics, we turned our attention to another question. Was it possible to produce polymerized biomembranes? Was it possible to obtain immobilized enzymes locked within a polymeric lipid membrane structure? We did this by introducing diacetylene groups into phospholipid hydrocarbon chains so that upon shining UV radiation or y-irradiation on the diacetylene phosphatidylcholine multilamellar vesicles we were able to cause polymerization to occur (Le. cross link) the lipids together42 (Figure 7a). The polymer so produced is polyconjugated and upon irradiation produces a visible color. The spectra of some LangmuirBlodgett (LB)layers of these diacetylene phosphatidylcholine (DAPC) monomers after various irradiation43times are shown in Figure 7b. The production of these LB films of polymerized phospholipids led us to consider how we might use such polymerized coatings on plastics or metal surfaces. We (42)Johnston, D. S.;Sanghera, S.; Pons, M.; Chapman, D. Biochim. Biophys. Acta 1980,602, 57. (43)Albrecht, 0.; Johnston, D. S.;Villaverde,C.; Chapman,D. Biochim. Biophys. Acta 1982,687,165.

began to consider the properties of existing biomaterials and to begin to develop new bi~materials.~~ The clinical application of devicesor biomaterials which contact blood is of major importance in modern medicine. Adverse reactions between foreign or prosthetic surfaces and blood components are the pre-eminent factors restricting the use of certain biomaterials. Typical examples of this are the inability to have biosensors to operate effectively for any length of time in blood and also the need for anticoagulants to be used during extracorporeal procedures such as dialysis and heart-lung bypass operations. There have been many attempts to understand why certain materials cause blood coagulation and many suggestions have been made as to how to overcome the problem. Andrade45has commented, “virtually every physical and chemical characteristic of materials has been suggested as being important in blood coagulation and thrombosis”. Our New Approach. Our approach to this problem is to mimic the outer lipid polar surface of the red blood cells and platelet cells.46 We have already pointed out that the lipid matrix of these cells has lipid class asymmetry. Furthermore we noted that while the inner lipid layer contains lipids, e.g. phosphatidylserine which on contact with blood can cause blood coagulationto occur, the outer lipid layer contains lipid classes which do not.47 The lipids of the outer biomembrane lipid matrix are lecithin and sphingomyelin. These lipid classes possess the same polar group so that the outer lipid polar surface of these biomembranes consists (90%) of only one polar group, the phosphorylcholine group. We next formed Langmuir-Blodgett films with lecithin molecules and diacetylene phospholipids which have this polar group and coated plastic surfaces43and also used a solution dipcoating method. Studies of blood coagulation characteristics where the phosphorylcholine polar group was in contact with blood gave excellentresults. For these studies we first used a simple thrombelastography test46and later examined platelet activation, platelet adhesion, and complement activation49with excellent results (see Figure 8). In further developments we showed that it is not the total phospholipid structure with lipid chains which is essential for hemocompatibility. It is the phosphorylcholine headgroup itself. We therefore began to attach phosphorylcholine polar groups in a number of different ways to a variety of surfaces and we were able to produce considerable improvements in hemo~ompatibility.~~ For example, film physisorbablephosphorylcholine-containing polymers have been synthesized for coating hydrophobic surfaces such as PVC, polyethylene, polypropylene, etc. These polymers, based on methacrylate chemistry, have high molecular weights and form stable coatings owing to their multipoint attachment. Materials such as celluloses and stainless steel have been successfully coated.49 Functionally active phosphorylcholine derivativeshave been synthesized, either as individual functionally active (44)Chapman, D. European Patent 32622, 1979. (45)Andrade, J. D.; Hlady, V. Adu. Polym. Sci. 1986,79,1. (46)Hayward, J. A.; Chapman, D. Biomaterials 1984,5, 135. (47)Zwaal, R. F. A.; Beavers, E. M. In Haemostasis Subcellular Biochemistry; Roodyn, D. B., Ed.; Plenum Press: New York, 1983;Vol. 9,p 299. (48)Bird, R.;Hall, B.; Chapman, D.; Hobbs, K. E. F. Thromb. Res. 1988,51, 471. (49)(a) Chapman, D.; Charles, S. A. Chem. Br. 1992,B (3),253. (b) Hall, B.; Pearce, D. J.; Campbell, E. J.; Sullivan, A. M.; New, R. R. C.; Charles, S. A. In Reference Materials of the European Communities: Test Results; Lemm, Ed.; Kluwer Academic Publishers: Dordrecht, in press. (50)Chapman, D.; Durrani, A. A. European patent 157469,1984.



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headgroupsw or as methacrylate based polymers. These molecules have atso been chemically attached to a variety of hydrophilic surfaces. Various surface modifications have been made to introduce partner functional groups where these groupsare not already present, e.g. introducing acid groups into stainless steel for the subsequent reaction of aminophosphorylcholine derivatives. Other new materials have been made either by introducing phosphorylcholine derivatives as plasticisere into polymers such as PVC and p0lyurethane~~*~2 or by copolymerising phoephorylcholine monomers into the polymer backbone of polyurethanes and polyesters.sg Phosphorylcholine treatments have now been successfully applied to a wide range of biomaterials. Medical devices,suchas intravascular catheters, drainage catheters, indwelling biosensors, extracorporeal circuits, clinical diagnostic kite, and filtration membraneshave been treated successfully. These in vitro observations have been recently substantiated by results from early animal and human clinical testa. Phosphorylcholine technology has now become established in a wide range of medical devices. We continue to ask basic fundamental questions about this new biomaterial technology. Why is the phosphorylcholine polar group so effective a t eliminating blood clotting processes? Is it due to the zwitterion structure which keeps it electrically neutral over a wide pH range (3-10) or to the large amount of water which is tenaciously held by this polar group. We studied the properties of the "bound water" many years ago using calorimetry and deuterium NMR spectroscopy and know that this polar group surrounds itaelf by such structured "unfreezing water".M We have also shown that the phosphorylcholine groupwhen in a tightly packed layer does not readily bind blood proteins, e.g. fibrinogen (see Figure 9) or even proteins such as lysozyme. Isolated phosphorylcholine groups can however bind to C-reactive protein and to (61) Hayward, J. A.; Durrani, A. A.; Lu, Y.; Clayton,C.; Chapman, D. Biomateriale 1986, 7, 262. (52) Valencia, G. P. European patent 247114,1985. (63)Durrani, A. A. European patent 276293,1986. (64) Snlebury,N.J.; Darke,A.; Chapman, D.Chem.Phys. Lipids 1972, 8, 142.

Post-flow Re-flow Figure 9. Extent of protein adsorption (fibrinogen) onto uncoatedand coated PVCwith phoepholipidpolymer. Reprinted with permission from ref 49b. Copyright 1992 Kluwer Academic Publishers.

certain antilipid antibodies. The fact that many proteins do not bind easily to this polar group led us to further technologicalapplications, i.e. to synthesize new hydrogel polymers containing phosphorylcholine groups which can be used to construct contact lenses. These lenses show no protein deposition and no drying out of the lens.

Conclusions Although considerable work by many scientists has clarified our views of biomembrane structures, many important questions remain which surface chemists and colloid chemists are considerably suited to rmolve, e.g. membrane transduction processes such as ion channel processes, membrane fusion, cell and membrane contact phenomena, and protein translocation across biomembranes. These proceases allrequire a deeper undemtanding than we have at present and involve an understanding of amphipathic molecules. We have demonstrated how molecular biology and a knowledge of biomembraneslinked with polymer chemistry can produce new coatings and new biomaterials. We envisage many other applications of this type including new approachesto delivery systems, new protein resistant and antifouling surfaces, gene transfer membranes, and new biosensor design. Langmuir himself said "Many radically new things for the home and elsewhere can come from fundamental research." Acknowledgment. I wish to thank the many colleagues both in the industrial environment, e.g. Unilever and Biocompatibles, Ltd.,and in the university environment who have worked with me on these topics. I also thank the Science & Engineering Research Council, the IRC on Medical Biomaterials, and the Wellcome Trust for their financial support for our studies.

Biomembranes and new hemocompatible materials - American

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