J Surfact Deterg (2010) 13:273280 DOI 10.

1007/s11743-009-1169-8

ORIGINAL ARTICLE

Effect of Sonication on the Thermotropic Behavior of DODAB Vesicles Studied by Fluorescence Probe Solubilization
Eloi Feitosa Carlos Roberto Benatti Tiera Marcio Jose

Received: 3 November 2008 / Accepted: 7 September 2009 / Published online: 12 December 2009 AOCS 2009

Abstract The effect of sonication on uorescence probe solubilization in cationic vesicles of dioctadecyldimethylammonium bromide (DODAB) was investigated by steady-state uorescence of pyrene (Py), trans-diphenylpolyenesdiphenylbutadiene (DPB), diphenylhexatriene (DPH), and their corresponding 4,40 -dialkyl derivatives 4B4A and 4H4A uorescence probes. The data indicate that sonication affects the bilayer polarity, the melting temperature (Tm), and the cooperativity of the melting process due to changes in vesicle morphology. The effect of temperature on the uorescence intensity and yielding Uf and anisotropy \r[ shows that the ionizable probes 4B4A and 4H4A are solubilized close to the vesicle interfaces, whereas the non-ionizable DPH and DPB are deeper in the bilayers. Py solubilization indicates that sonicated vesicles exhibit less densely packed bilayers. Keywords DODAB Pyrene trans-Diphenylpolyenes Fluorescence anisotropy Melting temperature Cationic vesicles Abbreviations DODAB Dioctadecyldimethylammonium bromide DSC Differential scanning calorimetry

DPB DPH ESR I1/I3 If Py \r[ t-Dpo Tm Uf

1,4-Diphenyl-1,3-butadiene 1,6-Diphenyl-1,3,5-hexatriene Electron spin resonance Ratio between the intensities of the rst and the third vibronic peaks Total uorescence intensity Pyrene Steady-state anisotropy trans-Diphenylpolyenes Melting temperature Fluorescence quantum yield

Introduction Vesicles are dynamic structures formed by the competition of the attractive and repulsive interactions between the polar and apolar portions of lipids, and are characterized by a packing parameter (v/al) close to unity [1]. Vesicles contain innerouter interfaces where the hydrophilic head groups are located, and an oil-like thin layer made up by the hydrocarbon chains. The lipid bilayer can host apolar solutes and small polar compounds can be entrapped in the inner aqueous compartment, which allows transport studies [2]. In excess water, double-chain lipids, such as DODAB, self-assemble as large unilamellar vesicles commonly used to mimic biological membranes and function as vehicles for drug delivery [3, 4]. Vesicle properties, such as stability, permeability, melting temperature, degree of ionization and size, among others, depend on the chemical composition of the lipids ionic, strength, temperature, pH, and method of vesicle preparation [310]. It has been reported that sonicated aqueous dispersions of a DODAB consist of one population of bilayer fragments (RH = 8.5 nm) and another

E. Feitosa (&) Physics Department, IBILCE/UNESP, Rua Cristovao Colombo do Rio Preto, SP 15054-000, Brazil o Jose 2265, Sa e-mail: eloi@ibilce.unesp.br C. R. Benatti o Paulo, SP, Brazil Physics Institute, USP, Sa M. J. Tiera Chemistry and Geoscience Department, IBILCE/UNESP, do Rio Preto, SP, Brazil o Jose Sa

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population of vesicles (RH = 22 nm) [9], while spontaneous DODAB aqueous dispersions contain two populations of vesicles with RH = 80 and 337 nm [10]. Such structures provide different solubilization compartments for small probe molecules for spectroscopic studies. Typical vesicles exhibit a gel to the liquidcrystalline state transition at a well-dened melting temperature (Tm) [11]. Below Tm the lipid chains are in a more extended (gel) state, whereas above Tm they are in a more exible (liquidcrystalline) state. The cooperativity of the phase transition may be inuenced by the lipid structural characteristics as the head-group or hydrocarbon chain composition and length [11], counterion type [12] as well as by the method of vesicle preparation [13]. A great effort has been made to understand better the effects of the lipid structure and composition and the bulk characteristics on the vesicle Tm as well as the correlation between the vesicle structure and general properties [1218]. For example, the bilayer barrier to water or small inorganic molecules is higher above than below Tm owing to different lipid packing constrains, and additives in general affect Tm and thus the vesicle permeability [18]. Fluorescence spectroscopy is useful for investigating microenvironments in vesicle bilayers whose local viscosity and polarity inuence the uorescence response of solubilized probes [14, 1921]. Fluorescence spectra of the probes give information about the bilayer organization and state of the lipid chains [19]. In the particular case of the trans-diphenylpolyene (t-Dpo) derivatives the excited uorophores solubilized in the bilayer are isomerized and the competition between the uorescence and the uorophore photoisomerization results in a decrease in the total uorescence intensity [14, 20]. Above Tm, in the liquid crystalline state, the higher mobility of the probes favors photoisomerization yielding a decrease in the total uorescence intensity (If), quantum yield (Uf) and anisotropy (\r[), that can be monitored as a function of temperature to obtain the vesicle Tm. The ratio of the uorescence intensities of the rst (I1) to the third (I3) vibronic bands of the Py emission spectrum has also been widely used to characterize the polarity of vesicle bilayers [19, 21]. In this communication, a number of trans-diphenylpolyenes (t-Dpo) uorescent probes were used to study the effect of sonication on the thermotropic behavior of DODAB vesicles. The data reported is thus complementary to those previously reported by Feitosa and Brown [9] and Benatti et al. [4] who investigated bath-sonicated DODAB vesicles by light scattering and electron spin resonance, respectively. Brito and Marques [5] and Feitosa et al. [18] also reported data on differential scanning calorimetry (DSC) for tip- and bath-sonicated DODAB vesicles. It is known that sonication and extrusion [17, 18, 22] yield formation of lens-shaped DODAB vesicles. The

amount of these structures depends on sonication power and time [17, 22], as well as on the nominal diameter of the extrusion membrane: the smaller the membrane pore diameter, the more lens-shaped vesicles are formed [22]. Since bath-sonication is less powerful than tip-sonication, a lower number of lens-like vesicles is expected in the former. The right choice of spectroscopic probe is thus important for the investigation of the thermal behavior of vesicles since it can be solubilized in different bilayer sites. t-Dpo are planar molecules with a high probability of being incorporated at the vesicle interfaces or within the bilayer [14, 20, 23, 24]. Although the probes in the vesicles may be distributed throughout different sites with different polarity, the spectra represent an average of the emission characteristic of the microenvironment felt by the probes. These probes have been used before to investigate DODAB and DODAC (dioctadecyldimethylammonium chloride) spontaneous vesicles [23, 24], and here they help us to gain insight into the effect of sonication on the thermal behavior of DODAB vesicles.

Experimental Section The uorescence measurements were made using pyrene (Py) and 1,4-diphenyl-1,3-butadiene (DPB), 1,6-diphenyl1,3,5-hexatriene (DPH), and their 4,40 -dialkyl- derivatives (4B4A and 4H4A) as uorescence probes, whose chemical structures are shown in Fig. 1. DODAB was used as purchased from Eastman Kodak. DPH (Aldrich) was recrystallized twice from acetone, and DPB was recrystallized three times from benzene/alcohol as described [20, 25, 26]. 4H4A and 4B4A were synthesized as reported [26]. Py (Aldrich Chemical Co.) was used as received. Milli-Q Plus high quality water was used in sample preparations.

Fig. 1 Chemical structures of the uorescence probes used in this work

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Vesicle Preparation Spontaneously formed vesicle dispersions were prepared by simply mixing DODAB (1 mM) in water at 55 C, as reported [10]. After preparation, the samples were cooled to room temperature and stored at this temperature for at least 24 h before the uorescence measurements. Bathsonicated vesicles were prepared by 90-min sonication of the DODAB dispersions at 55 C using a bath sonicator (Microsonic SX-10). The Probe Incorporation Solutions of DPH and DPB in cyclohexane and Py, 4H4A and 4B4A in chloroform were prepared. The incorporation of these probes in the vesicle compartments were made by placing a known amount of the probe organic solution on the bottom of a glass ask followed by evaporating the organic solvent under a N2 stream to form a lipid lm. The previously prepared vesicle dispersions were then added to the probe-containing ask and the dispersions were kept stirring overnight. The probed vesicles were prepared at very low amphiphile/probe molar ratios, like 200:1 or 250:1. Fluorescence Measurements The data were collected using a Hitachi F-4500 spectrouorometer. In order to check reproducibility, the experiments were measured two times and the mean values were used to construct the diagrams. Anisotropy and Quantum Yields Anisotropy measurements were performed in the range of 1566 C. A time interval of 20 min between two measurements was necessary to ensure the temperature equilibrium of the solution. The temperature of the water-jacketed cell holder in the spectrophotometer was controlled by circulating water connected to a bath circulator (Fisher-Scientic). All measurements were performed at 1.0 mM DODAB for the sonicated or 0.5 mM for the non-sonicated vesicles to minimize scattering effects. The uorescence spectra were obtained in the wavelength range of 370550 nm. Fluorescence quantum yield (Uf) was obtained by exciting the probes at 360 nm for DPH and 4H4A, and 321 nm for DPB and 4B4A. At these wavelengths, we measured the absorbance of the probes in the vesicle and reference solutions. Ur for DPB and DPH in cyclohexane (reference samples) at 25 C is 0.44 and 0.80, respectively [25]. Uf was obtained from the uorescence spectra according to Eq. 1 [20]:

 Uf Ur

Ar Fs As F r

 1

where As (Ar) and Fs (Fr) are, respectively, the solution (reference) absorbance at the excitation wavelength and the area under the emission spectra. The uorescence anisotropy \r[ was calculated using the equation [27] \r IVV GIVH [ IVV 2GIVH 2

where G = IHV/IHH is an instrumental factor and IVV, IVH, IHV and IHH account for the measured emission intensities polarized in the vertical or horizontal detection planes upon excitation with vertical or horizontally polarized light. The I1/I3 Ratio of the Pyrene Vibronic Bands Emission spectra of Py in DODAB vesicles were obtained at 90 to the excitation radiation. The excitation wavelength was 310 nm and the excitation and emission slit widths were adjusted to a resolution of 2.5 nm. The Py emission spectra were obtained around 550 nm in the absence of dimmers, and the I1/I3 ratio obtained, which is sensitive to the polarity of the microenvironment [19, 21, 28].

Results and Discussion The properties of DODAB vesicles, including size, structure (e.g., whether uni- or multilamellar), degree of counterion dissociation and polydispersity, depend on the sample preparation protocol [4, 5, 810, 15, 17, 18, 22, 29 32]. The t-Dpo probes incorporated in vesicles give information on the thermotropic behavior and melting temperature, Tm. They incorporate randomly in the vesicle bilayer and, upon excitation, have their uorescence quantum yield and anisotropy changed according to the conformational state of the lipid chains [25]. At room temperature, DODAB chains are in the gel state, while above Tm & 43 C they are in the liquidcrystalline state. The melting temperature, as well as the transition cooperativity, decreases when the dispersion is sonicated owing to changes in vesicle size and morphology, as shown below, what is in good agreement with data from the literature [4, 5, 18, 30]. Quantum Yield Results Figure 2 depicts typical uorescence emission spectra of 4H4A in sonicated DODAB vesicles at 16, 35 and 61 C, showing a decrease in the uorescence intensity when the

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800

600

15.8 35.4 61.2 C

giving different uorescence signals [20]. Deviation in the Uf curves may be related not only to the probe chemical structure but also to the vesicle size, lipid packing and the vesicle architecture as a result of sonication. Anisotropy Results

If
400

200

400

450

500

550

(nm)
Fig. 2 Typical uorescence emission spectra of 4H4A in DODAB/ water sonicated dispersions at selected temperatures

temperature is raised, indicating that the lipid chains acquire higher mobility on increasing temperature. The effect of temperature on the quantum yield (Uf) of 4B4A and 4H4A in sonicated and non-sonicated DODAB dispersions is shown in Fig. 3ad. For both probes Uf decreases linearly with temperature, and Tm can be obtained from the intercept of the tting curves. The curves for 4B4A give Tm = 43.4 and 47.3 C, for sonicated and non-sonicated DODAB vesicles, respectively (Fig. 3c and d), while those for 4H4A yield Tm = 43.5 C for sonicated and two intercepts for non-sonicated dispersions: Tp = 35.6 C and Tm = 41 C (Fig. 3a and b, respectively). This value for Tp is in good agreement with the pretransition temperature for non-sonicated DODAB vesicles obtained by DSC [18]. Furthermore, sonicated DODAB exhibit no pre-transition temperature [18]. ESR data [4] indicated that non-sonicated DODAB has a tighter bilayer packing than sonicated vesicles, indicating that 4H4A is solubilized along the hydrocarbon chains perpendicular to the vesicle interfaces, allowing it to monitor both the preand main transition temperatures. On heating the samples, as the temperature approaches Tm, Uf varies differently for 4B4A and 4H4A, suggesting that these probes are incorporated in different sites of the vesicle bilayers. Above Tm the probe isomerization increases yielding a linear decrease in Uf at a rate that depends on the probe structure and solubilization. Similar result was also reported by Allen et al. for 4B4A and 4H4A solubilized in phospholipid liposomes [20] and in mixtures of cationic lipids with co-surfactants [23, 24]. It was also observed that an increase in the separation length between the phenyl groups of t-Dpo reduces the probe ability to be aligned with the hydrocarbon chains, thus explaining the different accommodation of these probes in the bilayer

Figure 4 shows the anisotropy \r[ as a function of temperature for DPB, 4B4A, DPH and 4H4A in sonicated and non-sonicated DODAB vesicles. Relative to the ionizable 4H4A or 4B4A, the non-ionizable DPH and DPB incorporate deeper into the bilayer, thus exhibiting a higher mobility and lower \r[. For all vesicle dispersions, the anisotropy of 4B4A is larger than DPB which displays a higher mobility below and above Tm. It is expected that the charged carboxylic group favors the solubilization of 4B4A closer to the vesicle interfaces whereas DPB is deeper into the bilayer. The same behavior was observed for the pair DPH and 4H4A in DODAB (Fig. 4a and b) or in phospholipid vesicles [20]. The anisotropy of the pair DPH and 4H4A in DODAB dispersions (Fig. 4c and d) does not vary much when the temperature is raised to ca. 35 C, but decreases sharply beyond this temperature and around Tm, which was taken at the mid point of this variation. The sharpness of the jump in the anisotropy curve increases when the vesicle size is augmented, meaning that the cooperativity of the melting process increases with the vesicle size. Figure 5 illustrates the effect of sonication on the cooperativity of the melting transition of DODAB vesicles. The change in melting temperature, together with the shape of the transition curve, suggests that DODAB is packed differently in the sonicated and non-sonicated vesicles. Accordingly, the larger the vesicle size, the more compact is the bilayer and the more cooperative is the transition, and consequently the higher is Tm, in good agreement with the effect of size on Tm in extruded DODAB vesicles investigated by DSC [18]. Moreover, according to ESR [4] and dynamic light scattering [9] studies, the sonicated DODAB dispersions have two populations of aggregates, a highly uid and another with low mobility relative to the nonsonicated dispersions that exhibit more homogeneous lipid distributions. These two populations together may reduce the transition cooperativity. Tables 1 and 2 summarize the Tm values for sonicated and non-sonicated DODAB vesicles obtained from Figs. 3, 4, 5. The Tm obtained from the anisotropy of 4B4A and 4H4A are quite the same, indicating that these probes are submitted to similar strains. The Tm obtained from the Uf curves is slightly larger than that obtained by anisotropy. Overall, different methods may give different Tm since different correlation may exist between the melting transition and the physical phenomenon exploited

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J Surfact Deterg (2010) 13:273280 Fig. 3 Effect of temperature on the uorescence quantum yield for 4H4A (a and b) and 4B4A (c and d) in DODAB sonicated and non-sonicated vesicles

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1.0

(a)

(c)

1.0 0.8 0.6 0.4

0.9

0.8

0.7

f (normalized)

0.2 0.6 0.0 1.0 0.9 0.8 0.6 0.7 0.4 0.6 0.5 0.4 10 20 30 40 50 60 70 10 20 30 40 50 60 70 0.2 0.0

(b)

(d)

1.0 0.8

T (oC)

Fig. 4 Effect of temperature on the uorescence anisotropy of DPH, 4H4A, DPB and 4B4A in sonicated (a and c) and nonsonicated (b and d) DODAB vesicles, as indicated

0.42

0.30

Sonic.
0.35 0.28 0.21 0.14 0.07

(a)

Sonic.
4B4A

(c)
0.27

0.24

4H4A DPB

0.21

0.18

DPH
0.15

<r>

0.00

(b)
0.35 0.28 0.21 0.14

(d)
0.32

DPH

4B4A

0.28 0.24 0.20

4H4A
0.07 0.00 10 20 30 40 50 60 70 10 20 30 40 50 60 70

DPB

0.16 0.12

T (oC)

by these methods. For all probes and methods used in this work, however, Tm is higher for non-sonicated than for sonicated vesicles, as expected and reported for

dipalmitoylphosphatidylcholine [33] and DODAB sonicated [4, 5] and extruded [18] vesicles studied by DSC or ESR.

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I1/I3 Ratio of the Py Vibronic Bands Py exhibits a ne structure in the uorescence spectra of its monomers in aqueous solution. The intensity of the rst Py emission peak enhances with the solvent polarity, whereas the third peak shows a minimal intensity variation. Thus, the intensity ratio I1/I3 gives information on the measurement polarity of the Py microenvironment. In general, it is assumed that Py incorporates deeply into the bilayer [34]. However, Abuin et al. [35] reported that Py solubilizes near the bilayer interface, in good accordance with the data reported here. Previous ESR [4], DSC [18, 30] and uorescence [30] results indicate that the thermal behavior of DODAB vesicles depends on the way samples are prepared, that is, on the vesicle architecture. Figure 6 shows the effect of DODAB concentration in sonicated and non-sonicated dispersions on the I1/I3 ratio of Py 10-6 M, at 25 C. I1/I3 decreases to a minimum of 1.3 and 1.5 for DODAB concentrations around 0.1 and 0.6 mM for sonicated and non-sonicated dispersions, respectively, indicating that Py is deeper into the bilayer of sonicated than non-sonicated vesicles. The uorescence anisotropy together with our previous ESR results [4] indicates that the bilayers are less densely packed and the transition is less cooperative for sonicated than non-sonicated DODAB dispersions. In the latter, the transition is sharper due to deeper Py solubilization in the bilayer of sonicated vesicles, giving lower polarity signals. The tighter packing of the bilayers in non-sonicated vesicles has to do with the location of the Py molecules that are more exposed to the solvent (polar) molecules, erroneously indicating a more polar environment. These results are in agreement with those of ESR reported previously [4]. The tighter packing of non-sonicated vesicles reduces the number of water molecules at the bilayer interfaces, thus decreasing the polarity of the interface region relative to the sonicated vesicles.

0.28

0.21

<r>
0.14 0.07 0.00 10 20 30 40 50

60

70

T ( C)
Fig. 5 Effect of temperature on the uorescence anisotropy of DPH in DODAB sonicated and non-sonicated vesicles. Curves re-plotted from Fig. 4 Table 1 Melting temperature (Tm) obtained from uorescence quantum yield of 4B4A and 4H4A in sonicated and non-sonicated DODAB vesicles Sample Sonicated Non-sonicated 4B4A 43.4 48.4 4H4A 35.6 43.5 41.2

Table 2 Melting temperature (Tm) obtained by uorescence anisotropy of DPB, 4B4A, DPH and 4H4A in sonicated and non-sonicated DODAB vesicles Sample Sonicated Non-sonicated DPH 37.3 40.7 DPB 35.3 39.1 4H4A 38.1 39.3 4B4A 39.8 40.3

The Tm values of DODAB in sonicated and non-sonicated dispersions obtained by anisotropy of DPH or 4H4A thus corroborate the general trend of Tm to increase with vesicle size. For sonicated dispersions Tm lies between 37 and 40 C according to the DPH/4H4A and DPB/4B4A pair uorescence, respectively, whereas for non-sonicated dispersions Tm obtained by anisotropy is close to 40 C. Such a small difference in Tm obtained by quantum yield and anisotropy is due to the differing sensitivity of the monitored parameters since Uf reects small changes in the probe molecular geometry and strains the probes undergo [20], whereas any deviation in \r[ from its maximum value indicates tumbling of the probe about its long axis, which is inuenced mainly by the uorescence characteristics of the molecules as well as the surrounding moiety.

Concluding Remarks The uorescence data presented in this communication, together with previously reported results (Refs. [4, 18, 30]) indicate that the thermotropic behavior of DODAB vesicles depends on the way vesicles are prepared. Accordingly, sonication results in smaller vesicles with smaller Tm and a less cooperative melting transition as reported here and elsewhere [5, 9, 18]. The Tm data for sonicated DODAB vesicles here reported are in good agreement with previously reported values obtained by other techniques [4, 2932]. Small deviations in the experimentally obtained Tm may be

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279 7. Carmona-Ribeiro AM, Hix S (1991) pH effects on properties of dihexadecyl phosphate vesicles. J Phys Chem 95:18121817 8. Carmona-Ribeiro AM (1992) Synthetic amphiphile vesicles. Chem Soc Rev 21:209214 9. Feitosa E, Brown W (1997) Fragment and vesicle structures in sonicated dispersions of dioctadecyldimethylammonium bromide. Langmuir 13:48104815 10. Feitosa E, Karlsson G, Edwards K (2006) Extruded vesicles of dioctadecyldimethylammonium bromide and chloride investigated by light scattering and cryogenic transmission electron microscopy. Chem Phys Lipids 140:6674 11. Attwood D, Florence AT (1985) Surfactant systems: their chemistry, pharmacy and biology. Chapman and Hall, New York 12. Feitosa E, Alves F (2008) The role of counterion on the thermotropic phase behavior of DODAB and DODAC vesicles. Chem Phys Lipids 156:1316 13. Marsh D, Watts A, Knowles PF (1977) Cooperativity of the phase transition in single- and multibilayer lipid vesicles. Biochim Biophys Acta 465:500514 14. Allen MT, Miola L, Suddaby BR, Whitten DG (1987) Fluorescent stilbene, diene and triene surfactants as probes of reactivity in amylose inclusion complexes. Tetrahedron 43:14771484 15. Fendler JH (1980) Surfactant vesicles as membrane mimetic agents: characterization and utilization. Acc Chem Res 13:713 16. Blok MC, van Deenen LL, De Gier J (1976) Effect of the gel to liquid crystalline phase transition on the osmotic behaviour of phosphatidylcholine liposomes. Biochim Biophys Acta 433:112 m L, Edwards K (1995) Effect of 17. Andersson M, Hammarstro bilayer phase transitions on vesicle structure and its inuence on the kinetics of viologen reduction. J Phys Chem 99:14531 14538 18. Feitosa E, Barreleiro PCA, Olofsson G (2000) Phase transition in dioctadecyldimethylammonium bromide and chloride vesicles prepared by different methods Chem. Phys Lipids 105:201213 19. Kalyanasundaram K (1987) Photochemistry in microheterogenous systems. Academic Press, New York, p 194 20. Allen MT, Miola L, Whitten DG (1988) Host-guest interactions: a uorescence investigation of the solubilization of diphenylpolyene solute molecules in lipid bilayers. J Am Chem Soc 110:31983206 21. Winnik FM (1993) Photophysics of preassociated pyrenes in aqueous polymer solutions and in other organized media. Chem Rev 93:587614 22. Lopes A, Edwards K, Feitosa E (2008) Extruded vesicles of dioctadecyldimethylammonium bromide and chloride investigated by light scattering and cryogenic transmission electron microscopy. J Colloid Interface Sci 322:582588 23. Feitosa E, Bonassi NM, Loh W (2006) Vesicle-micelle transition in mixtures of dioctadecyldimethylammonium chloride and bromide with nonionic and zwitterionic surfactants. Langmuir 22:45124517 24. Barreleiro PCA, Olofsson G, Bonassi NM, Feitosa E (2002) Interaction of octaethylene glycol n-dodecyl monoether with dioctadecyldimethylammonium bromide and chloride vesicles. Langmuir 18:10241029 25. Allen MT, Miola L, Whitten DG (1987) Temperature effects on uorescence in diphenylpolyene derivatives: structure- and substituent-dependent changes in mechanisms and rates for nonradiative decay. J Phys Chem 91:60996102 26. Velsko SP, Fleming GR (1982) Photochemical isomerization in solution. Photophysics of diphenyl butadiene. J Chem Phys 76:35533562 27. Lakowicz JR (1983) Principles of uorescence spectroscopy, 2nd edn. Plenum Publishing Corporation, New York 28. Kalyanasundaram K, Thomas JK (1977) Environmental effects on vibronic band intensities in pyrene monomer uorescence and

1.9 1.8 1.7

sonicated non-sonicated

I1/I3

1.6 1.5 1.4 1.3 1.2 0.0 0.2 0.4 0.6 0.8 1.0

[DODAB] (mM)
Fig. 6 Effect of DODAB concentration on the I1/I3 ratio of the vibronic peaks of Py in bath-sonicated (circles) and non-sonicated (triangles) DODAB vesicles, at 25 C

attributed the different vesicle preparation protocols, lipid source, solvent conditions or method to obtain Tm. The melting temperature is an important property that can be monitored by different techniques to characterize and control the quality of vesicle dispersions. Fluorescence gives, in addition to Tm, information on the lipid packing into vesicles prepared by sonication or other methods that can be used for different purposes.
Acknowledgments E.F. thanks CNPq and FAPESP. Dr. Laerte Miola is acknowledged for kindly supplying the t-Dpo probes.

References
1. Israelachvili J (1992) Intermolecular and surface forces, 2nd edn. Academic Press, New York n M (2006) Diffusion of solutes in 2. Groth C, Tollgerdt K, Nyde highly concentrated vesicle solutions from cationic surfactants effects of chain saturation and ester function. Colloid Surf A Physicochem Eng Aspects 281:2334 3. Feitosa E, Jansson J, Lindman B (2006) The effect of chain length on the melting temperature and size of dialkyldimethylammonium bromide vesicles. Chem Phys Lipids 142:128132 4. Benatti CR, Feitosa E, Fernandez RM, Lamy MT (2001) Structural and thermal characterization of dioctadecyldimethylammonium bromide dispersions by spin labels. Chem Phys Lipids 111:93104 5. Brito RO, Marques EF (2005) Neat DODAB vesicles: Effect of sonication time on the phase transition thermodynamic parameters and its relation with incomplete chain freezing. Chem Phys Lipids 137:1828 6. Kano K, Romero A, Djermouni B, Ache HJ, Fendler JH (1979) Characterization of surfactant vesicles as membrane mimetic agents. 2. Temperature-dependent changes of the turbidity, viscosity, uorescence polarization of 2-methylanthracene, and positron annihilation in sonicated dioctadecyldimethylammonium chloride. J Am Chem Soc 101:40304037

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280 their application in studies of micellar systems. J Am Chem Soc 99:20392044 Cocquyt J, Olsson U, Olofsson G, der Meeren PV (2004) Temperature quenched DODAB dispersions: uid and solid state coexistence and complex formation with oppositely charged surfactants. Langmuir 20:39063912 Benatti CR, Tiera MJ, Feitosa E, Orlofsson G (1999) Phase behavior of synthetic amphiphile vesicles investigated by calorimetry and uorescence methods. Thermochimica Acta 328:137142 Feitosa E, Barreleiro PCA (2004) The effect of ionic strength on the structural organization of dioctadecyldimethylammonium bromide in aqueous solution. Prog Colloid Polym Sci 128:163 168 Alves FR, Zaniquelli MED, Loh W, Castanheira EMS, Real Oliveira MECD, Feitosa E (2007) Vesiclemicelle transition in aqueous mixtures of the cationic dioctadecyldimethylammonium and octadecyltrimethylammonium bromide surfactants. J Colloid Interface Sci 316:132139 Lichtenberg D, Freire E, Schmidt CF, Barenholz Y, Felgner PL, Thompson TE (1981) Effect of surface curvature on stability, thermodynamic behavior, and osmotic activity of dipalmitoylphosphatidylcholine single lamellar vesicles. Biochemistry 20:34623467 Kodama T, Ohta A, Toda K, Katada T, Asakawa T, Miyagishi S (2006) Fluorescence-probe study of vesicle and micelle formations in a binary cationic surfactants system. Colloids Surf A Physicochem Eng Aspects 277:2026

J Surfact Deterg (2010) 13:273280 35. Abuin E, Lissi E, Aravena D, Zanocco A, Macuer M (1988) A uorescent probe study of the effect of size on the properties of dioctadecyldimethylammonium chloride vesicles. J Colloid Inteface Sci 122:201208

29.

30.

Author Biographies
Eloi Feitosa received his PhD in physical chemistry from Sao Paulo State University, Brazil, where he is currently Adjunct Professor. His main research interests are association colloids, micelles, vesicles and microemulsions, polymer-containing systems and phase behavior of polymer and surfactant systems. Carlos Roberto Benatti received his MS and PhD at Sao Paulo State University, Brazil, under the supervision of Dr. E. Feitosa on cationic vesicle systems. His main interest is on the characterization and application of cationic liposomes. Tiera received his PhD degree in physical chemistry Marcio Jose from Sao Paulo State University, where he is currently a Research Professor. His main research interests are the synthesis and characterization of surfactants and polymer in colloidal systems.

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