COLLOIDSANDSURFAC~ A

ELSEVIERColloids and Surfaces

A: Physicochemical and Engineering Aspects 117 (1996) 1-5

Review

Recent trends in bilayer formation of synthetic amphiphiles

Joy T. Kunjappu. P. Somasundaran*Langmuir Center for Colloids and Interfaces. Henry Krumb School of Mines. New York. NY 10027. USA

Received 17 April 1995; accepted 7 March 1996

Abstnct

The property of synthetic dialkyl amphiphiles to form vesicular structures can be exploited in a variety of situations,such as mimick:ing biological membranes, drug delivery, studies related to artificial photosynthesis, aggregates onsolids, etc. The stability and thermotropic behavior of liposomes from natural lipids could be improved by syntheticallymodifying the alkyl chains of the lipids or by using totally synthetic structures. This article, which highlights theimportant milestones in the development of vesicular structures from synthetic amphiphilcs, encompasses a briefdescription of investigations reporting the introduction of totally synthetic dialkyl amphiphilcs, mixtures of monoalkylcationic and anionic amphiphiles, hyperextended alkyl amphiphiles, and so on. Furthermore, most of the relevantwork in the last decade relating to bilayer formation from synthetic amphiphiles is documented here.

Organized molecular assemblies have capturedthe attention of chemists, biologists, and physicistsever since Langmuir observed the behavior of long-chain fatty acids at the liquid-air interface. Byconfining the molecules in a thin unimolecular filmunder pressure, the hydrocarbon chains of the fattyacids were made to orient into air, which led tothe concept of the pressure-area (7t-A) isothermand to the calculation of molecular area. Later on,these molecular assemblies could be transferredonto a solid substrate by a simple dipping andcoating procedure known as the Langmuir-Blodgett technique [1], the repetition of whichenabled the creation of multilayered assemblies.Parallel to these were the developments in therealm of ampbipbilic molecules which aggregate

with a number of aesthetically pleasing and intel-lectually stimulating structural variations such asglobules, cylinders, bilayers and lamel1ae [2].

Biological membranes have been known to con-sist of amphiphilic lipid molecules. The first indi-cation of the bilayered nature of biologicalmembranes was obtained by Gorter and Grendel[3], who showed that the lipid from erythrocytemembrane occupied, on a Langmuir surface bal-ance, a surface area equal to double the externalarea of the cel1s as determined by optical micro-scopy. Although discrepancies were pointed outwith refined calculations of molecular area, thisview prevails even today [4]. The bilayer structureof biological membranes gained general acceptancewhen Singer and Nicholson [5] put forward theirfluid mosaic model of the structure of cel1membranes.. Corresponding author.

0927-7757/96/515.0001996 Elsevier Science B. V. All rights reservedPH 80927-7757(96 )03636-9

J. T. KJmjQppu. P. Soma.nmdaranlCol/oids Surfaces If: Physicochem. &g. ~ts J I 1996) 1-5

in a recent book by Lasic [13]. Such a syntheticstrategy can have benign effects in controlling thephase behavior and thennodynamic parameters ofresultant liposomes. In addition, Lasic collates inthis work a wealth of information on the physical,chemical, biological and engineering aspects ofliposomes.

Parallel to the use of natural bilayer forminglipids and pure synthetic compounds, severalattempts were made in modifying the natural struc-tures synthetically so as to achieve superior vesicu-lar properties needed for several applications.Improvements in the phannacokinetics and anti-tumor therapeutic efficiency of liposomes havebeen obtained by incorporating poly(ethyleneglycol) groups into phospholipids [14]. The pas-sage of ions through phospholipid vesicles couldbe facilitated by incorporating alkyl substituentsinto the lipid chain [15]; the effect of the size,position, and number of the substituents on theion flux during ion transport across bilayer mem-branes was monitored from the fluorescence of thepH sensitive dyes. The preparation of more highlystable liposomes than the conventional ones fordrug delivery and substances for controlling pros-taglandin levels is also reported by the same group[16]. They also adopted calorimetry to investigatesynthetically substituted phosphatidylcholines,correlated their thermotropic behavior with thenature of substituents on the hydrocarbon chainand threw light into the structure-activity relation-ship [17]. The much-expected performance oflipo-somes as safe drug carriers could not be realized,owing to the instability of these aggregates whichfailed to reach their targets. Lasic, in a recentreview [18], explains how this problem may becircumvented by tethering polymeric groups on tothe liposome structure, which act as a protectivecoating. A novel method for drug encapsulation insynthetically modified bilayers which took advan-tage of the pH gradient in the interior and exteriorof the liposomes was reported recently [19]. Thestructure-function relationship of PEG-derivatizedphosphatidylethanolamine in relation to its distri-bution in the blood stream has been reviewed byWoodle and Lasic [20]. The fonnation, stabilityand size distribution of liposomes have beentreated theoretically in a recent report [21].

A lipid bilayer structure with a spherical geome-try constituting the external membrane of cells hasbeen referred to as vesicles (liposomes). Vesiclescan be prepared by reconstituting lipid moleculessuch as dipalmitoyl phosphatidylcholine [6]. Thedialkyl chain was considered to be crucial for theorganization of the lipids into vesicles. In fact,unifying the role of geometry with those of molecu-lar forces and entropy, Israelachvili et al. [7]fonnulated the limiting condition for the sponta-neous aggregation of amphiphiles into vesicles asexpressed by the V fat parameter where V is thevolume of the hydrocarbon chain, a is the effectiveheadgroup area, and t is an optimum length corre-sponding to the fatty extended chain.

Vesicles generated immense interest due to theirpotential in mimicking biological membranes, inacting as microreactors for artificial photosynthesisand other chemical reactions, in drug delivery, andin the design of photonic, electronic and magneticfunctional units [8]. The crucial passage of lipo-somes from basic research to clinical practice hasbeen described by Lasic and Papabadjopoulos [9].They highlighted the advances in this area withspecial reference to gene therapy and drug delivery.Landmarks in liposome research paving the wayfor their therapeutic applications both in the fonnof oral administration and intravenous injectionshave been described in a series of articles dedicatedto Bangham who pioneered the preparation ofphospholipid vesicles [10,11].

Most of the studies on vesicle formation utilizedthe naturally occurring phospholipid moleculesuntil Kunitake and Okabata [12] demonstrated,using electron microscopy and NMR spectroscopy,the formation of a totally synthetic bilayer mem-brane from the double-chain surfactant didodecyl-dimethylammonium bromide in comparison withthe already well-established dipalmitoyl lecithinvesicles. Afterwards, a wealth of literature accumu-lated on the topic of bilayer and vesicle formationfrom synthetic amphiphiles. It is the aim of thisnote to highlight the pathbreaking recent contribu-tions in this field, since similar systems are gainingimportance in areas such as drug delivery, adsorp-tion at the solid-liquid interface, etc.

The possibility of preparing semi and fully syn-thetic double-chain polar lipids has been described~

J. T. Kunjappu. P. SomasundaranfColloids Surfaces A: Physicochem. Eng. Aspects 117 ( 19%j 1-5

All the four major classes of synthetic double-chain surfactants, i.e. cationic, anionic, non-ionic,and zwitterionic, are shown to aggregate into bilay-ered structures. Kunitake [22] pioneered thesestudies in which he introduced the concept ofinterposing the tail and head groups with connec-tor and spacer groups. Connectors and spacersserved to influence the alignment and molecularorientation in the bilayer. He further showed thatbilayer formation is exhibited by triple-chain [23]and quadruple-chain [24] amphiphiles.

A few groups have been active in finding novelefficient synthetic systems capable of forming vesic-ular bilayer structures. Some of the recent develop-ments in this area follow. The creation anddestruction of vesicles formed from didodecyldi-methylammonium bromide by octyl glucoside andsodium cholate, respectively were the theme of arecent study by Menger and Gabrielson [25].They documented the direct microscopic observa-tion of chemically induced evolutionary stages ingiant vesicles (aggregation, budding and fusion)caused by sodium sulfate using didodecyl cationicamphiphiles [26]. Bilayer phases in aqueousmixtures of dodecylpentaoxyethylene glycolmonoether -and sodium dodecyl sulfonate wereinvestigated by Douglas and Kaler [27]; themicrostructures of the phases were revealed bysmall-angle neutron scattering and classical lightscattering techniques. Double-chain ammoniumamphiphiles have been used by Wakayama andKunitake for the direct observation of orderedbilayers in cast films [28]. The thermal stabilityand specific dye binding of a hydrogen-bond-mediated bilayer membrane have also been thesubject of a recent study [29]. Self-assembledbilayers with novel molecular functions have beendesigned, and their efficacy in selectively bindingvarious guest molecules were the theme of anotherstudy [30]. The expectation that covalent linkingof component molecules would stabilize vesicles,facilitating their use in practical applications suchas controlled drug delivery, has been realized bythe polymerization of aqueous bilayers of gluta-mate-based double-chain ammonium amphiphiles[31]. Synthetic bilayer membranes with physico-chemical characteristics superior to those of natu-ral biolipid bilayer membranes are also reported;

some of these self-assembling systems are found tobe advantageous in making other molecular mem-branes such as cast multilayers, surface mono-layers, Langmuir-Blodgett multilayers, and planarlipid membranes, and they could also serve asmolecular templates [32]. One study is devotedto closely observing vesicles of synthetic amphiphi-lic ammonium bilayers by high-resolution scanningelectron microscopy and comparing the resultswith those from transmission electron microscopy[33]. Novel assemblies incorporating metal che-lates in synthetic bilayers linked either covalentlyor non-covalently are reported by Isshikawa andKunitake [34].

A bilayer forming capability was thought to bethe prerogative of double-chain amphiphiles.Recently, Kaler et al. [35] demonstrated sponta-neous vesicle formation in aqueous mixtures ofsingle-tailed surf act ants. They employed cationiccetyltrimethylammonium tosylate and anionicsodium dodecyl benzenesulfonate amphiphiles, andsubstantiated the vesicle formation (stable for atleast 1 year) using quasi-elastic light scattering,freeze-fracture transmission electron microscopy,and glucose entrapment experiments. They couldeven tune the vesicular charge and size by manipu-lating the amphiphile composition. This is the firstexample of spontaneous vesicle formation in vitrowithout expending energy in the form of sonicationor so, although vesicles are formed spontaneouslyin vivo. Such a theoretically bafDing phenomenonwas rationalized by assuming that the moleculesform anion-cation amphiphile pairs that may thenact as double-chain zwitterionic amphiphiles.

The idea of creating bilayer membranes fromsingle-chain amphiphiles was addressed some timeback. The effect of two alkyl chains in bilayer-forming amphiphiles is to restrict their conforma-tional mobility. The same effect could be achievedin single-chain surfactants by introducing a rigidsegment into the alkyl chain. Kunitake andOkahata [36] succeeded in this by incorporatinga liquid-crystal-forming moiety (a Schiff base unit)in the structure and by manipulating the alkylchain, the spacer and the rigid segment. Veryrecently, Menger and Yamasaki [37] achievedbilayer formation from single-chain amphiphilescontaining unusually long alkyl chains, two to

4 J. T. Klmjappu, P. Soma.nIIIdanIn, oIloidr Sllrfaces A: Physicochml. Eng. As~cts 11 1996)

three times longer than the normal chain (hyperex-tended amphiphiles). The accompanying solubilityproblem of the amphiphile molecule was circum-vented by making the molecule multi-ionicallyheaded (unsubstituted). They argued that thehydrophile-lipophile balance is also a determiningfactor in vesicle-forming capability apart from thegeometric packing parameter Vial (defined earlier).

Very recently, Zana and Talmon [38] predictedthe possibility of a spontaneous formation of vesi-cles from dimeric bisammonium compounds inwhich the quaternary ammonium groups are sepa-rated by methylene spacers. They investigated theaggregate morphology as a function of alkyl chainlength and methylene spacer length. Earlier,Kunitake et al. [39] related similar molecules ascapable of forming monolayer membranes. Lasicalso details the methods of preparing chemically,biologically and physically stable liposomes fromthe relatively new class of bolaamphiphiles [13].

An interesting new development in this area isthe discovery of reverse vesicles, the counterstruc-ture of biological membranes [40]. In reversevesicles, the hydrophilic part is located in theinterior of the bilayer. Such an orientation ofamphiphilic molecules as being completely oppo-site to "that in normal vesicles has been observedin stable reverse vesicle formation from sucrosemonoalkanoate, hexanol, and water in decane.Structures similar to reverse vesicles have beeninvoked to explain the stability of colloidal gasaphrons [41] that encapsulate a microbubble in adouble layer of amphiphilic molecules, retardingtheir coalescence.

The formation of stable bilayer aggregates hasbeen observed in a fluorocarbon medium [42]from amphiphiles with flexible fluorocarbon units.The molecules chosen contained double hydro-carbon chains as the solvophobic unit, glutamateor diethanolamine connectors to induce alignmentof the hydrocarbon chain, and a highly solvophilicfluorinated chain. The self-organization of solvo-phobic novel double-chained fluorocarbon deriva-tives has been investigated in organic solvents[43]; stable molecular bilayers are shown to beformed in organic media, though conventionalamphiphilic molecules such as hydrated lecithinform reversed micelles and gels under similar con-

ditions. Spontaneous vesicle formation is observedfrom ethoxylated perfluorocarbon alcohols at a1 wt.% surfactant concentration [44]. A new classof self-assembling amphiphiles has been reported[45] in which the construction of a bilayermembrane is achieved through complementaryhydrogen bonding, both in aqueous dispersionsand in cast films. The property of substitutedmelamines and isocyanuric acid derivatives to formextended arrays of complementary hydrogen bondsis explained in this mode of bilayer membraneformation [46].

The realization of bilayered and multilayeredamphiphilic structures on solid substrates (sup-ported bilayers and multilayers) has opened upnovel applications in thin-film technology [47]and is being actively pursued with the renewedpopularity of the Langmuir-Blodgett technique.The partake of structures similar to bilayers basbeen recognized in some separation techniqueswherein the hydrophobicity induced to the particlefines by the adsorbed layers [48] of amphiphilesat adsorption maxima plays a pivotal role inprocesses related to mineral enrichment.

The physics of aggregation of amphiphiles intovesicular and allied structures has a well-developedstatus. These structures are thought to be metasta-ble aggregates with high kinetic stability and lowthermodynamic stability derived from lamellarphases dispersed in a large excess of solvent.Theoretical interpretation of these systems isachieved by integrating concepts like bendingenergy, thermal roughening, etc. with thermo-dynamic and conformational analyses [49].Various mechanisms are enunciated to account forthe spontaneous formation of vesicles from syn-thetic amphiphiles: steric factors for the existenceof vesicles of singie-chain surfactants with rigidsegments in between and ion pairing for the forma-tion of vesicles from cationic and anionic surfac-tants. The energetics of these processes are verywell understood, and the physics of these processesis becoming clear, but the chemistry remainsobscure [51].

In conclusion, a significant amount of work hasbeen reported on novel synthetic amphiphiles capa-ble of forming vesicular structures under a varietyof conditions. In particular, some applications such

J. T. KWljappu, P. SomaslmdaranfCo/loids Surfaces A: Physicochem. Eng. Aspects 117 (1~) J-' s

as targeted drug delivery would benefit from theimproved stability and thermal properties of thesestructures. Thus, while attaining mild conditionsfor the formation of vesicular structures expendingminimum energy, recent work has revealed thecapability of hitherto unknown features of formingbilayered structures and of mimicking biologicalmembranes.

References

[23] T. Kunitake, N. Kimizuka, N. Higashi and N. Nakashima,J. Arn. Chern. Soc., 106 (1984) 1978.

[24] N. Kimizuka, H. Ohira, M. Tanaka and T. Kunitake,Chern. Leu., 29 (1990).

[25] F.M. Menger and K. Gabrielson, J. Am. Chern. Soc., 116(1994) 1567.

[26] F.M. Menger and N. Balachander, J. Am. Chern. Soc.,114 (1992) 5862.

[27] C.B. Douglas and E. W. Kaler, J. Chern. Soc. FaradayTrans., 90 (1994) 471.

[28] Y. Wakayama and T. Kunitake, Chern. Lett., (1993) 1425.[29) N. Kirnizuka, T. Kawasaki and T. Kunitake, Chern. Lett.,

(1994) 33.[30] T. Kunitake, Thin Solid Films, 210/211 (1992) 48.[31J S. Kato and T. Kunitake, Polym. J., 23 (l99l) 135.[32] T. Kunitake, Polym. J., 23 (1991) 613.[33] Y. Ishikawa, T. Nishimi and T. Kunitake, Chern. Leu.,

(1990) 25.[34J Y. Ishikawa and T. Kunitake, J. Macrornol. Sci., Chern.,

27 (1990) 1157.[35] E.W. Kaler, A.K. Murthy, B.E. Rodriguez and J.A.N.

Zasadzinski, Science, 245 (1989) 1371.[36J T. Kunitake and Y. Okahata, J. Arn. Chern. Soc., 102

(1980) 549.[37] F.M. Menger and Y. Yamasaki, J. Am. Chern. Soc., 115

(1993) 3840.[38] R. Zana and Y. Talmon, Nature, 362 (1993) 228.[39J T. Kunitake, A. Tsuge and K. Takarabe, Polym. J.

(Tokyo), 17 (1985) 633.[40J H. Kunieda, M. Akimaru, N. Ushio and K. Nakamura,

J. Colloid Interface Sci., 156 (1993) 446.[41] P.G. Chaphalkhar, K.T. Valsaraj and D. Roy, Sep. Sci.

Technol, 28 (1993) 1287.[42J H. Kuwahara, M. Hamada, Y.lshikawa and T. Kunitake,

J. Am. Chern. Soc., 115 (1993) 3002.[43J Y. Ishikawa, H. Kuwahara and T. Kunitake, Chern. Letl,

(1989) 1737.[44] J. Wurtz and H. Hoffmann, J. Colloid Interface Sci., 175

(1995) 304.[45J N. Kimizuka, T. Kawasaki and T. Kunitake, J. Am.

Chern. Soc., 115 (1993) 4387.[46J G.M. Whitesides, E.E. Simanek, J.P. Mathias, C.T. Seto,

D.N. Chin, M. Mammen and D.M. Gordon, Acc. Chern.Res., 28 (1995) 37.

[47] J.D. Swalen, D.L. Allara, J.D. Andrade, EA Chandross,S. Garoff, J.N. Israelachvili, TJ. McCarthy, R. Murray,R.F. Pease,J.F. Rabolt, KJ. Wynne and H. Yu, Langmuir,3 (1987) 932.

[48] J.T. Kunjappu and P. Somasundaran, J. Phys. Chern., 93(1989) 7744.

[49] J. Meunier, D. Langevin and N. Boccara (Eds.), Physicsof Amphiphilic Layers, Springer-Verlag, Berlin, 1987.

[50] M.-P. Krafft, F. Giulieri and J.G. Riess, Angew. Chern.Inl Ed. EngL, 105 (1993) 783.

[51] J. Maddox, Nature, 363 (1993) 205.

[1] K.B. Blodgett and I. Langmuir, Pbys. Rev., 51 (1937) 964.[2] B. Lindman and H. Wennerstrom, in Topics in Current

Chemistry, Springer-Verlag. Berlin, 87 (1980) 1-83.[3] E. Gorter and F. Grendel, J. Expt. Med., 41 (1925) 439.[4] D.M. Engelman, Nature, 223 (1969) 1279.[5] S.J. Singer and G.L. Nicholson, Science, 175 (1972) 720.[6] A.D. Bangham and R.W. Home, J. MoL BioL, 8

(1964) 660.[7] J.N. Israelachvili, D.J. Mitchell and B.W. Ninham,

J. Chem. Soc., Faraday Trans. 2, 72 (1976) 1525.[8] J.H. Fendler, Membrane Mimetic Chemistry, Wiley-

Interscience, New York, 1982.[9] D.D. Lasic and D. Papahadjopoulos, Science, 267

(1995) 1275.[10] H.M. Patel and NJ. Russell, Biochern. Soc. Trans., 16

( 1988)9{1).[11] D. Papahadjopoulos, Biochem. Soc. Trans., 16 (1988)910.[12] T. Kunitake and Y. Okahata, J. Am. Chern. Soc., 99

(1977) 3860.[13] D.D. Lasic, Liposomes: from Physics to Applications,

Elsevier, The Netherlands, 1993.[14] D. Papahadjopoulos, T.M. Allen, A. Gabizon, E. Mayhew,

K. Matthay, S.K. Huang. K.-D. Lee, M.C. Woodle, D.D.Lasic, C. Redemann and F J. Martin, Proc. Natl. Acad.Sci., 88 (1991) 11460.

[15] F.M. Menger and P. Aikens, Angew. Chern. Int. Ed.Engl., 31 (1992) 898.

[16] F.M. Menger and M.G. Wood, Jr., Angew. Chem. Int.Ed. EngL, 28 (1989) 1218.

[17] F.M. Menger, M.G. Wood, Jr. and Q.Z. Zhou, J. Am.Chern. Soc., 110 (1988) 6804.

[18] DD. Lasic, Angew. Chern. Int. Ed. Engl., 33 (1994) 1685.[19] DD. Lasic, P.M. Frederik and M.C.A. Stuart, FEBS

Lett., 312 (1992) 255.[20] M.C. Woodle and DD. Lasic, Biochim. Biophys. Acta,

1113 (1992) 171.[21] M. Winterhalter and DD. Lasic, Chem. Phys. Lipids,64

(1993) 35.[22] T. Kunitake, Angew. Chem. Int. Ed. Engl., 31 (1992) 709.