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Extruded vesicles of dioctadecyldimethylammonium bromide and chloride investigated by light scattering and cryogenic transmission electron microscopy
Journal of Colloid and Interface Science 322 (2008) 582–588 www.elsevier.com/locate/jcis
Extruded vesicles of dioctadecyldimethylammonium bromide and chloride 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, Portugal b Department of Physical Chemistry, Uppsala University, Uppsala, Sweden c 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 investigate extruded cationic vesicles of dioctadecyldimethylammonium chloride and bromide (DODAX, X being Cl− or Br− ). In salt-free dispersions the mean hydrodynamic diameter, Dh , and the weight average molecular weight, Mw , are larger for DODAB than for DODAC vesicles, and both Dh and Mw increase with the diameter (φ) of the extrusion filter. NaCl (NaBr) decreases (increases) the DODAB (DODAC) vesicle size, reflecting the general trend of DODAB to assemble as larger vesicles than DODAC. The polydispersity index is lower than 0.25, indicating the dispersions are rather polydisperse. Cryo-TEM micrographs show that the smaller vesicles are spherical while the larger ones are oblong or faceted, and the vesicle samples are fairly polydisperse in size and morphology. They also indicate that the vesicle size increases with φ and DODAB assembles as larger vesicles than DODAC. Lens-shaped vesicles were observed in the extruded preparations. Both light scattering and cryo-TEM indicate that 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 potential application as vehicle for drug delivery [1–3]. To mimic biological membrane or function as drug delivery vehicle, vesicles should exhibit narrow size distribution and be stable. Several vesicles preparation methods have thus been developed to attain these requirements. It includes sonication, organic solvent evaporation, extrusion, detergent-removal or simple mixing (spontaneous), among others [1–15]. In this communication we focus on the extrusion method used to prepare DODAX vesicles (X being usually the Br− or Cl− ions), which is currently one of the best to prepare unil* Corresponding author. Fax: +55 17 3221 22 47.
E-mail address: [email protected] (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, and the data are compared to those prepared by other methods (sonication, injection and spontaneous) [4,5,9,10,12,14] as well as extrusion [7,15]. Relative to extruded phospholipid liposomes, extruded DODAX vesicles have been poorly investigated probably because of technical limitation related to the relatively high melting temperature (Tm ), of these surfactants, which is usually higher than 45 ◦ C [7]. Extrusion is, however, very suitable to form smaller DODAX vesicles [7,15], although the vesicle characteristics may depend on the applied extrusion pressure [16], surfactant concentration [7] and the presence of co-solutes, according to this communication. Sonication has as well been used to prepare smaller DODAX vesicles although bilayer fragments and lens-shaped vesicles are mainly formed [9,10] while extruded and spontaneous vesicles form mainly smoothed vesicles, as shown in this communication and in Ref. [15].
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In spite of this, there is a lack of investigation on the relationship between the vesicle curvature and the pore diameter (φ) of the extrusion membrane. It is shown in this communication that the mean size of DODAX vesicles (obtained by static and dynamic light scattering and cryo-TEM) can be larger or smaller than φ, but it fits well to an optimal pore diameter φ ∗ ≈ 200 nm. The effect of NaCl and NaBr on the size of DODAX vesicles was 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 DODAC was obtained by counterion exchange from DODAB (Eastman Kodak, purity 99.5%) [8]. Mili-Q-Plus quality water 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 prepared by mixing DODAX and water to a desired final concentration (usually 1 mM) and then warming to 55 ◦ C, that is, above the melting temperature, Tm , of the surfactant to obtain an homogeneous dispersion [6,7,11–13]. Extruded vesicles were obtained using an extrusion system from Avanti Polar Lipids, Alabaster, AL. Extrusions were performed manually by forcing several times (typically 15 times) the DODAX spontaneous dispersion through two stacked 13 mm polycarbonate filters with nominal pore diameters, φ = 50, 100, 200 and 400 nm, keeping the dispersion temperature at 55 ◦ C, as described in [7]. After extrusion the vesicle dispersions were then cooled to room temperature and stored. DODAX vesicle dispersions at the desired surfactant concentration were obtained by direct dilution of the stock solutions. The measurements were performed 24 h after vesicle preparation.
Fig. 1. Zimm plot for extruded DODAB vesicle dispersions obtained with a filter pore diameter φ = 200 nm. Measurements made at 25 ◦ C.
for all DODAX dispersions under study. For each pair sample/scattering vector the hydrodynamic diameter (Dh ) distribution (histogram) was obtained with a CONTIN routine [17,18], where Dh is the hydrodynamic diameter extrapolated to zero surfactant concentration, c, and zero scattering angle, θ . Comparison of the radius of gyration, Rg , obtained from SLS, with the hydrodynamic radius, Rh = Dh /2, from DLS, reveals the geometry of the macromolecular aggregate based on the well known Rg /Rh ratio [18]. SLS allows the evaluation of the weight average molecular weight, Mw , the radius of gyration, Rg , and the second virial coefficient, A2 , of the aggregates present in dilute solutions by the usual Zimm method based on the Rayleigh–Gans–Debye theory [18]. Accordingly, the variation of the relative light scattering intensity, Is /Io , where Io is the intensity of the incident radiation, with the observation angle, θ , or scattering vector, q = 4π sin(θ/2)/λ, is related with Mw , Rg , and A2 , for a given sample concentration, c, through the equation [18]
2.3. Light scattering measurements
Kc 1 16π 2 n2o 2 2 θ = R sin + 2A2 c, 1+ g Rθ Mw 2 3λ2
Light scattering (LS) measurements were made with an apparatus from Brookhaven Instruments, Inc., Model 2030AT, equipped with a He–Ne laser (λ = 632.8 nm) and a 136 channel autocorrelator. The vesicle dispersions for LS measurements were centrifuged at approximately 1300g for 45 min to remove dust particles, and the solvent solutions or water used to dilute or to add salt into the vesicle dispersions were filtered through a Millipore membrane filter of 0.45 µm nominal pore diameter. Dynamic (DLS) and static (SLS) light scattering measurements were made at the scattering angles θ = 50, 70, 90, 110, 130◦ , and the Zimm plots made with a minimum of five logarithmically spaced concentrations. The optical constant of the apparatus was obtained with benzene prior to the measurements and the dn/dc values determined with a differential refractometer (Model G.M. Wood RF600) fall in the range 0.12–0.16 ml/g
where Rθ = d 2 (Is /Io ) sin2 θ is the Rayleigh factor, K = [4πn2o (dn/dc)2 / sin2 θ]/NA λ4 is an optical constant, no and n are, respectively, the refractive index of the solvent and solution, NA is the Avogadro constant, dn/dc is the differential rate of refractive index measured separately. The graphic of Kc/Rθ as a function of sin2 (θ/2) + c is known as Zimm plot [18] that allows one to obtain the vesicle Mw , Rg , and A2 , from the intercept and slope of the fitted curves (dashed lines in Fig. 1) to the c = 0 and θ = 0 extrapolated experimental points [18]. In this way vesicle aggregates can be characterized as a function of the surfactant concentration, temperature, ionic strength, method of vesicle preparation, etc. The scattering measurements were made at the sample temperature of 25 ± 1 ◦ C.
(1)
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Table 1 Hydrodynamic, Rh , and gyration, Rg , radii, weight average molecular weight, Mw , second virial coefficient, A2 , shape factor, Rg /Rh , and the average polydispersity index, P , obtained from light scattering for DODAB and DODAC extruded vesicles as functions of the filter pore diameter, φ, of the extrusion membrane (for comparison, Rh = 337 and 247 nm for DODAB and DODAC spontaneous vesicles, respectively [13]) Surfactant
φ (nm)
Rh (nm)
Rg (nm)
Mw (×107 )
A2 (cm3 mol/g2 )
Rg /Rh
P
DODAB
50 100 200 400
68 75 115 157
53 59 500 992
1.93 3.71 53.6 198
−9.1 1.03 0.42 0.46
0.80 0.85 4.32 6.37
0.15 0.21 0.18 0.13
DODAC
50 100 200 400
48 66 105 148
39 60 430 950
0.84 1.21 45.2 170
−12.0 0.00 0.45 0.44
0.83 0.91 4.10 6.47
0.12 0.21 0.15 0.17
2.4. Cryo-TEM measurements
Fig. 2. Histograms of the distribution of hydrodynamic diameter of 0.1 mM DODAB 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◦ .
Thin (10–500 nm) sample films were prepared under wellcontrolled temperature (25 ◦ C) and humidity conditions in an environmental chamber and vitrified by quickly freezing to 108 K in liquid ethane before being transferred to a Zeiss EM 902 transmission electron microscope for examination. To prevent sample perturbation the specimens were kept below 108 K during the transfer and viewing procedures. The observations were made in the zero loss bright-field mode and at an electron accelerating voltage of 80 kV. All samples were vitrified from the temperature of 25 ± 1 ◦ C. Further details on the cryo-TEM experimental procedure are found elsewhere [13,19].
Fig. 2 and Fig. SM1 show selected histograms of the distribution of hydrodynamic diameter, Dh , for θ = 90◦ , as function of the filter pore diameter φ = 50–400 nm and Br− or Cl− counterion. The representation of the mean hydrodynamic diameter as a function of the surfactant concentration is shown in Fig. 3. The effect of φ on Dh of DODAX vesicles in absence and presence of 5 mM NaCl or NaBr is shown in Fig. 4. Figs. 5–7 and Figs. SM3, SM4 show cryo-TEM micrographs for extruded vesicles of DODAX 1.0 and 5.0 mM in water, prepared with pore diameters φ = 100 or 400 nm. Cryo-TEM micrographs for the spontaneous [13] and sonicated [10] DODAX vesicles have already been reported.
3. Results
4. Discussion
Static and dynamic light scattering data, both in absence and presence of NaBr or NaCl (Figs. 1–4), as well as cryoTEM images of extruded DODAX vesicles (Figs. 5–7) are presented. Additional scattering (Figs. SM1 and SM2) and cryo-TEM (Figs. SM3 and SM4) data are shown as supplementary material. Dynamic light scattering data for DODAX spontaneous [13], sonicated [9] and injected [20] vesicles have been reported previously, as well as cryo-TEM images for DODAX spontaneous vesicles in the absence [13] and presence of NaBr [12]. This work supplies complementary information on size, molecular weight and shape of extruded DODAX vesicles through varying the membrane pore diameter, φ. Table 1 summarizes the values of Rh , Rg , Mw , A2 , the shape factor, Rg /Rh , and the average polydispersity index, P , obtained from static and dynamic light scattering, as functions of φ, for DODAX vesicles. Rg , Mw , and A2 were obtained from the Zimm plots, as shown in Fig. 1 for extruded DODAB vesicles 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 correlation coefficients higher than 0.98. Rh was obtained from the histograms of the distribution of hydrodynamic diameter, shown in Fig. 2 and Fig. SM1.
It is well known in the literature that the vesicle size increases with the pore size of the extrusion membrane, but to our knowledge there is no clear correlation between DODAX vesicle size and the pore dimension of the extrusion membrane. This work attempts to correlate these parameters for DODAX vesicles based on light scattering and cryo-TEM data and investigate 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 ) and molecular weight Mw of DODAX vesicles in salt-free water increases with φ. The polydispersity index, P , is always lower than 0.25, meaning that the dispersions are fairly polydisperse, although we found no clear correlation between φ and the polydispersity and size of DODAX vesicles (Table 1 and Fig. 4). Saveyn et al. [15] reported smaller Dh ≈ 125 nm for extruded DODAC through φ = 200 nm. These authors, however, prepared the vesicles in 0.02 wt% CaCl2 , that makes difficult a direct comparison with our reported data. In the presence of 5 mM single salts (Fig. 4) Dh is roughly the same as in absence of salt (Dh ≈ 210 nm in Table 1). These authors also reported that the osmotic response (yielded by sucrose and CaCl2 ) of
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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 hydrodynamic diameter for θ = 0◦ for DODAB (c) and DODAC (d) vesicles as a function of the DODAX concentration. Measurements made at 25 ◦ C. Symbols account for φ = 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 that the vesicle size is an important parameter in many vesicle applications [7]. Like the spontaneous vesicles [13], Dh and Mw of extruded vesicles in salt-free dispersions are always larger for DODAB than DODAC, and increases with φ (Table 1). It is worth noticing that for φ < 200 nm, Dh of DODAX vesicles is larger than φ, while for φ = 200 nm Dh almost matches that value, and for φ > 200 nm Dh is smaller than φ. This suggests that there is an optimal membrane pore size, φ ∗ ≈ 200 nm for preparation of extruded DODAX vesicles whose size matches well the pore diameter of the extrusion membrane. This is suitable technical information on the method to form DODAX extruded vesicles with a reasonable control of the vesicle size. These results matches those for the spontaneous (nonextruded) vesicles (Rh = 337 and 247 nm for DODAB and DODAC spontaneous vesicles, respectively [13]), since the size of the extruded vesicles increases with φ to attain the size of the spontaneous vesicles when φ > 400 nm. Furthermore, irrespective of φ, the extruded DODAX vesicles in water do not precipitate for months, indicating stability comparable to the spontaneous DODAX vesicles [7,13]. One should stress that Rg and Rh increase with φ in different ways, such that the shape factor Rg /Rh increases from 0.8 to ca. 6.5 for DODAB or DODAC (Table 1), indicating that the
Fig. 4. Mean hydrodynamic diameter of DODAB (E) and DODAC (1) extruded vesicles as a function of the filter pore diameter φ without salt. The broken line which corresponds to the equality Dh = φ is just a guide for the eye.
vesicle growth is followed by morphological changes due to the flexibility of the vesicle bilayer. In presence of salt, NaCl decreases while NaBr increases Dh of DODAB and DODAC vesicles, respectively (Table 1). The general behavior of the counterion exchange in the Stern layers reflects the prevailing trend of DODAB to form larger vesicles than DODAC, as already reported [13,20]. Overall, NaCl con-
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Fig. 5. Cryo-TEM micrographs of 1.0 mM DODAB in extruded vesicles prepared with φ = 100 nm. Bar equals 100 nm. Arrow points to a lens-shaped vesicle.
tracts DODAB vesicles while NaBr expands DODAC vesicles due to the specificity of the binding of these counterions to the vesicle interfaces [7,20]. Up to 10 mM single inorganic salts DODAX vesicles extruded trough φ = 200 nm does not change significantly the polydispersity index relative to the salt-free dispersion. However, DODAC vesicles prepared with φ = 50 nm tend to precipitate in presence of [Br− ] > 5 mM, while DODAB vesicles prepared with φ = 200 nm or smaller, in the presence of more than 5 mM Br− or Cl− , undergo an increase of about 20% in the polydispersity index. It thus indicates a trend of high smaller vesicles to be less stable than the corresponding larger vesicles, which is stable for months [9,13,21]. It was also observed 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 Zimm plots exhibit the same profile relative to the salt-free vesicles shown in Fig. 1 (results not shown), and the shape factor does not change significantly. The data thus suggest that smaller (higher curvatured) vesicles exhibit less densely packed bilayer that allow the counterion to interact strongly with the vesicle interfaces thus favoring vesicle aggregation followed by precipitation owing to electrostatic screening. 4.2. Cryo-TEM results Overall, the mean size and polydispersity information obtained from the cryo-TEM micrographs shown in Figs. 5–7 and Figs. SM3 and SM4 is in qualitative agreement with the light scattering results, indicating that at low concentrations DODAB molecules in water assemble as larger unilamellar vesicles than DODAC, as already reported for the spontaneous [13] and injected [20] vesicles. The cryo-TEM micrographs also indicate that extrusion tends to form smaller DODAX vesicles than the spontaneous, and the vesicle size increases with φ. Up to 1.0 mM (and above Tm ≈ 45 ◦ C) DODAB selfassembles in water as large unilamellar spherical, oblong or faceted 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 prepared with φ = 100 nm. Bar equals 100 nm.
Fig. 7. Cryo-TEM micrographs of 5.0 mM DODAC in extruded vesicles prepared with φ = 100 nm. Bar equals 100 nm. Arrow points to a lens-shaped vesicle.
phology similar to the spontaneous vesicles, and forms some lens-shaped vesicles. DODAX lens-shaped vesicles have been observed before in tip-sonicated [10] and extruded [15] dispersions. The images suggest that the smaller is φ, the higher is the amount of lens-shaped vesicles. For φ = 100 nm DODAB extruded dispersions consist mainly of quasi-spherical vesicles with the larger ones having diameter of ca. 160 nm, that is, larger than φ (Fig. 5). Smaller vesicles with diameter comparable to φ can also be discerned in the micrograph, as well as small lens-shaped vesicles. The micrograph also reveals that most of the vesicles have mean size larger than φ, indicating that extrusion membranes with φ smaller than the optimal φ ∗ ≈ 200 nm yield vesicles with mean size larger than φ in agreement with the scattering results (Fig. 4 and Table 1). For φ = 400 nm, DODAB vesicles are also quasi-spherical and unilamellar with dimension in the range of ca. 150– 350 nm (Fig. SM3). Thus, extrusion through φ = 400 nm gives DODAB vesicles smaller than φ, again in agreement with the scattering results. Note that no lens-shaped vesicle is seen in Fig. SM3, probably because for larger φ, a small amount of bilayer 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
and geometry (Figs. 6, 7 and Fig. SM4) [7,13]. Accordingly, DODAC tends to form smaller vesicles than DODAB either in spontaneous [7,11,13] or extruded dispersions [15], in good agreement with the scattering data reported above. DODAC also forms lens-shaped vesicles in extruded dispersions (Fig. 7 and Fig. SM4), as reported in [15]. Extrusion through φ = 100 nm results in spherical unilamellar DODAC vesicles with mean diameter in the range of ca. 80–200 nm (Fig. 6), that is, larger than φ. Extrusion through φ = 400 nm, on the other hand, gives spherical unilamellar vesicles with mean diameters in the range of ca. 250–350 nm (Fig. SM4), that is smaller than φ. In this sample we can also discern some small lens-shaped vesicles that are probably formed by fusion of pieces of fragmented bilayers, which come about after the extrusion or sonication, as reported [10,15]. One should note that the amount of DODAX lens-shaped vesicles is higher the higher is the surfactant concentration (Fig. 7). For this reason sonication produces more lens-shaped vesicles than extrusion, because the former gives more bilayer fragments. This is the case when we compare the present cryo-TEM pictures to those for sonicated DODAB [10] or extruded DODAC [15] vesicles. Extruded unilamellar DODAX vesicles can also be formed at higher surfactant concentrations [13], as shown in the cryoTEM micrograph of an extruded dispersion of 5.0 mM DODAC in water prepared through φ = 100 nm (Fig. 7). Accordingly, up to 5 mM the surfactant concentration plays no key role on the size and structure of extruded DODAC vesicles. The mean size of these vesicles is clearly larger than φ, but smaller than the mean size of the corresponding DODAB extruded vesicles. The larger vesicles have mean diameters of about 150 nm. One should also notice the presence of a higher amount of lensshaped vesicles with different size, indicating that smaller φ and higher surfactant concentration favor formation of lens-shaped vesicles. 5. Summary The vesicle size together with the melting temperature is a very important characteristic that should be well controlled in vesicle-mediated experiments. There is a relationship between the vesicle size and the melting temperature: Tm increases with DODAB or DODAC vesicle size [7]. Vesicles with different size are usually prepared by different methods, such as sonication, extrusion, injection or spontaneous [1–4]. Such methods, however, may influence not only the size but also the vesicle shape. It is well known that DODAB and DODAC assemble spontaneously above Tm as large unilamellar vesicles, and the size of the spontaneous vesicles is usually reduced by sonication or extrusion. The latter, however, is more suitable to form smaller vesicles because it causes less vesicle fragmentation (damage), and thus form more homogeneous vesicles with small amount of lens-shaped vesicles. The vesicle size can be changed as well by varying the solvent conditions like temperature or ionic strength. This work deals with two ways to modify the size of DODAB and DODAC vesicles: extrusion and ionic strength. It reports on the
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proportionality between the vesicle size and the pore diameter (φ) of the extrusion membrane, and the dependence of the vesicle size on the counterion type, indicating that DODAB forms larger vesicles than DODAC. It also shows that lens-shaped vesicles are also formed during the extrusion process, with the amount of lens-shaped vesicles being higher the smaller is φ, probably because the smaller φ gives higher amounts of bilayer fragments that form lens-shaped vesicles. The difference in DODAB and DODAC vesicle size [13] and melting temperature [7] can be explained by the specificity of the counterion binding to the vesicle interfaces. Sonicated DODAX vesicles are as well smaller than the spontaneous [9], but their Tm is larger [7], indicating that sonication not only reduces the size but also changes the vesicle shape (spherical- to lens-shaped). Extrusion, on the other hand, reduces the size and Tm [7] and leaves the vesicle shape unchanged, according to the cryo-TEM images shown in this communication. The specificity of the counterion binding to DODAB and DODAC vesicles is clear from previous reports that point to the lower curvature [9,13] and Tm [7,20] of DODAB vesicles. Since DODAB and DODAC vesicles differ only by the counterion (Br− or Cl− ) the differing size and Tm might be due to counterion specificity to binding the vesicles [7,20]. The data reported here for extruded vesicles are in good agreement with the reported ones for spontaneous, sonicated and injected dispersions [7,9,13,20], indicating that Br− binds to DODAB vesicles yielding larger vesicles (than DODAC) but smaller Tm , that is, more densely packed bilayer, thus allowing Br− to bind more tightly to the vesicle interfaces than Cl− , as reported [7,20]. Particularly interesting for vesicle application is that, irrespective of the presence of small amount of added salt (up to 5 mM), the polydispersity index is rather low (<0.25). NaBr and NaCl increase and decrease, respectively, the size of DODAC and DODAB vesicles, indicating the general trend of vesicles to be larger for DODAB than DODAC as well as the specificity of the counterion binding to the vesicle interfaces. Furthermore, the mean size of the extruded DODAX matches the optimal φ ∗ ≈ 200 nm; below (above) φ ∗ , the main vesicle diameter is larger (smaller) than φ. The data also reveal that besides the ordinary smoothed vesicles, the extrusion process forms lens-shaped vesicles with varying size as already observed for tip-sonicated DODAB [10] and more recently for extruded DODAC vesicles [15]. However, lens-shaped vesicles were observed in smaller amount in extrusion than in sonicated dispersions [10]. The amount of DODAX lens-shaped vesicles is higher in the extruded vesicles through the smaller φ (100 nm) and at higher surfactant concentrations (Fig. 7), probably because the smaller φ and the higher concentration produce more bilayer fragments. The fine-tuning of vesicle size and shape in general claims for narrow size distribution and long term stability. Extrusion rather than sonication seems to be more suitable to form smaller and smooth vesicles relative to the spontaneous method as required in many applications.
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Acknowledgments E.F. thanks CNPq for research grant (Grant 304543/2006-3). The authors are indebted to Dr. J.G. Martinho for many helpful discussions regarding the interpretation of LS data and G. Karlsson for performing the cryo-TEM experiments. Supplementary material The online version of this article contains additional supplementary material. Please visit DOI: 10.1016/j.jcis.2008.03.015. References [1] D.D. Lasic, Liposomes. From Physics to Applications, Elsevier, Amsterdam, 1993. [2] G. Cevc (Ed.), Phospholipid Handbook, Marcel Dekker, New York, 1993. [3] J.M. Fendler, Membrane Mimetic-Chemistry, Wiley–Interscience, New York, 1982. [4] J.B.F.N. Engberts, D. Hoeskstra, Biochim. Biophys. Acta 1241 (1995) 323. [5] A.M. Carmona-Ribeiro, Chem. Soc. Rev. 21 (1992) 207.
[6] E. Feitosa, J. Jansson, B. Lindman, Chem. Phys. Lipids 142 (2006) 128. [7] E. Feitosa, P.C.A. Barreleiro, G. Olofsson, Chem. Phys. Lipids 105 (2000) 201. [8] I.M. Cuccovia, A. Sesso, E.B. Abuin, P.F. Okino, P.G. Tavares, J.F.S. Campos, F.H. Florenzano, H. Chaimovich, J. Mol. Liquids 72 (1997) 323. [9] E. Feitosa, W. Brown, Langmuir 13 (1997) 4810. [10] M. Andersson, L. Hammarström, K. Edwards, J. Phys. Chem. 99 (1995) 14531. [11] C.R. Benatti, E. Feitosa, R.M. Fernandez, M.T. Lamy-Freund, Chem. Phys. Lipids 111 (2001) 93. [12] E. Feitosa, P.C.A. Barreleiro, Prog. Colloid Polym. Sci. 128 (2004) 163. [13] E. Feitosa, G. Karlsson, K. Edwards, Chem. Phys. Lipids 140 (2006) 66. [14] R.O. Brito, E.F. Marques, Chem. Phys. Lipids 137 (2005) 18. [15] P. Saveyn, J. Cocquyt, P. Bomans, P. Frederik, M. De Cuyper, P. Van der Meeren, Langmuir 23 (2007) 4775. [16] P.J. Patty, B.J. Frisken, Biophys. J. 85 (2003) 996. [17] M. Nichifor, A. Lopes, A. Carpov, E. Melo, Macromolecules 32 (1999) 7078. [18] W. Brown (Ed.), Light Scattering: Principles and Developments, Clarendon Press, Oxford, 1996. [19] J.R. Bellare, H.T. Davis, L.E. Scriven, Y. Talmon, J. Electron Microsc. Tech. 10 (1988) 87. [20] D.B. Nascimento, R. Rapuano, M.M. Lessa, A.M. Carmona-Ribieiro, Langmuir 14 (1998) 7387. [21] I.M. Cuccovia, E. Feitosa, H. Chaimovich, L. Sepulveda, W.F. Reed, J. Phys. Chem. 94 (1990) 3722.
Extruded vesicles of dioctadecyldimethylammonium bromide and chloride 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, Portugal b Department of Physical Chemistry, Uppsala University, Uppsala, Sweden c 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 investigate extruded cationic vesicles of dioctadecyldimethylammonium chloride and bromide (DODAX, X being Cl− or Br− ). In salt-free dispersions the mean hydrodynamic diameter, Dh , and the weight average molecular weight, Mw , are larger for DODAB than for DODAC vesicles, and both Dh and Mw increase with the diameter (φ) of the extrusion filter. NaCl (NaBr) decreases (increases) the DODAB (DODAC) vesicle size, reflecting the general trend of DODAB to assemble as larger vesicles than DODAC. The polydispersity index is lower than 0.25, indicating the dispersions are rather polydisperse. Cryo-TEM micrographs show that the smaller vesicles are spherical while the larger ones are oblong or faceted, and the vesicle samples are fairly polydisperse in size and morphology. They also indicate that the vesicle size increases with φ and DODAB assembles as larger vesicles than DODAC. Lens-shaped vesicles were observed in the extruded preparations. Both light scattering and cryo-TEM indicate that 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 potential application as vehicle for drug delivery [1–3]. To mimic biological membrane or function as drug delivery vehicle, vesicles should exhibit narrow size distribution and be stable. Several vesicles preparation methods have thus been developed to attain these requirements. It includes sonication, organic solvent evaporation, extrusion, detergent-removal or simple mixing (spontaneous), among others [1–15]. In this communication we focus on the extrusion method used to prepare DODAX vesicles (X being usually the Br− or Cl− ions), which is currently one of the best to prepare unil* Corresponding author. Fax: +55 17 3221 22 47.
E-mail address: [email protected] (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, and the data are compared to those prepared by other methods (sonication, injection and spontaneous) [4,5,9,10,12,14] as well as extrusion [7,15]. Relative to extruded phospholipid liposomes, extruded DODAX vesicles have been poorly investigated probably because of technical limitation related to the relatively high melting temperature (Tm ), of these surfactants, which is usually higher than 45 ◦ C [7]. Extrusion is, however, very suitable to form smaller DODAX vesicles [7,15], although the vesicle characteristics may depend on the applied extrusion pressure [16], surfactant concentration [7] and the presence of co-solutes, according to this communication. Sonication has as well been used to prepare smaller DODAX vesicles although bilayer fragments and lens-shaped vesicles are mainly formed [9,10] while extruded and spontaneous vesicles form mainly smoothed vesicles, as shown in this communication and in Ref. [15].
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In spite of this, there is a lack of investigation on the relationship between the vesicle curvature and the pore diameter (φ) of the extrusion membrane. It is shown in this communication that the mean size of DODAX vesicles (obtained by static and dynamic light scattering and cryo-TEM) can be larger or smaller than φ, but it fits well to an optimal pore diameter φ ∗ ≈ 200 nm. The effect of NaCl and NaBr on the size of DODAX vesicles was 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 DODAC was obtained by counterion exchange from DODAB (Eastman Kodak, purity 99.5%) [8]. Mili-Q-Plus quality water 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 prepared by mixing DODAX and water to a desired final concentration (usually 1 mM) and then warming to 55 ◦ C, that is, above the melting temperature, Tm , of the surfactant to obtain an homogeneous dispersion [6,7,11–13]. Extruded vesicles were obtained using an extrusion system from Avanti Polar Lipids, Alabaster, AL. Extrusions were performed manually by forcing several times (typically 15 times) the DODAX spontaneous dispersion through two stacked 13 mm polycarbonate filters with nominal pore diameters, φ = 50, 100, 200 and 400 nm, keeping the dispersion temperature at 55 ◦ C, as described in [7]. After extrusion the vesicle dispersions were then cooled to room temperature and stored. DODAX vesicle dispersions at the desired surfactant concentration were obtained by direct dilution of the stock solutions. The measurements were performed 24 h after vesicle preparation.
Fig. 1. Zimm plot for extruded DODAB vesicle dispersions obtained with a filter pore diameter φ = 200 nm. Measurements made at 25 ◦ C.
for all DODAX dispersions under study. For each pair sample/scattering vector the hydrodynamic diameter (Dh ) distribution (histogram) was obtained with a CONTIN routine [17,18], where Dh is the hydrodynamic diameter extrapolated to zero surfactant concentration, c, and zero scattering angle, θ . Comparison of the radius of gyration, Rg , obtained from SLS, with the hydrodynamic radius, Rh = Dh /2, from DLS, reveals the geometry of the macromolecular aggregate based on the well known Rg /Rh ratio [18]. SLS allows the evaluation of the weight average molecular weight, Mw , the radius of gyration, Rg , and the second virial coefficient, A2 , of the aggregates present in dilute solutions by the usual Zimm method based on the Rayleigh–Gans–Debye theory [18]. Accordingly, the variation of the relative light scattering intensity, Is /Io , where Io is the intensity of the incident radiation, with the observation angle, θ , or scattering vector, q = 4π sin(θ/2)/λ, is related with Mw , Rg , and A2 , for a given sample concentration, c, through the equation [18]
2.3. Light scattering measurements
Kc 1 16π 2 n2o 2 2 θ = R sin + 2A2 c, 1+ g Rθ Mw 2 3λ2
Light scattering (LS) measurements were made with an apparatus from Brookhaven Instruments, Inc., Model 2030AT, equipped with a He–Ne laser (λ = 632.8 nm) and a 136 channel autocorrelator. The vesicle dispersions for LS measurements were centrifuged at approximately 1300g for 45 min to remove dust particles, and the solvent solutions or water used to dilute or to add salt into the vesicle dispersions were filtered through a Millipore membrane filter of 0.45 µm nominal pore diameter. Dynamic (DLS) and static (SLS) light scattering measurements were made at the scattering angles θ = 50, 70, 90, 110, 130◦ , and the Zimm plots made with a minimum of five logarithmically spaced concentrations. The optical constant of the apparatus was obtained with benzene prior to the measurements and the dn/dc values determined with a differential refractometer (Model G.M. Wood RF600) fall in the range 0.12–0.16 ml/g
where Rθ = d 2 (Is /Io ) sin2 θ is the Rayleigh factor, K = [4πn2o (dn/dc)2 / sin2 θ]/NA λ4 is an optical constant, no and n are, respectively, the refractive index of the solvent and solution, NA is the Avogadro constant, dn/dc is the differential rate of refractive index measured separately. The graphic of Kc/Rθ as a function of sin2 (θ/2) + c is known as Zimm plot [18] that allows one to obtain the vesicle Mw , Rg , and A2 , from the intercept and slope of the fitted curves (dashed lines in Fig. 1) to the c = 0 and θ = 0 extrapolated experimental points [18]. In this way vesicle aggregates can be characterized as a function of the surfactant concentration, temperature, ionic strength, method of vesicle preparation, etc. The scattering measurements were made at the sample temperature of 25 ± 1 ◦ C.
(1)
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Table 1 Hydrodynamic, Rh , and gyration, Rg , radii, weight average molecular weight, Mw , second virial coefficient, A2 , shape factor, Rg /Rh , and the average polydispersity index, P , obtained from light scattering for DODAB and DODAC extruded vesicles as functions of the filter pore diameter, φ, of the extrusion membrane (for comparison, Rh = 337 and 247 nm for DODAB and DODAC spontaneous vesicles, respectively [13]) Surfactant
φ (nm)
Rh (nm)
Rg (nm)
Mw (×107 )
A2 (cm3 mol/g2 )
Rg /Rh
P
DODAB
50 100 200 400
68 75 115 157
53 59 500 992
1.93 3.71 53.6 198
−9.1 1.03 0.42 0.46
0.80 0.85 4.32 6.37
0.15 0.21 0.18 0.13
DODAC
50 100 200 400
48 66 105 148
39 60 430 950
0.84 1.21 45.2 170
−12.0 0.00 0.45 0.44
0.83 0.91 4.10 6.47
0.12 0.21 0.15 0.17
2.4. Cryo-TEM measurements
Fig. 2. Histograms of the distribution of hydrodynamic diameter of 0.1 mM DODAB 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◦ .
Thin (10–500 nm) sample films were prepared under wellcontrolled temperature (25 ◦ C) and humidity conditions in an environmental chamber and vitrified by quickly freezing to 108 K in liquid ethane before being transferred to a Zeiss EM 902 transmission electron microscope for examination. To prevent sample perturbation the specimens were kept below 108 K during the transfer and viewing procedures. The observations were made in the zero loss bright-field mode and at an electron accelerating voltage of 80 kV. All samples were vitrified from the temperature of 25 ± 1 ◦ C. Further details on the cryo-TEM experimental procedure are found elsewhere [13,19].
Fig. 2 and Fig. SM1 show selected histograms of the distribution of hydrodynamic diameter, Dh , for θ = 90◦ , as function of the filter pore diameter φ = 50–400 nm and Br− or Cl− counterion. The representation of the mean hydrodynamic diameter as a function of the surfactant concentration is shown in Fig. 3. The effect of φ on Dh of DODAX vesicles in absence and presence of 5 mM NaCl or NaBr is shown in Fig. 4. Figs. 5–7 and Figs. SM3, SM4 show cryo-TEM micrographs for extruded vesicles of DODAX 1.0 and 5.0 mM in water, prepared with pore diameters φ = 100 or 400 nm. Cryo-TEM micrographs for the spontaneous [13] and sonicated [10] DODAX vesicles have already been reported.
3. Results
4. Discussion
Static and dynamic light scattering data, both in absence and presence of NaBr or NaCl (Figs. 1–4), as well as cryoTEM images of extruded DODAX vesicles (Figs. 5–7) are presented. Additional scattering (Figs. SM1 and SM2) and cryo-TEM (Figs. SM3 and SM4) data are shown as supplementary material. Dynamic light scattering data for DODAX spontaneous [13], sonicated [9] and injected [20] vesicles have been reported previously, as well as cryo-TEM images for DODAX spontaneous vesicles in the absence [13] and presence of NaBr [12]. This work supplies complementary information on size, molecular weight and shape of extruded DODAX vesicles through varying the membrane pore diameter, φ. Table 1 summarizes the values of Rh , Rg , Mw , A2 , the shape factor, Rg /Rh , and the average polydispersity index, P , obtained from static and dynamic light scattering, as functions of φ, for DODAX vesicles. Rg , Mw , and A2 were obtained from the Zimm plots, as shown in Fig. 1 for extruded DODAB vesicles 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 correlation coefficients higher than 0.98. Rh was obtained from the histograms of the distribution of hydrodynamic diameter, shown in Fig. 2 and Fig. SM1.
It is well known in the literature that the vesicle size increases with the pore size of the extrusion membrane, but to our knowledge there is no clear correlation between DODAX vesicle size and the pore dimension of the extrusion membrane. This work attempts to correlate these parameters for DODAX vesicles based on light scattering and cryo-TEM data and investigate 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 ) and molecular weight Mw of DODAX vesicles in salt-free water increases with φ. The polydispersity index, P , is always lower than 0.25, meaning that the dispersions are fairly polydisperse, although we found no clear correlation between φ and the polydispersity and size of DODAX vesicles (Table 1 and Fig. 4). Saveyn et al. [15] reported smaller Dh ≈ 125 nm for extruded DODAC through φ = 200 nm. These authors, however, prepared the vesicles in 0.02 wt% CaCl2 , that makes difficult a direct comparison with our reported data. In the presence of 5 mM single salts (Fig. 4) Dh is roughly the same as in absence of salt (Dh ≈ 210 nm in Table 1). These authors also reported that the osmotic response (yielded by sucrose and CaCl2 ) of
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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 hydrodynamic diameter for θ = 0◦ for DODAB (c) and DODAC (d) vesicles as a function of the DODAX concentration. Measurements made at 25 ◦ C. Symbols account for φ = 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 that the vesicle size is an important parameter in many vesicle applications [7]. Like the spontaneous vesicles [13], Dh and Mw of extruded vesicles in salt-free dispersions are always larger for DODAB than DODAC, and increases with φ (Table 1). It is worth noticing that for φ < 200 nm, Dh of DODAX vesicles is larger than φ, while for φ = 200 nm Dh almost matches that value, and for φ > 200 nm Dh is smaller than φ. This suggests that there is an optimal membrane pore size, φ ∗ ≈ 200 nm for preparation of extruded DODAX vesicles whose size matches well the pore diameter of the extrusion membrane. This is suitable technical information on the method to form DODAX extruded vesicles with a reasonable control of the vesicle size. These results matches those for the spontaneous (nonextruded) vesicles (Rh = 337 and 247 nm for DODAB and DODAC spontaneous vesicles, respectively [13]), since the size of the extruded vesicles increases with φ to attain the size of the spontaneous vesicles when φ > 400 nm. Furthermore, irrespective of φ, the extruded DODAX vesicles in water do not precipitate for months, indicating stability comparable to the spontaneous DODAX vesicles [7,13]. One should stress that Rg and Rh increase with φ in different ways, such that the shape factor Rg /Rh increases from 0.8 to ca. 6.5 for DODAB or DODAC (Table 1), indicating that the
Fig. 4. Mean hydrodynamic diameter of DODAB (E) and DODAC (1) extruded vesicles as a function of the filter pore diameter φ without salt. The broken line which corresponds to the equality Dh = φ is just a guide for the eye.
vesicle growth is followed by morphological changes due to the flexibility of the vesicle bilayer. In presence of salt, NaCl decreases while NaBr increases Dh of DODAB and DODAC vesicles, respectively (Table 1). The general behavior of the counterion exchange in the Stern layers reflects the prevailing trend of DODAB to form larger vesicles than DODAC, as already reported [13,20]. Overall, NaCl con-
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Fig. 5. Cryo-TEM micrographs of 1.0 mM DODAB in extruded vesicles prepared with φ = 100 nm. Bar equals 100 nm. Arrow points to a lens-shaped vesicle.
tracts DODAB vesicles while NaBr expands DODAC vesicles due to the specificity of the binding of these counterions to the vesicle interfaces [7,20]. Up to 10 mM single inorganic salts DODAX vesicles extruded trough φ = 200 nm does not change significantly the polydispersity index relative to the salt-free dispersion. However, DODAC vesicles prepared with φ = 50 nm tend to precipitate in presence of [Br− ] > 5 mM, while DODAB vesicles prepared with φ = 200 nm or smaller, in the presence of more than 5 mM Br− or Cl− , undergo an increase of about 20% in the polydispersity index. It thus indicates a trend of high smaller vesicles to be less stable than the corresponding larger vesicles, which is stable for months [9,13,21]. It was also observed 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 Zimm plots exhibit the same profile relative to the salt-free vesicles shown in Fig. 1 (results not shown), and the shape factor does not change significantly. The data thus suggest that smaller (higher curvatured) vesicles exhibit less densely packed bilayer that allow the counterion to interact strongly with the vesicle interfaces thus favoring vesicle aggregation followed by precipitation owing to electrostatic screening. 4.2. Cryo-TEM results Overall, the mean size and polydispersity information obtained from the cryo-TEM micrographs shown in Figs. 5–7 and Figs. SM3 and SM4 is in qualitative agreement with the light scattering results, indicating that at low concentrations DODAB molecules in water assemble as larger unilamellar vesicles than DODAC, as already reported for the spontaneous [13] and injected [20] vesicles. The cryo-TEM micrographs also indicate that extrusion tends to form smaller DODAX vesicles than the spontaneous, and the vesicle size increases with φ. Up to 1.0 mM (and above Tm ≈ 45 ◦ C) DODAB selfassembles in water as large unilamellar spherical, oblong or faceted 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 prepared with φ = 100 nm. Bar equals 100 nm.
Fig. 7. Cryo-TEM micrographs of 5.0 mM DODAC in extruded vesicles prepared with φ = 100 nm. Bar equals 100 nm. Arrow points to a lens-shaped vesicle.
phology similar to the spontaneous vesicles, and forms some lens-shaped vesicles. DODAX lens-shaped vesicles have been observed before in tip-sonicated [10] and extruded [15] dispersions. The images suggest that the smaller is φ, the higher is the amount of lens-shaped vesicles. For φ = 100 nm DODAB extruded dispersions consist mainly of quasi-spherical vesicles with the larger ones having diameter of ca. 160 nm, that is, larger than φ (Fig. 5). Smaller vesicles with diameter comparable to φ can also be discerned in the micrograph, as well as small lens-shaped vesicles. The micrograph also reveals that most of the vesicles have mean size larger than φ, indicating that extrusion membranes with φ smaller than the optimal φ ∗ ≈ 200 nm yield vesicles with mean size larger than φ in agreement with the scattering results (Fig. 4 and Table 1). For φ = 400 nm, DODAB vesicles are also quasi-spherical and unilamellar with dimension in the range of ca. 150– 350 nm (Fig. SM3). Thus, extrusion through φ = 400 nm gives DODAB vesicles smaller than φ, again in agreement with the scattering results. Note that no lens-shaped vesicle is seen in Fig. SM3, probably because for larger φ, a small amount of bilayer 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
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and geometry (Figs. 6, 7 and Fig. SM4) [7,13]. Accordingly, DODAC tends to form smaller vesicles than DODAB either in spontaneous [7,11,13] or extruded dispersions [15], in good agreement with the scattering data reported above. DODAC also forms lens-shaped vesicles in extruded dispersions (Fig. 7 and Fig. SM4), as reported in [15]. Extrusion through φ = 100 nm results in spherical unilamellar DODAC vesicles with mean diameter in the range of ca. 80–200 nm (Fig. 6), that is, larger than φ. Extrusion through φ = 400 nm, on the other hand, gives spherical unilamellar vesicles with mean diameters in the range of ca. 250–350 nm (Fig. SM4), that is smaller than φ. In this sample we can also discern some small lens-shaped vesicles that are probably formed by fusion of pieces of fragmented bilayers, which come about after the extrusion or sonication, as reported [10,15]. One should note that the amount of DODAX lens-shaped vesicles is higher the higher is the surfactant concentration (Fig. 7). For this reason sonication produces more lens-shaped vesicles than extrusion, because the former gives more bilayer fragments. This is the case when we compare the present cryo-TEM pictures to those for sonicated DODAB [10] or extruded DODAC [15] vesicles. Extruded unilamellar DODAX vesicles can also be formed at higher surfactant concentrations [13], as shown in the cryoTEM micrograph of an extruded dispersion of 5.0 mM DODAC in water prepared through φ = 100 nm (Fig. 7). Accordingly, up to 5 mM the surfactant concentration plays no key role on the size and structure of extruded DODAC vesicles. The mean size of these vesicles is clearly larger than φ, but smaller than the mean size of the corresponding DODAB extruded vesicles. The larger vesicles have mean diameters of about 150 nm. One should also notice the presence of a higher amount of lensshaped vesicles with different size, indicating that smaller φ and higher surfactant concentration favor formation of lens-shaped vesicles. 5. Summary The vesicle size together with the melting temperature is a very important characteristic that should be well controlled in vesicle-mediated experiments. There is a relationship between the vesicle size and the melting temperature: Tm increases with DODAB or DODAC vesicle size [7]. Vesicles with different size are usually prepared by different methods, such as sonication, extrusion, injection or spontaneous [1–4]. Such methods, however, may influence not only the size but also the vesicle shape. It is well known that DODAB and DODAC assemble spontaneously above Tm as large unilamellar vesicles, and the size of the spontaneous vesicles is usually reduced by sonication or extrusion. The latter, however, is more suitable to form smaller vesicles because it causes less vesicle fragmentation (damage), and thus form more homogeneous vesicles with small amount of lens-shaped vesicles. The vesicle size can be changed as well by varying the solvent conditions like temperature or ionic strength. This work deals with two ways to modify the size of DODAB and DODAC vesicles: extrusion and ionic strength. It reports on the
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proportionality between the vesicle size and the pore diameter (φ) of the extrusion membrane, and the dependence of the vesicle size on the counterion type, indicating that DODAB forms larger vesicles than DODAC. It also shows that lens-shaped vesicles are also formed during the extrusion process, with the amount of lens-shaped vesicles being higher the smaller is φ, probably because the smaller φ gives higher amounts of bilayer fragments that form lens-shaped vesicles. The difference in DODAB and DODAC vesicle size [13] and melting temperature [7] can be explained by the specificity of the counterion binding to the vesicle interfaces. Sonicated DODAX vesicles are as well smaller than the spontaneous [9], but their Tm is larger [7], indicating that sonication not only reduces the size but also changes the vesicle shape (spherical- to lens-shaped). Extrusion, on the other hand, reduces the size and Tm [7] and leaves the vesicle shape unchanged, according to the cryo-TEM images shown in this communication. The specificity of the counterion binding to DODAB and DODAC vesicles is clear from previous reports that point to the lower curvature [9,13] and Tm [7,20] of DODAB vesicles. Since DODAB and DODAC vesicles differ only by the counterion (Br− or Cl− ) the differing size and Tm might be due to counterion specificity to binding the vesicles [7,20]. The data reported here for extruded vesicles are in good agreement with the reported ones for spontaneous, sonicated and injected dispersions [7,9,13,20], indicating that Br− binds to DODAB vesicles yielding larger vesicles (than DODAC) but smaller Tm , that is, more densely packed bilayer, thus allowing Br− to bind more tightly to the vesicle interfaces than Cl− , as reported [7,20]. Particularly interesting for vesicle application is that, irrespective of the presence of small amount of added salt (up to 5 mM), the polydispersity index is rather low (<0.25). NaBr and NaCl increase and decrease, respectively, the size of DODAC and DODAB vesicles, indicating the general trend of vesicles to be larger for DODAB than DODAC as well as the specificity of the counterion binding to the vesicle interfaces. Furthermore, the mean size of the extruded DODAX matches the optimal φ ∗ ≈ 200 nm; below (above) φ ∗ , the main vesicle diameter is larger (smaller) than φ. The data also reveal that besides the ordinary smoothed vesicles, the extrusion process forms lens-shaped vesicles with varying size as already observed for tip-sonicated DODAB [10] and more recently for extruded DODAC vesicles [15]. However, lens-shaped vesicles were observed in smaller amount in extrusion than in sonicated dispersions [10]. The amount of DODAX lens-shaped vesicles is higher in the extruded vesicles through the smaller φ (100 nm) and at higher surfactant concentrations (Fig. 7), probably because the smaller φ and the higher concentration produce more bilayer fragments. The fine-tuning of vesicle size and shape in general claims for narrow size distribution and long term stability. Extrusion rather than sonication seems to be more suitable to form smaller and smooth vesicles relative to the spontaneous method as required in many applications.
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Acknowledgments E.F. thanks CNPq for research grant (Grant 304543/2006-3). The authors are indebted to Dr. J.G. Martinho for many helpful discussions regarding the interpretation of LS data and G. Karlsson for performing the cryo-TEM experiments. Supplementary material The online version of this article contains additional supplementary material. Please visit DOI: 10.1016/j.jcis.2008.03.015. References [1] D.D. Lasic, Liposomes. From Physics to Applications, Elsevier, Amsterdam, 1993. [2] G. Cevc (Ed.), Phospholipid Handbook, Marcel Dekker, New York, 1993. [3] J.M. Fendler, Membrane Mimetic-Chemistry, Wiley–Interscience, New York, 1982. [4] J.B.F.N. Engberts, D. Hoeskstra, Biochim. Biophys. Acta 1241 (1995) 323. [5] A.M. Carmona-Ribeiro, Chem. Soc. Rev. 21 (1992) 207.
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