ORIGINAL ARTICLE

Effect of Sonication on the Thermotropic Behavior of DODABVesicles Studied by Fluorescence Probe Solubilization

Eloi Feitosa • Carlos Roberto Benatti •

Marcio Jose Tiera

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

� AOCS 2009

Abstract The effect of sonication on fluorescence probe

solubilization in cationic vesicles of dioctadecyldimethy-

lammonium bromide (DODAB) was investigated by

steady-state fluorescence of pyrene (Py), trans-diphenyl-

polyenes—diphenylbutadiene (DPB), diphenylhexatriene

(DPH), and their corresponding 4,40-dialkyl derivatives

4B4A and 4H4A fluorescence 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 fluorescence 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 1,4-Diphenyl-1,3-butadiene

DPH 1,6-Diphenyl-1,3,5-hexatriene

ESR Electron spin resonance

I1/I3 Ratio between the intensities of the first and the

third vibronic peaks

If Total fluorescence intensity

Py Pyrene

\r[ Steady-state anisotropy

t-Dpo trans-Diphenylpolyenes

Tm Melting temperature

Uf 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 inner–outer 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 [3–10]. 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

2265, Sao Jose do Rio Preto, SP 15054-000, Brazil

e-mail: eloi@ibilce.unesp.br

C. R. Benatti

Physics Institute, USP, Sao Paulo, SP, Brazil

M. J. Tiera

Chemistry and Geoscience Department, IBILCE/UNESP,

Sao Jose do Rio Preto, SP, Brazil

123

J Surfact Deterg (2010) 13:273–280

DOI 10.1007/s11743-009-1169-8

population of vesicles (RH = 22 nm) [9], while spontane-

ous 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 liquid–crystalline

state transition at a well-defined melting temperature (Tm)

[11]. Below Tm the lipid chains are in a more extended

(gel) state, whereas above Tm they are in a more flexible

(liquid–crystalline) state. The cooperativity of the phase

transition may be influenced by the lipid structural char-

acteristics as the head-group or hydrocarbon chain com-

position 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 [12–18]. 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 vis-

cosity and polarity influence the fluorescence response of

solubilized probes [14, 19–21]. 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

fluorophores solubilized in the bilayer are isomerized and

the competition between the fluorescence and the fluoro-

phore photoisomerization results in a decrease in the total

fluorescence intensity [14, 20]. Above Tm, in the liquid–

crystalline state, the higher mobility of the probes favors

photoisomerization yielding a decrease in the total fluo-

rescence 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 fluorescence

intensities of the first (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-diphenyl-

polyenes (t-Dpo) fluorescent 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 impor-

tant 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 polar-

ity, the spectra represent an average of the emission char-

acteristic 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 fluorescence measurements were made using pyrene

(Py) and 1,4-diphenyl-1,3-butadiene (DPB), 1,6-diphenyl-

1,3,5-hexatriene (DPH), and their 4,40-dialkyl- derivatives

(4B4A and 4H4A) as fluorescence 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 synthe-

sized 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 fluorescence probes used in this

work

274 J Surfact Deterg (2010) 13:273–280

123

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 fluorescence measurements. Bath-

sonicated 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 flask followed by evaporating the

organic solvent under a N2 stream to form a lipid film. The

previously prepared vesicle dispersions were then added to

the probe-containing flask 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 spectro-

fluorometer. In order to check reproducibility, the experi-

ments 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

15–66 �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 circu-

lator (Fisher-Scientific). 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

fluorescence spectra were obtained in the wavelength range

of 370–550 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 fluorescence spectra

according to Eq. 1 [20]:

Uf ¼ Ur

Ar

As

� Fs

Fr

� �ð1Þ

where As (Ar) and Fs (Fr) are, respectively, the solution

(reference) absorbance at the excitation wavelength and the

area under the emission spectra.

The fluorescence anisotropy \r[ was calculated using

the equation [27]

\r ¼ IVV � GIVH

IVV þ 2GIVH

[ ð2Þ

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 wave-

length 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, struc-

ture (e.g., whether uni- or multilamellar), degree of

counterion dissociation and polydispersity, depend on the

sample preparation protocol [4, 5, 8–10, 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 fluorescence

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 liquid–crystalline state.

The melting temperature, as well as the transition coop-

erativity, 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 lit-

erature [4, 5, 18, 30].

Quantum Yield Results

Figure 2 depicts typical fluorescence emission spectra of

4H4A in sonicated DODAB vesicles at 16, 35 and 61 �C,

showing a decrease in the fluorescence intensity when the

J Surfact Deterg (2010) 13:273–280 275

123

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. 3a–d. For both probes Uf

decreases linearly with temperature, and Tm can be

obtained from the intercept of the fitting curves. The

curves for 4B4A give Tm = 43.4 and 47.3 �C, for soni-

cated 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 dis-

persions: Tp = 35.6 �C and Tm = 41 �C (Fig. 3a and b,

respectively).

This value for Tp is in good agreement with the pre-

transition 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 pre-

and 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

giving different fluorescence 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

Figure 4 shows the anisotropy \r[ as a function of tem-

perature 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 incor-

porate 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 investi-

gated 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

fluid and another with low mobility relative to the non-

sonicated 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

400 450 500 550

200

400

600

800

15.8 35.4 61.2 °C

I f

λ (nm)

Fig. 2 Typical fluorescence emission spectra of 4H4A in DODAB/

water sonicated dispersions at selected temperatures

276 J Surfact Deterg (2010) 13:273–280

123

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 soni-

cated [4, 5] and extruded [18] vesicles studied by DSC or

ESR.

0.6

0.7

0.8

0.9

1.0 (a) (c)

(d)(b)0.0

0.2

0.4

0.6

0.8

1.0

10 20 30 40 50 60 700.4

0.5

0.6

0.7

0.8

0.9

1.0

10 20 30 40 50 60 700.0

0.2

0.4

0.6

0.8

1.0

T (oC)

Φ f (no

rmal

ized

)

Fig. 3 Effect of temperature on

the fluorescence quantum yield

for 4H4A (a and b) and 4B4A

(c and d) in DODAB sonicated

and non-sonicated vesicles

0.00

0.07

0.14

0.21

0.28

0.35

0.42

(a) (c)

(d)(b)0.15

0.18

0.21

0.24

0.27

0.30

10 20 30 40 50 60 700.00

0.07

0.14

0.21

0.28

0.35

T (oC)

< r >

10 20 30 40 50 60 700.12

0.16

0.20

0.24

0.28

0.32

DPH

4H4A

DPB

4B4A

Sonic. Sonic.

DPH

DPB

4B4A

4H4A

Fig. 4 Effect of temperature on

the fluorescence anisotropy of

DPH, 4H4A, DPB and 4B4A in

sonicated (a and c) and non-

sonicated (b and d) DODAB

vesicles, as indicated

J Surfact Deterg (2010) 13:273–280 277

123

The Tm values of DODAB in sonicated and non-soni-

cated 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 fluorescence, 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 reflects 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 influenced mainly by the fluorescence character-

istics of the molecules as well as the surrounding moiety.

I1/I3 Ratio of the Py Vibronic Bands

Py exhibits a fine structure in the fluorescence spectra of its

monomers in aqueous solution. The intensity of the first 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 measure-

ment 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 fluorescence [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 fluorescence 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.

Concluding Remarks

The fluorescence 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, 29–32]. Small

deviations in the experimentally obtained Tm may be

10 20 30 40 50 60 700.00

0.07

0.14

0.21

0.28

0.35

sonicated non-sonicated

< r >

T ( °C)

Fig. 5 Effect of temperature on the fluorescence anisotropy of DPH

in DODAB sonicated and non-sonicated vesicles. Curves re-plotted

from Fig. 4

Table 1 Melting temperature (Tm) obtained from fluorescence

quantum yield of 4B4A and 4H4A in sonicated and non-sonicated

DODAB vesicles

Sample 4B4A 4H4A

Sonicated 43.4 — 43.5

Non-sonicated 48.4 35.6 41.2

Table 2 Melting temperature (Tm) obtained by fluorescence anisot-

ropy of DPB, 4B4A, DPH and 4H4A in sonicated and non-sonicated

DODAB vesicles

Sample DPH DPB 4H4A 4B4A

Sonicated 37.3 35.3 38.1 39.8

Non-sonicated 40.7 39.1 39.3 40.3

278 J Surfact Deterg (2010) 13:273–280

123

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.

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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.

Marcio Jose Tiera received his PhD degree in physical chemistry

from Sao Paulo State University, where he is currently a Research

Professor. His main research interests are the synthesis and charac-

terization of surfactants and polymer in colloidal systems.

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