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DPPC vesicles
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Chemistry and Physics of Lipids 152 (2008) 38–45
Characterization of DODAB/DPPC vesicles Cec´ılia N.C. Sobral a , Marco A. Soto b , Ana M. Carmona-Ribeiro a,∗ a
b
Departamento de Bioqu´ımica, Instituto de Qu´ımica, Universidade de S˜ao Paulo, P.O. Box 26077, 05513-970 S˜ao Paulo, SP, Brazil Departamento de Qu´ımica F´ısica, Facultad de Qu´ımica, Pontif´ıcia Universidad Cat´olica de Chile, Casilla 306, Correo 22, Santiago, Chile Received 18 June 2007; received in revised form 23 November 2007; accepted 20 December 2007 Available online 12 January 2008
Abstract Dioctadecyldimethylammonium bromide (DODAB)/dipalmitoylphosphatidylcholine (DPPC) large and cationic vesicles obtained by vortexing a lipid film in aqueous solution and above the mean phase transition temperature (Tm ) are characterized by means of determination of phase behaviour, size distribution, zeta-potential analysis and colloid stability. The effect of increasing % DODAB over the 0–100% range was a nonmonotonic phase behaviour. At 50% DODAB, the mean phase transition temperature and the colloid stability were at maximum. There is an intimate relationship between stability of the bilayer structure and colloid stability. In 1, 50 and 150 mM NaCl, the colloid stability for pure DPPC or pure DODAB vesicles was very low as observed by sedimentation or flocculation, respectively. In contrast, at 50% DODAB, remarkable colloid stability was achieved in 1, 50 or 150 mM NaCl for the DODAB/DPPC composite vesicles. Vesicle size decreased but the zeta-potential remained constant with % DODAB, due to a decrease of counterion binding with vesicle size. This might be important for several biotechnological applications currently being attempted with cationic bilayer systems. © 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: Dipalmitoylphosphatidylcholine; Dioctadecyldimethylammonium bromide; Large vesicles; Zeta-average diameter; Zeta-potential; Phase transition; Colloid stability
1. Introduction Cationic lipid-like compounds can be used to deliver DNA to cells aiming at gene therapy and drug delivery (Felgner and Ringold, 1989; Zhu et al., 1993; Felgner, 1997). A large variety of synthetic cationic amphiphiles or detergents that are physically similar but chemically different from natural polar lipids has been synthesized such as interesting phosphatidylcholine tri-esters that can be metabolized by cells (Solodin et al., 1996; MacDonald et al., 1999a,b; Rosenzweig et al., 2000) or highly efficient cetylpiridinium amphiphiles (Zuhorn et al., 2002; Zuhorn and Hoekstra, 2002). However, finding optimal lipid phases for transfection is still a matter of considerable interest (Koltover et al., 1998; Koynova et al., 2006). Characterization of mixtures of cationic lipids with other neutral lipids is important not only to determine the effect of helper lipids on lipoplex structure and activity, but also to evaluate the lipid phases arising when cellular lipids interact
∗
Corresponding author. Tel.: +55 11 3091 3810x237; fax: +55 11 3818 5579. E-mail address: [email protected] (A.M. Carmona-Ribeiro).
0009-3084/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.chemphyslip.2007.12.004
with cationic lipids during DNA transfection. Our group has been finding many applications in drug and vaccine delivery for the simple, inexpensive and synthetic cationic lipid dioctadecyldimethylammonium bromide (DODAB) over the years (Carmona-Ribeiro, 1992a,b; Carmona-Ribeiro, 2001; CarmonaRibeiro, 2006). However, its cytotoxicity (Carmona-Ribeiro, 2003) needs to be circumvented possibly by mixing DODAB with a natural zwitterionic lipid. In this work, a systematic study of binary DODAB/dipalmitoylphosphatidylcholine (DPPC) mixed vesicles is performed in order to characterize these dispersions in aqueous solution. A search in the literature reveals that the self-assembly of DODAB/DPPC composite bilayer vesicles in aqueous dispersion was seldom systematically studied. The pioneer work by Linseisen et al. (1996) investigated the phase behaviour of these vesicles using differential scanning calorimetry (DSC) and reported a higher melting temperature than each of the pure components together with a narrow coexistence region for the gel and liquid–crystalline phases at 50% DODAB. Recently, an electrostatic model for mixed cationic-zwitterionic lipid bilayers was developed by Mbamala et al. (2006) based on an extended Poisson-Boltzmann theory. This model recovered the reorientation of the zwitte-
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rionic lipid headgroups, the nonmonotonic behaviour of the average cross-sectional area per lipid, and the absence of these properties for a mixed anionic-zwitterionic membrane. In contrast to the monotonic increase in mean area per molecule in the mixed anionic-zwitterionic bilayer, the cationic-zwitterionic bilayer theoretically displayed contraction followed by expansion as a function of the % cationic lipid in the binary mixture (Mbamala et al., 2006). In this work, a systematic evaluation of phase behaviour, size distribution, zeta-potential and colloid stability is undertaken for DODAB/DPPC vesicles prepared by hydration and vortexing of a lipid film in aqueous solution. The results confirm Linseisen et al. (1996) data on the highest mean phase transition temperature around 50% DODAB, agree with theoretical predictions by Mbamala et al. (2006) and, in addition, suggest that the highest colloid stability for DODAB/DPPC vesicles occurs precisely at the molar ratio where stability of the bilayer structure (molecular packing) is at highest. 2. Materials and methods 2.1. Materials Dioctadecyldimethylammonium bromide (DODAB), and dipalmitoylphosphatidylcholine (DPPC) were obtained from Sigma Chemical Co. and used as such without further purification. Extrinsic fluorescent probes 1,6-diphenyl-1,3,5-hexatriene (DPH) and 6-dodecanoyl-2-dimethyl aminenaphthalene (Laurdan) were purchased from Molecular Probes (Oregon, USA). One should recall that DPH senses the inner parts of the lipid bilayer (Repakova et al., 2005) whereas Laurdan reports the hydrophobic/hydrophilic region (Parasassi et al., 1995). Chemical structures for lipids and probes are given in Table 1. All other reagents were of analytical grade. Water was Milli-Q quality. 2.2. Preparation of lipid dispersions Films of DODAB, DPPC or mixtures of both were prepared from chloroformic stock solutions of the lipids by evaporating the solvent under N2 flux. In order to remove the residual solvent, the films remained under vacuum overnight at room temperature. Vesicles were prepared by hydrating the films with 15 mL of NaCl aqueous solution (1, 50 or 150 mM) at 70 ◦ C by vortexing dispersions until they became homogeneously dispersed. Final lipid concentration was 2 mM. 2.3. Determination of temperature effects on turbidity and fluorescence properties of probes in lipid dispersions The gel-to-liquid-crystalline phase transition of lipid vesicles was followed spectrophotometrically from turbidity dependence on temperature. The dispersions were placed inside a thermostatted quartz cuvette and the temperature was varied at a constant rate of 0.3 ◦ C/min by means of a circulating bath connected to the cuvette holder. Temperature inside the cuvette was determined by means of a Ni/Cr (+) and Ni/Al (−) thermocouple in direct contact with the dispersion. The thermocouple was connected
Fig. 1. The effect of temperature (◦ C) on turbidity at 400 nm for 100% DPPC (on top), or 100% DODAB bilayer dispersions in NaCl 1 mM at pH 6.3 and 2 mM total lipid (in the middle and on the bottom).
to a digital thermometer. Vesicles turbidity at 400 nm was then recorded as a function of temperature with mean phase transition temperatures (Tm ) determined from inflection points in the heating/cooling curves. 2.4. Incorporation of the fluorescent probes to vesicle dispersions and determination of steady state fluorescence measurements as a function of temperature DPH dissolved in DMF, and Laurdan, dissolved in ethanol, were incorporated after the vesicles preparation. A small aliquot
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Table 1 Chemical structures of lipids and probes Lipid or probe
Chemical structure
Dipalmitoylphosphatidylcholine (DPPC)
Dioctadecyldimethylammonium bromide (DODAB)
1,6-Diphenyl-1,3,5-hexatriene (DPH)
6-Dodecanoyl-2-dimethylaminenaphthalene (Laurdan)
(less than 0.5% in volume) of a concentrated solution was added to the dispersions, and incubated by 60 min at temperatures above the bilayer transition temperature. The molar ratio probe/lipid was 1:800. The steady state fluorescence measurements such as fluorescence anisotropy of DPH or generalized polarization (GP) of Laurdan were determined as a function of temperature as previously described (Soto et al., 2002). 2.5. Determination of zeta-average diameter and zeta-potential for lipid dispersions The size distributions for DODAB, DPPC and DODAB/ DPPC vesicles (mean zeta-average diameter, Dz ) were determined by means of a ZetaPlus Zeta-Potential Analyzer (Brookhaven Instruments Corporation, Holtsville, NY, USA) equipped with a 570 nm laser and dynamic light scattering at 90◦ for particle sizing (Grabowski and Morrison, 1983). The mean diameters referred in this work from now on should be understood as the mean hydrodynamic diameter Dz . Zeta-potentials (ζ) were determined from the electrophoretic mobility μ and Smoluchowski’s equation, ζ = μη/ε, where η and ε are medium viscosity and dielectric constant, respectively. All samples were analyzed in polystyrene cuvettes at 25 ◦ C. 2.6. Determination of colloid stability for lipid dispersions The effect of time, % DODAB and ionic strength on colloidal stability of lipid dispersions was evaluated from turbidity at 400 nm as a function of time and photographs.
3. Results and discussion 3.1. The effect of % DODAB on the phase transition of the composite bilayers Turbidimetry has often been used to assess the phase transition of aqueous bilayer dispersions (Lee, 1977; CarmonaRibeiro and Chaimovich, 1983; Nascimento et al., 1998; Vieira et al., 2006). In Fig. 1, turbidity at 400 nm displays steep changes over narrow ranges of temperature as typically expected from cooperative phase transitions in large and closed bilayer vesicles. For pure DPPC dispersions in 1 mM NaCl, two transitions were obtained upon heating: the first around 33.1 ◦ C and the second around 41.1 ◦ C (Fig. 1 on top; Table 1). This closely agrees with previously reported mean temperatures for transitions of the DPPC bilayer vesicles such as 41.2 ◦ C for the main transition determined by fluorescence of Laurdan (Soto et al., 2002) or 34 ◦ C for the pre-transition and 41.0 ◦ C for the main transition determined by differential scanning calorimetry (DSC) (Hayashi et al., 2005). For pure DODAB dispersions in 1 mM NaCl, upon heating, only one main transition took place over a narrow range of temperatures around 44.2 ◦ C as seen from turbidimetry (Fig. 1 middle) and 44.7 ◦ C by Laurdan fluorescence (Fig. 1 bottom). These values closely agree with previously reported 43.5 ◦ C (Nascimento et al., 1998) for a similar DODAB dispersion prepared by chloroform vaporization (Carmona-Ribeiro and Chaimovich, 1983) yielding unilamellar large vesicles with about 500 nm mean hydrodynamic diameter (Nascimento et al., 1998).
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Fig. 2. The effect of temperature (◦ C) on turbidity at 400 nm for mixed DODAB/DPPC bilayer dispersions at 10, 20, 30, 40, 50, 60, 70, 80, 90% DODAB dispersions in NaCl 1 mM at pH 6.3 and 2 mM total lipid.
The effect of % DODAB on the phase transition of DODAB/DPPC large vesicles is shown in Fig. 2 and Table 2. The most remarkable feature for the phase behaviour of these vesicles was the occurrence of a maximum value of the mean phase transition temperature (Tm ), 55.6 ◦ C, around 50% DODAB (Table 2), precisely as theoretically predicted by Mbamala et al.
(2006). For DODAB/DPPC monolayers at the air–water interface, a similar result was obtained by Gonc¸alves da Silva and Romao (2005) around 50% DODAB. This was understood from the large calculated negative excess for the Gibbs free energy of mixing that took place around 50% DODAB, a strong evidence of optimal miscibility between DODAB and DPPC around
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Table 2 The effect of % DODAB on mean phase transition temperatures (Tm ) and physical properties (zeta-average diameter (Dz ), zeta-potential (ζ) and polydispersity (P)) of mixed DODAB–DPPC dispersions %
Tm (◦ C) Heating
0 10 20 30 40 50 60 70 80 90 100
Dz (nm)
ζ (mV)
P
1534 ± 515 ± 385 ± 431 ± 260 ± 421 ± – 336 ± – 370 ± 260 ±
– 66 ± 65 ± 69 ± – 68 ± – 65 ± – 64 ± 56 ±
0.325 ± 0.285 ± 0.268 ± 0.289 ± – 0.280 ± – 0.257 ± – 0.293 ± 0.246 ±
Cooling
Turb400
Laurdan
DPH
33.6/41.1 44.2 46.1 54.4 55.6 55.2 52.1 36.1/51.4 34.9/44.2/50.5 36.0/43.3/48.4 44.2
41.2* – 46.8 – 57.5 55.7 56.0 – 52.0 – 44.8
38.8* – 46.9 – 57.5 54.2 55.2 – 51.6 – 44.0
40.8 46.1 48.1 54.0 54.7 54.4 51.3 50.5 43.4/46.7 41.4 39.9
20 10 4 7 3 3 2 3 3
2 3 4 4 3 4 3
0.020 0.010 0.010 0.010 0.010 0.004 0.010 0.010
Total lipid concentration is 2 mM, final NaCl concentration is 1 mM and pH is 6.3. The phase transitions were followed by means of turbidimetry at 400 nm or fluorescence of Laurdan or diphenylhexatriene (DPH) as a function of temperature. (*) refers to Tm taken from Soto et al. (2002). P refers to sample polydispersity regarding size distribution.
the 1:1 molar ratio. The interactions between adjacent DODAB and DPPC molecules in the monolayer or in the bilayer lead to a tight packing at 50% DODAB because the cationic headgroup of DODAB interacts electrostatically with the negatively charged phosphate moiety of the zwitterionic DPPC headgroup promoting reorientation of the latter and strong hydrophobic interaction between adjacent hydrocarbon chains (Gonc¸alves da Silva and Romao, 2005; Mbamala et al., 2006). At low % DODAB, the average orientation of the zwitterionic DPPC headgroup dipoles is more parallel to the bilayer interface, whereas at 50% DODAB it is perpendicular with choline moieties extended and protruding straight to the outer medium. Thereby, from 0 up to 50% DODAB, molecular packing becomes more tight and Tm increases (Fig. 3). However, further increasing % DODAB beyond 50% represents an increase in lateral electrostatic pressure which ultimately leads to phase separation beyond 70% DODAB (Figs. 2 and 3). Fig. 2 and Table 2 clearly show the occurrence of increasing extents of phase separation for the composite DODAB/DPPC vesicles at and above 70% DODAB as depicted from reappearance of the typical pre-transition usually presented by DPPC domains around 32–35 ◦ C (Kaasgaard et al., 2003). The pre-transition from gel to a rippled phase around 33.6 ◦ C for pure DPPC bilayers shown in Fig. 2 agrees with 33.4 ◦ C previously obtained by means of high-sensitive differential scanning calorimetry (Hayashi et al., 2005). Furthermore, at 80% DODAB and above, it is possible to identify from the transition curves in Fig. 2, the typical phase transition for DODAB separate domains around 43.0–44.0, which had disappeared from the curves obtained at lower % DODAB. From Laurdan fluorescence and turbidity data for large DODAB vesicles (100% DODAB), the main transition occurred at 44.2 and 44.5 ◦ C, respectively (Fig. 2; Table 2), in good agreement with a Tm of 43.5 ◦ C previously reported for a very similar vesicle system (Nascimento et al., 1998). Interestingly enough, in this work, upon increasing % DODAB in the composite vesicles over the 0–50% DODAB range, vesicle size decreased but the zeta-potential remained
practically constant considering the limits of the experimental error (Table 2). This was also observed for DODAB–DPPC vesicles prepared by the ethanolic injection method over the 0–20% DODAB range (Moura and Carmona-Ribeiro, 2007).
Fig. 3. The effect of % DODAB on Tm (◦ C) for DODAB/DPPC dispersions (on top). Data previously obtained by Linseisen et al. (1996) are also plotted as triangles. Tm values taken from heating and cooling curves in Fig. 2 are shown as filled and open circles, respectively. Tm from fluorescent probes Laurdan and DPH are represented by leangles (() Laurdan; (♦) DPH). The effect of % DODAB on colloid stability of mixed DODAB/DPPC bilayer dispersions at 0 (upper line) and 24 h (lower line) after preparation in 1 mM NaCl, pH 6.3 and 2 mM total lipid (on the bottom).
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This suggests that increasing % DODAB, and thereby decreasing vesicle size, does not increase the surface charge density on vesicles, possibly because counterion binding to cationic moieties at the composite bilayer surface is decreasing over the 0–100% DODAB range. Two arguments are in favour of this hypothesis. The fact that pure DODAB vesicles have the lowest size among all composite DODAB/DPPC vesicles (Table 2) and the direct measurements of bromide counterion binding by Scarpa et al. (2002) reporting that counterion binding to DODAB vesicles increases with vesicle size. 3.2. The relationship between stability of the bilayer structure and colloid stability The stability of the bilayer structure as a function of % DODAB is given by Tm whereas the colloid stability for the large composite vesicles is depicted from absence of aggregation and/or sedimentation for the lipidic dispersions over time. At maximal bilayer packing (50% DODAB), composite vesicles present also excellent colloid stability in contrast to the poor colloid stability of pure DPPC or DODAB vesicles. In the case of DPPC vesicles, the poor colloid stability in dispersion is due to occurrence of multilamellar vesicles that sediment in the bottom of the glass tube. Regarding the large DODAB vesicles prepared by vortexing, although electrostatic repulsion prevents formation of multilamellar liposomes, intervesicle aggregation at the secondary minimum of the interaction energy curve has been proposed as a possible cause for their low colloid stability (Carmona-Ribeiro, 1992a,b, 1993). Fig. 3 suggests an interesting correlation between bilayer structure stability and colloid stability lending support to the hypothesis that intrabilayer interactions are very similar to interbilayer ones. The optimal stabilization of the DODAB/DPPC system at 50% DODAB described in Fig. 3 at 1 mM NaCl was further checked against two other NaCl concentrations, 50 and 150 mM. The phase transition for DODAB/DPPC vesicles at 50% DODAB remained very steep and apparently unaffected by ionic strength as depicted from Fig. 4 and Table 3. Nevertheless, the negative difference in turbidity at 400 nm before and after the heating–cooling cycle, which was null at 1 mM NaCl, substantially increased in modulus for vesicles in 50 and 150 mM NaCl (Fig. 4). Therefore, the annealing procedure diminished intervesicle aggregation as usually happens when intervesicle aggregation takes place at the secondary minimum of the interaction energy as a function of the separation distance between interacting vesicles. Although the depth of the secondary minimum has been shown to increase upon increasing ionic strength, secondary minimum are usually shallow so that aggregation is reversible upon increasing temperature. Therefore, aggregation at the secondary minimum would be reversible upon annealing DODAB/DPPC vesicles at 50 and 150 mM NaCl. The effect of ionic strength on self-assembly of DODAB/ DPPC at 50% DODAB to form vesicles was also evaluated by means of dynamic light scattering for vesicles prepared in 1, 50 and 150 mM NaCl. Vesicle size substantially increased amounting to mean hydrodynamic diameters of 389, 878 and 1445 nm at 1, 50 and 150 mM NaCl, respectively (Table 3). This means that
Fig. 4. The effect of temperature (◦ C) on turbidity at 400 nm of mixed DODAB/DPPC bilayer dispersions at 50% DODAB in 1, 50 or 150 NaCl solutions at pH 6.3 and 1 mM total lipid.
decreasing intrabilayer electrostatic repulsion from screening of surface charge is increasing vesicle size. This was also observed for the turbidities at 400 nm and 25 ◦ C for vesicles prepared in the three different ionic strengths (Fig. 4). By dispersing the DPPC lipid alone in water, multilamellar vesicles (liposomes) are formed spontaneously (New, 1990). By dispersing the DODAB lipid in water, single bilayer vesicles or bilayer fragments are formed depending on the dispersion method (Carmona-Ribeiro, 1992a,b; Nascimento et al., 1998; Feitosa et al., 2000; Cocquyt et al., 2004). As a result of the multilamellarity and high density of DPPC vesicles, sedimentation
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Table 3 The effect of [NaCl] on mean phase transition temperatures (Tm ) of DODAB/DPPC dispersions at 50% DODAB and physical properties (zetaaverage diameter (Dz ), zeta-potential (ζ) and polydispersity) [NaCl] (mM) 1 50 150
Tm (◦ C) Heating
Cooling
55.2 55.3 56.2
54.4 54.2 54.8
Dz (nm)
ζ (mV)
Polydispersity
389 ± 08 878 ± 12 1445 ± 43
66 ± 1 – –
0.267 ± 0.008 0.330 ± 0.015 0.343 ± 0.010
The total lipid concentration is 1 mM and pH is 6.3.
Fig. 5. Colloid stability of mixed DODAB/DPPC bilayer dispersions at 0, 50, 100% DODAB in 1, 50 or 150 mM NaCl at pH 6.3.
takes place over time at 1, 50 or 150 mM NaCl whereas single bilayer DODAB vesicles remain well dispersed in aqueous solution only at 1 mM NaCl (Fig. 5). At 50 and 150 mM NaCl, aggregation is visible in the DODAB dispersions (Fig. 5). This low colloid stability for DPPC or DODAB vesicles contrasts remarkably with the high colloid stability of DODAB/DPPC composite vesicles at 50% DODAB at 1, 50 and 150 mM NaCl once again indicating an intimate relationship between stability of the bilayer structure and colloid stability. Acknowledgments FAPESP and CNPq are gratefully acknowledged for financial support. CNCS is the recipient of an undergraduate CNPq fellowship. MAS thanks VRAID Project 2006/31 of Pontificia Universidad Cat´olica de Chile for financial support. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.chemphyslip.2007. 12.004. References Carmona-Ribeiro, A.M., 1992a. Does the DLVO account for interactions between charged spheric vesicles? J. Phys. Chem. 96, 9555–9557. Carmona-Ribeiro, A.M., 1992b. Synthetic amphiphile vesicles. Chem. Soc. Rev. 21, 209–214.
Carmona-Ribeiro, A.M., 1993. Interactions between charged spheric vesicles. J. Phys. Chem. 97, 11843–11846. Carmona-Ribeiro, A.M., 2001. Bilayer vesicles and liposomes as interface agents. Chem. Soc. Rev. 30, 241–247. Carmona-Ribeiro, A.M., 2003. Bilayer-forming synthetic lipids: drugs or carriers? Curr. Med. Chem. 10, 2425–2446. Carmona-Ribeiro, A.M., 2006. Lipid bilayer fragments and disks in drug delivery. Curr. Med. Chem. 13, 1359–1370. Carmona-Ribeiro, A.M., Chaimovich, H., 1983. Preparation and characterization of large dioctadecyldimethylammonium chloride liposomes and comparison with small sonicated vesicles. Biochim. Biophys. Acta 733, 172–179. Cocquyt, J., Olsson, U., Olofsson, G., Van der Meeren, P., 2004. Temperature quenched DODAB dispersions: fluid and solid state coexistence and complex formation with oppositely charged surfactant. Langmuir 20, 3906–3912. Feitosa, E., Barreleiro, P.C.A., Olofsson, G., 2000. Phase transition in dioctadecyldimethylammonium bromide and chloride vesicles prepared by different methods. Chem. Phys. Lipids 105, 201–213. Felgner, P.L., 1997. Nonviral strategies for gene therapy. Sci. Am. 276, 102–106. Felgner, P.L., Ringold, G.M., 1989. Cationic liposome-mediated transfection. Nature 337, 387–388. Gonc¸alves da Silva, A.M., Romao, R.I.S., 2005. Mixed monolayers involving DPPC, DODAB and oleic acid and their interaction with nicotinic acid at the air–water interface. Chem. Phys. Lipids 137, 62–76. Grabowski, E.F., Morrison, I.D., 1983. Particle size distributions from analyses of quasi-elastic light-scattering data. In: Dahneke, Barton E. (Ed.), Measurements of Suspended Particles by Quasi-elastic Light Scattering. Wiley, New York, pp. 199–236. Hayashi, H., Tozaki, K., Ikeuchi, C., Inaba, H., 2005. Phase transitions in dipal mitoylphosphatidylcholine–water and dipalmitoylphosphatidylcholine– CaCl2 aqueous solution system by means of a high resolution and high-sensitive differential scanning calorimeter. Thermochim. Acta 431, 205–211. Kaasgaard, T., Leidy, C., Crowe, J.H., Mouritsen, O.G., Jorgensen, K., 2003. Temperature controlled structure and kinetics of ripple phases in one- and two-component supported lipid bilayers. Biophys. J. 85, 350–360. Koltover, I., Salditt, T., Radler, J.O., Safinya, C.R., 1998. An inverted hexagonal phase of cationic liposome–DNA complexes related to DNA release and delivery. Science 281, 78–81. Koynova, R., Wang, L., MacDonald, R.C., 2006. An intracellular lamellar–nonlamellar phase transition rationalizes the superior performance of some cationic lipid transfection agents. Proc. Natl. Acad. Sci. U.S.A. 103, 14373–14378. Lee, A.G., 1977. Lipid phase transitions and phase diagrams I. Lipid phase transitions. Biochim. Biophys. Acta 472, 237–281. Linseisen, F.M., Bayerl, S., Bayerl, T.M., 1996. 2H NMR and DSC study of DPPC–DODAB mixtures. Chem. Phys. Lipids 83, 9–23. MacDonald, R.C., Ashley, G.W., Shida, M.M., Rakhmanova, V.A., Tarahovsky, Y.S., Pantazatos, D.P., Kennedy, M.T., Pozharski, E.V., Baker, K.A., Jones, R.D., Rosenzweig, H.S., Choi, K.L., Qiu, R., McIntosh, T.J., 1999a. Physical and biological properties of cationic triesters of phosphatidylcholine. Biophys. J. 77, 2612–2629. MacDonald, R.C., Rakhmanova, V.A., Choi, K.L., Rosenzweig, H.S., Lahiri, M.K., 1999b. O-ethylphosphatidylcholine: a metabolizable cationic phospholipid which is a serum-compatible DNA transfection agent. J. Pharm. Sci. 88, 896–904. Mbamala, E.C., Fahr, A., May, S., 2006. Electrostatic model for mixed cationiczwitterionic lipid bilayers. Langmuir 22, 5129–5136. Moura, S.P., Carmona-Ribeiro, A.M., 2007. Adsorption behavior of DODAB/DPPC vesicles on silica. J. Colloid Interf. Sci. 313, 519–526. Nascimento, D.B., Rapuano, R., Lessa, M.M., Carmona-Ribeiro, A.M., 1998. Counterion effects on properties of cationic vesicles. Langmuir 14, 7387–7391. New, R.R.C., 1990. Liposomes: A Practical Approach. Oxford University Press, Oxford, pp. 1–291. Parasassi, T., Giusti, A.M., Raimondi, M., Gratton, E., 1995. Abrupt modifications of phospholipid bilayer properties at critical cholesterol concentrations. Biophys. J. 68, 1895–1902.
C.N.C. Sobral et al. / Chemistry and Physics of Lipids 152 (2008) 38–45 Repakova, J., Holopainen, J.M., Morrow, M.R., McDonald, M.C., Capkova, P., Vattulainen, I., 2005. Influence of DPH on the structure and dynamics of a DPPC bilayer. Biophys. J. 88, 3398–3410. Rosenzweig, H., Rakhmanova, V.A., McIntosh, T.J., MacDonald, R.C., 2000. O-alkyl dioleoylphosphatidylcholinium compounds: the effect of varying alkyl chain length on their physical properties and in vitro DNA transfection activity. Bioconjug. Chem. 11, 306–313. Scarpa, M.V., Maximiano, F.A., Chaimovich, H., Cuccovia, I.M., 2002. Interfacial concentrations of chloride and bromide and selectivity for ion exchange in vesicles prepared with dioctadecyldimethylammonium halides, lipids, and their mixtures. Langmuir 18, 8817–8823. Solodin, I., Brown, C.S., Heath, T.D., 1996. Synthesis of phosphotriester cationic phospholipids. Cationic lipids 2. Synth. Lett. 5, 457–458. Soto, M.A., Sotomayor, C.P., Lissi, E.A., 2002. Effect of gramicidin addition upon the physicochemical properties of dipalmitoyl phosphatidyl
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choline large unilamellar vesicles. J. Photochem. Photobiol. A 152, 79– 93. Vieira, D.B., Pacheco, L.F., Carmona-Ribeiro, A.M., 2006. Assembly of a model hydrophobic drug into cationic bilayer fragments. J. Colloid Interf. Sci. 293, 240–247. Zhu, N., Liggitt, D., Liu, Y., Debs, R., 1993. Systematic gene expression after intravenous DNA delivery into adult mice. Science 261, 209–211. Zuhorn, I.S., Hoekstra, D., 2002. On the mechanism of cationic amphiphilemediated transfection. To fuse or not to fuse: is that the question? J. Membr. Biol. 189, 167–179. Zuhorn, I.S., Oberle, V., Visser, W.H., Engberts, J.B.F., Bakowsky, N.U., Polushkin, E., Hoekstra, D., 2002. Phase behavior of cationic amphiphiles and their mixtures with helper lipid influences lipoplex shape, DNA translocation, and transfection efficiency. Biophys. J. 83, 2096– 2108.
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Characterization of DODAB/DPPC vesicles Cec´ılia N.C. Sobral a , Marco A. Soto b , Ana M. Carmona-Ribeiro a,∗ a
b
Departamento de Bioqu´ımica, Instituto de Qu´ımica, Universidade de S˜ao Paulo, P.O. Box 26077, 05513-970 S˜ao Paulo, SP, Brazil Departamento de Qu´ımica F´ısica, Facultad de Qu´ımica, Pontif´ıcia Universidad Cat´olica de Chile, Casilla 306, Correo 22, Santiago, Chile Received 18 June 2007; received in revised form 23 November 2007; accepted 20 December 2007 Available online 12 January 2008
Abstract Dioctadecyldimethylammonium bromide (DODAB)/dipalmitoylphosphatidylcholine (DPPC) large and cationic vesicles obtained by vortexing a lipid film in aqueous solution and above the mean phase transition temperature (Tm ) are characterized by means of determination of phase behaviour, size distribution, zeta-potential analysis and colloid stability. The effect of increasing % DODAB over the 0–100% range was a nonmonotonic phase behaviour. At 50% DODAB, the mean phase transition temperature and the colloid stability were at maximum. There is an intimate relationship between stability of the bilayer structure and colloid stability. In 1, 50 and 150 mM NaCl, the colloid stability for pure DPPC or pure DODAB vesicles was very low as observed by sedimentation or flocculation, respectively. In contrast, at 50% DODAB, remarkable colloid stability was achieved in 1, 50 or 150 mM NaCl for the DODAB/DPPC composite vesicles. Vesicle size decreased but the zeta-potential remained constant with % DODAB, due to a decrease of counterion binding with vesicle size. This might be important for several biotechnological applications currently being attempted with cationic bilayer systems. © 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: Dipalmitoylphosphatidylcholine; Dioctadecyldimethylammonium bromide; Large vesicles; Zeta-average diameter; Zeta-potential; Phase transition; Colloid stability
1. Introduction Cationic lipid-like compounds can be used to deliver DNA to cells aiming at gene therapy and drug delivery (Felgner and Ringold, 1989; Zhu et al., 1993; Felgner, 1997). A large variety of synthetic cationic amphiphiles or detergents that are physically similar but chemically different from natural polar lipids has been synthesized such as interesting phosphatidylcholine tri-esters that can be metabolized by cells (Solodin et al., 1996; MacDonald et al., 1999a,b; Rosenzweig et al., 2000) or highly efficient cetylpiridinium amphiphiles (Zuhorn et al., 2002; Zuhorn and Hoekstra, 2002). However, finding optimal lipid phases for transfection is still a matter of considerable interest (Koltover et al., 1998; Koynova et al., 2006). Characterization of mixtures of cationic lipids with other neutral lipids is important not only to determine the effect of helper lipids on lipoplex structure and activity, but also to evaluate the lipid phases arising when cellular lipids interact
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Corresponding author. Tel.: +55 11 3091 3810x237; fax: +55 11 3818 5579. E-mail address: [email protected] (A.M. Carmona-Ribeiro).
0009-3084/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.chemphyslip.2007.12.004
with cationic lipids during DNA transfection. Our group has been finding many applications in drug and vaccine delivery for the simple, inexpensive and synthetic cationic lipid dioctadecyldimethylammonium bromide (DODAB) over the years (Carmona-Ribeiro, 1992a,b; Carmona-Ribeiro, 2001; CarmonaRibeiro, 2006). However, its cytotoxicity (Carmona-Ribeiro, 2003) needs to be circumvented possibly by mixing DODAB with a natural zwitterionic lipid. In this work, a systematic study of binary DODAB/dipalmitoylphosphatidylcholine (DPPC) mixed vesicles is performed in order to characterize these dispersions in aqueous solution. A search in the literature reveals that the self-assembly of DODAB/DPPC composite bilayer vesicles in aqueous dispersion was seldom systematically studied. The pioneer work by Linseisen et al. (1996) investigated the phase behaviour of these vesicles using differential scanning calorimetry (DSC) and reported a higher melting temperature than each of the pure components together with a narrow coexistence region for the gel and liquid–crystalline phases at 50% DODAB. Recently, an electrostatic model for mixed cationic-zwitterionic lipid bilayers was developed by Mbamala et al. (2006) based on an extended Poisson-Boltzmann theory. This model recovered the reorientation of the zwitte-
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rionic lipid headgroups, the nonmonotonic behaviour of the average cross-sectional area per lipid, and the absence of these properties for a mixed anionic-zwitterionic membrane. In contrast to the monotonic increase in mean area per molecule in the mixed anionic-zwitterionic bilayer, the cationic-zwitterionic bilayer theoretically displayed contraction followed by expansion as a function of the % cationic lipid in the binary mixture (Mbamala et al., 2006). In this work, a systematic evaluation of phase behaviour, size distribution, zeta-potential and colloid stability is undertaken for DODAB/DPPC vesicles prepared by hydration and vortexing of a lipid film in aqueous solution. The results confirm Linseisen et al. (1996) data on the highest mean phase transition temperature around 50% DODAB, agree with theoretical predictions by Mbamala et al. (2006) and, in addition, suggest that the highest colloid stability for DODAB/DPPC vesicles occurs precisely at the molar ratio where stability of the bilayer structure (molecular packing) is at highest. 2. Materials and methods 2.1. Materials Dioctadecyldimethylammonium bromide (DODAB), and dipalmitoylphosphatidylcholine (DPPC) were obtained from Sigma Chemical Co. and used as such without further purification. Extrinsic fluorescent probes 1,6-diphenyl-1,3,5-hexatriene (DPH) and 6-dodecanoyl-2-dimethyl aminenaphthalene (Laurdan) were purchased from Molecular Probes (Oregon, USA). One should recall that DPH senses the inner parts of the lipid bilayer (Repakova et al., 2005) whereas Laurdan reports the hydrophobic/hydrophilic region (Parasassi et al., 1995). Chemical structures for lipids and probes are given in Table 1. All other reagents were of analytical grade. Water was Milli-Q quality. 2.2. Preparation of lipid dispersions Films of DODAB, DPPC or mixtures of both were prepared from chloroformic stock solutions of the lipids by evaporating the solvent under N2 flux. In order to remove the residual solvent, the films remained under vacuum overnight at room temperature. Vesicles were prepared by hydrating the films with 15 mL of NaCl aqueous solution (1, 50 or 150 mM) at 70 ◦ C by vortexing dispersions until they became homogeneously dispersed. Final lipid concentration was 2 mM. 2.3. Determination of temperature effects on turbidity and fluorescence properties of probes in lipid dispersions The gel-to-liquid-crystalline phase transition of lipid vesicles was followed spectrophotometrically from turbidity dependence on temperature. The dispersions were placed inside a thermostatted quartz cuvette and the temperature was varied at a constant rate of 0.3 ◦ C/min by means of a circulating bath connected to the cuvette holder. Temperature inside the cuvette was determined by means of a Ni/Cr (+) and Ni/Al (−) thermocouple in direct contact with the dispersion. The thermocouple was connected
Fig. 1. The effect of temperature (◦ C) on turbidity at 400 nm for 100% DPPC (on top), or 100% DODAB bilayer dispersions in NaCl 1 mM at pH 6.3 and 2 mM total lipid (in the middle and on the bottom).
to a digital thermometer. Vesicles turbidity at 400 nm was then recorded as a function of temperature with mean phase transition temperatures (Tm ) determined from inflection points in the heating/cooling curves. 2.4. Incorporation of the fluorescent probes to vesicle dispersions and determination of steady state fluorescence measurements as a function of temperature DPH dissolved in DMF, and Laurdan, dissolved in ethanol, were incorporated after the vesicles preparation. A small aliquot
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Table 1 Chemical structures of lipids and probes Lipid or probe
Chemical structure
Dipalmitoylphosphatidylcholine (DPPC)
Dioctadecyldimethylammonium bromide (DODAB)
1,6-Diphenyl-1,3,5-hexatriene (DPH)
6-Dodecanoyl-2-dimethylaminenaphthalene (Laurdan)
(less than 0.5% in volume) of a concentrated solution was added to the dispersions, and incubated by 60 min at temperatures above the bilayer transition temperature. The molar ratio probe/lipid was 1:800. The steady state fluorescence measurements such as fluorescence anisotropy of DPH or generalized polarization (GP) of Laurdan were determined as a function of temperature as previously described (Soto et al., 2002). 2.5. Determination of zeta-average diameter and zeta-potential for lipid dispersions The size distributions for DODAB, DPPC and DODAB/ DPPC vesicles (mean zeta-average diameter, Dz ) were determined by means of a ZetaPlus Zeta-Potential Analyzer (Brookhaven Instruments Corporation, Holtsville, NY, USA) equipped with a 570 nm laser and dynamic light scattering at 90◦ for particle sizing (Grabowski and Morrison, 1983). The mean diameters referred in this work from now on should be understood as the mean hydrodynamic diameter Dz . Zeta-potentials (ζ) were determined from the electrophoretic mobility μ and Smoluchowski’s equation, ζ = μη/ε, where η and ε are medium viscosity and dielectric constant, respectively. All samples were analyzed in polystyrene cuvettes at 25 ◦ C. 2.6. Determination of colloid stability for lipid dispersions The effect of time, % DODAB and ionic strength on colloidal stability of lipid dispersions was evaluated from turbidity at 400 nm as a function of time and photographs.
3. Results and discussion 3.1. The effect of % DODAB on the phase transition of the composite bilayers Turbidimetry has often been used to assess the phase transition of aqueous bilayer dispersions (Lee, 1977; CarmonaRibeiro and Chaimovich, 1983; Nascimento et al., 1998; Vieira et al., 2006). In Fig. 1, turbidity at 400 nm displays steep changes over narrow ranges of temperature as typically expected from cooperative phase transitions in large and closed bilayer vesicles. For pure DPPC dispersions in 1 mM NaCl, two transitions were obtained upon heating: the first around 33.1 ◦ C and the second around 41.1 ◦ C (Fig. 1 on top; Table 1). This closely agrees with previously reported mean temperatures for transitions of the DPPC bilayer vesicles such as 41.2 ◦ C for the main transition determined by fluorescence of Laurdan (Soto et al., 2002) or 34 ◦ C for the pre-transition and 41.0 ◦ C for the main transition determined by differential scanning calorimetry (DSC) (Hayashi et al., 2005). For pure DODAB dispersions in 1 mM NaCl, upon heating, only one main transition took place over a narrow range of temperatures around 44.2 ◦ C as seen from turbidimetry (Fig. 1 middle) and 44.7 ◦ C by Laurdan fluorescence (Fig. 1 bottom). These values closely agree with previously reported 43.5 ◦ C (Nascimento et al., 1998) for a similar DODAB dispersion prepared by chloroform vaporization (Carmona-Ribeiro and Chaimovich, 1983) yielding unilamellar large vesicles with about 500 nm mean hydrodynamic diameter (Nascimento et al., 1998).
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Fig. 2. The effect of temperature (◦ C) on turbidity at 400 nm for mixed DODAB/DPPC bilayer dispersions at 10, 20, 30, 40, 50, 60, 70, 80, 90% DODAB dispersions in NaCl 1 mM at pH 6.3 and 2 mM total lipid.
The effect of % DODAB on the phase transition of DODAB/DPPC large vesicles is shown in Fig. 2 and Table 2. The most remarkable feature for the phase behaviour of these vesicles was the occurrence of a maximum value of the mean phase transition temperature (Tm ), 55.6 ◦ C, around 50% DODAB (Table 2), precisely as theoretically predicted by Mbamala et al.
(2006). For DODAB/DPPC monolayers at the air–water interface, a similar result was obtained by Gonc¸alves da Silva and Romao (2005) around 50% DODAB. This was understood from the large calculated negative excess for the Gibbs free energy of mixing that took place around 50% DODAB, a strong evidence of optimal miscibility between DODAB and DPPC around
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Table 2 The effect of % DODAB on mean phase transition temperatures (Tm ) and physical properties (zeta-average diameter (Dz ), zeta-potential (ζ) and polydispersity (P)) of mixed DODAB–DPPC dispersions %
Tm (◦ C) Heating
0 10 20 30 40 50 60 70 80 90 100
Dz (nm)
ζ (mV)
P
1534 ± 515 ± 385 ± 431 ± 260 ± 421 ± – 336 ± – 370 ± 260 ±
– 66 ± 65 ± 69 ± – 68 ± – 65 ± – 64 ± 56 ±
0.325 ± 0.285 ± 0.268 ± 0.289 ± – 0.280 ± – 0.257 ± – 0.293 ± 0.246 ±
Cooling
Turb400
Laurdan
DPH
33.6/41.1 44.2 46.1 54.4 55.6 55.2 52.1 36.1/51.4 34.9/44.2/50.5 36.0/43.3/48.4 44.2
41.2* – 46.8 – 57.5 55.7 56.0 – 52.0 – 44.8
38.8* – 46.9 – 57.5 54.2 55.2 – 51.6 – 44.0
40.8 46.1 48.1 54.0 54.7 54.4 51.3 50.5 43.4/46.7 41.4 39.9
20 10 4 7 3 3 2 3 3
2 3 4 4 3 4 3
0.020 0.010 0.010 0.010 0.010 0.004 0.010 0.010
Total lipid concentration is 2 mM, final NaCl concentration is 1 mM and pH is 6.3. The phase transitions were followed by means of turbidimetry at 400 nm or fluorescence of Laurdan or diphenylhexatriene (DPH) as a function of temperature. (*) refers to Tm taken from Soto et al. (2002). P refers to sample polydispersity regarding size distribution.
the 1:1 molar ratio. The interactions between adjacent DODAB and DPPC molecules in the monolayer or in the bilayer lead to a tight packing at 50% DODAB because the cationic headgroup of DODAB interacts electrostatically with the negatively charged phosphate moiety of the zwitterionic DPPC headgroup promoting reorientation of the latter and strong hydrophobic interaction between adjacent hydrocarbon chains (Gonc¸alves da Silva and Romao, 2005; Mbamala et al., 2006). At low % DODAB, the average orientation of the zwitterionic DPPC headgroup dipoles is more parallel to the bilayer interface, whereas at 50% DODAB it is perpendicular with choline moieties extended and protruding straight to the outer medium. Thereby, from 0 up to 50% DODAB, molecular packing becomes more tight and Tm increases (Fig. 3). However, further increasing % DODAB beyond 50% represents an increase in lateral electrostatic pressure which ultimately leads to phase separation beyond 70% DODAB (Figs. 2 and 3). Fig. 2 and Table 2 clearly show the occurrence of increasing extents of phase separation for the composite DODAB/DPPC vesicles at and above 70% DODAB as depicted from reappearance of the typical pre-transition usually presented by DPPC domains around 32–35 ◦ C (Kaasgaard et al., 2003). The pre-transition from gel to a rippled phase around 33.6 ◦ C for pure DPPC bilayers shown in Fig. 2 agrees with 33.4 ◦ C previously obtained by means of high-sensitive differential scanning calorimetry (Hayashi et al., 2005). Furthermore, at 80% DODAB and above, it is possible to identify from the transition curves in Fig. 2, the typical phase transition for DODAB separate domains around 43.0–44.0, which had disappeared from the curves obtained at lower % DODAB. From Laurdan fluorescence and turbidity data for large DODAB vesicles (100% DODAB), the main transition occurred at 44.2 and 44.5 ◦ C, respectively (Fig. 2; Table 2), in good agreement with a Tm of 43.5 ◦ C previously reported for a very similar vesicle system (Nascimento et al., 1998). Interestingly enough, in this work, upon increasing % DODAB in the composite vesicles over the 0–50% DODAB range, vesicle size decreased but the zeta-potential remained
practically constant considering the limits of the experimental error (Table 2). This was also observed for DODAB–DPPC vesicles prepared by the ethanolic injection method over the 0–20% DODAB range (Moura and Carmona-Ribeiro, 2007).
Fig. 3. The effect of % DODAB on Tm (◦ C) for DODAB/DPPC dispersions (on top). Data previously obtained by Linseisen et al. (1996) are also plotted as triangles. Tm values taken from heating and cooling curves in Fig. 2 are shown as filled and open circles, respectively. Tm from fluorescent probes Laurdan and DPH are represented by leangles (() Laurdan; (♦) DPH). The effect of % DODAB on colloid stability of mixed DODAB/DPPC bilayer dispersions at 0 (upper line) and 24 h (lower line) after preparation in 1 mM NaCl, pH 6.3 and 2 mM total lipid (on the bottom).
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This suggests that increasing % DODAB, and thereby decreasing vesicle size, does not increase the surface charge density on vesicles, possibly because counterion binding to cationic moieties at the composite bilayer surface is decreasing over the 0–100% DODAB range. Two arguments are in favour of this hypothesis. The fact that pure DODAB vesicles have the lowest size among all composite DODAB/DPPC vesicles (Table 2) and the direct measurements of bromide counterion binding by Scarpa et al. (2002) reporting that counterion binding to DODAB vesicles increases with vesicle size. 3.2. The relationship between stability of the bilayer structure and colloid stability The stability of the bilayer structure as a function of % DODAB is given by Tm whereas the colloid stability for the large composite vesicles is depicted from absence of aggregation and/or sedimentation for the lipidic dispersions over time. At maximal bilayer packing (50% DODAB), composite vesicles present also excellent colloid stability in contrast to the poor colloid stability of pure DPPC or DODAB vesicles. In the case of DPPC vesicles, the poor colloid stability in dispersion is due to occurrence of multilamellar vesicles that sediment in the bottom of the glass tube. Regarding the large DODAB vesicles prepared by vortexing, although electrostatic repulsion prevents formation of multilamellar liposomes, intervesicle aggregation at the secondary minimum of the interaction energy curve has been proposed as a possible cause for their low colloid stability (Carmona-Ribeiro, 1992a,b, 1993). Fig. 3 suggests an interesting correlation between bilayer structure stability and colloid stability lending support to the hypothesis that intrabilayer interactions are very similar to interbilayer ones. The optimal stabilization of the DODAB/DPPC system at 50% DODAB described in Fig. 3 at 1 mM NaCl was further checked against two other NaCl concentrations, 50 and 150 mM. The phase transition for DODAB/DPPC vesicles at 50% DODAB remained very steep and apparently unaffected by ionic strength as depicted from Fig. 4 and Table 3. Nevertheless, the negative difference in turbidity at 400 nm before and after the heating–cooling cycle, which was null at 1 mM NaCl, substantially increased in modulus for vesicles in 50 and 150 mM NaCl (Fig. 4). Therefore, the annealing procedure diminished intervesicle aggregation as usually happens when intervesicle aggregation takes place at the secondary minimum of the interaction energy as a function of the separation distance between interacting vesicles. Although the depth of the secondary minimum has been shown to increase upon increasing ionic strength, secondary minimum are usually shallow so that aggregation is reversible upon increasing temperature. Therefore, aggregation at the secondary minimum would be reversible upon annealing DODAB/DPPC vesicles at 50 and 150 mM NaCl. The effect of ionic strength on self-assembly of DODAB/ DPPC at 50% DODAB to form vesicles was also evaluated by means of dynamic light scattering for vesicles prepared in 1, 50 and 150 mM NaCl. Vesicle size substantially increased amounting to mean hydrodynamic diameters of 389, 878 and 1445 nm at 1, 50 and 150 mM NaCl, respectively (Table 3). This means that
Fig. 4. The effect of temperature (◦ C) on turbidity at 400 nm of mixed DODAB/DPPC bilayer dispersions at 50% DODAB in 1, 50 or 150 NaCl solutions at pH 6.3 and 1 mM total lipid.
decreasing intrabilayer electrostatic repulsion from screening of surface charge is increasing vesicle size. This was also observed for the turbidities at 400 nm and 25 ◦ C for vesicles prepared in the three different ionic strengths (Fig. 4). By dispersing the DPPC lipid alone in water, multilamellar vesicles (liposomes) are formed spontaneously (New, 1990). By dispersing the DODAB lipid in water, single bilayer vesicles or bilayer fragments are formed depending on the dispersion method (Carmona-Ribeiro, 1992a,b; Nascimento et al., 1998; Feitosa et al., 2000; Cocquyt et al., 2004). As a result of the multilamellarity and high density of DPPC vesicles, sedimentation
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Table 3 The effect of [NaCl] on mean phase transition temperatures (Tm ) of DODAB/DPPC dispersions at 50% DODAB and physical properties (zetaaverage diameter (Dz ), zeta-potential (ζ) and polydispersity) [NaCl] (mM) 1 50 150
Tm (◦ C) Heating
Cooling
55.2 55.3 56.2
54.4 54.2 54.8
Dz (nm)
ζ (mV)
Polydispersity
389 ± 08 878 ± 12 1445 ± 43
66 ± 1 – –
0.267 ± 0.008 0.330 ± 0.015 0.343 ± 0.010
The total lipid concentration is 1 mM and pH is 6.3.
Fig. 5. Colloid stability of mixed DODAB/DPPC bilayer dispersions at 0, 50, 100% DODAB in 1, 50 or 150 mM NaCl at pH 6.3.
takes place over time at 1, 50 or 150 mM NaCl whereas single bilayer DODAB vesicles remain well dispersed in aqueous solution only at 1 mM NaCl (Fig. 5). At 50 and 150 mM NaCl, aggregation is visible in the DODAB dispersions (Fig. 5). This low colloid stability for DPPC or DODAB vesicles contrasts remarkably with the high colloid stability of DODAB/DPPC composite vesicles at 50% DODAB at 1, 50 and 150 mM NaCl once again indicating an intimate relationship between stability of the bilayer structure and colloid stability. Acknowledgments FAPESP and CNPq are gratefully acknowledged for financial support. CNCS is the recipient of an undergraduate CNPq fellowship. MAS thanks VRAID Project 2006/31 of Pontificia Universidad Cat´olica de Chile for financial support. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.chemphyslip.2007. 12.004. References Carmona-Ribeiro, A.M., 1992a. Does the DLVO account for interactions between charged spheric vesicles? J. Phys. Chem. 96, 9555–9557. Carmona-Ribeiro, A.M., 1992b. Synthetic amphiphile vesicles. Chem. Soc. Rev. 21, 209–214.
Carmona-Ribeiro, A.M., 1993. Interactions between charged spheric vesicles. J. Phys. Chem. 97, 11843–11846. Carmona-Ribeiro, A.M., 2001. Bilayer vesicles and liposomes as interface agents. Chem. Soc. Rev. 30, 241–247. Carmona-Ribeiro, A.M., 2003. Bilayer-forming synthetic lipids: drugs or carriers? Curr. Med. Chem. 10, 2425–2446. Carmona-Ribeiro, A.M., 2006. Lipid bilayer fragments and disks in drug delivery. Curr. Med. Chem. 13, 1359–1370. Carmona-Ribeiro, A.M., Chaimovich, H., 1983. Preparation and characterization of large dioctadecyldimethylammonium chloride liposomes and comparison with small sonicated vesicles. Biochim. Biophys. Acta 733, 172–179. Cocquyt, J., Olsson, U., Olofsson, G., Van der Meeren, P., 2004. Temperature quenched DODAB dispersions: fluid and solid state coexistence and complex formation with oppositely charged surfactant. Langmuir 20, 3906–3912. Feitosa, E., Barreleiro, P.C.A., Olofsson, G., 2000. Phase transition in dioctadecyldimethylammonium bromide and chloride vesicles prepared by different methods. Chem. Phys. Lipids 105, 201–213. Felgner, P.L., 1997. Nonviral strategies for gene therapy. Sci. Am. 276, 102–106. Felgner, P.L., Ringold, G.M., 1989. Cationic liposome-mediated transfection. Nature 337, 387–388. Gonc¸alves da Silva, A.M., Romao, R.I.S., 2005. Mixed monolayers involving DPPC, DODAB and oleic acid and their interaction with nicotinic acid at the air–water interface. Chem. Phys. Lipids 137, 62–76. Grabowski, E.F., Morrison, I.D., 1983. Particle size distributions from analyses of quasi-elastic light-scattering data. In: Dahneke, Barton E. (Ed.), Measurements of Suspended Particles by Quasi-elastic Light Scattering. Wiley, New York, pp. 199–236. Hayashi, H., Tozaki, K., Ikeuchi, C., Inaba, H., 2005. Phase transitions in dipal mitoylphosphatidylcholine–water and dipalmitoylphosphatidylcholine– CaCl2 aqueous solution system by means of a high resolution and high-sensitive differential scanning calorimeter. Thermochim. Acta 431, 205–211. Kaasgaard, T., Leidy, C., Crowe, J.H., Mouritsen, O.G., Jorgensen, K., 2003. Temperature controlled structure and kinetics of ripple phases in one- and two-component supported lipid bilayers. Biophys. J. 85, 350–360. Koltover, I., Salditt, T., Radler, J.O., Safinya, C.R., 1998. An inverted hexagonal phase of cationic liposome–DNA complexes related to DNA release and delivery. Science 281, 78–81. Koynova, R., Wang, L., MacDonald, R.C., 2006. An intracellular lamellar–nonlamellar phase transition rationalizes the superior performance of some cationic lipid transfection agents. Proc. Natl. Acad. Sci. U.S.A. 103, 14373–14378. Lee, A.G., 1977. Lipid phase transitions and phase diagrams I. Lipid phase transitions. Biochim. Biophys. Acta 472, 237–281. Linseisen, F.M., Bayerl, S., Bayerl, T.M., 1996. 2H NMR and DSC study of DPPC–DODAB mixtures. Chem. Phys. Lipids 83, 9–23. MacDonald, R.C., Ashley, G.W., Shida, M.M., Rakhmanova, V.A., Tarahovsky, Y.S., Pantazatos, D.P., Kennedy, M.T., Pozharski, E.V., Baker, K.A., Jones, R.D., Rosenzweig, H.S., Choi, K.L., Qiu, R., McIntosh, T.J., 1999a. Physical and biological properties of cationic triesters of phosphatidylcholine. Biophys. J. 77, 2612–2629. MacDonald, R.C., Rakhmanova, V.A., Choi, K.L., Rosenzweig, H.S., Lahiri, M.K., 1999b. O-ethylphosphatidylcholine: a metabolizable cationic phospholipid which is a serum-compatible DNA transfection agent. J. Pharm. Sci. 88, 896–904. Mbamala, E.C., Fahr, A., May, S., 2006. Electrostatic model for mixed cationiczwitterionic lipid bilayers. Langmuir 22, 5129–5136. Moura, S.P., Carmona-Ribeiro, A.M., 2007. Adsorption behavior of DODAB/DPPC vesicles on silica. J. Colloid Interf. Sci. 313, 519–526. Nascimento, D.B., Rapuano, R., Lessa, M.M., Carmona-Ribeiro, A.M., 1998. Counterion effects on properties of cationic vesicles. Langmuir 14, 7387–7391. New, R.R.C., 1990. Liposomes: A Practical Approach. Oxford University Press, Oxford, pp. 1–291. Parasassi, T., Giusti, A.M., Raimondi, M., Gratton, E., 1995. Abrupt modifications of phospholipid bilayer properties at critical cholesterol concentrations. Biophys. J. 68, 1895–1902.
C.N.C. Sobral et al. / Chemistry and Physics of Lipids 152 (2008) 38–45 Repakova, J., Holopainen, J.M., Morrow, M.R., McDonald, M.C., Capkova, P., Vattulainen, I., 2005. Influence of DPH on the structure and dynamics of a DPPC bilayer. Biophys. J. 88, 3398–3410. Rosenzweig, H., Rakhmanova, V.A., McIntosh, T.J., MacDonald, R.C., 2000. O-alkyl dioleoylphosphatidylcholinium compounds: the effect of varying alkyl chain length on their physical properties and in vitro DNA transfection activity. Bioconjug. Chem. 11, 306–313. Scarpa, M.V., Maximiano, F.A., Chaimovich, H., Cuccovia, I.M., 2002. Interfacial concentrations of chloride and bromide and selectivity for ion exchange in vesicles prepared with dioctadecyldimethylammonium halides, lipids, and their mixtures. Langmuir 18, 8817–8823. Solodin, I., Brown, C.S., Heath, T.D., 1996. Synthesis of phosphotriester cationic phospholipids. Cationic lipids 2. Synth. Lett. 5, 457–458. Soto, M.A., Sotomayor, C.P., Lissi, E.A., 2002. Effect of gramicidin addition upon the physicochemical properties of dipalmitoyl phosphatidyl
45
choline large unilamellar vesicles. J. Photochem. Photobiol. A 152, 79– 93. Vieira, D.B., Pacheco, L.F., Carmona-Ribeiro, A.M., 2006. Assembly of a model hydrophobic drug into cationic bilayer fragments. J. Colloid Interf. Sci. 293, 240–247. Zhu, N., Liggitt, D., Liu, Y., Debs, R., 1993. Systematic gene expression after intravenous DNA delivery into adult mice. Science 261, 209–211. Zuhorn, I.S., Hoekstra, D., 2002. On the mechanism of cationic amphiphilemediated transfection. To fuse or not to fuse: is that the question? J. Membr. Biol. 189, 167–179. Zuhorn, I.S., Oberle, V., Visser, W.H., Engberts, J.B.F., Bakowsky, N.U., Polushkin, E., Hoekstra, D., 2002. Phase behavior of cationic amphiphiles and their mixtures with helper lipid influences lipoplex shape, DNA translocation, and transfection efficiency. Biophys. J. 83, 2096– 2108.