Articles

Vesicle Formation from Dimeric Ion-Paired Amphiphiles.Control over Vesicular Thermotropic and Ion-Transport

Properties as a Function of Intra-amphiphilic HeadgroupSeparation†

Santanu Bhattacharya* and Soma De

Department of Organic Chemistry, Indian Institute of Science, Bangalore 560 012, India

Received July 14, 1998. In Final Form: October 26, 1998

A novel series of vesicle-forming ion-paired amphiphiles, bis(hexadecyldimethylammonium)alkanedipalmitate (1a-1h), containing four chains were synthesized with two isolated headgroups. In each ofthese amphiphiles, the two headgroup charges are separated by a flexible polymethylene spacer chain-[(CH2)m]- of varying lengths (m) such that the length and the conformation of the spacer chain determinethe intra-“monomer” headgroup separation. Transmission electron microscopy indicated that each of theseforms bilayer membranes upon dispersion in aqueous media. The vesicular properties of these aggregateshavebeenexaminedbydifferential scanningcalorimetryandtemperature-dependent fluorescenceanisotropymeasurements. Interestingly, their Tm values decreased with the increase in the m value. Thus while theapparent Tm of the lipid with m ) 2 (1a) is 74.1 °C, the corresponding value observed for the lipid withm ) 12 (1h) is 38.9 °C. The fluorescence anisotropy values (r) for 1b-1g were quite high (r ∼ 0.3) comparedto that of 1h (r ∼ 0.23) at 20-30 °C in their gel states. On the other hand, the r value for vesicular 1bbeyond melting was higher (0.1) compared to any of those for 1c-1h (∼0.04-0.06). X-ray diffraction ofthe cast films was performed to understand the nature and the thickness of these membrane organizations.The membrane widths ranged from 30 to 51 Å as the m values varied. The entrapment of a small water-soluble solute, riboflavin, by the individual vesicular aggregates, and their sustenance under an imposedtransmembrane pH gradient have also been examined. These results show that all lipid vesicles entrapriboflavin and that generally the resistance to OH- permeation decreases with the increase in m value.Finally, all the above observations were comparatively analyzed, and on the basis of the calculated structuresof these lipids, it was possible to conclude that membrane properties can be modulated by spacer chainlength variation of the ion-paired amphiphiles.

Introduction

Lipids form the bilayer membrane core (BLM) ofbiological cells.1 Almost all biosyntheses and vectorialreaction chains such as photosyntheses, respiration, nerveexcitation, and so forth are carried out within BLMsbecause of the presence of membrane-associated proteinswith definite orientations and their ability to sustain andtransport water-soluble solutes and various ions.2-5

However, due to difficulties associated with their isolation,effective purification, and characterization, it has beennecessary to develop synthetic analogues of lipids. Inaddition to their utilities in reconstitution of enzymes andmembrane properties in functional studies, lipidic am-phiphiles have been widely employed to understand theirbehavior at the molecular level upon self-organization.Moreover, synthetic amphiphiles that form micellar orvesicular aggregates in aqueous media are also attractivefor their potential in diverse applications such as drugdelivery, catalysis, and synthesis of nanomaterials.6-8

Recently, a new class of bipolar amphiphiles has beendeveloped which are made up of two hydrophobic chainsand two ionic headgroups covalently attached through aspacer at the level of the headgroups.9,10 Upon dispersionin water, while hydrocarbon segments in a surfactant unit

† Dedicated to Professor C. N. R. Rao on the occassion of his 65thbirthday.

* To whom correspondence should be addressed. Fax: 91-080-334-1683. E-mail: sb@orgchem.iisc.ernet.in. Also at the ChemicalBiology Unit, Jawaharlal Nehru Centre for Advanced ScientificResearch, Jakkur, Bangalore 560 064, India.

(1) (a) Merz, K. M., Jr.; Roux, B. Biological Membranes. A MolecularPerspective from Computation and Experiment; Birkhauser: Boston,1996. (b) Slater, J. L.; Huang, C.-H. In The Structure of BiologicalMembranes; Yeagle, P., Ed.; CRC Press: Boca Raton, FL, 1992; p 175-210. (c) Marsh, D. CRC Handbook of Lipid Bilayers; CRC Press: BocaRaton, FL, 1990.

(2) (a) Grunner, S. M.; Jain, M. K. Biochim. Biophys. Acta 1985, 818,352. (b) Bittman, R.; Clejan, S.; Jain, M. K.; Deroo, P. W.; Rosenthal,A. F. Biochemistry 1981, 20, 2790. (c) Friedberg, S. J.; Holpert, M. J.Lipid. Res. 1978, 19, 57.

(3) (a) De Rosa, M.; Gambacorta, A.; Gliozzi, A. Microbiol. Rev. 1986,50, 70. (b) Langworthy, T. A. In The Bacteria. The Treatise on Structureand Function; Woese, C. R., Wolfe, R. S. Eds.; Academic: Orlando, FL,1985; pp 459-497.

(4) Setter, K. O. Nature 1982, 300, 258.(5) Brock, T. D.; Brock, K. M.; Belley, R. T.; Weiss, R. L. Arch.

Mikrobiol. 1972, 84, 54.

(6) Ringsdorf, H.; Schlarb, B.; Venzmer, J. Angew. Chem., Int. Ed.Engl. 1988, 27, 113.

(7) (a) Bhattacharya, S.; Snehalatha, K.; George, S. K. J. Org. Chem.1998, 63, 27. (b) Bhattacharya, S.; Snehalatha, K. Langmuir 1997, 13,378. (c) Bhattacharya, S.; Snehalatha, K. J. Org. Chem. 1997, 62, 2198.(d) Bhattacharya, S.; Snehalatha, K. Langmuir 1995, 11, 4653. (e)Scrimin, P.; Tecilla, P.; Tonellato, U. J. Am. Chem. Soc. 1992, 114,5086.

(8) (a) Schnur, J. M. Science 1993, 262, 1669. (b) Whitesides, G. M.;Mathias, J. P.; Seto, C. T. Science 1991, 254, 1312.

(9) (a) Aswal, V. K.; De, S.; Goyal, P. S.; Bhattacharya, S.; Heenan,R. K. Phys. Rev. 1998, 57E, 776. (b) De, S.; Aswal, V. K.; Goyal, P. S.;Bhattacharya, S. J. Phys. Chem. B 1998, 102, 6152. (c) De, S.; Aswal,V. K.; Goyal, P. S.; Bhattacharya, S. J. Phys. Chem. 1996, 100, 11664.(d) Zana, R.; Benrraou, M.; Rueff, R. Langmuir 1991, 7, 1072.

3400 Langmuir 1999, 15, 3400-3410

10.1021/la9808770 CCC: $18.00 © 1999 American Chemical SocietyPublished on Web 04/21/1999

tend to minimize water exposure and thus prefer to self-organize and aggregate closely, the twin headgroups ofidentical charge tend to stay away from each other as aresult of electrostatic repulsion. The morphology of thesemicellar aggregates has been shown to strongly dependon the spacer chain length (m value). These bipolaramphiphiles are architecturally related to bolaphiles.Synthetic bolaphiles are mimics of archaebacterial mem-branes which can sustain extreme physiological conditions(85 °C, pH ∼ 1) and form ultrathin “monolayer” mem-branes.11,12 Due to these exceptional properties, a lot ofinterest is growing toward the development of their mimicsto sort out their unusual tolerance to external stimulicompared to that of the membrane present in otherorganisms.

Despite manifestation of impressive properties withdimeric micelle-forming surfactants, the scope of corre-sponding membrane-forming analogues has not beenexamined adequately. On the basis of these considerations,we decided to develop dimeric vesicle-forming surfactantsand investigated the role of the variation of the spacerbetween polar headgroups on various properties of thelipid aggregates. In a preliminary communication, wereported vesicle formation via ion-pairing of an anionicamphiphile with biscationic geminis.13 Here, we presentin detail the synthesis, vesicle-forming properties andthermotropic behavior of a series of bis(hexadecyldi-methylammonium)alkane dipalmitate amphiphiles, 1a-1h, in which the two headgroups of the bolaamphiphiliccounterions are attached by a flexible spacer polymeth-ylene chain (Figure 1). Differential scanning calorimetryand fluorescence depolarization studies have been per-formed to establish their thermal phase transition prop-erties. Cast film X-ray diffraction studies on multilayershave been carried out to secure insight about the possiblenature of the bilayer organization in these membranes.The dye entrapment abilities and permeability propertiesof these vesicles under an imposed transmembrane pHgradient were also looked upon. Energy-minimized struc-tures of these ion pairs were calculated to rationalize theexperimental findings. Interestingly, the thermal transi-

tion profiles and the permeabilities of these vesicularaggregates were found to be significantly influenced bythe length of the spacer chain in the gemini amphiphilicdications.

Experimental Procedure

General Methods. Melting points were recorded inopen capillaries and are uncorrected. 1H NMR spectrawere recorded on Bruker SEM-200 (200 MHz) and BrukerWH-270 (270 MHz) NMR spectrometers. Chemical shifts(δ) are reported in ppm downfield from the internalstandard, TMS. IR spectra were recorded on a Perkin-Elmer Model 781 spectrometer and are reported inwavenumbers (cm-1). Microanalyses were performed ona Carlo Erba elemental analyzer model 1106. Steam-distilled water was used for all physical measurements,and pH measurements were made with a SystronicsDigital pH meter 335.

Materials.Cetyltrimethylammonium bromide (CTAB),cetyl bromide, R,ω-alkanedibromides, palmitic acid, 1,6-diphenyl-1,3,5-hexatriene (DPH), riboflavin, pyrene, andAmberlite IRA-900 ion-exchange resin (strongly basic,macroreticular resin of moderately high porosity) werepurchased from Aldrich Chemical Co. N-n-hexadecyl-N,N-dimethylamine was obtained by refluxing cetyl bromidewith dimethylamine (Merck, 40% solution in water) indry ethanol at 80 °C for 24 h. The ion-exchange resin wasfirst washed with MeOH several times and then convertedto its OH- form by stirring it with 3 N NaOH solutionovernight at room temperature. Thin-layer chromatog-raphy was performed on silica gel-G (Merck)-coated plates.Column chromatography was done on silica gel (60-120mesh) obtained from Merck. All the reagents and solventsthat were used in this study were of the highest gradeavailable commercially and used purified, dried, or freshlydistilled as required according to a literature procedure.14

Synthesis of Bis(quaternary ammonium) Surfac-tants (2a-2h). The bis(quaternary ammonium) surfac-tants 2a-2h were synthesized according to a literatureprocedure.9c

GeneralProcedurefortheSynthesisof Ion-PairedAmphiphiles (1a-1h). First, the dibromide counterionsof 2a-2h were converted to the hydroxide (OH-) form bythe passage of a methanolic solution of freshly recrystal-lized 2a-2h (1 equiv) through a column (9 cm × 1.5 cm)filled with freshly washed ion-exchange resin (AmberliteIRA-900, OH- form). The eluents were collected together,to this was added 2.0 equiv of palmitic acid (freshlyrecrystallized), and the reaction mixture was stirred atroom temperature for 12 h under a nitrogen inlet. Solventwas evaporated under vacuum and the solid obtained wasrecrystallized several times from a mixture of MeOH/EtOAc (1:10) (yield ∼ 90-95%). IR showed the absenceof a carboxylic acid moiety (1710 cm-1) and the appearanceof an ammonium carboxylate group (Me2N+‚‚‚-O2C) (1560cm-1) in these ion pairs.

All compounds (1a-1h) were found to be hygroscopicand crystallized as hydrates despite prolonged dryingunder vacuum. IR and repetitive elemental analyses alsoconfirmed the presence of water molecules in them. High-resolution 1H NMR spectra of 1a-1h confirmed thepresence of an additional peak due to hydrate formation.However, this additional peak could vanish upon treat-ment with D2O, as they were found to be fully exchangeablewith D2O. They gave satisfactory 1H NMR, IR, and C, H,

(10) (a) Oda, R.; Huc, I.; Candau, S. J. Chem. Commun. 1997, 2105.(b) Sommerdijk, N. A. J. M.; Hoeks, T. H. L.; Synak, M.; Feiters, M. C.;Nolte, R. J. M.; Zwanenburg, B. J. Am. Chem. Soc. 1997, 119, 4338. (c)Duivenvoorde, F. L.; Feiters, M. C.; van der Gaast, S. J.; Engberts, J.B. F. N. Langmuir 1997, 13, 3737. (d) Menger, F. M.; Littau, C. A. J.Am. Chem. Soc. 1991, 113, 1451.

(11) Bolaphiles (bolaamphiphiles), in contrast to their monopolarcounterparts (single-chain/single-polar-headgroup amphiphile) consistof two hydrophilic headgroups linked to the hydrophobic core at the R,ωpositions. See for reviews: (a) Escamilla, G. H.; Newkome, G. R. Angew.Chem., Int. Ed. Engl. 1994, 33, 1937. (b) Fuhrhop, J.-H.; Bach, R. InAdvances in Supramolecular Chemistry; Gokel, G. W., Ed.; JAI Press:Greenwich, CT, 1992; Vol. 2, pp 25-63.

(12) (a) Gulik, A.; Luzatti, V.; De Rosa, M.; Gambacorta, A. J. Mol.Biol.1985, 182, 131. (b) Gliozzi, A.; Rolandi, R.; De Rosa, M.; Gambacorta,A. J. Membr. Biol. 1983, 75, 45.

(13) Bhattacharya, S.; De, S. J. Chem. Soc., Chem. Commun. 1995,651.

(14) Perrin, D. A.; Armerego, W. L.; Perrin, D. R. Purification ofLaboratory Chemicals, 3rd ed.; Pergamon: New York, 1990.

Figure 1. Chemical structures of the dimeric ion-paired lipids.

Vesicle Formation from Dimeric Ion-Paired Amphiphiles Langmuir, Vol. 15, No. 10, 1999 3401

N analyses. Pertinent spectroscopic (1H NMR) and ana-lytical (elemental analyses) data are given below.

Bis(hexadecyldimethylammonium)ethane Dipalm-itate (1a). Mp 73 °C (soften), 102 °C (clear melt); 1H NMR(270 MHz, CDCl3) δ 0.88 (t, 12H, CH3), 1.25-1.59 (s +“br” m, 108H, CH2), 1.74 (s, exch with D2O), 2.24 (t, 4H,CH2COO-), 3.34 (s, 12H, N+CH3), 3.51-3.61 (m, 4H, alkylchain CH2N+), 4.66 (t, 4H, spacer CH2N+). Anal. Calcd forC70H144N2O4‚H2O (1095.92): C, 76.72; H, 13.43; N, 2.56.Found: C, 77.02; H, 13.24; N, 2.28.

Bis(hexadecyldimethylammonium)propane Di-palmitate (1b). Mp 73 °C (soften), 123 °C (clear melt);1H NMR (200 MHz, CDCl3) δ 0.86 (t, 12H, CH3), 1.15-1.85 (m + s, 108H, CH2), 2.19 (t, 4H, CH2COO-), 2.56 (s,exch with D2O), 2.70-2.90 (“br” m, 2H, spacer CH2CH2N+),3.20 (s, 12H, N+CH3), 3.45-3.45 (m, 4H, alkyl chainCH2N+), 3.84-3.94 (m, 4H, spacer CH2N+). Anal. Calcdfor C71H146N2O4‚2 H2O (1127.95): C, 75.60; H, 13.40; N,2.48. Found: C, 75.75; H, 13.00; N, 2.07. Repeatedmicroanalytical examinationsdidnotgivebetterelementalanalysis presumably because of the persistent hygroscopicnature of 1b. Consequently, we also examined the purityof 1b by HPLC using a normal-phase Shimadzu silica gelcolumn (250 mm×4.6 mm) with 5% CHCl3/MeOH solutionas eluent. It was found to be >97% pure.

Bis(hexadecyldimethylammonium)butane Dipal-mitate (1c). Mp 82 °C (soften), 148 °C (clear melt); 1HNMR (200 MHz, CDCl3) δ 0.88 (t, 12H, CH3), 1.19-1.58(“br” m + s, 112H, CH2), 1.85 (s, exch with D2O), 2.19 (t,4H, CH2COO-), 3.17 (s, 12H, N+CH3), 3.28-3.40 (m, 4H,alkyl chain CH2N+), 3.85-4.00 (crude t, 4H, spacerCH2N+). Anal. Calcd for C72H148N2O4‚2 H2O (1141.97): C,75.73; H, 13.42; N, 2.45. Found: C, 75.97; H, 13.07; N,2.19.

Bis(hexadecyldimethylammonium)pentane Di-palmitate (1d). Mp 70 °C (soften), 147 °C (clear melt);1H NMR (270 MHz, CDCl3) δ 0.88 (t, 12H, CH3), 1.15-1.75 (m + s, 112H, CH2), 1.95-2.10 (m, 2H, spacer CH2-CH2CH2N+), 2.15 (t, 4H, CH2COO-), 2.21 (s, exch withD2O), 3.21 (s, 12H, N+CH3), 3.27-3.35 (m, 4H, alkyl chainCH2N+), 3.65-3.80 (m, 4H, spacer CH2N+). Anal. Calcdfor C73H150N2O4‚2 H2O (1156.00): C, 75.85; H, 13.43; N,2.42. Found: C, 76.03; H, 13.23; N, 2.15.

Bis(hexadecyldimethylammonium)hexane Dipal-mitate (1e). Mp 55 °C (soften), 135 °C (clear melt); 1HNMR (200 MHz, CDCl3) δ 0.88 (t, 12H, CH3), 1.16-1.94(“br” m + s, 116H, CH2), 2.18 (t, 4H, CH2COO-), 2.40 (s,exch with D2O), 3.15-3.37 (s + m, 16H, N+CH3 and alkylchain CH2N+), 3.55-3.70 (crude t, 4H, spacer CH2N+).Anal. Calcd for C74H152N2O4‚2 H2O (1170.03): C, 75.96;H, 13.44; N, 2.39. Found: C, 76.05; H, 13.17; N, 2.09.

Bis(hexadecyldimethylammonium)octane Dipalm-itate (1f). Mp 60 °C (soften), 125 °C (clear melt); 1H NMR(200 MHz, CDCl3) δ 0.88 (t, 12H, CH3), 1.25-1.80 (m +s, 120H, CH2), 2.13 (t, 4H, CH2COO-), 2.81 (s, exch withD2O), 3.21-3.30 (s + m, 16H, N+CH3 and alkyl chainCH2N+), 3.38-3.50 (m, 4H, spacer CH2N+). Anal. Calcdfor C76H156N2O4‚2 H2O (1198.08): C, 76.19; H, 13.46; N,2.34. Found: C, 76.30; H, 13.16; N, 2.07.

Bis(hexadecyldimethylammonium)decane Dipal-mitate (1g). Mp 50 °C (soften), 118 °C (clear melt); 1HNMR (200 MHz, CDCl3) δ 0.88 (t, 12H, CH3), 1.17-1.80(m + sharp s, 124H, CH2), 1.98 (s, exch with D2O), 2.18(t, 4H, CH2COO-), 3.20-3.38 (s + m, 16H, N+CH3 andalkyl chain CH2N+), 3.40-3.50 (m, 4H, spacer CH2N+).Anal. Calcd for C78H160N2O4‚2 H2O (1226.13): C, 76.41;H, 13.48; N, 2.28. Found: C, 76.44; H, 13.13; N, 2.12.

Bis(hexadecyldimethylammonium)dodecane Di-palmitate (1h). Mp 48 °C (soften), 92 °C (clear melt); 1H

NMR (270 MHz, CDCl3) δ 0.88 (t, 12H, CH3), 1.17-1.80(m + s, 128H, CH2), 1.97 (s, exch with D2O), 2.18 (t, 4H,CH2COO-), 3.25-3.39 (s + m, 16H, N+CH3 and alkyl chainCH2N+), 3.40-3.50 (m, 4H, spacer CH2N+). Anal. Calcdfor C80H164N2O4‚2 H2O (1254.19): C, 76.61; H, 13.50; N,2.23. Found: C, 76.54; H, 13.37; N, 1.98.

Vesicle Preparation. The modified reverse-phaseevaporation (REV) method was applied for the preparationof vesicles for transmission electron microscopy (TEM)studies.15 A given ion pair (2.5 mmol) was dissolved in 1mL of CHCl3. To this was added 1 mL of water (pH 6.8),andthe two-phasemixturewasbrieflybath-sonicated (15-20 min) at ambient temperature to produce a stableemulsion. The organic solvent from this emulsion wasgradually evaporated under reduced pressure. The re-sulting suspension was then subjected to freeze-thawcycles several times. Then a brief bath-sonication (5 min)above 70 °C afforded stable, translucent suspensions. Forfluorescence anisotropy and X-ray diffraction (XRD)experiments, the required amount of a lipid was taken ina container and the solid was dissolved completely inchloroform. Solvent was evaporated by keeping it con-tinuously in high vacuum for 12 h to produce a film of thelipid. It was then hydrated with deionized water andsubjected to four or five freeze-thaw cycles (above thephase-transition temperature). The resulting opalescentsolution was finally sonicated to give stable aggregates.For entrapment experiments, bath-sonicated vesicles wereused. In all the cases, clear and optically translucentdispersions were obtained. Vesicle stability was measuredin a Shimadzu Model 2100 UV-vis recording spectro-photometer equipped with a TCC-60 temperature con-troller. As judged by their turbidity measurements at 450nm, it was found that the vesicles prepared by either ofthese procedures remained optically stable for severalweeks, except for 1a and 1b, which were stable for 2-3days. Gel filtration of aqueous dispersions of 1a-1hthrough a sephadex G-50 (Pharmacia) column led to >70%recovery, as seen by UV-vis spectroscopy.

Transmission Electron Microscopy (TEM) Stud-ies. Vesicular suspensions (2.5 × 10-3 M) were preparedfor TEM studies. Uranyl acetate solution (0.5% w/v) hasbeen used as the staining agent. Optically stable, opal-escent aqueous dispersions were obtained in all the cases.One drop of the above dispersion was placed onto a carbon-formvar-coated copper grid (400 mesh). Filter paper wasemployed to wick away the excess water. It was then keptunder mechanical vacuum for approximately 0.5 h. AJEOL-TEM-200 CX electron microscope typically with anaccelerating voltage of 120 keV was used. Micrographswere recorded at magnifications of 38 000, 57 000, 70 000,and 96 000.

Light-Scattering Experiments. Mean hydrodynamicdiameters were determined by laser light scattering usinga Zetasizer 3000 (Malvern Instrument Ltd., Malvern,U.K.). Light scattering employed a He-Ne laser sourceat a wavelength of 633 nm, keeping the detector angle at90°. Each suspension was generated by reverse-phaseevaporation, as described previously. The data wereanalyzed using internal instrument software involving aMalvern 7132 digital (16-bit) autocorrelator. A 220 nmlatex standard was used for calibration.

Differential Scanning Calorimetry (DSC). (a) Prepa-ration of Lipid Suspension for Calorimetry. Individual lipiddispersions in pure water (Millipore) were prepared by

(15) DuZguenes, N.; Wilschut, J.; Hong, K.; Fraley, R.; Perry, C.;Friend, D. S.; James, T. L.; Papahadjopoulos, D. Biochim. Biophys.Acta 1983, 732, 289.

3402 Langmuir, Vol. 15, No. 10, 1999 Bhattacharya and De

taking an appropriate amount of solid ion-paired lipidinto the DSC pan containing 12 µL of water. The pan wasimmediately sealed, transferred to the calorimeter, andkept at 80-90 °C to accomplish proper hydration. Typicallya 100-110 mmol solution of each lipid was used for DSCmeasurements.

(b) Calorimetric Measurements. DSC measurementswere carried out with a Perkin-Elmer Model DSC-4differential scanning calorimeter at a sensitivity of 1 mcal/sand a scanning rate of 5 K/min. A pan containing anidentical volume of water (Millipore) was used as referencein all the cases. Heating and cooling thermograms foreach sample were repeated several times to get reproduc-ible thermograms. The peak in the excess heat capacityversus temperature plot was taken as the main transitiontemperature Tm. At different scanning rates, comparablevalues of enthalpy, transition temperature and half-heightwidth were obtained. The transition enthalpy ∆H wasobtained from the computer attached with it and was theaverage of at least three separate isolated runs. Thisintegral was used to calculate both the calorimetric (∆Hcal)and van’t Hoff (∆HvH) enthalpies of the transition. ∆Hcaland ∆HvH were used to determine the “cooperativity unit”(CU) of the transition, the number of monomers under-going the phase transition, from the relationship CU )∆HvH/∆Hcal.16 All the thermodynamic parameters are theaverages of at least two independent sample preparations.The main phase-transition entropy ∆S in units of caloriesper mole Kelvin was determined on the basis of theassumption that at Tm the gel and the liquid-crystal-likephases were in equilibrium. ∆S was measured from theequation ∆S ) ∆Hcal/Tm, where ∆Hcal is expressed incalories per mole and Tm in Kelvin. Phase-transitiontemperatures and enthalpies are accurate up to (0.5 °Cand (1.0 kcal‚mol-1 respectively. The van’t Hoff enthalpy∆HvH was obtained from the relation ∆HvH ) 6.9Tm

2/∆T1/2,where ∆T1/2 is the width at the half-maximum excessspecific heat and is accurate within (10%.

Fluorescence Anisotropy Studies. Steady-state fluo-rescence measurements were carried out in a HitachiF-4500 fluorescence spectrophotometer equipped withpolarizers. Temperature was regulated with a constant-temperature water-circulating bath (model Julabo F10)which heated or cooled the sample cell holder. Sampleswere allowed to equilibrate for 10 min prior to each run.Both the heating and cooling scans were run at 4 °Cintervals. The excitation wavelength was fixed at 360 nm,and the emission spectra in the wavelength region 390-480 nm were studied. A band-pass of 5 nm was used. Thefluorescence anisotropies (r) of DPH (9 × 10-8 M), dopedin individual vesicular solutions (2.5 mM), were measured.At each temperature, the fluorescence emission spectrawere recorded by adjusting the polarizers at four differentpositions. At different temperatures, r for each vesicularsolution was calculated by employing Perrin’s equation,r ) (III - I⊥‚G)/(III + 2I⊥‚G), where III and I⊥ are the observedintensities measured with polarizers parallel and per-pendicular to the vertically polarized exciting beam,respectively. G is a factor used to correct for the inabilityof the instrument to transmit differently polarized lightequally. The r versus T plot for each aggregate gaveinformation about its gel to liquid-crystalline phase changeas a function of temperature. The values reported hereinfor r are the average values of three independent

measurements. The systematic gel to liquid-crystallinephase transition temperatures were calculated from themidpoints of the breaks related to the temperature-dependent anisotropy values.

Phase Transition Temperature of Pyrene. A meth-anolic solution of pyrene was introduced into the vesicularsuspension of an ion-paired amphiphile, generated asdescribed previously. The lipid and pyrene concentrationswere 5 × 10-4 M and 7.9 × 10-8 M, respectively. A Hitachi650 spectrofluorimeter was used to determine the sys-tematic micropolarities and apparent phase-transitiontemperatures (Tm). The temperature of the sample cellholderwasregulatedbycirculatingwaterusingaconstant-temperature water bath (model Julabo F10). The excita-tion wavelength was fixed at 337 nm, and the emissionspectrum in the region 360-400 nm was recorded.Bandwidths were fixed at 5 nm for both the emission andexcitation spectra. The samples were allowed to thermallyequilibrate for 10 min prior to each measurement. At eachtemperature, the ratio I3/I1, where I3 and I1 are theintensities of the third and the first vibronic peaks in thefluorescence emission spectrum due to pyrene was cal-culated and plotted against temperature T. The measure-ments were done in the temperature range 25-80 °C.

X-ray Diffraction (XRD) Studies. Self-supported castfilms for the XRD studies were prepared by dispersingthe lipids in water (10 mg/1.0 mL), as described above. Afew drops of this suspension were dispersed on a pre-cleaned glass plate and then air-dried at room temper-ature. Finally the plate was kept under vacuum for 15min. Reflection XRD studies were carried out using a X-raydiffractometer (model XDS-2000, Scintag Inc., USA). TheX-ray beam was generated with a Cu anode, and thewavelength of KR1 beam was 1.5406 Å. The X-ray beamwas directed toward the film edge, and the scanning wasdone up to the 2θ value of 20°.

Entrapment of Riboflavin and Gel Filtration.Vesicles were prepared by the bath sonication of theamphiphiles (1 mg/1 mL) in aqueous riboflavin solution(concentration 2 × 10-5 M) at pH 6.8 for 30 min at ∼60°C. Since the lamellar phases formed from ion-paired lipidsare often sensitive to the presence of salt, the trappingexperiments were performed in water alone. After it wascooled to room temperature, the vesicle suspension (3 mL)was loaded onto a pre-equilibrated sephadex G-50 M(Pharmacia) column (26 cm × 1 cm) and eluted with water(pH 6.8). Fractions containing most of the vesicles werepooled. These contained riboflavin molecules that werebound to the vesicles at the outer surface and also onesthat were entrapped in the vesicular inner pools.

Kinetics of Transmembrane Permeation. An ali-quot of 1 mL (pH 6.8) of the above gel-filtered riboflavin-entrapped and riboflavin-adhered vesicular solution wasplaced in a quartz cuvette. The decrease of the fluorescenceintensity at 514 nm (excited at 374 nm, excitation andemission slit widths were 10 and 20 nm, respectively)with time at 25 ( 0.5 °C was determined using a HitachiF-4500 fluorescence spectrophotometer upon adjustmentof the pH to 10.2 by the addition of an aliquot of 66.3 µLof a 0.034 M KOH solution. So, time-dependent quenchingof the fluorescence intensity at 514 nm due to OH-

permeation across the bilayer vesicles under an imposedtransmembrane pH gradient of 3.4 pH units was exam-ined. Rate constants were obtained from the monoexpo-nential, time-dependent portion of the loss of the fluo-rescence intensity.

Molecular Modeling Studies. All the eight ion-pairedlipids were drawn, and their energy-minimized, preferredconformations were calculated using the INSIGHT II 2.3.5

(16) (a) Chung, Y.-C.; Regen, S. L. Langmuir 1992, 8, 2843. (b)Mabrey-Gaud, S. In Liposomes: From Physical Structure to TherapeuticApplications; Knight, C. G., Ed.; Elsevier/North-Holland BiomedicalPress: Cambridge, 1981; p 105. (c) Mabrey, S.; Sturtevant, J. M. MethodsMembr. Biol. 1978, 9, 237.

Vesicle Formation from Dimeric Ion-Paired Amphiphiles Langmuir, Vol. 15, No. 10, 1999 3403

package (DISCOVER, Biosym. Technologies). DISCOVERis a molecular simulation program that performs energyminimization to optimize the initial geometries of differentmolecules constructed from appropriate fragments inINSIGHT. The minimization path followed conjugategradients optimization. A consistent valence force fieldwas selected for computations.

Results and Discussion

Molecular Design and Synthesis. It is known that,by mixing single-chain amphiphiles of the same charge inwater, one can obtain mixed micelles.17 However, whenequivalent amounts of single-chain amphiphiles of op-posite charge are mixed, lamellar or bilayer type ag-gregates are formed.18 Presumably, the electrostaticinteractions at or near the headgroup region and hydro-phobic association of the nonpolar chains result in theformation of “tight” complexes which eventually developinto lamellae or bilayers. So far these studies have beenconfined only to single-chain surfactants, and the corre-sponding mixtures of monopolar amphiphile and bipolargeminis have not been examined for vesicle formation.The “dimeric” ion-paired lipids in the present study donot mimic predominant components of natural biologicalmembranes which are composed of single phospholipidmolecules. Instead, they simulate the components ofmitochondrial and bacterial membranes which includetetraacyl phospholipids (i.e. “dimeric”) such as cardiolipinor diphosphatidylglycerol.19 Dialkylglycerol based tetra-ether lipids are also known to exist in thermophilicarchaebacteria, for example, Sulfolobus Solfataricus,through formation of a cubic phase under extremephysiological conditions (85 °C, pH 3).12

To realize bilayer-like assemblies from cationic dimericamphiphile-fatty acid ion pairs and to explore whetherheadgroup separation in the starting ion pairs is reflectedon the properties of their bilayer assemblies, we performeda systematic study on a series of ion-paired lipids whichare structurally analogous except in their spacer chainlengths. We synthesized altogether eight ion-paired am-phiphiles, as shown in Scheme 1.

We initiated the synthesis of the “dimeric” ion-pairedamphiphiles 1a-1h via the preparation of eight dicationicamphiphiles, n-C16H33N+Me2-(CH2)m-N+Me2-n-C16H33, 2Br-

(m ) 2-6, 8, 10, and 12), 2a-2h. Then the bromidecounterions of these amphiphiles were exchanged withOH-. To each of the reaction mixtures containing 1 equivof 3a-3h was added 2 equiv of freshly recrystallizedpalmitic acid in dry methanol, and the resulting mixtureswere stirred at room temperature for 12 h under nitrogen.Subsequent removal of solvent afforded solid residues,which were recrystallized several times from EtOAc/MeOH (10:1). Each of them gave expected analytical andspectroscopic data consistent with their structures (cf.Experimental Section). Due to their strong hygroscopicnature, only freshly recrystallized ion pairs were employedfor vesicle preparations and other subsequent character-izations.

Electron Microscopy. To ensure the nature of thesesuspensions, all aggregates were examined under atransmission electron microscope. Vesicular suspensionswere generated with reverse-phase evaporation methodand uranyl acetate was used as the staining agent. TEMexamination of the air-dried aqueous suspensions of 1a-1h revealed the existence of closed aggregate structuresin all the cases. Four representative micrographs for 1b,1d, 1f, and 1h are shown in Figure 2. All but 1h formedpredominantly multilamellar spherical vesicles havingdiameters 600-1150, 1000-1300, and 600-850 Å, re-spectively. 1h generated mostly ellipsoidal vesicles withthe diameter 950-3000 Å. Closer scrutiny of the micro-graphs showed apparent discrete bilayers within multi-lamellar structures in all the cases. For vesicular 1b, thewidth of each lamellae ranged from ∼43 to 47 Å. Similarly,vesicles of 1f and 1h had lamellar widths of ∼36 and 34Å, respectively, on an average. These results are consistentwith that obtained from XRD measurements (see below).

Having demonstrated the feasibility of vesicle formationfrom presynthesized, ion-paired systems, we then soughtto explore whether mere physical mixing of a cationicmicellar solution of a gemini directly with an anionic fattyacid such as palmitate affords vesicular aggregates. Thisstrategy of vesicle formation, if successful, could beconveniently extended to vesicle preparation of widelyvarying cation/anion ion pairs. Thus we tried to generatevesicles by taking 2a-2h and palmitic acid in a 1:2 ratioin the presence of an equimolar amount of Tris buffer. Itis gratifying to note that this method also generatesvesicular aggregates, as evident from TEM. Figure 3ashows micrographs of vesicles generated by mixing 2band palmitic acid in a 1:2 ratio in the presence of anequimolaramountofTrisbuffer.Here, thevesiclediameteris in the range 850-950 Å and the bilayer width is 41-47Å, which are comparable with the values obtained for 1b.

Interestingly, a 2.5 × 10-3 M vesicular solution of 1bformed a gel-like substance when left for 2 days at roomtemperature and this gel was stable for several days.

(17) Tanford, C. The Hydrophobic Effect: Formation of Micelles andBiological Membranes; Wiley: New York, 1980.

(18) (a) Fukuda, H.; Kawata, K.; Okuda, H.; Regen, S. L. J. Am.Chem. Soc. 1990, 112, 1635. (b) Kaler, E. W.; Murthy, A. K.; Rodrigueg,B. A.; Zasadzinski, J. A. N. Science 1989, 245, 1371.

(19) Grunner, S. M.; Jain, M. K. Biochim. Biophys. Acta 1985, 818,352. Recently, Regen and co-workers made biomimetic analogues ofdiphosphatidylglycerol through a disulfide connection at the headgrouplevels. (a) Davidson, S. M.; Liu, Y.; Regen, S. L. J. Am. Chem. Soc. 1993,115, 10104. (b) Krisovitch, S. M.; Regen, S. L. J. Am. Chem. Soc. 1992,114, 9828.

Scheme 1a

a Conditions: (a) n-C16H33Br (3 equiv), EtOH (dry), reflux,48 h, 70%; (b) n-C16H33NMe2 (3 equiv), EtOH (dry), reflux, 48h, 70-90%; (c) Amberlite IRA-900, OH- form, MeOH; (d)n-C15H31CO2H (2 equiv), MeOH, 12 h, 90-95%.

3404 Langmuir, Vol. 15, No. 10, 1999 Bhattacharya and De

Examination with a bright field optical microscopeindicated the existence of tubular aggregates in thishydrogel-like substance (Figure 3b). A similar kind oftubular phase was exhibited by dimeric micelles of otherbiscationic surfactants with short spacer chain lengths,and its stability increased with the lengthening of itshydrophobic tails.10b

Dynamic Light Scattering. Mean hydrodynamicdiameters were determined by laser light scattering. Eachsuspension was generated by a reverse-phase evaporationmethod and examined for its ability to scatter. For 1b,vesicles of multiple size (at least three) populations wereseen. These were 700, 900, and 1200 Å. For 1d and 1f,however, the mean hydrodynamic diameters were 1200( 50 Å (>97%) and 800 ( 100 Å (>97%), respectively.Very minor populations (<3%) with suspensions of 1dand 1f consisting of larger aggregates (∼2000 Å) werealso seen. Again a suspension of 1h showed the existenceof larger vesicles that ranged from 1500 to 3000 Å.

Differential Scanning Calorimetry. To furtherascertain the presence of lamellar organizations and alsoto understand the relative tightness of packing in suchaggregates, vesicular suspensions of 1a-1h were exam-ined by DSC. A well-defined, sharp, and single-phasetransition (gel f liquid crystalline) was readily observedfor each dispersion during the heating runs. All lipidsuspensions showed virtually reversible phase transitionbehavior during heating as well as cooling runs. Tworepresentative thermograms for 1a and 1f are shown inFigure 4. The phase transition temperatures (Tm’s),

calorimetric enthalpies (∆Hcal’s), van’t Hoff enthalpies(∆HvH’s), and cooperativity units (CUs) are given in Table1. Strikingly, the phase transition temperatures (Tm’s)were found to be strongly dependent on the m value of thespacer chain [-(CH2)m-]. Thus, when the apparent Tm ofthe aggregates of 1a with m ) 2 was found to be 74.1 °C,the corresponding Tm for vesicles of ion pair 1h with m )12 was found to be 38.9 °C. In addition, the transitionprocess becomes less cooperative as the m-value increasesbeyond 3. A comparison of the thermal phase transitiontemperature (Tm) and the cooperativity unit (CU) withspacer chain length (m value) is given in Figure 5. Thiscan be explained in light of molecular modeling studies(see below). When the two Me2N+ centers in an amphiphileare reasonably close to each other, as in the cases of 1aand 1b, the counterion carboxylates can stabilize boththe Me2N+ centers through electrostatic interaction. Thishelps the lipid molecules to pack in the bilayer efficiently.In contrast, with an increase in the headgroup separation,the COO- anions can interact only with the nearestquaternary ammonium cations. The polymethylene chainalso starts to loop into a hydrophobic membrane core inorder to avoid water contacts, thereby impairing thepacking within the assembly. Thus, the phase transitiontemperature as well as the number of “dimeric” am-phiphiles that cooperatively participate in this processdecreases with increased headgroup separation. However,there is little variation in the calorimetric enthalpies asa function of m value.

Fluorescence Anisotropy Measurements. To fur-

Figure 2. Uranyl acetate-stained transmission electron micrographs of vesicles prepared by REV: (a) 1b; (b) 1d; (c) 1f; (d) 1h.

Vesicle Formation from Dimeric Ion-Paired Amphiphiles Langmuir, Vol. 15, No. 10, 1999 3405

ther probe the nature of these bilayers, we then measuredthe fluorescence anisotropy as a function of temperature.This technique has been extensively used for probing theinterior of the vesicle bilayer and for measuring thetemperature (Tm) at which the bilayer melts from a gel-like solid phase to a fluid state.20 We measured thefluorescence anisotropy (r) as a function of temperature(in the range 10-75 °C) using 1,6-diphenylhexa-1,3,5-E,E,E-triene (DPH), a hydrophobic fluorescent probewhich intercalates between the alkyl chains in thehydrophobic interior core of the vesicle bilayer.21

As is evident from Figure 6, vesicular solutions of 1b-1h showed rather well-defined and cooperative phasetransitions. Further examination of Figure 6 revealed thatwhile r values for 1b-1g were quite high (r ∼ 0.30) at20-30 °C in their gel states, 1h showed considerably lowerr value (0.23) in this temperature range. Since theanisotropy is directly related to the fluidity of bilayers, alower anisotropy value indicates more freedom of move-ment of DPH in the bilayer of 1h. Thus the intramonomervan der Waals interaction between the chains in vesicular1h is weaker than that of its other m value counterpartsin the gel state. On the other hand, the r value for vesicular1b beyond melting was higher (0.1) compared to any of1c-1h (∼0.04-0.06). This suggests that the bilayers of

1b are quite rigid in its fluid state compared to others.The increased rigidity may be due to the presence of asmall spacer unit, -(CH2)3- in 1b, which assists in themaintenance of reasonably tighter packing in this bilayer.Details of fluorescence anisotropy values and apparentphase transition temperatures for 2.5 mM 1a-1h areincluded in Table 2. This experiment could not, however,be carried out with precision due to rapid onset of turbiditywith vesicular 1a.

Phase Transition Temperature of Pyrene. Tosecure additional information pertaining to the observedspacer chain length dependence of the bilayer properties,we also measured the monomer vibrational intensity ratio(I3/I1) of bilayer-doped pyrene as a function of temper-

(20) (a) Shinitzky, M.; Barenholz, Y. Biochim. Biophys. Acta 1978,515, 367. (b) Andrich, M. P.; Vanderkooi, J. M. Biochemistry 1976, 15,1257.

(21) Shobha, J.; Balasubramanium, D. Proc. Ind. Acad. Sci. (Chem.Sci.) 1987, 98, 469.

Figure 3. (a) Uranyl acetate-stained transmission electronmicrographs of vesicles obtained by mixing 2b and palmiticacid prepared by REV. (b) Bright field optical micrograph of gelobtained from 1b.

Figure 4. Gel to liquid crystalline phase transition profiles ofthe vesicles of the lipids (a) 1a and (b) 1f.

Table 1. Gel to Liquid Crystalline Main PhaseTransition Properties of Ion-Paired Lipids 1a-1h by

Differential Scanning Calorimetrya

Tm (°C)lipid heat cool

∆Hcalb

(kcal/mol)∆Sc

(cal/(K‚mol))∆HvH

d

(kcal/mol)CUe

(molecules)

1a 74.1 71.7 19.32 55.67 1047.51 541b 66.2 64.6 15.64 46.10 766.00 491c 47.4 45.7 15.83 49.41 553.63 351d 45.6 42.9 19.66 61.72 586.32 301e 45.6 42.6 20.07 62.98 541.25 271f 42.8 40.4 20.36 64.45 494.30 241 41.1 38.4 18.86 60.06 655.89 251h 38.9 37.7 20.51 65.75 523.18 24

a The concentration of the gel was 100-110 mM. b The ∆Hcalvalues quoted are the average of the total enthalpies for successiveruns. We estimate that transition enthalpies are accurate to (0.5kcal‚mol-1. c ∆S values were calculated by dividing ∆Hcal/Tmassuming the phase transition as a first-order process. d ∆HvHvalues were calculated from the relation ∆HvH ) 6.9Tm

2/∆T1/2, where∆T1/2 is the spread of the temperature at half-height. e CU valuesare the average for successive runs.

3406 Langmuir, Vol. 15, No. 10, 1999 Bhattacharya and De

ature22 and determined the apparent Tm from the break

of the I3/I1 versus T plot for various lipid dispersions.This method involves the use of a hydrophobic fluorescencedye, pyrene, which senses the polarity of the solubilizingmedium and thus exhibits different fluorescence behaviorwhen a vesicular suspension is gradually heated beyondthe phase transition temperature.

In all the cases, I3/I1 versus T gave sigmoidal plots, asexpected; that is, the I3/I1 value sharply increased duringthe melting phase transition, and then once the bilayersmelted into the liquid crystalline state, it became constant(1.14-1.21) with any further increase in temperature(Figure 7). The relevant data are summarized in Table 2.Pyrene experiences a polar environment when the bilayersare still in their gel state. As bilayers start to melt, theirrigidity decreases and pyrene gets incorporated betweenthe hydrophobic segments of the lipid due to morefavorable hydrophobic interaction.22 This is accompaniedby an increase in the I3/I1 value. Once the bilayer reachesthe liquid crystalline state, there is no significant changein the I3/I1 value with temperature.

X-ray Diffraction Studies. To understand the ori-entation of these ion pairs in the membranes and also togain knowledge about their lamellar packing, XRD studieswere performed on supported, cast films of 1a-1h.Reflections up to 20° were analyzed and interpreted interms of higher order reflections of stacked bilayerstructures.23 A series of reflections mostly up to eighthorder reflections were obtained for 1a-1h, the highestintensity peak being the long spacing (corresponds to thelowest 2θ value) (Table 3).

The diffraction patterns of 1a-1e showed membranethicknesses (d’s) of 49.5, 48.6, 49.3, 51.2, and 51.5 Å,respectively, corresponding to 2θ values in the range∼1.7-1.8 on the basis of higher order reflections (n )3-7). Assuming the long hydrocarbon and fatty acid chainsin these lipids have an all s-trans conformation, theirbilayer widths were estimated to be ∼41 Å from the CPKmodel of two molecules oriented parallel to the bilayernormal (see below). The above-measured values suggesthydrated bilayer organizations for 1a-1e (Figure 8a).

(22) Kalyanasundaram, K. Photochemistry in MicroheterogeneousSystems; Academic Press: New York, 1987; p 177.

(23) (a) Kimizuka, N.; Kawasaki, T.; Kunitake, T. J. Am. Chem. Soc.1993, 115, 4387. (b) Kunitake, T.; Shimomura, M.; Kajiyama, T.; Harada,A.; Okuyama, K.; Takayanagi, M. Thin Solid Films 1984, 121, L89.

Figure 5. Dependence of the thermotropic phase transitiontemperature (Tm) and the cooperativity unit (CU) of differentlipids 1a-1h on m value.

Figure 6. Fluorescence anisotropy (r) versus temperature (°C)plots for different lipid vesicles: 1b (O), 1d (0), 1f (4); 1h (3).

Table 2. Phase Transition Temperatures (Tm/°C) ofVesicular 1a-1h by Fluorescence Spectroscopy Using

DPH and Pyrene Probesa and Fluorescence Anisotropy(r) and I3/I1 for Comparisonb

maintransitionc (°C)

anisotropy (r)d I3/I1d

lipidDPH

methodpyrenemethod

1a e 76.2 0.27 (30) e 0.97 (25) 0.98 (80)1b 61.5 62.5 0.30 (30) 0.10 (68) 0.95 (25) 1.21 (74)1c 47.5 43.1 0.29 (20) 0.06 (68) 0.94 (25) 1.14 (57)1d 45.7 45.0 0.25 (20) 0.06 (60) 0.91 (25) 1.17 (66)1e 44.0 44.6 0.27 (20) 0.05 (55) 0.91 (25) 1.15 (54)1f 42.0 42.2 0.28 (20) 0.04 (53) 0.92 (25) 1.13 (54)1g 40.0 40.0 0.28 (20) 0.04 (53) 0.93 (25) 1.13 (54)1h 35.1 47.7 0.23 (20) 0.04 (62) 0.91 (25) 1.15 (54)DPPCf 41.2 41.3 0.29 (20) 0.08 (60) 0.91 (25) 1.15 (54)

a The fluorescence anisotropy method employs DPH as a probe,while the other method (I3/I1) employs pyrene as a probe. b Theconcentration of the vesicular solutions in all the cases was 2.5mM. c The average deviation is (1 °C for the fluorescence anisotropymethod and (2 °C for the pyrene method. d The temperatures (°C)at which fluorescence anisotropy and I3/I1 are reported are shownin parentheses. e Experiment could not be carried out due to rapidonset of turbidity during the experiment. f Dipalmitoyl phospha-tidylcholine.

Figure 7. Plots of I3/I1 due to vesicle doped pyrene versus T(°C); 1a (O); 1d (0); 1g (4); 1h (3).

Vesicle Formation from Dimeric Ion-Paired Amphiphiles Langmuir, Vol. 15, No. 10, 1999 3407

These cast films did not, however, show any evidence oflipid polymorphism.

In contrast to the above observations, 1f and 1g showedmainly two different kinds of molecular packing in theiraggregates. Two series of reflections could be marked:the one corresponding to the long spacing value of 41.2 Åbeing the weaker and the other corresponding to the longspacing value of 27.0 Å being the stronger. This points toboth tilted and fully interdigitated bilayer orientations oflipid molecules with respect to the bilayer normal,respectively, in these vesicles (Figure 8b). Since the spacerchain can loop now, the propensity of these modes of bilayerorganization increases, the latter one being more probablefor helping to fill up the “void” places inside the bilayer.

1h exhibited only one series of reflections up to n ) 6,the bilayer width being 30.3 Å. It indicates the presenceof the exclusively tilted, interdigitated nature of bilayerorganization, which is presumably an outcome of anincrease in length and, thereby, flexibility of the spacerchain (Figure 8c).

Entrapment of Fluorescent Dye in Vesicles. Afterstudying the thermodynamic properties of these ion pairs,we then decided to examine whether the vesicles wereclosed and in that situation whether they can sustain animposed transmembrane pH gradient. Generally, themembrane permeability can be measured with suitablewater-soluble markers. Although several methods basedon radiochemical, redox, or enzymatic techniques areknown in the literature, one utilizing fluorescent probesis widely used due to its simplicity and convenience.24

Thus a number of probes such as pyranine,25 carboxy-fluorescein,26 calcein,27 and so forth have been used toestimate membrane permeability. However, due to theionic nature of these probes, it is possible that they couldcompete for ion pairing with the amphiphilic systemsduringsuchexperiments.Dueto this concern,weemployeda neutral water-soluble molecule, riboflavin, which isstrongly fluorescent in its neutral form but becomesnonfluorescent upon deprotonation (pKa ∼ 10.2). It canalso withstand low ionic strength and, as a result, can bestudied even in water. The usefulness of riboflavin hasalready been demonstrated independently by Bhatta-charya,28 Kunitake,29 and Regen18a for the estimation ofentrapment capacity and for the determination of trans-

membrane pH gradients with unrelated vesicle-formingsurfactants of diverse surface charges.

To examine their entrapment abilities, vesicles werefirst generated from 1a-1h by dispersing them in watercontaining riboflavin. Light scattering indicated theformation of a translucent suspension of uniform particlesize with 1b-1h. However, 1a did not form a clear, stablesuspension even after sonication beyond 1 h. Gel filtrationchromatography was performed to separate vesicle-entrapped riboflavin from the free unbound riboflavin.As observed from instances reported in the literature,28,29

while some riboflavin molecules adhered to the membra-nous interfaces, few got entrapped in the inner aqueouspools of vesicles. Importantly in all the cases, riboflavin-associated vesicles could be separated out from the freeriboflavin molecules. The information pertaining to thepercentage entrapment of riboflavin in each vesicle is givenin Table 4.

Kinetics of Transmembrane Permeation. Thefluorescence emission due to the membrane-bound andentrapped riboflavin was measured at 514 nm uponexcitation at 374 nm at 25 ( 0.5 °C. As the pH of thevesicle dispersion was raised from 6.8 to 10.2, thefluorescence intensity (514 nm) decreased initially “in-stantaneously” to about 80 ( 5% for 1b-1d, 86 ( 4% for1e-1g, and 70 ( 3% for 1h of the original value (recordedprior to pH adjustment). We attribute this “instantaneous”loss in the fluorescence intensity to the deprotonation ofriboflavin molecules that are bound to the outer surfacesof the vesicles; that is, at the exposed exovesicular surface,riboflavin molecules underwent immediate ionization asthe pH was increased. The residual fluorescence intensity,however, disappeared as a function of time for vesicular1c-1f (Figure 9) but remained virtually unchanged for1b and 1h. Since the residual loss in fluorescence intensitywas time-dependent for 1c-1f, we calculated the half-time of this process (t1/2) for these vesicles, and the relevantdata are presented in Table 4. Time-dependent loss offluorescence intensity due to the conversion of neutralriboflavin into its deprotonated form upon pH adjustment(6.8 f 10.2) demonstrated that these vesicles containedinternal aqueous compartments. Using their respectivebilayer thicknesses based on XRD results and assumingtime-dependent loss of riboflavin fluorescence as a rate-limiting permeation of exovesicular OH- into the vesicle’sinterior aqueous compartment, we calculate the perme-ation constant P (in cm‚s-1) of OH- toward vesicles at pH10.2 (Table 4).30

At 25 °C, that is, <Tm, at which the kinetic assays wereconducted, the riboflavin-loaded vesicular suspensionswere all in their more ordered, less permeable, rigid “gel”phases. Although the differences in permeability amongthe various types of vesicular systems are not enormous,they are significant and provide useful informationpertaining to the vesicular packing. The permeabilityproperties of the vesicular systems clearly appear to bedependent on the length of the spacer chain connectingthe two NMe2

+ groups. As the spacer chain lengthincreases, the rate of OH- permeation increases, presum-ably due to the loss of “tightness” in the molecular packingand the increased propensity of formation of a tilted andinterdigitated bilayer. So, we see while 1b is not at allpermeable, 1h cannot sustain the pH difference. In thecase of 1c-1f, t1/2 decreases as the m value increases (Table4).

In the case of vesicular 1b, the initial fluorescenceintensity underwent a rapid decay upon pH adjustment

(24) Brunner, J.; Graham, D. E.; Hauser, H.; Semenza, G. J. Membr.Biol. 1980, 57, 133.

(25) Kano, K.; Fendler, J.-H. Biochim. Biophys. Acta 1978, 509, 289.(26) (a) Neumann, R.; Ringsdorf, H.; Patton, E. V.; O’Brien, D. F.

Biochim. Biophys. Acta 1987, 898, 338. (b) Weinstein, J. N.; Yoshikami,S.; Henkart, P.; Blumenthal, R.; Hagins, W. A. Science 1977, 195, 489.

(27) Matzuzaki, K.; Harada, S.; Funakoshi, N.; Fujii, K.; Miyajima,K. Biochim. Biophys. Acta 1991, 1063, 162.

(28) Bhattacharya, S.; Haldar, S. Langmuir 1995, 11, 4748.(29) Kunitake, T.; Okahata, Y.; Yasunami, S. Chem. Lett. 1981, 1397.

(30) Dorn, K.; Klingbiel, R. T.; Specht, D. P.; Tyminski, P. N.;Ringsdorf, H.; O’Brien, D. F. J. Am. Chem. Soc. 1984, 106, 1627.

Table 3. Unit Layer Thicknesses Obtained from X-rayDiffraction of Self-Supporting Cast Films of 1a-1h and

Their Proposed Packing

lipid

unit layerthickness(Å) (obsd)a proposed packing

1a 49.5 hydrated C16 bilayer1b 48.6 hydrated C16 bilayer1c 51.4 hydrated C16 bilayer1d 51.0 hydrated C16 bilayer1e 51.5 hydrated C16 bilayer1f 41.2 (w), 27.0 (s)b tilted and tilted interdigitated bilayer1g 41.0 (w), 31.1 (s)b tilted and tilted interdigitated bilayer1h 30.3 tilted interdigitated bilayer

a As obtained from reflection XRD of cast films. b w and s denotethe weak and strong intensity peaks, respectively.

3408 Langmuir, Vol. 15, No. 10, 1999 Bhattacharya and De

(6.8 f 10.2). However, the subsequent time-dependentloss of fluorescence in this instance was not observed,unlike what was seen with other vesicles. This could bedue to the “rigid” nature of the bilayer in 1b at 25 °C,which could sustain the transmembrane pH gradientduring pH shock. Indeed, when the riboflavin-loaded

vesicles were heated, a time-dependent loss in fluorescencewas observed.

In contrast to the above situation, vesicular 1h, afteran initial loss of fluorescence intensity, did not show anydecrease in fluorescence intensity with time. Since itadopts an interdigitated bilayer structure, its molecularpacking is probably not such that it can withstand thetransmembrane pH gradient during pH shock.

Molecular Modeling Studies. To rationalize thepossible origin of such strong control of the spacer chainconstraints of bolaphilic segments in these ion pairs overthe biophysical properties of vesicular 1a-1h, we alsocarried out molecular mechanics studies. All the ion pairs1a-1h were drawn with a computer using INSIGHT II,31

and their energies were minimized. Examination ofstructural features reveals that the packing mode in 1aand 1b is strikingly different from that of the otheramphiphiles (figure not shown). In 1a and 1b, wherecationic Me2N+ centers are separated by 2 and 3 methyleneunits, respectively, the counterion carboxylates are avail-able for mutual electrostatic interaction with both theMe2N+ centers. Such organization leads to efficientpacking. In contrast, as the headgroup separation in-creases with higher m values, the COO- anions becomeconfined to individual quaternary ammonium cations.Additionally, the other conformational states of the spacerpolymethylene chain arise from rotations about the C-Cbond as the m value surpasses 3. With the increase in thenumber of gauche conformers, the polymethylene chainstarts to loop into the membrane interior in order tominimize water contacts and, thus, it impairs the packingwithin the assembly. As a result, the phase transitiontemperature as well as the cooperativity unit decreaseswith increased headgroup separation. We also studiedthe energy minimization of the assemblies of 1a and 1hwhen they are soaked with few water molecules. We indeedsee the looping of the spacer chain in 1h toward thehydrophobic core of the bilayer. In the presence of bulkwater and aqueous interfaces at the periphery of exo- andendovesicular leaflets, the two headgroups will be sur-rounded by many more water molecules. In that case, thistendency will be much more pronounced.

Packing Parameter Calculations. We calculated thepacking parameter of 1a-1h to confirm the feasibility offormation of vesicular type aggregates by them. Thepacking parameter p introduced by Israelachvili is relatedto aggregate morphology in aqueous solutions by theequation p ) v/la, where v is the volume occupied by thehydrophobic moiety of the surfactant molecule, l is thecritical length in the fully extended conformation, and ais the surface area occupied by the surfactant headgroup.32

(31) For details, please consult BIOSYM programs, which areavailable from Biosym Technologies, 9685 Scranton Road, San Diego,CA 92121-3752.

(32) Israelachvili, J. N.; Marcelja, S.; Horn, R. G. Q. Rev. Biophys.1980, 13, 121.

Figure 8. Schematic representation of modes of packing in bilayers. The arrows indicate the periodicity observed in the XRDexperiment on stacked bilayers.

Table 4. Riboflavin Entrapment and Estimated OH-

Permeability in Different Bilayer Membranes fromIon-Paired Lipids 1a-1h at 25 °Ca

lipidlipid conc

(mM)%

entrapmentbt1/2

(min)103kperm

(s-1)108Pc,d

(cm‚s-1)

1a 0.91 e1b 0.89 1.0 f1c 0.88 3.5 3.5 3.3 1.191d 0.86 2.0 3.86 3.0 1.081e 0.85 1.2 3.0 3.85 1.391f 0.83 1.8 1.4 8.25 3.491h 0.80 0.7 g

a The time-dependent decay of the fluorescence intensity at 514nm was monitored as a function of time. The decay process followedapparentpseudo-first-orderkinetics.Rateconstantvaluesrepresentaverages of three independent experiments, and reproducibilitywas (4%. In each case, the extent of fluorescence decay was followedbeyond 90%. b Entrapment of riboflavin given in percentages hasbeen corrected for an apparent absorption of marker molecules ineach case. c Considering the diameter of the vesicle to be ∼1000 Å,which is also supported by TEM. d Bilayer thicknesses have beenobtained from XRD studies. We interpret the time-dependent lossof fluorescence intensity as a permeation-limited deprotonation ofriboflavin entrapped in the internal aqueous compartment by OH-.e A clear stable suspension could not be obtained even aftersonication beyond 1 h. f Time-dependent loss of fluorescence in thisinstance was not observed. g Fluorescence loss in this case was“instantaneous”.

Figure 9. OH- permeability profiles of riboflavin-entrappedvesicular 1b (O), 1c (0), 1d (4), 1e (3), 1f (]), and 1h (b).

Vesicle Formation from Dimeric Ion-Paired Amphiphiles Langmuir, Vol. 15, No. 10, 1999 3409

Both l and v can be calculated using Tanford’s equation.33

Specific values of p are associated with spherical micelles(p < 0.33), wormlike micelles (0.33 < p < 0.5), vesicles(0.5 < p < 1), flat bilayers (p ) 1), and inverted micelles(p > 1). Since l is constant for 1a-1h, v and a primarilydetermine the morphology of the resulting aggregates.While v increases monotonically with an increase in mvalue, a changes in a nonmonotonic fashion with m value.34

p was calculated for all the amphiphiles. It was found tobe 0.63-0.65 for 1a and 1b. Since the spacer chain loopsinto the membrane as the m value increases, a reliableestimate of p values could not be arrived at for 1c-1h,but they also fall in the range 0.6-0.7. Such an order ofp values (p > 0.5) supports the vesicle formation by them.32

ConclusionIn summary, we have synthesized eight gemini bisca-

tionic surfactants and their corresponding ion pairs withpalmitic acid. All of these afforded vesicular aggregatesupon dispersion in water. Detailed examination of theirmorphological and bilayer properties by a variety ofphysical methods indicates that the characteristics of thebilayerorganizationscan indeedbemodulatedbyvariationof the length of the polymethylene spacer unit connecting

the two headgroups in different bolaphiles. Manifestationof the unusual thermal stability observed with 1a and 1bmight have possible practical ramifications.35 Thus, thepresent findings illustrate a novel approach to fine-tunevesicular properties and suggest new but easy to achieverecipes for stable vesicular structures that may be ofpractical value. We are now planning to examine theinfluence of headgroup structure alteration, chain tether-ing, and incorporation of stiff spacers in the ion-pairedamphiphiles. In particular, mixed bilayers composed ofbacterial lipid (bolaamphiphile) and fatty acids or mam-malian lipids could be tailored to have useful biologicalproperties. Some of these mixtures may also produce atransient membrane-like milieu. In addition, interlipidmixing via ion pairing is an important step that leads toDNA release during transfection.36 These aspects arecurrently under examination.

Acknowledgment. This work was supported byDepartment of Science and Technology Grant SP/S1/G-32/91. We thank Dr. G. N. Subanna for performing theTEM and SERC for giving us access to the computationalfacilities.

LA9808770

(33) Tanford, C. J. Phys. Chem. 1972, 76, 3020.(34) (a) Diamant, H.; Andelman, D. Langmuir 1994, 10, 2910. (b)

Alami, E.; Beinert, G.; Marie, P.; Zana, R. Langmuir 1993, 9, 1465.(35) Sprott, G. D. J. Bioenerg. Biomembr. 1992, 24, 555.(36) Bhattacharya, S.; Mandal, S. S. Biochemistry 1998, 37, 7764.

3410 Langmuir, Vol. 15, No. 10, 1999 Bhattacharya and De