Langmuir 1998, 14, 7387-7391
7387
Counterion Effects on Properties of Cationic Vesicles D. B. Nascimento, R. Rapuano, M. M. Lessa, and A. M. Carmona-Ribeiro* Departamento de Bioquı´mica, Instituto de Quı´mica, Universidade de Sa˜ o Paulo, CP 26077, 05599-970 Sa˜ o Paulo, SP, Brazil Received July 8, 1998. In Final Form: September 28, 1998 The effect of counterion nature and concentration on phase transition, bilayer structure, vesicle size, vesicle internal volume per mole of amphiphile, and surface potential is evaluated for cationic vesicles composed of dioctadecyldimethylammonium (DODA) acetate, chloride, or bromide. Over a range of ionic strengths (0-5 mM monovalent salt), no interdigitation was detected in the bilayer structure for the three DODA counterions. The preferential type of aggregate formed from self-assembly of DODA salts is a large vesicle composed of a single traditional and noninterdigitated bilayer. Vesicle size and zeta-potentials were inversely related, i.e., an increase in zeta-potential was accompanied by a decrease in vesicle size. The largest zeta-potentials and smallest sizes were obtained for bilayer vesicles of DODA acetate which have the largest and more hydrated counterion. The effect of ionic strength (0-5 mM NaAc, NaCl, or NaBr as monovalent salt) was a slight decrease followed by a significant increase in vesicle size as a function of salt concentration. The results for counterion effects on vesicle size agree with predictions from the selfassembly model by Israelachvili and co-workers.
Introduction Since their introduction as bilayer-forming synthetic compounds,1,2 some synthetic lipoid amphiphiles as dihexadecyl phosphate or dioctadecyldimethylammonium salts have found many different uses in strategic applied areas.3 In particular, synthetic cationic liposomes have been sucessfully employed to interact with negatively charged surfaces or biomolecules such as prokaryotic4-6 or eukaryotic cells,7 antigenic proteins,8 nucleic acids,9,10 synthetic polymers and latex,11-14 mineral surfaces,15,16 viruses,17,18 etc. Despite their practical utility as immunoadjuvants, gene, protein, or drug carriers, bactericides, flocculants, dispersing agents, membrane models, matrix for supporting biocomponents in biosensors, etc., they still * To whom correspondence may be addressed. Phone: 55-118187964. Fax: 55-11-8155579. (1) Kunitake, T.; Okahata, Y.; Tamaki, K.; Kumamaru, F.; Takayanagi, M. Chem. Lett. 1977, 387. (2) Mortara, R. A.; Quina, F. H.; Chaimovich, H. Biochem. Biophys. Res. Commun. 1978, 81, 1080. (3) Carmona-Ribeiro, A. M. Chem. Soc. Reviews 1992, 21, 209. (4) Ta´pias, G. N.; Sicchierolli, S. M.; Mamizuka, E. M.; CarmonaRibeiro, A. M. Langmuir 1994, 10, 3461. (5) Sicchierolli, S. M.; Mamizuka, E. M.; Carmona-Ribeiro, A. M. Langmuir 1995, 11, 2991. (6) Martins, L. M. S.; Mamizuka, E. M.; Carmona-Ribeiro, A. M. Langmuir 1997, 13, 5583. (7) Carmona-Ribeiro, A. M., Ortis, F.; Schumacher, R. I.; Armelin, M. C. S. Langmuir 1997, 13, 2215. (8) Tsuruta, L. R.; Quintilio, W.; Costa, M. H. B.; Carmona-Ribeiro, A. M. J. Lipid Res. 1997, 38, 2003. (9) Felgner, P. L. Adv. Drug Delivery Rev. 1990, 5, 163. (10) Behr, J. P. Acc. Chem. Res. 1993, 26, 274. (11) Carmona-Ribeiro, A. M.; Midmore, B. R. Langmuir 1992, 8, 801. (12) Lessa, M. M.; Carmona-Ribeiro, A. M. J. Colloid Interface Sci. 1996, 182, 166. (13) Tsuruta, L. R.; Carmona-Ribeiro, A. M. J. Phys. Chem. 1996, 100, 7130. (14) Tsuruta, L. R.; Lessa, M. M.; Carmona-Ribeiro, A. M. J. Colloid Interface Sci. 1995, 175, 470. (15) Rapuano, R.; Carmona-Ribeiro, A. M. J. Colloid Interface Sci. 1997, 193, 104. (16) Salay, L. C.; Carmona-Ribeiro, A. M. J. Phys. Chem. B 1998, 102, 4011. (17) Katz, D.; Kraaijeveld, C. A.; Snippe, H. In The Theory and Practical Application of Adjuvants; Stewart-Tull, D. E. S., Ed.; John Wiley & Sons: Chichester, New York, Brisbane, Toronto, Singapore. 1994. (18) Smith, R H.; Ziola, B. Immunology 1986, 58, 245.
are poorly characterized from the point of view of important physicochemical properties that determine their utility. Understanding the structure, stability, and dynamics of vesicles is important in elucidating biological self-assembly and in devising strategies for practical use of stable vesicles. Intra- and interlayer interactions between bilayers of quaternary ammonium surfactants exhibit a pronounced counterion specificity.19-22 The smaller the hydrated anion, the more contracted the monolayer compression isotherm becomes.20 Expanded phases for monolayers20 and interdigitated thin bilayers of dihexadecyldimethylammonium acetate23,24 have been proposed as possible structures for DODA salts assemblies. In pure water, dioctadecyldimethylammonium chloride (DODAC) vesicle adhesion onto DODAC bilayer-covered microspheres was reported, in contrast to DODAB vesicles that underwent disruption at contact with the bilayer-covered latex.25 Further understanding of this vesicle adhesion in pure water, with or without vesicle rupture, requires further experimental and theoretical insights on intra- and interlayer interaction forces determining bilayer structure and colloid stability of cationic vesicles at low ionic strength. Whether the bilayer structure is a traditional or an interdigitated one is still a matter of debate in the literature as is the counterion effect on important physical properties of the aggregate such as bilayer structure and packing, vesicle size and entrapment efficiency, and surface potential. In this work, the effect of counterion nature and concentration on gel-to-liquid-crystalline phase transition, bilayer structure, vesicle size, internal volume, and surface potential is systematically investigated for cationic vesicles (19) Ninham, B. W.; Evans, D. F.; Wei, G. J. J. Phys. Chem. 1983, 87, 5020. (20) Marra, J. J. Phys. Chem. 1986, 90, 2145. (21) Pashley, R. M.; McGuiggan, P. M.; Ninham, B. W.; Brady, J.; Evans, D. F. J. Phys. Chem. 1986, 90, 1637. (22) Claesson, P. M.; Carmona-Ribeiro, A. M.; Kurihara, K. J. Phys. Chem. 1989, 93, 917. (23) Tsao, Y.; Evans, D. F.; Rand, R. P.; Parsegian, V. A. Langmuir 1993, 9, 233. (24) Parsegian, V. A.; Rand, R. P.; Fuller, N. L. J. Phys. Chem. 1991, 95, 4777. (25) Tsuruta, L. R.; Lessa, M. M.; Carmona-Ribeiro, A. M. Langmuir 1995, 11, 2938.
10.1021/la980845c CCC: $15.00 © 1998 American Chemical Society Published on Web 12/03/1998
7388 Langmuir, Vol. 14, No. 26, 1998
composed of dioctadecyldimethylammonium (DODA) acetate, chloride, or bromide. There is a clear correlation between vesicle size and surface potential: large vesicles having small surface potentials and vice versa. Sizes are at smallest and potentials, at largest, for the largest and more hydrated counterions. In other words, vesicle size is electrostatically controlled whereas electrostatics is determined by counterion nature and concentration. The largest vesicles, formed when bromide is the counterion, have the smallest zeta-potential. Furthermore, comparison between sizes and entrapment efficiencies allows the conclusion that vesicles are composed by one single, noninterdigitated bilayer for the three counterions tested over 0-5 mM of monovalent salt concentration.
Nascimento et al.
Chemicals. Dioctadecyldimethylammonium bromide, 99.9% pure (DODAB), was obtained from Sigma and used as such without further purification. Dioctadecyldimethylammonium chloride (DODAC) or acetate (DODAAc) was obtained by ion exchange from DODAB using AMBERLYST A-26 from E. Merck (Darmstadt, Germany) in the chloride or acetate form, respectively, as previously described.12 1-Palmitoyl-2-pyrenedecanoylsn-glycero-3-phosphocholine (PyPC) was obtained from Molecular Probes (Eugene, OR). All other reagents were analytical grade and were used without further purification. Water was Milli-Q quality. Vesicle Preparation. Large DODAX vesicles, where X stands for acetate, chloride, or bromide as counterions, were prepared by vaporization of a chloroformic DODAX solution in water solution as previously described.26 The chloroform vaporization method yields large cationic vesicles that are much more stable in a colloidal sense than the smaller vesicles obtained by sonication with tip.26 Unfortunately, this holds for vesicles in pure water because preparing these synthetic charged vesicles in salt solutions (as we did in the present work), even in small ionic strength, may considerably decrease colloidal stability due to screening of double layer forces. Therefore, we performed the measurements for very fresh preparations though the necessity remains of a more systematic study of colloidal stability. Certainly, vesicles are not stable in a thermodynamical sense (they are metastable). DODAB or DODAC concentrations were determined by microtitration of the halide counterion.27 DODAAc concentration was determined by solubilization of a dye-amphiphile complex in nonionic micelles.28 Phase Transition Determination. The vesicles for dispersion composed of DODAX were prepared in water or in 1 mM NaAc, NaCl, or NaBr and were placed inside a quartz cuvette. The temperature of the vesicle dispersion inside the cuvette was varied using a circulating bath connected to the cuvette holder and measured from a thermocouple in direct contact with the dispersion that was connected to a Jencons digital thermometer, Model 2003. Turbidity at 280 nm was recorded as a function of temperature with mean phase transition temperatures (Tm) determined from inflection points in the heating/cooling curves. The temperature variation rate was constant and equal to 0.2 °C/min. Determination of Fluorescence Spectra for the Pyrenyl Derivative of Phosphatidylcholine (PyPC) Incorporated in the DODAX Bilayer. An 83 µL portion of a stock PyPC chloroformic solution (0.1 mg PyPC/mL or 0.12 mM PyPC) was added to 100 µL of a stock DODAX chloroformic solution (10 mM), and the volume was completed with chloroform up to 1 mL. The final molar proportion was ca. 1 PyPC:100 DODAX. One milliliter of this chloroformic solution was injected and vaporized in 10 mL of pure water for a final DODAX concentration in the dispersion of ca. 1 mM. After a further 10 times dilution, 0.1 mM DODAX dispersions containing ca. 0.001 mM PyPC had their fluorescence emission spectra scanned from 370 to 400 nm using
a spectrofluorometer SPEX FLUOROLOG, model 1681, with excitation wavelength at 325 nm. Intensity ratios calculated from intensities at peaks III and I are related to polarity of the bilayer region where the pyrenyl moiety is inserted.29 Changing the pyrenyl environment from the middle of the bilayer to the interdigitated bilayer/water interface would cause a significant change in the polarity seen by the probe with concomitant changes in III/I intensity ratios. Any counterion effect leading to interdigitation in the bilayer would cause an increase in polarity in the medium seen by the probe.29 Vesicle Electrophoretic Mobilities (EM) and Calculation of Zeta-Potentials. Mobilities were determined at 25 °C using a Rank Brothers Mark II particle microelectrophoresis apparatus with a flat cell. Just after preparation, the vesicle sample was successively diluted for performing both EM and size measurements. The sample to be measured was placed into the electrophoresis cell, electrodes were connected, and a voltage of (60 V was applied across the cell. Velocities of individual vesicles over a given tracking distance were recorded, as was direction of vesicle movement. Average velocities were calculated from data on at least 20 individual vesicles. EM was calculated according to the equation EM ) cm(u/V)(1/t), where u is the distance over which the vesicle is tracked (micrometers), cm is the interelectrode distance (7.27 cm), V is the voltage applied ((60 V), and t is the average time in seconds required to track one vesicle a given distance u. Because of the large effect of amphiphile concentration on EM,30 mobilities were determined as a function of DODAX concentration for each NaX concentration. The EM values used for calculating zeta-potentials (ζ) were those extrapolated to zero amphiphile concentration (EM0) in order to eliminate any effect of interacting vesicles on the surface potential of a single vesicle. EMo values were transformed into reduced mobilities which were finally inserted in the theory of O’Brien and White31 to calculate zeta-potentials30 that are expressed in millivolts. Determination of Mean z-Average Diameters. Sizes were measured simultaneously with mobilities for each vesicle sample inside 1 h after obtaining the vesicle dispersion using a Malvern 4700c PCS apparatus employing a Coherent Innova 90 laser. The size quoted throughout is the mean harmonic z-average diameter (Dz) of at least five independent measurements at 25 °C. Although vesicles were prepared at 1 mM DODAX concentration, immediately thereafter the dispersion was diluted (1: 10, v:v) in the same salt solution where vesicles were prepared so that any problems with colloid instability were avoided. This was seen from the constancy in turbidity at 400 nm over at least 1 week after vesicle preparation and 1:10 dilution. Determination of Entrapment Efficiency from Incorporation of Methylene Blue as a Marker of the Internal Vesicle Compartment and Calculation of Entrapment Efficiency from Vesicle Size. Vesicles were prepared in salt solutions that contained 0.5 mM of methylene blue (MB). Samples of vesicles prepared in MB solution (5 mL) and a control sample of the MB solution were dialyzed against 2 L of salt solution (3×, over 72 h) with vigorous stirring. Before dialysis, the MB solution or the vesicles prepared in the MB solution were diluted 1:50 and aliquots of these diluted solutions were reserved for determining MB entrapment in the vesicles. After dialysis, 1 mL of each sample was added to 50 µL of BRIJ (20 mM) and kept at 50 °C for 10 min before taking absorbance readings. Absorbance at 660 nm was determined for vesicle samples and for the control MB solution before and after dialysis. The entrapment efficiency (ENT) was obtained from MB entrapment and quantitative analysis of DODA molar concentration (Ca) in the vesicle sample.28 ENT can be taken as25 ENT ) (1/Ca)(A2/A1 - A2c/A1c) where A1 and A2 are absorbances at 660 nm of vesicle samples before and after dialysis, respectively; A1c and A2c are absorbances at 660 nm of the 0.5 mM MB solution before and after dialysis, all corrections for dilution of samples before dialysis being considered. ENT is therefore expressed in liters per mole of DODA. ENTc was calculated from vesicle size and limiting mean area per molecule at the air-water interface for DODAX assuming
(26) Carmona-Ribeiro, A. M.; Chaimovich, H. Biochim. Biophys. Acta 1983, 733, 172. (27) Schales, O.; Schales, S. S. J. Biol. Chem. 1941, 140, 379. (28) Stelmo, M.; Chaimovich, H.; Cuccovia, I. M.. J. Colloid Interface Sci. 1987, 117, 200.
(29) Komatsu, H.; Rowe, E. S. Biochemistry 1991, 30, 2463. (30) Carmona-Ribeiro, A. M.; Midmore, B. R. J. Phys. Chem. 1992, 96, 3542. (31) O’Brien, R.; White, L. R. J. Chem. Soc., Faraday Trans. 2 1978, 74, 1607.
Material and Methods
Counterion Effects on Vesicles
Langmuir, Vol. 14, No. 26, 1998 7389 Table 1. Mean Phase Transition Temperature (Tm) for Cationic DODA Vesicles with Acetate, Chloride, or Bromide as Counterions Prepared in Water or in 1 MM Monovalent Sodium Salt (NaAc, NaCl or NaBr, respectively)a DODAX/ concentration DODAAc/1.0 mM DODAC/0.9 mM DODAB/0.9 mM
NaX Tm Tm concentration/ (heating)/ (cooling)/ hysteresis/ M °C °C °C 0 1 0 1 0 1
45.0 47.5 47.5 47.0 43.5 43.5
33.5 37.0 42.0 42.0 39.0 40.0
11.5 10.5 5.5 5.0 4.5 3.5
a T was obtained from inflection points in turbidity vs temm perature curves (not shown).
Figure 1. Turbidity at 280 nm as a function of temperature for large DODAX vesicles in water at DODA concentrations specified in Table 1. Heating is represented by squares and cooling by circles. a traditional bilayer model with a 1:1 ratio for lipids in the outer and inner monolayer of the bilayer.
Results and Discussion 1. Mixed Composition for Alkyl Chains against Counterion Effects on Phase Transition of Cationic Vesicles. From the temperature effect on turbidity at 280 nm for DODAX vesicles obtained either in water or in 1 mM NaX (Figure 1), the mean phase transition temperatures were determined for Br-, Cl-, and Ac- as counterions (Table 1, Figure 1). One should notice that the DODAX amphiphiles used in this work are very pure dialkyl (99.9% C18:99.9% C18) dimethylammonium salts, in contrast with the DODAC used previously.26,32 DODAC having a trade name of Herquat 2HT-75 from Herga Indu´strias Quı´micas do Brasil was shown to be a mixture of dialkyl (75% C18:25% C16) dimethylammonium chlo(32) Carmona-Ribeiro, A. M.; Chaimovich, H. Biophys. J. 1986, 50, 621.
ride by mass-spectral analysis.33 This mixed composition for the alkyl chains generated a mean phase transition temperature (Tm) equal to 38.6 °C (heating) and 34.8 °C (cooling).26 In this work, Tm for pure DODAC is shown to be much higher: 47.5 °C (heating) and 42.0 °C (cooling) (Table 1, Figure 1). Hysteresis for heating and cooling curves is very large for DODAAc vesicles, decreasing as a function of counterion size and hydration (Table 1). The path for the transition from the gel state to the liquidcrystalline state is not identical to that for the transition from the liquid-crystalline state to the gel state. At temperatures close to that of the transition, domains of one phase will be present within a bulk matrix of the other phase. Hysteresis will then follow because the free energy of a domain of liquid-crystalline-like lipid within a matrix of gel phase lipid will be different from that of a domain of gellike lipid: since the molar volume of lipid in the liquid-crystalline phase is greater than that in the gel phase, a domain of liquid-crystalline-like lipid will be under compression, whereas a domain of gel phase lipid will be under tension. If instead of a large number of separate domains of one phase within the bulk second phase, all the domains were to collect together to give one large domain, then effects of hysteresis would be very much reducedsin the case of a domain of macroscopic dimensions, strain energy would be very small. However, the cooperative changes required within the structure to bring all the domains together would have to be of very much greater magnitude than appears kinetically possible. Consistently, the largest hysteresis was observed for the smallest vesicle type (DODAAc vesicles) and the smallest one was obtained for the largest vesicle (DODAB vesicles) (Figure 1 and Table 1). The effect of counterion nature on Tm is also in Table 1. Bromide, the smallest and least hydrated counterion, yields the smallest Tm value. Tm remains unaffected by preparing vesicles in water or in 1 mM salt but decreases by 4 °C by preparing DODAB instead of DODAC or DODAAc vesicles. The smaller counterion yields less tightly packed, “softer” bilayers than those obtained with the larger, more hydrated and less tightly bound counterions. Bromide as a counterion generates the smallest electrostatic repulsion between adjacent polar heads (as shown from zeta-potential data in the next section) causing a less tightly packed hydrocarbon region than the one occurring when counterions are chloride or acetate. 2. Bilayer and Vesicle Structure from PyPC Fluorescence Spectra Plus Determination of Entrapment Efficiency. Pyrene is a fluorescence probe (33) Cuccovia, I. M.; Aleixo, R. M. V.; Mortara, R. A.; Filho, P. B.; Bonilha, J. B. S.; Quina, F. H.; Chaimovich, H. Tetrahedron Lett. 1979, 33, 3065.
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Nascimento et al. Table 2. Effect of Monovalent Salt Concentration (C) on Mean Zeta-Average Diameter (Dz) of Dioctadecyldimethylammonium Acetate (DODAAc), Chloride (DODAC), or Bromide (DODAB) Vesicles Prepared by the Chloroform Vaporization Method in Sodium Acetate, Chloride or Bromide, Respectivelya D z/ DODAX C/mM nm
Figure 2. Fluorescence emission spectra of PyPC in bilayers of large DODAX vesicles prepared in water. All curves were normalized to band I. The concentration of PyPC was 1.2 µM. The excitation wavelength was 325.0 nm (excitation and emission slits of 1 nm).
useful for membrane studies because its vibrational band intensities are sensitive to the polarity of its environment. However, it has significant water solubility, which can complicate the interpretation of results. Pyrene-labeled lipids, i.e., lipids with the pyrenyl moiety covalently attached to the lipid hydrocarbon chain, have the advantage that they are located in the membrane and the location of the pyrene moiety is known. In particular, PyPC has been used to detect ethanol-induced interdigitation in phospholipid vesicles.29 Interdigitation for the synthetic cationic vesicles is a hypothesis sporadically raised in the literature to explain vesicle adhesion or fusion at low salt32,35 or a decrease in bilayer thickness indirectly measured using a surface force apparatus for the acetate derivative.23 Figure 2 shows spectra obtained for PyPC incorporated in DODAX bilayer vesicles prepared in water. Intensity ratios for peaks III and I as a function of the counterion vary between 0.35 and 0.52. The smallest value (0.35) is coincident with the one obtained for PyPC inserted in dipalmitoylphosphatidylcholine (DPPC) bilayer vesicles that are in the gel state at room temperature and recognized as traditional bilayers for which interdigitation is absent.29 Larger ratios for the cationic vesicles mean polarities seen by the probe that are still smaller than the environmental polarity seen by PyPC in traditional DPPC bilayers (29). This speaks against any possibility of interdigitation in the cationic synthetic bilayers. Further confirmation of the traditional bilayer structure for the three counterions comes from comparison between measured and calculated entrapment efficiencies (ENT). Although the variation of vesicle volume due to interdigitation is expected to be negligible, the volume entrapped per mole of amphiphile can be considerably increased if interdigitation occurs. Considering vesicles 1 and 2 of the same size, entrapping the same volume and having one single lipid layer of 5 and 2.5 nm thick, respectively, vesicle 1 has a traditional bilayer whereas vesicle 2 has a fully interdigitated monolayer. For vesicle 1, the volume entrapped per mole will be half the volume entrapped by vesicle 2. This is so only because the total number of amphiphile molecules in vesicle 1 is double the number of molecules in vesicle 2. Therefore, by measuring (34) Lee, A. G. Biochim. Biophys. Acta 1977, 472, 237. (35) Carmona-Ribeiro, A. M. J. Phys. Chem. 1993, 97, 11843.
P
EM0/ µcm A/ ENTc/ ENT/ ξ/ 2 nm (L/mol) (L/mol) V-1 s-1 mV
DODAAc 0 0.001 0.01 0.1 0.5 1 5 DODAC 0 0.001 0.01 0.1 0.5 1 5 DODAB 0
217 201 186 209 242 217 257 321 250 264 311 371 301 372 345
0.59 0.57 0.54 0.57 0.61 0.70 0.65 0.59 0.70 0.40 0.40 0.50 0.52 0.52 0.48 0.50 0.60 0.61
0.001 0.01 0.1 0.5 1 5
315 323 446 479 508 711
0.41 0.47 0.45 0.40 0.34 0.90 0.54
7 7 6 7 7 7
7 7 6 6 4
8
14 11 9 8
7 8 8 9
14 19
15 ( 6 12
4.3 3.4 4.2 3.5 3.1 4.1 1.9 3.0 3.5 3.7 3.5 1.8 3.6 1.8 3.2
81 64 100 80 54 80 28 54 59 85 73 28 57 24 58
3.9 2.8 1.8 1.6 1.3 0.2
80 58 31 24 19 6
a P is the polydispersity index. ENT is the entrapment efficiency c calculated from Dz and from limiting area per monomer at the air-water interface, A (A values taken from refs 34 and 35), and the measured Dz. ENT is the entrapment efficiency measured using entrapment of methylene blue. EM0 is the electrophoretic mobility extrapolated at infinite DODA dilution and ξ is the zeta-potential calculated from the ionic strength, vesicle size, and EM0 data. ENT measurements were done at 25 °C. b Mean value obtained from eight measurements using [14C]-sucrose as a marker of the internal aqueous compartment.
ENT as well as vesicle size, it is theoretically possible to distinguish between interdigitated and noninterdigitated structures. ENT was measured for the three vesicle types and compared with ENTc calculated from vesicle size assuming entrapment due to a traditional bilayer. Interdigitation would considerably increase ENT in comparison to ENTc since the same amount of lipid would entrap a larger volume for an interdigitated structure in comparison to the volume entrapped by a traditional bilayer vesicle. In Table 2 is the comparison between experimental ENT and ENTc. Inside the limits of experimental error there is no difference between ENT and ENTc, a result that supports the traditional bilayer structure for the three counterions tested. DODAAc vesicles, which were the strongest candidates to an interdigitated structure due to the very large electrostatic intralayer repulsion at low ionic strength, which could have been causing interdigitation, gave very similar figures for ENT and ENTc (Table 2). 3. Counterion Effects on Vesicle Size and ZetaPotential. The effect of counterion type and concentration on mean z-average diameter (Dz) of cationic vesicles is shown in Table 2 and in Figure 4B. From a geometric model for the self-assembly,36 the largest area per molecule A at the air-water interface in Table 2 should yield the smallest geometric parameter v/(lA)36 and the smallest aggregation number for the aggregate. This is indeed the case for DODAAc, which has the largest area per molecule (36) Israelachvili, J. N. Intermolecular & Surface Forces; Academic Press: London, 1992.
Counterion Effects on Vesicles
Langmuir, Vol. 14, No. 26, 1998 7391
Figure 4. Effect of monovalent salt concentration (NaX) on zeta-potentials and mean z-average diameters for large DODAX vesicles. One should notice data for Herquat vesicles23 represented as open triangles for comparison with the pure DODA salts. Dz determinations were done at 25 °C.
Figure 3. Electrophoretic mobility of large DODAX vesicles as a function of DODAX concentration at 0 mM of monovalent salt, NaX. EM measurements were done at 25 °C.
at the air-water interface and yields the smallest vesicles (Table 2). Consistently, the experimentally determined entrapment efficiency is also smallest for acetate as counterion (Table 2). The effect of amphiphile concentration on electrophoretic mobilities for DODAX vesicles prepared in water is shown in Figure 3. It was previously shown that large surface potentials for charged vesicles may result in EM values well above those that can be fitted from any theory connecting mobility measurements to zeta-potentials.30 In the case of large vesicles, electrophoretic mobilities may be affected by overlap of ionic atmospheres so that extrapolations to infinite DODAX dilution were performed to determine mobilities of isolated vesicles. This procedure generated mobility values that could be fitted in the O’Brien and White theory to obtain zeta-potential values (Table 2, Figure 3). One should notice that this procedure was also adopted for each NaX concentration in which large DODAX vesicles were prepared (data not shown). All EM0 values were found to lie below the maximum EM value predicted by the theory. The counterion effect on zeta-potentials is shown in Figure 4A. The largest (smallest) zeta-potentials are those obtained for Ac- (Br-) as counterion. The zeta-potential decreases with increasing vesicle radius (Figure 5). For the cationic DODAC vesicles produced from Herquat (see open triangles in Figure 4), some experimental evidence for such a relationship has already been presented.30 In this work, the effect is
Figure 5. Inverse relationship between surface potential and vesicle size for DODAX vesicles suggesting the existence of an electrostatic control of vesicle size.
systematically confirmed for the three counterions over a range of NaX concentrations and for pure double C18 amphiphiles. There is an electrostatic control of vesicle size so that large intralayer electrostatic repulsion between adjacent monomers in the bilayer produces small vesicles. This agrees with previous findings for another synthetic amphiphile system, anionic dihexadecyl phosphate vesicles, for which changes in size as a function of pH and ionic strength were inversely related to the surface potential.30 Acknowledgment. D.B.N. and R.R. thank FAPESP and CNPq, respectively, for undergraduate fellowships. FAPESP and CNPq are gratefully acknowledged for financial support. LA980845C
