Journal of Colloid and Interface Science 322 (2008) 582–588www.elsevier.com/locate/jcis

Extruded vesicles of dioctadecyldimethylammonium bromide andchloride investigated by light scattering and cryogenic transmission

electron microscopy

António Lopes a, Katarina Edwards b, Eloi Feitosa c,∗

a ITQB – Inst. Tecn. Química e Biológica, Univ. Nova de Lisboa, Oeiras, Portugalb Department of Physical Chemistry, Uppsala University, Uppsala, Swedenc Department of Physics, IBILCE/UNESP, São José do Rio Preto, SP, Brazil

Received 11 January 2008; accepted 4 March 2008

Available online 18 March 2008

Abstract

Combined dynamic and static light scattering (DLS, SLS) and cryogenic transmission electron microscopy (cryo-TEM) were used to investigateextruded cationic vesicles of dioctadecyldimethylammonium chloride and bromide (DODAX, X being Cl− or Br−). In salt-free dispersions themean hydrodynamic diameter, Dh, and the weight average molecular weight, Mw, are larger for DODAB than for DODAC vesicles, and both Dhand Mw increase with the diameter (φ) of the extrusion filter. NaCl (NaBr) decreases (increases) the DODAB (DODAC) vesicle size, reflectingthe general trend of DODAB to assemble as larger vesicles than DODAC. The polydispersity index is lower than 0.25, indicating the dispersionsare rather polydisperse. Cryo-TEM micrographs show that the smaller vesicles are spherical while the larger ones are oblong or faceted, and thevesicle samples are fairly polydisperse in size and morphology. They also indicate that the vesicle size increases with φ and DODAB assemblesas larger vesicles than DODAC. Lens-shaped vesicles were observed in the extruded preparations. Both light scattering and cryo-TEM indicatethat the vesicle size is larger or smaller than φ when φ is smaller or larger than the optimal φ∗ ≈ 200 nm.© 2008 Elsevier Inc. All rights reserved.

Keywords: DODAB; DODAC; Cationic vesicle; Extruded vesicle; Lens-shaped vesicle; Light scattering; Cryo-TEM

1. Introduction

Vesicles are natural membrane model systems with potentialapplication as vehicle for drug delivery [1–3]. To mimic bio-logical membrane or function as drug delivery vehicle, vesiclesshould exhibit narrow size distribution and be stable. Severalvesicles preparation methods have thus been developed to attainthese requirements. It includes sonication, organic solvent evap-oration, extrusion, detergent-removal or simple mixing (sponta-neous), among others [1–15].

In this communication we focus on the extrusion methodused to prepare DODAX vesicles (X being usually the Br− orCl− ions), which is currently one of the best to prepare unil-

* Corresponding author. Fax: +55 17 3221 22 47.E-mail address: eloi@ibilce.unesp.br (E. Feitosa).

0021-9797/$ – see front matter © 2008 Elsevier Inc. All rights reserved.doi:10.1016/j.jcis.2008.03.015

amellar vesicles with well-controlled size and geometry, andthe data are compared to those prepared by other methods (son-ication, injection and spontaneous) [4,5,9,10,12,14] as well asextrusion [7,15].

Relative to extruded phospholipid liposomes, extruded DO-DAX vesicles have been poorly investigated probably becauseof technical limitation related to the relatively high melting tem-perature (Tm), of these surfactants, which is usually higher than45 ◦C [7]. Extrusion is, however, very suitable to form smallerDODAX vesicles [7,15], although the vesicle characteristicsmay depend on the applied extrusion pressure [16], surfactantconcentration [7] and the presence of co-solutes, according tothis communication. Sonication has as well been used to pre-pare smaller DODAX vesicles although bilayer fragments andlens-shaped vesicles are mainly formed [9,10] while extrudedand spontaneous vesicles form mainly smoothed vesicles, asshown in this communication and in Ref. [15].

A. Lopes et al. / Journal of Colloid and Interface Science 322 (2008) 582–588 583

In spite of this, there is a lack of investigation on the relation-ship between the vesicle curvature and the pore diameter (φ) ofthe extrusion membrane. It is shown in this communication thatthe mean size of DODAX vesicles (obtained by static and dy-namic light scattering and cryo-TEM) can be larger or smallerthan φ, but it fits well to an optimal pore diameter φ∗ ≈ 200 nm.The effect of NaCl and NaBr on the size of DODAX vesicleswas also investigated.

2. Materials and methods

2.1. Materials

DODAB from Avanti Polar Lipids (purity higher than 99%)was used without further purification, and recrystallized DO-DAC was obtained by counterion exchange from DODAB(Eastman Kodak, purity 99.5%) [8]. Mili-Q-Plus quality wa-ter was used to prepare the vesicle dispersions. All the salts(Merck) used were of analytical grade.

2.2. Vesicle preparation

Stock solutions of the spontaneous vesicles were preparedby mixing DODAX and water to a desired final concentra-tion (usually 1 mM) and then warming to 55 ◦C, that is, abovethe melting temperature, Tm, of the surfactant to obtain anhomogeneous dispersion [6,7,11–13]. Extruded vesicles wereobtained using an extrusion system from Avanti Polar Lipids,Alabaster, AL. Extrusions were performed manually by forcingseveral times (typically 15 times) the DODAX spontaneous dis-persion through two stacked 13 mm polycarbonate filters withnominal pore diameters, φ = 50,100,200 and 400 nm, keepingthe dispersion temperature at 55 ◦C, as described in [7]. Afterextrusion the vesicle dispersions were then cooled to room tem-perature and stored. DODAX vesicle dispersions at the desiredsurfactant concentration were obtained by direct dilution of thestock solutions. The measurements were performed 24 h aftervesicle preparation.

2.3. Light scattering measurements

Light scattering (LS) measurements were made with an ap-paratus from Brookhaven Instruments, Inc., Model 2030AT,equipped with a He–Ne laser (λ = 632.8 nm) and a 136 channelautocorrelator. The vesicle dispersions for LS measurementswere centrifuged at approximately 1300g for 45 min to removedust particles, and the solvent solutions or water used to diluteor to add salt into the vesicle dispersions were filtered througha Millipore membrane filter of 0.45 µm nominal pore diameter.Dynamic (DLS) and static (SLS) light scattering measurementswere made at the scattering angles θ = 50,70,90,110,130◦,and the Zimm plots made with a minimum of five logarithmi-cally spaced concentrations. The optical constant of the appa-ratus was obtained with benzene prior to the measurements andthe dn/dc values determined with a differential refractometer(Model G.M. Wood RF600) fall in the range 0.12–0.16 ml/g

Fig. 1. Zimm plot for extruded DODAB vesicle dispersions obtained with afilter pore diameter φ = 200 nm. Measurements made at 25 ◦C.

for all DODAX dispersions under study. For each pair sam-ple/scattering vector the hydrodynamic diameter (Dh) distribu-tion (histogram) was obtained with a CONTIN routine [17,18],where Dh is the hydrodynamic diameter extrapolated to zerosurfactant concentration, c, and zero scattering angle, θ . Com-parison of the radius of gyration, Rg, obtained from SLS, withthe hydrodynamic radius, Rh = Dh/2, from DLS, reveals thegeometry of the macromolecular aggregate based on the wellknown Rg/Rh ratio [18].

SLS allows the evaluation of the weight average molecularweight, Mw, the radius of gyration, Rg, and the second virialcoefficient, A2, of the aggregates present in dilute solutions bythe usual Zimm method based on the Rayleigh–Gans–Debyetheory [18]. Accordingly, the variation of the relative light scat-tering intensity, Is/Io, where Io is the intensity of the incidentradiation, with the observation angle, θ , or scattering vector,q = 4π sin(θ/2)/λ, is related with Mw,Rg, and A2, for a givensample concentration, c, through the equation [18]

(1)Kc

Rθ

= 1

Mw

(1 + 16π2n2

o

3λ2R2

g sin2 θ

2

)+ 2A2c,

where Rθ = d2(Is/Io) sin2 θ is the Rayleigh factor, K =[4πn2

o(dn/dc)2/ sin2 θ ]/NAλ4 is an optical constant, no andn are, respectively, the refractive index of the solvent and so-lution, NA is the Avogadro constant, dn/dc is the differentialrate of refractive index measured separately. The graphic ofKc/�Rθ as a function of sin2(θ/2) + c is known as Zimmplot [18] that allows one to obtain the vesicle Mw, Rg, and A2,from the intercept and slope of the fitted curves (dashed linesin Fig. 1) to the c = 0 and θ = 0 extrapolated experimentalpoints [18]. In this way vesicle aggregates can be character-ized as a function of the surfactant concentration, temperature,ionic strength, method of vesicle preparation, etc. The scat-tering measurements were made at the sample temperature of25 ± 1 ◦C.

584 A. Lopes et al. / Journal of Colloid and Interface Science 322 (2008) 582–588

Table 1Hydrodynamic, Rh, and gyration, Rg, radii, weight average molecularweight, Mw, second virial coefficient, A2, shape factor, Rg/Rh, and the av-erage polydispersity index, P , obtained from light scattering for DODAB andDODAC extruded vesicles as functions of the filter pore diameter, φ, of the ex-trusion membrane (for comparison, Rh = 337 and 247 nm for DODAB andDODAC spontaneous vesicles, respectively [13])

Surfactant φ

(nm)

Rh(nm)

Rg(nm)

Mw(×107)

A2(cm3 mol/g2)

Rg/Rh P

DODAB 50 68 53 1.93 −9.1 0.80 0.15100 75 59 3.71 1.03 0.85 0.21200 115 500 53.6 0.42 4.32 0.18400 157 992 198 0.46 6.37 0.13

DODAC 50 48 39 0.84 −12.0 0.83 0.12100 66 60 1.21 0.00 0.91 0.21200 105 430 45.2 0.45 4.10 0.15400 148 950 170 0.44 6.47 0.17

2.4. Cryo-TEM measurements

Thin (10–500 nm) sample films were prepared under well-controlled temperature (25 ◦C) and humidity conditions in anenvironmental chamber and vitrified by quickly freezing to108 K in liquid ethane before being transferred to a Zeiss EM902 transmission electron microscope for examination. To pre-vent sample perturbation the specimens were kept below 108 Kduring the transfer and viewing procedures. The observationswere made in the zero loss bright-field mode and at an electronaccelerating voltage of 80 kV. All samples were vitrified fromthe temperature of 25 ± 1 ◦C. Further details on the cryo-TEMexperimental procedure are found elsewhere [13,19].

3. Results

Static and dynamic light scattering data, both in absenceand presence of NaBr or NaCl (Figs. 1–4), as well as cryo-TEM images of extruded DODAX vesicles (Figs. 5–7) arepresented. Additional scattering (Figs. SM1 and SM2) andcryo-TEM (Figs. SM3 and SM4) data are shown as supple-mentary material. Dynamic light scattering data for DODAXspontaneous [13], sonicated [9] and injected [20] vesicles havebeen reported previously, as well as cryo-TEM images for DO-DAX spontaneous vesicles in the absence [13] and presence ofNaBr [12]. This work supplies complementary information onsize, molecular weight and shape of extruded DODAX vesiclesthrough varying the membrane pore diameter, φ.

Table 1 summarizes the values of Rh, Rg, Mw, A2, the shapefactor, Rg/Rh, and the average polydispersity index, P , ob-tained from static and dynamic light scattering, as functionsof φ, for DODAX vesicles. Rg, Mw, and A2 were obtained fromthe Zimm plots, as shown in Fig. 1 for extruded DODAB vesi-cles prepared using a φ = 200 nm (for the other values of φ

the Zimm plots exhibit similar pattern and are not shown here).The Zimm plots were fitted to quadratic curves with correlationcoefficients higher than 0.98. Rh was obtained from the his-tograms of the distribution of hydrodynamic diameter, shownin Fig. 2 and Fig. SM1.

Fig. 2. Histograms of the distribution of hydrodynamic diameter of 0.1 mMDODAB extruded vesicles in water, as a function of the filter pore diameter,φ = 50,100,200, and 400 nm. Measurements made at 25 ◦C and θ = 90◦ .

Fig. 2 and Fig. SM1 show selected histograms of the distrib-ution of hydrodynamic diameter, Dh, for θ = 90◦, as function ofthe filter pore diameter φ = 50–400 nm and Br− or Cl− coun-terion. The representation of the mean hydrodynamic diameteras a function of the surfactant concentration is shown in Fig. 3.The effect of φ on Dh of DODAX vesicles in absence and pres-ence of 5 mM NaCl or NaBr is shown in Fig. 4.

Figs. 5–7 and Figs. SM3, SM4 show cryo-TEM micrographsfor extruded vesicles of DODAX 1.0 and 5.0 mM in water,prepared with pore diameters φ = 100 or 400 nm. Cryo-TEMmicrographs for the spontaneous [13] and sonicated [10] DO-DAX vesicles have already been reported.

4. Discussion

It is well known in the literature that the vesicle size in-creases with the pore size of the extrusion membrane, but toour knowledge there is no clear correlation between DODAXvesicle size and the pore dimension of the extrusion membrane.This work attempts to correlate these parameters for DODAXvesicles based on light scattering and cryo-TEM data and inves-tigate the effect of NaBr and NaCl on the vesicle size.

4.1. Light scattering results

According to Table 1 and Fig. 4, the size (Rh and Rg) andmolecular weight Mw of DODAX vesicles in salt-free water in-creases with φ. The polydispersity index, P , is always lowerthan 0.25, meaning that the dispersions are fairly polydisperse,although we found no clear correlation between φ and the poly-dispersity and size of DODAX vesicles (Table 1 and Fig. 4).

Saveyn et al. [15] reported smaller Dh ≈ 125 nm for ex-truded DODAC through φ = 200 nm. These authors, however,prepared the vesicles in 0.02 wt% CaCl2, that makes difficulta direct comparison with our reported data. In the presence of5 mM single salts (Fig. 4) Dh is roughly the same as in absenceof salt (Dh ≈ 210 nm in Table 1). These authors also reportedthat the osmotic response (yielded by sucrose and CaCl2) of

A. Lopes et al. / Journal of Colloid and Interface Science 322 (2008) 582–588 585

Fig. 3. Mean hydrodynamic diameter of 0.5 mM DODAB (a) and DODAC (b) extruded vesicles as a function of sin(θ/2)2. Extrapolated mean hydrodynamicdiameter for θ = 0◦ for DODAB (c) and DODAC (d) vesicles as a function of the DODAX concentration. Measurements made at 25 ◦C. Symbols account forφ = 50 (!), 100 (P), 200 (E) and 400 nm (1).

the extruded DODAC vesicles depends on the bilayer rigidity,which in turn is determined by the vesicle size, meaning thatthe vesicle size is an important parameter in many vesicle ap-plications [7].

Like the spontaneous vesicles [13], Dh and Mw of extrudedvesicles in salt-free dispersions are always larger for DODABthan DODAC, and increases with φ (Table 1). It is worth notic-ing that for φ < 200 nm, Dh of DODAX vesicles is largerthan φ, while for φ = 200 nm Dh almost matches that value,and for φ > 200 nm Dh is smaller than φ. This suggests thatthere is an optimal membrane pore size, φ∗ ≈ 200 nm forpreparation of extruded DODAX vesicles whose size matcheswell the pore diameter of the extrusion membrane. This issuitable technical information on the method to form DO-DAX extruded vesicles with a reasonable control of the vesiclesize.

These results matches those for the spontaneous (non-extruded) vesicles (Rh = 337 and 247 nm for DODAB andDODAC spontaneous vesicles, respectively [13]), since the sizeof the extruded vesicles increases with φ to attain the size ofthe spontaneous vesicles when φ > 400 nm. Furthermore, irre-spective of φ, the extruded DODAX vesicles in water do notprecipitate for months, indicating stability comparable to thespontaneous DODAX vesicles [7,13].

One should stress that Rg and Rh increase with φ in differentways, such that the shape factor Rg/Rh increases from 0.8 toca. 6.5 for DODAB or DODAC (Table 1), indicating that the

Fig. 4. Mean hydrodynamic diameter of DODAB (E) and DODAC (1) ex-truded vesicles as a function of the filter pore diameter φ without salt. Thebroken line which corresponds to the equality Dh = φ is just a guide for theeye.

vesicle growth is followed by morphological changes due tothe flexibility of the vesicle bilayer.

In presence of salt, NaCl decreases while NaBr increases Dh

of DODAB and DODAC vesicles, respectively (Table 1). Thegeneral behavior of the counterion exchange in the Stern layersreflects the prevailing trend of DODAB to form larger vesiclesthan DODAC, as already reported [13,20]. Overall, NaCl con-

586 A. Lopes et al. / Journal of Colloid and Interface Science 322 (2008) 582–588

Fig. 5. Cryo-TEM micrographs of 1.0 mM DODAB in extruded vesicles pre-pared with φ = 100 nm. Bar equals 100 nm. Arrow points to a lens-shapedvesicle.

tracts DODAB vesicles while NaBr expands DODAC vesiclesdue to the specificity of the binding of these counterions to thevesicle interfaces [7,20].

Up to 10 mM single inorganic salts DODAX vesicles ex-truded trough φ = 200 nm does not change significantly thepolydispersity index relative to the salt-free dispersion. How-ever, DODAC vesicles prepared with φ = 50 nm tend to pre-cipitate in presence of [Br−] > 5 mM, while DODAB vesiclesprepared with φ = 200 nm or smaller, in the presence of morethan 5 mM Br− or Cl−, undergo an increase of about 20%in the polydispersity index. It thus indicates a trend of highsmaller vesicles to be less stable than the corresponding largervesicles, which is stable for months [9,13,21]. It was also ob-served a phase separation (but without vesicle precipitation) for[DODAX] > 0.32 mM and φ = 400 nm when [Br−] > 5 mM.In spite of the increase in the polydispersity index, the Zimmplots exhibit the same profile relative to the salt-free vesiclesshown in Fig. 1 (results not shown), and the shape factor doesnot change significantly. The data thus suggest that smaller(higher curvatured) vesicles exhibit less densely packed bilayerthat allow the counterion to interact strongly with the vesicleinterfaces thus favoring vesicle aggregation followed by pre-cipitation owing to electrostatic screening.

4.2. Cryo-TEM results

Overall, the mean size and polydispersity information ob-tained from the cryo-TEM micrographs shown in Figs. 5–7 andFigs. SM3 and SM4 is in qualitative agreement with the lightscattering results, indicating that at low concentrations DODABmolecules in water assemble as larger unilamellar vesicles thanDODAC, as already reported for the spontaneous [13] and in-jected [20] vesicles. The cryo-TEM micrographs also indicatethat extrusion tends to form smaller DODAX vesicles than thespontaneous, and the vesicle size increases with φ.

Up to 1.0 mM (and above Tm ≈ 45 ◦C) DODAB self-assembles in water as large unilamellar spherical, oblong orfaceted vesicles, rather polydisperse in size and geometry [12,13]. The cryo-TEM images (Figs. 5–7 and Figs. SM3 and SM4)show that extrusion decreases the vesicle size leaving their mor-

Fig. 6. Cryo-TEM micrographs of 1.0 mM DODAC in extruded vesicles pre-pared with φ = 100 nm. Bar equals 100 nm.

Fig. 7. Cryo-TEM micrographs of 5.0 mM DODAC in extruded vesicles pre-pared with φ = 100 nm. Bar equals 100 nm. Arrow points to a lens-shapedvesicle.

phology similar to the spontaneous vesicles, and forms somelens-shaped vesicles. DODAX lens-shaped vesicles have beenobserved before in tip-sonicated [10] and extruded [15] disper-sions. The images suggest that the smaller is φ, the higher is theamount of lens-shaped vesicles.

For φ = 100 nm DODAB extruded dispersions consistmainly of quasi-spherical vesicles with the larger ones havingdiameter of ca. 160 nm, that is, larger than φ (Fig. 5). Smallervesicles with diameter comparable to φ can also be discernedin the micrograph, as well as small lens-shaped vesicles. Themicrograph also reveals that most of the vesicles have meansize larger than φ, indicating that extrusion membranes with φ

smaller than the optimal φ∗ ≈ 200 nm yield vesicles with meansize larger than φ in agreement with the scattering results (Fig. 4and Table 1).

For φ = 400 nm, DODAB vesicles are also quasi-sphericaland unilamellar with dimension in the range of ca. 150–350 nm (Fig. SM3). Thus, extrusion through φ = 400 nm givesDODAB vesicles smaller than φ, again in agreement with thescattering results. Note that no lens-shaped vesicle is seen inFig. SM3, probably because for larger φ, a small amount of bi-layer fragments (and thus of lens-shaped vesicles) are formed,being more scarce to be observed in the cryo-TEM images.

Like DODAB, DODAC molecules assemble (above Tm ≈48 ◦C) as large unilamellar vesicles, rather polydisperse in size

A. Lopes et al. / Journal of Colloid and Interface Science 322 (2008) 582–588 587

and geometry (Figs. 6, 7 and Fig. SM4) [7,13]. Accordingly,DODAC tends to form smaller vesicles than DODAB eitherin spontaneous [7,11,13] or extruded dispersions [15], in goodagreement with the scattering data reported above. DODACalso forms lens-shaped vesicles in extruded dispersions (Fig. 7and Fig. SM4), as reported in [15].

Extrusion through φ = 100 nm results in spherical unilamel-lar DODAC vesicles with mean diameter in the range of ca.80–200 nm (Fig. 6), that is, larger than φ. Extrusion throughφ = 400 nm, on the other hand, gives spherical unilamellarvesicles with mean diameters in the range of ca. 250–350 nm(Fig. SM4), that is smaller than φ. In this sample we canalso discern some small lens-shaped vesicles that are probablyformed by fusion of pieces of fragmented bilayers, which comeabout after the extrusion or sonication, as reported [10,15]. Oneshould note that the amount of DODAX lens-shaped vesiclesis higher the higher is the surfactant concentration (Fig. 7).For this reason sonication produces more lens-shaped vesiclesthan extrusion, because the former gives more bilayer frag-ments. This is the case when we compare the present cryo-TEMpictures to those for sonicated DODAB [10] or extruded DO-DAC [15] vesicles.

Extruded unilamellar DODAX vesicles can also be formedat higher surfactant concentrations [13], as shown in the cryo-TEM micrograph of an extruded dispersion of 5.0 mM DODACin water prepared through φ = 100 nm (Fig. 7). Accordingly,up to 5 mM the surfactant concentration plays no key role onthe size and structure of extruded DODAC vesicles. The meansize of these vesicles is clearly larger than φ, but smaller thanthe mean size of the corresponding DODAB extruded vesicles.The larger vesicles have mean diameters of about 150 nm. Oneshould also notice the presence of a higher amount of lens-shaped vesicles with different size, indicating that smaller φ andhigher surfactant concentration favor formation of lens-shapedvesicles.

5. Summary

The vesicle size together with the melting temperature is avery important characteristic that should be well controlled invesicle-mediated experiments. There is a relationship betweenthe vesicle size and the melting temperature: Tm increases withDODAB or DODAC vesicle size [7]. Vesicles with differentsize are usually prepared by different methods, such as sonica-tion, extrusion, injection or spontaneous [1–4]. Such methods,however, may influence not only the size but also the vesicleshape. It is well known that DODAB and DODAC assemblespontaneously above Tm as large unilamellar vesicles, and thesize of the spontaneous vesicles is usually reduced by soni-cation or extrusion. The latter, however, is more suitable toform smaller vesicles because it causes less vesicle fragmenta-tion (damage), and thus form more homogeneous vesicles withsmall amount of lens-shaped vesicles.

The vesicle size can be changed as well by varying the sol-vent conditions like temperature or ionic strength. This workdeals with two ways to modify the size of DODAB and DO-DAC vesicles: extrusion and ionic strength. It reports on the

proportionality between the vesicle size and the pore diameter(φ) of the extrusion membrane, and the dependence of the vesi-cle size on the counterion type, indicating that DODAB formslarger vesicles than DODAC. It also shows that lens-shapedvesicles are also formed during the extrusion process, with theamount of lens-shaped vesicles being higher the smaller is φ,probably because the smaller φ gives higher amounts of bi-layer fragments that form lens-shaped vesicles. The differencein DODAB and DODAC vesicle size [13] and melting temper-ature [7] can be explained by the specificity of the counterionbinding to the vesicle interfaces. Sonicated DODAX vesiclesare as well smaller than the spontaneous [9], but their Tm islarger [7], indicating that sonication not only reduces the sizebut also changes the vesicle shape (spherical- to lens-shaped).Extrusion, on the other hand, reduces the size and Tm [7] andleaves the vesicle shape unchanged, according to the cryo-TEMimages shown in this communication.

The specificity of the counterion binding to DODAB andDODAC vesicles is clear from previous reports that point tothe lower curvature [9,13] and Tm [7,20] of DODAB vesicles.Since DODAB and DODAC vesicles differ only by the coun-terion (Br− or Cl−) the differing size and Tm might be dueto counterion specificity to binding the vesicles [7,20]. Thedata reported here for extruded vesicles are in good agreementwith the reported ones for spontaneous, sonicated and injecteddispersions [7,9,13,20], indicating that Br− binds to DODABvesicles yielding larger vesicles (than DODAC) but smallerTm, that is, more densely packed bilayer, thus allowing Br−to bind more tightly to the vesicle interfaces than Cl−, as re-ported [7,20].

Particularly interesting for vesicle application is that, irre-spective of the presence of small amount of added salt (up to5 mM), the polydispersity index is rather low (<0.25). NaBrand NaCl increase and decrease, respectively, the size of DO-DAC and DODAB vesicles, indicating the general trend ofvesicles to be larger for DODAB than DODAC as well as thespecificity of the counterion binding to the vesicle interfaces.Furthermore, the mean size of the extruded DODAX matchesthe optimal φ∗ ≈ 200 nm; below (above) φ∗, the main vesiclediameter is larger (smaller) than φ.

The data also reveal that besides the ordinary smoothedvesicles, the extrusion process forms lens-shaped vesicles withvarying size as already observed for tip-sonicated DODAB [10]and more recently for extruded DODAC vesicles [15]. How-ever, lens-shaped vesicles were observed in smaller amount inextrusion than in sonicated dispersions [10]. The amount ofDODAX lens-shaped vesicles is higher in the extruded vesi-cles through the smaller φ (100 nm) and at higher surfactantconcentrations (Fig. 7), probably because the smaller φ and thehigher concentration produce more bilayer fragments.

The fine-tuning of vesicle size and shape in general claimsfor narrow size distribution and long term stability. Extrusionrather than sonication seems to be more suitable to form smallerand smooth vesicles relative to the spontaneous method as re-quired in many applications.

588 A. Lopes et al. / Journal of Colloid and Interface Science 322 (2008) 582–588

Acknowledgments

E.F. thanks CNPq for research grant (Grant 304543/2006-3).The authors are indebted to Dr. J.G. Martinho for many help-ful discussions regarding the interpretation of LS data andG. Karlsson for performing the cryo-TEM experiments.

Supplementary material

The online version of this article contains additional supple-mentary material.

Please visit DOI: 10.1016/j.jcis.2008.03.015.

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